RESEARCH PROPOSAL UNDER PARB CGS SYSTEM
PROJECT ID NO. 144
1.
PROJECT TITLE
Transgenic approach to improve drought and
salinity tolerance in wheat
2.
PARB THEME UNDER WHICH
THIS PROJECT FALLS
Theme-1: Enhancing Productivity on
Sustainable Basis of Major Farming
Systems
3.
PARB SUB-THEME UNDER
WHICH THIS PROJECT FALLS
Sub-theme 1.1: Rice-wheat System
4.
PARB PROJECT GROUP FOR
WHICH THIS PROJECT
MATCH
Project groups 1.1.1:Improve salt tolerance in
rice and wheat
5.
OBJECTIVE OF THE
PROJECT(Mission statement)
Development of transgenic wheat with
improved resistance against drought and
salinity.
6.
ORGANIZATION SUBMITTING THE PROJECT
a. Name of the Host Organization:
University of Agriculture, Faisalabad
b. Host Institute:
Centre of Agricultural Biochemistry and
Biotechnology (CABB)
c. Administrative Contacts
i. Head of the Host Organization (VC/DG/DIRECTOR/etc.)
Name:
Title:
Telephone:
Email:
Professor Dr. Iqrar Ahmad Khan
Vice Chancellor
041-9200200
vc@uaf.pk
Signature with date and seal
2
ii. Head of the Host Institute (Director/Chairman/Division Head etc.)
Name
Professor Dr. Iqrar Ahmad Khan
Title:
Director, CABB
Telephone 041-9201087: 041-9200161-70Ext 2925
Email:
vc@uaf.pk
Signature with date and seal
7.
COLLABORATING ORGANIZATION (S) NIL
8.
PROJECT MANAGER
Name: Dr. Nisar Ahmed
Title:
Assistant Professor
Institute: Centre of Agricultural Biochemistry and Biotechnology (CABB),
University of Agriculture, Faisalabad.
Qualification and Relevant Experience: Ph.D.(CV attached)
Telephone:
041-9201087: 041-9200161-70 Ext 2925
Mobile:
0300-6652334
E mail:
drmiannisar@yahoo.com
Signature with date and seal
9.
COLABBORATING SCIENTIST(S)
1- Professor David A. Lightfoot
The Department of Plant, