COMMON FUND FOR COMMODITIES Banana Improvement Project

advertisement
COMMON FUND FOR COMMODITIES
Technical Paper No. 5
Banana Improvement Project
COMMON FUND FOR COMMODITIES
Technical Paper No. 5
Banana Improvement Project
Common Fund for Commodities
Stadhouderskade 55, 1072 AB Amsterdam, The Netherlands
Postal Address: P.O. Box 74656, 1070 BR Amsterdam, The Netherlands
Tel: (31 20) 5754949
Telex 12331 cfc nl
Fax: (31 20) 6760231
e-mail:Managing.Director@common-fund.org
Copyright - Common Fund for Commodities 2001
The contents of this report may not be reproduced, stored in a data retrieval system
or transmitted in any form or by any means without prior written permission of the
Common Fund for Commodities; except that reasonable extracts may be made for
the purpose of comment or review provided the Common Fund for Commodities is
acknowledged as the source.
Table of Contents
Foreword…………………………………... ………………………………………………1
Acknowledgements………………..……………………………………………………...3
Executive Summary……………………………………………………………………….5
Part I: Banana Improvement Project Completion Report
Introduction .............................................................................................................. 15
Project Structure ...................................................................................................... 15
Research Portfolio .................................................................................................... 18
Project Management ................................................................................................ 40
Public/Private Sector Collaboration ......................................................................... .42
Impact Assessment .................................................................................................. 42
Conclusion ............................................................................................................... 44
Part II: Project Evaluation and Detailed Research Findings
Introduction .............................................................................................................. 47
Background .............................................................................................................. 49
Review of the Project ............................................................................................... 54
Field and Laboratory Evaluation of Diploid Bananas for their Use
in Breeding Schemes (BIP 01) ..................................................................... 54
Field Crosses for Understanding the Inheritance of Black Leaf
Streak Resistance to Bananas (BIP 02) ........................................................ 60
Collection, Characterisation and Evaluation of Nendran Banana in
India (BIP 03) ............................................................................................... 65
Collection, Evaluation and Characterisation of Genetic Resources
and Improved Banana Crop Plants in Vietnam (BIP 04) ............................... 70
Banana Breeding in Brazil (BIP 05) ............................................................... 76
Banana Biotechnology Consortium: Novel Genes for Fungal
Resistance and Post-Harvest Quality (BIP 08) .............................................. 85
i
Development of Transgenic Bananas with Resistance to Banana
Bunchy Top and Banana Mosaic Viruses (BIP 09) ........................................ 92
Genetic Transformation of Prototype Bananas for Black Sigatoka
Resistance (BIP 10) ....................................................................................... 99
Genetic Engineering of Ethylene Biosynthesis in Bananas (BIP 11) ........... 104
Use of Biotechnology to Produce Transgenic Bananas Resistant to
Banana Bunchy Top Virus Infection (BIP 12) .............................................. 108
Elimination of Banana Streak Badnavirus (BSN) from Improved Musa
Germplasm and related Studies on Transmission of Host Plant/Virus/Vector
Interactions (BIP 14) .................................................................................... 112
Variability and Relationships within Populations of Fusarium Oxysporum
FSP Cubense for its Centre of Origin (BIP 15) ............................................ 117
Origin and Distribution of Fungicide-Resistant Strains of
Mycosphaerella Fijensis in Banana Plantations in Costa Rica (BIP 17) ...... 122
Study of Resistance of Banana and Plantains to Nematodes (BIP 18) ....... 128
Identification of Durable Nematode Resistance Sources in Banana and
Plantain (BIP 19) ......................................................................................... 132
Identification of Durable Nematode Resistance Sources in Banana and
Plantain (BIP 20) ......................................................................................... 136
Identification of Durable Nematode Resistance Sources in Banana and
Plantain (BIP 21) ......................................................................................... 141
Breeding Hybrid Musaceae with Resistance to Multiple Diseases,
Especially Black Sigatoka and Panama Disease (BIP 22) .......................... 143
Conclusions and Recommendations ...................................................................... 146
Annex 1: Original Terms of Reference of the BIP .................................................. 153
Annex 2: List of Acronyms ...................................................................................... 154
Part III: Impact Assessment of the Banana Improvement Project
Executive Summary, Impact Assessment of Banana Improvement Project
prepared by P.D. Chudleigh for the World Bank .................................................... 159
Introduction ............................................................................................................ 164
Description of Banana Improvement Programme .................................................. 164
ii
Summary of Principal Outputs of the Programme .................................................. 169
Summary of Actual and Potential Outcomes and Benefits ..................................... 172
Investment Analysis ............................................................................................... 179
Conclusion ............................................................................................................. 188
References ............................................................................................................. 189
Annex 1: Terms of Reference ................................................................................ 190
Annex 2: Summary and Impacts of Individual Projects .......................................... 191
iii
iv
FOREWORD
Bananas (Musa spp.) are cultivated in over 100 countries in the tropical and subtropical regions of the world, where they constitute a major staple food crop for over
120 million people as well as providing a valued source of income through local and
international trade. Musa is the developing world’s fourth most important global food
crop after rice, wheat and maize in terms of gross value of production. The vast
majority of producers are small-scale farmers growing the crop either for home
consumption or for local markets.
Average productivity of bananas in small-scale production of 8 tons per hectare per
year is very low, compared to large-scale plantations of 40 tons per hectare per year.
The low productivity is mainly due to the adverse effects of pests and diseases,
which are most severe for small-scale farmers, who do not have the economic or the
technical capacity to manage such problems. Chemical control methods employed
by large plantations are beyond the means of the majority of smallholder banana
farmers and, moreover, the intensive use of pesticides is likely to have a detrimental
effect on the environment and human health.
The Banana Improvement Project (BIP) was conceived to address this critical
problem of the banana industry and to develop improved varieties for commercial as
well as for staple food production. Eighteen research institutions were involved in the
BIP funded research and development work carried out from January 1994 to
December 1998. The BIP was among the most important projects financed by the
Common Fund for Commodities (CFC), both in terms of financial exposure as well
as in terms of the scope and geographical spread of the work. It was also a unique
project which was based on a collaborative effort of the CFC as financier, the World
Bank as the Project Executing Agency (PEA) and the Food and Agriculture
Organisation (FAO) as the initiator and supervisor of the project.
The BIP was launched with the intention of “…channelling of funds to precisely
targeted research objectives…” with a view to attaining new knowledge with strategic
goals and new profitable products. The specific objectives that the project aspired to
achieve were: (i) to develop and evaluate a range of improved banana varieties with
export potential, incorporating increased productivity with disease resistance; and (ii)
to develop more efficient and integrated disease management practices for Black
Sigatoka disease, based on an improved understanding of disease epidemiology
and pathogen variability in different areas and over different seasons. While the
attainment of these objectives proved to be too ambitious, the BIP has produced
many scientific advances and achieved excellent return on investment. The results
obtained from the project activities can be summarised as follows:
(1)
New knowledge has been produced in the following areas:
•
Collection and characterisation of banana germplasm that will allow
increased effectiveness of the banana breeding programs around the
world.
1
•
Additional information gathered through use of genetic markers,
particularly for disease resistance to Sigatoka, Panama disease, and
nematodes, will be valuable in future work.
•
Improved and refined technology for genetic manipulation, including
genetic transformation. Use of this knowledge will enhance banana and
plantain production whether for cooking or for dessert banana exports.
(2)
Applied outputs with immediate value to banana producers:
•
Black Sigatoka-resistant clones further developed are being used to
help control an outbreak of the disease in the Amazonas region of
Brazil.
•
Strains of the Fusarium Wilt pathogen in a given location can now be
typed using new molecular and genetic techniques. This will be
particularly beneficial to new banana enterprises in determining where
and where not to plant in relation to damaging races of panama
disease.
Although the real value of the BIP can only be assessed over a longer period in
terms of the strategic outputs of the project, the above outputs are significant. Using
the strategic outputs, it will not be too long before diverse banana varieties which are
disease-resistant and have desirable properties for commercial production are
produced. Furthermore, present and future successes will have positive
environmental and health benefits.
Among other benefits that resulted from the project is the identification of research
projects through an internationally competitive research grants scheme carried out
by a Scientific Advisory Panel on the basis of merit-based criteria. Furthermore, BIP
fostered an informal network of the research work being conducted under the BIP,
which will hopefully be sustained after the termination of the project.
It is our strong belief that the project
produced and the applied outputs,
beneficiaries of the BIP, namely the
countries around the globe. The CFC
objective.
results, both the strategic new knowledge
will generate benefits for the intended
smallholder producers in more than 100
will continue with its efforts to realise this
Rolf W. Boehnke
Managing Director
Common Fund for Commodities
2
ACKNOWLEDGEMENTS
The three parts of this report have been prepared by the World Bank, with whose
kind permission this report is being presented, and by consultants employed by the
World Bank. The work performed, which is reflected in this report, has been largely
financed by the Common Fund for Commodities.
The Common Fund for Commodities greatly values the important international
collaboration effort of this project, firstly with the World Bank and the Food and
Agriculture Organisation, secondly with the principal investigators and their
collaborators for their valuable contributions to the success of the project. The
Common Fund for Commodities also pays tribute to the Scientific Advisory Panel
(SAP) and the consultants who evaluated the project; namely:
-
Dr. Edmond de Langhe - University of Leuven – Belgium
-
Professor Günther Kahl – Johann Wolfgang Goethe University –
Germany
-
Dr. Franklin Rosales – the International Network for the Improvement
of Bananas and Plantains (INIBAP); and
-
Dr. P. D. Chudleigh – Agtrans Research, Queensland, Australia for his
Impact Assessment of the project under the sponsorship of the World
Bank
3
4
EXECUTIVE SUMMARY
1. Introduction
“Careful channelling of funds to precisely targeted research objectives with the right
programs will lead to new, useful knowledge and to new profitable products. New
innovative breeding approaches combined with advances in plant biotechnology
offer really new dimensions to banana improvement”.
The Banana Improvement Project (BIP) has in large measure met the challenge
above, quoted from the 1994 background paper on the status of global research on
banana and plantain. This paper, which was commissioned by the World Bank,
provided the backdrop for the 5 year BIP program that brought together some of the
best research talent world-wide to work on bananas.
BIP operated throughout 1994 to 1998 and this project completion report documents
the results of the 18 individual research projects, plus the details of an independent
economic impact assessment that reported a return on investment of 20-33% The
uniquely conceived, and innovatively run, program has achieved its main goals. The
structure of the project, its management, and the competitive grant style of selecting
and funding individual research projects, are described in full detail in this report.
2. Background
Bananas and plantain are grown mainly by smallholder farmers in developing
countries. Bananas are of major importance to food security in the tropics, and their
production provides a vital source of income through local and international trade.
Bananas and plantains are the fourth most important global food crop and export
commodity. They rank first as a fruit.
Only in very recent years has research been able to improve banana breeding
techniques, thus overcoming some of the barriers to genetic improvement of this
crop. Producers are still facing serious pest and disease problems, which are
controlled in large measure by the use of chemical sprays. Resource-poor farmers
often cannot afford expensive pesticides, so they suffer damaging losses. The use of
pesticides is a major health concern in developing countries, so the development of
disease- and pest-resistant varieties is viewed as urgent to protect a vital industry
and the health of banana producers.
A significant contribution to this global effort has been through BIP, an international
5-year research program that used some innovative scientific and management
approaches, notably a competitive grants structure, to harness the talents of
outstanding researchers. BIP started in January 1994, and contracted research
concluded in December 1998, with some of the individual research projects
continuing their activities into 1999 .for completion of their work.
The Project is co-sponsored by the Common Fund for Commodities (CFC), the FAO
Inter-Governmental Group on Bananas (FAO/IGB), and the World Bank. The CFC
committed funding of SDR 2,500,000 (approximately US$3.5m) to the project
portfolio and management of the program. The World Bank, as the Project Executing
5
Agency (PEA), has prepared this completion report covering the management and
research activities resulting from this investment.
BIP had two main objectives:
(1)
(2)
To develop and evaluate improved banana varieties with export
potential, which would incorporate increased productivity and durable
disease resistance through conventional and non-conventional breeding
techniques.
To develop more efficient and integrated disease management practices,
especially for black Sigatoka disease.
BIP funded 18 research projects in four technical areas: (1) Biotechnology, (2)
Germplasm collection and characterisation, (3) Plant improvement, and (4) Pest and
disease management. The intended beneficiaries were producers of dessert
bananas, particularly smallholders. The potential spillovers to producers of plantain
for subsistence were increasingly recognised during the course of the Project.
3. Project Structure
Management:
The Cosponsors and their technical representatives constituted the governing body
of the Project; with the FAO/IGB as the Supervisory Body in accordance with CFC
normal practices.
In order to meet their obligations to the CFC, FAO convened a Technical Advisory
Committee (TAC) to assess project progress. The World Bank acted as the Project
Executing Agency, handling the daily management of the project, and convened a
Scientific Advisory Panel (SAP) to assist in the planning, selection, and monitoring of
research activities. The Bank reported to both the CFC and the FAO/IGB on project
activities by way of twice-yearly Progress and Annual Reports. Annual Meetings of
the Cosponsors were convened to discuss project progress, and met at the same
time and venue as the Annual Scientific Meetings.
Research:
The eighteen projects were chosen through a merit-based process of internationally
competitive research grants. Proposals were reviewed by the SAP against a set of
merit-based criteria, and were selected to form a cohesive and complementary
portfolio to work in parallel toward the project objectives. Projects were monitored by
the SAP on a six-month basis through a review of Progress and Annual Reports.
SAP recommendations were submitted to the PEA for acceptance, and Principal
Investigators were notified of the review’s comments and outcome. Three Annual
Scientific Meetings permitted Principal Investigators to present their work to the
Cosponsors, their technical panels and scientific peers.
6
Reporting:
FAO also produced reports of the Technical Advisory Committee and these were
provided separately to the CFC. A BIP Newsletter was also circulated to report on
progress to interested parties.
4. Research Highlights
Results of research activities are set out below against the two BIP objectives,
followed by results against technically related activities.
4.1
Results against BIP Objectives:
a.
To develop and evaluate improved banana varieties with export
potential, which would incorporate increased productivity and durable
disease resistance through conventional and non-conventional
breeding techniques.
i.
ii.
Refined and utilised two methods of genetic transformation.
Genetically engineered expression of resistance to two viruses, BBTV
and BBMV, into Cavendish and other varieties.
Collected and preserved valuable germplasm, and biotypes of
nematodes and pathogens.
Reversed serious decline in conventional breeding capacities in two
regions.
Contributed to the safe international transportation of important genetic
stocks.
Supplied breeding programs with useful diploid lines as parental
stocks.
iii.
iv.
v.
vi.
b.
To develop more efficient and integrated disease management
practices, especially for black Sigatoka disease.
i.
Expanded options for pest management choices by:
• Identifying important components of durable host plant resistance;
and,
• Adding understanding to the consequences of pesticide use.
ii.
iii.
iv.
Identified sources of resistance in germplasm collections.
Mapped genetic resistance to the black Sigatoka pathogen.
Completed a global map of geographical distribution of Fusarium races
for risk assessment.
Developed an early screening protocol for nematode-resistant
varieties.
v.
4.2
Results against BIP technically-related activities.
AREA I: Biotechnology
7
BIP succeeded in its strategy to develop a molecular toolbox. Numerous methods for
reliably transforming Musa are now a reality. By working in a network of partners,
scientists supported by BIP have exchanged transformation protocols, and verified
results. We now know much more about the molecular manipulation of Musa
including:
•
•
•
•
Receptivity of different types of plant materials;
Dependability of DNA introduction methods;
Gene expression;
Time necessary to transform Musa.
As a consequence of transformation technology, BIP-sponsored research has
produced banana clones with resistance to BBTV and BBMV that are undergoing
glasshouse evaluation and are ready for field testing.
AREA 2: Germplasm Collection and Evaluation
Valuable genetic stocks of wild and domesticated Musa species have been placed in
accessible collections in India and Vietnam through the sponsorship of BIP. The
future value of these preserved stocks will only be fully realised as scientists
evaluate their genetic attributes and combine traits using conventional and molecular
tools. Had these stocks not been preserved they would likely have been lost to future
generations.
AREA 3: Plant Improvement
The projected demise of two critically important banana breeding programs in the
Americas was reversed by the timely awarding of BIP funding. These grants
permitted plant breeders to continue to generate improved progeny and to evaluate
genetic selections for industry and subsistence farmers alike. The practical
implication of this outcome is the critical need for conventional genetics programs to
help find the solutions to production and protection problems faced by banana
producers.
AREA 4: Pest and Disease Management
BIP’s dual strategy of applying genetic transformation and DNA selection marker
methods for adding host plant resistance to banana clones for black Sigatoka
resistance made significant progress. Transformation technologies (see Area 1) are
now available to introduce single gene resistance should suitable candidate genes
be found. Secondly, marker assistance selection technologies for multigenic
resistance for black Sigatoka appear promising. The value of this strategy is in the
expectation that multigenic forms of resistance should be more durable. Strategies
for managing black Sigatoka pest resistance in M. fijiensis proved unreliable in those
spray-free periods failed to let the pathogen rebound to sensitive forms. This
research outcome removes pesticides as a sustainable solution to black Sigatoka
and increases the need for nonpesticide-based IPM programs.
Resistance to nematodes is a high priority for bananas in as much as chemical
controls are costly and hazardous. The sources of resistance identified, as well as
8
the screening methods developed through BIP-sponsored research are a significant
success.
5. Impact Assessment
The World Bank commissioned an economic impact study of BIP in late 1998. The
key comments and findings below have been extracted from that report.
1.
New knowledge has been produced in the following areas:
•
•
•
•
2.
Collection and characterisation of banana germplasm that will allow
increased effectiveness of the banana breeding programs around the
world.
Increased efficiency of breeding programs through improved testing,
screening and typing methods, and development of protocols. Additional
information gathered through use of genetic markers, particularly for
disease resistance to Sigatoka, Panama Disease and nematodes, will also
be valuable in future work.
Improved and refined technology for genetic manipulation, including
transformation processes for banana and improved knowledge of disease
resistance mechanisms in banana.
Use of this knowledge will enhance banana and plantain production,
whether for cooking, domestic consumption as dessert bananas, or for
export. Applying this knowledge in future to provide real improvements for
producers and exporters will require further R&D investment.
Applied outputs of some of the projects will have immediate value to and
impact on industry. The following stand out:
•
•
Black Sigatoka-resistant clones further developed in BIP projects are
being used to help control an outbreak in the Amazonas region of Brazil.
Strains of Fusarium in a given location can now be typed using BIPgenerated techniques. This will be particularly beneficial to new banana
enterprises in determining where and where not they can be planted in
relation to damaging races of Panama disease.
Although these outcomes are significant, the real value of BIP-supported work will be
the long-term strategic knowledge flowing from the BIP projects. Landmark events
using some of this new knowledge might be the development of a Cavendish or
alternative export banana type that is disease resistant. Other developments that
might result include socio-economic, environmental, and health benefits. The
economic benefits would include reduced production costs (few chemicals required);
the environment would be improved through less chemical usage; and banana
producers would suffer fewer health problems by not having to handle as much toxic
chemical material (organ damage to an estimated 10% of workers according to
Swennen, 1997). Improvements would lead to better health of workers, higher
productivity, arid an overall increase in the quality of life of communities in bananaproducing areas. The BIP-supported research has been complemented by other
significant investments in R&D.
9
3.
Leverage of Funds
BIP also leveraged substantial other investments in banana improvement:
•
•
•
•
4.
Building on the original CFC investment, further funds have been attracted
to BIP, both within the projects and outside: i.e. WOB and BDC, Belgium
for the nematode consortium personnel positioned around the world in
various breeding programs.
Funding from the New Zealand Government for post-harvest practices in
Vietnam.
Funding from the University of Hong Kong and the Governors Fund in
Hawaii.
Conservative estimate of funds contributed is US$ 2 million.
Increased Collaboration
A longer-term benefit to industry will be the enhanced scientific co-operation in
banana improvement work as a result of the creation of a Biotechnology Consortium
(Katholeike Universiteit Leuven, Belgium, Boyce Thompson Institute, USA, and
Queensland University of Technology, Australia). This grouping resulted in staff
exchanges and the transfer and refinement of particle bombardment and
Agrobacterium transformation and regeneration technology. In a similar way BIP
fostered the creation of a "nematode consortium”, which has resulted in improved
screening techniques leading to a better understanding of nematode resistance.
•
•
•
•
•
•
•
•
Formation of consortium in biotechnology enabling the refinement of both
particle bombardment and Agrobacterium transformation techniques.
Formation of nematode consortium strengthened during the project with
funding from VVOB and BDC that will continue past the completion of BIP.
Collaboration between Australian and Vietnamese scientists, exemplified
in the training of Vietnamese scientists by institutes in Australia.
Collaboration between Australian and CIRAD scientists in screening for
Fusarium Wilt on CIRD breeding diploids.
Collaboration between Costa Rica and Brazil in screening breeding
materials.
Attraction of new research providers into banana improvement.
Additional permanent staff position created in CIRAD focused on banana
improvement.
Continued generic resources provided by Belgium through nematode
consortium.
The impact assessment study indicated that BIP should probably provide a rate of
return on investment of at least 20% to 33%. Conservative assumptions were used
in the analyses, and additional analyses showed robustness of the results. This
demonstrates that further investments in banana improvement research will be well
justified.
10
6.
Conclusion
BIP results have provided methods for transformation and regeneration of bananas,
and have moved world-wide banana producers closer to a disease and pestresistant transgenic banana being a reality. Considerable progress has been made
in identifying new germplasm suitable for breeding, and ways have been developed
to improve the breeding
BIP has, therefore, made an important contribution in both the provisions of research
results, and catalysing collaborative activities and co-financing through the
innovative process of utilising a competitive research grants scheme. This enabled
the identification of research providers of excellent quality, and the investment of
resources in a portfolio of research projects of direct relevance to the objectives of
the program.
The World Bank’s overarching commitment is to the alleviation of poverty. The
contribution this Project has made toward the development of resistant varieties, and
new control methods of banana and plantain production, is a positive step in
reaching this goal.
11
12
PART I
Banana Improvement Project Completion Report
13
14
1. Introduction
The Banana Improvement Project (BIP) commenced on 1 January 1994, with
contracted research activities reaching completion on the 31st December 1998. The
Project is cosponsored by the Common Fund for Commodities (CFC), the FAO InterGovernmental Group on Bananas (FAO/IGB), and the World Bank (IBRD). The CFC
committed funding of SDR 2,500,000 (approximately US$3.5 million) toward the
research portfolio and management of the program. This report, prepared by the
World Bank, documents the project management structure, research activities under
a competitive grants program, and results achieved from this investment.
Objectives
BIP was conceived and specially structured to make a significant contribution toward
the improvement and productivity of banana, by using higher-yielding, diseaseresistant varieties, and seeking ways to reduce the cost of production, especially the
cost of pesticide application. A two-pronged approach was taken, based on
commissioning research concerned with:
(1) Genetic improvement, by collecting, developing, and evaluating new
banana varieties with export potential, incorporating higher yields and
durable disease resistance.
(2) Improved and integrated pest management practices to reduce pesticide
use.
The intended beneficiaries of the Project were producers of dessert bananas,
particularly smallholders, and subsistence farmers growing plantain and banana for
their own or local consumption. Other targeted beneficiaries were plantation workers,
because of the focus on improved pest management practices, and the public at
large, by decreasing environmental degradation through a reduction in pesticide use
and establishment of plantations in soils and regions that are not optimal to support
banana production. Consumers world-wide will benefit through access to a selection
of banana varieties grown under more environmentally neutral conditions.
2. Project Structure
2.1
Management: The Cosponsors and their technical representatives constituted
the governing body of the Project, with the FAO/IGB as the Supervisory Body
in accordance with CFC normal practices. The FAO convened a Technical
Advisory Committee (TAC) to assess overall Project progress. The World
Bank acted as the Project Executing Agency, handling the daily management
of the Project, and convened a Scientific Advisory Panel (SAP) to assist in the
planning, selection, and monitoring of the research activities. The Bank
provided twice-yearly progress and annual reports, to the CFC and the
FAO/IGB on Project activities. Annual Meetings of the Cosponsors were held
to discuss progress at the time of the Annual Scientific Meeting.
15
2.2
Project Planning and Implementation:
2.2.1 Planning:
In the early 1980s, FAO and its Intergovernmental Group on Bananas
identified the need for greater investment in banana research in response to
the increasing cost of production and falling returns to growers. More frequent
fungicide applications were required because the major disease, black
Sigatoka, continued to spread into banana- and plantain-growing areas. At the
same time, fungicide-resistant strains were appearing. Consumers were
becoming more concerned about environmental degradation resulting from
the increased spraying. Nematicides in particular were being more widely
used in an effort to control this major nematode pest, and new strains of
Fusarium Wilt were emerging that were completely destroying new plantations
in the tropics and subtropics. New virus diseases were also being recognised,
and banana streak virus in particular was threatening the viability of the
international exchange of Musa germplasm to breeding programs. At the
same time, markets were expanding, and presenting an opportunity to
producing countries to increase their export trade. This was of particular
importance to the smaller exporting countries, particularly the ACP countries,
which traded banana as their most significant export commodity.
The World Bank supports banana and plantain research through its
contributions to the Consultative Group on International Agricultural Research
(CGIAR). The Bank had undertaken a review of international support to
banana and plantain research by participating in a CGIAR Task Force whose
recommendations were complementary to the plans of FAO. The FAO/IGB’s
overriding interest in Musa was focused on the banana export crop, but they
also shared the interest of the World Bank in addressing the problems of
growers of banana and plantains for subsistence, and the importance of both
domestic and export production for poverty alleviation.
The banana and plantain export crop accounts for approximately 10% of total
production, the remainder being a subsistence crop grown widely in Africa
and the Asia/Pacific region. The two organisations therefore collaborated in
designing and presenting a project proposal to the 12th Banana Improvement
Project, Completion Report, December 1998 CFC, with the resultant Project
Agreement being signed during International Centres Week of the CGIAR in
Washington, D.C., in October 1993.
2.2.2 Implementation:
The BIP Secretariat was located in the Bank within the Agricultural Research
and Extension Group (ESDAR). A Technical Manager was appointed with
responsibility for Project implementation, supported by a Project Co-ordinator.
Initial activities of the Bank in the first half of 1994 were the preparation and
dissemination of a call for proposals, and the identification of members of a
Scientific Advisory Panel (SAP). A review of current banana and plantain
improvement programs was also commissioned by the World Bank and a
16
report was prepared on the activities of the current banana improvement
programs, and opportunities and innovative ideas identified that could be
useful to exploit within the new Project. This was of particular importance due
to the lack of success in breeding a disease-resistant commercial dessert
banana over the past 70 years.
The Bank consulted with representatives of the private sector and the donor
community during the initiation of BIP. Consultative workshops were held
immediately preceding meetings of the Scientific Advisory Panel (SAP) in
June and December 1994. The June meeting in Washington, D.C., confirmed
the relevance of the aims of BIP, with black Sigatoka being the major
constraint, and the use of pesticides, involving increasing costs,
environmental degradation, and worker health and safety concerns.
Discussion also focused on the newly available techniques of biotechnology,
with industry indicating that a genetically engineered Cavendish would be of
great interest to them. The use of biotechnology was the focus at the second
meeting held in Houston, Texas, where the issue of the likelihood of success
in using transformation techniques with subsequent regeneration was
discussed.
2.3
Research Strategy:
As a result of these activities, the research strategies considered best able to
address the objectives of BIP were agreed as follows:
i.
New banana varieties:
•
•
•
ii.
Biotechnology-based breeding to genetically modify presently
cultivated clones (especially Cavendish types) for a few key
characteristics such as black Sigatoka resistance, resistance to banana
bunchy top virus (BBTV), and delayed ripening to extend shelf life.
Conventional breeding based on the crossing of improved diploid to
generate new hybrids.
Novel breeding schemes based on triploid resynthesis, to develop new
banana types with export potential, suitable for niche markets.
Better disease control practices:
•
•
Identification of the variation in the major pathogens causing black
Sigatoka disease and Fusarium Wilt, to underpin conventional and
non-conventional breeding.
Epidemiology and ecology of black Sigatoka disease, as a basis for
better management of the disease aimed at reducing the frequency of
fungicidal applications, while maintaining effective control.
The Project used the innovative mechanism of an internationally competitive
research grants program to identify research projects. Two calls for proposals
resulted in over 100 submissions from leading research institutes world-wide.
Scientists from national, regional, and international research institutes were
invited to submit proposals, and combined proposals were particularly
17
encouraged. Proposals were received from institutes in both banana importing
and exporting countries, and the public and private sector.
3. Research Portfolio
Proposals were reviewed by the SAP against a set of merit-based criteria, and were
selected in order to form a cohesive and complementary portfolio to work in parallel
toward Project objectives. A brief outline of individual project results is given below,
organised in technically related areas of activity.
A number of institutes will be continuing BIP experiments into 1999 due to the need
for materials to mature prior to analysis. Field testing of transgenic plants produced
during BIP is currently under final negotiation to ensure that regulatory procedures
are addressed. Further results will therefore become available in mid-1999.
AREA I: Biotechnology
PN8: Biotechnology Consortium: Novel genes for fungal resistance and post-harvest
quality.
Boyce Thompson Institute for Plant Research, USA
Activity: The researchers sought to create a “molecular toolbox” in collaboration with
PN9 and PNl0. The ‘toolbox’ is for use in isolating and manipulating new genetic
elements from Musa SPP. The toolbox will include Grand Nain genomic libraries,
cDNA libraries representing mRNAs present in a variety of banana tissues at
different developmental stages or under pathogen attack, and a variety of
Agrobacterium strains for Musa transformation. Work at BTI was related to genes
associated with fruit ripening and fungal resistance.
Background: Development of the toolbox will allow the creation of new banana
germplasm, with resistance to virus and fungal diseases that now limit production
and increase production costs. It will also provide a better fundamental
understanding of the mechanisms controlling qualitative and quantitative changes in
gene expression that occur during banana fruit development or under pathogen
attack.
Results:
•
•
•
•
•
Identification and characterisation of eleven distinct classes of differentially
expressed cDNAs from the pulp of ripening Grand Nain banana fruit. Efforts
continue toward the isolation of regulatory elements governing the expression
of mRNAs.
Transformation cassettes incorporating these regulatory elements were built.
Identification of previously uncharacterised mRNAs from banana roots and
leaves.
Development of a transient assay system based on protoplasts derived from
readily available banana pulp.
cDNA libraries were generated from leaves of Gros Michel, Malaccensis, and
a Cavendish variety that were challenged by Mycosphaerella fijiensis.
18
•
Rapidly doubling Grand Nain embryonic cell suspensions have been
developed.
Outcome: This project was completed in February 1997. Since then further work has
been carried out in the various areas above. Cloned genes have been distributed to
other laboratories both within the consortium and to the wider research community.
Further refinement of the Agrobacterium-mediated transformation system has been
undertaken by identification of alternative sources of starting materials, the aim being
to reduce the number of chimeric plants obtained. Several banana-derived promoter
elements are being isolated and characterised that will allow for the tissue- or
developmental-specific expression of foreign genes in transgenic banana plants. The
project also gained a better understanding of the molecular events that occur during
banana fruit development and ripening, and those events that are associated with
pathogen challenge in fungal-resistant and non-resistant banana varieties. The
‘molecular toolbox’ has provided many laboratories with molecular biology materials.
Impact: The major impact of this project has been the additional knowledge
generated on ethylene control, and the further development of the Agrobacterium
method of transformation. The project contributed to strengthening the collaborative
activities of the BTI group as a member of the biotechnology consortium, with visiting
scientists, and through sharing of technologies.
PN9: Biotechnology Consortium: The development of transgenic bananas with
resistance to banana bunchy top virus (BBTV) and banana bract mosaic virus
(BBMV).
Queensland University of Technology (QUT), Australia
Activity: In collaboration with other members of the Biotechnology Consortium (KUL,
Leuven; BTI, USA); and with the University of Hawaii, (USA), the scientists
attempted to control banana bunchy top virus (BBTV) and banana bract mosaic
viruses (BBMV) through the development of multivirus resistant transgenic bananas.
The team utilised current and emerging knowledge of BBTV resistance, and
characterisation of the BBMV genome, to develop transformation cassettes.
Transformation proceeded initially on single resistance transgenes, and ultimately to
multiple resistance transgenes. Transformed material was screened for resistance,
and promising transgenic lines prepared for field trials.
Background: BBTV is largely controlled in Australia by removal of infected plants, a
costly undertaking. Cavendish is particularly susceptible to the disease, and can
reduce yields by up to l00%, but averages at about 30%. BBMV creates losses of
between 20% and 40%. A range of cassettes has been generated that potentially
confer resistance to BBTV and BBMV in transgenic bananas, and have been used in
the transformation and regeneration of Bluggoe and Cavendish embryos. Three
different BBTV, and one BBMV, resistance cassette were used, resulting in
regenerated plantlets that are ready for virus challenge in the field. A range of
promoters for transgene expression continues to be analysed for use in generating
virus resistance.
19
Results:
•
•
•
•
•
•
Identification of potentially three different BBTV resistance genes, and one
BBMV gene.
Cavendish and Goldflnger plants have been regenerated from embryonic cell
suspensions, using the particle bombardment transformation method.
Cavendish “Grand Nain” embryos have been transformed and regenerated
with GUS, GFP, two different BBTV resistance cassettes, and one BBMV
resistance cassette.
Additional embryonic cell suspensions have been generated, and new virus
resistance constructs have been designed that are expected to be more
efficient in generating resistance.
Planning for initial greenhouse evaluation and field trials is progressing to
ensure that biosafety regulations are addressed, and to minimise the
possibility of loss of material.
Three unexpected problems arose during the project that have delayed the
realisation of final results: Cavendish transformation and regeneration is
slower and less efficient than Bluggoe; the erratic rate of multiplication of
transgenic lines for virus challenge, and identification of a suitable screening
site for BBMV proved more difficult than expected, but this has now been
overcome.
Outcome: The technology developed under the project is directly applicable to the
generation of transgenic resistance to other diseases. The development of an
efficient transformation and regeneration protocol for Cavendish, and identification of
promoters to drive high level transgene expression, can be utilised together to
express novel genes in banana. It is expected that of the current transgenic lines
produced, 50% will test resistant. This will increase as the technique is further
refined.
Impact: Technology to generate potentially virus-resistant Musa varieties, including
transformation, regeneration, and expression of new genes, has been demonstrated.
Confirmation of resistance has yet to be tested in the field, and it is expected that
resistance breakdown will be in the order of 5-10% within a period of 10 years of
release of resistant types. Technologies developed are relevant not only to viral
resistance, but also to other diseases of banana and plantain types. Access to BIP
influenced the QUT approach, therefore playing a positive role in the successful
results.
PNlO: Biotechnology Consortium: Genetic transformation of prototype bananas for
black Sigatoka and Fusarium resistance.
Katholeike University, Leuven, Belgium
Activity: In collaboration with PNS and PN9, the main focus of the project was the
investigation and optimisation of a reliable and transferable transformation system.
Agrobucterium-mediated transformation and particle bombardment techniques were
tested for their suitability and efficiency for large-scale applications. Other project
activities were the isolation of a strong banana promoter, high molecular weight DNA
for large insert genomic libraries; the preparation of a leaf cDNA library; and the
20
construction of expression vectors with selectable marker genes for banana
transformation.
Background: The impetus for this project came with the significant progress that had
been made in in-vitro manipulation of banana in the early 1990s. Highly regenerable
embryonic cell suspensions (ECSs) were established at KUL from vegetative
meristematic tissue. The material enabled the demonstration, for the first time, of
banana transformation at the level of transient gene expression in electroporated
protoplasts. At the same time, regenerable ECSs also showed a great potential for
direct generation of transgenic banana plants by particle bombardment. The BIP
project, therefore, built on this previous work to develop or adapt these new
biotechnological tools for genetic manipulation of banana and plantain, and apply
them to genetic improvement.
Results:
•
•
•
•
•
•
•
Particle bombardment of embryonic cell suspensions proven to be the
superior transformation method.
Plant expression vectors containing chimeric selectable marker genes for the
two best selection systems of banana cells were constructed.
Several heterologous promoters driving high gene expression in banana were
identified.
New assays and bacterial test strains were developed to allow for the study of
interaction between banana and Agobacterium tumefaciens.
cDNA library was prepared from banana leaves for the isolation and
characterisation of specifically expressed genes.
Partial isolation and characterisation of an actin gene from a Grand Nain
genomic library was carried out.
A protocol was described for the isolation of high molecular weight DNA
banana leaf nuclei, resulting in library-quality DNA for the preparation of large
insert genomic libraries.
Outcome: The particle bombardment method was used by collaborative projects
within BIP, and made available for molecular breeding of banana in independent
laboratories world-wide. Optimal conditions are now defined for the most critical
steps of a technology for particle bombardment of embryonic banana and plantain
cell suspensions. This has many practical applications, the paramount one being the
production of material resistant to major pests and diseases. The Agro bacteriummediated transformation system needs significant improvement, although work on
this activity has supplied some useful information. New transformation vectors have
been designed that are useful for sequential transformations to introduce multiple
genes, and also provide a readily available backbone to insert any potentially
interesting selectable marker gene for testing in banana. These vectors enable more
versatile and routine applications for molecular breeding of banana.
Impact: BIP accelerated the ability to transform banana by refining the current
systems, and widening the parameters of exploration. BIP assisted in knowledge
generation of the techniques enabling other laboratories to utilise the technologies.
This BIP project was the catalyst for the collaborative link between the participating
laboratories, which extended to the University of Hawaii (PN12). Through this
21
project, and others in the biotechnology consortium, BIP has played a role in the
acceptance of potential biotechnology solutions to the major problems of banana
production by the private sector.
PN11: Genetic engineering of ethylene biosynthesis in bananas.
Hong Kong University of Science & Technology, Hong Kong
Activity: The aim of this project was to clone genes involved in ethylene biosynthesis
in banana, the genetic transformation of banana with reduced ethylene production
capacity, and the evaluation of transformed plants in relation to reduced disease
development and fruit quality. Due to time constraints on the project, only the first
objective was addressed.
Background: The plant hormone ethylene is produced by the banana plant and fruit,
and plays an important role in disease susceptibility and fruit ripening. Ethylene
production is induced by pathogen attacks resulting in accelerated leaf senescence
and yellowing, and can also cause premature ripening of the fruit resulting in
reduced storage life and poor quality. It has been established that 1aminosyclopropane-1-darboxylic acid (ACC) synthase and ACC oxidase genes
participate in the regulation of and catalyse the last two steps in the pathway of
ethylene biosynthesis. While cloned in a number of other species, cloning in banana
had not been achieved. The recent advances in genetic engineering of higher plants
presented the possibility of using biotechnology to genetically engineer crop plants
with endogenous ethylene production in fruits resulting in prolonged storage life.
Results:
•
•
•
•
•
Construction of a banana genomic library and identification of some ACC
synthase (ACS) and ACC oxidase (ACO) clones.
Characterisation of the clones by restriction enzyme digestion and partial DNA
sequencing.
Complete sequencing of one ACS and two ACO genomic DNA clones.
Elucidation of the regulation of ACS and ACO gene expression by
developmental and environmental factors by performing an RNA gel blot
analysis.
Delivery of an antisense ACS and ACO gene construct to KUL for
investigation.
Outcome: This project has generated substantial new knowledge on the issue of
ethylene production, and highlights the potential that, with further investment, postharvest losses during transportation and storage can be significantly reduced. KUL
have advised that genetic transformation was successfully performed on the gene
construct, but a mite problem on the resultant material has delayed work. A
rehabilitation program is now underway. Further support is required for this activity to
build on the results in linking the gene structure to their functions in disease
resistance and fruit ripening.
Impact: The increased knowledge of the genetic information associated with
ethylene biosynthesis will be useful in future research aimed at post-harvest qualities
of fruit, as well as disease control. The study has laid the foundation for further
22
analysis of the fruit ripening-specific promoter, and provided the basis for genetic
engineering of ethylene biosynthesis and metabolic pathways in banana.
PN12: Use of biotechnology to produce transgenic bananas that are resistant to
banana bunchy top virus (BBTV) infection.
University of Hawaii, U.S.A.
Activity: The objective of this project was to produce transgenic banana resistance
to BBTV by introducing viral genes into the genome. The project utilised and
modified both systems developed in the “molecular toolbox” of the consortium,
particle bombardment, or “gene gun” technology, and the Agrobucterium methods.
Although not a member of the Biotechnology Consortium, the UH group has worked
closely with its members.
Background: BBTV is the most devastating virus disease of bananas in many
producing areas, including Asia, Africa, and South Pacific. Since 1989 the Hawaii
Dept. of Agriculture has been attempting to eradicate the virus, an approach that
worked well in Australia. This approach appears insufficient in Hawaii, indicating that
additional control strategies and long-term solutions to the problem must be found.
Development of resistant varieties based on breeding efforts has been complicated
by ploidy level differences and sterility in the commercial clones. Recent
developments in biotechnology offered a new avenue.
Results:
•
•
•
•
•
Two banana transformation and regeneration systems were evaluated for
development of transgenic banana plants.
Several transformation experiments were conducted with various BBTV-gene
constructs.
Transformed Cavendish ‘Williams’ bananas containing the BBTV gene
constructs were obtained, using a modified version of the Agrobucteriummediated transformation system developed by the BTI group.
Transgenic banana plants were screened in the greenhouse, resulting in the
identification of BBTV-resistant material.
Evaluation in field trials is planned. However, the BIP-funded research is now
completed, and funds are required to undertake field experiments.
Outcome: This project has provided evidence that pathogen-derived resistance
works for BBTV-like viruses, and, with further support, varieties can be developed for
banana growers in Asia, Africa and the South Pacific, where BBTV is a major
problem at production levels. The modified Agrobacterium system will also be useful
for other researchers working on resistance to other diseases, including black
Sigatoka. Sixty-five transgenic plants were evaluated in the greenhouse for BBTV
resistance, and of these; three plants did not develop any BBTV-specific symptoms,
and registered negative in ELISA tests. The project has also succeeded in refining a
protocol to identify transgenic cells. Gene constructs from this project have been
made available to KUL and CATIE in Costa Rica, and are available to other
researchers on request.
23
Impact: Field experiments remain to be conducted to challenge the transformed
plants against BBTV. Further work needs to be undertaken using BBTV virus groups
from other areas where the disease occurs. Current results, however, are highly
significant in that it has been demonstrated that viral-resistant plants can be
produced, and that further refinement of the technique could translate into
considerable reduction in the losses caused by the virus in Asia, Africa, and South
Pacific countries.
AREA 2: Germplasm Collection & Evaluation
PN3: Developing improved banana varieties with pest and disease resistance, postharvest superiority and maximum export potential.
Kerala Agricultural University, India
Activity: The collection, characterisation, and evaluation of Nendran banana in India
undertaken in close collaboration with the national banana program (NRCB), to
establish reference data for Nendran for use by future researchers. Selected clones
from the collection were evaluated for dwarfness, short cycling, and disease
resistance, particularly for yellow Sigatoka, and to a lesser extent weevils and
nematodes.
Background: The project concentrated on collection and characterisation of the
Nendran type AAB plantain banana and wild relatives. Nendran varieties are the
most important type grown in the South Indian peninsula, and are exclusively
cultivated on commercial lines, but to date have not been systematically evaluated
for optimal breeding characteristics. Characterisation of the Nendran-type
germplasm is based on morphological and isozyme markers.
Results:
•
•
•
•
•
•
•
•
125 accessions available for characterisation. 111 Nendran, and three wild
types were collected from eight different locations. Fourteen accessions from
the germplasm bank at the Banana Research Station, Kannara, were included
in the study.
The collection was established in the field at the Banana Research Station,
Kannara, and the passport data for each of the collections have been
prepared.
Morphological characterisation was carried out using the INIBAP descriptors.
Differences have been observed in colour, height, and aspect of pseudostem,
inflorescence, and bunch characters.
Tentative identification of 14 types has been made, and a key developed.
A database has been established containing information on the collection.
Procedures for isozyme polymorphism studies have been standardised, with
five enzyme systems surveyed in various accessions. Results indicated a
variation in the banding pattern as well as intensity of individual bands.
Evaluation was also undertaken using a combination of enzyme systems in an
attempt to ensure classification into distinct groups.
The reaction of accessions to yellow Sigatoka was undertaken and reported,
with further trials underway.
The reaction of accessions to nematode attack was assessed and reported.
24
Outcome: Variations in Nendran types collected are being documented on a
reference database. This will enable identification of varieties with superior
agronomic traits, such as tolerance to drought and water logging, which is of
particular interest in Southern India. Accessions collected and established at
Kannara will be available for future collaborative programs.
Impact: Southern Indian breeding programs are now able to select material better
suited to the various farming systems. The possibility of identifying a short statured
variety has been increased. This will assist in lowering production costs since
propping would not be required. Germplasm has been collected that could be useful
for breeding programs world-wide.
PN4: Collection, evaluation and characterisation of genetic resources and improved
banana crop plants in Vietnam.
Institute for Agricultural Genetics, Hanoi, Vietnam
Activity: The major focus of this project was the collection of banana germplasm,
and its evaluation for productivity and disease resistance to enable selection of
varieties with high export quality. The project also focused on micropropagation
techniques for elimination of virus diseases. Cultivars displaying desirable agronomic
and fruit qualities are being selected for use in the Vietnamese breeding program.
Background: Located in the centre of origin of banana, the Vietnamese crop is
highly diversified with many low-yielding and disease-susceptible cultivars being
grown, resulting in a low annual yield of approximately 15 tons/hectare and below
standard quality of export fruit. Even so, banana is reported as being the most
important fruit crop in Vietnam, contributing significantly to the income of millions of
farm households. Studies have shown that genotypes in the collection are not
represented in existing collections anywhere in the world. This presents a valuable
source of parent material for breeding programs. The collection has been
established in vitro. The project was strengthened through training visits by selected
scientists to laboratories in Queensland, Australia, to learn techniques of
identification of banana diseases, and banana production technology. A further
positive activity was the convening of an international seminar in 1995 that attracted
the participation of a wide range of Vietnamese officials. This culminated in the
formation of an internal banana research network, with close contacts between AGI
and the Fruit Research Centre in Phu Ho, Vinh Phu, for banana genetic resource
studies.
Results:
•
•
•
A large number of banana cultivars, clones, and wild species were collected,
preserved, and characterised.
Material displaying the most important agrobiological characters was released for
production. Guidelines for field management based on individual agrobiological
traits of each cultivar were recommended to farmers in an effort to achieve the
highest economic returns.
Improved banana micropropagation technologies were attained, aimed at a
reduction in production costs of micropropagated plants. The technology and
25
•
•
disease-free materials were transferred to a number of production laboratories in
Vietnam, enabling them to supply large quantities of high-quality microplants for
production.
Information gained on levels of disease susceptibility and pathogens of banana
genotypes will enable the study of biocontrol and genetic improvement of disease
resistance in banana.
The addition of genetic resources from introduced material, and the addition of
tetraploids to the Vietnamese collection by in vitro polyploidation are providing
valuable new materials for future banana breeding programs.
Outcome: Higher economic returns for farmers are expected from improved
understanding of production practices. Genetic resource activities are providing
information to enable selection of potentially useful cultivars for breeding programs
both in Vietnam and internationally. Many of the accessions being. analysed have
not previously been collected, therefore this project is providing a valuable source of
material with potential for disease and pest resistance and quality characteristics.
The project provided the impetus required in Vietnam to focus on this important
commodity, including the training of scientists, which is a valuable resource for future
activities on banana improvement in Vietnam.
Impact: The project has allowed scientists in Vietnam to address issues of banana
research for the first time. New material has been collected that is potentially useful
for world-wide breeding programs. An internal network has been established with
other Musa research institutes, and strong linkages have been formed with Australia
as a result of a scientific training course arranged with PN 15 at the Queensland
Department of Primary Industries (QDPI).
AREA 3: Plant Improvement
Banana genetic improvement schemes that have been selected by BIP rely on either
conventional or molecular approaches to crop breeding (or both). The BIP Research
Portfolio consists of four projects addressing the availability and improvement of
naturally occurring accessions for use in breeding programs.
PN 1: Field and laboratory evaluation of diploid bananas for their use in breeding
schemes.
Centre de co-operation internationale en recherche agronomique pour le
développement (CIRAWFLHOR), Guadeloupe
Activity: The objective of this project is to supply breeding programs with reliable
information on diploid lines with desirable characteristics for use as parent stock.
Agronomic and laboratory characterisation methods are being used, as are wild and
cultivated clones.
Background: Collaborative arrangements for evaluation were undertaken between
CIRAD-FLHOR Guadeloupe, Montpellier, and New Caledonia; WIBDECO, Saint
Lucia; and the Queensland Department of Primary Industries (QDPI, PN15),
Australia. Evaluations focused on agronomic and morphological traits, molecular
characterisation, and resistance to black and yellow Sigatoka and Fusarium Wilt
26
Race 4. Accessions were obtained from the CIRAD-FLHOR collection and the
INIBAP Transit Centre.
Results:
•
•
•
•
Indexed and virus-free accessions have been multiplied and sent to prepare
field sites for disease resistance evaluation. Evaluation protocols have been
devised for New Caledonia (black Sigatoka resistance trials) and Saint Lucia
(yellow Sigatoka resistance trials). Accessions are screened for Fusarium Wilt
disease in Australia at QDPI. Accessions are tested for their agronomic
expression in Guadeloupe due to the absence of most major diseases.
Isozyme, RFLP, and microsatellite analyses have been performed on all
existing accessions in Guadeloupe, and morphotaxonomic descriptions
carried out. Complete characterisation of the diploid clones is being
undertaken using all available markers.
Accessions new to the Guadeloupe collection have been assessed for their
ploidy level by flow cytometry methods, and are currently being evaluated
using all available markers. This work will result in the classification of clones
by genotypic and phenotypic characters, and will assist in the identification of
relations between wild and cultivated diploids.
Indexation results are being awaited from Australia to enable the fmal batch of
accessions to be dispatched for Fusarium Wilt and yellow Sigatoka screening
trials.
Outcome: This project is providing helpful information on diploid material used by all
breeding institutes, and is a valuable addition to the existing information on banana
accessions held in genebanks around the world. This rational basis is important to
identify clearly the diploid material of the greatest potential within the available
germplasm, natural or not, in order to optimise the use of resources available to
breeding programs.
Impact: Information has been supplied on diploid materials that are useful within all
breeding programs. This has been of immediate use in the CIRAD and CRBP
programs, and is also available to others. It is expected that use of this information
will result in a shorter timeframe to produce suitable hybrids with the required traits.
CIRAD has conservatively estimated that this new knowledge will result in a ten-fold
increase in the number of dessert bananas produced.
PN2: Field cross experiments for the purpose of understanding the mechanism
governing the inheritance of Black Leaf Streak resistant characters in banana.
Centre de co-operation intemationale en recherche agronomique pour le
développement (CIRAD-FLHOR), Guadeloupe/Montpellier
Activity: All breeding programs are concerned with the development of varieties
showing resistance to black leaf streak disease (BLSD) in order to lessen the use of
fungicides, and therefore the cost of production, environmental damage and human
health concerns. Field resistance screenings against BLSD led to the identification of
two forms of resistance reported as highly resistant (HR) and partially resistant (PR).
Within the frame of this project, experimental crosses were undertaken with three
wild Musa acuminata accessions expressing the HR, PR, and susceptible (S)
27
reactions against BLSD. Moreover, the project aimed at establishing linkages
between field observations and molecular studies in order to identify quantitative trait
loci (QTL), and to localise the main resistance genes on a saturated genetic map of
bananas.
Background:
Ongoing field test screening for resistance indicates that the
environmental effect on gene expression is significant, and that controlled
environment testing is necessary for a precise analysis of the inheritance of the HR
and PR resistance forms. Therefore, research on the PR form of resistance was
dropped because it was found that field evaluation procedures routinely used were
not precise enough in this case for genetic analysis. The project focus on
observation of resistance pattern of hybrids arose from crosses involving the HR
resistant parent and their correlation with molecular data for the localisation of the
main resistance genes.
Results:
•
•
•
The phenotype of most of the 153 clones of the F2 progeny from the HR x S
cross has been confirmed. Questions remain about the phenotype of some
hybrids (HR versus highly PR). This was undertaken by field evaluation on at
least two cycles in two locations, Njombe and Ekona, CRBP, Cameroon.
Molecular characterisation of the F2 progeny has been undertaken in CIRAD
where the genotype has been characterised for 39 polymorphic codominant
markers (RFLP I microsatellites) and 110 polymorphic dominant AFLP
markers. Results showed significant allelic distorted segregations (for 58
markers) in the F2 progeny that highlight the use of molecular study for the
inheritance analysis. A first mapping was concluded to join the markers in 11
linkage groups. Correlation made with field observations ranked in six classes
of resistance, and a second RFLP marker mapped on a different linkage
group with a lower significance level.
Observations made on the F-progeny from the HR x PR cross-concluded
unequivocally that the PR phenotype is dominant over the HR phenotype in
bananas.
Outcome: For a precise identification of resistance genes, protocols for glasshouse
trials are being developed by CIRAD. This activity is designed first to confirm, in
controlled conditions, the resistance phenotype of the hybrids, and second to identify
all different genes involved in the resistance, even minor genes, for a durable
resistance. The main genes/QTLs involved localised on the genetic molecular map,
will enable the early screening for BLS resistance plants or hybrids to be done
directly on vitroplants with molecular markers (marker assisted selection). The
banana genetic molecular map first developed by CIRAD is being completed, and
many codominant markers have been mapped and information is available for any
gene / QTLs localisation and identification. This work contributes to the breeding of
BLS-resistant plants that are economical and ecological necessities.
Impact: Molecular marker assistance in the identification of resistance genes for
black Sigatoka will initially be useful for conferring partial resistance to black
Sigatoka through breeding programs. The assumption is that this will decrease the
spraying frequency for black Sigatoka by 50% or more. The understanding and
28
identification of genetic resistance mechanisms will be a critical component of future
strategies to reduce the economic and environmental impact of existing chemical
control methods.
PN5: Banana breeding in Brazil
Empresa Brasileira de Pesquisa Agropecusiria (EMBRAPA), Brazil
Activity: The aim of this project was to breed high-yielding varieties of banana
resistant or tolerant to the main pests and diseases, with agronomic qualities of low
plant height, short production cycle, and optimal fruit bunch and fruit size. This was
attempted by the production of superior diploid and tetraploid cultivars and
identification of new varieties; and the development of techniques for in vitro
fertilisation and for propagation with reduced somoclonal variation. With the arrival of
black Sigatoka into the Amazon region in 1998, the objectives of this project became
even more critical than previously.
Background: Banana production is an important agricultural activity in Brazil, with
high economic and social benefits. The cultivated area covers about 516,000
hectares. The industry is based on smallholder farmers and is an important source of
employment. BIP enabled the Brazilian program to be recognised internally and
internationally. Valuable diploids were evaluated and selected as breeding parents to
create diploid and tetraploid cultivars, as well as AAB dessert cultivars. This material
was evaluated for resistance to rhizome weevil borer, Fusarium Wilt, Moko disease,
and yellow and black Sigatoka. In tests to evaluate for nematode and insect
resistance, researchers have found one hybrid that is tolerant to the rhizome weevil
(Cosmopolites sordidus), and two hybrids that are tolerant to the nematode
Radopholus similis.
Results:
•
•
•
•
•
•
•
Diploid hybrids are being obtained by introducing pre-selected genotypes
and/or new material from other breeding programs.
Diploid and tetraploid hybrids were evaluated for resistance to black and
yellow Sigatoka, Fusarium Wilt, and nematodes, as well as for agronomic
characteristics and fruit quality.
The resultant hybrids were evaluated for 23 desirable characteristics. 28
diploid hybrids and 13 tetraploids (12 Prata and one Maca type) were
selected.
The tetraploid (AAAB) hybrid PV-03-44 and the cultivar Caipira were
recommended for commercial planting in the Amazon region.
Techniques have been refined for in vitro fertilisation, somatic embyrogenesis,
micro-propagation, somaclonal variation, molecular characterisation of
germplasm, and somatic hybridisation.
Generation of AAAB seeds and seedlings from crosses of Maca AAB and AA
diploid PA Musore 2.
Development of a device to measure the resistance to fruit detachment for
use during germplasm evaluation.
Outcome: The arrival of black Sigatoka in Northern and Northwest Brazil made this
project one of considerable urgency. Two new varieties, a cultivar Caipira and hybrid
29
PV03-44, partly developed under BIP and resistant to black Sigatoka, are being used
as a barrier in an attempt to avoid the spread of the disease to the plantation growing
areas in Southern Brazil. These arid other materials that are being developed under
BIP will provide valuable breeding lines for international breeding programs. Viable
seeds and seedlings of AAAB types are considered an important result of the
project, since no Maca type tetraploid hybrid had been generated previously.
Impact: The project resulted in improved selection of breeding lines for EMBRAPA
programs for resistance to black Sigatoka, and other pests and diseases of banana
and plantain. BIP enabled the Brazilian breeding program to accelerate trials in
various ecosystems, assisting in the development of a Sigatoka-resistant clone. This
clone is currently being planted in the Amazon as part of a program to prevent
further distribution within Brazil of this newly arrived disease, which is potentially
devastating for the industry.
PN22: Breeding hybrid Musacea with resistance to multiple diseases, especially
black Sigatoka and Panama disease.
Fundacion Honduretia de Investigation Agricola (FHIA), Honduras
Activity: This project builds on the existing breeding program to obtain hybrid
bananas with resistance to multiple diseases, including black Sigatoka and Panama
disease, by conventional breeding.
Background: Pollinations were made onto established and new plantings of the
seed-fertile Lowgate (the shortest dwarf mutant of Gros Michel) and Dwarf Prata
triploid parental lines for the production of tetraploid hybrids. The two main diploid
pollen parents used in these 3x x 2x cross-pollinations were the burrowing
nematode-resistant SH-3142 and the Race 4 of Panama disease-resistant SH-3362.
The SH-3444 tetraploid was also pollinated in 4x x 2x crosses to produce tiploid
hybrids. It has previously been verified that the 4x x 2x approach is a highly effective
way of breeding new cooking bananas. It is therefore anticipated that breeding for a
triploid export dessert banana will also be successful.
Results:
•
•
•
Of the 2200 Lowgate bunches harvested, 16 seed were produced and 10 had
embryos that were cultured for germination. Four of these 10 embryos were
viable, and three of the plants produced have been evaluated in the field.
These three mature tetraploid plants with Lowgate parentage had the
desirable Grand Nain-type plant height, but were susceptible to black
Sigatoka and had mediocre bunches.
These results illustrate that much greater numbers of Lowgate bunches
should be pollinated to produce larger populations from which selections
could be made. Such larger segregating populations would greatly increase
the possibility of selecting a hybrid with black Sigatoka tolerance and bunch
qualities of FHIA-23, but which would also have the desired shorter plant
height. FHIA-23, which was derived from Highgate x SH-3362, is tolerant to
black Sigatoka and has excellent bunch features.
From the 450 Dwarf Prata bunches harvested, a total of 685 seeds were
produced and 250 had embryos. From these 250 cultured embryos, 123
30
•
germinated and most of these hybrid plantlets were transplanted to the field
for subsequent evaluation.
The 600 bunches of SH-3444 yielded 84 seeds and 20 cultured embryos.
From these 20 cultured embryos, 17 germinated. Two of these 17 plants have
the gene for dwarfness and only these dwarf plants will be transplanted to the
field for further evaluation.
Outcome: The results of this project demonstrated that dwarf tetraploid export
bananas could be bred. However, larger segregating populations must be produced
to expect subsequent selection of dwarf plants with the desired bunch qualities and
resistance to diseases. In view of the proven effectiveness of both the 3x x 2x and 4x
x 2x breeding schemes for producing tetraploid and triploid commercial hybrids, it is
critical that these activities be continued.
Impact: The ability of FHIA to produce varieties displaying the desirable traits of pest
and disease resistance, short stature, and good fruit quality was assisted by BIP,
and will have a major impact on banana production when achieved. Field trials will
be continuing on the various crosses undertaken by the project.
AREA 4: Pest and Disease Management
4.1 Disease Control:
Fusarium Wilt (Panama disease)
PN15: Variability and relationships within populations of Fusarium oxysporum f.sp.
cubense from its centre of origin.
Queensland Department of Primary Industries, Australia
Activity: This project was intended to provide information on the genetic diversity
within populations of Fusarium oxysporum f.sp. cubense (Foe) in various areas in
the Asia/Pacific region, and the risk this poses to banana production. The reliability
of using tissue culture-derived plants as opposed to conventional planting materials
when assessing pathogen resistance was also addressed. The project will enable
breeding and selection programs to screen genotypes against a more complete
representation of the pathogen. Isolates lyophilised during the study will also be
available for use in virulence and pathogenicity tests in countries that are able to test
nonendemic strains.
Background: There are no control mechanisms for Panama disease, meaning that
once infected the plantation has to be abandoned, with the pathogen remaining in
the soil for long periods. Studies have been undertaken on genetic diversity of
Fusarium as well as on field evaluations of potential breeding materials. Specific
parts of the ribosomal DNA within the Fusarium Wilt fungus were characterised and
sequenced, and this information was used to identify genetic variation among the
different races of the fungus. Associated activities included the planning and
execution of a six-month training course for four scientists from the BIP-funded
project in Vietnam, and collaboration with CIRAD-FLHOR, Guadeloupe, in screening
diploid breeding materials for Fusarium Wilt.
31
Results:
•
•
•
•
•
•
•
•
Establishment of a world-wide collection of over 2000 isolates of Foe (Africa,
South and Southeast Asia, North and South America, Australia) that have
been lyophilised and will serve as a reference collection.
Diversity of Foe determined through the use of vegetative compatibility groups
(VCGs), DNA fingerprinting, and volatile groups.
Determined the global distribution of strains of Foe. Conducted in-depth
studies on the pathogens from the presumed centre of co-evolution. Tropical
Race 4 populations (VCGs 012 13-01216), which are Cavendish-competent in
tropical regions, were restricted to Southeast Asia (Taiwan, Indonesia,
Malaysia, and Irian Jaya) and far northern Australia.
Identified strain-specific PCR primers for development of PCR-based
detection systems for Foe. Developed a library of DNA banding patterns.
Characterised and sequenced the internal transcribed space (ITS) region and
the intergenic spacer region (IGS) of the ribosomal DNA (rDNA) genes of Foe.
Identified genetic variation among different races and VCGs based on IGS
haplotype. Constructed a phylogeny for the genotypes of Foe based on DNA
fingerprinting and IGS sequence information.
Developed protocols for field evaluation of hybrids and diploids for resistance
to Fusarium Wilt. Provided information on their performance to major
pathogen groups.
Demonstrated the greater susceptibility of plants derived from tissue culture to
Fusarium Wilt when compared to conventional seed pieces. Studies have
commenced to clarify the cause and to increase their resistance using
rhizosphere bacteria, endophytes, mycorrhizae and abiotic inducers.
Fusarium Wilt-suppressive soils have been identified to assist in this study.
Evaluated a Muss acuminata ssp. population that is segregating for resistance
to subtropical Race 4.
Outcome: Information has been provided on the pathogenic and genetic diversity of
Foe in various regions, enabling breeding and selection programs to screen Musa
genotypes against a more complete representation of the pathogen. The project
established that maximum variability was found in the Asia region, considered the
centre of origin for Foe. Until this study, Fusarium Wilt in Cavendish cultivars was
thought to be confined to the subtropics due to cool winter temperatures. Since no
predisposing factors have been recognised in Southeast Asia and northern Australia,
isolates in VCGs 0 12 13 and 0 12 16 appear to constitute a tropical Race 4 that has
the capacity to threaten the world export trades. The only way to control Fusarium is
through host resistance. Therefore disease-resistant germplasm is essential to any
breeding program, together with knowledge of the genetic nature of resistance.
Consequently CIRAD-FLHOR have sent their breeding diploids to Australia for field
evaluation. Work on the identification of soil-borne resistance organisms will enable
inoculation regimes to be designed for micropropagated plants prior to field planting.
Impact: The collection of various strains of Foe will enable the more efficient
identification of the pathogen, being especially useful in the identification of suitable
plantation locations. The increased knowledge on genetic diversity will be useful to
breeding programs to screen against a more complete profile of the pathogen, as will
the protocols for field evaluation of hybrids and diploid materials. Quarantine or
32
exclusion procedures need to be quickly implemented in the Asia/Pacific region to
confine tropical Race 4 populations to their existing areas of distribution. The finding
of a Muss acuminata ssp. population segregating for resistance to subtropical Race
4 should be useful in developing molecular markers leading to the identification of
host resistance genes. BIP has developed and strengthened professional links
between the project and the world’s major breeding programs, particularly FHIA,
CIIUD and EMBRAPA.
Sigatoka Disease (Black Leaf Streak)
PN17: Origin and distribution of fungicide-resistant strains of M. fijiensis in banana
plantations in Costa Rica.
Corporacion Bananera National (CORBANA), Costa Rica
Activity: This project sought to increase knowledge on the dynamics of isolates of M.
fijiensis (the causal agent of black Sigatoka) resistant to fungicides, to allow the
design of better strategies for use of fungicides that will enhance the efficacy of
control strategies. The project also investigated the biology and epidemiology of
resistance to two major fungicides (benomyl and propiconazole) and less sensitive
strains to improve the understanding of the effect that genes for resistance have on
some important components of fitness of M. fijiensis.
Background: Results to date have shown that the genes controlling resistance to
fungicides in the pathogen do not reduce its aggressiveness. It has also been
confirmed that two fungicides, propiconazole and tridemorph, are synergistic in their
effect on the fungus, and thus may give better control in combination. Most
importantly, it is now known that the previously proposed strategies for managing
fungicide resistance to the more important pesticides will not work. The practice of
rotating pesticides to allow a decline in the frequency of resistance to favoured
pesticides will only result in failure, as the expected reduction does not occur.
Apparently the expected selection forces that would cause the change to occur do
not function as expected. Once resistance to a pesticide has occurred there is little
hope of it once again becoming effective. This has serious consequences for banana
producer’s world-wide.
Results:
•
•
•
The sensitivity of progenies from 28 crosses of isolates of M. fijiensis with
different sensitivities to propiconazole has been determined.
Acquisition of genes for resistance to chemicals does not have an effect on
the fitness of the pathogen populations. Resistance patterns remain although
fungicide use is stopped.
A greater understanding of the relationship between acquisition of resistance
and the ability of isolates of M. fijiensis less sensitive to propiconazole to
produce conidia in culture.
Outcome: Results from conidial sporulation between propiconazole less sensitive,
and propiconazole-sensitive strains of M. fijiensis, are important to understand the
effect that gaining genes for resistance has on fitnesses of the pathogen. This would
help to explain why some strategies, and what strategies, would help prevent or
33
diminish the resistance population, thereby improving the control of the disease.
Results from crosses and inheritance of the resistance to propiconazole will help
understand the potential for triazole-resistant genes to flow in the population and, in
conjunction with other aspects of fitness, will help clarify the efficacy of strategies for
resistance management in the population of M. fijiensis.
Impact: Project activities have confirmed that a chemical-free period does not lessen
the impact of populations of pathogens resistant to fungicides. Although this was a
negative finding in some respects, it did confirm that strategies for improved use of
chemicals to control the black Sigatoka pathogen are not easy to develop, and that
continuing development of new chemicals, together with associated costs, is the
principal avenue by which some retention of chemical control can be managed.
Banana Streak Virus (BSV)
PN14: Elimination of banana streak badnavirus (BSV) from improved Muss
germplasm and related studies on transmission and host plant/virus/vector
interactions.
International Institute of Tropical Agriculture, Nigeria
Activity: This project first aimed to develop reliable therapeutic methods to eliminate
BSV from improved Musa germplasm. This objective was later altered to the
identification of Musa plant tissues that are free of both genomic and episomal BSV,
when it was found that the virus had the ability to integrate into the Musa genome.
The project focused heavily on the need for a greater understanding of the host
plant/virus/vector relationships, and BSV transmission and identification of its
vectors, when it was realised that the virus was far more insidious than originally
thought. The project also undertook to provide a protocol for the elimination of BSV
from infected genotypes.
Background: IITA undertook to seek more reliable methods of detection of BSV in
naturally infected plants. BSV is a difficult virus inasmuch as it has the capacity to
insert itself into the host genome, thus making detection difficult. IITA researchers
and their collaborating partners have confirmed that the IC-PCR method is detecting
viral (episomal) DNA, and they are continuing to work toward refining this detection
technique. They are also continuing to gather more information about the insect
vectors of the virus, and the best protocols for evaluating disease symptoms.
Results:
•
•
•
•
Diagnostic techniques were developed for BSV detection in infected. banana
and plantain.
BSV transmission through suckers was proven, and transmission through true
seed implicated. It was also found that materials propagated through in vitro
culture were highly susceptible to the virus.
Several mealybug species that are potential vectors were identified and
colonies initiated, but transmission was not confirmed.
The researchers developed a better understanding of the symptom
expression of BSV-infected improved IITA hybrids and local landraces, under
natural and controlled-environment conditions.
34
•
The project added significantly to the knowledge base of screening for
resistance and characterising field resistance and/or tolerance.
Outcome: This project resulted in an increased understanding of BSV: its etiology,
epidemiology, and possibly control. As a direct result of the activities undertaken,
informed decisions regarding the safe movement of germplasm can now be taken.
The project has also generated knowledge that will greatly assist future breeding
efforts and quarantine activities.
Impact: The project has produced increased knowledge and understanding of BSV.
Information relevant to the safe movement of germplasm and appropriate quarantine
activities was produced, although the virus remains a serious constraint to the
movement of germplasm due to its incorporation into the genome. BSV is now also
increasingly recognised as a production constraint in certain banana-growing areas.
4.2
Pest Control:
Nematode Resistance
PN18: Study of tolerance/resistance of bananas to nematodes.
Centre Regional Bananiers et Plantains (CRBP), Cameroon
Activity: This project attempted to identify sources of resistance to the major
nematode species of banana and plantain from the Musa germplasm collection at
CRBP, and the provision of information on the susceptibility levels of plantain and
other cooking bananas, as well as identifying less tolerant plantains that can be
recommended to farmers. The project will assist the CRBP breeding program, by
providing information on the susceptibility levels of parental materials and their
progenies to two of the most important nematodes of bananas and plantains
(Radopholus similis and Pratylenchus goodeyi).
Background: Banana and plantain are important staple foods for a significant
proportion of the Cameroon population, and are an important source of export
revenue. Radopholus similis is a major pest in commercial plantations at low
altitudes, whereas Pratylenchus goodeyi is dominant in smallholder plantations in
highland areas with cool temperatures. No control of nematodes is made in
smallholder plantations, whereas in commercial plantations the use of nematicides is
one of the main costs of production. This project has a close association with the
CIRAD Musa improvement program, and established a collaborative linkage with the
nematode consortium spearheaded by the KULeuven Project, PN19.
Results:
•
•
Data collection continues in the trial concerning the verification of field
resistance of accessions selected during pot experiments for resistance to
Radopholus similis on two sites (Njombe and Mbalmao). The sites have
different soil and climatic characteristics, but R. similis is the dominant
nematode pest species found in the two locations.
Results of the analysis of root samples collected nine months after planting
confirmed the resistance of four accessions to R. similis, comparable to that of
Yangambi Km5.
35
Outcome: Thirteen plantain hybrids and nine diploid clones have been compared for
resistance to R. similis. All but one was susceptible, with one diploid showing a low
level of resistance. Tests to compare resistance in tissue culture versus conventional
planting material to the nematode P. goodeyi showed that there was no difference
between the two. The project is contributing to the pool of knowledge on the
resistance of Musa to nematodes, the development of new procedures for the
selection of Musa clones for resistance to lesion nematodes, and in the identification
of new sources of resistance to R. similis. Sources of identified resistance are
available to breeding programs.
Impact: As well as contributing to our overall knowledge on resistance, results of this
project will impact mainly on the breeding program at CRBP. This will enable the
selection of hybrid parents displaying resistance to nematodes, therefore increasing
the rate of genetic gain with respect to resistance. A number of the selected clones
also display resistance to other banana pests and pathogens, enhancing the
possibility that multiple resistance can be achieved. The development of an early
screening method will help in the rapid selection of resistant material.
PN19: Identification of durable nematode resistance sources in banana and plantain.
Katholieke Universiteit Leuven (KUL), Belgium
Activity: This project was aimed at the identification of durable nematode resistance
sources in banana and plantain leading to improved nematode management
beneficial to both the producer and the environment through the increase in yields;
decreasing production costs; and a reduction in the number of applications of
nematicides. The intention is to provide a nematological component to existing
breeding programmes, and to screen banana and plantain genotypes for nematode
resistance based on the development of early, rapid and reliable screening methods.
Background: Nematodes constitute an important limiting factor of banana and
plantain throughout the world. Both the high cost and toxicity of nematicides underlie
the urgent need for the identification of nematode-resistant sources and an early and
rapid screening method for use in breeding programs. The principal investigator of
this project was active in obtaining co-financing through the Belgium Administration
for Development Co-operation (BADC) and the Flemish Association for Development
Co-operation and Technical Assistance (VVOB) to fund nematological research
associates at breeding programs in FHIA Honduras, IITA in Nigeria and Uganda,
VASI in Vietnam, and recently CORBANA, Costa Rica. The project also worked
closely with PN21 in Spain during the initial year of activities. More recently, a
memorandum of understanding was signed for closer collaboration with CRBP in
Cameroon.
Results:
•
•
6 Radopholus similis, 3 Praiylenchus coffeae and 6 Meloidogyne spp.
populations were initially established for use in screening experiments.
Approximately 30 greenhouse experiments were conducted at KUL, and 75
Musa genotypes were evaluated for resistance to these populations.
36
•
•
•
•
•
The capacity to undertake large-scale nematode resistance/tolerance
screening of Musa germplasm and comparison screening methods was
provided at FHIA and IITA.
Host plant responses obtained by early greenhouse screening (in soil in pots)
have been compared with host plant responses under field conditions.
Host plant responses of in vitro micropropagated plants screened in the
greenhouse were compared with the host response of suckers. Preliminary
experiments demonstrated that resistance can be observed under in vitro
conditions, but due to contamination of the nematode population, this
screening method has been compromised.
Multiple nematode species resistance screening of Musa genotypes from
various sources was undertaken with no new sources of resistance found.
Several genotypes were identified that are much less susceptible to R. similis
compared with a susceptible reference genotype.
Outcome: This work will continue following the completion of BIP, with information
being generated on the host response of Musa genotypes to infection at different
levels of nematode screening. New techniques are currently being investigated to
obtain contamination-free R. similis for the routine in vitro screening of Musa
germplasm for nematode resistance. Work also continues at KUL on the host
response of R. similis of a series of diploids and triploids from the Philippines.
Although work continues, the data collected to date already enable a more reliable
interpretation of the host response of Musa genotypes to nematode infection.
Impact:
A major impact is the establishment of the Musa Nematologists’
Consortium, which will continue to function following the completion of BIP. It is
envisaged that additional partners from South America, the Caribbean, Africa, Asia,
and Australia will be members in the near future. This is a unique consortium in the
field of nematological research. For banana production, the consortium provides a
reliable nematological evaluation under different agroecological conditions of existing
or new varieties. Under the project, KUL was able to assist FHIA in the evaluation of
the genotypes used or produced in their Musa breeding program. This is the first
time that nematode resistance of these genotypes has been reliably and thoroughly
documented, allowing the identification of the most interesting parent lines for
crossings aimed at nematode resistance. Valuable information has also been
obtained on the host response of Musa genotypes to nematode infection at the
different levels of nematode screening: in vitro, under greenhouse conditions, and in
the field.
PN20: Identification of durable pest and disease resistance sources in banana and
plantain.
Fundacion Hondurena de Investigation Agricola (FHIA), Honduras
Activity: In collaboration with KUL, Leuven, the researchers sought to improve
nematode management on banana and plantain to benefit both the producer and the
environment through yield increases, production cost decreases, and a reduction in
the use of nematicides.
Background: Nineteen genotypes of Musa have been screened for resistance to R.
similis and P. coffeae, the two major nematode pests. Some genotypes with a high
37
degree of resistance to one or both were identified for further resistance breeding.
Results showed that tissue culture plants could be used to differentiate between
different degrees of resistance and tolerance in the genotypes tested.
Results:
•
•
•
•
•
Carrot dish cultures of R. similis and P. coffeae are being maintained in the
laboratory for a continuous supply of nematodes for inocula to infest pests in
the shadehouse trials.
Six shadehouse trials were completed, and four further trials are underway
that include 29 genotypes.
Data collection and management of two field trials established in 1997
continued with 17 test genotypes.
Data from the completed shadehouse and ongoing field trials were entered
into a computerised database and statistical analysis is in progress.
The multiplication of 22 additional genotypes from FHIA’s collection was
started in the tissue culture laboratory for use in the final phase of the project.
Outcome: Technicians were trained to evaluate nematode resistance in subsequent
hybrid developed in the ongoing breeding program. Most of the genotypes available
at FHIA have been evaluated for resistance and tolerance to R. similis and P.
coffeae, and are undergoing re-evaluation because results varied with type of
planting material, and the test was not performed in optimal conditions. Field trials
should provide the necessary information to determine if early testing in shadehouse
conditions is a reliable tool for nematode resistance screening. This knowledge will
assist in the selection of improved genotypes for breeding programs, resulting in
varieties with increased yield and lower production costs due to reduced use of
nematicides, and should also be beneficial to the environment.
Impact: Prior to BIP there had been no validation of resistant material in breeding
programs. The information from this project provided knowledge of resistance when
selecting parents for crossing. New sources of resistance to nematodes were found,
which have the potential to increase knowledge on the mechanism for resistance.
This could have considerable value if a biotechnological approach is considered for
resistance. Evaluations will continue, linked within the nematology consortium.
PN21: Identification of durable nematode resistance sources in banana and plantain.
Institut de Recerca i Tecnoligia Agroalimentaries (IRTA), Spain.
Activity: The specific aim of this project was to establish a live collection of migratory
endoparasitic and root knot nematodes of the main nematode species that attack
bananas and plantains. A second activity was the preparation of banana plant
material for resistance evaluation and the initiation of screening trials under
greenhouse conditions.
Background: The project was initiated as a single-year component of the nematode
consortium with the same objectives.
38
Results:
•
•
A total of 27 isolates comprising 3 genera and 6 important Musa-attacking
nematode species (Radopholus similis, Pratylenchus goodeyi, P. coffeae, and
Meloidogyne spp.) were isolated and established in monoxenic cultures, with
the exception of Meloidogyne spp., which were established and maintained on
alternate hosts.
Plant material and screening tests of Musa germplasm of interest to the
Canary Islands against Pratylenchus goodeyi and Meloidogyne spp. under
greenhouse conditions were organised.
Outcome: A pure, abundant, and highly infective inoculum was made available for
plant material evaluation and disseminated to consortium researchers for use in
screening trials.
Impact: As part of the nematode consortium activities, the germplasm screening for
resistance will enable more effective breeding programs to be initiated through
selection of resistant material. Such development should reduce costs of nematode
control and lower the use of nematicides.
Research Achievements
In summing up at the Third Scientific Meeting, the SAP Chairman observed that the
BIP-funded research projects have made exciting discoveries, contributed
considerable useful new knowledge, and established strong collaboration and
linkages that will continue into the future.
“Important advances have been made in the understanding of the genetics of
resistance to black Sigatoka. We now have a much greater understanding of what
we face with the Fusarium threat. Molecular tools to transform bananas have been
developed, and key banana breeding programs have been strengthened as an
investment in problem solving. Resistance to nematodes is now better understood,
and we have in hand the means of controlling some significant virus diseases of
banana. BIP has also demonstrated that chemical control strategies aimed at
managing resistance to fungicides of black Sigatoka will not work. We also now
know that tissue culture apparently predisposes plants to Fusarium and nematodes.
BIP has fostered an informal but solid network of scientists that should continue for
many years. Areas of research that need to be addressed within any future
framework are listed below in random order:
1.
2.
3.
Field testing of transformed Musa materials must begin as soon as
possible. The scientists involved are encouraged to make sure that this
happens.
There is a need to address the emerging biosafety issues regarding
field-testing of the transformed material. This requires immediate
attention.
All of the institutions involved should address the intellectual property
rights questions that revolve around the discoveries associated with
BIP-sponsored research.
39
4.
5.
6.
7.
8.
9.
10.
We need to develop a better understanding of industry’s resistance to
cultivar replacement, because the banana research community needs
industry people as partners.
The Musa research community needs to develop a strategy to address
consumer acceptance; i.e., issues associated with transformed
cultivars. Ignoring this issue could doom some otherwise successful
research.
There needs to be an investment in understanding the genetics of the
pathogen of black Sigatoka (M. fijiensis). This research should parallel
studies of the genetics of host resistance and the development of
quantitative trait loci (QTL) probes to enable breeders to breed for
durable resistance to black Sigatoka.”
There is a need to develop an understanding of how to use antifungal
proteins (AFP) as a part of a black Sigatoka resistance strategy. Will
they function as single gene resistance mechanisms and therefore
represent a genetic system susceptible to being overcome by pathogen
race variation?
There is a need to merge the excellent Fusarium research work with
molecular approaches to resistance to banana disease. This applies as
well to nematode research.
Investment is required in biological control strategy research, given the
tantalising results with Fusarium and nematodes.
Maintenance of conventional breeding capacity is crucial, as it will be
needed to put together all of the above.
BIP, through the project researchers, has delivered many research projects, with
many significant scientific advances. Important new methods for enabling others to
do research have been devised. A community of Musa scholars has been formed,
which has generated a renewed interest in what can be done in Musa improvement
that can no longer be ignored. The work has produced an excellent return on
investments, and all principal investigators and collaborators are to be commended
for their outstanding work.
Substantial advances have been achieved in Musa research, but we must build on
these successes by generating new funds for both medium and long-term programs.
The programs need to focus on the most promising results and pressing issues to
maximise benefits of the research, which should be based on a market-driven
approach. The diversification of products to provide choices for diversified demand
patterns should be available, and should meet the criteria of consumer markets. The
need for software and materials from BIP was highlighted as a response to the
urgent need in the Latin American and Caribbean area for improving the livelihood of
growers, both in cash value and nutritional value.”
4.
Project Management
4.1
BIP Research Management
The World Bank convened a Scientific Advisory Panel (SAP) to assist in the
planning, selection and monitoring of research activities.
40
The 18 projects were identified through an internationally competitive research
grants scheme. Proposals were reviewed by SAP against a set of merit-based
criteria, and were selected to form a cohesive and complementary portfolio to work in
parallel toward the project objectives. Projects were monitored by the SAP against
progress and annual reports, and following acceptance of their recommendations by
the PEA, principal investigators were notified of the review outcome. Continuation of
financial support was based on acceptance of reports. Annual Scientific Meetings
were also held to enable Principal Investigators to present their work to the
cosponsors, their technical panels, and scientific peers.
The members of the Scientific Advisory Panel over the course of the Project are
listed in Annex I.
4.2
BIP Annual Meetings
The First Cosponsors meeting was held in Washington, D.C., in March 1995, with
further meetings held in Belgium in February 1996, and in Guadeloupe in March
1997. Reports of these meetings have been provided previously. The convening of
AnnuaI Scientific Meetings was extremely beneficial for information exchange for the
Cosponsors and Technical and Scientific Advisory Panels, and they enhanced the
monitoring process. The principal investigators were able to present and discuss
their work with their peers. The meetings were also an obvious venue for discussing
and planning current project activities within the two consortiums, and for interacting
with other collaborators within the BIP portfolio. This helped establish new and
productive external collaborative and networking opportunities.
The Fourth Cosponsors and Third Scientific Meetings were held in Douala,
Cameroon, on 5-6 November 1998, followed by a field trip to the CRBP facilities in
Njombe. The meeting was timed to coincide with the impending completion of project
activities. As in previous years, the meeting venue was selected to take advantage of
the presence of Musa researchers and the development community who were
participating in a ProMusa Workshop and International Seminar sponsored by
INIBAP, CIRAD and CRBP immediately following the BIP meetings.
4.3
Scientific Advisory Panel (SAP)
A meeting of the Scientific Advisory Panel was convened on 4-5 August 1998. This
was the final meeting in a series over the life of BIP, where the Panel convened on a
biannual basis to monitor the progress of contracted research by reviewing the
Progress and Annual Reports supplied by BIP principal investigators. In addition to
reviewing the research portfolio, members of SAP also discussed the methodology
to be followed for the impact assessment study, and the planned annual meetings.
Membership of SAP is contained in Annex I. The original Panel consisted of six
scientists, with the Program Manager serving as scientific secretary to the Panel.
Their breadth of experience and expertise covered a wide range of scientific
disciplines and knowledge of the banana industry. Following the portfolio selection
phase of BIP, the original Panel was reconstituted to comprise three members, with
additional technical expertise being provided by observers from the CFC and
FAO/IGB, and a representative of the International Network for the Improvement of
41
Banana and Plantain (INIBAP). INIBAP is an international group within the
International Plant Genetic Resources Institute (IPGRI) of the Consultative Group on
International Agricultural Research (CGIAR).
The members of SAP gave generously of their time and expertise during the life of
the Project. The World Bank would like to acknowledge that the success of BIP is
due in no small part to their dedication and input.
4.4
Financial Statement
The Bank received the final disbursement from the CFC during September 1998,
amounting to US$ 245,669.56 (equivalent SDR l79,120.81). Funding received over
the life of the project amounted to US$ 3,325,205.84, of which US$ 2,659,876.00
was invested in research activities. The balance of funds (US$ 665,329) supported
project management, annual meetings, and research monitoring activities.
Expenditures in the last semester, June - December 1998, amounted to US$
181,353.81. The balance of funding is held against commitments to the annual
meetings, a final publication, and project management costs. On closure of the Trust
Fund a final financial report will be provided, and an external audit will be carried out
and results forwarded to the CFC. Any funds remaining in the Trust Fund will be
returned to the CFC.
A copy of the financial statement and signed auditors report from the 1998 financial
year is included in Section 2 of this report.
4. Public/Private Sector Collaboration
The World Bank established an on-going dialogue with independent producers and
multinational companies in the export banana industry from the start of the BIP. The
purpose was to obtain input from the industry as to the constraints to sustainable
banana production and industry R&D needs. At recent meetings, the potential
applications of biotechnology to solve the major disease problems were discussed.
As a result of recent wider consultations with a broad range of companies, producer
co-operatives and other industry representatives, the Bank is examining the
feasibility of establishing a banana consortium, “BioBanana”, to jointly fund further
applications of biotechnology to solve the major disease problems, especially black
Sigatoka disease.
6.
Impact Assessment
1.
Strategic Knowledge:
Collection and characterisation of a wide range of banana germplasm for use in
breeding programs world-wide.
•
Increased efficiency of breeding programs through improved testing,
screening, and typing methods, development of protocols; additional
information on genetic markers for resistance to nematodes/Fusarium
Wilt/black and yellow Sigatoka.
42
•
•
•
2.
Identification of resistance genes for BBTV and I&IV.
Improved and refined technology for genetic manipulation, including
transformation systems (Agrobacterium-mediated and particle bombardment).
Greater understanding of BSV vectors and etiology.
Applied Outputs Beneficial to Producers in the Short Term:
•
•
•
•
•
3.
Transgenic Cavendish and Bluggoe plants going into glasshouse and field
trials.
Support to breeding programs, i.e. enabling EMBRAPA to refine a clone with
black Sigatoka resistance, currently being planted in the Amazonas (NW)
region. Continuation of work at FHIA Honduras on disease-resistant, shortstatured varieties.
Evaluation of Indian Nendran varieties has identified more desirable types for
Southern Indian conditions.
Ability to type strains of Fusarium Wilt in given locations.
Information regarding the continued resistance to chemicals once fungicides
are no longer applied.
Leverage of Funds:
•
•
•
•
4.
Building on the original CFC investment, further funds have been attracted to
BIP, both within the projects, and outside: i.e. WOB and BDC, Belgium for the
nematode consortium personnel positioned around the world in various
breeding programs.
Funding from the New Zealand Government for post-harvest practices in
Vietnam.
Funding from the University of Hong Kong and the Governors Fund in Hawaii.
Conservative estimate of funds contributed is US$ 2 million.
Increased Collaboration
•
•
•
•
•
•
•
•
Formation of consortium in biotechnology enabling the refinement of both
particle bombardment and Agrobacterium transformation techniques.
Formation of nematode consortium strengthened during the project with
funding from VVOB and BDC that will continue past the completion of BIP.
Collaboration between Australian and Vietnamese scientists, exemplified in
the training of Vietnamese scientists by institutes in Australia.
Collaboration between Australian and CIRAD scientists in screening for
Fusarium Wilt on CIRAD breeding diploids.
Collaboration between Costa Rica and Brazil in screening breeding materials.
Attraction of new research providers into banana improvement.
Additional permanent staff position created in CIRAD focused on banana
improvement and,
Continued generic resources provided by Belgium through nematode
consortium.
Although these outcomes are significant, the real value of BIP-supported work will be
the long-term strategic knowledge flowing from the BIP projects. Landmark events
43
using some of this new knowledge might be the development of a Cavendish or
alternative export banana type that is disease resistant. Other developments that
might result at least in part from BIP research include socio-economic,
environmental, and health benefits. The economic benefits would include reduced
production costs (few chemicals required); the environment would be improved
through less chemical usage; and banana producers would suffer fewer health
problems by not having to handle as much toxic chemical material (organ damage to
an estimated 1 % of workers according to Swennen 1997). Improvements would lead
to better health of workers, higher productivity, and an overall increase in the quality
of life of communities in banana-producing areas.
A longer-term benefit to industry will be the enhanced collaboration in banana
improvement work as a result of the creation of a Biotechnology Consortium
(Katholeike Universiteit Leuven, Belgium, Boyce Thompson Institute, USA, and
Queensland University of Technology, Australia). This grouping resulted in staff
exchanges and the transfer and refinement of particle bombardment and
Agrobacterium transformation and regeneration technology. In a similar way, BIP
fostered the creation of a “nematode consortium”, which has resulted in improved
screening techniques leading to a better understanding of nematode resistance.
The impact assessment study indicated that BIP should probably provide a rate of
return on investment of at least 20% to 33%. Conservative assumptions were used
in the analyses, and additional analyses showed robustness of the results. This
demonstrates that further investments in banana improvement research will be well
justified.
7.
Conclusion
BIP results have provided methods for transformation and regeneration of bananas,
and have world-wide banana producers closer to a disease and pest-resistant
transgenic banana being a reality. Considerable progress has been made in
identifying new germplasm suitable for breeding, and ways have been developed to
improve the breeding programs. The independent impact assessment clearly
indicates that the most important output from BIP is strategic new knowledge that
can be used for future research and development projects.
BIP has, therefore, made an important contribution in both the provisions of research
results, and in catalysing collaborative activities and co-financing through the
innovative process of utilising a competitive research grants program. This enabled
the identification of research providers of excellent quality, and investing resources in
a portfolio of research projects of direct relevance to the objectives of the program.
The World Bank’s overarching commitment is to the alleviation of poverty. The
contribution this Project has made toward the development of resistant varieties and
new control methods of banana and plantain production is a positive step in reaching
this goal.
44
PART II
Project Evaluation and Detailed Research Findings
45
46
1.
Introduction
In 1993, the COMMON FUND FOR COMMODITIES (CFC) agreed to finance a fiveyear project, entitled “Banana Improvement Project” (BIP), for a total of
US$ 3,566,000.
The Agreement was signed between CFC, the World Bank as the “Project Executing
Agency” (PEA), and the “Intergovernmental Group on Bananas” (FAO) as the
Supervisory Body.
The BIP officially started on January 1, 1994.
Its objectives were:
1. To develop and evaluate, over a five-year period, a range of improved banana varieties with export
potential, incorporating increased productivity with durable disease resistance, using conventional
and non-conventional breeding techniques;
2. To develop more efficient and integrated disease management practices for black Sigatoka
disease based on an improved understanding of disease epidemiology and pathogen variability in
different areas and over different seasons.
The expected outputs and desired activities are reported in Annex 1.
During the preparatory phase to the Agreement, the Intergovernmental Group (IGG)
had agreed on the proposal for this project, with the same objectives, and had even
indicated a number of leading research institutes (such as the FHIA) and
laboratories for possible implementation.
The Agreement was manifestly focussed on (1) export bananas and (2) the Sigatoka
diseases, but left some flexibility for interpretation. Terms such as ‘new Musa
genetic material’ (Output 1), ‘related pests and pathogens’ (Output 2), and some
terms in the activity titles allowed to a certain extent for other research objectives.
The PEA had the difficult task of distributing the operational funds among a number
of selected research bodies, in the most efficient way, but still maintaining the
agreed objectives. The PEA took two major decisions:
•
•
In 1994, it commissioned a “review of banana research needs, banana
improvement programs and banana research laboratories”. The report of that
review (October 1994) ended with a number of recommendations
representing a research spectrum far exceeding the scope of the Agreement.
The recommendations were nevertheless accepted.
It addressed a “Call for Applications” in two rounds to probably the widest
forum of bodies, actually or potentially involved in Musa research, ever
approached for possible research involvement. The call referred to the
objectives of the Agreement.
On the basis of the commissioned review’s recommendations, the PEA eventually
selected 18 of the 95 proposals received. These became the projects reviewed in
the present document. The initiative has since been called a “Competitive Research
Grant System”.
47
A cursory glance through the project titles makes clear that a number of research
subjects do not match the original objectives and expected outputs: virus diseases
(Projects BIP 09, 12 and 14), nematode pests (Projects BIP 18, 19, 20 and 21), and
Fusarium Wilt (Project BIP 15). The only project addressing Objective (2) of the
Agreement is, in a remote sense, Project BIP 17.
This important change in the orientation of the BIP was implicitly accepted by the
sponsors, probably in the first place for the following major practical reason: In the
long period between the initiation of the concept (in 1989 within the IGG) and its
shaping at the end of 1994, a change had taken place with respect to diseases and
pests encountered by banana growers. It was felt that the black Sigatoka disease,
while still a menace, could cause a less serious problem ‘in the end’ than the killing
Fusarium Wilt and virus diseases, for which no sources of resistance had been
detected for (conventional) resistance breeding.
There was also the growing and increasingly shared perception that the nematode
pests could be responsible to a great extent for the decline in banana production in
many areas throughout the world.
The actual operational phase of the BIP started by March 1995 for the majority of
the 18 projects with the contracts signed and funds received soon afterwards. A
major consequence of this late start was that only three to three and a half years
were left for implementation.
Another consequence of the revised concept of the BIP was the wide spectrum of
research subjects covered by the 18 projects. A challenge was how to group the 18
topics with due respect to the original objectives and outputs? The PEA eventually
used the following classification:
A. BREEDING AND GENETIC RESOURCES: projects 01 to 05 and 22
B. BIOTECHNOLOGY: projects 07 to 12
C. PEST AND DISEASE CONTROL
Disease Control: projects 14, 15 and 17
Nematode Control: projects 18 to 21.
After due consideration, the present Review Team found that the following grouping
is more practical for discussion, especially with respect to the Chapters
“Background” and “Conclusions and Lessons Learned”:
I
II
GENETIC IMPROVEMENT: including:
Conventional breeding: projects 02, 05 and 22
Non-conventional breeding: projects 08 to 12
Genetic resources and resistance sources: projects 01, 03, 04, 18 to 21.
PEST AND DISEASE CONTROL: projects 14, 15 and 17
48
It should be noted that this classification still recognises that projects 14 and 15 do
1
have implications for genetic improvement in their results .
2.
Background
This chapter provides information on the state of the art in Musa research at the time
the BIP was shaped.
Genetic Improvement
Conventional Breeding
The major goal of the current banana/plantain breeding programs is to produce
disease/pest-resistant hybrids with good performance in the field and meeting local,
regional or even international preferences, so as to be market competitive.
The terms “durable resistance” and “multiple disease/pest resistance” can be a
source of confusion. Any breeder naturally wishes to produce hybrids with a longstanding resistance to more than one disease and/or pest, but is also aware of the
practical limits.
The most practical goal is to obtain hybrids that could be satisfactory alternatives for
certain regions/preferences in the immediate future. During the distribution-selectionintegration process of some such initial hybrids, the breeder can in the meantime
work further on the construction of better performing hybrids, or hybrids that would
meet the requirements of other regions, or resistance to other diseases or strains
and so on. Such repetitive processes, if maintained for a long time, would hopefully
meet the various requirements over the world and meanwhile tackle new strains,
diseases, and pests.
The actual goal of the breeder is to produce a range of hybrids, allowing for
selection at any place according to the local/regional requirements.
Not many breeding schemes are feasible in the case of Musa. When it comes to
improving dessert bananas, the possibilities are even more restricted due to the long
list of post-harvest and consumption criteria to be met.
The program at FHIA (Honduras) has a long history in banana breeding, and its
current efforts are based on the considerable capital in intermediate hybrids as well
as in expertise, accumulated over three decades. It is widely accepted that this
Institute offers the best opportunities for creating a resistant alternative to the
famous ‘Gros Michel’ dessert cultivar, and Project BIP 22 was shaped with that
purpose in mind.
The EMBRAPA (Brazil) embarked on a banana-breeding program during the
eighties with the aim to improve two popular dessert varieties in the country: ‘Prata’
and ‘Maça’. Both cultivars belong to a group (AAB) genetically quite distant from the
1
The ‘missing’ project numbers need comment. Projects 06, 13, and 16 were originally to be implemented, but were not
contracted for various practical reasons (but see also Project 15 regarding Project 16). Project 07 was to deal with the
‘Biotechnology Consortium’ (Projects 08 to 10) but its budget implications were incorporated within the latter projects.
49
‘Cavendish’ or the ‘Gros Michel’. Very different, and previously not used, genetic
material had to be explored, and specific intermediary hybrids created. Disease
resistance is focussed on Fusarium Wilt and the Sigatokas. At the time project BIP
05 was launched at EMBRAPA, breeding expertise had been built around the
promoter Dr. Ken Shepherd, who had been a banana breeder of international repute
at the Banana Board in Jamaica for many years.
The breeding program at Guadeloupe (CIRAD-FHLOR) started in the seventies and
is based on an entirely new strategy. While the programs at FHIA and EMBRAPA
are built around the key crosses (3nx2n 4n; 4nx2n 3n; i.e. starting from existing
triploid cultivars), the Guadeloupe program aims at de novo construction of the
desired triploid hybrids with disease resistance. Suitable edible diploids are selected
for the purpose, and manipulated (colchicine treatment) so as to obtain tetraploids
which are then crossed for triploid obtention. Crosses with resistance sources are
implemented during this procedure.
The program proved to be valid, but the access to rather homozygous resistance
sources is of paramount importance. Hence, Project BIP 02 was initiated to focus on
the genetics of black Sigatoka-resistance.
Non-Conventional Breeding
The term ”non-conventional breeding” encompasses both molecular marker-assisted
selection of cultivars with desirable agronomic traits, and the transfer of foreign
genes into selected genotypes with a favourable genetic background, so as to
engineer new traits. As such, non-conventional breeding has been a reality for a
series of crops for a long time. However, the genus Musa remained largely outside
the mainstream of biotechnology, and, in genetic terms, was an orphan crop at the
outset of the BIP. It is beyond the scope of this review to explain this obvious
deficiency, but certainly the general biotechnological interest focussed on crops of
moderate climates, and on crops with a good regenerative potential (e.g. potatoes,
rape seed), thus mostly excluding bananas and plantains. This situation has
changed since 1990; a series of projects on the non-conventional improvement of
bananas is now funded by various donors, and a network of collaborating institutions
exists world-wide. The BIP with its limited funds and time has contributed to this
improved situation, which contrasts to the pre-BIP situation in several aspects.
Though there had been experiments on the transformation of banana cell cultures
effected at Texas A & M University and KULeuven, no reliable transformation and
regeneration protocols were at hand, nor were meaningful genes or promoters
available. The general strategy was aimed at transgenic bananas producing edible
vaccines. Doubtless, the development of the ”molecular toolbox” by Projects BIP 08,
09, and 10 was to catalyse progress in this important area.
There was, and still is, no meaningful natural source of virus resistance in the
germplasm. Again, basic research into genetically engineered virus resistance had
started before BIP (e.g. at QUT, aiming at a coat protein protection strategy). This
research would have continued without BIP, though at a lower speed and with less
international co-operation. The same holds for Project BIP 12, and for the BIPenhanced research into virus resistance so that transgenic plants resistant to BBTV
50
and BMV can be expected soon in the field, a major aim of Projects BIP 09 and BIP
12.
Although transgenic bananas expressing ectopic resistance genes under the control
of regulated promoters were discussed before the BIP, none of the tools were
developed: no resistance genes and no inducible promoters were available, and
even the transformation process was far from being optimised, not to speak of
reproducible regeneration of transformed cells or tissues. Project BIP 10, with some
loose co-operation with Projects BIP 08 and 09, has changed this situation.
Some time before the BIP started, research intensified in the area of fruit ripening
and its control. A plethora of scientific papers appeared on the biosynthesis of
ethylene, its regulation, and its function(s) before and amidst the fruit ripening. The
corresponding genes were isolated and sequenced, and their expression in the
ripening process tested. However, the genus Musa was largely excluded from this
progress. Yet the rapid increase in the knowledge of ripening in plants
simultaneously stimulated several groups to work on bananas, amongst them BTI
and HKUST. At the onset of BIP, banana genes (e.g. ACC synthase) were already
isolated and sequenced, and experimentation into the molecular processes
underlying banana fruit ripening was underway. Project BIP 08 therefore enhanced
an otherwise already established research initiative.
Genetic Resources and Resistance Sources
Unexplored resources
It can be stated that by the year 1990, and under the auspices of international and
co-ordinated national organisations, a core Musa germplasm of the whole tropical
world had been collected and investigated, with a few exceptions. But the few
exceptions remained important gaps because they were dealing with germplasm
generated within the wild Musa area.
Germplasm in Vietnam, Cambodia Laos and Myanmar had never been thoroughly
studied.
In India, and despite earlier important work achieved in different research stations,
the status of the “Nendran-complex”, which is most important for food supply in
South-India, was still unclear. They mainly belong to the AAB-Plantain group
(Simmonds), but there are indications that the complex contains yet unknown
cultivars and even wild relatives. An important group of AAB cultivars - the “Maia
maoli/Popoulu/Iholena group”- exists in Polynesia, and is being investigated with
modest support of ACIAR.
The Projects BIP 03 and BIP 04 had the objective to fill the identified gaps for India
and Vietnam respectively.
Breeding potential
Characterisation (morphological as well as molecular) and (agronomic) evaluation of
the collected and conserved Musa germplasm had not made great progress by the
year 1990. The problem does not so much concern the popular triploids, such as the
51
so-called dessert-AAA, AAB and ABB. These have almost automatically been
th
studied in various research programs during the 20 century. Most of the thus
accumulated knowledge had been published, especially in N. W. Simmonds’
handbook, “Bananas”, the three editions of which remain standard works for
consultation world-wide.
With a few exceptions (e.g. Pisang Lilin, Pisang Jari Buaya) the numerous collected
edible diploids had not been subject to systematic characterisation, let alone
agronomic evaluation. The situation was equally unsatisfactory for the wild diploids.
During the eighties, French scientists (CIRAD) embarked on a breeding program
with the aim to construct new triploids starting from selected diploids which were
inter-crossed, with colchicine treatment for polyploidisation as a convenient
instrument at appropriate moments during that operation (cfr “Conventional
breeding”). The first products actually have been evaluated in some of the BIP
projects.
Although the diploids involved had been studied for female/male fertility, their
potential for the construction of the desired triploids (in morphological and
agronomical terms) was a matter of speculation in most cases.
Project BIP 01 was an attempt at meeting this requirement. This project had a
second purpose, which is explained in the subsection on "Resistance sources".
Resistance sources
Since the breeding programs are mainly focussed on resistance to diseases and
pests, the access to resistant diploid sources and precise knowledge on the genetics
of resistance/susceptibility genes during hybridisation became a paramount need.
The objectives of Project BIP 01 included the screening of the numerous diploids for
resistance to Fusarium races 1 and 4, as well as to yellow and black Sigatoka (the
latter disease also called “Black Leaf Streak”).
Screening of breeding material for resistance to Fusarium Wilt also formed part of
the Project BIP 15, although not exclusively focussed on diploids.
Resistance screening against nematodes had been conducted in some instances in
the past (e.g. at FHIA), but, again, not systematically on diploid sources. Besides,
there was the need to develop standardised early screening techniques that would
be easily manageable and would reflect performances in the field.
The Projects BIP 18, 19 ,20 and 21 form the first attempt in this direction, with
regular contact among the Principal Investigators within a “Musa Nematology
Consortium”.
52
Pest and Disease Control
Black Sigatoka
The expensive chemical control of this fungal disease is increasingly facing the
difficulty that the pathogen is acquiring resistance to the chemicals used. The
genetic background of this resistance-building, and its implications for control, form
the objectives of Project BIP 17.
Study of the Mycosphaerella species within the area of origin (SE Asia and Pacific),
a topic of fundamental importance for understanding the generation of new and illdefined strains in various regions (e.g. India), was unfortunately not the subject of a
BIP project.
Fusarium Wilt
The development of race 4 of Fusarium oxysporum f. sp. cubense during the recent
decades became a serious menace: the race attacked the ‘Cavendish’ bananas
(resistant to race 1), and, although it was not active in equatorial regions, a “move”
into the whole of the tropics could not be excluded. Hence, the urgent need for much
more data on the pathogen within its area of origin, as well as for a better
understanding of the genetic background of its evolution. Project BIP 15 was mainly
shaped for that purpose.
Banana Streak Badnavirus (BSV)
The Banana Bunchy Top Virus (BBTV) had been causing much damage in Asia and
the Pacific for a long time, but was sparsely present in Africa and not at all in
America. The more recent outbreak of the Banana Bract Mosaic Virus (BBMV)
disease seems at present to be confined to some regions in Asia. Control of both
these viruses is feasible and screening techniques for exchange of plant material (in
vitro) are available.
In contrast, the BSV appeared as a menace only two decades ago, is present in
America and Africa (and perhaps in Asia as well), and seemed to escape the
standard control measures. Specific screening techniques were still not available by
the end of the eighties.
Consequently, the BSV was seriously compromising any exchange of breeding
material and promising final products, especially when generated in America and
Africa, where the major breeding programs are established.
A proposal from the IITA to tackle this problem was thus most welcome, with Project
BIP 14 as the consequence.
53
3.
Review of the Projects
BIP 01
Field and Laboratory Evaluation of Diploid Bananas for their
Use in Breeding Schemes
Description
Co-ordinates:
Organisation: CIRAD-FLHOR Guadeloupe
Principal Investigator:
Dr. Ch. Jenny
Dr. F. Carreel (associate)
Partners:
Dr. V. Kagi, CIRAD-FLHOR New Caledonia
Dr. H. J. Fagan, WIBDECO St Lucia
Dr. K. Pegg, QDPI Australia
Duration:
3 years, starting April 1995
Objectives
The stated objectives of the project initially were:
1.
The objective of the project was to obtain preliminary evaluation data on land
race diploid cultivars and on bred hybrid diploids to assist in the selection of
potentially useful material in breeding programs and/or production.
Preliminary evaluation data in Guadeloupe will include information on basic
agronomic characters such as cycle duration, plant height, ratooning, bunch
characteristics (shape, weight, number of hands and fruits), and fruit
characteristics (shape, length, taste). The diploids will also be evaluated for
resistance to Black Leaf Streak in New Caledonia and Fusarium oxysporum
f.sp. cubense race 4 in Australia.
2.
The evaluations for agronomic characteristics and disease resistance will be
supplemented by laboratory studies in Guadeloupe, to determine ploidy levels
and molecular characteristics.
It should be noted that while the above objectives appear
interim/annual reports, they have not appeared since the 1997
However, a shortened objective “to obtain preliminary evaluation
accessions” figured as sub-item (4) of an Executive Summary.
objectives has been reported.
54
in the earlier
Annual Report.
data on diploid
No revision of
Activities
Activities have not been listed systematically, but are reported under items in the
Executive Summary or under Research Activities as “achievements’. The planned
means/methods for the implementation of activities have not been explained, except
partly for “molecular characterisation”. However, activities are expressed in the
Milestone/timechart as follows:
1)
2)
3)
4)
5)
6)
7)
8)
9)
Recording of agronomic characteristics over 3 cycles ( CIRAD-FHLOR Guadeloupe)
Determination of the ploidy level (CIRAD-FHLOR Guadeloupe)
Molecular characterisation (cytoplasmic and nuclear data) (CIRAD-FHLOR
Guadeloupe)
In vitro duplication and mailing of the clones not available at the ITC (CIRAD-FHLOR
Guadeloupe)
Indexation of the clones not available at ITC (QDPI)
Evaluation for their resistance to Fusarium race 1 and 4 (QDPI
Evaluation for their resistance to yellow Sigatoka (QDPI)
Evaluation for their resistance to Black Leaf Streak (CIRAD-FHLOR New Caledonia)
Evaluation for their resistance to yellow Sigatoka (WIBDECO)
On the other hand, the same activities are listed in a different way within the
“Executive Summary” of later reports, as follows:
Different sets of activities must be distinguished in this project:
a. Multilocality evaluation
- Multiplication, shipment and reception of the plants
- Disease resistance evaluation in remote sites
b. Guadeloupe evaluation of already existing accessions
- Agronomic and morphotaxonomic evaluation
- Laboratory evaluation (molecular markers)
c. Guadeloupe evaluation of new accessions
- Field planting
- Agronomic and morphotaxonomic evaluation
- Laboratory evaluation (molecular markers)
It should be noted that some activities might not be viewed as “research” in a strict
sense: for example, the activities (4) and (5) in the first list; the first activity (a) of the
second list. They are routine procedures, although their importance was crucial for
the running of the project.
Achievements
The present review of achievements is based on the progress reports as well as on
the Final Completion Report.
Because of the confused reporting of the planned activities (as shown above), the
items under which achievements were reported changed from one report to another
(e.g. what is in the 1995-96 Annual Report under “Implications/ Results”, as a sub-
55
item of “Research Activities”, re-appears in the Interim Completion Report under
“Relations with other Research Programs”, as a sub-item of “Implications/Results”).
Nevertheless, the Final Completion Report is well structured and provides the
necessary information with profuse details.
For a clear reflection of the actual achievements, it was found more useful to refer to
the purpose of the project: field and laboratory evaluation of diploids (for optimal use
in breeding programs). The vast majority of the accessions are from Papua New
Guinea (PNG).
The following articulation of activities and achievements is used for practical reasons
when the actual results are considered:
1. Field evaluation:
a.
b.
Morphotaxonomical description; preliminary performance evaluation (at
Guadeloupe).
Disease resistance evaluation: black and yellow Sigatoka, respectively, at NewCaledonia -FHLOR- and St Lucia – Winban Islands, WIBDECO; Fusarium
Races 1 and 4 at QDPI (Australia)
2. Laboratory evaluation:
a.
b.
Ploidy assessment with flow-cytometry
Molecular markers: isozymes; mitochondrial DNA (mtDNA), chloroplast DNA
(cpDNA) and nuclear DNA markers, by using the RFLP technique.
The achievements can then be reported as follows:
1. Field evaluation
a. Morphotaxonomic description.
The INIBAP descriptor system was used and the results compiled in a CIRAD
designed software system. The Completion Report does not provide details but
states that the data are available through the MGIS program of the INIBAP. On
a request for such data, it appeared that this was not the case at the time the
Review was finalised.
b. Preliminary performance evaluation (i.e. Agronomic Evaluation).
This exercise is not completed. The delay is partly due to the late start of this
exercise (late ‘96) for about 60 of the accessions introduced from the ITC. While the
report provides an extensive table with data such as bunch weight, finger
dimensions, cycle length, these are not discussed and no performance evaluation is
provided. The few comments in the reports deal with morpological characters rather
than with agronomical ones and are part of the INIBAP descriptor list. They would
thus be accessible via the above-mentioned MGIS system.
56
A glance at the table shows very interesting features, which would have been worth
some discussion; For example, the contrast among PNG diploids in bunch and fruit
characters is spectacular and deserves a wide diffusion of information for breeder’s
use. One case suffices here: in the ratoon stage (second cycle after planting) and
following about the same growth rhythm, cultivar ‘Gulum’ produces a 4-hand bunch
of 6kg, while ‘Guyod’ bunches reach 16kg with 8 hands.
b. Disease resistance evaluation
Regarding in vitro propagation and shipment, considerable effort has been spent in
the successful propagation and dispatching of about 250 accessions to the selected
evaluation sites over the world. Although this is not a research activity per se, it has
been a heavy burden on the project.
The actual disease resistance evaluation is still in progress, due to the late
establishment of the trials. Yellow Sigatoka-resistance evaluation had already been
carried out at QDPI in field collection conditions.
2. Laboratory evaluation
a. Ploidy assessment was an important achievement of the project. The carefully
conducted flow-cytometric measurements and interpretations led to the general
confirmation of what was found by previous morphotaxonomic work, with a few
remarkable exceptions.
Analysis of genome sizes points to significative differences between the Sections
Australimusa and Eumusa, and between M. balbisiana and M. acuminata within the
latter section. The results are the subject of a paper submitted for publication (of
which the text was provided in the report).
For the first time, a non-Eumusa triploid was detected: ‘Asupina’ was proved to be a
M. textilis triploid. The deep significance of the result in the context of the
phylogenesis of cultivars should be underlined.
A few presumed diploid PNG accessions (see the INIBAP Musalogue for the PNG
accessions) turned out to be triploids, with important breeding implications.
b. Molecular markers
The first reports deal with isozyme analysis, which was in fact effected during the
pre-BIP period.
The impressive cytoplasm and nuclear RFLP work was an extension of what had
been performed before the onset of the project (Carreel, PhD 1994). More than 50
homologous probe-enzyme combinations were used for nuclear genome analysis,
43 among them presenting a simple pattern that may be assimilated to a locus.
Eleven heterologue combinations were used for cytoplasmic analysis.
Resulting dendrological structures were established, and the combined results on 94
edible diploid clones allowed for the detection of identical/proximal cultivars, largely
confirming the presumptions based on morhological observation.
57
STMS polymorphism study led to the choice of eight markers with high
discrimination potential and clearly localised on the different linkage groups. Most
clones revealed different patterns this way too.
No evaluation of the combined performance of the two methods (RFLP versus
STMS) is reported, and the amount of plus-value of STMS is thus not evident from
the report.
Evaluation
Methodology
A desk study was carried out on the basis of the available interim reports. The Final
Completion Report was due in June 1999.
The project had been visited by one of the present evaluators during the CFC MidTerm Review exercise at the end of 1995. The molecular geneticist (F. Carreel) had
just arrived at that time and the facilities for her research were nearly completely
established.
Results
Evident achievements from BIP 01, are:
-
-
-
A massive flow over long distances of diploid material with potential for
breeding. It certainly was a sound performance to transfer 140 accessions
in good condition to New Caledonia, 77 to St Lucia and 67 to Australia.
The establishment of a network for multilocational disease resistance
assessment. A critical mass of accessions has been tested in local trials at
four locations throughout the world. Although the results will only be
available by the end of calendar 1999, this can be considered as a major
achievement of the project.
The considerable substantiation of the INIBAP MGIS system for cultivar
identification at diploid level.
Unequivocal ploidy assessment by means of flow cytometry has been
realised for all the accessions.
A powerful basis has been established for any further molecular marker
research on the genus Musa, with deep implications for genetic
improvement.
Surprising findings, such as the above-mentioned triploid textilis, open
entirely new prospects in the study of the phylogenesis of bananas, with,
again, far-reaching implications for genetic improvement.
One other rather exciting result was that the diploid accessions from the Comores
and Madagascar do form a special group with perhaps no comparable genotype
anywhere else in the world. Their potential in relation with the equally unique EastAfrican Highland bananas deserves special investigation.
58
However, and despite the abundant positive results, some shortcomings should be
noted. Work on bred diploids – as specified in the original project goals - is not
reported.
On the molecular side, the PI admits that the RFLP method has but a limited
efficiency in detecting differences between diploids, which can be similar in plant
behaviour but widely different in inflorescence structure. This “cries” for the AFLP
method (of which the discriminating performance among the Plantains has been
proved), and one wonders why the method was not adopted in the course of the
project.
Conclusions
The objectives have been partly met. It also appears from the reports that some of
the activities involved were redundant:
(a)
Objective 1 was over-ambitious and partly redundant. The
considerable effort of propagating and dispatching 310 accessions was
a real (but non-scientific) achievement, resulting in multi-location
disease resistance testing which, however, could not have been
achieved within the active BIP 01 period. The intended agronomic
performance evaluation over 3 cycles was an unrealistic goal. The
expected results for all of the PNG accessions are still to be compared
with the similar work achieved at the QDPI in Australia, for the
establishment of a firm agronomical assessment. It is regrettable that
the morphotaxonomic data of all these accessions are not accessible
through the MGIS system of the INIBAP.
In addition, the intention to evaluate “bred hybrid diploids”, formulated in this
objective, does not appear to have been performed, judging from the reports.
(b)
Objective 2 was met with great satisfaction. Ploidy assessment and
genome size evaluation were major achievements and even led to
spectacular findings. The not-planned AFLP method would certainly
have been of great help to better discriminate the bewildering range of
PNG diploids, and it is recommended that this method be essayed in
the immediate future.
Notwithstanding the shortcomings, the project outputs have great significance.
When the results on disease resistance are available, genetic improvement
programs will be fully informed on all major aspects of the germplasm in order to
select the most suitable diploids for particular objectives.
59
BIP 02
Field Crosses for Understanding the Inheritance of Black
Leaf Streak Resistance to Bananas
Description
Co-ordinates:
Principal Investigator:
Dr. F. BAKRY
CIRAD Montpellier
Collaborators:
Dr. F. CARREEL
Dr. C. ABADIE
Dr. K. TOMEKPE
Dr. J. CARLIER
CIRAD Guadeloupe
CIRAD Cameroon
CIRAD Cameroon
CIRAD Montpellier
Administrative Contact:
H. TEZENAS du MONTCEL, CIRAD Montpellier
Duration:
3.5 years, starting April 12, 1995
Objectives
1.
2.
3.
Understanding of the genetic determinism of High Resistance (HR) form and
Partial Resistance (PR) form against Black Leaf Streak Disease in bananas
and relations among both resistance forms.
The establishment of linkages between field observations, segregation and
molecular studies to map the banana genome and locate the genes and
quantitative trait loci (QTLs) coding for resistance versus susceptibility (S) to
the disease.
Develop the use of molecular marker-assisted selection in the banana
breeding programmes.
Modification to objectives
Research on the PR form was dropped at a late stage of the project, because of the
(previously underestimated) significant influence of environment on the PR
phenotype, leading to variable assessment of a supposed same degree in
resistance.
Activities
The Milestone/time chart (as produced by the Principal Investigator in the first 6month report) distinguishes the following activities (grouped and titled by the present
evaluator):
60
A. Study of the resistance inheritance with cross (HRxS)
1. Cross (HRxS) and field observation of the F1 population
2. Selfing a selected F1 issued from (HRxS) cross
3. In vitro growth and duplication of the F2 population
4. Planting and field evaluation (resistance + agronomic data)
Of the F2 population in two locations
B. Study of the resistance inheritance with cross (PRxS)
5. Cross (PRxS) and field observation of the F1 population
6. Selfing a selected F1 issued from (PRxS) cross
7. In vitro growth and duplication of the F2 population
8. Planting and field evaluation (resistance + agronomic data)
Of the F2 population in two locations
C. Study of the resistance inheritance in cross (PRxHR)
9. Cross (PRxHR) and field observation of the F1 population
D. Molecular genetics
10. Search for polymorphic markers in parents and F1
11. Molecular analysis of the F2 (HRxS) population
12. Molecular analysis of the F2 (PRxS) population
13. Genetic mapping and QTL analysis
Activity Groups A, B, C were to be carried out at CRBP, Cameroon, and Activity
Group D at CIRAD-FHLOR, Guadeloupe. The work in CRBP was carried out at
Nyombe station (altitude 60m) with duplication at Ekona (altitude 400m).
All activities were originally planned for the period 1/04/95 to 31/12/98. A 6-month
delay was arranged for Activity Group (D) because of the molecular geneticist (F.
Carreel) arriving in the project only in late 1995.
Achievements
Background information included:
The parents for the crosses were 3 wild accessions:
-
for HR: clone ‘Calcutta 4’ (M. acuminata subspecies burmannicoïdes);
for PR: clone ‘Zebrina’ (M. acuminata subspecies zebrina);
for S: clone ‘Madang’ (M. acuminata subspecies banksii).
The three parent clones are considered homozygous in a morphological sense, but
molecular markers indicate some heterozygosity in ‘Calcutta 4’ (26%) and in
‘Madang’ (9%), which could point to a certain degree of heterozygosity in
physiological traits of potential influence on the resistance mechanism.
The distinction between HR (High Resistance) and PR (Partial Resistance)
emanates from the CIRAD concept. In HR plants, the disease symptoms are hardly
developing and the attacked tissue seems to “die off”, leaving the leaf intact as a
whole. In PR plants, the symptoms develop up to a variable extent but necrosis is
always late (if appearing at all), allowing the plant to develop a rather normal bunch.
61
Other institutes (such as the IITA) consider both HR and PR as expression of the
same (but complex) resistance genes set. Hence the Project BIP 02 could be of
basic significance in that its results could settle this issue.
Review of the achievements
The logical and transparent Activity Scheme enabled a good appreciation of the
achievements, which were extensively reported.
The achievements can be briefly reviewed in parallel with the Activity Groups.
A. Cross (HRxS)
All the 35 F1 plants obtained initially showed HR, but 6 plants began to display a PR
phenotype at later stages. The “molecular marker heterozygosity” of the ‘Calcutta 4’
parent could have played a role in this variation.
The F2 population (153 plants) showed a wide variation in resistance (but no S), with
only 21 plants looking firmly HR, and the rest either HR (uncertain) or PR in various
degrees. The uncertain HR evaluation is due to the less vigorous growth of plants,
whereby the faster-than-normal leaf decay may not have allowed for the disease
symptoms to reach final stages.
B. Cross (PRxS)
The 205 F1 plants differed widely in resistance but no HR was detected (perhaps a
point in favour of the CIRAD theory in its clear distinction between HR and PR).
However, the considerable spectrum in PR was much larger than the slight (9%)
molecular heterozygosity of the ‘Zebrina’ parent would have led one to expect.
Further observation revealed that the classical symptomatological field method led to
highly varying degrees in PR assessment for the same F1 plant at different periods
of the year.
It was thus not possible to distinguish environmental from genetic factors in the PR
assessment.
CIRAD (Montpellier) also found (via molecular markers) a genetic diversity in
Mycosphaerella fijiensis for a sample of one field and collected from the one plant.
CIRAD is presently developing a method under controlled environmental (e.g. glasshouse) conditions for a more accurate resistance analysis in the case of segregating
populations (in the understanding that the F1 population was already segregating,
due to some heterozygosity of one parent).
Consequently no F2 population was created and the B group of activities was halted.
62
C. Cross (HRxPR)
Observation of the 156 F1s revealed two important facts:
-
No S (susceptible) plant was identified;
The PR phenotype was dominant over the HR one.
D. Molecular Genetics
Because of the above-explained difficulty with the B group activities, the study was
concentrated on the (HRxS) cross products.
Considerable efforts produced 40 RFLP and 40 STMS markers (of which a majority
appeared to characterise the F2 progeny), and more than 200 AFLP markers, of
which 149 have been analysed to construct the genetic map. Results showed
significant allelic distorted segregation in the F2 progeny for 58 of these markers,
which proves how a molecular genetics study can complement the field observation
results. Indeed, cytogenetic studies on meiosis of the F1 selfed parent showed its
heterozygosity for at least three reciprocal translocations inferring four sets of
chromosomes.
In a mapping effort, the 91 remaining markers were joined in 11 linkage groups, with
a high correlation constraint (a Lod score of 6). A Kruskal and Wallis analysis, made
with field observations ranked in 6 classes of infection severity, led to the
identification of one RFLP and one AFLP marker strongly associated to the
resistance, and a second RFLP one, mapped on a different linkage group, with
lower significance.
Evaluation
Methodology
A desk study was carried out on the basis of the reports, including the Final
Completion Report.
The project had been visited by one of the present evaluators during the CFC MidTerm Review exercise at the end of 1995. The molecular geneticist (F. Carreel) had
just arrived at that time and the facilities for her research were nearly completely
established.
Results
This project is of a high quality in several aspects. The objectives were clear-cut and
realistic and the layout of the activities was very clear. The achievements are
impressive, considering the pioneering character of the research. The reports
systematically and carefully reflect the progress made for each activity.
A major breakthrough has been made in the understanding of the genetics of
resistance to Black Leaf Streak (also called black Sigatoka) Disease.
63
The three groups of field activities A, B and C produced results of basic importance:
-
-
-
The CIRAD approach of distinguishing HR (High Resistance) from PR (Partial
Resistance) remains a valid instrument for investigation. No HR was found
among the 205 F1s from (PRxS) which would mean that the HR-genes were
absent in the (slightly heterozygotic) PR parent. However, the Group A results
point to the existence of PR-genetic components in the HR parent, since F2
segregation shows a wide range of PR besides the relatively few HR plants.
But this distorted segregation is explainable since PR appeared to be
dominant over HR, as the Group C results showed.
The absence of S (susceptible) plants in the F1 population from (PRxHR)
seems to prove that the resistance mechanism is linked to the existence of
specific resistance genes.
The discovery that environmental factors strongly interfere with genetic ones
in the resistance assessment (Group B results) has far reaching implications
for the future of field observations.
As for the Group D results, molecular marker analysis proved to be a powerful tool
for unravelling the HR-PR interaction. The ultimate results of the project will almost
certainly produce the set of markers for early screening of HR and PR, a tool which
is badly needed in breeding programs.
Conclusion
Objective (1) has been met in a very satisfactory way. Great progress was made in
the understanding of the genetics of resistance to black Sigatoka. Objective (2) was
only partially attained but the identified RFLP markers for resistance can now be
tested for performance. Objective (3) is not yet met, but rapid progress can be
expected on the basis of the present results. The delay is a direct consequence of
the very complex nature of the resistance mechanism and the genes involved.
The prospects for the future of this research are evident. The BIP 02 project has laid
a powerful basis for further fundamental and applied research on resistance to black
Sigatoka or Black Leaf Streak Disease.
64
BIP 03
2
Collection, Characterisation and Evaluation of Nendran Banana in India
Description
Co-ordinates
Organisation:
Kerala Agricultural University, Vellanikkara
Principal Investigators:
Dr. Rema Menon, Associate Professor (Hort.)
Dr. K. Aravindakshan, Associate Professor (Hort.)
Duration:
18 months, starting July 1, 1997
Objectives
1.
2.
To collect, catalogue and characterise clones of Nendran banana and wild
relatives, in close collaboration with the program at the National Research
Centre for Banana (NRCB) with the purpose of establishing reference data for
Nendran for use by future researchers.
To evaluate clones for dwarfness, short cycling and disease resistance,
particularly for yellow Sigatoka and to a lesser extent to weevils and
nematodes.
Activities
1.
2.
3.
4.
5.
Exploration (July ‘97 to March ‘98): Collection of Nendran germplasm planting
material in and around Kerala State, and at the Banana Research Station;
Field Collection, Kanarra: Establishment of the Field Collection at Banana
Research Station, Kanarra. Starting August ’97, through first half 1998;
Morphological characterisation, using the INIBAP Descriptor List;
Evaluation: Initial agronomic evaluation of phenotype, i.e. pseudostem
height/girth, vegetative cycle, number of functional leaves at flowering stage,
resistance to yellow Sigatoka disease (using the Gauhl scoring method for
degree of attack) and to nematodes and weevils (no particular techniques
explained);
Molecular characterisation: Isozyme analysis/zymograms with following
enzymes: peroxidase, malate dehydogenase, catalase, esterase, superoxide
dismutase, shikimate dehydrogenase and acid phosphatase.
Achievements
2
The project originally had another title and purpose: “Developing improved banana varieties with pest
and disease resistance, post-harvest superiority and maximum export potential: Kerala, Krishnan Nair.”
It was realized that the objectives were too ambitious for a BIP-type undertaking and had to be reformulated and concentrated on a feasible program. Hence the rather late start of the actual project.
65
1. Exploration was effected in 8 different locations which roughly cover the following
regions: Southern Kerala and Southern Tamil Nadu (Location 1), Cardamom Hills
(L2), South-Central Kerala (L3), North-Central Kerala (L4), Nilgiri hills (L5),
Coimbatore and Thiruchirapilly districts, i.e. Central Tamil Nadu (L6), Northern
Kerala (L7), and the forest areas of Western Ghats (L8).
The 14 already existing accessions at the Kanarra Station (L9) were added to the
collection.
Thus, the entire envisaged area has been explored. A total of 128 Nendran cultivars
were collected, with 34 from Central Kerala, and relatively few (4) from Central Tamil
Nadu. Three wild relatives were found in Location 8. Only one Horn Plantain was
found (Northern Kerala) and no False Horn could be collected (one “Horn” and one
“False Horn” existed in the Kanarra collection).
2. Field Collection
By June 1998, 125 accessions had been planted. They were given the accession
numbers 001 to 125.
3, 4. Morphological Characterisation/Evaluation
About half of the French Plantain accessions had achieved the first vegetative cycle
by mid 1998 and were tentatively grouped in 12 categories, based on the following
discriminative characters:
-
Persistent versus deciduous male bracts;
Bunch orientation (pendant versus oblique);
Compact versus loosely packed bunches;
Plant stature: tall, medium, small (“short”), and one dwarf; pseudostem colour.
By the end of 1998, most but not all of the accessions will have fructified and a
firmer classification will be possible.
Yellow Sigatoka-resistance appears to be low for all observed accessions with the
possible exception of Category x (the “shorter” French Plantains). Nematode and
weevil resistance is under investigation.
5. Molecular Characterisation
Polyphenol inhibition of enzyme activity could be overcome by adding PVP to the
extraction buffer. A standardised technique for peroxidase provided zymograms
where 3 of the 30 cultivars analysed so far showed a distinct band pattern. These
appear to belong to the “short French Plantain” category (accession numbers
017,045 and 048) but other “shorts” (043,044) could not be distinguished. It is likely
that a comparative study of zymograms with other enzymes will enable further
discrimination up to a certain extent. Work on esterase and superoxide dismutase
was initiated.
66
Evaluation
Methodology
A desk study was performed on the basis of the three reports on the BIP project to
the World Bank. These reports were examined and commented upon by the SAP.
The project was visited from 31 December, 1998 - 5 January, 1999, and included:
-
Review of the project; review of the banana research at the University;
Visit to the Department of Pomology and Floriculture (with the biotech- and
isozyme analysis labs);
Several visits to the field collection with discussion of morphological
characterisation;
Visits to farmers’ fields at Chalakudy and adjoining areas;
Address by evaluator to academic and scientific staff and students on Musa
genetic improvement;
Discussion of the BIP 03 achievements, the pests and diseases
resistance/control work, and the future prospects.
Results
Exploration and field planting were achieved in a short time. Further exploration of
plantains looks unproductive; discussions and visits to farmers proved that the PI
and his associates have a profound knowledge of plantain diversity and that they
would not have overlooked other cultivars.
Morphological characterisation
The INIBAP Descriptor Lists have been established by a scientist (Dr. Rema Menon)
with “expert eyes”. The Nendran (Plantain) germplasm in the Kerala region looks
quite different in composition from that in humid Central Africa, not only because it
consists exclusively of French Plantains, but also because the spectrum of the latter
subgroup is a narrow one.
The identification of “short” French Plantains is of interest because these may turn
out to be semi-dwarfs (their vegetative cycle is not different from that of the
“medium” categories). Such cultivars have been found in Africa only in the central
rainforest zone. Initial evaluation tends to show them as more Yellow Sigatokaresistant than the main group.
The one dwarf accession turned out to be similar (if not identical to) the ‘Njock kon’
of West Central Africa, a dwarf mutant of a Giant Plantain.
Agronomic evaluation
A potentially very important achievement was the collection and characterisation of a
few French Plantain cultivars with some resistance to drought or to flooding
conditions. Such traits have not been reported for the African French Plantains, and
67
study of their physiological background could lead to the identification of structural
components (such as root anatomy) with high potential for genetic improvement.
Molecular Characterisation
It should be stressed that DNA markers for differentiation at inter-cultivar level are
not yet satisfactory, and that better knowledge of isozyme patterns is not only an
alternative, but could help in the construction of more adapted DNA markers (via
RNA). The Kerala attempt at using the combined result of different isozyme
analyses as a marker at inter-cultivar level looks promising and this research should
be encouraged further.
“Yellow Sigatoka (YS)”-resistance
The study of Nendran at KAU shows that the majority of the cultivars are susceptible
3
to this ill-defined Leaf Spot disease , but that the “shorter” cultivars of category X are
more resistant. It is suggested to change the name of the disease into “pseudoyellow Sigatoka” (because M. musicola is not the agent) for as long as the exact
pathogen has not clearly been identified.
It is recommended that the KAU seeks close operational contact with the CIRAD
laboratories (via INIBAP), which are actively studying the different Mycosphaerella
strains.
Resistance to nematodes and weevil
No striking results were reported for this item and its discussion was limited to
generalities. On the basis of what is known for plantains in Africa, the large majority
of the Nendran cultivars should be susceptible to Radopholus and Pratylenchis
coffea because they mostly grow at low altitude. But since the Nendran complex has
been cultivated in Kerala for perhaps two millennia or more, and considering the
results of the agronomic evaluation, there is a chance for identifying resistant
accessions (see Simmonds’ “Bananas”).
Conclusions
The KAU team has achieved important results of international significance in a very
short time. There exist enough indications that the Nendran complex in Kerala State
contains several cultivars of economic importance of which some have not been
reported elsewhere in the world to date.
3
Plantains are known to be resistant to YS, at least in Africa at sea level, and to be susceptible to black Sigatoka
(BS). Thus the Nendran (Plantain) suffering from a Leaf Spot disease in India calls for special attention.
According to David Jones (Infomusa Vol. 4, No 2) the same symptoms of this disease are seen in other parts of
India, as well as in Sri Lanka, Thailand and Malaysia. Examination of the samples (including from Kerala) could
reveal neither Mycosphaerella musicola nor M. fijiensis but there were indications that “a Mycosphaerella sp. with
Septoria as the imperfect state” could be the agent. Because the symptoms in Malaysia are now becoming
combined with BS ones, and since in West-Africa something similar may have been the case at early stages of
the BS invasion, there exists the great danger of black Sigatoka appearing sooner or later in the Kerala zone.
68
Further taxonomic work should concentrate on standardisation of nomenclature, in
4
view of the desired exchange of information and material with the rest of the world .
The promising effort in finding isozyme markers should proceed further and
morphologic counterparts should be sought for each cultivar, in view of a solid
combined identification, which could be of much help for the screening of
appropriate DNA markers.
Agronomic evaluation should move towards physiological research on the key
factors of “drought” and “flooding” tolerance discovered in some cultivars. Among the
far-reaching implications of such research is the subsequent possibility of finding
corresponding DNA-sequences (the expertise and facilities of the visited
biotechnology laboratory clearly offers such opportunity).
Regarding Disease Studies, and apart from the above recommended collaboration
with CIRAD on the “pseudo-yellow Sigatoka”, the need for intensive research on
resistance to Banana Bract Mosaic Virus (BBMV) disease (a vastly growing menace
in Kerala) is becoming urgent. Forging operational contacts with the QUT (Jim Dale’s
lab) would mean a very productive spin-off from the BIP (with the BIP 09 project on
genetic transformation in view of BBMV resistance).
4
In his book “Bananas”, N. W .Simmonds very briefly describes the previously collected French Plantains in
South India, but discussion at Kerala of his proposed names revealed that they can lead to confusion because of
use by people speaking different local languages. Both ‘ethan’ and ’nendran’ are rather generic names, even
within the same location. Do farmers distinguish sub-subgroups within French plantains?
69
BIP 04
Collection, Evaluation and Characterisation of Genetic Resources and
Improved Banana Crop Plants in Vietnam
Description
Co-ordinates
Organisation:
Institute of Agricultural Genetics (AGI), Tuliem-Hanoi
Collaborators:
Phu Ho Fruit Research Centre
Centre of Biocontrol (at the National Institute for Plant
Protection)
Principal Investigator:
D. Do Nang Vinh
Duration:
3 years, plus 9 month extension (by Contract Variation 1,
signed by the PEA on March 16, 1998; no budget
implications), starting March 1, 1995; ending 31
December, 1998
Objectives
A. Original Objectives:
1.
2.
3.
Collection of banana germplasm and evaluation for quality, productivity
and disease resistance;
Development of techniques for micropropagation of high quality
exports varieties of banana, and techniques for elimination of virus
diseases;
Improvement of banana cultivars by selection and crossing of
somaclonal variants.
It can be seen in the subsection “Activities” that the actual objectives covered a
wider research field than the above objectives (e.g. study of diseases, germplasm
conservation). The wider objectives were already being applied at the start of the
project and thus before the October 1995 Workshop.
B. New Objectives (following Workshop in October 1995):
1.
2.
To establish a molecular diagnostic capability for the major virus
diseases of banana (initially for BBTV) in material used for
micropropagation for growers, and for testing in vitro and in situ
collections of banana germplasm;
To test and evaluate methods for the eradication of BBTV from
meristem and callus tissues;
70
3.
4.
5.
6.
7.
To establish a diagnostic capability for Fusarium Wilt and Sigatoka;
To establish Fusarium Wilt and Sigatoka screening plots in which to
select resistant variants of major bananas grown in Vietnam, and
screen diploid, triploid, and tetraploid cultivars available from
collections elsewhere;
To establish DNA fingerprinting methods and apply these to banana
germplasm used in breeding programs for increasing disease
resistance, field condition tolerance, and fruit quality parameters;
To analyse germplasm from Vietnamese banana collections for ploidy
and genotype;
To double the chromosome complement of useful diploids to use as
parents capable of contributing diploid gametes in breeding
experiments.
The reports show that the “new” objectives were added to the previous ones, and
had no influence on the original list of activities. The original list was reproduced as
such in subsequent reports in the form of a milestone/timetable, with a footnote:
“The workplan for the project is currently undergoing change following
recommendations” -i.e. by the Workshop for “new” objectives.
Activities
The reports do not explain the activities in a systematic way. The “Milestone/time
chart” shows the following list of activities:
(1) VARIETY IMPROVEMENT
3. Improving micrpropagation technologies;
4. Developing industrial propagation;
5. Callus, suspension and shoot culture for somaclonal variation induction;
6. Developing protoplast culture procedure for genetic manipulation;
7. Tetraploid-diploid test-crossing and establishing selection procedures;
8. Field competition trials of varieties;
9. Selection of desirable traits;
10. Application of RFLP for molecular mapping of important traits
(10) GERMPLASM STUDY
11. Germplasm collection in North & South;
12. Establishing field collection, Haihung Province;
13. Morphological and cytological study on genotypes;
14 Classification and characterisation of germplasm;
15. Establishing in vitro collection;
16. Study on in vitro conservation by slow growth;
17. International exchange of germplasm
(18) DISEASE STUDY
19. Identification and characterisation of major diseases (fungus and virus);
20. Identification and selection of resistant varieties;
21. Study of methods for virus identification and virus elimination.
71
Most of the objectives B1-B7 can be considered as activities implied in the above
list, sub 19 to 21.
Achievements
Introduction
The final report is to be received by the PEA in the near future. Meanwhile, the
review of the achievements is based on the interim/annual reports. Achievements
are not systematically reported, and it is impossible to review them in parallel with
the planned activities; the latter also being reported in a rather random way.
However, an attempt at extracting the essentials of the reported achievements
proved that they have been very important, and the following attempt is made to
provide a general picture of the reported facts.
Germplasm Study
1. Collection:
The field collection at Phu Ho presently comprises about
125 cultivars. Most of the accessions were apparently
realised within the BIP project.
2. Characterisation:
All the accessions have been characterised by using the
standard INIBAP Descriptor List. Ploidy of all these
accessions was assessed by chromosome counting.
Quite a number of newly discovered species and local
cultivars have the potential of becoming important at an
international level for breeding purposes.
3. Evaluation:
Agronomic evaluation of many accessions has been
performed. Because evaluation took place in two different
environments (Phu Ho for midland; Chaugiang for Red
River Delta), it was possible to qualify cultivar-specific
habitats.
4. In vitro collection:
Duplicates of all the Phu Ho accessions are kept in vitro
under slow-growth conditions.
5.Germplasm Exchange: Several accessions were introduced from the INIBAP
International Transit Centre and are growing at the Phu
Ho collection.
Disease Study
1. Virus diseases
Banana Bunchy Top Virus (BBTV) was clearly identified as the agent of the disease
attacking the commercial bananas (Cavendish group). Cucumber Mosaic Virus
(CMV) and Banana Streak Virus (BSV) were found in a few cases.
72
2. Fungal diseases
Fusarium Wilt: This study was made possible with a 6-month training of three
Vietnamese scientists in Australia (at the laboratory of Dr. Ken Pegg, PI of another
BIP project). The situation has been clarified by VGC (vegetative compatibility
group) analysis, and it was found among others that the isolate VGC 0123 was
infecting the bananas in South Vietnam, while VGC 0124 was dominant in North
Vietnam, together with VGC 0124/5 and 0125.
A list of FW sensitive accessions could be compiled and it turned out that many
ABBs were very susceptible. On the other hand, Race 4 does not appear to be
present in Thailand, and protective measures in germplasm introduction were
advised.
Sigatoka: Both black and yellow Sigatoka diseases have been identified in some
areas, but pressure is apparently not yet high. The diseases were more frequently
observed in South Vietnam.
Other Fungi: A number of other (leaf, fruit) fungal diseases (such as Freckle, leaf
speckle, fruit speckle diseases and anthracnose) were identified, with various
intensities depending on the regions.
3. Bacterial Wilt.
The symptoms of this disease (important in some Cavendish gardens) could be
clearly differentiated from those of Fusarium Wilt. The isolated pathogen was
identified as a bacterium which needs further classification.
4. Disease Control
Virus elimination. The ELISA test has been successfully used for BBTV, BSV and
BMV indexing, and a number of accessions were found positive, but no virusinfected samples were found in the in vitro collection.
Fungus control. Antibiotics were tried with apparent success on in vitro plants for
controlling black Sigatoka and Fusarium Wilt, but field tests have not yet been
performed.
Variety Improvement
1. Micropropagation of selected material:
The technique poses no problem anymore and material has been sent to local
stations for further propagation.
2. Screening for resistant cultivars:
Fifteen commercial cultivars (belonging to the different genomic groups AA, AAA,
AAB, ABB) were tested for resistance to Fusarium oxysporum f.sp. cubense on an
experimental site incubated with Race 1 pathogen. After 24 months, all of the 8
73
ABBs died or were heavily attacked, while the other groups showed no symptoms,
except the AAB, which was lightly infected.
3. Pre-Breeding Work:
-
Cytological analysis of pollen viability was effected on a large number of
accessions;
High pollen sterility was observed on many diploids;
A list of seedy cultivars was compiled;
Based on experimental results, cultivars of potential use in crossbreeding
have been identified;
Test-crosses (attempted for the first time in Vietnam) confirmed the sterility of
the Cavendish group, but revealed that the ‘Chuoi tay’ (Pisang Awak) did not
produce any seed with 6 pollen donors (either AAs or BBs), which is intriguing
since ‘Pisang awak’ is known for its incidental seed production elsewhere.
Evaluation
Methodology
A desk study was effected on the basis of the interim and annual reports of the PI,
before visiting the project from 26 to 28 December 1998. The visit was well
organised by the PI. One day was spent on a 3-hour exposé of the activities and
results, followed by a 4-hour discussion on the progress made and the outlook for
further research. All the collaborators of the AGI participated in the meeting.
Laboratories were also visited. A visit to the Phu Ho collection took another day.
Results
The achievements of this project should be seen in a special context. No banana
research program existed previously and the PI had to rely on staff and facilities of
several AGI laboratories (which had been equipped with support from the IAEA and
from the Rockefeller Foundation).
Therefore, a key aspect of the results is that an actual banana research program
5
has been initiated by this project, which thus had a stimulatory effect.
Germplasm study
The taxonomy of the Vietnamese species and cultivars is firmly established. The
Descriptor Lists of all the accessions have been compiled with great care and at
least two staff members are actual specialists in the matter. The number of
synonyms was reduced to a minimum. The in vitro slow-growth conservation of the
accessions is under full control. The product (information and material) needs now to
be made available to the world in a practical way (and fruitful discussions took place
on this issue).
5
Germplasm collection and characterisation benefited from supplementary support from INIBAPUNDP.
74
Disease
Several staff members proved to be particularly versed in virology and/or research
on fungal diseases. The achievements with respect to Fusarium Wilt are impressive.
Variety Improvement
A good basis for breeding work has been established, but much needs to be
realised for the improvement to be productive. The lack of operational contacts with
other programs in the world is evident.
Conclusions
The lack of a clear strategy at the outset of this project finds its expression in the
multitude of objectives and activities, which actually would be more consistent with a
fully-fledged Banana Research Program. Instead, the project had only a rather
modest budget of $150,000.
It is therefore remarkable that only Objectives A3 and B5, 7 have not been met in a
desirable way.
The project has consequently been carried out in a satisfactory manner, due to the
high motivation and skill of the staff involved, and despite the modest support upon
which it could rely. Guidance from experts abroad was only real during the 1995
Workshop, and via the training of 3 scientists in Australia. For the rest, the PI and his
staff had to rely on their own capacity, which impressed the present evaluator.
The real significance of the BIP 04 project is that it has created a momentum for the
development of banana research in Vietnam.
The institutes involved appear to be in real need of further support, and it would be
deplorable if the momentum already gained were lost.
75
BIP 05
Banana Breeding in Brazil
Description
Co-ordinates
Organisation:
Brazilian Organisation for Agricultural Research
(EMBRAPA-CNPMF)
Cruz das Almas-BA
Principal Investigator:
Dr. S. O. E Silva
Dr. Aristóteles Pires de Matos
Duration:
Three years, starting January 1996
Objectives
a) Breeding of more productive varieties resistant or tolerant to the main pest
disease problems in Brazil;
b) Reduction of plant stature and duration of the production cycle;
c) Increase in the number of fruits per bunch and in fruit size;
d) Production and evaluation of 10,000 diploid hybrids and 1,000 tetraploid
hybrids, resulting in the availability of 10 superior diploid and five tetraploid
cultivars resistant to pests and diseases, with fruit quality and yield better than
test cultivars belonging to the Prata-type; identification of five new varieties
showing superior characteristics; development of techniques for in vitro
fertilisation and for propagation with reduced somaclonal variation (lower than
5%).
Activities
1.
2.
3.
4.
5.
Production and evaluation of AA diploid hybrids;
Production and evaluation of tetraploid hybrids;
Evaluation of genotype reactions to Panama Disease;
Evaluation of resistance to yellow/black Sigatoka;
Advanced tests of promising varieties and tetraploid hybrids in different
ecosystems;
6. Evaluation of banana genotypes with respect to Radopholus similis;
7. Evaluation of banana genotypes with respect to the rhizome weevil borer;
8. Chemical analysis of fruits of banana genotypes;
9. Behaviour of promising banana genotypes in widely different levels of soil
fertility;
10. Use of molecular markers for monitoring somaclonal variation;
11. Chromosomal aberrations in relation to somaclonal variation;
12. In vitro fertilisation;
13. Determination of ploidy levels in banana pollen by diameter measurements.
76
Notes:
•
•
•
•
•
•
Evaluation of triploid hybrids was added to Activity 2, after the beginning of
the project.
Activities 3-7 are evaluations to ensure that hybrids are screened readily
before possible release.
Activity 9 was not conducted as planned. Rather, it was substituted by an
experiment oriented to study the effects of different levels of minor elements
as a means to avoid fruit detachment in the Pionera variety. Pionera is being
promoted by EMBRAPA, but it suffers the limitation of fruit detachment, which
is preventing the adoption of such a variety by farmers.
Activity 11 was eliminated since it was suggested that the same result could
be obtained by using RAPDs.
Activity 12 was cancelled. The cancellation of the work was due to the
following: a) No success in the ovule development under in vitro conditions; b)
Success in obtaining viable seeds from natural hybridisation (pollination) of
the cultivar Maçã; c) Some success in the process of somatic hybridisation as
a tool to perform crosses among genotypes that do not produce seeds
through pollination.
Activity 13 was eliminated at the beginning of the project since CIRAD had
already found out that there was not much practical use for this type of study.
Achievements
1. Production and evaluation of AA diploid hybrids
During the life of the project, 55,612 hybrid seeds from different genetic
combinations were produced and more than 15,000 were evaluated under field
conditions. Less than 2% were selected for clonal evaluation.
In addition, one hundred bunches were pollinated during 1998, but are not yet ready
for harvest. It is expected that 900 hybrid seeds will be generated from these
pollinations. Currently, 368 diploid hybrids are undergoing single plant evaluation
and 97 are under clonal evaluation.
2. Production and evaluation of tetraploid and triploid hybrids
Tetraploids. During the life of the project, 72,217 female flowers of triploid (AAB)
cultivars were pollinated, generating 979 tetraploid seeds. Under laboratory
conditions, embryos of 804 seeds were rescued and cultured in vitro.
Under field conditions and in both phases of single plant and clonal evaluation, one
Maçã-type hybrid (YB 42-21), as well as several Prata-type hybrids, were selected.
Currently, several other tetraploid hybrids are undergoing single and clonal
evaluation for further selection.
Fifty new seedlings are expected in the near future, from the 160 pollinations
conducted during 1998. These seedlings have not yet been harvested. In addition,
77
forty tetraploid hybrids are currently under the phases of either embryo
extraction/culture or hardening.
The production of viable seeds and seedlings (AAAB), resulting from crossings
between the cultivar Maçã (AAB) and the AA diploid Pa Musore 2 (Musa acuminata
ssp. malaccensis), is one of the most relevant results of this project, since no Maçãtype tetraploid hybrid had been generated before. Around twenty such hybrids will
be evaluated in coming years. The synthesis of Maçã-type tetraploid hybrids will
continue.
It is also worth mentioning that 300,000 plants of the tetraploid PV 0344 (Pacovan x
Calcutta IV) have been multiplied for distribution.
Triploids. On 14,429 female flowers of AAAB Prata-type hybrids crossed with diploid
parents, 156 triploid seeds were obtained. Currently, seven triploid hybrids are under
single plant evaluation, and several seedlings are under hardening to be brought to
the field.
The production of triploid hybrids is an activity that was born with this project. No
attempt had been made before. Seven triploid hybrids are under single plant
evaluation, and more than thirty additional ones are under hardening awaiting
transplanting to the field.
As a result of the strengthening of the diploid crossing block and the planting of
more tetraploids, it is expected that the number of triploid hybrids generated will
increase in the near future.
3. Evaluation of genotype reactions to Panama Disease
Sixteen diploid hybrids, 15 of them generated at EMBRAPA-CNPMF and one from
Honduras (SH 3263), already evaluated for reaction to black Sigatoka/yellow
Sigatoka and showing superior agronomic characteristics, are undergoing evaluation
for Fusarium Wilt. No external symptoms of Panama disease have been detected up
to the date of this report. Internal symptoms of this disease, however, have been
detected in the rhizome of one plant of SH-3263. Very severe symptoms have been
observed for the reference cultivar Maçã. Considering that most of the diploids
behaved as resistant to the pathogen, the experiment was installed once more in an
area showing high inoculum potential.
Very severe symptoms were observed on plants of the susceptible cultivar Maçã six
months after planting. The following tetraploid hybrids and cultivars are undergoing
evaluation: Maçã-type, YB 42-21; Prata-type, PV 42-53, PV 42-68, PV 42-81, PV 4285, PV 42-129, PV 42-143, ST 12-31, ST 42-08, SH 3640; and the cultivars FHIA-03
and Figue Pomme Naine.
4. Evaluation of resistance to yellow/black Sigatoka
The 368 diploid and 31 tetraploid hybrids that are undergoing single plant evaluation
are also under evaluation for resistance to yellow Sigatoka. All hybrids and/or
78
varieties selected during the conduction of this project expressed resistance to
yellow Sigatoka.
At the beginning of the project black Sigatoka had not been detected in Brazil. The
project contemplated a study of pathogenic variability of Mycosphaerella musicola
(yellow Sigatoka), in which thirty-four isolates were collected from different varieties
and ecosystems. A highly significant differential interaction for the evaluated
parameters was observed. These data, which suggest a high pathogenic variability
within the species, were used for the Ph.D. thesis of Dr. Zilton Cordeiro, who is now
back on the CNPMF staff.
The project also contemplated the testing of all improved hybrids at CORBANA´s
fields in Costa Rica. Now that black Sigatoka is present in Brazil, only diploid hybrids
(17) are being tested at CORBANA. Ten tetraploid hybrids were sent to a black
Sigatoka-affected area in Manaos to be evaluated in field conditions in 1999.
5. Advanced tests of promising varieties and tetraploid hybrids in different
ecosystems
A trial was planted at Cruz das Almas to evaluate the reaction to Fusarium Wilt
attack of three cultivars, one tetraploid hybrid and five chemically derived Maca
mutants. The results of the study of the reaction to Fusarium Wilt of nine Maca-type
genotypes showed a high incidence of the fungus in all genotypes. Only the hybrid
YB42-21 showed acceptable scores of Panama disease.
Regarding the testing of nine cultivars in the fifteen different sites, it was found out
that in most of the evaluation sites Grande Naine, FHIA-01 and FHIA-18 expressed
the highest yields. It is worth noting that in the evaluation sites, black Sigatoka is still
not present. In addition, the majority of the fields have irrigation systems. Regarding
FHIA-01 and FHIA-18, despite their high yield, they expressed two characteristics
that, according to the researchers in charge of the evaluation, would limit their
commercial use: fruit detachment, and fruit taste different from the Prata-type.
Other experiments: Eleven tetraploid hybrids are being tested in six farmers’ fields,
but have not yet been harvested.
6. Evaluation of banana genotypes with respect to Radopholus similis
Eighteen diploid hybrids, 11 tetraploids, and eight cultivars were evaluated to
Meloidogyne sp. under greenhouse conditions. Another five triploid and four diploid
cultivars are under evaluation in Petrolandia, State of Pernambuco.
The diploids 1318-01, 1319-01 and 4223-06; the tetraploid PV42-68; and cultivars
Pioneira, Prata Comum and Prata Anã did not allow a large multiplication of R.
similis. The data provided for this test allowed for the selection of genotypes to
conform the diploid-crossing block.
The cultivars selected at Cruz das Almas were taken for further testing against
Helicotylenchus sp and M. incognita to Petrolandia, State of Pernambuco. The test
79
confirmed that the cultivars were resistant to Meloidogyne sp., but had high
populations of Helicotylenchus sp. However, there was no economic damage.
7. Evaluation of banana genotypes with respect to the rhizome weevil borer
Four tetraploids and four diploids were evaluated under laboratory conditions. The
data collected showed that the shortest cycles of the insect were observed when
individuals were kept on Terra, Caipira, Lidi, Nanicão, Grande Naine and Nanica,
thus suggesting that these genotypes are the most favourable to the pest’s
development.
Another trial related to the pest preference was installed using the diploids Tuugia,
Lidi and M53, the triploid (AAB) Pacovan, triploids (AAA) Nam, Nanica, Nanicão,
Caipira and Grande Naine, and the tetraploid (AAAB) Pioneira, with the Plantain
Terra as the control treatment. Preliminary results showed that adults of C. sordidus
expressed higher preference for the cultivar Grande Naine, while Pioneira was the
least preferred cultivar.
Most of the data collected indicate that there are different C. sordidus larvae
multiplication rates among cultivars and also different preference levels. Up to now,
these data cannot be used as a tool in selection schemes to assess resistance to the
weevil borer. Therefore, the CNPMF personnel decided to start research on a
biocontrol strategy using Beauveria bassiana.
So far, six strains of Beauveria bassiana out of 31 have been selected as biocontrol
agents of C. sordidus, which kill over 50% of the pest population in 6 to 9 days.
These promising strains are under evaluation in commercial banana plantations.
8. Chemical analysis of fruits of banana genotypes
Sensorial analyses were performed on two Prata Ana and two Pacovan tetraploids,
and twelve more tetraploids (Prata-types) are being submitted. The sensorial
analyses showed that the hybrids PA 12-03, PA 03-22, PV 03-44, and PV 03-76 had
a high consumer’s preference.
The results on ripening showed that cultivars Nanicão and Caipira expressed the
highest firmness values and longer post-harvest longevity, while Prata Anã showed
the longer shelf-life period. Fruits from Prata Anã, Nanicão and Thap Maeo showed
the highest contents of soluble solids, while the lowest values were detected in
Caipira and FHIA-18.
Vitamin C analysis performed on 14 cultivar showed that Prata Anã, PV 03-44,
FHIA-01, FHIA-18, Pioneira and the cultivar Mysore had the highest vitamin C
contents, all of them higher than Gran Nain.
Analysis of eleven mineral nutrients were conducted in fruit pulps of seven banana
cultivars and six tetraploids. Results showed that potassium and nitrogen were the
nutrients found in higher amounts in the pulp of the banana fruits, and that there
were differences between genotypes, regardless of the genomic group. High
amounts of potassium are found in cultivars belonging to the AAA group.
80
All studies related to fruit quality traits have been published, three in ACORBAT
1998, and the others in “Brazilian Magazine of Fruticulture”.
9. Behaviour of promising banana genotypes in widely different levels of soil
fertility (Study of Fruit Detachment)
Activity 9 was changed from what was originally planned. It was changed to a study
of fruit detachment on the Pioneira cultivar. The experiment consisted of evaluating
minor element effects on fruit detachment during two years.
The soil fertility study to overcome fruit detachment by supplying different levels of
minor nutrients with two different levels of N (300 and 400 kg/ha/yr), P (100 and 120
kg/ha/yr), and K (300 and 500 kg/ha/yr) did not show significant differences among
treatments. Thus, it can be inferred that fruit detachment on Pioneira is genetically
determined, with a low environmental effect.
10. Use of molecular markers for monitoring somaclonal variation
A study to detect somaclonal variation through RAPDs in several subcultures of the
diploid hybrids is currently in place. Preliminary results in samples of 10 subcultures
show the presence of somaclonal variation, especially in the last subcultures. For
this purpose, a set of primers has been selected because they show good potential
to detect somaclonal variation, starting from the fifth subculture. Differences in
fingerprinters have also been observed.
It should be mentioned that detecting somaclonal variation in tetraploid and triploid
hybrids exposed to tissue culture was the main goal of the study mentioned above.
However, it was not possible to detect such variation by using RAPDs, as has
occurred with the diploid hybrids, since it has been impossible to get primers related
with somaclonal variation in tetraploid and triploid genotypes. The use of RAPDs to
detect somaclonal variation in diploid genotypes, however, has allowed for the
application of such techniques in other related areas: to detect variability in Sigatoka,
perform molecular characterisation of germplasm, and moko disease
characterisation.
11. Chromosomal aberrations in relation to somaclonal variation
(dropped, see Notes sub “Activities”)
Evaluation
Methodology
A desk study was performed based on the progress reports for June - December 6,
January – June 1997, and January - June 1998, and on the Final Report. Other
studies consulted were:
Martins, V. et al. 1998. Evaluación de las características de la maduración
poscosecha de genótipos de banano. ACORBAT Meeting, Ecuador 1998.
81
Borges, A.L. 1998. Adubacao quimic do solo sobre a producao e resistencia ao
despencamento de frutos de banana “pioneira”. EMBRAPA: 65: 1-4.
The project was visited on March 23 and 24, 1999, and included:
•
•
•
•
Visit to EMBRAPA´s Director of CNPMF;
Review of the project with Principal Investigators;
Visit to the fields to observe breeding and progeny blocks and demonstration
plots;
Wrap-up session with project’s leaders.
Results
The goal of obtaining and evaluating 10,000 diploid hybrids was surpassed, since
more than 55,612 hybrid seeds were obtained and more than 15,000 hybrids were
evaluated under field conditions, during the lifespan of the project. The main product
of this large effort is the strengthening of CNPMF´s breeding blocks, consisting now
of thirty improved diploids, ten of which were in existence before the beginning of the
project.
The project was aimed at the production and evaluation of 1,000 tetraploid hybrids
that could result in the selection of five tetraploids. This latter goal has been
surpassed since the first 13 tetraploid hybrids are under field evaluation, in
seventeen locations.
The main objective of the project of obtaining high-yield varieties resistant or tolerant
to the main pests and diseases in Brazil by conventional breeding has definitely
been achieved. The tetraploid (AAAB) hybrid PV 03-44 and the Caipira cultivar, that
express resistance to Fusarium Wilt, yellow and black Sigatokas, besides showing
excellent agronomic characteristics, have been recommended for commercial
planting in the Amazon Region.
Besides the above releases, several other genotypes (diploid, tetraploid, and triploid
hybrids) are in the pipeline and will be ready for recommendations in the near future.
This is rather remarkable. In addition, the execution of the project allowed the
breeding program at EMBRAPA/CNPMF to considerably strengthen its structure,
allowing for further substantial progress in the near future. The project provided
ample opportunity for scientists to obtain experience in genotype evaluation, an
expertise that was lacking at the beginning of the project. Other important outputs
are related to the development of screening techniques to be used in genotype
evaluation. Screening techniques have been developed/adapted/adjusted for
Sigatoka, weevil borer, nematode, moko disease, Fusarium Wilt, and fruit quality
and post-harvest assessment.
It is worth mentioning that seeds from the Maca cultivar were obtained for the first
time. This is remarkable since it opens the doors for the genetic improvement of the
most preferred banana cultivar in Brazil.
82
It should be pointed out that the above results, especially the massive multiplication
and field evaluation of improved genotypes, are very important since black Sigatoka
is now present in Brazil.
Some of the results of the project have already been published or presented in
scientific journals and meetings, respectively. Thirteen articles have already been
published.
In general, the expertise and technical preparation of the scientists involved proved
to be adequate, and the excellent performance of the project is to be commended.
Conclusions
The project´s original expected outputs were:
(a)
(b)
(c)
Identification of 10 superior diploid hybrids;
Identification of 5 new tetraploid hybrids resistant to the principal
diseases and pests and with better productivity and fruit quality than
their traditional equivalents; and
Official launching of at least two new varieties resistant to Panama
disease and to one or both Sigatoka leaf spots.
These outputs have been accomplished beyond the original expectations as can be
observed in the sections “Achievements” and “Evaluation: Results” of the present
review.
It is important to point out that with the financial support and results obtained from
this project, the Banana Breeding Program at EMBRAPA/CNPMF now has a strong
basis to systematically release more banana hybrids in the near future. It was
observed during the evaluation of the project that the breeding program is being
managed scientifically, including all the ideal components for improving a
vegetatively propagated crop: single plant evaluation, clonal evaluation, early and
advanced generation testing, and crossing blocks based on a set of improved diploid
hybrids.
The EMBRAPA Banana Breeding Program is presently associated with a private
tissue culture enterprise (“CAMPO”), which has already established laboratory
facilities close to the Programs offices and fields. Three hundred thousand seedlings
of each Caipira and PV03-44 have been produced by this company already.
Prospects. In order to better achieve or to continue to achieve the main objective,
there is a need to dedicate more time and funds to obtaining larger segregating
populations in order to increase the possibilities of obtaining more banana hybrids
with desirable commercial traits, as was planned in this project. Indeed, since black
Sigatoka is beginning to spread in Brazil, and could destroy the export banana trade,
the activities undertaken in this project should be greatly enlarged.
83
The following activities need to be or will be considered in order to increase or
broaden the impact of a project of this kind:
a) Continue to evaluate existing genetic material (2x and 4x) as well as material
emerging from the breeding activities;
b) Characterise the diploid gene pool located in the crossing block as well as
new acquisitions for the collection, to assess their potential use in a breeding
program as sources of resistance to major pests and diseases;
c) The EMBRAPA/CNPMF has about fifty non-improved natural genotypes that
are being intercrossed to determine their fertility potential for breeding
purposes. Their potential use as female or male parents needs to be
determined;
d) The project needs additional personnel for the breeding work, since this is
being carried out by only one person at the moment.
84
BIP 08
Banana Biotechnology Consortium:
Novel Genes for Fungal Resistance and Post-Harvest Quality
Description
Co-ordinates
Leading Institute:
Boyce Thompson Institute for Plant Research, Plants and
Human Health Program, Cornell University
Principal Investigator(s):
Dr. Charles J. Arntzen
Dr. Gregory D. May
Duration:
March 1, 1995 - February 28, 1997
Objectives
1.
To create and make available to co-operating investigators a “molecular
toolbox”6 comprised of a reliable and efficient banana genetic transformation
system, including vectors for different gene introduction strategies, and
genetic libraries for isolation and manipulation of genetic elements from Musa
spp. to create economically useful phenotypes.
2.
To identify the genes in banana which encode enzymes participating in
ethylene biosynthesis, characterise the members of this gene family, which
are expressed in response to fungal pathogen stress and to the onset of
ripening, and create prototype transgenic bananas which have increased
fungal resistance traits or enhanced fruit ripening traits as a result of modified
expression of the genes or members of the gene family.
Notes
1.
6
Objective (1) also appears in the BIP 10 project. For this objective, both
projects formed (together with BIP 9) the so-called “Biotechnology
Consortium”. Consequently, the relevant achievements in BIP 8 are to be
considered in combination with those reviewed sub BIP 10 for this item.
The Molecular Toolbox simply represents a collection of protocols that describe optimized procedures for
the production of transgenic banana plants in detail. These protocols encompass:
1. Optimized procedure(s) for the regeneration of complete plants from embryogenic cell cultures of
different banana cultivars;
2. Optimized procedure(s) for the permanent transformation (i.e. transfer and integration of
agronomically relevant genes) of such embryogenic cell cultures;
3. Ready-to-use and easy-to-handle constructs for the transformation process, including vectors for the
transfer and integration of the foreign genes, optimal selectable marker genes and promoters for their
strong expression in the transgenic environment.
85
2.
Tissue culture research became an important activity of BIP 08, when it was
realised that the existing transformation method produced a number of
chimeric transformed plants. The meristem tips were abandoned as
transformation tissue, and replaced with success by the embryogenic cell
suspension technique, which was previously established in the BIP 10 (for
biolistic transformation). The “Biotechnology Consortium” again proved to be
very instrumental. Some achievements in this sector do belong to the postBIP period, but are nevertheless reviewed, partly because of their key
importance, but also because they prove that the BIP effort had a most
stimulating effect on this research.
Activities
Two major areas dominated research at BTI: tissue culture and molecular biology.
Tissue Culture
1.
2.
3.
Development of techniques to isolate protoplasts (for transient expression
assays), to transform and regenerate banana cells, and to produce
somaclonal mutants;
Establishment of embryogenic cell suspensions for the regeneration of nonchimeric transformants;
Screening of Agrobacterium tumefaciens strains for efficient transformation of
banana cells.
Molecular Biology
1.
2.
3.
4.
Study of gene expression during fruit ripening, specifically at different stages
of pulp and peel development;
Establishment of cDNA libraries from pulp, peel, leaf, and diseased leaf;
Construction of selectable marker and reporter gene fusions to heterologous
promoters and tests for function in transient expression assays;
Besides these activities, but not forming part of the objectives, an intense
research effort on the use of transgenic banana plants for the production of
antigenic proteins (“edible vaccines”) involved several scientists (among them
Drs. Hugh S. Mason and Tsafir S. Mor). The productivity of this group is
documented by a series of publications in top journals (PNAS, Nature
Medicine) and promises future breakthroughs, but is beyond the scope of this
review on activities.
Achievements
Tissue Culture
1.
A transient expression assay to test for promoter function and strength has
been developed from banana pulp protoplasts, isolated from ripening fruits at
86
stage six and seven. Reporter gene constructs (e. g. GUS) with heterologous
promoters (e. g. 35S promoter of Cauliflower mosaic virus).
2.
Embryogenic cell suspensions from male flower tissues of Grand Naine were
established, that served as substrates for Agrobacterium-mediated
transformation(s) and regenerations (Dr. Joyce M. Van Eck, Nicole S. Higgs).
3.
In a type of technology transfer, Dr. T. R. Ganapathi (Trombay, India)
regenerated plants (AAA Grand Naine, AAB, Rasthali) from embryogenic
tissues of flower buds. He employed co-cultivation of micro-cross sections of
these tissues (ideally containing a single layer of cells) from Grand Naine and
several lines of Rasthali with Agrobacterium tumefaciens to produce
transgenic plantlets expressing a ß-glucuronidase-intron-rice and ßglucuronidase-ubiquitin-promoter fusion. Insertion of these constructs into the
target genomes was verified with PCR. The time from co-cultivation to
regeneration of plantlets is only four months. A chloroplast transformation
protocol is presently being developed.
4.
A series of Agrobacterium tumefaciens strains were tested for their potential
to transform banana cells. EHA 105 proved to be the best transformer in 5
cultivars, especially Grand Naine (Dr. Greg D. May).
Molecular Biology
1.
cDNA libraries from fruits of different ripening stages (separately from pulp
and peel), roots, leaves, and leaves challenged with Mycosphaerella fijiensis
were established. Two strategies were followed: cDNAs differentially
expressed during the ripening process of the banana fruit were isolated and
eleven of them characterised, and cDNAs induced in infected leaves were
identified. Among the eleven ripening-associated cDNAs are two
metallothionein transcripts, a ß-1,3-glucanase, thaumatin-like protein and
class III acidic chitinase transcripts. Northern analysis revealed a tissuespecific steady-state level of the different mRNAs, e. g. thaumatin-like protein,
endochitinase and ß-1,3-glucanase transcripts are only present in pulp and
peel tissue, but not in roots, corms, or leaves (Drs. S. K. Clendennen, G. D.
May, J. Narvaez-Vasquez, Colombia).
2.
A previously established Musa acuminata genomic library was screened with
ripening-specific cDNAs, and genes encoding ACC-synthase and -oxidase as
well as a polygalacturonase isolated, restriction-mapped and sequenced.
Moreover, genomic clones of metallothionein and ß-1,3-glucanase genes
have been characterised, and in both cases the respective promoters (up to
1500) isolated and sequenced (Dr. G. D. May).
Evaluation
Methodology
87
An intense desk study was performed on 9 scientific papers, the Termination Report,
and two Annual Reports of the Boyce Thompson Institute for Plant Research. A total
of two days was spent to become familiar with the various laboratories of the Boyce
Thompson Institute and the scientists who were and are involved in BIP research, as
well as some scientists who will continue research on banana improvement. Among
the former category were Drs. Greg D. May, Hugh S. Mason, Tsafir S. Mor, T. R.
Ganapathi, Joyce M. Van Eck, and Nicole S. Higgs; among the latter were Drs.
Peter Balint-Kurti, Alice Churchill, and Peter Beetham. Basically, Dr. Greg May
organised seminars and interviews for the reviewer.
Results
To begin with, it should be stressed that the research at Boyce Thompson Institute
for Plant Research:
(1) Had a good start on the basis of previous research (partly done at Texas A &
M University); and
(2) Was fuelled from several resources; but
(3) Had only 2 years to achieve the results reported in this review.
For the type of experiments necessary for the BIP, spanning from tissue culture
work over molecular biology to gene technology, two years was an extremely short
time. Nevertheless, major progress was made, though some of the topics of the
project (e. g. promoter isolation) have only been touched on to date, or were
achieved post-BIP.
Molecular toolbox
This toolbox, in the view of the reviewer, the centre of the whole project, comprises
a collection of A. tumefaciens strains for Musa transformation (the best of which is
EHA 105), a genomic library (from Grand Naine), and cDNA libraries from a variety
of tissues at different developmental stages or different times after pathogen attack
(here: Mycosphaerella fijiensis).
Originally, the toolbox also included promoters or promoter elements with or without
intron insert-ion between them and the gene, driving either a constitutive or tissuespecific gene expression in transgenic banana plants, combined with visual
transformation markers, selectable markers, and reporter genes. The toolbox is now
available (means: the different ingredients have been worked out), except that the
different promoters are just being characterised. In any case, all the work for the
production of the toolbox was very successful, despite the relatively short period of
time. The toolbox itself, if fully available to other laboratories especially in
developing countries, will certainly catalyse banana research in this area, since the
time-consuming and costly development of the toolbox ingredients have already
been done.
Genes for banana improvement
A series of coding sequences (as cDNAs or genomic clones) for scientifically or
agronomically interesting proteins (or traits) is now available. Especially genes
involved in defence reactions (encoding e. g. ß-1,3-glucanase, chitinase, thaumatin88
like protein, metallothionein, and others) can be used: (1) to characterise their
structure; (2) to study their expression and its regulation; and (3) to transform
susceptible banana plants to complement the deficiency. The last aspect will
certainly dominate continuing work, and also interest many other laboratories,
especially in banana growing (i.e. developing) countries. Since genes of ethylene
biosynthesis are also available, a down-regulation approach via ACC-S-antisense
technology could be used to manipulate ripening.
The molecular toolbox and improvement gene aspects of the project were
satisfactorily accomplished, yet there are deficiencies regarding the various
promoters. Again, the short project time prevented completion of this important
part, though promising work is in progress at BTI.
Tissue culture work (including transformation and transient expression
assays)
For all the above work, reliable transformation and regeneration protocols are
prerequisites. Therefore, first embryogenic cell suspension cultures were developed
that reproduce rapidly. Second, a reliable regeneration protocol is at hand that
should be applicable for various banana (and plantain) cultivars. Third, protoplasts
can be isolated and maintained for a short period sufficient for transient expression.
Fourth, a highly effective Agrobacterium tumefaciens strain has been selected for
transient and permanent transformation. Fifth, a transformation protocol has been
worked out, and sixth, a transient expression assay has been successfully
established, using GUS reporter fused to heterologous promoters (e. g. CaMV35S,
actin).
The results of all this work are at least part of already published papers (e. g.
BioTechnology 13: 486-492, 1995) or are being prepared for publication. Given the
problems with regeneration of monocotyledonous plants generally, with regenerative
potential differences between different banana cultivars, and the limited host range
of A. tumefaciens (not normally including monocots), the above research now allows
banana plants to be managed in different areas of tissue culture. This conclusion is
confirmed by work in at least two other laboratories. The available protocols, once
fully published, will certainly trigger extensive research on other banana cultivars not
used in the present project, but nevertheless economically as important as the ones
employed here.
Conclusions
Project goals
Most of the project goals as listed in the CONTRACT FOR SERVICES of March 1,
1995, were reached. However, the promoter sequences to be used for various
purposes and the proposed Internet “bulletin board” for information exchange could
not be finished, because of limited project time. Altogether, the achievements are
impressive.
All these BTI activities witness the competence of the BTI researchers in banana
89
research. In conclusion, the BIP funding of research at BTI definitely catalysed
progress in this somewhat neglected area. In view of the documented success, it is
strongly recommended to grant funds for a longer period of time (e. g. 3-4 years),
and to check all proposals in a future BIP, which I most strongly suggest, for
overlaps, so that identical research at two supported institutions is a priori excluded,
which was not the case in the present BIP.
General performance
The technical and scientific environment at the BTI was, and still is, excellent. A
number of renowned plant pathologists, plant breeders and plant and fungal experts
are working at Cornell University. The BTI itself is in top condition, extremely well
equipped, and owns ample laboratory and greenhouse space. More importantly
though, the scientists involved in the BIP, as far as I could ascertain, are
exceptionally motivated and experts in their fields. As judged from the short visit, BTI
is a good place for good research. Further, the efficiency is very high, as gauged by
the time available and papers published (or in preparation). Also the fact, that the
second PI was recently hired by NOBEL Foundation, adds to this very positive
impression of the personnel at BTI.
Training and technology transfer was not explicitly a requirement fixed in the
Contract with the PEA. However, a fairly strong training component existed and
should be appreciated, with Dr. P. Rowe (Honduras), but more so with Colombian,
Mexican, Malaysian and Indian scientists.
Prospects
The high quality of the BIP research at BTI led to a series of spin-off activities, which
are partly extending research of the BIP, partly focussing on new, but nevertheless
related areas:
•
•
•
•
•
•
Isolation, characterisation and use of banana promoter sequences for
transient expression and stable transformation(s), driving tissue- or
development-specific expression or elicitation of target genes in transgenic
banana plants;
Differentially expressed cDNA clones from ripening fruit and
Mycosphaerella-elicited leaves will be further characterised and mapped on
segregating populations (in co-operation with CIRAD, France, or INIBAP,
whose populations will possibly become available later in 1999;
Transformation and regeneration protocols for a series of other banana
cultivars will be developed (e.g. for Grand Naine);
Establishment of Musa BIBAC libraries (in the view of the reviewer, a central
aspect of all labs working with Musa species);
Non-destructive reporter gene tests will be developed (e.g. with GFP or its
mutated derivates);
More stress will be on Mycosphaerella fijiensis and M. musicola research per
se. First, the role of fungal “secondary” metabolites in black Sigatoka
disease development and the regulation of synthesis of these compounds
(e. g. induction by starvation) as well as toxin identification will be dealt with
more intensively. Secondly, GFP-transformed M. fijiensis and M. musicola
90
•
•
•
•
will allow study of the mechanism of infection of the host in colourful
detail(s);
cDNA array technology will be used to identify genes of M. fijiensis induced
by plant compounds (Dr. P. Balint-Kurti, Dr. A. Churchill);
Another spin-off was the detection of retrotransposons of the gypsy and
copia types in the banana genome (~ 1000 copies/haploid genome), that are
either specific for the A- or the B-genome. These can be used for various
aspects (e. g. mapping purposes);
A banana research meeting was organised by BTI for March 22-25, 1999,
where all groups working on various aspects of banana/ plantain or
Mycosphaerella research came together;
One of the components of the Cornell University Comparative Genomics
Program is a Banana Genome Initiative proposed by BTI centering around
establishing an expressed sequence tag (EST) project and the physical
mapping of the banana genome.
91
BIP 09
Development of Transgenic Bananas with Resistance to Banana
Bunchy Top and Banana Bract Mosaic Viruses
Description
Co-ordinates
Organisation:
Queensland University of Technology (QUT) Brisbane,
Australia
Principal Investigator(s):
Prof. Dr. J. Dale; Dr. R. Harding
Duration:
July 1, 1995 - June 30, 1998
Objectives
1.
Development of transformation cassettes for banana bunchy top virus
resistance based on current and developing knowledge for stable resistance
to all strains;
Characterisation of the banana bract mosaic virus genome and development
of transformation cassettes for resistance;
Transformation of bananas with a single resistance transgene and ultimately
multiple resistance transgenes, resistance screening of transformants, and
preparation of promising transgenic lines for field trials.
2.
3.
Activities
The activities can be grouped into three different categories:
-
A molecular biology part (virus characterisation,
transformation cassettes, isolation of viral promoters);
A transformation and regeneration part;
Testing of transgenics in the greenhouse.
construction
of
The PI additionally tried to involve the Indian Agricultural Research Institute (New
Delhi) to conduct glasshouse trials with transgenic Bluggoe and Cavendish and
BBMV in India, but Biosafety implications are still hampering the operation.
1. Characterisation of viral sequences
Through part of the molecular toolbox consortium, QUT performed all the activities
listed below with only little contact with the other partners:
92
1.
2.
3.
4.
The ssDNA genomes of banana bunchy top virus (BBTV) were further
characterised functionally.
The genome of banana bract mosaic virus (BBMV) was further characterised.
Promoter regions of the six ssDNA components of BBTV were isolated and
functionally tested. The so-called BBTV6.1 promoter was more intensely
characterised.
Four transformation cassettes were produced using BBTV gene sequences
and binary vectors, and tested in transient expression assays.
2. Transformation and regeneration of various banana cultivars
1.
2.
3.
Three different transformation protocols were developed at QUT:
Agrobacterium-mediated gene transfer, biolistic transfer in embryogenic cell
suspensions (with the help of the KULeuven (see Project BIP 10), and
transformation of embryogenic callus derived from immature male flower
buds.
Both Agrobacterium-mediated and biolistic gene transfers were used to
transform:
Bluggoe plants (reporters: GUS, sGFP);
Bluggoe embryos (four different BBTV and one BBMV gene cassette);
Grand Nain embryos (reporters: GUS, sGFP, two different resistance
BBTV gene cassettes, and one BBMV resistance gene cassette).
Embryogenic cell suspensions from male flower explants of Cavendish
(Williams and Grand Nain) and Goldfinger were developed, and plants
regenerated from these embryos (Douglas K. Becker).
3. Greenhouse testing of transgenic banana for tolerance towards BBTV
Characterisation, multiplication and glasshouse challenging of potentially virusresistant transgenic Bluggoe and Grand Nain plants.
Achievements
Characterisation of viral sequences
1. The viral genomes:
a) BBTV:
The BBTV genome consists of six (or more) circular ssDNA
components each about 1 kb in size and each containing at least one
ORF (BBTV DNA-1: two ORFs). The major DNA-1 gene encodes a
replication initiation protein (Rep), DNA-3 the viral coat protein (cp),
and DNA-4 a movement protein. The function(s) of the other ORFs are
still unknown. The intergenic (non-coding) regions of DNA 1-6 share
three regions of homology: 1. the 66 - 92 nt common region, CR-M, the
binding site for an endogenous ssDNA primer for complementary
strand synthesis; 2. the stem-loop common region, CR-SL, a target for
the replicase; and 3. a TATA-box. These results were published in a
total of 6 publications.
93
b)
BBMV: The BBMV genome was further characterised, and the coat
protein gene isolated and sequenced.
2. The viral promoters:
a)
Intergenic regions of BBTV DNA-1 to DNA-6 were structurally and
functionally characterised.
Structure: three regions of homology (see above), and TATA-like sequences
present.
b) Function: The potential promoter sequences of all intergenic regions were fused
to β-glucuronidase (GUS) and green fluorescent protein (sGFP) genes,
and their functions tested in transient expression assays using tobacco
and banana cells (Bluggoe, Grand Nain). All the promoters are active,
but the different activities vary considerably in one and the same
system (e. g. BBTV 4, 5 and 6 are more active than the others are),
and between tobacco and banana. The promoter of BBTV DNA-6 was
analysed by deletions generated by restriction or PCR, and contains Gand I-boxes in a 272 bp minimal promoter region. This promoter (BBTV
6.1) was also used for stable transformation of banana.
3. The transformation cassettes
A series of transient expression and transformation cassettes was constructed. The
different BBTV resistance cassettes used either: (1) the full-length replication
initiation protein (rep) gene; (2) a truncated rep gene with an introduced stop codon
(“mutated rep gene”); (3) a DNA-1 internal ORF; (4) a DNA-5 ORF; or (5) a DNA-5
UT ORF. The BBMV resistance cassette was based on the coat protein gene of this
virus. All the viral genes were driven by the 35S promoter. The NPT II selectable
marker gene was controlled by the Nos promoter.
Transformation and regeneration of various cultivars
a)
A series of transgenic Bluggoe lines was established expressing GUS
and sGFP under the control of the 35S promoter and the npt II gene
driven by either the 35S promoter or the BBTV 6.1 promoter.
b)
A series of transgenic Bluggoe lines was established containing the
various virus resistance genes (PCR positive):
Construct
Number of lines
BBTV DNA-1 rep
BBTV DNA-1 internal ORF
BBTV DNA-5 ORF
BBTV DNA-5 UT ORF
BBMV cp
94
33
40
4
37
41
c)
A series of transgenic Williams and Grand Nain lines was established
expressing GUS, sGFP, BBTV DNA-1 rep, BBTV DNA-1 internal ORF,
and BBMV coat protein gene under the control of the maize
polyubiquitin promoter. The selective marker gene for NPT II was
driven by the BBTV 6.1 promoter.
PCR-positive Grand Nain lines
Construct
Number of lines
BBTV DNA-1 internal ORF
BBTV DNA-5 UT ORF
BBMV cp
16
55
9
Molecular analysis of transgenic plants
A fraction of the potentially transgenic bananas were analysed for transgene
presence and copy number by Southern blotting hybridisation.
a)
b)
Bluggoe: A total of 28 independent lines contained at least one,
maximally seven copies of the transgene.
Grand Nain: A total of 12 independent lines contained at least one,
maximally 14 copies of the transgene.
Performance of the transgenic plants
Three transgenic plants were challenged with BBTV-carrying aphids in the
greenhouse (amongst them a Cavendish line expressing BBTV-5 UT ORF, and a
Bluggoe line expressing BBTV DNA-1 internal ORF).
Evaluation
Methodology
A desk study was performed on 6 scientific publications, the two interim reports, the
Termination Report and a manuscript (accepted for Plant Cell Reports). Two days
were spent at the Centre for Molecular Biotechnology, School of Life Sciences,
QUT. This relatively short time was a consequence of a severe influenza brought
from Hong Kong, and a cold that prevented Dr. Dale from coming to work. During
Day 1, Dr. Robert M. Harding and Dr. Douglas K. Becker were interviewed. They
also showed me the Carseldine Campus and the transgenic bananas. Day 2 was
used to speak to Dr. James L. Dale, see the laboratories and growth chambers with
the transgenics, and discuss some aspects of the project with Dr. Becker’s work.
Results
It should be stressed, that Dr. J. Dale and his group have more than ten years of
experience with BBTV, two years with BBMV, well-equipped laboratories, and
several funding sources for their research. Moreover, his research in the frame of
BIP has been supported for three full years.
95
1. Molecular toolbox
The contribution of QUT to the molecular toolbox comprises:
a)
b)
(c)
Several transformation cassettes with genes, or gene variants, thought
to confer partial or total resistance to BBTV or BBMV;
A total of six different viral promoters, of which BBTV 6.1 is used in
stable transformation of bananas;
Integration of three different transformation protocols , developed in
other laboratories of the Biotech Consortium (see BIP 8 and 10), of
which Agrobacterium tumefaciens (strain LBA 4404) and micro-particle
bombardment transformation was used.
The components of the toolbox have been worked out in parallel with University of
Hawaii, but no direct contacts were established between the two institutions. In any
case, all the work for the development of the above toolbox had already began
before BIP, but was continued and accelerated by BIP, and altogether is very
successful. Since no construct has yet proven to confer BBTV or BBMV resistance,
the perspectives remain vague. However, once a viral gene or gene variant has
proven to be effective, the toolbox, if available to researchers world-wide, will trigger
activities to generate various cultivars with resistance to BBTV, BBMV, and other
viruses as well.
2. Transformation and regeneration of cultivars
For the application of the toolbox, reliable transformation and effective regeneration
protocols are needed. Therefore, various cultivars were regenerated first (cv.
Bluggoe, cv. Williams and cv. Grand Nain). Second, the function of potential
promoters, derived from the intergenic region of the virus, could be successfully
tested in transient expression assays using GUS and sGFP reporter genes. Third, a
series of transformants could be generated with various constructs and resistance
cassettes, which were partially characterised in molecular terms (Southern analysis,
copy number). All these achievements prove that QUT has the capacity to produce
the constructs, to transform the target cells (either with particle gun or
Agrobacterium), and to regenerate the transformed cells. Moreover, a regeneration
scheme has been developed for Williams and Goldfinger (male flower explants,
apparently an adaptation of a similar CIRAD technique). Also here, the results have
already been published in part (e. g. J. Gen. Virol, 79: 2301 - 2311, 1998), or are
prepared for publication. So, QUT has been adding to the success of the BIP
considerably.
3. Performance of transformants in the greenhouse:
The extremely small number of transgenic bananas challenged with BBTV in the
glasshouse and the negative results (no resistance) represent a deficiency of the
project. Certainly more transgenics could have been tested from the beginning in
collaboration with the KULeuven, as a part of the Consortium activities, and it is to
be expected that resistance will be found with increasing numbers of plants, but only
if the general concept is correct (resistance conferred by viral proteins).
96
Conclusions
Project goals
Most of the project goals were reached. However, it was a priori not proven that the
general strategy of expressing a viral gene in the host plant and thereby conferring
virus resistance, holds also for gemini viruses, as e.g. BBTV. Therefore, BIP-funded
research at QUT aimed at testing this strategy with different BBTV genes and one
BBMV gene was important. So far, no resistance to BBTV (or BBMV) could be
observed in transgenic plants. However, since only three plants were challenged, the
results cannot be conclusive, especially since only three of a total of 65 transgenic
plants were resistant to BBTV in Dr. Hu’s experiments (University of Hawaii).
Nevertheless, the achievements at QUT are altogether impressive, though final
proof for the resistance concept is missing. It is for this reason, that multiple
resistance transgenes could not be tested either.
General performance
There is absolutely no doubt, that much competence is accumulated at QUT. The
scientific environment is therefore excellent. The Department of the PI is extremely
well equipped and organised, although the greenhouse with the transgenic plants is
remote from the laboratory. The scientists at QUT, of whom I met only few, are
experts in their areas and very motivated. The efficiency of the scientific work is
extremely high (published papers per time unit).
All the QUT achievements are a consequence of the high competence and
motivation of the researchers, and it is to be expected that QUT will play a major role
in developing transgenic bananas resistant to BBTV and BBMV in future. However, it
is advisable in the future that more attention is given to checking all future proposals
for overlaps, so that identical research at two supported institutions will be excluded
(specifically, QUT and UH).
Prospects
Research at QUT on virus resistance in bananas started before BIP, continued with
the help of BIP, and will focus on related research topics in future:
-
Further characterisation of the BBMV genome and isolating more genes with
potential for resistance to viruses;
The cloning, sequencing and elucidation of functions of the BBTV genes,
especially DNA-1 internal ORF, DNA-2, -4, -5, and -6 ORFs;
The “erratic rate of multiplication of transgenic lines” (habituation: no
response to hormones) has to be overcome;
Many more transgenic lines have to be regenerated from different
bombardments, and challenged with BBTV or BBMV, respectively, in the
glasshouse;
97
-
Organisation of field trials in India, using transgenic plants whose resistance
has been proven in glasshouse trials.
98
BIP 10
Genetic Transformation of Prototype Bananas for Black
Sigatoka-Resistance
Description
Co-ordinates
Organisation:
Katholieke Universiteit Leuven
(KUL) Leuven, Belgium
Principal Investigator(s):
Prof. Dr. R. Swennen
Dr. L. Sagi
Duration:
November 1, 1994 - October 31, 1996
Objectives
1.
To create and make available to co-operating investigators a “molecular
toolbox” comprised of a reliable and efficient banana genetic transformation
system, including vectors for different gene introduction strategies, with the
aim to create economically useful phenotypes.
2.
To identify heterologous regulatory DNA elements to drive constitutive or
tissue-specific gene expression and construct efficient expression cassettes
for stable transformation of bananas.
Activities
The activities at KUL can be broadly categorised into:
1.
2.
3.
4.
Optimisation of Agrobacterium tumefaciens-mediated and biolistic gene
transfer techniques;
The construction of banana transformation vectors with (twin) selectable
marker genes and strong promoters;
Study of the efficiency of heterologous promoters in transient expression
assays;
The production of many independent transgenic lines of several different
cultivars using the optimised techniques.
99
Achievements
Gene transfer techniques
1.
A systematic study on chemotaxis and attachment of A. tumefaciens, strain
LBA 4404, to wounded banana tissues was performed, and the
transformation efficiency tested with a GUS reporter system. Results were not
conclusive during the BIP project period, but the system was improved
afterwards.
2.
Optimum conditions for biolistic transformations of banana embryogenic cell
suspensions (ECSs) of cv. Bluggoe, cv. Williams and cv. Three Hand Planty
were established. The optimised particle bombardment of ECS was superior
to A. tumefaciens, when the latter was performed on meristems and allowed
to produce a large number of independent transgenic lines.
Banana transformation vectors
For consecutive transformation steps, plant expression vectors containing (twin)
selectable marker genes (geneticin and hygromycin resistances) driven by a rice
actin promoter and flanked by a poly(A) signal from A. tumefaciens gene 7 were
constructed.
Heterologous and homologous promoters
1.
Several heterologous promoters for high gene expression in bananas have
been identified by fusing them with the gusA gene and introducing these
fusions into target cells by the improved particle bombardment technique.
Among these promoters were the maize polyubiquitin gene promoter
containing the first intron, the rice Act 1 gene promoter, the recombinant Emu
promoter, the duplicated CaMV 35S promoter together with the untranslated
leader sequence of AMV, and a duplicated CaMV 35S promoter. The
promoter performance decreased in the same order, if tested in transient
expression assays with the GUS reporter.
2.
For the isolation of homologous promoters, a genomic Grand Nain library was
screened with polyubiquitin and actin cDNA clones, and a vectorette
approach used to isolate the corresponding promoters with vague results only
for the actin promoter. Probably to the same end, a leaf cDNA library was
established, but not further used.
Transgenic banana lines
The improved particle bombardment procedure allowed the transformation of ECSs
from cvs. Williams, Three Hand Planty, Bluggoe, Cardaba, Monthan, and Musa
balbisiana to produce many independent transgenic lines ( as proven by transient
GUS expression).
Stable transformations were achieved with cv. Williams, Bluggoe and cv. Three
Hand Planty.
100
Evaluation
Methodology
On two occasions the laboratory of the PI at KUL was visited. Additionally, an
intensive desk study was performed on the Termination Report and 7 scientific
papers. Also, several contacts by phone and e-mail helped the understanding of
specific details.
Results
From the above study and personal experiences with the PI’s laboratory, it can be
stated that research at KUL on bananas: (1) had already proceeded for more than a
decade; (2) has accumulated a lot of expertise; (3) produced a series of pacemaking results in banana improvement; (4) was fuelled from several sources; and (5)
was accomplished by a series of excellent scientists. It should be stressed, that KUL
had only two years of BIP funding, but nevertheless made major progress.
1. Gene transfer techniques
Monocotyledons are not natural hosts for Agrobacterium tumefaciens, so that their
transformation has to be triggered (by e. g. acetosyringon or other virA effectors), or
to be optimised. This has successfully been achieved with only relatively few
monocots. Consequently, the trial to establish an effective Agrobacterium-based
transformation protocol with banana host cells originally remained inconclusive,
though the bacteria were obviously attracted by wounded banana tissues and
attached. The team has improved the technique in the post-BIP period, with
satisfactory results.
In contrast, particle bombardment of banana tissues proved to be effective, has
been optimised considerably, and been used successfully to transform a series of
Musa cultivars. Though in neither case the integration of the introduced DNA, or its
physical integrity, or its copy number has been shown, particle bombardment seems
nevertheless to be the method of choice for banana transformation.
2. Banana transformation vectors
The idea of twin selectable marker genes for consecutive transformation steps is not
new, but has first been shown to be applicable to banana cells. Since such cells are
sensitive to the corresponding antibiotics (geneticin, hygromycin), such dual
selection vectors will certainly be very useful in future banana transformation
experiments (if available to institutions not funded by BIP).
3. Heterologous and homologous promoters
Since banana promoters were not available at the start of the BIP, a logical objective
was to search for effective promoter sequences from other plants. From the outset
of a research program it is also acceptable to test only constitutive promoters (as the
ones selected here), but clearly inducible (or regulated) promoters are needed for
101
realistic banana improvement. Nevertheless, the promoters identified to work in
banana cells served and will serve to optimise constructs, cassettes and expression
conditions. Although it might have been advisable to give more attention to the
isolation of banana promoters that are, for example, induced by pathogens, this
aspect was not part of the contract with the PEA. Notwithstanding this critical
remark, there is no doubt that the promoter work at KUL was very successful.
4. Transgenic banana lines
The successful transformation of several cultivars with the optimised biolistic transfer
technique demonstrates its potential. The scientists at KUL have already
successfully transferred this technique to eight students from other laboratories, and
it is to be expected that it will be used more frequently in banana research.
The various aspects of the KUL project have been competently and successfully
accomplished, in spite of the fact that work at KUL has only been funded for two
years by BIP.
Conclusions
Project goals
If the objectives as stated in this report are taken as guidelines, then most of the
project goals were reached. However, tissue-specific gene expression was only
marginally addressed, though it is highly important, for example in resistance
breeding. Nevertheless, the achievements at KUL are impressive.
General performance
The institution of the PI has long-standing experience in banana research, and the
PI himself is one of the leading experts in this field. The scientific environment at
KUL is also stimulating, and links to industry are established (via Dr. B. Cammue).
The scientists involved in BIP-funded research, as far as is known to the reviewer,
are altogether highly motivated and active. Again, the efficiency of KUL research
(publications per unit time) is very high, and compares well with BTI and QUT.
There was also a strong training, technology transfer and co-operation aspect in the
BIP-funded research at KUL.
All these activities will certainly continue and are proof of the competence of the KUL
scientists in banana research.
Prospects
The future research at KUL will continue in the following areas:
-
Improvement of the Agrobacterium-mediated gene transfer system for
bananas;
102
-
Control of somaclonal variation in the generative phase of newly established
cell suspensions;
Development of large insert genomic libraries for the isolation of
agronomically interesting genes;.
Isolation of tissue-specific and pathogen-inducible promoters via differential
display techniques;
Characterisation of the isolated polyubiquitin and actin genes to isolate their
promoters.
103
BIP 11
Genetic Engineering of Ethylene Biosynthesis in Bananas
Description
Co-ordinates
Organisation:
Hongkong University of Science and Technology
(HKUST) Biology Department
Principal Investigator(s):
Prof. Dr. S. F. Yang
Dr. N. Li
Dr. Yi-Min Chen
Duration:
August 1, 1995 - August 31, 1997.
Objectives
1.
2.
3.
4.
5.
Isolation of full length ACC-synthase and ACC-oxidase encoding sequences
from banana genomic libraries;
Northern blot analysis of expression of both genes during fruit ripening and
after attack of leaves by Mycosphaerella (fijiensis, musicola), and roots by
Fusarium oxysporum f.sp. cubense;
Isolation and sequencing of ACC-synthase and ACC-oxidase promoters for
transformation experiments;
Engineering constructs of sense and antisense ACC-synthase and ACCoxidase genes driven by the above promoters;
Transformation of banana plants with these constructs, and molecular
(Northern analysis), biochemical (ACC-synthase/ACC-oxidase activities,
levels of ACC and/or ethylene), and agronomic characterisation of the
transformants (shelf life of fruits, post-harvest behaviour, and response to
exogenous ethylene, response to Mycosphaerella and Fusarium attack).
This project aimed at engineering bananas with reduced ethylene production and
reduced disease susceptibility, but enhanced fruit quality.
Activities
The activities of HKUST were restricted to three areas: the isolation of ACC
synthase and ACC oxidase genes from bananas (Musa acuminata cv Grand Nain);
determination of their steady-state transcript levels in pulp and peel tissues, and
construction of sense and anti-sense overexpression vectors for banana
transformation.
104
1.
2.
3.
ACC synthase/ACC oxidase genes: Genomic phage λ libraries were
established from green M. acuminata cv. Grand Nain leaves, screened with
RT-PCR generated cDNA clones of both genes from ripening fruits, positive
clones restriction-mapped and some partially sequenced.
Both ACC synthase and ACC oxidase gene expression were tested by
conventional Northern blotting analysis in pulp and peel tissues, using ACC
synthase and ACC oxidase gene probes.
Construction of an ACC synthase / ACC oxidase sense and anti-sense gene
fusion driven by a doublet 35S promoter and 3‘ flanked by a nos terminator.
The construct was sent to KUL for transformation of embryogenic plantain cell
suspensions.
Achievements
1. Isolation and sequencing of ACC synthase and ACC oxidase genes
Genomic DNA from cv. Grand Nain was partially digested with Sau 3 A, the
fragments size-selected for 12 - 23 kb, ligated into an EMBL 3/Bam HI vector and
packaged. The titers were medium, and insert sizes low. The primary libraries were
screened with RT-PCR-generated cDNA clones encoding ACC synthase and ACC
oxidase, a total of 5 positive clones detected, cloned, and restriction mapped. A total
of 3 distinct ACC synthase, and 2 distinct ACC oxidase genes were finally available,
one representative of each family sequenced, and parts of the flanking regions
determined by primer walking.
2. Expression analysis
Northern blot hybridisation was used to determine the regulation of ACS and ACO
gene expression in pulp and peel tissues at various developmental stages. Both
ACS 1 and ACO 1 genes were expressed in pulp at the preclimacteric mature green
stage, and this basic level of both transcripts was made responsible for the basal
level of ethylene production (S. F. Yang’s system 1 ethylene biosynthesis). Both
genes are ethylene-inducible, and the stimulation is drastic. MA-ACO 1 transcript
levels in peel and pulp tissues peaked prior to the climacteric ethylene production (S.
F. Yang’s system 2 ethylene production), and were linked to the rise of ethylene
synthesis during banana fruit ripening. The increase in MA-ACO 1 mRNA levels in
pulp tissue of green mature fruits preceded the increase in peel tissue upon ethylene
induction, which conflicts with published results, but is logical in a ripening process
from inside out.
3. Gene fusion
A gene fusion was constructed from MA-ACS 1 and MA-ACO 1 genes in both a
sense and an anti-sense orientation driven by the 35S promoter. These constructs
were sent to KUL as part of a direct co-operation in early 1998, but so far no
transgenic plants (here: plantain “Three Hand Planty”) have been received by
HKUST for molecular characterisation and greenhouse evaluation.
105
Evaluation
Methodology
A desk study was performed on 6 scientific papers, the Termination Report, and a
manuscript to be submitted for publication in a scientific journal. A total of 2 days
were devoted to familiarisation with the Hong Kong University of Science and
Technology Campus, the Department of Biology, and the various laboratories of Dr.
Ning Li. Dr. Li gave an exhaustive overview on his part in the BIP, whereas Dr. Wai
M. Chung (Development Manager, Technology Transfer Centre) provided
information on budgetary issues.
Results
It should be stressed that Dr. Li, a former student of Professor S. F. Yang (Taiwan)
and still linked to him, has long-standing experience in the field of fruit ripening and
ethylene. Moreover, Dr. Li had experimented with ACC synthase genes before the
BIP program. Last, but not least, he has laboratories packed with sophisticated and
new instruments at his disposal, but almost no human resources. His research in the
BIP has only been supported for one single year, which is close to nothing in view of
the objectives. As a consequence, only part of the proposed work could be
completed.
1. ACC synthase and ACC oxidase genes
Gene techniques nowadays are elaborate and relatively easy to adapt. Therefore
the straightforward establishment of an EMBL 3 genomic library from M. acuminata
cv. “Grand Nain” DNA, the screening of this bank with 32 P-labelled cDNAs from
ACC-S and ACC-O genes, the restriction mapping of the isolated clones, and their
sequence analysis did not meet any major difficulties. This task then was performed
satisfactorily, and adds three banana ACC-S and two ACC-O genes and their
sequences and internal structure to the list of corresponding genes from other
plants.
2. Expression of ACC synthase and ACC oxidase genes
Again, techniques for expression analysis are so advanced that Northern
experiments with ACC-S and ACC-O gene probes were successfully performed. The
results support the ethylene autocatalysis model (in the author’s view). At the least,
ethylene induces both ACS and ACO genes in pulp, and in peel with a delay. The
results of the isolation and characterisation of both genes and their expression
analysis were compiled in a manuscript (submitted).
3. Gene constructs
The sense and anti-sense ACS 1 and ACO 1 gene fusions, though not originally
mentioned, could not be tested in a transgenic environment, since a serious mite
problem at KUL delayed the regeneration of the transgenic plants. As a result,
nothing can be said of the fidelity of the constructs or their function(s) in vivo.
106
Conclusions
Project goals
The number of project goals was forcefully reduced by limiting the financial support
to only one single year. While this peculiar decision is surprising, it is nevertheless
good to see that HKUST at least performed one task: the isolation of two classes of
genes catalysing ethylene biosynthesis in bananas. It is the view of the reviewer that
this would not have been achieved without additional support (e.g. HKUST granted
about US$ 10,000 for a three-month fellowship) and some previous work. Therefore
the major goals of this particular project could not be reached: transgenic banana
plants with reduced ethylene production capacity and their characterisation as well
as evaluation in the field (reduced disease development, and enhanced fruit quality).
General performance
The technical environment at HKUST is excellent. All the most modern instruments
are available, and, as far as was ascertained, well maintained. There are also some
basic funds available for Dr. Ling Ni’s research. However, human resources are
limited (e.g. only one Ph. D. student is currently working in Dr. Ni’s lab), partly due to
the high salaries at HKUST. Dr. Li has proven competence in the field of gene
technology, but the time within the BIP was simply not enough to achieve more.
Also, there could have been more contact with, for example, QUT (Australia). For
example, the QUT group was able to regenerate not only cv. Bluggoe, but also
Cavendish-type bananas, and maintains experience in transformation. Also, they
would have been prepared to insert the ACS-ACO fusion into their lines. In any
case, substantially more could have been achieved, if the funds had not been limited
to one single year.
Prospects
-
-
-
The performance of the sense and anti-sense ACS and ACO fusions in target
plants and the effect(s) of their expression on fruit ripening and host
resistance to pathogens await further experimentation.
A major goal remains the transformation of several (regenerable) Cavendish
cultivars of economical importance for Southeast Asia with genes controlling
ethylene biosynthesis with the aim of increasing shelf life of fruits.
The role of pathogen and wound-induced ethylene synthesis in bananas will
be evaluated further.
The ultimate aim of all this work will be transgenic banana plants with reduced
ethylene production in fruits (and also leaves).
The over-expression of CTR (constitutive triple response) gene(s) in ripening
fruits and the inhibition of ethylene response with concomitant increase in
post-harvest fruit quality will remain a future challenge.
107
BIP 12
Use of Biotechnology to Produce Transgenic Bananas Resistant
to Banana Bunchy Top Virus Infection
Description
Co-ordinates
Organisation:
University of Hawaii, Department of Plant Pathology
Principal Investigator(s):
Dr. J. S. Hu
Dr. R. M. Manshardt
Duration: March 1, 1995 – June 30, 1998
Objectives
1.
2.
3.
Transformation of Cavendish bananas, cv. Williams with BBTV gene
constructs, using a modified Agrobacterium tumefaciens and a biolistic
transfer protocol;
Molecular characterisation and challenge of transgenic plants with BBTV in
the greenhouse;
Field trials with identified resistant transgenic banana plants.
Activities
The activities fell into two broad categories: the production of BBTV-resistant
(tolerant) transgenic banana plants, and their evaluation in the greenhouse.
1. Production of transgenic banana plants
This activity encompassed several approaches listed below, and was based on the
molecular toolbox developed by BTI and KUL:
1.
2.
3.
4.
5.
Isolation and cloning of the rep and coat protein (cp) genes of BBTV isolates
from Hawaii;
Isolation and cloning of cp, movement protein, and rep genes from Chinese
and Philippine BBTV isolates;
Construction of transformation and expression vectors for the rep and cp
genes;
Optimisation of biolistic and Agrobacterium-mediated transformation
procedures developed at Leuven (KUL) and Cornell (BTI), respectively;
Optimisation of the antibiotic selection procedure for putative transformants;
108
6.
7.
Transformation of Cavendish ‘Williams’ with an improved Agrobacteriummediated system developed at BTI;
Screening of the transgenics in the greenhouse.
2. Evaluation of transgenic bananas for tolerance towards BBTV
Achievements
Transgenic banana plants
1.
2.
3.
A series of constructs containing the BBTV rep gene, truncated versions,
antisense-rep, and a gene encoding a 19 kDa protein, as well as a coat
protein gene from banana mosaic virus (cucumber mosaic virus: CMV) have
been produced. It was not disclosed what promoter(s) was (were) used to
drive transcription of the transgenes.
Particle gun and Agrobacterium-mediated transformation protocols were
compared for their relative transformation efficiency, modified and refined.
The original biolistic technique developed at KUL, though improved and
optimised, was not used, because contamination occurred in the suspension
cultured cells, which in turn took too long to grow. The changes in the
agrotransformation technique (transformation of adventitious meristems
growing on corm tissue, and induction of multiple shoots from the above
meristems) made it suitable for Cavendish transformation.
Optimal antibiotic concentration during recovery from transformation and
subsequent regeneration was determined (10 mg/l kanamycin).
Evaluation of transgenic banana plants
A total of 300 putative transgenic plants were generated, of which 65 were screened
for BBTV tolerance in the greenhouse. Of these, 21 plants exhibited a 2 - 3 weeks
delay of symptoms, and only 3 plants showed no BBTV-specific symptoms at all.
ELISA testing for BBTV was negative on these plants.
Evaluation
Methodology
The evaluation was based exclusively on the Termination Report, direct contacts
with Dr. Hu, and cross-references in Reports of other BIP partners.
Results
The group at the University of Hawaii was an informal part of the Biotechnology
Consortium, and therefore links existed to BTI at Cornell University (transformation
technology) and KULeuven (constructs, transformation technology). This cupertino
certainly favoured progress, though the major goal of testing BBTV resistant
transgenic banana plants in the field could not be reached in the three years of the
project.
109
Transgenic banana plants:
1.
Gene technology is nowadays standard, so that the production of various
constructs for engineering BBTV resistance obviously was not difficult.
The work also included cloning cp, mp and rep genes from Chinese and Philippine
BBTV isolates, which means that one had to engineer resistance to both BBTV
groups in one step: double transformation. This was regrettably not done.
Therefore, the transgenic banana plants obtained in this project may only be of use
in the Pacific area. Another problem, the instability of monogenic traits, has not been
addressed in the relatively short time. It was not mentioned what type of promoter
was used; though rapidly inducible promoters certainly would have advantages.
2.
A dozen gene transfer techniques are now available, from which the scientists
at UH have selected particle gun and Agrobacterium-mediated transfer. Both
techniques had already been used for banana transformation at BTI and KUL,
but had to be adapted for the specific needs at UH (Grand Nain, meristems).
Therefore, much time was devoted to improving the original protocols. In the
end, a modified version of Agrotransfer was employed.
The use of cell suspension cultures from cv. Williams and cv. Grand Nain was
obviously avoided, since endophytic bacteria were present. There was, however, no
effort made to tackle this problem.
3.
Optimisation of selection procedures as part of
transformation/regeneration protocol was achieved rapidly.
the
whole
4.
The main outcome of the project are 300 potentially transgenic banana plants
(cv. Williams), of which 65 were tested under greenhouse conditions, and
found partly (32%) or fully resistant (5%) to BBTV infection (vectors: aphids).
It is to be expected that a number of the non-tested transgenics are false
positives, especially since it was shown that not all of the tested genomes
contain the BBTV gene (Southern hybridisation).
The three fully resistant plants have probably been multiplied by now, and evaluation
of the remaining putative transgenics for BBTV resistance may be complete. The
researchers regrettably do not comment on the partly resistant plants (symptom
delay by several weeks), because partial resistance may be the preferred and
durable type of virus tolerance.
As a whole, the construction of various gene fusions, their transfer by Agrobacterium
vectors into target plants, their regeneration on selective media, and testing a
fraction of the obtained transgenic plants in the greenhouse were accomplished.
Though the resistant positives are few, the general approach of engineering
resistance to ssDNA viruses such as BBTV in bananas is now promising.
Nevertheless, questions remain: Did the experiments with an anti-sense construct
fail? Is a single gene-based resistance durable enough, and if not, why was there no
effort made to combine several genes? It is also disappointing that no real field tests
were possible in the relatively short time of three years.
110
The availability of virus resistance gene constructs, conferring partial or complete
resistance to BBTV, and the whole technology of transformation and regeneration of
Cavendish “Williams” bananas has certainly opened this area to many banana
researchers world-wide.
Conclusions
Project goals
If one reduces the goals of this project to produce transgenic bananas with tolerance
to BBTV by inserting viral sense or anti-sense genes into the host’s genome, then
the project goals have been reached. Given the difficulties encountered with
establishment of cell suspension cultures, the time spent on improvement or
adaptation of already existing transformation and regeneration techniques, and the
problems with the monocot banana in particular, the results are satisfying, and
certainly will stimulate further research. On the other hand, there remain
deficiencies, especially the missing double or triple transformations with different
viral genes, the transformation with a combination of genes from the two BBTV
groups, and the lack of field-testing.
General performance
The group at University of Hawaii co-operated intensely with BTI and KUL, but
apparently by long-distance cupertino only (except for the project meetings). A
laboratory training session, e.g. in cell suspension culture at KUL, or in Agrobacterium-mediated transformation at BTI would certainly have been catalytic.
Nevertheless, the work at UH demonstrates competence in different fields ranging
from gene technology over tissue culture to transformation and regeneration
methodology.
Prospects
Basically, three spin-off activities are planned:
•
•
•
First, the remaining putative transgenic plants have to be tested for
BBTV resistance in the greenhouse, the promising candidates to be
propagated and field tests (in different environments) to be
conducted. The resistant plants (including the partially resistant ones)
have to be molecularly characterised (e. g. Southern and Western
analysis, stability of the transgenes) and the heritability of the trait to
be confirmed.
Second, resistance to other viruses (e. g. banana streak badna virus,
BSV, and cucumber mosaic virus, CMV) can and will be engineered
in several co-operative projects.
Third, continuing collaborations with the BIP partners BTI and KUL,
as well as a Philippine group and Chinese groups, will focus on
producing BBTV-resistant bananas for world-wide use (and possibly
distribution).
111
BIP 14
Elimination of Banana Streak Badnavirus (BSV) from Improved Musa
Germplasm and Related Studies on Transmission and Host Plant/Virus/Vector
Interactions
Description
Co-ordinates
Leading Institute:
IITA
Principal Investigators:
G. Dahal
J. d’A. Hughes
G. Thottappilly
J. H. Crouch
Project Virologist7
Virologist
Biotechnologist/Virologist
Biotechnologist
IITA
IITA
IITA
IITA
Collaborating
Institutions8:
Several IITA Scientists
John Innes Centre, UK; QDPI, Australia;
University of Minnesota, USA;
KULeuven, Belgium; Cocoa Research Institute, Ghana;
Museum National d’Histoire Naturelle, France.
Duration:
3 years (with 3 months no-cost extension),
starting Nov 1, 1994
Objectives
1.
2.
3.
To develop reliable therapeutic methods to eliminate BSV from Musa
germplasm;
To determine if BSV is transmitted through pollen, embryo, endosperm;
To study host plant/virus/vector relationships.
Modification:
In the course of the project, Objective 1 was adapted to the discovery of the
episomal versus genomic nature of the virus:
1.
7
8
Identify Musa plant tissues, which are free of both genomic and episomal
BSV and consequently produce ‘virus-free’ Musa germplasm.
Recruited for this project
No budget implications. Linking operations funded by other instances.
112
Activities
Initial formulation:
1. Develop virus-free materials;
2. Develop and validate indexing method;
3. Transmission studies;
4. Vector identity studies;
5. Symptom expression and systemicity studies;
6. Screen for resistance.
Definitive formulation (without budget implications):
1. Development of protocols for sensitive detection of BSV in Musa tissue
(Objective 1).
2. Development of a protocol for virus elimination through meristem dissection,
chemotherapy, thermotherapy, or a combination of all three (Objective 1).
3. Transmission of BSV through in vivo seed germination and in vitro embryo rescue
(Objective 2).
4. Laboratory and field-tests to determine the identity of BSV vector(s) (Objective 2).
5. Field and laboratory experiments to study vector transmission in relation to natural
dissemination through vegetative material (Objective 2).
6. Field and laboratory experiments to determine the effect of environmental factors
7. Identification of genotypes with tolerance or resistance to BSV,
especially temperature and host genotype on virus systemicity, persistence, and
expression of symptoms (Objective 3).
The reformulation reflects the progress made during the implementation of this
project, as well as the care taken by the PIs to optimise it.
Achievements
The PIs always took care to report extensively on each activity. This facilitated the
understanding of the achievements to a great degree.
1. BSV detection
Considerable efforts were made to perform this activity in a successful way.
Collaboration of the University of Minnesota (Prof. Ben Lockhart) played a crucial
role in this process.
The basic reason for this painstaking work is the elusive character of this
Badnavirus, which at one time can be integrated in the genomes of a Musa plant
(and be inoffensive), and become episomal (and thus harmful) at another time, on
the same plant.
The said elusive character of the virus misled the researchers for some time, in that
apparently well functioning detection techniques afterwards turned out to be
unreliable.
113
Eventually, the following (briefly sketched) battery of techniques, applied in a
cascade way for precise detection, was found to be quite efficient:
-
A first ELISA (enzyme-linked immunoabsorbent assay) using sap samples;
Negatively tested plantlets to undergo a second ELISA test (using both sap
and partially purified virus preparations);
Negatively tested plants to be tested by ISEM (immunoabsorbent electron
microscopy) (same type of material);
Negatively tested plantlets to be tested by IC-PCR (immuno-capture
polymerase chain reaction) (same type of material).
The entire procedure takes at least 6 months (visual inspections included), and has
been internationally accepted as a reliable Indexing Technique for BSV.
2. Virus elimination
The elusive nature of BSV discovered in the course of the project explains why not
one of the techniques tried out under this activity could eliminate the virus. Even the
plants generated from embryogenic cell suspensions (provided by the KULeuven
laboratory) tested positive.
3. Transmission through in vivo seed germination and in vitro embryo rescue
The studies (600 plants from 12,500 seeds in vivo; 1750 plantlets from 18,000
embryos in vitro) suggest that BSV is transmitted through both in vivo and in vitro
germination.
Genomic DNA extracts from flower stigmas with pollen from BSV symptomatic
plants also indicated (PCR amplification) the presence of BSV. The PIs also
checked what happens in the case of propagation through suckers: the majority of
suckers tested positive for BSV by ISEM within a few months.
4. and 5. Vector transmission studies
Among the different mealybug populations found active in bananas, even the two
most aggressive ones (Dysmococcus brevipes and Ferrisia virgata) could not be
proved to be vectors of BSV. In the field, there was no evidence of radial or jump
spread that is usually associated with mealybug vectored viruses. Attempts at
mechanical transmission were unsuccessful. Tissue culture material exhibited more
severe symptoms when compared with suckers (in vitro stress).
The PIs conclude:
“Circumstantial evidence suggests that there is very little, if any, vector transmission
or radial spread and that the outbreaks are often associated with plants that are, or
were, stressed (e.g. drought stress).”
114
6. Effect of environmental factors
A scoring system on a 0-3 scale for the symptom severity was devised and different
types and combinations of symptoms grouped into five major categories. The
system is likely to be adopted internationally.
By using these systems, the complex dynamics in relation to the environment were
revealed (e.g. different symptom-combinations in the rainy versus dry season).
Symptom expression varied within the same plant and even on the same leaf. This
suggests that the virus may be incompletely or partially systemic, and the different
concentrations of BSV found in different plant parts (TAS-ELISA) seem to confirm
this.
Yield loss attributable to BSV depended on time of infection and crop cycle. A
significant cultivar x environment interaction was found.
7. Resistance/tolerance to BSV
A relatively cool environment with low rainfall and potential evapotranspiration
appeared to be favourable for BSV and enabled good screening of cultivars for
resistance or tolerance. The important field collection at IITA was thus screened.
A differentiation at genomic level is tempting but not clear:
-
The observed M.acuminata group (AA and AAA ) did not show symptoms;
The ABBs and the AAB French Plantains seldom showed BSV-like
symptoms;
But the AAB False Horn Plantain showed the symptoms in most of the
environments;
All plantain hybrids showed the symptoms, although significant differences
among the hybrids were observed.
It was tentatively concluded that:
-
Transgressive segregation (->epistasis) may be the main non-additive gene
interactive system controlling the response to virus(es) in plantain hybrids;
The overall variation in a field would be determined by a combination of
additive clonal and environmental effects.
Evaluation
Methodology
A desk study was performed on the basis of the Termination Report. Visits to the
IITA laboratories involved at Ibadan and Onne, discussions with the virologists Dr.
G. Dahal and Dr. J. d’A.Hughes, who prepared the Terminal Report in place of Dr. J.
Crouch (who left the Institute in the course of the project), as well field visits (August
‘98) allowed familiarisation with details of the project.
115
Results
All of the seven activities were pertinent to the concept of the project and led to
major breakthroughs in knowledge on this virus which is presently menacing banana
cultivation in at least two continents, Africa and America.
As a consequence of the project, whereby the PIs worked in collaboration with
several specialists and laboratories over the world, it became clear that:
-
The “nuclear phase” of the virus is responsible for its elusive character;
A reliable BSV detection method was nonetheless developed;
The virus cannot be eliminated by any of the available techniques.
Vector transmission is unlikely;
Rather precise environmental factors (weather) have been found to be
triggering factors for BSV outbreaks in the field;
Conventional breeding for BSV-resistance is possible, although considerably
more knowledge on genetic aspects are required;
Genetic manipulation (in combination with breeding) may again be the more
useful method for obtaining resistance/tolerance.
Conclusions
The three objectives have been met satisfactorily. The entire initial domain for
investigation of the disease (which did not become important before the eighties)
has been covered, and the results are of basic importance for the future. The
international scientific community is now prepared for well-focussed further research.
While the project formed an integral part of IITA’s program on BSV research, it
undoubtedly played the motor-role in the progress made so far. No less than 22
papers in international journals were one of its product outputs.
One spin-off of the project is the continued close collaboration of several Institutes
and Universities (John Innes Centre, University of Minnesota, KULeuven) which
have now attracted support from donors, such as the Gatsby Foundation, in an
attempt to achieve resistance/tolerance through genetic manipulation.
116
BIP 15
Variability and Relationships within Populations of Fusarium Oxysporum F.
SP. Cubense from its Centre or Origin
Description
Co-ordinates
Leading Institute:
Plant Protection Unit, Dept. of Primary Industries,
Indooroopilly, Australia
Principal Investigator:
Ken G. Pegg
Collaborating Institutes:
Queensland Department of Primary Industries (QDPI)
with Dr. N. Y. Moore, Dr, M. K. Smith, Mr P. W. Langdon,
Mr R. D. Davis
CRC for Tropical Plant Pathology, University of
Queensland
Duration:
3 years (1 March, 1995 to 28 February, 1998)
Objectives
Original Objectives:
The project was to be implemented in very close collaboration with the University of
Florida (Dr. Randy Ploetz). Both institutes were in fact to carry out two similar
projects (BIP15 and BIP16) with the same objectives:
1. To collect isolates of Fusarium oxysporum f.sp. cubense (Foc) in the Asia/Pacific Region
to assess diversity in the centres of origin for Musa acuminata and Musa balbisiana.
2. To assess relationships within and among these populations of Foc (and populations
from elsewhere) using phenetic and genetic procedures.
3. To develop early screening methods whereby host:pathogen responses could be quickly
and reliably ascertained in a quarantine environment.
4. To study the influence of host genotype on VCG (vegetative compatibility groups)
composition in the field.
5. To evaluate diploid bananas being used in breeding programs, as well as their hybrids,
for resistance to Fusarium Wilt.
117
Final Objectives:
Because the Project Executing Agency decided to drop proposal BIP16, the
following objectives were agreed upon:
1. To collect isolates of Foc in the Asia/Pacific region in order to assess phenetic and
genetic variability in the pathogen. Isolates will be assessed using production of volatile
aldehydes, vegetative compatibility and arbitrarily primed PCR (RAPD and DAF
techniques) to generate DNA fingerprints. A PCR-based DNA detection and diagnostic
system will be developed using the SCARs (Sequence Characterised Amplified Regions)
approach.
2. To assess the reliability of using plants derived from tissue culture as opposed to
conventional planting material (bits/suckers) when evaluating banana genotypes in the
field for resistance or susceptibility to Foc.
3. To evaluate breeding diploids and hybrids, from international breeding programs, in the
field against Australian and Asian populations of Foc.
This change meant:
-
-
-
The original Objectives (3) and (4) were dropped (which caused concern for
the PI because (3) would have optimised a handy technique of general
importance);
Objectives (1) and (2) were merged, with the essential difference that the
RFLP method (in which Dr. Ploetz’s group was specialised) was no longer
included. The decision had important implications for the classification of the
isolates at the end of the project (as will be shown in “Results”). The “new”
Objective (1) is spelled out in a number of activities;
A genuinely new Objective (2) was inserted, dealing with the interesting
problem of the “non-hardy” tissue culture plants (which frequently show
susceptibility to diseases and pests for plants that become resistant in later
stages of their development).
Surprisingly, a fourth objective appears in the Completion Report only:
4. To increase the disease resistance and hardiness of plantlets derived from tissue
culture using mycorrhizae, fungal endophytes and rhizosphere bacteria.
Activities
It was apparently not found opportune by the PI to spell out the 3 objectives in a
number of relevant activities. The present reviewers admit that such would be very
difficult with objectives that are, in fact, activities themselves. The effort would lead
to the description of the very techniques, repeatedly used in close sequences within
each objective for the “waves” of collected Foc isolates and of assayed Musa
genotypes.
Activities for Objective (1) are mentioned within the paragraph on Objectives (see
above).
118
Discussion of the timetable produced by the PI is unproductive in the present
context because all of the objectives have been met.
The PI preferred to highlight in each of the interim reports the progress made for
each of the objectives, which makes reading quite captivating.
Achievements
Achievements are reviewed here by objective for the reason explained in “Activities”
above.
1. Understanding the variability in Foc within the primary centre of Musa
diversity
Considerable progress was achieved in this domain, as the interim reports and the
Completion Report clearly show. This is not surprising since the PI is an authority of
world-wide reputation in research on Foc.
The most salient achievements were:
-
-
-
-
-
-
The host Institute of the PI is now depositor of the most important Foc
collection in the world, with more than 2000 isolates from the centre of origin
as well as from various parts of the world (leaving quite some cumbersome
work to screen this huge collection for the duplicates);
77 Foc specimens were collected during an expedition to Indonesia (1996);
About 400 isolates were subject to a DNA fingerprint study of which the
results confirmed the validity of the VCG classification;
Nine clonal lineages were established on the basis of DNA fingerprinting (and
referred to since as DNA fingerprint groups (DFGs). Five of these DFGs
contained numerous closely related VCGs and genotypes, while the
remaining four DFGs (or lineages) each contained only a single VCG or
genotype;
The 9 lineages were then divided into two major groups, by means of
phenetic
analysis.
These
two
groups
seem
to
have
a
phylogenetic/geographical significance (i.e. two areas of Foc diversification
within the primary centre of Musa diversity). Tropical race 4 populations which
are Cavendish competent in tropical regions were restricted to south-east
Asia and far northern Australia;
The hypothesis of co-evolution (Foc-Musa) within the primary centre of origin
of Musa was confirmed and can be accepted as a reliable theory. The
absence of Foc in PNG (at least in the traditional context), where the
susceptible species Musa schizocarpa and M.acuminata ssp banksii are
endemic, is of great practical importance in this respect (quarantine rationale);
Strain-specific PCR primers for development of PCR-based detection
systems for Foc were identified and a DNA library (banding patterns)
established;
In the study on rDNA, the internal transcribed spacer (ITS) region and the
intergenic spacer region (IGS) of the rDNA genes were characterised and
119
-
sequenced. Genetic variation could be identified among different races and
VCGs based on the IGS haplotype;
A phylogeny was eventually constructed for the genotypes of Foc based on
DNA fingerprinting and IGS sequence information.
2. The value of tissue culture plantlets
By contrast with Objective (1), achievements for this objective within the BIP project
have not been very impressive, beyond the confirmation that such plantlets are more
susceptible to Fusarium Wilt than conventional material such as suckers and “bits”.
Attached to the Completion Report is a copy of a paper reporting a strong indication
that this greater susceptibility cannot be the consequence of any lower
photosynthetic activity nor sink-source dynamics in photosynthates. It thus becomes
clear (to the present reviewers at least) that the cause of the susceptibility should be
sought either in the structural (histological) elements of these plants (lack of silicate
compounds?) or in a lack of beneficial associations with endophytes.
But the paper is the product of work carried out with non-BIP support (Queensland
Banana Industry Protection Board; and the Horticultural Research and Development
Corporation). This support is acknowledged in the paper.
3. Screening of breeding material for resistance to Foc
A large number of edible diploids and improved diploids and triploids were
introduced mostly from CIRAD (Guadeloupe) and FHIA, and a couple of hybrids
from IITA.
The operation of testing all that material for resistance to Foc proved to be a very
time-consuming one. Strict quarantine regulations, virus indexing, propagation,
careful selection of fields infected with the critical VCGs, eventually led to the bulk of
the material being planted not before 1997.
Since reliable testing of resistance versus susceptibility to Foc cannot be achieved
within the first two years after planting (two cycles are needed), the majority of the
results will be obtained this year (1999),and thus no longer can be reported within
the BIP framework.
The operation extends to countries other than Australia and the first “waves” of the
material were already planted in Indonesia and Malaysia.
A finding of fundamental importance to breeding was the discovery in Indonesia of a
population of Musa acuminata ssp malaccensis, which segregated for resistance to
the subtropical race 4. While several acuminata subspecies are resistant, this is the
first opportunity to study the genetics of resistance in a precise genetic context and
perhaps to understand the molecular basis of it.
120
Evaluation
Methodology
A desk study was performed on the basis of the Completion Report as well as of all
the interim reports.
Results and Conclusions
Because the activities coincide with the objectives, the present review embraces
both aspects.
Objective 1 has been amply met, as the above long list of achievements
demonstrates. The project enabled the establishment of a solid basis for all further
research on the origin, the diversification and the significance of the pathogen.
While the results of the massive DNA fingerprinting work do provide a firm rationale
for further molecular research, it is unfortunate that this endeavour could not be
backed by the RFLP analysis carried out separately at Florida University (due to the
BIP16 proposal being dropped): the conjunction would by now have produced a
great theory of international authority in this domain.
One element of the objective has not yet been achieved: the detection kit for fast
resistance-to-Foc screening. But a convenient PCR primer was found and the kit is
likely to be ready in the near future.
Objective 2 was but partly met, and some reported results appear to be the product
of non-BIP support. Much work still lies ‘down the road’ and the impromptu addition
by the end of this project of a fourth objective dealing exactly with that research is
significant in the context.
Objective 3 will be met in a satisfactory way during the post-BIP period. Even the
reported first results are useful. The relatively short time-frame of 3 years may have
been insufficient to accomplish such a task, given its time-consuming nature.
With respect to Objective 1, the prospects for further progress are manifest. Many
operational links with institutes over the world were established and are likely to
remain active. The research involved now has a rather routine character (further
collection of isolates, continued DNA fingerprinting, interpretation and
completion/modification of the established classification). The BIP project attracted
ample co-financing in Australia, and part of this has a good chance of success in the
light of very promising achievements to date.
Still, international interest should be maintained because the Asian mainland
(beyond Malaysia, Thailand and Vietnam) remains a vast area of investigation for
the study of Foc.
121
BIP 17
Origin and Distribution of Fungicide-Resistant Strains of Mycosphaerella
Fijiensis in Banana Plantations in Costa Rica
Description
Co-ordinates
Organisation:
Corporación Bananera Nacional de Costa Rica
(CORBANA)
Principal Investigator:
Ronald Romero, Project Leader
Mauricio Guzmán-Quesada
Alexander Jiménez-Loria
Randall Vargas
Note:
Eng. M. Guzmán-Quesada became PI after the departure
of Dr. R. Romero and managed the project in a very
satisfactory way.
Duration:
Three years, starting March 1, 1995
Objectives
1.
To study changes in the dynamics of isolates of Mycosphaerella fijiensis
resistant to benomyl and propiconazole in commercial banana fields
subjected to different fungicide regimes;
To determine the production of conidia of benomyl and propiconazoleresistant or sensitive isolates in culture. Similarly, to study conidia production
of isolates that are resistant to both benomyl and propiconazole;
To study the parasitic fitness (aggressiveness) of benomyl and propiconazoleresistant isolates of Mycosphaerella fijiensis on young banana plants in the
greenhouse. In the same way, study the aggressiveness of isolates that are
both benomyl and propiconazole-resistant;
To study the ability of fungicide-resistant isolates of Mycosphaerella fijiensis
to produce conidia and ascospores, and to study the inheritance of the
character.
2.
3.
4.
Note:
-
Objectives 2 and 3 are hereafter combined due to their similarity and
convenience in reporting. They are reviewed under Activity/Achievement (2),
while Objective (4) is dealt with under Activity/Achievement (3).
122
Activities
1.1
Testing the effect of a propiconazole-free period on the changes in
sensitivity of Mycosphaerella fijiensis to this fungicide
The original plan which contemplated the use of four commercial farms was
accomplished. Three treatments were considered: DMI-free period of six
months, DMI-free period of three months, and no DMI-free period.
The sensitivity to propiconazole was determined by calculating the effective
dose that caused 50% inhibition of colony growth (EC50), by regressing
growth against the logarithm of the fungicide concentration as well as by using
the germ tube inhibition method.
1.2
Determining the effect of a mixture of propiconazole with mancozeb on
the control of the disease, and on the changes of Mycosphaerella
fijiensis sensitivity to propiconazole.
This experiment was conducted on commercial farms as well.
2.
Evaluation of isolates of Mycosphaerella fijiensis that are resistant to
both benomyl and propiconazole for their ability to produce conidia in
culture
Three runs of this experiment were conducted, each using a different set of
isolates of M. fijiensis, with a range of sensitivities to the fungicide. The
number of conidia produced for each isolate was determined by transferring
the individual colonies to a test tube and counting the number of conidia in a
hemocytometer.
A related activity was to determine whether the acquisition of resistance to
fungicides affects the ability of these strains to sporulate, which is an
important component of fitness.
3.
Inter-crossing isolates with different degrees of sensitivity and testing
their progenies (monoascosporic isolates) for their sensitivity to
propiconazole.
Achievements
Effect of fungicide regimes
1.
No significant differences in the EC50 of populations (due to a DMI-free
period) were observed by either method of sensitivity test used. The
population of less sensitive isolates was not affected by any duration of a
triazole-free period carried out for two consecutive years. The treatments did
not affect the level of practical control of the disease, since the Infection Index
and all other variables related to disease did not show any differences among
treatments.
123
Consequently, according to the results of this activity, the common practice of using
a triazole-free period as a way of controlling black Sigatoka seems to have no
important implications on the dynamics of the fungicide-resistant populations, since
a DMI or a triazole-free period of six months did not show any effect.
However, this practice still has importance in regions where M. fijiensis has
appeared just recently or in regions with no shift or where only a small shift has been
observed. In fact, CORBANA has been promoting it as an important factor to
consider in any black Sigatoka control strategy.
2.
The most interesting result is that with doses of fungicide concentration of
0.05 and 0.1 µgml of each fungicide tested, the mixtures of propiconazole and
tridemorph (each of them with the same concentration as the fungicide alone)
consistently gave better control than each of its components separately. This
identified synergism would allow safe use of important fungicides, which
should be of great value for any integrated pest management program, as
well as cost-effective. The use of fungicide mixtures at low concentrations
should be studied further to determine the potential in any integrated pest
management program.
Parasitic fitness and ability to produce conidia
The results indicate that there is no loss of parasitic fitness associated with the
acquisition of resistance genes, since no relationship was found between the level of
resistance to propiconazole and the ability of the isolate to produce conidia in
culture.
Significant variation in conidia production among isolates was observed, and is
possibly due to the existing natural genetic variation in the population. In general,
isolates that produced the highest number of conidia were from the fungicide treated
areas, and they possessed intermediate levels of resistance to propiconazole, but
isolates from the wild type population did not have any isolate producing high
numbers of conidia.
These preliminary results may help explain the high frequency of individuals with
resistance to propiconazole found in the fungus population in several commercial
banana plantations.
Although the DMI-fungicides are facing a widespread resistance in M. fijiensis, they
are still used because their performance has not completely failed. Possibilities to
continue using DMI-fungicides against black Sigatoka in bananas would strongly
depend on the effectiveness of strategies aimed at reducing the frequency of the
resistant strains.
Inter-crossing ability of M. fijiensis isolates
It was proven that crosses between different M. fijiensis isolates are possible.
Crosses of triazole-sensitive versus resistant isolates generated progenies with low
and intermediate levels of sensitivity, and only a few isolates that appeared to
accumulate higher levels of resistance than the original parents.
124
Crosses of resistant x resistant, sensitive x sensitive, and intermediate x
intermediate isolates gave progenies with resistant, sensitive, and intermediate
levels of sensitivity, respectively. Conidia were obtained from any of the crosses
involving different isolates of M. fijiensis. Monoascosporic isolates were also
obtained from all possible cross combinations. In general, most crosses in which a
resistant isolate was used against an intermediate or a sensitive one, the progeny
rendered intermediate levels of resistance, some with high resistance. Similar results
were observed when isolates with intermediate levels of sensitivity were crossed
between them and with sensitive isolates.
Evaluation
Methodology
Desk Study:
A desk study was performed based on the Project´s Original Contract, the Annual
Reports for March 1995 to March 1996, March 1996 to December 1996, January
1997 to December 1997, a Six-Month Progress Report of January 1998 to June
1998, and on the Final Report.
Field Visit:
The project was visited on February 12, 1999 and included:
-
Official visit to Corbana´s Research Director at Guapiles, Costa Rica;
Formal presentation of results by the project´s leader;
Discussion with personnel in charge of project;
Visit to Pest Control Laboratory facilities.
Results
All the project objectives were met and their activities to achieve them were
conducted effectively.
Crosses of isolates with different degrees of sensitivity to propiconazole were
performed on banana leaves. Isolates were obtained from a work collection existing
at CORBANA, with known EC50 to this fungicide. Isolates with high, intermediate
and low EC50 values were crossed in all possible combinations, except selfing.
Then, monoscosporic isolates were obtained from these crosses (progenies) and
their sensitivity to propiconazole tested.
Regarding fitness, a great deal of information has resulted from this project and its
use should be promoted to design environmentally sound strategies to control black
Sigatoka. In consequence, this will result in the reduction and safer use of fungicides
as well as less costly applications. It should be taken into consideration that this can
only be possible if less sensitive propiconazole populations can be obtained. It is
worth mentioning that fungicide-free periods are no longer the only methods to
reduce resistance.
125
Conclusions
CORBANA’s principal investigators merit special recognition for the excellent work
achieved in studying M. fijiensis under strictly controlled growth conditions and for
having been able to cross different strains of the fungus and obtain monioscosporic
isolates from the various combinations (RxR; SxS; IxI and SxR).
The technical and scientific environment at CORBANA as well as the expertise of
the principal investigators was adequate to conduct the project.
The co-operation provided by the multinational companies Dole, Del Monte and
Geest Caribbean (now called CARIBANA) for the conduct of the experiment is to be
commended.
During the conduct of this project, inter-institutional co-operation was present. For
example, CORBANA provided isolates of M. fijiensis to CIRAD to study genetic
variability and to KUL for testing transgenic plants.
Prospects
According to the desk study and the field visit, the following studies should be
considered to broaden the results of this project:
1.
2.
3.
4.
5.
Research on the resistance of M. fijiensis to triazoles needs to be undertaken
further. From our point of view, we believe that the problem of increased
triazole resistance still exists and studies to decipher its origin should be
strengthened and funded.
Synergism between tridemorph and propiconazole, which may be important to
improve the control of the disease, should be studied further. This BIP project
did not have the necessary time to devote to this aspect of the research,
however, it represents an avenue for further research to improve the control
of the pathogen in populations with a shift in sensitivity to triazoles.
Evaluate all possible alternatives for black Sigatoka control within an
Integrated Pest Management strategy, including: mixtures of fungicides;
different levels of fungicides; interactions of fungicide by environment; DMIfree periods, fungicide rotations.
Related to number 3, it will be important to study the economic implications of
the use of a more environmentally-based control strategy in contrast to the
chemically-oriented one. Parameters studied could be the possible
economical losses at the beginning of the strategy implementation, the time
needed to recover control of the disease once the strategy has been in place,
and the actual cost of implementing such a strategy.
“Sensibility Gene Pools”—this refers to the relative value of having pools of
sensitive genes to fungicides within commercial plantations to allow sexual
recombination with resistant strains in order to bring down the sensibility value
of the population to fungicides. The effective percentage of “non-treated
areas” nearby or within commercial plantations should be determined in order
to allow an efficient recombination of susceptible and resistant isolates of M.
fijiensis.
126
6.
Studies to measure the movement/migration of isolates and their
recombination percentage with neighbouring populations. Genetic and
inheritance studies of M. fijiensis, such as those already initiated by
CORBANA, should be encouraged in order to shed more light on the wellknown complicated case of black Sigatoka.
127
BIP 18
Study of Resistance of Bananas and Plantains to Nematodes
Description
Co-ordinates
Organisation:
Centre Régional de Recherches sur Bananiers et
Plantains (CRBP) NYOMBE,CAMEROON
Principal Investigator:
Dr. Roger Fogain
Duration:
March 1995 - December 1998
Objectives
1. To identify sources of resistance to R. similis and P. goodeyi in the CRBP Musa
germplasm in Nyombe (Cameroon).
2. To identify cultivars with lower levels of susceptibility to these nematodes.
3. To evaluate improved materials from breeding programs for resistance to
nematodes.
Activities
1. Collection of roots, isolation of nematodes for axenic culture;
2. Pot screening for resistance to Radopholus similis, using diploid Musa, for rapid
selection of interesting materials for field studies;
3. Field screening of diploids for resistance to R. similis and P. goodeyi;
4. Pot screening of triploids for tolerance to R. similis;
5. Pot evaluation of hybrids from breeding programs (IITA, FHIA, CRBP, CIRADFHLOR).
Achievements
The three objectives implied the same methods/techniques which were not subject
to research per se beyond the check of ‘pot versus field’ results for consistency.
Screening for ‘tolerance’, i.e. non-resistant genotypes allowing for nematode
colonisation without much suffering, was left out early in the course of the project
when it appeared that root damage, using the root lesion index (RLI), was not a
good indicator (see BIP 20 for the same difficulty ). The pot technique is rather
labour and space-consuming, since each pot contains one sucker and allows the
growth of normal roots for more than two months. but this cannot be considered as a
handicap in tropical countries. the technique is not time-consuming, the nematode
development being already measured two months after inoculation.
128
Because the mass of the inoculum (1000 individuals) is the same as in the BIP
projects 19 and 20, results can be compared for the same Musa genotypes tested.
Consequently, the Achievements are reviewed ‘across-objectives’ and they only deal
with resistance/susceptibility.
1. Pot Experiments
Radopholus similis (dominant in the lowlands, i.e. altitude less than 900m in
Cameroon)
Few of the 61 tested diploids (AA, AB, BB) showed good resistance, comparable to
that of the control ‘Yangambi Km5’. The resistance of ‘Pisang Jari Buaya’(PJB) and
the lower susceptibility of the tested BBs were confirmed. Of the diploids used as
male parents in current breeding programmes, only the wild AA ‘Calcutta 4’ had
lower susceptibility. The 5 accessions from Papua New Guinea and the wild
M.acuminata ssp banksii (resistant to BS) were susceptible as well.
Of a total of 62 triploids (inclusive of the 34 plantain cultivars previously tested and
several East-African AAAs), only the ‘Ibota’-group (presumably synonyms of, or at
least akin to, ‘Yangambi Km5’) proved to be resistant. No significant variation could
be detected within the plantains or the East-African AAAs which are all susceptible.
Equally susceptible were the 13 plantain hybrids tested (from CRBP) and the 4
CIRAD hybrids IRFA 909, 910, 911, 912.
Pratylenchus goodeyi (dominant on highlands, i.e. altitude more than 900m in
Cameroon)
The unusually low reproduction level of nematodes (even on clones known for their
susceptibility such as the plantains), did not allow for differentiation in
resistance/susceptibility of the 10 accessions tested. The PI offers as explanations:
(a) that the inoculum (1000 nematodes/plant) is too low in the case of P. goodeyi
or/and (b) that the counting should be effected at a later time (which may be a
consequence of (a).
2. Field Experiments and Pot/Field Consistency
Radopholus similis
The pot-evaluation indicated that 23 of about 142 screened accessions could be
moderately susceptible or resistant. These were planted, together with three FHIA
hybrids at two sites (Njombè and Mbalmayo; randomised blocks, five replications),
together with the susceptible standards ‘Grande Naine’ (Cavendish) and ‘French
Sombre’ (Plantain) and the resistant one ‘Yangambi Km5’. Nematodes were counted
at 3, 6, 9 and 12 months. The 6-month data proved to be reliable at both sites as
they were confirmed by the later observations (classical statistics with LSD and
Confidence Interval 95%).
A positive correlation (R2= 0.650) was found between the data from the pot trials and
those from the field experiments. The moderately susceptible/resistant status of
129
these accessions was thus confirmed in general. The PI also concludes that “the
results of pot studies are valid and consequently the early screening method used in
this study can be recommended”, but see subsection on ‘Evaluation’.
Pratylenchus goodeyi
The same accessions were planted on an infested field at Bova (highland). The
nematode counting at 3 months appeared to have been destructive as most of the
plants died because of the excavation of more than 50% of their total root mass, and
the experiment was stopped. First indications pointed to the BB and ‘Calcutta 4’
having the same moderate susceptibility/resistance as to R. similis, but PJB being
susceptible.
Evaluation
Method
A desk study was based mainly on the very comprehensive Completion Report (with
17 tables). During a visit to the CRBP (August ’98) interim results were briefly
discussed with the Principal Investigator.
Results
This project was focussed on screening of Musa material for resistance to the
nematodes R. similis and P. goodeyi, and not on screening methodology/techniques
(as with Projects BIP19 and 20).
The experiments were carefully carried out and the data duly analysed, so that their
results are of great importance.
Remarkable
progress
was
thus
achieved
in
the
knowledge
on
resistance/susceptibility to P. similis. It becomes clear that convenient sources for
resistance breeding are scarce. The resistant triploid Yangambi Km5 can hardly be
considered as a convenient source (female sterile and erratic pollen production) and
the wild ‘Calcutta 4’ turns out (again, see BS work) to be the most practical source,
despite its poor bunch characteristics (the other resistant wild AA ‘truncata’ and
‘malaccensis’ bear even less attractive bunches).
The PI’s conclusion that pot-evaluation is a reliable fast technique for screening
cannot be shared. Indeed, the positive correlation found between pot and field data
does not coincide with a strict regression. The tables show for example that the
hybrid ‘IRFA 903’, found to be resistant in the pot-trial (log-value of nematode
number per 100g roots = 2.680 < 3 ) was quite susceptible in the field (log-value =
4.5 > 4). Less pronounced, but still significant discrepancies can be noticed for
‘Pisang Tongat’, ‘P.trimulin’, ‘Palimberry’ and even for Musa balbisiana.
Discussions at early stages of the project and within the ‘Nematode Consortium’, on
methodology and techniques could have been helpful, but there are no indications
that these took place.
130
It is unfortunate that almost no progress could be made in the investigation on
resistance/susceptibility to P. goodeyi. This lesion nematode dominates on the
highlands and is a particular menace for the bananas in East-Africa. Breeding of the
East-African AAA for nematode-resistance remains handicapped since no certain
sources are available beyond the ‘Calcutta 4’.
Conclusions
Although the objectives have only partly been met (i.e. the gap in knowledge about
resistance to P. goodeyi), the considerable work carried out at CRBP within the BIP
project has produced results of international importance.
Research on techniques for reliable fast screening (of resistant/susceptible Musa
genotypes) should be pursued and preferably be conceived in collaboration with
other nematode specialists within the now active Nematode Consortium. Much
research on the Musa-P. goodeyi interaction remains to be achieved to the benefit of
banana/plantain growers in Africa.
131
BIP 19
Identification of Durable Nematode Resistance Sources in Banana and
Plantain
Description
Co-ordinates
Organisation :
Laboratory of Tropical Crop Improvement,
Catholic University of Leuven (KULeuven), Belgium
Principal Investigator :
Dr. D. De Waele
Collaborating :
Associated :
Dr. R. Swennen
Ir. R. Stoffelen
J. Reynders
Duration : 1 March, 1995 - 31 December, 1998
Objectives
1. To provide the existing classical breeding programs for Musa improvement of
FHIA in Honduras and of IITA in Africa (Nigeria and Uganda) with a
nematological component.
2. To adapt or develop early, rapid and reliable methods for nematode resistance
screening.
3. To screen banana and plantain genotypes for nematode resistance.
Activities
1.
2.
3.
4.
5.
Establishment and maintenance of stock cultures of Musa nematodes;
Characterisation of nematode populations by reproductive fitness on carrot discs;
In vitro screening experiments for nematode resistance;
In vitro culture of Radopholus similis on carrot callus (inserted afterwards);
Greenhouse screening experiments for nematode resistance.
Achievements
Stock culture
Populations from various parts of the world were established on carrot discs for
Radopholus similis (6), Pratylenchus coffeae (3) and Pratylenchus goodeyi (3).
Populations of the root-knot Meloidogyne spp. (6) were established on transformed
tomato roots. The P. goodeyi populations were not used in the project.
132
Characterisation
The reproductive fitness of R. similis populations was higher than that of the P.
coffeae populations, with the sample from Cuba showing the fastest multiplication.
In vitro screening
The method has a great potential: it is very precise, has to be carried out in fully
controlled conditions, and requires a minimum of space within a single room. The
techniques involved have been established within this project (probably for the first
time in this research area). Many components of the screening had to be adapted.
An example is the inoculum mass where good performance was found with 25
mature female nematodes (per tested plantlet), individually collected with a
micropipette and placed in a drop of sterile water on the surface of the growth
medium.
The results are promising but the method still needs further investigation. A serious
problem was the microbial contamination of the nematodes collected from the carrot
discs and ‘boosted’ by the rich in vitro growth media. It prompted the PI to search for
an alternative (next activity).
Musa genotypes of known resistance/susceptibility against R. similis were tried out :
‘Pisang Jari Buaya’ and Yangambi Km5 (resistant); ‘Gros Michel’ and ‘Grande Naine’
(respectively moderately and highly susceptible). Tentative results are in agreement,
but the resistance of Yangambi Km5 was only partly expressed, and since that
resistance is clear in the classical testing conditions (pots, fields), the in vitro
technique needs to be adjusted.
In vitro culture of R. similis on carrot callus
Callus tissue developed from carrot discs enabled nematode proliferation (200-fold
increase in 10 weeks which is, however, 10 times lower than with carrot discs) and
no further microbial contamination was reported. But the nematodes did not
proliferate further on banana plants in the greenhouse pots, while those from carrot
discs multiplied 7 times. An alternative culturing method for rearing sterile
nematodes is thus still needed.
Screening for resistance in greenhouse conditions
In vitro plantlets (roots developed) were placed in plastic pots (diameter 12cm) filled
with autoclaved soil, and progressively exposed (4 weeks, flexible plastic cover) to
the greenhouse atmosphere, whereupon inoculation took place and observation was
effected 56 days later. Data were expressed in number of nematodes per gram of
fresh roots. The method proved to be efficient (see also BIP 20).
About 80 Musa genotypes were screened, including more than 30 edible AA from
Papua New Guinea, plantains, one Highland (East-African AAA), accessions from
Vietnam and Malaysia, 14 cultivars considered to belong to the ‘Pisang Jari Buaya’
(PJB)-subgroup, various AAB and ABB, a couple of new hybrids and even ‘Fe’I’
(Australimusa) clones.
133
The “PJB-subgroup” is probably an artefact since some cultivars were quite
susceptible.
The standard PJB as well as the ‘Yangambi Km5’, both resistant to R. similis, were
susceptible to P. coffeae and Meloidogyne spp. Results of resistance screening
against one nematode cannot be extrapolated. New sources of resistance to R.
similis were found in the ‘Fe-I’ group.
Evaluation
Method
A desk study was performed on the basis of the very comprehensive reports. The
project was visited regularly and the PI consulted for technical details.
Results
The way the objectives have been met needs some explanation in order to evaluate
the achievements.
Objective (2) is the key one. Two early-screening methods were established of which
one, in vitro screening, is still under investigation and has consequently not been
applicable as yet. The other method, small-pot screening in greenhouse, proved to
be efficient and was applied for Objective (3) as well as Objective (1) of which the
FHIA component was in fact a major element of Project BIP 20, and of which the
IITA component is not part of any BIP project.
Consequently, the achievements within this project are concerned with Objectives
(2) and (3) only.
Objective (2) was only partly met but the first results are still promising: the small-pot
method ‘works’ and the in vitro method has a good chance to become applicable in
the near future. Finding an alternative nematode rearing method (to the carrot disc
method) remains a feasible goal.
Objective (3) has been met with satisfaction for the R. similis case. The results also
made clear that the Musa germplasm has to be screened for probably each of the
nematode species concerned, a most important factor in the future organisation of
the screenings of hybrids.
However, and beyond the stated objectives, the BIP 19 project played a motor role
in the establishment and the running of a ‘Musa Nematologists Consortium’. The
partners (FHIA, IITA, KULeuven) are presently forging operational links with
specialists in South America, Caribbean, Asia and Australia, and are thus preparing
a precious instrument for reliable nematological evaluation under different agroecological conditions of cultivars and new hybrids.
134
Conclusions
Due to Project BIP 19, a mechanism with specialised laboratories and proper
methods and techniques for nematode resistance screening of Musa genotypes has
successfully been initiated.
It can be expected that the mechanism will be developed further and become
productive in the coming years, mainly under the impulse of the newly created ‘Musa
Nematologists Consortium’.
135
BIP 20
Identification of Durable Nematode Resistance Sources in Banana and
Plantain
Description
Co-ordinates
Organisation:
Fundación Hondureña de Investigación Agrícola (FHIA)
Principal Investigators:
Dr. Phillip Rowe
Dr. Nicole Viaene
Dr. Mauricio Rivera
Duration:
Three years, starting March 1, 1995
Objectives
1. To provide the existing conventional breeding program for Musa improvement of
FHIA with a nematological component.
2. To adapt or develop early, rapid and reliable methods for nematode resistance
and/or tolerance screening in banana and plantain.
3. To screen banana and plantain genotypes for nematode resistance and
tolerance.
Activities
1. Development and maintenance of nematode cultures in the laboratory to provide
a continuous supply of fresh inocula used to infest soil of pots and bags in the
shadehouse experiments.
2. Shadehouse testing of 35 genotypes of interest, following INIBAP´s guidelines for
recording data, to evaluate resistance and tolerance to R. similis and P. coffeae
in comparison to several reference genotypes of known reaction to the nematode
R. similis.
3. Field testing of fifteen genotypes for resistance and tolerance to nematodes was
conducted in a field located at Guaruma, La Lima, Cortés, well known for the
predominant occurrence of P. coffeae, although Meloidogyne sp., R. similis and
H. multicinctus are also found occurring in very low numbers. The field was
planted on August 1996 with corms of banana cv. Grand Nain as a blank crop to
increase the natural populations of P. coffeae in the field.
4. A second field trial was planted on October 1997 in lot C3N, Finca La Paz, a
commercial banana plantation at Isletas, Colón, for testing the resistance and
tolerance to nematodes of 20 genotypes (including the 15 genotypes in the test
mentioned in item 3).
136
Achievements
Nematode Cultures:
The technique used for in vitro development of the nematode populations proved to
be satisfactory in the case of R. similis. P. coffeae showed to be less amenable for
in vitro multiplication under local conditions. Culturing of Meloidogyne sp. on
bananas (FHIA-03 and Williams) gave excellent results: high quantities of eggs were
obtained and maintenance of the plants was very easy. Rearing Meloidogyne on
eggplants also gave good results. Establishment of a culture of Meloidogyne sp. in
Petri dishes on excised tomato roots did not succeed due to contamination with
bacteria and fungi. No tests were conducted with M. incognita as a result of not
being able to develop in vitro cultures and besides this nematode is of no economic
significance in the region.
Shadehouse Tests
Significant differences in resistance to R. similis and P. coffeae were found among
genotypes. Materials which consistently showed resistance to either one, or both of
the nematodes were identified, some of them promising for current of future use in
FHIA’s breeding program.
Complete resistance to R. similis was shown by the reference genotype Pisang Jari
Buaya (source FHIA), the natural diploids Pisang Tunjuk II-28, Mundan AVP-36, and
the improved parental diploids SH-2095 and SH-3142. The reference genotype
Yangambi Km5 (source FHIA), together with the improved parental diploids, SH3723, SH-3362 and SH-3624, and the wild diploids Pisang Jari Buaya (source ITC),
Calcutta 4, Pisang Batuau II-95, Biu Batu II-92, Siang Hil II-82 and Tangung II-77 all
showed to be highly resistant to R. similis. Moderately resistant to R. similis were
SH-3648, reference genotype Yangambi Km5 (source ITC), FHIA-01, FHIA-18, SH3386, SH-3437, FHIA-23 and Pelipita. As for P. coffeae, the lack of reference
genotypes of known reaction to this nematode forced the investigators to consider
the resistance reaction grading with some reserve. Nevertheless, a consistent,
continuous variation in reaction was evident in the genotypes. Complete resistance
to P. coffeae was found only in Calcutta 4. The genotypes SH-3437, SH-3217,
Yangambi Km5 (both sources), Lowgate, Biu Batu II-92, Siang Hil II-82, and
Tangung II-77 were highly resistant to P. coffeae. Moderate resistance to P. coffeae
was found in Highgate, SH-3624, FHIA-17, Prata Enana, Mundan AVP-36 and
Pisang Batuau II-195. Distinct high to complete resistance to both R. similis and P.
coffeae in the same genotypes did occur in the natural diploids Calcutta 4,
Yangambi Km5 (FHIA source), Biu Batu II-92, Siang Hil II-82 and Tangung II-77.
The natural diploids Mundan AVP-36 and Pisang Batuau II-195, the cv. Prata
Enana, and also the improved diploids SH-3624 and SH-3437 appeared to carry
resistance to both nematodes, although in variable levels.
Field Tests
In the field, in Guarumas specifically, collection of samples three months after
planting, at shooting and at harvest was completed during the first cycle. During the
second cycle, sampling at shooting and harvest covered only 35% and 3%,
137
respectively, due to the destruction of the trial by floods brought about by Hurricane
Mitch. The first sampling date was the only one performed at Finca La Paz, due also
to the destruction of the trial by the floods. Sampling at first shooting and first
harvest was only 74% and 30%, respectively.
At Guarumas 1, P. coffeae was the predominant banana nematode present. At all
three sampling dates at this site, there were differences in nematode populations
and damage between main lots (much lower in nematicide-treated than in nonnematicide treated lots) and among genotypes within main lots. Like in the
shadehouse tests, lack of reference genotypes of known reaction to P. coffeae
forced us to consider the resistance reaction grading with some reserve. Based on
the results of all three sampling dates, regardless of applying or not applying
nematicide, Yangambi Km5 and Highgate appeared as completely resistant and
highly resistant, respectively; FHIA-01, FHIA-02, FHIA-03, FHIA-18 and SH-3624 fell
in a dubious category that included what would be considered moderately resistant
and moderately susceptible. FHIA-20, FHIA-21, Grand Nain, SH-3723, SH-3697,
SH-3142 and SH-3362 were moderate to highly susceptible. These data are still
preliminary considering that at least two cropping seasons in the field are required to
obtain conclusive data. Nevertheless, the data clearly showed the tendency of most
of the materials to have smaller nematode populations and lower damage ratings, in
close agreement in most cases with the rankings or the resistance grading resulting
from the shadehouse tests.
At La Paz, it was confirmed that R. similis was the predominant nematode species,
although Helicotylenchus sp. and Meloidogyne sp also occurred in very low
numbers. At this site the results were not as clear as at Guarumas 1, most likely as a
result of the natural variation of nematode populations in the soil as well as the
problem of frequent failure of plant germination at the initial planting date and in
replants done later. As a result, no major attempt was made to try to interpret the
data. In spite of their short duration, the results of the field trial at Guarumas 1
provided strong evidence that there is a close correlation and agreement between
field results and results obtained from shadehouse tests. Although more information
is necessary in order to fully document this correlation, these results partially
validate the results obtained in the shadehouse for most genotypes. In fact,
shadehouse tests should provide a better estimate of the reaction of the genotypes
since their environment is free of the problems of aggregation of nematode
populations that naturally occur in cultivated soils and which undoubtedly provoke
unexpected results in materials of known reaction, as occurred in some cases with
the field trials.
Evaluation
Methodology
A desk study was performed based on two interim reports for activities in 1996, a
1997 annual report, and an interim report for the first semester of 1998, as well as
the final report.
138
The project was visited on February 16, 1999 and included:
-
Review of the project;
Visit to the shadehouse where testing was conducted;
Visit to the Guaruma field where testing was conducted (heavily affected by
Hurricane Mitch).
Results
1. Nematode Cultures:
In vitro development of nematode cultures of R. similis was accomplished readily. To
the contrary, in vitro nematode cultures of P. coffeae were not easily accomplished.
Having enough quantities of these cultures is very important for the pathologist and
breeder testing for resistance to these nematodes. Therefore, it will be important to
continue to develop the best protocol to develop cultures of P. coffeae more
efficiently.
2. Shadehouse and Field Genotype Testing for Resistance to Nematodes:
The genotypes tested showed significant differences for resistance to R. similis and
P. coffeae. Similar rankings for genotype resistance were observed for the tests in
the shadehouse and in the field. This is a rather important finding for the plant
breeder who is searching for genotype resistance in segregating populations.
Regarding the planting material used, essentially the same degree of resistance to
nematodes occurred whether the genotypes were tested as tissue culture (TC) or as
corm (CO) plants.
Besides obtaining information on resistance of the Musa genotypes, the results of
the screening tests have also provided insight into the influence of experimental
conditions (such as initial nematode infestation level, type and age of planting
material, influence of environment) and on nematode reproduction and nematode
damage caused to the plants. This information will be useful in future interpretation
of results and in improving screening methods.
Because field testing was partly affected by the flooding caused by Hurricane Mitch,
it is advisable to perform more field tests in order to corroborate the high correlation
observed between the resistance in the field and in the shadehouse.
Conclusions
By implementing this project, FHIA has now developed the expertise and resource
capability to perform on a routine basis all the laboratory, shadehouse and field
activities required for determination of Musa resistance to Radopholus similis and
Pratylenchus coffeae. The techniques and methods used in determination of the
reaction of the genotypes to both nematodes proved to be quick and also reliable
since repetitive results were obtained. In addition, improved and natural genotypes
were identified which consistently showed resistance to either nematode. This
139
knowledge will enable the breeder at FHIA to design the crossing schemes in a way
that gives maximum chance to the resulting progenies to express resistance to both
nematodes.
Therefore, all the three project objectives were met.
This is all the more remarkable since the field tests were hampered for some time
because of the flooding caused by Hurricane Mitch. The field data that it was
possible to obtain were judiciously analysed and results could thus be included in
the final report, with the necessary care in interpretation.
The expertise and technical preparation of the scientists involved was very
adequate.
Prospects
The following studies will be undertaken or considered in the near future to
strengthen the results of this project:
1. Determination of the possible differences in pathogenicity that may occur
between inocula of P. coffeae obtained from continued subculturing in laboratory
and inocula obtained from roots of infected banana plants.
2. Determination of the relationship between nematode populations, magnitude of
damage on corm and roots and their effect on total plant fresh weight as a
means of measuring tolerance to nematodes in potted or bagged plants in
shadehouse conditions.
3. Determination at the end of the exposure time of the relationship that might exist
between nematode populations in the roots, nematode populations in the soil,
and the magnitude of damage expressed in roots and corms. This research is
aimed at trying to explain the occurrence of high nematode damage in some
genotypes and yet contrastingly very low nematode populations in the roots.
Error could be occurring in the quantification of the true nematode population as
a result of migration taking place to the surrounding soil once they have
exhausted their source of food.
4. Research is planned in order to define a set of reference genotypes with known
reaction to P. coffeae.
5. Formal determination of the response of Musa genotypes to P. coffeae when
using TC and CO plants.
6. Comparison of the reaction expressed by the reference genotypes of the FHIA
and ITC sources.
7. Retesting (for conclusiveness) those genotypes in which the available evidence
of their reaction to the nematodes was based on one, or at the most two, tests.
8. Provided financial resources are available, it has been considered to establish a
field study with select nematode-resistant genotypes to determine the durability
of their resistance to those nematodes after continued exposure for several
production cycles.
140
BIP 21
Identification of Durable Nematode Resistance Sources in Banana
and Plantain
Description
Co-ordinates
Organisation:
Instituto de Recerca I Tecnologia Agroalimentares (IRTA)
Collaborating Institute:
Insituto Canario de Investigaciones Agrarias (ICIA)
Principal Investigator:
Dr. Jorge Pinochet
Collaborating:
Dr. Montserrat Jaumot (IRTA) and
Mariá de Carmen Jaizme (ICIA)
(abroad) Dr. D. De Waele (KULeuven),
Dr. P. Rowe (FHIA), Dr. P. Speyer (IITA)
Duration:
1 March 1995 - August 1996
Objectives
1.
2.
3.
To provide the existing classical breeding programs for Musa improvement of
FHIA in Honduras and of IITA in Africa (Nigeria and Uganda) with a
nematological component.
To adapt or develop early, rapid and reliable methods for nematode
resistance screening.
To screen banana and plantain genotypes for nematode resistance.
Note:
Although the three objectives are stated in the Completion Report, they are in fact
only reflecting what was originally proposed by the PI, and conform to the intention
to work in close collaboration with the ‘Nematological Consortium’ for three years
(see same objectives for BIP 19). The project did not, however, receive the required
funds for the total operation and had to be terminated after 1.5 years. Only the
activities related to Objectives (2) and (3) could be undertaken (in part).
The practical purpose of the project was to screen relevant Musa genotypes at the
ICIA (Canary Islands), and this could eventually be achieved with other funds
(personal communication: Dr.De Waele).
Activities
1. Nematode culture and inocula preparation;
141
2. Resistance screening in pots, under greenhouse conditions.
Achievements
Nematode culture
A total of 21 isolates were established and maintained at IRTA. They belong to the
species R. similis, P. coffeae, P. goodeyi and Meloidogyne spp. The rearing of the
nematodes on carrot discs was difficult (see same problem in BIP 20).
Around 10 to 15 cultures per isolate were made readily available for immediate use
(20,000 to 100,000 nematodes per culture). Several cultures were sent to members
of the ‘Nematological Consortium’.
Resistance screening in Greenhouse
By mid-1996, screening trials for lesion and root-knot nematodes were running.
Evaluation
Method
A desk study was carried out with the Completion Report as the only source.
Results
Considering the time constraint and the fact that funds were not received before
September 1995, leaving hardly one year for implementation, the work achieved can
be regarded as very satisfactory. In this short time:
-
Many abundant and pure nematode cultures were established, maintained,
and even distributed to partners;
Greenhouse tests were initiated, thus inaugurating a program of great
importance for banana research in the Canary Islands.
Had the project been supported as was proposed, it would have been very beneficial
for the ‘Musa Nematological Consortium’. The PI was already one of the only Musa
nematode specialists in the world at the onset of the project, and had previously
been active in Musa resistance screening (at FHIA).
Conclusions
The first results of the project were very promising and it is unfortunate for the
‘Nematology Consortium’ that the project could not evolve beyond the scope of the
BIP.
142
BIP 22
Breeding Hybrid Musaceae with Resistance to Multiple Diseases, especially
Black Sigatoka and Panama Disease
Description
Co-ordinates
Organisation:
Fundación Hondureña de Investigación Agrícola (FHIA)
Principal Investigator:
Dr. Phillip Rowe
Duration:
Two years, starting January 1, 1997
Objectives
To develop hybrid bananas with resistance to multiple diseases, especially black
Sigatoka (Mycosphaerella fijiensis) and Panama Disease (Fusarium oxysporum f.sp.
cubense) by conventional breeding
Activities
Pollinations were made between two diploid parents (SH-3142 and SH-3362) and
two triploid banana cultivars, the seed-fertile Lowgate and Dwarf Prata, for the
production of tetraploid hybrids.
The SH-3444 tetraploid was pollinated in 4x x 2x crosses to produce triploid hybrids.
Achievements
Pollinations between diploid and triploid parents
From the 2,200 pollinated bunches of Lowgate, 16 hybrid seeds were obtained. Four
embryos were viable, and three hybrid plants were evaluated in the field (the other is
still in the nursery).
Three tetraploids were derived from the Lowgate x SH-3362 crosses and were
evaluated in the field. All these hybrids had the Gran Nain-like short plant height, but
were susceptible to black Sigatoka and had mediocre bunch features.
A total of 685 seeds were produced on 450 pollinated bunches of Dwarf Prata. A
total of 250 hybrid seeds had embryos. From these 250 cultured embryos, 123 new
hybrid plants have been transplanted to the field. At the end of the project, the 123
plants derived from crosses onto Dwarf Prata had not yet flowered in the field.
143
Pollinations between SH-3444 and diploid parents:
A total of 84 seeds were obtained from 600 pollinated bunches of SH-3444. A total
of 20 embryos were cultured. From these 20 cultured embryos, 17 hybrid plants
were obtained. Two of these 17 have the gene for dwarfness and only these two
dwarf plants will be transplanted to the field for further evaluation (these two plants
are presently in the nursery).
Evaluation
Methodology
A desk study was performed based on the annual report for 1997 and a Progress
Report for the period January 1, 1998 to June 30, 1998, as well as the Final Report.
The project was visited on February 15, 1999 and included:
-
Review of the project with Principal Investigator;
Visit to the fields where the hybrid evaluation is being conducted (heavily
affected by Hurricane Mitch);
Visit to the crossing blocks in the field (heavily affected by Hurricane Mitch).
Results
The results in this project have shown that very few seeds are produced from
pollinating Lowgate: one hybrid per 500 bunches. This indicates that several
thousand bunches of Lowgate should be pollinated annually in order to obtain a
large segregating population to increase the possibilities for selection of commercial
hybrids with the desired combinations of disease resistance, productivity, and fruit
qualities.
Although low numbers of viable embryos were obtained, the 3x x 2x and 4x x 2x
crossing schemes used in this project have proven to be effective in the
development of disease-resistant hybrids. There is no question about whether these
crossing schemes work. It is just a matter of time and larger segregating populations
from which to select the best hybrids.
Conclusions
The objective of the project to develop hybrid bananas with resistance to multiple
diseases especially black Sigatoka (Mycosphaerella fijiensis) and Panama Disease
(Fusarium oxysporum f.sp. cubense) by conventional breeding was not achieved, as
was expected, in the time frame defined for the project. The eventual infertility of
some of the genetic material used handicapped progress, especially in the
development of large segregating populations. However, it was proven that the
crossing schemes (3x x 2x and 4x x 2x) are valid.
144
It is critically important that all the activities in this project be continued, the FHIA
scheme being the only one with the proper plant material and expertise to create
resistant hybrids that would reproduce the organoleptic qualifications of the
‘Lowgate’. This cultivar belongs to the ‘Gros Michel’ subgroup of dessert bananas, of
which the fruit quality is commonly recognised as superior to that of the ‘Cavendish’
subgroup.
The time frame defined to achieve the main objective of developing excellent hybrid
dessert bananas with resistance to multiple diseases especially black Sigatoka
(Mycosphaerella fijiensis) and Panama Disease (Fusarium oxysporum f.sp.
cubense) by conventional breeding was not sufficient for a project of this kind.
The expertise and scientific knowledge of the Principal Investigator is well known
and of excellent quality.
Prospects
In order to better achieve the objective, there is a need to dedicate more time and
funds to obtaining larger segregating populations in order to increase the
possibilities of obtaining hybrid bananas with desirable commercial traits, as was
planned in this project. Indeed, since black Sigatoka could destroy the export
banana trade, the activities undertaken in this project should be greatly enlarged.
FHIA will continue limited work on genetic improvement of bananas with its own
funds. However, these reduced activities will be only a fraction of what needs to be
undertaken. Therefore, a next phase for this project should be planned and funded.
145
CONCLUSIONS AND RECOMMENDATIONS
The Concept
As was explained in the Introduction, the Terms of Reference of this Project evolved
in the course of its preparation, with the main result that it eventually covered a
much larger research field than was initially conceived.
One is consequently dealing with a large research program, rather than with a
project. The program consisted of eighteen projects which together did not form a
coherent and fully-fledged entity since several relevant research domains were
missing (e.g. the multi-faceted work on important pests and diseases, such as the
Borer Weevil, the Banana Bunchy Top Virus, the Banana Bract Mosaic Virus;
research on post-harvest technology; and research on various agronomic issues).
The only project directly addressing the black Sigatoka disease (BIP 17) was
restricted to one aspect of the resistance of M. fijiensis to fungicides. The entire
domain of the host-pathogen relation and of its co-evolution was left untouched.
A key question, therefore, is whether the actual implementation of the BIP
addressed the following two original objectives, entirely and correctly:
1. To develop and evaluate, over a five-year period, a range of improved banana
varieties with export potential, incorporating increased productivity with durable
disease resistance, using conventional and non-conventional breeding
techniques.
2. To develop more efficient and integrated disease management practices for
black Sigatoka disease, based on an improved understanding of disease
epidemiology and pathogen variability in different areas and over different
seasons.
The answer appears to be: “Partially yes, but several positive results concern
subjects that were not implied by the above objectives, while other relevant activities
are missing”.
This “skewed” product is the result of the change in orientation of the BIP during the
initial implementation phase as explained in the Introduction. The present Review
Team does not consider it as its task to evaluate the judiciousness of such reorientation, which apparently was effected by the PEA with the implicit agreement of
the CFC and the Supervisory Body. The Team had to evaluate the implementation
and the products of the BIP as it was eventually established.
But it should be borne in mind that the re-orientation, while addressing very
important research subjects that were not in the original Terms of Reference, had as
a consequence the abandonment (or at least the considerable “shrinking”) of the
following three major activities:
-
Testing and post-harvest evaluation and propagation of new hybrids;
146
-
Research on the Sigatoka diseases;
9
Information, documentation and training.
IMPLEMENTATION AND RESULTS
Genetic Improvement
It can be stated that the actual BIP, although no longer exclusively focussed on the
development and evaluation of “improved banana varieties with export potential”,
has nevertheless produced important results to that end. Project BIP 22 directly
addressed this theme, while the results of most other projects, especially the
‘biotechnology’ ones, will be of great (future) help for such improvement.
The BIP projects dealing with Conventional Breeding can be considered as an
interim support to long-term programs. They nevertheless had a direct (BIP 05, 22)
or indirect (BIP 02) boosting effect on the breeding effort. The EMBRAPA project
(BIP 05) produced the most striking results, since black Sigatoka-resistant hybrids
are presently propagated right in the zone of the recent attack in Brazil. Project BIP
22 at FHIA had a duration of 2 years only, and adequate further support for the effort
to develop improved ‘Highgate’ commercial export bananas is an absolute necessity
for the breeding program to become fully productive. While progress was made in
the understanding of the genetics of resistance to Black Leaf Streak (black Sigatoka)
with the help of project BIP 02, the results reveal the complexity of that research
theme as well as the need for still more improved observation techniques in highly
controlled environments in order to make the promising results applicable for
breeders.
Indeed, it can be stated that the results of these three projects risk becoming
obsolete unless the programs involved receive substantial further support. This
leads to the question of whether such long-term efforts can survive on projects with
limited duration, such as the BIP. The Review Team advances the opinion that in
this particular research area a support system based on projects of limited duration
is less productive in the end, because of the production of fragmented and often
inapplicable results. The Team hopes that sponsors and supporters will reconsider
the short duration approach to funding.
As documented in the different reviews on the biotechnological part of the BIP, i.e.
the Non-Conventional Breeding (encompassing BIP 08, 09, 10, 11 and 12), the
results were a mixture of deficiencies and remarkable achievements. Even if viewed
very critically, the biotechnology projects of the BIP were together productive, and
some of the outcomes will doubtlessly be of future importance for the nonconventional genetic improvement of banana varieties.
A few examples should illustrate this point. First, successful culture of tissues and
cells, and reproducible regeneration of complete plants is no more restricted to one
(or at best a few) cultivars. On the basis of the established regeneration protocols, it
is fair to expect, that any cultivar will be regenerable in future. Second, the
9
Conferatur Annex 1, Objective (1), sub Outputs 1 and 3; Objective (2).
147
transformation of banana has left the purely experimental stage, and the BIPsupported development of transformation technologies will make stable banana
transformation less of a trial-and-error type of experiment. Third, the scientific
community now knows which promoters perform best in transgenic bananas, and
which genes are available that confer meaningful agronomic traits. Fourth, cDNA
libraries are available from different tissues that await exploitation beyond the
isolation of fruit ripening-associated messages. Genomic libraries allow the isolation
of full-length genes and relevant promoters. Fifth, some, not always encouraging,
experiences have been made with transgenic banana plants, from which lessons for
the future can be learned. For example, the number of proven transgenics should
definitely be higher (> 50 plants) than available at the end of BIP. There are other
achievements of the BIP, that will last and positively influence banana research in
this area (see the individual project reviews).
The ground for all of these achievements was prepared before the BIP started, and
was supported by various other sources. In some cases, BIP funding was taken as
an additional, yet welcome increase in financial potential; in other cases it was
catalytic. In all BIP groups working on the biotechnology aspects, research on the
above topics will continue with other, as yet limited and short-duration funds only.
So, was BIP only an episode in an otherwise already established banana
improvement program? Certainly yes! As far as volume and supported activities are
concerned, the BIP was no more than any other research program: too short, too
spartanic. However, it shares with many other programs, the provision of an
incentive to the banana research community to focus and to collaborate, albeit with
limited success, as might be expected in view of the limited time and resources
applied.
In the area of Genetic Resources and Resistance Sources, the BIP produced results
of unequal value, for several reasons. Positive is the newly acquired and
multifaceted knowledge on diploids as breeding sources (BIP 01), the much better
understanding on plantains elsewhere than in Africa (BIP 03), the establishment of a
good-performing banana research capacity in Vietnam (BIP 04), and the
considerable progress made in the screening of breeding material for nematoderesistance (BIP 18 to 21).
Shortcomings in BIP 01 were mainly due to over-ambitious objectives (e.g.
agronomic evaluation over three cycles is an unrealistic goal within three years of
implementation; the link between morphological and molecular markers remains to
be established).
Unrealistic objective setting was a weakness also in BIP 04. The present review also
shows that this project evolved in a rather isolated scientific environment, despite the
BIP Workshop organised there in its initial stages. Lack of scientific advice from
abroad was a handicap for project BIP 03 as well, on top of its extremely short
duration (18 months). Still, both these projects, implemented by modest but skilful
local scientists, paved the way for solid further research of great importance. Further
support is, however, again the problem.
The BIP has been instrumental in the initiation of an international network for
mematode-resistance screening methods. In particular, the implementers of BIP 20
148
can be commended for the remarkable progress made in the methodology for
assessing resistance to nematodes. But much further work is necessary for the
improvement of the relevant techniques, particularly at laboratory level, as project
BIP 19 reveals. Yet, the momentum created by the BIP risks stalling by lack of
further support. The Musa Nematology Consortium, which proved to be an
instrumental innovation, cannot rely in the future solely on the goodwill of its
members and calls for sufficient international support.
Pest and Disease Control
With the exception of project BIP 17, no progress can be registered for the BIP in
research on the Sigatoka diseases. The reason for this shortcoming was explained
earlier (“The Concept”).
On the other hand, the BIP can be considered as “the architect” of the considerable
progress made in research on Fusarium Wilt (BIP 15) and on Banana Streak Virus
(BIP 14). The results of both projects do have far-reaching and long-lasting positive
implications for banana breeding. The projects were also instrumental in some
informal network building among specialists and collaborators at international level.
IMPACT
As is the case for many research projects, the impact of the BIP on the future of
banana/plantain production can hardly be formulated in economic terms, because
the integration of its results into the production and the marketing sectors needs
quite some time.
The Review Team is of the opinion that the impact on further research is what
interested parties (including donors) would like to see in the first place.
In this respect, the BIP’s significance is undeniable. Entire panels of research have
been catalysed through the BIP in each of the domains where it was active. On the
basis of the above conclusions regarding “Implementation and Results”, one can
predict with confidence that many results will positively and deeply influence further
research.
A “spin-off” is already discernible in the following cases:
-
-
-
For the first time in Brazil, black Sigatoka-resistant hybrids, suited to the local
market, have been propagated for further testing and many newly produced
resistant diploid hybrids have been introgressed for the development of still better
performing end-products, which will play an important role in banana production
for local consumption in Latin-America and elsewhere.
Research on the genetics of the resistance to black Sigatoka as well as the
characterisation/ evaluation of numerous diploid cultivars are likely to culminate
during the coming years in the production of appropriate “building stones”
(improved diploids) of all sorts of resistant hybrids.
Genetic transformation of Musa tissues and subsequent plant regeneration is
presently implemented in a wide cultivar spectrum and the catalytic role of the
149
-
-
BIP in this evolution is undeniable. The cDNA- and genomic libraries established
via the BIP now offer the opportunity to extend the production of “transgenics” for
different purposes.
Systematic screening of breeding material for resistance to nematodes has been
achieved for the first time on an international scale and the established basic
methodology has been improved.
The considerable progress realised in the study for Fusarium Wilt has already
had an impact on resistance-building adapted to local host-pathogen conditions.
The better understanding of the Banana Streak Virus in all its aspects is of great
assistance to breeders.
The BIP “came in time”.
However, interested parties should be aware of the fragile character of this potential
impact, in a world where financial support from the public sector for such research is
apparently much less certain than it was only a decade ago. For nearly all of the
eighteen projects, the cessation or even reduction of financial support would
seriously compromise the impact of further research. The BIP has brought banana
research near the point of multi-purpose application, and a decrease in public sector
interest in this research could run the risk of being viewed in the future as a historical
error.
RECOMMENDATIONS
A major lesson for the future is that a 5-year project such as the BIP should not be
compromised in its implementation by a fundamental reconsideration of its agreed
activities leading to a two-year delay in “action on the field”. Many subprojects
dealing with genetic improvement would have achieved the set objectives with a
four-to-five year duration.
The argument that the competitive bidding system was a guarantee for an
equilibrated performance of the whole BIP is only partly valid. A major part of the
funds was allocated anyway to institutes with existing expertise, and these institutes
would have achieved a lot more within the agreed five-year framework if more direct
support had been given.
Competitive bidding (for a substantial remaining part of the total funding) could then
still have had a meaning for institutes where research on Musa was/is being
developed. That part would probably have attracted necessary co-financing, which
apparently and most unfortunately has been lacking in the present BIP.
Instead, the BIP tried to involve too many different aspects. While this was desirable
in a sense, it led to a dilution of funds with the result that that no single activity could
be sufficiently supported. For example, the inclusion of HKUST in the BIP for only
one single year would not have had any effect on banana research, if HKUST had
not invested considerable funds to save the project. Another example is the
excellent but very incomplete work on plantains in India (BIP 03) for which only 18
months were planned, due to previous insufficient investigation in local research
capacities and needs.
150
Regarding biotechnology research, genomics and transcriptomics (i. e. the huge
repertoire of techniques for genome analysis and gene expression analysis) should
be involved to isolate more agronomically interesting genes (e. g. resistance genes,
genes for fruit quality, or genes for yield increase), and promoters to drive these
genes under various environmental regimes. These genes and promoters should
then be used to improve commercially important local cultivars for a particular trait,
and transgenics should be studied for trait inheritance and stability. Cornell
University (among others) has already started a similar program very much along the
same lines. It is certain, that industry would be involved in such a Banana Genome
Initiative (BGI). So, less (projects) can be more (progress) in research.
It is recommended that projects of the importance of the BIP should not only
10
restrict the objectives to a minimum , but should assure sufficient time (5
years) and funds for the actual implementation of the (previously) carefully
selected11 activities involved.
An important aspect of the BIP was its involvement in long-term research
(conventional breeding and fundamental aspects in several subprojects, as, for
example, the genetics of black Sigatoka-resistance, BIP 02). It should be made clear
to interested parties that this type of research, so vital for the future, should not have
to rely on projects with limited duration, but rather form part of long-term core
support. While the BIP was very beneficial for that research, the question now is
whether the effort will be continued (if funds still have to come via short-duration
projects).
To paraphrase this: Everybody mentions “durable resistance/productivity/etc”; where
is the “durable support”?
It is recommended that conventional breeding and connected genetics
research (including the search for proper molecular markers) benefit from
appropriate long-term core support in an institutional context.
Pathogens and their host plant evolved and still evolve simultaneously in a dynamic
and rather closed system (co-evolution). Therefore, it is obligatory to study both in
order to predict the consequences of any intervention in the system. While research
on virus resistance within the BIP met this criterion in the case of the BSV,
analogous work on the important black Sigatoka (and yellow Sigatoka) diseases, for
example, was clearly under-represented. The productivity of the BIP suffered from a
serious discrepancy in efforts between “resistance-building” and “understanding
what this resistance actually means”12. As a result, genetic improvement is still short
of precise and adequate physiological criteria (and of involved genetic markers)
which are needed for the construction of a reliable resistance to the disease: failures
and shortages appear on hybrids grown in different environments, and these can as
yet not be explained for the breeder to adapt methods and techniques.
10
As was actually the case for the BIP.
That is: envisaged and then selected during the preparatory phase.
12
In physiological and ecological terms.
11
151
It is recommended that genetic improvement for resistance to a particular
disease or pest be flanked by sufficient research on the host-pathogen/pest
interaction. The INIBAP-promoted PROMUSA system, with its working groups
articulated on genetic improvement as well as on the relevant diseases and pests,
could offer a convenient forum.
In the experience of the Review Team, contact between the different participants in
the BIP existed, but in no case was intense co-operation established. Such an
interaction would have prevented some duplication, which generally is not at all
rewarding. And such duplication existed in several cases in the biotechnology part of
the BIP.
It is recommended that projects, during their preparatory phase, encourage
the desired co-operation between groups with similar interests (e.g. BBTV
resistance research) to submit joint and interlinked research proposals. The
INIBAP should be instrumental in such endeavours.
Though transgenic bananas were considered a product from the beginning of the
BIP, obviously issues of deliberate release, availability of national guidelines, and
public perception were not considered sufficiently at the onset of the program. As a
consequence, even after termination of the BIP funding, no clear decision was
possible as to the release of transgenics in, for example, India.
It is recommended that sensitive issues in relation to the production of
transgenic bananas be positively considered prior to, or early in, a
project/program.
152
ANNEX 1 :
ORIGINAL TERMS OF REFERENCE OF THE BIP
Objectives:
1. To develop and evaluate, over a five-year period, a range of improved banana
varieties with export potential, incorporating increased productivity with durable
disease resistance, using conventional and non-conventional breeding
techniques;.
2. To develop more efficient and integrated disease management practices for
black Sigatoka disease, based on an improved understanding of disease
epidemiology and pathogen variability in different areas and over different
seasons.
Outputs and Activities
Objective 1:
Output 1. New banana varieties with export potential
Activity 1: Conventional breeding of improved Musa germplasm
Activity 2: Multi-locational testing of new varieties
Activity 3: Rapid propagation of new varieties
Activity 4: Post-harvest evaluation
Activity 5: Disease screening methodologies
Activity 6: Use of modern biotechnologies in banana breeding.
Output 2. Musa germplasm collection and conservation
Activity 1: Germplasm collecting, conservation and utilisation
Activity 2: Collection of pest, pathogens and biocontrol agents
Output 3. Documentation, information and training
Activity 1: Information and documentation
Activity 2: Workshop and training
Objective 2:
Output 1. Sigatoka Disease Forecasting System
Activity 1: Epidemiological studies
Output 2. Sigatoka variability description
Activity 1: Genetic and ecological studies on the Sigatoka disease complex in
different regions and over different seasons.
153
ANNEX 2:
LIST OF ACRONYMS
ACIAR:
AGI:
Australian Centre of International Agricultural Research
Institute of Agricultural Genetics (Vietnam)
BBMV:
BBTV:
BIP:
BMV:
BS:
BSV:
BTI:
Banana Bract Mosaic Virus
Banana Bunchy Top Virus
Banana Improvement Project
Banana (bract) Mosaic Virus
Black Sigatoka
Banana Streak Virus
Boyce Thompson Institute (Cornell University, USA)
CFC:
CIRAD-FLHOR:
Common Fund of Commodities
Centre de coopération Internationale en Recherche
Agronomique pour le Développement – Département
productions fruitières et horticoles
Cucumber Mosaic Virus
Corporación Bananera Nacional de Costa Rica
Centre de Recherche sur les Bananiers et les Bananiers
Plantains
CMV:
CORBANA:
CRBP:
EMBRAPA:
Brazilian Organisation for Agricultural Research
FAO:
FHIA:
Food and Agriculture Organisation
Fundación Hondureña de Investigaciõn Agricola
GFP:
GUS:
Green Fluorescence Protein
β- Glucuronidase
HKUST:
Hongkong University of Science and Technology
IAEA:
ICIA:
IGG:
IITA:
INIBAP:
IRTA:
ITC:
International Atomic Energy Agency
Instituto Canario de Investigaciones Agrarias (Spain)
Intergovernmental Group on Bananas
International Institute of Tropical Agriculture
International Network for the Improvement of Banana and
Plantain
Instituto de Recerca I Tecnologia Agroalimentares (Spain)
INIBAP Transit Center
KAU:
KUL:
Kerala Agricultural University (India)
Katholieke Universiteit Leuven (Belgium)
NRCB:
National Research Center for Banana (India)
PEA:
PI:
Project Executing Agency
Principal Investigator
154
PNG:
QDPI:
QUT:
Papua New Guinea
Queensland Department of Primary Industries (Australia)
Queensland University of Technology (Australia)
UH:
University of Hawaii
WIBDECO:
Winban Development Corporation
YS:
Yellow Sigatoka
155
156
PART III
Impact Assessment for Banana Improvement Project
157
158
EXECUTIVE SUMMARY
This report provides a brief assessment of the impact of the Banana Improvement
Project (BIP) co-sponsored by the Common Fund for Commodities, the InterGovernmental Group on Bananas and the World Bank. The BIP consisted of 18
individual projects funded between 1994 and 1998. The original target audience for
these projects was the world export banana industry with the focus altering during the
life of the program to take in the needs of small holder farmers. The principal objective
was to increase the productivity of banana production through the development and use
of higher yielding disease-resistant varieties and by reducing the cost of production,
especially the cost of pesticide applications.
The BIP was managed by the World Bank with the assistance of a Program Manager
and a Scientific Advisory Panel. The Panel was involved in industry consultation, the
definition of research priorities and project selection. The Panel also met regularly to
review the progress of the portfolio after projects had commenced.
Before the projects were called for and selected, the World Bank commissioned a report
to investigate the then current status of banana research and priority areas for the BIP.
The report (Buddenhagen, 1996) highlighted the need for combining innovative
breeding approaches with advances in plant biotechnology. This influenced the
composition of the program portfolio.
Most of the projects fell into two categories, breeding and biotechnology and many of
the projects were long term and strategic in nature, where knowledge was the primary
output. The knowledge produced has been in the following areas:
•
•
•
The collection and characterisation of a wide range of banana germplasm that
will allow increased effectiveness of the banana-breeding programs around the
world;
Increased efficiency of breeding programs through improved testing, screening
and typing methods and development of protocols; use of additional information
assembled through use of genetic markers, particularly for disease resistance to
Sigatoka, Panama Disease and nematodes;
Improved and refined technology for genetic manipulation including
transformation processes for banana and improved knowledge of disease
resistance mechanisms in banana.
The use of this knowledge will provide positive outcomes for plantains and all types of
banana production whether for cooking, domestic consumption as dessert bananas or
for production of export bananas. However, the application of this knowledge in the
future to provide tangible outcomes for producers and exporters will require further R&D
investment.
While the program did not specifically target applied outputs, there were a number of
areas where the program has had or will have direct industry impacts in the short term.
159
These include some black Sigatoka-resistant clones produced in the BIP currently being
used to assist control of the recent black Sigatoka incursion into the Amazonas region
of Brazil. Also, the ability to now type strains of Fusarium in a given location when
considering new banana developments will be beneficial to new banana enterprise
developments.
The BIP enhanced co-ordination of effort and increased collaboration in international
banana plant improvement. This was particularly evident in relation to what was termed
the ‘biotechnology consortium’ (a joint effort between KUL, BTI and QUT). This alliance
fostered staff exchanges between the three institutions involved and assisted the
transfer and refinement of both particle bombardment and Agrobacterium
transformation and regeneration technology. Another area in which increased
collaboration was fostered was that of nematode screening for resistance through what
was termed the ‘nematode consortium’. This consortium will continue after the BIP is
completed and will be important in the screening and understanding of nematode
resistance in the future (de Waele, 1995). A number of other collaborative efforts have
been stimulated by the BIP.
The BIP was successful, through the competitive grants approach, in attracting new
research providers into banana improvement. Moreover, due to the necessity for many
research institutions to attract external funding, such as through the BIP, the attraction
of more resources into banana plant improvement was achieved. While some of the
resources attracted into BIP projects would have probably been committed to banana
improvement anyway, it is likely that, due to the BIP, there were additional resources
attracted into the overall effort on banana improvement.
In summary, the BIP has provided a unique set of outputs in terms of both content and
process. The BIP has produced knowledge of a strategic nature as well as some
technologies that have been, and can be, applied in the short term. In terms of process,
the competitive bid system for identifying the best projects to fund within a strategic
vision has proved successful, as has the leverage of resources into the banana R&D
world, as well as increased collaboration and co-ordination among research teams.
Outcomes of the BIP are defined as the activities or events that have occurred, or are
likely to occur, as a result of the outputs from the BIP. Outcomes are directly relevant to
producers of bananas and to others in the banana value chain including consumers.
Outcomes can be positive or negative and are usually associated with benefits and
costs. These benefits and costs may fall differentially on the various participants in the
banana value chain.
Two important short-term outcomes stand out. The first was the ability to type Fusarium
strains before locating new banana developments. The world-wide collection of the
various strains of Fusarium will serve as a reference collection in the future enabling the
strains causing an outbreak of Panama disease to be quickly identified. New banana
developments can be more safely implemented through determining the strains present
in any given location before planting begins. For example, recent outbreaks of Fusarium
160
Wilt in Cavendish plantations in the eastern tropics (peninsular Malaysia, Sumatra and
Halmahera) caused enormous damage. These outbreaks have ominous implications for
the durability of western trades that are based on Cavendish clones. The avoidance of
future losses will be one of the short-term benefits from the BIP.
The second was the Brazilian genetic material used in Amazonas to combat the spread
of black Sigatoka as it enters Brazil. Some banana clones resistant to black Sigatoka
have been produced in Brazil with assistance from the BIP and are being used to slow
the rate of advance of the disease in northern Brazil. This would have a major reduction
in the costs of those who can potentially afford to spray with fungicides as well as
reduce yield losses of those who cannot afford chemical control technologies.
While the short-term outcomes and associated benefits are likely to have been
significant the most important outcomes and contributions from the BIP are likely to be
derived from the strategic knowledge contributions of BIP.
Outcomes associated with knowledge generated from the BIP are inherently uncertain.
However, in terms of assessing the impact of the BIP, it is possible to make some
assumptions about the future and the role that the BIP will have played in the future
development of the export banana industry.
The key events shaping the future use of knowledge from the BIP with respect to export
banana industries would include the development (in full or in part) of a Cavendish or
alternative export banana type that is resistant to disease. Benefits that might be
attributed at least in part to the BIP can be grouped into economic, environmental, and
social and health benefits.
The principal economic benefit most likely associated with the development of a
disease-resistant banana is the reduced cost of production of export bananas through
chemical replacement. The principal environmental benefit associated with the BIP is
the likelihood of lesser amounts of pesticides being used if resistant or more tolerant
varieties of export bananas are developed. The lower the pesticide applications, the
lower the potential damage to the environment. As with the environment, a range of
statements has been made that fungicides and nematicides used in banana production
are associated with negative health impacts on banana plantation workers, and those
living in banana-producing communities. Some of the perceived benefits related to
health that would arise from developing disease-resistant bananas would be improved
health of banana industry workers and higher labour productivity, as well as improved
health and quality of life of communities in banana-producing regions.
In order to evaluate the past investment in the BIP in quantitative terms, it is necessary
to construct future scenarios that assume that the particular knowledge generated from
the BIP is used in further R&D efforts that are eventually successful with the resulting
technologies or products being adopted by industry. This requires a number of
assumptions to be made about the type of future investment and associated costs, the
time period before disease-resistant bananas will actually be produced, the likelihood
161
that the technologies will be produced within the timeframe assumed, and the proportion
of total benefits that might be attributed to the BIP.
The two key future scenarios assumed were:
1. The production of a transgenic Cavendish that is reasonably resistant to black
Sigatoka and nematodes;
2. The development of export banana types that are resistant to black Sigatoka and
nematodes. This is assumed to be achieved through a combination of
transformation and regeneration technologies together with breeding
components and has resulted in a robust form of resistance.
Each of these scenarios was analysed in terms of potential benefits and costs that
might be expected ‘with’ and ‘without’ the R&D program.
The analysis was effected through a discounted cash flow analysis that ran for a period
of thirty-five years, commencing in the 1994 calendar year. All costs and benefits were
discounted back to 1994 using a discount rate of 7%. All values were expressed in 1998
US dollars. Investment criteria estimated included the Net Present Value (NPV), the
Benefit-Cost Ratio (B/C Ratio), and the Internal Rate of Return (IRR). Criteria were
estimated for each of the two scenarios separately.
The investment criteria estimated using the base assumptions for Scenario 1 are
summarised below:
Expected Investment Criteria for Scenario 1 (Transgenic Cavendish)
(Discount rate 7%)
Total Investment
BIP Investment only
NPV ($ million)
578
27.1
B/C Ratio
9:1
10:1
IRR (%)
21.4
21.9
Expected Investment Criteria for Scenario 2 (Alternative Banana Type)
(Discount rate 7%)
Total Investment
BIP Investment only
NPV ($ million)
484
22.8
B/C Ratio
8:1
8:1
IRR (%)
20.0
20.5
The estimation of benefits is conservative in two ways. First, the actual parameters used
in the quantitative analysis could be considered to be conservative. Second, there are a
number of other benefits, outlined in earlier sections, that have not been included in the
quantitative analysis. Environmental and human health benefits are two such benefits
162
that are usually difficult to value but are likely to be high and significant. Such benefits
could also provide justification for public investment in banana improvement since
benefits could be captured by communities, as opposed to the private sector, in
banana-exporting countries.
Given the assumptions made in the analysis, the results suggest that the BIP could be
expected to provide a rate of return on funds invested of at least 20%. Conservative
assumptions have been used through the analyses, and the sensitivity analyses and
break-even parameters estimated show that the results are robust across a credible
range of assumptions. The results of the investment analysis demonstrate that the
payoffs being sought in terms of disease-resistant bananas can justify a large
investment in banana improvement research.
The BIP has provided the methods for the transformation and regeneration of bananas
and has moved the world banana industry closer to the reality of a transgenic banana.
Transformation and regeneration have now been accomplished. There has also been
significant progress demonstrated in terms of new germplasm for use in breeding
programs and methods for making breeding programs more effective in the future. The
most important output from the BIP was strategic knowledge that can be used in further
R&D projects in the future.
163
1.
Introduction
1.1
Background and Methods Employed
This report provides an impact assessment of the Banana Improvement Project (BIP)
managed by the World Bank over the five-year period 1994 to 1998. The BIP is
hereafter sometimes referred to as the ‘program’ rather than the ‘project’ so as not to
confuse the ‘total portfolio of projects’ with the 18 individual research projects. The
terms of reference for the impact assessment are provided in Annex 1.
Most of the information assembled in this report was gained from progress and
termination reports for individual projects. Most of this material was made available by
the Program Co-ordinator before and during the consultant’s visit to the World Bank in
Washington, D.C. In addition, personal visits were made to six individual project sites
(two in Australia, two in Belgium and two in France), where discussions on specific
projects were held with Principal Investigators.
Discussions with each of the three members of the BIP Advisory Panel and further
exchanges with Advisory Panel members also took place. These discussions and a visit
to the World Bank in Washington, D.C. were held over the period 5 to 17 October, 1998.
In addition, various industry personnel associated with the world export banana industry
were also contacted to assist with various information requirements for the assessment.
1.2
Structure of Report
Section 2 of the report provides a brief background to the BIP, including its aims and
objectives, and a description and classification of the constituent projects that were
funded.
Section 3 provides a summary of the principal outputs from the program, while Section
4 describes the actual and likely outcomes of the research, as well as the benefits that
have been derived to date and those that are likely to ensue in the future.
The assumptions and results of an investment analysis for the funds invested in the BIP
are reported in Section 5, and a brief conclusion of the report is contained in Section 6.
2.
Description of Banana Improvement Program
2.1
Introduction and Background
The BIP was sponsored by the Common Fund for Commodities (CFC), the FAO InterGovernmental Group on Bananas (FAO/IGB), and the World Bank. Resources of
approximately US$ 3.5 million were provided by the CFC to fund the research portfolio.
The BIP consisted of 18 projects located in a range of R&D organisations around the
164
world. The projects were selected through a competitive bidding system whereby over
200 national and international research organisations were invited to prepare and
submit project proposals to the World Bank.
The World Bank managed the BIP with the assistance of a Program Manager and a
Scientific Advisory Panel. The Panel was involved in industry consultation, the definition
of research priorities and project selection. The Panel also met regularly to review the
progress of the portfolio after projects had commenced.
All BIP projects will be completed by the end of December 1998.
2.2
Aims, Objectives and Priorities
The goal addressed by the BIP is to make banana production more profitable and to
reduce the use of pesticides. The aim of the program is to increase the productivity of
the crop through the development and use of higher yielding, disease-resistant varieties
and by reducing the cost of production, especially the cost of pesticide applications.
The two key objectives of the BIP are to support research which will:
1. Develop and evaluate a range of improved banana varieties with export potential,
incorporating increased productivity and durable disease resistance through
conventional and non-conventional breeding techniques;
2. Develop more efficient and integrated disease management practices, especially
for black Sigatoka disease.
The objectives of the BIP were consistent with the strategies of the FAO/IGB, which
included:
•
•
•
•
•
2.3
Making bananas a more profitable crop for smallholder producers by increasing
productivity and reducing the costs of production, especially by improved disease
control based on durable disease resistance;
Reducing pesticide use, thus both reducing costs of production and limiting the
environmental damage and human health risks caused by excessive use of
fungicides and nematicides;
Improving the reliability of supply of high–quality products;
Expanding the export market, including opening up niche markets for speciality
bananas;
Improving access to markets.
Projects Funded
Before the projects were called for and selected, the World Bank commissioned a report
during 1994/95 to investigate the then current status of banana research and priority
areas for the BIP. The report (Buddenhagen, 1996) highlighted the need for combining
165
innovative breeding approaches with advances in plant biotechnology. This influenced
the composition of the program portfolio.
The projects funded concentrated on banana improvement rather than agronomy or
integrated pest management. Both breeding and biotechnical projects were funded.
A list of projects funded within the program is provided in Table 2.1. Further details are
provided in Annex 2.
Table 2.1: Constituent Projects of BIP
Project ID
PN1
PN2
PN3
PN4
PN5
PN8
PN9
PN10
PN11
PN12
PN14
PN15
Title
Field and laboratory evaluation of diploid
bananas for their use in breeding
schemes
Field crosses for understanding the
inheritance of black leaf streak resistance
in bananas
Collection, characterisation and
evaluation of Nendran bananas in India
Collection, evaluation and
characterisation of genetic resources and
improvement of banana in Vietnam
Banana-breeding in Brazil
Novel genes for fungal resistance and
post-harvest quality
The development of transgenic bananas
with resistance to banana bunchy top and
banana mosaic viruses
Molecular tool box
Genetic engineering of ethylene
biosynthesis in bananas
Use of biotechnology to produce
transgenic bananas that are resistant to
BBTV infection
Elimination of banana streak badnavirus
(BSV) from improved Musa germplasm
and related studies on transmission and
host plant/virus/vector interactions
Variability and relationships within
populations of Fusarium oxysporum f sp
cubense from its centre of origin
166
Research Provider
CIRAD-FLHOR, France
CIRAD-FLHOR, France
Kerala Agricultural
University, India
Institute of Agricultural
Genetics, Vietnam
EMBRAPA, Brazil
Boyce Thompson
Institute for Plant
Research, USA
QUT, Australia
KUL, Belgium
Hong Kong University of
Science and
Technology, Hong Kong
University of Hawaii,
USA
IITA, Nigeria
QDPI, Australia
PN17
PN18
PN19
PN20
PN21
PN22
2.4
Origin and distribution of fungicideresistant strains of M. Fijiensis in banana
plantations in Costa Rica
Study of tolerance and resistance of
banana in nematodes
Identification of durable resistance
sources in banana and plantain
Identification of durable resistance
sources in banana and plantain
Identification of durable resistance
sources in banana and plantain
Breeding of hybrid Musaceae with
resistance to multiple diseases, especially
black Sigatoka and Panama Disease
CORBANA, Costa Rica
CRBP, Cameroon
KUL, Belgium
FHIA, Honduras
IRTA, Spain
FHIA, Honduras
Type of Research
Table 2.2 shows the projects presented by project number, title, whether strategic or
applied and whether the project was biotechnology or breeding-orientated in nature. A
project was defined as strategic when the intended output from the project could not be
used directly by industry at the present time, and where the output was intended as an
input to further research. On the other hand, a project was considered applied if the
outputs could be used directly by industry. Some projects had a mix of both applied and
strategic components.
Strategic projects usually produce knowledge as their primary outputs. Such information
or knowledge can be used in other projects to eventually produce applied outputs which
are implementable, by industry. Strategic projects are usually long term in nature.
As can be seen from Table 2.2, the portfolio of projects funded by the program is
predominantly strategic in nature.
167
Table 2.2: Classification of Constituent Projects of BIP
Project ID
PN1
PN2
PN3
PN4
PN5
PN8
PN9
PN10
PN11
PN12
PN14
PN15
PN17
PN18
Title
Field and laboratory evaluation of
diploid bananas for their use in
breeding schemes
Field crosses for understanding
the inheritance of black leaf streak
resistance in bananas
Collection, characterisation and
evaluation of Nendran bananas in
India
Collection, evaluation and
characterisation of genetic
resources and improvement of
banana in Vietnam
Banana-breeding in Brazil
Novel genes for fungal resistance
and post-harvest quality
The development of transgenic
bananas with resistance to banana
bunchy top and banana mosaic
viruses
Molecular tool box
Genetic engineering of ethylene
biosynthesis in bananas
Use of biotechnology to produce
transgenic bananas that are
resistant to BBTV infection
Elimination of banana streak
badnavirus (BSV) from improved
Musa germplasm and related
studies on transmission and host
plant/virus/vector interactions
Variability and relationships within
populations of Fusarium
oxysporum f sp cubense from its
centre of origin
Origin and distribution of fungicideresistant strains of M. Fijiensis in
banana plantations in Costa Rica
Study of tolerance and resistance
of banana in nematodes
168
Type
Strategic
Target Activity
Breeding with
biotechnology
component
Breeding with
biotechnology
component
Strategic
Strategic with
an applied
component
Breeding
Strategic with
an applied
component
Applied with a
strategic
component
Breeding
Breeding with
biotechnology
component
Strategic
Biotechnology
Strategic
Strategic
Biotechnology
Biotechnology
Strategic
Biotechnology
Strategic
Biotechnology
Strategic
Biotechnology
and breeding
Strategic
Breeding and
biotechnology
Applied with a
strategic
component
Pest and
disease control
methods
Strategic
Breeding
PN19
PN20
PN21
PN22
Identification of durable resistance
sources in banana and plantain
Identification of durable resistance
sources in banana and plantain
Identification of durable resistance
sources in banana and plantain
Breeding of hybrid Musaceae with
resistance to multiple diseases,
especially black Sigatoka and
Panama Disease
Strategic
Breeding with
biotechnology
component
Strategic
Breeding
Strategic
Breeding
Applied
Breeding
Another observation was that projects focused more strongly on the first objective of the
program (developing the improved banana types) compared with the second objective
(more efficient and integrated disease management practices for black Sigatoka).
3.
Summary of Principal Outputs of the Program
3.1
Introduction
The outputs of the program have been:
•
•
•
Increased knowledge that will input into further projects which are targeted
towards producing disease-resistant export bananas;
Some applied and more immediate outputs that can already be used by industry;
Leverage of additional resources into banana R&D, and increased collaboration
and international co-ordination in plant improvement R&D on bananas.
A summary of the achievements of the BIP in each of these areas follows.
3.2
Strategic Knowledge
Strategic knowledge has been the principal output from the program. This knowledge
has been in the following areas:
•
•
The collection and characterisation of a wide range of banana germplasm that
will allow increased effectiveness of the banana-breeding programs around the
world (PN3 and PN4);
Increased efficiency of breeding programs through improved testing, screening
and typing methods, development of protocols; and use of additional information
assembled through use of genetic markers, particularly for disease resistance to
169
•
Sigatoka, Panama Disease and nematodes (PN1, PN2, PN14, PN15, PN18,
PN19, PN20, and PN21);
Improved and refined technology for genetic manipulation, including
transformation processes for banana and improved knowledge of disease
resistance mechanisms in banana (PN5, PN8, PN9, PN10, PN11,PN12, PN14
and PN18).
The use of this knowledge will provide positive outcomes for plantains and all types of
banana production whether for cooking, domestic consumption as dessert bananas or
for production of export bananas. The application of this knowledge in the future to
provide tangible outcomes for producers and exporters is addressed in Section 5.
3.3
Applied Outputs
While the program did not specifically target applied outputs, there were a number of
areas where the program may have direct industry impacts, albeit not necessarily in the
export banana industries. These include projects PN3, PN5, PN15, PN17 and PN22.
PN5 and PN22 were projects embedded in the breeding programs of EMBRAPA
(Brazil) and FHIA (Honduras) respectively. Black Sigatoka clones produced in the BIPsupported part of the EMBRAPA breeding program are currently being used to assist
control of the recent black Sigatoka incursion into the Amazonas region of Brazil. Also,
the FHIA attempt to develop disease-resistant bananas with a short stature could result
in such types being commercially accepted.
Project PN3 evaluated Indian germplasm and the results may have implications for the
use of more short-statured bananas and a cost reduction in propping in South Indian
production systems.
Project PN15 will have direct application by industry in being able to type strains of
Fusarium in a given location when considering new banana developments.
The finding in Project PN17 concerning the continuation of resistance to chemicals once
fungicides are no longer applied is of direct industry relevance.
These applied outputs should be viewed as direct benefits from the BIP investment and
are considered again later in a description of outcomes.
3.4
Leverage
Other investments in banana research have been leveraged by the BIP into the banana
realm. This has not only been achieved within many of the 18 projects themselves, but
also outside the projects. For example, the establishment of the BIP has leveraged the
Flemish Association for Development Co-operation and Technical Assistance (VVOB)
170
and the Belgium Development Agency (BDA) to provide significant funding to the
Nematode Consortium through positioning scientific personnel in various breeding
programs around the world. This will be ongoing after the BIP is completed.
A conservative estimate of the funds contributed to the BIP projects by the host
institutions (co-financing) was $2 million.
Other instances of leverage and attraction of other resources into banana research
include NZ support in Vietnam for post-harvest production studies and additional funds
outside the original project contributed by the University of Hong Kong and the
University of Hawaii.
3.5
Co-ordination and Collaboration
Because of the nature of the program that was established, there was enhanced coordination of effort and increased collaboration in international banana plant
improvement. This was particularly evident in relation to what was termed the
‘biotechnology consortium’ (a joint effort between KUL, BTI and QUT). This alliance
fostered staff exchanges between the three institutions involved and assisted the
transfer and refinement of both particle bombardment and Agrobacterium
transformation and regeneration technology. However, in the latter stage of the BIP, this
consortium faded in activity due to the pursuit of more specific goals by each research
provider involved in the biotechnology initiative within the BIP.
The other area in which increased collaboration was fostered was that of nematode
screening for resistance through what was termed the ‘Nematode Consortium’. This
consortium will continue after the BIP is completed and will be important in the
screening and understanding of nematode resistance in the future (de Waele, 1995).
A number of other collaborative efforts have been stimulated by the BIP. These include
continuing interaction between Australian and Vietnamese scientists and Australian
involvement in testing genetic material for a range of breeding programs. Unfortunately,
attempts to integrate the classical breeding approaches in Honduras with molecular aids
to breeding have not been successful. Integration between the FHIA approach to
breeding and that of the French breeding program would have been beneficial to both
areas of endeavour.
The BIP was successful, through the competitive grant approach, in attracting new
research providers into banana improvement. Moreover, due to the necessity for many
research institutions to attract external funding, such as through the BIP, the attraction
of more resources into banana plant improvement was achieved. While some of the
resources attracted into BIP projects would have probably been committed to banana
improvement anyway, it is likely that significant additional resources were encouraged
into banana improvement due to the BIP.
171
Two examples suffice:
•
•
3.6
CIRAD have now secured an additional permanent position for banana
improvement due to the program;
Generic resources provided by VVOB in Belgium have been committed to
banana improvement through the Nematode Consortium
Conclusion
The BIP has provided a unique set of outputs in terms of both content and process. The
BIP has produced knowledge of a strategic nature as well as some technologies that
have been, and can be, applied in the short term. In terms of process, the competitive
bid system for identifying the best projects to fund within a strategic vision has proved
successful, as has the leverage of resources into the banana R&D world, as well as
increased collaboration and co-ordination among research teams.
4.
Summary of Actual and Potential Outcomes and Benefits
4.1
Introduction
Section 3 has provided a brief summary of the reported outputs of the program up to
November 1998. There are still a number of projects to be completed due to a range of
reasons and a number of research providers will be continuing with research instigated
under the BIP. It will be important for such future outputs to be reported even after the
official end of the BIP in 1999. How this will be achieved is uncertain, but this issue
highlights the unsatisfactory nature of short-term funding such as provided by the BIP.
Outcomes of the BIP are defined as the activities or events that have occurred, or are
likely to occur, as a result of the outputs from the BIP. Outcomes are directly relevant to
producers of bananas and to others in the banana value chain including consumers.
Outcomes can be positive or negative and are usually associated with benefits and
costs. These benefits and costs may fall differentially on the various participants in the
banana value chain.
Outcomes can be short term or long term. Short-term outcomes are often preferable,
but not always easy to achieve in agricultural research.
Outcomes can also be classified into those of content (products, technologies, and
knowledge), and those of a structural nature (e.g. changed priorities and resource
allocation, attraction of additional resources).
The following describes the outcomes and associated benefits and costs that have
arisen due wholly or partially to the BIP.
172
4.2
Short-Term Outcomes and Benefits
Short-term outcomes and benefits have arisen from the BIP. Two important outcomes
were:
•
•
The ability to type Fusarium strains before locating new banana developments;
The Brazilian genetic material used in Amazonas to combat the spread of black
Sigatoka as it enters Brazil .
Typing Fusarium strains
The world-wide collection of the various strains of Fusarium will serve as a reference
collection in the future, enabling the strains causing an outbreak of Panama disease to
be quickly identified. New banana developments can be more safely implemented
through determining the strains present in any given location before planting begins. For
example, recent outbreaks of Fusarium Wilt in Cavendish plantations in the eastern
tropics (peninsular Malaysia, Sumatra and Halmahera) caused enormous damage.
These outbreaks have ominous implications for the durability of western trades that are
based on Cavendish clones. The avoidance of future losses will be one of the shortterm benefits from the BIP.
Brazilian material
Some banana clones resistant to black Sigatoka have been produced in Brazil with
assistance from the BIP and are being used to slow the rate of advance of the disease
in northern Brazil. This would have a major reduction in the costs of those who can
potentially afford to spray with fungicides as well as reduce yield losses of those who
cannot afford spraying control technologies.
4.3
Long-Term Outcomes and Benefits
While the short-term outcomes and associated benefits are likely to be significant, the
most important outcomes and contributions from the BIP are likely to be derived from
the strategic knowledge contributions.
Outcomes associated with knowledge generated from the BIP are inherently uncertain.
However, in terms of assessing the impact of the BIP, it is necessary to make some
assumptions about the future and the role that the BIP may play in the future
development of the export banana industry.
The key events shaping the future use of knowledge from the BIP with respect to export
banana industries would include the development (in full or in part) of a Cavendish or
alternative export banana type that is resistant to the two principal fungal diseases of
black Sigatoka and Panama Disease, as well as to nematodes and viral diseases.
173
These events may utilise both conventional breeding technologies and biotechnologies
such as genetic markers or genetic engineering.
The key uncertainties associated with events of this type would include the following:
•
•
•
•
•
•
•
•
•
The number of years until they will occur;
The annual investment required for them to occur within a given period with a
specific probability of research success;
The applicability of the resistant material (across pathogens and strains within a
pathogen);
The durability of resistance inherent in the new material;
The level of use of chemicals still required;
Any interaction or enhancement with cultural techniques including integrated pest
management;
The level of maintenance of resistance through breeding/biotechnology required
on an ongoing basis after the events are realised;
The cost and level of availability to industry of any new material produced;
The levels of adoption of new export banana types.
There are still serious constraints to be able to effectively introduce disease-resistant
genes and have them expressed in banana plants. A major constraint is still the degree
of somaclonal variation in the recipient banana material. The cell target tissue needs to
be able to be produced in vitro fast and effectively to reduce the degree of variation.
Promoters need to be more specific so that they are not expressed in all tissue types
including the fruit. Specific promoters that are only expressed in certain tissues (e.g.
roots) or when an infection is present are required. However, it is not known how
precisely specific promoters need to be.
Target genes need to be identified and evaluated so that they can be combined in a
manner that confers robust protection.
Finally, it is not known how strongly expression of the gene in the laboratory will be
related to resistance in the field.
These technical issues will influence the time, resources and probability of success of
the events actually occurring. For example, time and resources will vary depending on
whether a short-term solution is assumed or one of a more durable nature incorporating
multiple sources of resistance.
There are also considerable commercial constraints to the development of transgenic or
disease-resistant bananas. One of these is the intellectual property issue. Viral transfer
constraints (banana streak virus) (Frison and Sharrock, 1998), and the need to test
against multiple strains of black Sigatoka will also reduce the rate of adoption of any
transgenic banana across the whole export industry.
174
In addition, there are legal issues that might retard progress in introducing a transgenic
banana into the field. These may be related to consumer concerns about genetically
engineered food crops. However, the area of genetically engineered food crops is
increasing every year and it is likely that concerns will dissipate with education and clear
labelling. The reduction in chemical use associated with the production of genetically
engineered bananas, if promoted, is likely to outweigh any negative perceptions.
If such a transgenic banana is not a Cavendish type, then there may be additional
difficulties of market development and a slower rate of adoption throughout the industry.
The greater the investment in biotechnology R&D, the broader and more robust may be
the resulting form of resistance. Pathogens can become resistant to gene products,
especially if the transgenic banana plants are used intensively and the genes bestowing
the resistance properties are not broad based in their action. Combining different forms
of resistance in one or more transgenic plants, combined with the use of some chemical
spraying, is likely to provide a more enduring strategy. In any case, the extent of
spraying is likely to be considerably reduced.
On the other hand resistance against Fusarium is likely to be more robust as the fungal
organism in this case has less chance of recombination due to its asexual form of
propagation.
While Sigatoka-resistant genetic material should result in significant chemical savings
and, in the case of production for domestic markets, significant quality improvements,
the technology is not likely to be made available outside the private interests that are
most likely to develop the clones in the first instance. If the development is through the
public sector there will still be the royalties that need to be paid to the owners of the
embedded technologies. This may result in significant costs for the resistant planting
material. Any increase in the cost of the planting material is still likely to be less than the
chemical costs saved, thus conferring a net benefit to the industry. Additional benefits to
society should result from the reduced impact of chemicals on the environment as well
as the reduced impact on human health.
The following provides a qualitative description of the type of long-term benefits that
might be attributed in part to the program. The benefits are classified as economic,
environmental, and social and health benefits.
4.3.1
Economic Benefits
The principal benefit most likely to be associated with such key events as specified
earlier is the reduced cost of production of export bananas. This might be achieved
through:
•
•
Reduced chemical spraying costs;
Reduced importation costs of chemicals in banana-exporting countries;
175
•
•
•
•
Higher yields (which can be translated into a cost reduction);
Higher quality fruit resulting in less wastage along the value chain (which can be
translated into a cost reduction);
Reduced other control costs, such as saved rouging costs in the case of bunchy,
top or lowered replanting and relocation costs in the case of Panama Disease;
The ability to reclaim land lost for banana production due to disease.
The economic benefits should accrue to both large plantations producing for export as
well as smallholder banana producers producing for local markets. Smallholders often
cannot afford chemical treatments so losses due to disease such as black Sigatoka can
be significant. With higher productivity, less land may be required to produce a given
level of banana production or more bananas can be produced, both of which will result
in improved profitability to individual producers, regions and countries.
The replacement of chemicals is required due to the ever-increasing costs of chemical
control. Some chemicals have been, or are being, banned from use. Resistance to
chemicals is increasing and new chemicals are needing to be developed which involves
more costs.
Other benefits can flow on from a more profitable banana export industry, as the sector
is very important in a number of countries in terms of its contributions to export income.
Improved consumer confidence and a higher level of food security in banana consuming
countries from less use of chemicals can also provide benefits to banana-producing and
exporting countries.
4.3.2
Environmental Benefits
The principal environmental benefits associated with the BIP are those resulting from
the use of lesser amounts of pesticides applied if resistant or more tolerant varieties of
export bananas are used or if a more efficient chemical regime is identified. The lower
the pesticide applications, the lower the potential damage to the environment.
The damage to the environment has not been verified or precisely quantified for the
whole of the banana export industry around the world, but there have been various
statements made in relation to specific countries that suggest that at least some
negative impact is occurring (Andreatta, 1997; Colburn 1997). In addition to ecosystem
damage, and the possible reduction in biodiversity, there is one instance of a potential
causal negative impact on another export industry through fungicide runoff into prawn
farming locations in Ecuador (Colburn, 1997). Short-term impacts on the environment
are serious but can be dealt with more easily than potential long-term impacts which are
uncertain and less focused and disparate. In both cases, there is difficulty in dealing
with these issues in terms of government policies in many banana-exporting countries
due to the conflict between economic development goals and environmental
sustainability.
176
The objectives of the BIP pertaining to developing disease-resistant types requiring less
chemicals takes on even more importance if the political and government policy
vehicles are incapable of resolving such issues.
Hence, benefits from even partial success in developing resistant banana varieties
should have a positive environmental impact through:
•
•
•
•
•
4.3.3
Reduced impact of chemicals on industries relying on water quality in rivers and
estuaries (e.g. fishing, prawn farming);
Reduced impact of chemicals on other agricultural industries;
Improved conservation of ecological systems in banana-producing countries with
associated amenity and aesthetic benefits;
Improved environment for tourism activities;
Lesser need for increased land areas for bananas so potentially conserving
native forests.
Social and Health Benefits
As with the environment, a range of statements have been made that fungicides and
nematicides used in banana production are associated with negative health impacts on
banana plantation workers and those living in banana-producing communities. Again
this is claimed by many to be due to the use of various chemicals, and particularly those
applied through spraying where overuse and misuse is reported as occurring. For
example, Swennen (1997) claims that people associated with banana production have a
10% higher chance of damage to their vital organs. Both Andreatta (1997) and Murray
(1994) suggest that chemical use in banana production is associated with human health
problems.
Some of the perceived benefits related to health that would arise from developing
disease-resistant bananas would be improved health on the part of banana industry
workers and higher labour productivity, as well as improved health and quality of life of
communities in banana-producing regions.
Other social improvements from increasing productivity in the banana industry are the
maintenance of, or increase in employment in banana-producing and exporting
countries.
4.4
Process and Structural Outcomes and Benefits
A number of process benefits have arisen from the BIP investment. As identified in
Sections 3.4 and 3.5, there has been leverage of additional resources into banana
research as well as improved co-ordination and collaboration between research
providers due to the BIP. Both these outputs should contribute to more effective
177
investment in banana improvement in the future and should result in lessening the time
that it might take to produce the disease-resistant types in the future.
4.5
A Framework for Analysis
The following framework has been provided as a guide to analysing the value for money
obtained by the Common Fund for Commodities (CFC) in its investment in the Banana
Improvement Project. Effecting an investment analysis for a program of what is really
strategic research is not a simple or easy undertaking. However, it is possible to effect a
quantitative analysis using some of the benefits that were described in Section 4.3.
Clearly there is a wide range of scenarios for the future. Assessing the impact of the
past BIP investment will require assumptions about the future and needs to take into
account the uncertainty concerning the success and timing of any scenarios postulated.
Potential gross benefits are those benefits that can be derived from the outcomes such
as the development of disease-resistant export bananas. Additional costs involved in
realisation of defined potential gross benefits need to be subtracted from the potential
gross benefits. For example, additional costs of planting material that might arise due to
intellectual property rights for transgenic material, or due to the additional value chain
costs of exporters changing to alternative export banana types, would need to be
subtracted from the production cost reductions that might occur due to resistant banana
types being introduced. Once additional costs incurred are subtracted, the remaining
benefits can be termed potential net benefits.
Potential net benefits need to be assessed relative to what would have been likely to
have happened to industry costs in the event of these positive outcomes not having
taken place at some stage in the future. It is reasonable to assume a ‘no intervention’
strategy where there is no R&D investment. In this case, assumptions would have to be
made about escalating costs of chemical control, banning of nematicides, or increasing
environmental impacts over time.
The term ‘potential net benefit’ is used to signify that the benefits may not necessarily
be realised. When multiplied by some estimate of the probability of the event actually
occurring then the resulting parameter may be termed ‘expected net benefits’. Expected
net benefits are those that might be realised by the industry and consumers of export
bananas.
The distribution of benefits to producers, exporters, retailers and consumers is not part
of the framework specified here. Such distributional aspects require a more
sophisticated framework and more detailed assumptions than can be made in the
current impact assessment.
Expected net benefits may not be fully realised if the technologies produced are not
applicable to all geographic regions or all export banana industries, all pathogen strains
178
etc. The actual benefits that might be ascribed in part to the BIP will depend on how the
technologies are actually adopted by industry. In other words, even though expected net
benefits to multinational companies might be positive, there may be other constraining
factors that might retard or negate the technologies being adopted by industry. Hence
the rate of adoption of any ensuing technologies needs to be taken into account.
A quantitative analysis using this framework is provided in Section 5.
5.
Investment analysis
5.1
Approach
In order to evaluate the past investment in the BIP in quantitative terms, it is necessary
to construct future scenarios that assume that the particular knowledge generated from
the BIP is used in further R&D efforts that are eventually successful with the resulting
technologies or products being adopted by industry. This requires a number of
assumptions to be made about the type of future investment and associated costs, the
time period before disease-resistant bananas will actually be produced, and the
likelihood that the technologies will be produced within the timeframe assumed.
A part of the benefits that might arise in the future can then be attributed to the BIP and
the remaining benefits to the non-BIP investment. Assessing the relative contributions is
difficult. One way is to ascertain the proportion of the BIP investment to the total
investment that will be required and attribute benefits to the BIP according to that
proportion. This attribution method was the principal method used in the investment
analysis described in this section.
Another way in which the likely returns of the BIP investment can be estimated is
through an assumption that the BIP ‘sped up’ the delivery of the final products that are
achieved. For example, it could be assumed that the development and
commercialisation of a disease-resistant export banana would have occurred in the year
2017, and that the additional investment and impetus provided by the BIP would allow it
to occur in 2012. An exercise using this method has also been used in the present
analysis and is hereafter known as the ‘earlier impact’ method. In this case it is
assumed that investment in R&D would have occurred without the BIP but that the BIP
contributed to the effectiveness of the mainstream R&D investment through speeding
up of the realisation of scenarios envisaged, that is, the benefits from the introduction of
the transgenic and alternative banana would have been received earlier.
The two key future scenarios specified earlier were:
1. The production of a transgenic Cavendish that is reasonably resistant to black
Sigatoka and nematodes;
179
2. The development of export banana types that are resistant to black Sigatoka and
nematodes. This is assumed to be achieved through a combination of
transformation and regeneration technologies together with breeding
components and has resulted in a robust form of resistance.
For the application of the attribution method, each of these scenarios is analysed in
terms of potential benefits and costs that might be expected ‘with’ and ‘without’ the R&D
program. In the case of benefits for the ‘with’ R&D situation, these are principally the
reduction (not necessarily elimination) in chemical costs and any reduction in yield
losses from improved disease control. These benefits may differ in each of the two
scenarios analysed.
In the case of costs for the ‘with’ R&D situation, these will include the costs of R&D to
develop the disease-resistant bananas. In the case of the necessity to move away from
Cavendish (Scenario 2), it will also be necessary to include the cost of producing and
exporting systems for changing to another banana type.
These benefits and costs for the ‘with’ R&D situation are then compared with the
‘without’ R&D situation where, while there are no R&D costs, there are assumed to be
future increases in chemical costs and yield losses.
In the case of the ‘earlier impact’ method, the costs of the BIP are placed against the
difference in benefits expected in two situations. The benefits in the ‘with BIP’ situation
were assumed to commence in 2012, but in the ‘without BIP’ scenario, the benefits
were assumed to commence later. It is the difference in the benefits between these two
situations that is assumed to be the benefit due to the BIP investment.
5.2
Assumptions
Assumptions used in the analysis using the attribution method are summarised in Table
5.1.
Table 5.1: Assumptions Used in Investment Analysis for BIP
Item
Without and R&D
Program
Annual banana exports (million t)
12.6
(a)
Banana yield (t/ha)
40
(b)
Export banana production costs ($/t)
169
(c)
Cost of fungicides ($/ha/annum)
1150
(d)
Cost of nematicides ($/ha/annum)
225
(d)
Yield loss due to black Sigatoka with efficient chemical 10.0
(d)
control (%)
Yield loss due to nematodes with efficient chemical control 17.5
(d)
(%)
180
Annual increase in cost of fungicide (% per annum)
Annual increase in cost of nematicide (% per annum)
Resource cost of BIP ($ million per annum) (1994-1998)
Other resources devoted to banana improvement worldwide (scientist years per annum) (1994-1998)
Other resources devoted to banana improvement worldwide (scientist years per annum) (1999-2012)
Resource cost of a scientist year ($ per annum)
Year in which transgenic disease-resistant Cavendish
commercialised and adopted by growers
Probability of development and commercialisation of
transgenic Cavendish (%)
Reduction in cost of fungicides used per ha after transgenic
Cavendish introduced (%)
Reduction in yield loss due to black Sigatoka after
transgenic Cavendish introduced (%)
Reduction in cost of nematicides after transgenic
Cavendish introduced (%)
Reduction in yield loss due to nematodes after transgenic
Cavendish introduced (%)
Maximum adoption level of transgenic Cavendish (%)
Year in which new disease-resistant banana type
commercialised and adopted by growers
Probability of development and commercialisation of new
disease-resistant banana type (%)
Reduction in cost of fungicides used per ha after new
banana type introduced (%)
Reduction in yield loss due to black Sigatoka after new
banana type introduced (%)
Reduction in cost of nematicides after new banana type
introduced (%)
Reduction in yield loss due to nematodes after new banana
type introduced (%)
Maximum adoption level of new banana type (%)
Cost to export banana industry of changing banana type ($
million)
181
1
(e)
1
(e)
With and R&D
Program
0.7
(f)
30
(g)
35
(e)
180 000 (h)
Outcome: Scenario
One (Transgenic
Cavendish)
2012
(I)
50
(I)
60
(I)
30
(I)
60
(I)
30
(I)
80
(I)
Outcome : Scenario
Two (Alternative
Banana Type)
2012
(i)
50
(i)
80
(i)
50
(i)
80
(i)
50
(i)
50
100
(i)
(j)
(a) CIRAD (1998)
(b) Estimated from CIRAD statistics for major exporting countries
(c) Estimated from figures supplied by Corbana, Costa Rica
(d) Mid-point of range supplied by Corbana, Costs Rica
(e) Estimate by Agtrans Research
(f) Estimate of annual investment by the BIP
(g) Estimate by Agtrans after discussions with CIRAD
(h) Estimate by Agtrans after discussions with Advisory Panel
(i) Estimate by Agtrans and Advisory Panel
(j) Estimate by Advisory Panel; this cost will depend on what level of existing banana
production will need to be ploughed out due to the changeover, the degree to which
the new banana type resembles Cavendish for post-production activities, and
whether, and to what degree, market research and promotion is required.
The cost of applying for and holding patents was assumed to be only 1-2 million dollars
and was not included in the analysis due to its relative insignificance in comparison with
other costs. However, the cost of royalty payments was considered potentially
significant. Such royalty payments could be considered part of the total benefits
estimated in the analysis. Assuming such royalty payments do not inhibit the adoption of
the technologies, the total benefits will remain the same, but may not accrue solely to
banana producers, exporters or banana consumers. No estimate has been made of the
size of any royalty payments or the distribution benefits between the banana export
industry, and research organisations or biotechnology companies. Further, there has
been no attempt to distribute the total benefits that might accrue between banana
producers and consumers.
The analysis was effected through a discounted cash flow analysis that ran for a period
of thirty-five years, commencing in the 1994 calendar year. All costs and benefits were
discounted back to 1994 using a discount rate of 7%. All values were expressed in 1998
US dollars.
Investment criteria estimated included the Net Present Value (NPV), the Benefit-Cost
Ratio (B/C Ratio), and the Internal Rate of Return (IRR). Criteria were estimated for
each of the two scenarios separately.
Sensitivity analyses were carried out to assess how investment criteria changed, with
changes in the value of a range of assumptions including adoption level, probability of
development and commercialisation, and the reduction in the fungicide and chemical
costs after the disease-resistant type is developed.
In the case of the ‘earlier impact’ approach, the basic assumption was that the BIP
caused the impact to occur two years earlier than without the BIP. However, a
sensitivity analysis was carried out on the length of this time period.
182
5.3
Attribution of Benefits to BIP
Attribution of benefits to the BIP are made on the basis of the present value of costs of
the BIP in relation to the present value of other funding available from 1994 to 1998 as
well as that required from 1998 onwards to achieve the outcomes specified. It is
assumed that 30 scientist years per annum were contributed outside of the BIP over the
period 1994 to 1998. In addition, it is assumed that a further 35 scientist years per
annum will need to be committed between 1998 and 2012 for the two scenarios
specified in the assumptions table to be achieved. Valued at $180,000 per scientist year
(full cost), this assumption means that the investment in the period 1998 to 2012 will be
of the order of $6.3 million per annum and will include both private and public sector
investments.
5.4
Results Using the Attribution Method
5.4.1
Results for Scenario 1 – The Transgenic Cavendish
The investment criteria estimated using the base assumptions for Scenario 1 are shown
in Table 5.2.
Table 5.2: Expected Investment Criteria for Scenario 1 (Transgenic Cavendish)
(Discount rate 7%)
Total Investment
BIP Investment only
NPV ($ million)
578
27.1
B/C Ratio
9:1
10:1
IRR (%)
21.4
21.9
The results show that, even with the conservative assumptions used, there is an
expected positive NPV from the BIP investment of $27 million, with a B/C Ratio of 10:1.
This is despite the time lag of 18 years assumed between the commencement of the
BIP and the introduction of a disease-resistant Cavendish banana. The $27.1 million
NPV for the BIP investment could be larger if a higher level of benefits from the
transgenic Cavendish is attributed to the BIP. The leverage of additional resources into
banana improvement as a result of the BIP has not been included in the current
attribution; in addition, the influence of the BIP on the degree of interest from the private
sector has been significant, but the implications of this influence have not been included
in the attribution of benefits made to the BIP.
The estimation of benefits is conservative in two ways. First, the actual parameters used
in the quantitative analysis could be considered to be conservative. Second, there are a
number of other benefits outlined in earlier sections that have not been included in the
quantitative analysis. Environmental and human health benefits are two such benefits,
which are usually difficult to value but are likely to be high and significant. Such benefits
could also provide justification for public investment in banana improvement since
183
benefits could be captured by communities as opposed to the private sector in bananaexporting countries.
Table 5.3 shows the sensitivity of investment criteria to small chemical cost increases
that are likely due to an increasing amount of chemicals applied to control banana
diseases as well as the increase in per unit costs of chemicals due to the need to
continually develop new products to overcome chemical resistance. While the influence
of the rate of increase of this cost is significant, it would have been even greater if not
for the lag between the BIP investment and the likely introduction of a disease-resistant
variety. Up until the year 2012, the cost increases are assumed to apply in both the
‘with’ and ‘without’ R&D situations so there is no benefit to the R&D until the year 2012.
It is only after the year 2012 that a difference is apparent.
Table 5.3: Sensitivity of Investment Criteria to Rate of Chemical Cost Increase
Rate of increase (%
pa)
1 (base)
2
3
5
10
NPV ($ million)
B/C Ratio
27.1
31.3
36.4
50.5
125.5
10:1
11:1
13:1
17:1
42:1
Table 5.4 shows that the investment criteria are highly sensitive to the probability of
success of the R&D. The base level probability assumed was 0.5. The break-even
probability (where the BIP investment would break even at a 7% discount rate) was
0.05, indicating that the chances of development and commercialisation do not have to
be very high in order for the investment to provide a worthwhile expected payoff.
Table 5.4: Sensitivity of Investment Criteria to Probability of Success
Probability of success by
2012
0.10
0.25
0.50 (base)
0.75
1.00
NPV ($ million)
B/C Ratio
3.0
12.0
27.1
42.2
57.3
2:1
5:1
10:1
15:1
20:1
Table 5.5 shows that the maximum level of adoption of the transgenic Cavendish needs
to be above about 8% before the investment will break even. Since the changeover
costs of adopting a disease-resistant Cavendish are likely to be minimal, as the
changeover could be phased in over a period, it is likely that the adoption level among
banana-exporting producers will be quite high. This is reflected in the base assumption
used, which was 80% adoption.
184
Table 5.5: Sensitivity of Investment Criteria to Level of Adoption
Level of
adoption (%)
10
25
50
80 (base)
100
NPV ($ million)
B/C Ratio
0.7
6.4
15.8
27.1
34.7
1.2:1
3:1
6:1
10:1
12:1
The level of reduction of chemicals due to the use of a transgenic Cavendish is difficult
to assess without further assumptions about the source of resistance and the
mechanisms involved in conferring such resistance. It is assumed that at least some
chemicals will still need to be used. The base level reduction was assumed to be 60%
of what the use would have been without the transgenic Cavendish. As illustrated in
Table 5.6, the investment criteria were very sensitive to changes in this assumption.
The NPV is still positive even if there is no reduction in chemical usage, due to the
assumption that yield losses will still be reduced.
Table 5.6: Sensitivity of Investment Criteria to Level of Reduced Chemical Usage
Reduction
usage
0
10
30
60 (base)
100
in
chemical NPV ($ million)
8.7
11.8
17.9
27.1
39.4
B/C Ratio
4:1
5:1
7:1
10:1
14:1
Investment criteria changed significantly when the assumption concerning the reduction
in yield loss due to the transgenic Cavendish was varied (Table 5.7).
Table 5.7: Sensitivity of Investment Criteria to Reduced Yield Losses
Reduction in level of yield
loss (%)
0
10
30 (base)
60
100
NPV ($ million)
B/C Ratio
15.3
19.4
27.1
37.8
50.5
6:1
7:1
10:1
13:1
17:1
185
5.4.2
Results for Scenario 2 – The Alternative Banana Type
The investment criteria estimated using the base assumptions for Scenario 2 are shown
in Table 5.8.
Table 5.8: Expected Investment Criteria for Scenario 2 (Alternative Banana Type)
(Discount rate 7%)
NPV ($ million)
Total Investment
484
BIP Investment only 22.8
B/C Ratio
8:1
8:1
IRR (%)
20.0
20.5
The results show that, again with the conservative assumptions used, there is a highly
positive expected rate of return from the BIP, with a B/C Ratio of 8:1. The main
difference between this future possibility and the transgenic Cavendish outcome is that
Scenario 2 assumes that there is a cost to the industry of changing the banana type
away from Cavendish. The magnitude of this cost is difficult to estimate as it will
depend, in part, on the similarity of the new export banana type to Cavendish.
Depending on the market development strategy and how quickly change is
accommodated, there may be some replanting costs (additional replanting costs to what
would have been the case with continuing with Cavendish). But handling, storage and
shipping facilities may also need to be altered and the associated costs could be
significant. While the base assumption is a cost of $100 million, this variable is subject
to a sensitivity analysis. Results are shown in Table 5.9. The break-even changeover
cost that could be sustained and still provides a 7% return on R&D investment was
estimated at $1.75 billion.
Table 5.9: Sensitivity of Investment Criteria to Changeover Cost
Changeover cost ($
million)
100 (base)
500
1000
2000
NPV ($ million)
B/C Ratio
22.8
17.2
10.3
-3.5
8:1
7:1
4:1
-0.14:1
The other two major differences between Scenarios 1 and 2 were:
•
•
The maximum adoption level was assumed to be lower for Scenario 2;
The reductions in chemical costs and yield losses were assumed to be higher in
Scenario 2.
Most other sensitivity analyses carried out for the new banana type scenario showed
similar trends to those for Scenario 1.
186
The analyses to date have assumed that either one or the other of Scenarios 1 and 2
could occur. Although it is possible that both Scenarios 1 and 2 could occur, to assess
the combined effect would require recasting the assumptions regarding adoption rates.
5.5
Results Using the Earlier Impact Method
5.5.1
Results for Scenario 1 – The Transgenic Cavendish
Results for the investment analysis using the ‘earlier impact’ method and assuming an
earlier impact of the BIP of two years (Scenario 1 occurring in 2012 instead of 2014) are
shown in Table 5.10.
Table 5.10: Expected Investment Criteria for Scenario 1 (Transgenic Cavendish)
(Discount rate 7%)
BIP Investment
NPV ($ million)
120
B/C Ratio
40:1
IRR (%)
33
A sensitivity analysis of investment criteria to the time period for the earlier impact is
shown in Table 5.11.
Table 5.11: Sensitivity Analysis of Investment Criteria to Time Impact of BIP
Difference in impact timing
(years)
1
2 (base)
3
5
5.5.1
NPV ($ million)
B/C Ratio
61
120
175
275
21:1
40:1
58:1
90:1
Results for Scenario 2 – The Alternative Banana Type
Results for the investment analysis for the ‘earlier impact” method for the alternative
banana type scenario are shown in Table 5.12. This result is based on an assumption of
a two-year speeding up of the realisation of benefits.
Table 5.12: Expected Investment Criteria for Scenario 2 (Alternative Banana Type)
(Discount rate 7%)
BIP Investment
NPV ($ million)
104
187
B/C Ratio
35:1
IRR (%)
31%
5.6
Conclusion
Given the assumptions made in the analysis, the results reported in this section suggest
that the BIP could be expected to provide a rate of return on funds invested of over
20%. Conservative assumptions have been used throughout the analyses, and the
sensitivity analyses and break-even parameters estimated show that the results are
robust across a credible range of assumptions.
The benefits estimated from this analysis are only a part of the total benefits that would
accrue to the investment in banana improvement and, in this regard, the results are
likely to be an underestimate of the performance of the investment.
The attribution method of analysis gave a lower expected rate of return than the ‘earlier
impact’ approach, even when the impact of the transgenic Cavendish or the
development of the alternative banana type was assumed only one year earlier (2012
instead of 2013) due to the BIP. This is not surprising since under the attribution
method, the BIP’s share of the benefits from the total investment was less than 5%,
whereas with the earlier impact method the full benefits of the total investment for two
years were credited to the BIP investment.
6.
Conclusion
The BIP has provided the methods for transformation and regeneration of bananas and
has moved the world banana industry closer to the reality of a transgenic banana.
Transformation and regeneration have now been accomplished. There has also been
significant progress demonstrated in terms of new germplasm for use in breeding
programs and methods for making breeding programs more effective in the future. The
most important output from the BIP was strategic knowledge, which can be used in
further R&D projects in the future.
Short-term and long-term benefits, and both content and process benefits, are likely to
be derived from the BIP. The long-term benefits include those related to reduced costs
of production, the environment and health. Some of the economic benefits were
quantified and used as input to a benefit-cost analysis of the BIP investment.
The results of the investment analyses demonstrate that the expected payoffs from
development of disease-resistant bananas can justify a large investment in banana
improvement research. Assumptions were generally conservative and only limited sets
of benefits were included in the analysis. Based on the various assumptions used, the
expected rate of return was estimated at 20% to 33%, the NPV at $ 22 to $ 120 million
and the B/C Ratio at 8:1 to 40:1.
188
References
Andreatta, S. L. (1997) ‘Bananas, Are They the Quintessential Health Food? A
Global/Local Perspective’, Human Organisation Vol. 56, No 4, pp. 437-449.
Buddenhagen (1996) ‘Banana Research Needs and Opportunities’. In Persley G. J. and
George P. (eds) ‘Banana Improvement Research Challenges and Opportunities’, World
Bank, USA, pp. 1-20.
CIRAD (1998) ‘Banana World Production and Trade Estimates’, FruiTrop, No 51, Oct,
CIRAD-FLHOR, France.
Colburn, F. D. (1997) ‘Shrimp or Bananas’, Journal of Business Research, 38, 97-103.
De Waele, D. (1995) ‘Musa Hematologists’ Consortium: A Step Towards Global
Collaboration’, INIBAP Annual Report, Montpellier, France.
Frison, E. A. and Sharrock, S. L. (eds) (1998) ‘Banana Streak Virus: A Unique Virus
Musa Interaction’, Proceedings of a Workshop of the ProMusa Virology Working Group,
held in Montpellier, France, INIBAP.
Murray, D. L. (1994) ‘Cultivating Crisis: The Human Cost of Pesticides in Latin America’,
University of Texas Press, Austin, USA.
Speijer and De Waele, D. (1997) ‘Screening of Musa Germplasm for Resistance and
Tolerance to Nematodes’, INIBAP Technical, IPGRI.
Swennen, R. (1997) ‘Breeding our Future’, Second National Banana Industry Congress,
Gold Coast, Australia.
189
ANNEX 1:
TERMS OF REFERENCE
To prepare an impact assessment statement on the results of the contracted research
undertaken by the BIP portfolio of projects on the future production techniques and
costs of the export banana industry.
In undertaking this objective, the consultant will:
•
•
•
•
•
Analyse and summarise program and project documentation;
Develop a framework for benefit-cost analysis for the program;
Visit project locations as appropriate for consultation with Principal Investigators
of individual projects;
Visit the World Bank Office in Washington DC to meet with the Project Coordinator to discuss the report and access further documentation and write the
initial report;
Interact with members of the BIP Scientific Advisory Panel.
190
ANNEX 2:
SUMMARY AND IMPACTS OF INDIVIDUAL PROJECTS
PN1: Field and laboratory evaluation of diploid bananas for their use in breeding
schemes
CIRAD-FLHOR, France
Introduction
This project focused on producing basic information on diploids in order to increase the
efficiency of breeding programs. The project characterised new genetic material as well
as material that was already used in breeding programs, but was not well characterised.
The additional information provided by this project can be used to select parents for
crossing that are more likely to provide improved clones with desirable characteristics.
The information assembled included information from both the field and laboratory. Field
data assembled included agronomic and fruit characteristics, morphological data and
disease resistance. With respect to disease resistance, material was tested for black
Sigatoka in New Caledonia, for Panama Disease in Australia, and for yellow Sigatoka in
St Lucia. Laboratory data included information on ploidy level and molecular markers.
Results
Information for about 150 clones has been assembled from the project. Assembly of
agronomic and morphological data and the information on ploidy levels and molecular
markers will be completed by the end of calendar 1998. However, the resistance
information will not be completed by that time. This is because there were delays in
obtaining and multiplying the material for the field trials. In addition, not all clones
arrived at the same time in some locations, and problems in growing plants from tubes
occurred in one location. These constraints led to only 150 clones being tested (original
intention was 200) and were the main reasons for the delayed production of results.
Overall these problems, as well as the necessity to index for viruses, demonstrated the
difficulties in establishing and implementing international network activities.
It is likely that all disease resistance information will be available by about June 1999.
The information will be provided to all banana breeders through the MGIS (Musa
Germplasm Information System) sponsored by INIBAP.
Impact
While the information is provided on diploids only, it can be useful for all breeding
programs, as diploid material has to be used in all programs including the classical
breeding approach. Nevertheless, the information is likely to be most useful in the
CIRAD and CRBP programs and will most certainly be used in such programs. There is
also some chance that the information will be used in the EMBRAPA, IITA and FHIA
programs. While additional information produced will refer in some instances to dessert
bananas, the evaluation showed that the new material was more related to cooking
191
bananas.
Where the information is used in the selection of diploid parent material for crossing, it is
likely that such use will increase the probability of producing improved clones from any
particular crossing. This is likely to increase the total amount of improved material
produced over a given time period, or alternatively, decrease the time to produce a
hybrid of a particular type that has been targeted.
It has been conservatively estimated by CIRAD that the use of the information could
result in ten times the number of improved dessert type bananas, compared with
breeding strategies that did not utilise the additional information. This may mean that for
any given set of resources applied to breeding it may take only one tenth of the time to
reach a specific target compared to non-use of the information. However, it should be
recognised that there are different philosophies in breeding programs and that the
information, while relevant to all breeding programs, is particularly targeted at the
CIRAD program.
A breeding cycle for bananas is approximately five years. What may be achieved in five
years with the information, therefore, may have taken up to 50 years without
information. There will also be a cumulative effect over time if further information is
assembled as it is produced from crosses made.
The value of the information produced from this BIP project will depend to a large extent
on the success of the CIRAD breeding program itself. While this success is not
guaranteed, there are signs that the program will be able to produce dessert bananas
that may be suitable for export. Again the issue is one of time and how significant
constraints on market development and adjustment along the value chain for alternative
Cavendish types can be overcome.
PN2 : Field crosses for understanding the inheritance of black leaf streak
resistance in bananas
CIRAD-FLHOR, France
Introduction
This project focused on understanding the basis of the resistance of banana types to
black Sigatoka. This entailed the testing of resistance in the field of crosses of specific
wild types of bananas for which resistance characteristics were already known, and
correlating resulting observations with molecular studies in the laboratory. The idea was
to use the resulting information to use molecular markers to quickly determine the likely
resistance levels of germplasm that might be used in future breeding strategies (marker
assisted selection).
192
Results
Due to environmental interactions in the field, it was difficult to accurately assess the
degree of resistance of crossed material to black Sigatoka. This required the
development of a leaf test (SLP test) that could be carried out under controlled
conditions. This test allowed the determination of the level of resistance for each cross
to be made. The necessity to develop this test within the project delayed the progress of
the project.
Molecular markers for highly resistant material will be in place by the end of calendar
1998. Highly resistant material appears to be controlled by a recessive gene so making
it difficult to introduce to new plant material through breeding programs.
The work on partial resistance will not be completed by the end of calendar 1998. The
completion of this work may take another 12 months, but should be completed outside
of the project by the end of calendar 1999. This is because finding molecular markers
for the set of genes involved will be quite difficult, but this information will be critical.
The major output from the project will be increased knowledge about the mechanisms
involved in resistance in bananas to black Sigatoka. This may allow a higher rate of
progress in producing black Sigatoka clones in breeding programs for both cooking and
dessert type bananas. Further, the knowledge may allow the identification of natural
resistance genes or gene segments that may be used in transformation processes.
Impact
Molecular markers are not yet available to screen for resistance, but should be available
by the end of the 1999 calendar year. There will probably be a low probability of being
able to confer total resistance to black Sigatoka through breeding programs; this may
not be desirable in the long term anyway due to the ease of the black Sigatoka agent in
overcoming resistance to a single gene. However, prospects for conferring partial
resistance appear bright if and when molecular markers that correlate with resistance
are identified. This outcome will therefore be time related.
Banana hybrids with resistance to black Sigatoka have already been bred outside the
BIP and are now in the validation phase. More hybrids will be selected soon and there
are currently 10 hybrids being tested. The BIP has had some part in the development of
these later bred hybrids.
Even with the development of genetically resistant bananas, spraying will still most likely
be necessary. However, one assumption is that spraying frequency for black Sigatoka
could be reduced by at least 50% and possibly more. It is likely also that partial
resistance will need to be retained through a continuous breeding program. In this
respect the molecular marker technology could be of significant value in future breeding
programs.
193
It is likely that a wide range of resistant material will be required over space and time,
due to the diversity of the fungus and its ability to recombine. Hence, the understanding
and identification of genetic resistance mechanisms will be a critical component of
future strategies to reduce the negative economic and environmental impact of existing
chemical control methods.
The BIP will not be able to claim any specific direct commercial impact with respect to
the development of resistant material. The contribution through the BIP supported
project has been in the main one of additional knowledge. However, it may be
appropriate to attribute up to 5% of the benefits that might eventuate from black
Sigatoka-resistant clones that could be produced and adopted by industry under the
CIRAD program. The appropriate level of attribution could be roughly equated to the
relative financial contribution of the BIP project to that of the total CIRAD breeding
program aimed at producing resistant types.
The benefits from the breeding program as a whole will depend on the program
producing export type bananas, the timing and cost of such activities, and the rate and
level of adoption of such types by industry.
Note: The figure for two BIP projects is US$ 450,000 for three years, compared with
CIRAD’s contribution over 16 years of 6 million francs per year or 96 million francs; this
is about US$ 20 million; relative contribution is about 2.25%.
PN3: Collection, characterisation and evaluation of Nendran bananas in India
Kerala Agricultural University, India
Introduction
The objective of this project was to collect and categorise genetic material on Nendran
bananas and their wild relatives in southern India. The idea was to establish reference
data for further research and to make the germplasm available world-wide for input into
breeding programs.
Results
A total of 128 accessions were collected and evaluated for dwarfism, short-cycling and
disease resistance, particularly for yellow Sigatoka and to lesser extent weevils and
nematodes.
Impact
There may be some local impact on Indian banana production system in terms of using
the more suitable genotypes for different farming systems. The evaluation may result in
the use of more short-stature bananas, which in turn may reduce the cost of propping
as well as increasing suitability to fit into annual rotation systems. Moreover, the
194
germplasm may be very useful in terms of expanding the range of germplasm available
to breeding programs around the world. However, it is not clear as to whether the
germplasm has yet been made available to the international germplasm collection.
PN4: Collection, evaluation and characterisation of genetic resources and
improvement of banana in Vietnam
Institute of Agricultural Genetics, Vietnam
Introduction
This project was aimed at the collection and evaluation of new germplasm for improved
productivity and disease resistance. Other components were also included in the
original objectives of the project, but the main emphasis was on germplasm collection.
Results
The collection of the germplasm was effected and much of the material has already
been evaluated and classified. Characterisation of morphological and agrobiological
traits of local cultivars has been completed, with a number of valuable clones selected
and recommended for production. The collections have been established in tissue
culture, but some accessions have exhibited difficulty in tissue culture multiplication,
which is delaying their availability to contribute to the genetic improvement of Musa.
Impact
The new material will eventually be available for use in breeding programs. As this
region, together with south China is near to the origin of the banana, it is likely that the
material does contain new genetic types. The material could provide an important
resource for future banana improvement.
PN5: Banana-breeding in Brazil
EMBRAPA, Brazil
Introduction
This project was associated with the Brazilian banana-breeding program and was aimed
at obtaining high yielding varieties resistant to the main pests and diseases in Brazil.
The project included the production and evaluation of 10,000 diploid hybrids and 1000
tetraploid hybrids. Although yellow Sigatoka is present in Brazil, black Sigatoka has only
just been detected in the Amazonas region; hence it is likely that this project will assist
the efforts to control the spread of the disease.
195
Results
Diploid hybrids are being produced and evaluated. Breeding techniques to utilise sterile
material are currently being developed. Molecular markers for germplasm
characterisation are being identified and used.
Impact
The project should allow improved breeding for resistance to black Sigatoka and other
pests and diseases in banana. Of more immediate impact is that two black Sigatokaresistant clones, to whose development and testing the BIP project contributed, are
being used to assist control of the black Sigatoka outbreak in the Amazonas. These
clones may slow down the rate of spread of the disease. This would have a major
reduction in the cost of those who can potentially afford to spray with fungicides as well
as reduce yield losses of those who cannot afford spraying control technologies.
PN8: Novel genes for fungal resistance and post-harvest quality
Boyce Thompson Institute for Plant Research (BTI), USA
Introduction
This project was a component of the molecular tool box project and was aimed at
identifying and characterising genes controlling ethylene metabolism.
Results
The project allowed an improved understanding of the molecular events that occur
during banana fruit development and ripening and those events that are associated with
pathogen challenge in fungal resistant and non-resistant banana varieties.
While some genes were successfully identified, there were still some problems in
successfully applying the Agrobacterium method in the transformation process. It is
understood that these issues have now been partly overcome as the group at the
University of Hawaii is currently using a modified version of the technique for gene
introduction after collaborating with the group at BTI.
Impact
The major impact of this project will be through the additional knowledge generated on
ethylene control and the further development of the Agrobacterium method of
transformation.
196
PN9: The development of transgenic bananas with resistance to Banana Bunchy
Top (BBTV) and Banana Mosaic (BBMV) viruses
Queensland University of Technology (QUT), Australia
Introduction
The aim of the project was to control BBTV and BBMV viruses through transformation
with resistance genes. These viral diseases are costly to control, largely through pulling
out infected plants. While this is effective in Australia, such control needs a strong legal
backing and responsive producers.
If bunchy top is not controlled, it can reduce yields by up to 100%. Losses may average
about 30%. Cavendish is one of the most susceptible clones to bunchy top and shows
serious symptoms. Some other banana clones are difficult to infect. Banana Bract
Mosaic virus can create losses in yield of up to 20-40%.
Results
The particle bombardment transformation process was used to successfully transform
both Bluggoe and Cavendish banana types with anti-viral genes. This has been
achieved for four different BBTV-potentially resistant genes and one gene for BBMV.
Regeneration was achieved but propagation of some of the regenerated cells proved
troublesome. It is expected that about 50% of the transgenic lines produced will be
resistant. This proportion is expected to be increased to 90% within a year as the
process is further refined.
Impact
The technology to generate potentially virus-resistant Cavendish plants, including
transformation, regeneration, and expression of the new genes that should confer
resistance, has now been demonstrated. Transgenic plants generated so far need to be
tested in the field to ensure that the expression of the gene does confer resistance. It
was estimated by the Principal Investigator that the chance of the resistance breaking
down will be only 5-10% within a period of 10 years from release. Field trials may take
some two years to complete and will face the potential constraint of biosafety
regulations.
The technologies developed will not only be relevant to viral resistant transgenics but
will also be relevant to the generation of transgenic resistance to other banana
diseases.
Without the BIP project, efforts would have continued in this area at QUT, but a different
approach would have been taken and the result would not have been achieved. Hence,
there is a direct relationship between the BIP and the achievement of these results.
197
PN10: Molecular tool box
KUL, Belgium
Introduction
PN10 consisted of two phases: a generic phase involving three different institutions
working in collaboration with one another; and a second phase where the project was
split up into two specific components, one associated with ethylene and one associated
with viruses. A project component targeting anti-fungal biotechnologies did not proceed
due to intellectual property issues.
The idea of the molecular toolbox was to improve the collaboration of a range of efforts
to produce transgenic bananas with commercial application. The idea was to produce
synergy so that efforts were focused and the rate of gain was increased.
Results
The first phase of the project, the development of a molecular toolbox, was successfully
completed and demonstrated effective co-operation among the three research providers
making up the consortium.
The project resulted in the refinement and optimisation of the transformation process
using the particle bombardment process. However, there is still some doubt about the
repeatability of the Agrobacterium process. Isolation of promoters has not been
completed, although some genes eliciting high expression have been defined. The
project could not work with the antifungal gene as it had already been patented by
Zeneca. The DNA library has been made available to other interested laboratories.
After the completion of the generic part of the project, the synergy exhibited earlier in
the project weakened.
Impact
Transformation was possible before the molecular toolbox project, but the project
allowed systems to be refined and additional parameters to be specified. In this regard,
the BIP accelerated the ability to transform banana. The BIP also helped to publish
some of the previous work from KUL and Texas A&M.
This has resulted in others using transformation technologies such as those at QUT.
BIP partners at QUT, Hawaii and Hong Kong supplied constructs to KUL, where they
were inserted into banana according to the refined protocol.
Due in part to the BIP, there has been increased confidence within the private sector in
the ability to capture the potential gains that biotechnology has to offer. It is now
understood that a number of the multinational banana-producing and exporting
companies are pursuing biotechnology solutions, mostly in conjunction with private
198
technology companies. It is likely that the BIP project has contributed to this increasing
interest on the part of the private sector. Most private sector groups are concentrating
on Cavendish transformation. If these initiatives are successful, the BIP could claim
maybe 5-10% of the total benefits through the indirect triggering of such private
investment. Alternatively, the BIP project could be attributed to bringing forward the
benefits of the transgenic bananas by some two to three years.
Any benefit that can be attributed to the BIP will rely on the actual production and use of
transgenic bananas in the future, whether produced by the private sector or through
other investment taking place via public research institutions.
PN11: Genetic engineering of ethylene biosynthesis in bananas
Hong Kong University of Science and Technology, Hong Kong
Introduction
This project set out to clone genes involved in ethylene biosynthesis in banana as well
as to genetically engineer bananas with reduced ethylene production capacity and
evaluate transformed plants in relation to reduced disease development and enhanced
fruit quality.
Results
The project achieved only the first objective of identifying and cloning two genes
involved in ethylene biosynthesis. The project constructed a banana genomic library
and identified several genes. However, the results were somewhat inconclusive and
much more work is needed to link the gene structure to their functions in disease
resistance and fruit ripening.
Impact
The project results produced knowledge of the genetic information associated with
ethylene biosynthesis. This may be useful in future research aimed at post-harvest
qualities of fruit as well as in disease control.
PN12: Use of biotechnology to produce transgenic bananas that are resistant to
BBTV infection
University of Hawaii, USA
Introduction
The project was aimed at developing further transformation technology using a modified
version of the Agrobacterium-mediated banana transformation system developed by
BTI at Cornell University. The project focused on BBTV gene constructs and their
199
incorporation into ‘Williams’ bananas.
Results
Much of the project was associated with the transformation technologies and, in that
regard, it appears that the Agrobacterium process was refined, although some problems
still remain. Three hundred transgenic plants were produced using pathogen-derived
genes and 65 of these were evaluated. This resulted in three of the 65 showing no
BBTV-specific symptoms and this was confirmed with ELISA testing. A further 21
transgenic plants showed two to three week delays in symptom development.
Impact
Field experiments still remain to be conducted to test the field performance of the
plants. In addition, the isolate used was that found in Hawaii and it is uncertain whether
the transgenic plants would also be resistant to the other BBTV virus group endemic in
other parts of Asia. However, the impact of these results could be highly significant in
that it has now been demonstrated that it is possible to produce viral resistant plants.
Further refinement of the process could result in considerable savings in losses from
bunchy top virus disease, which is present in Asia, the Pacific and in African countries.
PN14: Elimination of banana streak badnavirus (BSV) from improved Musa
germplasm and related studies on transmission and host plant/virus/vector
interactions
IITA, Nigeria
Introduction
BSV (banana streak virus) is present in many banana cultivars and in germplasm used
in breeding programs. The presence of the virus has serious implications for the safe
movement of germplasm around the world. This project was aimed at identifying Musa
tissue that was free of both genomic and episomal BSV to test the hypothesis that some
cells could be free of both BSV types. The project was also aimed at testing whether
BSV could be eliminated from germplasm, and how it is transmitted.
Results
Diagnostic techniques were developed and transmission mechanisms defined.
Transmission through seeds and suckers was confirmed as was propagation through
tissue culture. No evidence of spread via mealybugs or field spread of BSV was
confirmed. Elimination of BSV does not appear possible using conventional virus
elimination methods.
200
Impact
The project produced increased knowledge and understanding of BSV. This included its
etiology, epidemiology and possibility of control. Information relevant to the safe
movement of germplasm and appropriate quarantine activities was produced. The virus
remains a serious constraint to the movement of germplasm due to its incorporation into
the banana genome. BSV is now also increasingly recognised as a (field) production
constraint in certain banana growing areas.
PN15: Variability and relationships within populations of Fusarium oxysporum f
sp. cubense from its centre of origin
Queensland Department of Primary Industries (QDPI), Australia
Introduction
The essence of this project was an assessment of variability in the pathogen causing
Fusarium Wilt (Panama Disease) in bananas. Other aims of the project were to assess
the reliability of using plants derived from tissue culture as opposed to conventional
planting materials when assessing resistance of banana germplasm to the pathogen. A
third component was the assessment of breeding material against the Australian and
Asian populations of the pathogen.
There is no control mechanism for Panama disease. Infection means the planted area
has to be abandoned. The pathogen will remain in the soil for long periods.
Results
The QDPI now has a world-wide collection of over 2000 isolates of the pathogen, as
well as a DNA database for more than 500 isolates representing the 20 recorded VCGs
and the 33 known genotypes, and would be able to provide a service in the future for
typing specific pathogen types. Protocols have been drawn up for field evaluation of
host genotypes against pathogen strains. One banana type is resistant to tropical race 4
of the pathogen (strain 4 is the Asian pathogen to which Cavendish is extremely
susceptible). However, strain 4 is not yet in Central and Latin America or the Caribbean
areas.
The pathogen displays significant genetic variability and is the product of co-evolution in
Asia and has moved with the planting material around the world. Some data provides
evidence for the pathogen’s origin being outside the banana’s centre of origin.
Plants derived from tissue culture are more susceptible when compared to conventional
planting material. The idea of using this to advantage in attempting to increase
resistance with beneficial and bio-control organisms is being pursued.
The project has been screening material from the CIRAD program and from the
201
Brazilian and FHIA breeding programs for resistance against the Australian and Asian
strains of the pathogen.
Impact
The world-wide collection of the various strains of the pathogen will serve as a
reference collection in the future, enabling the strains causing an outbreak of Panama
disease to be quickly identified. The information on the genetic diversity of the pathogen
will also be useful in breeding programs to screen against a more complete profile of the
pathogen.
New banana developments can be more safely implemented through determining the
strains present in any given location before planting begins. For example, recent
outbreaks of Fusarium Wilt in Cavendish plantations in the eastern tropics (peninsular
Malaysia, Sumatra and Halmahera) caused enormous damage. These outbreaks have
ominous implications for the durability of western trades that are based on Cavendish
clones.
Protocols for the field evaluation of hybrids and diploid material for resistance against
Panama Disease have been developed and these should be useful in future breeding
programs.
Professional links with CIRAD, FHIA,EMBRAPA and other breeding programs around
the world have been developed and strengthened due to the BIP. The Australian group
at QDPI is collaborating with these other initiatives as a result of the program.
PN17: Origin and distribution of fungicide-resistant strains of M. fijiensis in
banana plantations in Costa Rica
CORBANA, Costa Rica
Introduction
This project was aimed at providing information that would be useful in developing
management strategies for chemical control of black Sigatoka disease. Part of the
project was associated with identifying inheritance of resistance in the progenies of the
black Sigatoka pathogen. The population dynamics of the pathogens that are resistant
to two major fungicides (benomyl and propiconazole) were studied and attempts made
to determine how many genes were involved in the resistance to fungicides.
Results
It was found that the acquisition of genes for resistance to chemicals did not have an
effect on the fitness of pathogen populations. It was found that resistance patterns
persisted even after fungicide use was stopped.
202
The project also provided a greater understanding of the parasitic fitness of resistant
strains (conidial sporulation, aggressiveness, and ability to cross) and on the ability of
the pathogen to carry double resistance (to benzimidazoles and triazones).
Impact
The absence of an effect of a chemical-free period is useful information in the
development of strategies to more effectively use chemicals and lessen the impact of
populations of pathogens resistant to fungicides. While this was a negative finding in
some respects, it has confirmed that strategies for improved use of chemicals for control
of the black Sigatoka pathogen are not easy to develop and that the continuing
development of new chemicals, together with its associated costs, is the principal
avenue by which some retention of chemical control can be managed.
PN18: Study of tolerance and resistance of banana to nematodes
CRBP, Cameroon
Introduction
The primary aim of the project was to identify sources of resistance of bananas from the
CRBP Musa germplasm collection at CRBP to major nematode species. Screening and
testing were carried out with trials in greenhouses, with young suckers, as well as in the
field. The greenhouse inoculation was artificial, but that in the field was natural.
A total of 142 accessions from different genotypes were screened including diploids,
triploids and hybrids from CIRAD, CRBP, and FHIA. The aims were:
(i)
(ii)
To identify types to use in the breeding program in CRBP;
To look for less susceptible plantains that can be recommended to farmers to
replace the susceptible ones
Results
It was found that many genotypes displayed resistance but not real immunity. As the
resistance was in different genotypes, it was suspected that many genes were involved.
It was likely therefore that a range of barriers could provide sources of resistance e.g.
penetration, biochemical, physiological etc. The suspected source of resistance is
polygenic and therefore, if built further into banana types, it would be less susceptible to
being overtaken by the organism. However, polygenic resistance would be less simple
to incorporate into future types through breeding.
It was also shown that the plantain clones are all susceptible to the major nematode
species, indicating that there is need to incorporate nematode resistance into fertile
plantains.
203
Impact
The most important impact will be through the selection of parents in the breeding
program in CRBP. It is likely that an increased rate of genetic gain with respect of
resistance could therefore be forthcoming. Further, increased knowledge on the
resistance of Musa to nematodes has been generated. Some of the resistant clones
also have resistance to other banana pests and pathogens. This will have implications
for the effectiveness of strategies to incorporate multiple resistance into banana types.
An early screening method using suckers was developed and validated. This method
will help to rapidly discard susceptible breeding lines in the selection process.
PN19: Identification of durable nematode-resistance sources in banana and
plantain
KUL, Belgium
Introduction
The project PN19 examined Musa germplasm for nematode resistance. The original
project proposal was submitted by FHIA, IITA, IRTA (Spain - Canary Islands) and KUL.
The project (PN19) was part of this original project concept with other projects also
being separately funded. A broad network of collaboration developed between several
BIP projects on nematodes as well as other projects outside BIP.
Results
PN19 confirmed that there are durable resistance types for nematodes (PJB types), and
gave validity of screening resistance at the pot level and across locations. An important
finding was that if sufficient time (eight weeks) was allowed for roots to have developed
sufficiently, then the test in the pots was reliable. The publication of the screening
protocol was a particular output from the BIP.
The project found that it was possible to use in vitro screening and thus save a few
months in time, as opposed to growing material out in plots. However, it was found that
contamination with bacteria was high and there was a need to improve the culture
preparations to minimise contamination. Hence the project is not quite completed.
Screening that might have taken eight to nine months can now be effected in three to
four months and would take less space so that more material can be evaluated for a
given set of resources in breeding programs. In this regard the project output will
increase the efficiency of future breeding programs.
The project also found new sources of resistance, mainly in genotypes from PNG.
These materials may not be very useful in breeding programs as they generally
displayed low productivity, were rather infertile, and were not of the Cavendish type.
However, the materials may be useful in understanding the mechanisms of resistance
204
to nematodes.
A major accomplishment of the project was the establishment of the Nematode
Consortium, which is continuing today.
Impact
The efficiency of future breeding programs will be increased through the findings and
protocol developed within this project and through PN20.
The IRTA initiative would not have started without the BIP, but some of the other
initiatives might have commenced in spite of BIP. Further, FHIA screening would not
have started without BIP. BIP also triggered a whole network of research through the
consortium, including leveraging further resources from VVOB (Flemish government)
into bananas.
The impact of the consortium will continue under the IMPT3. (The International Musa
Trial Program supported by INIBAP and UNDP). IMPT3 will benefit from the similar
testing of genotypes against nematodes on a world-wide basis. The original BIP nucleus
will be the mainstay of this testing program; without the BIP this network would have
had to start from scratch.
Before this project, in vitro methods were not available for testing for nematode
resistance. Both PN19 and PN20 have improved existing methods and resulted in a
better understanding of the reliability of the use of different methods of testing for
resistance (both in vitro and in vivo) and also improvement of nematode inoculum.
PN19 provided a high level of leverage for the introduction of other resources into
nematode-related research. It has been estimated that for every dollar put into the
nematode projects PN19 and PN20 by BIP a further $ 6.20 was invested by others,
including the Flemish and Belgian governments.
PN20: Identification of durable nematode-resistance sources in banana and
plantain
FHIA, Honduras
Introduction
PN20 was carried out in Honduras by FHIA. Screening was against two of the most
important nematodes, and both corms and tissue culture were used. Both parents and
hybrids were screened to enable breeders to follow what they were doing in their
crossing program. The FHIA evaluation initiative would not have happened without the
BIP. KUL personnel experienced with nematodes provided supervision for PN20. It has
been estimated that losses due to nematodes may average around 20%.
205
Results
The project produced systematic information on resistance and brought forward the
possibility of screening for resistance. Also some sources of resistance were found that
were not known to exist beforehand.
Impact
Before PN20, there had been no validation of resistance materialised in breeding
programs. The information from this project provided knowledge of resistance when
selecting parents for crossing within the breeding program. This would have the
potential of increasing the rate of genetic gain with respect to nematode-resistant
clones. Also new sources of resistance to nematodes were found, and this could lead to
more information on the mechanism for resistance, which could have implications for
biotechnological approaches. However, the nematode-resistant varieties of other crops
which are reported in the literature were developed by conventional breeding.
The PN20 findings are being used in the breeding program and the new material from
PNG is currently being prepared. However, there has been no information provided on
genetic mechanisms for resistance.
It will probably be necessary to keep breeding for resistance. There is not only a range
of nematode types, but some of the less aggressive types may become more
aggressive when the more dominant ones are controlled. Such 'new' dominants may not
be resistant to the banana type produced for resistance to the previous dominant strain.
This possibility indicates a need to breed for multiple resistance, that is, against a range
of nematode types. Little is known about how robust any resistance mechanisms to the
existing principal nematodes might be in this regard.
Nematicides may not have to be used once resistance is built into the genome. There is
also a possibility that this research will lead to the identification of resistant genes,
although due to the mechanism of feeding of the nematode, any target toxin will have to
be tested via an engineered living plant cell.
The work effected under PN19 and PN20 may have shortened the time until a
nematode-resistant banana type is available. This would apply to export bananas that
would still need to be developed and commercialised. Cavendish is highly susceptible to
the major nematodes and a biotechnology solution may need to be sought in the case
of providing a nematode-resistant Cavendish. However, if a nematode-resistant
alternative to Cavendish is targeted instead, then the FHIA breeding program which has
developed agronomically-advanced, nematode-resistant diploid parental lines which are
readily usable in cross pollinations for breeding resistant, commercial type hybrids for
export, may well be successful. The FHIA-23 ‘Gros Michel’ type hybrid is currently being
cultivated on about 2000 hectares in Cuba. It is replacing Cavendish as rapidly as
planting material of the hybrid can be multiplied.
206
The evidence that conventional breeding works in breeding nematode-resistant
commercial hybrids has been confirmed in the FHIA-19 apple-flavoured hybrid. FHIA-18
was derived from crossing the burrowing nematode-resistant SH-3142 diploid onto the
Dwarf Prata triploid, and this resultant nematode-resistant tetraploid is now being
cultivated on about 900 hectares in Cuba.
PN21: Identification of durable nematode-resistance sources in banana and
plantain
IRTA, Spain
Introduction
This project was a one-year project aimed at evaluating Musa genotypes for durable
resistance against the main nematode pests that affect banana production. This was
effected by establishing a live collection of migratory nematodes and initiating screening
trials.
Results
The live collection was established and the material prepared for resistance evaluation.
Screening trials were initiated and were continuing at the time of the termination report.
Impact
The germplasm screening for resistance will enable more effective breeding programs
to be initiated through selection of resistant material. The development of more resistant
material should reduce costs of nematode control and lower the use of nematicides.
PN22: Breeding hybrid Musaceae with resistance to multiple diseases, especially
black Sigatoka and Panama disease
FHIA, Honduras
Introduction
The objective of the project is to produce commercial-type dessert bananas with a short
stature and which are resistant to both black Sigatoka and Panama Disease. Already
some success has been obtained with resistant types but the plant has been too tall for
ready acceptance by commercial producers, except in Cuba where it is reported that the
area of the resistant type is increasing.
207
Results
Cross pollinations have been made with improved diploids crossed onto seed fertile
triploids. The first tetraploid hybrids produced under this strategy have not yet been
evaluated.
Impact
If varieties with appropriate stature and disease resistance are produced, this could
have a major impact on the banana export industry. This positive impact will only occur
if the resulting hybrids are seedless, productive, exhibit desirable bunch features, and
maintain disease resistance over time.
The black Sigatoka-tolerant tetraploid (FHIA-23) being cultivated in Cuba illustrates that
conventional breeding can work for development of disease-resistant alternatives to
Cavendish.
However, as the sources of resistance have not been identified through molecular
studies, the nature of resistance conferred will not be understood. This would be
relevant if biotechnical solutions are to be pursed as well as conventional breeding
solutions.
208
Download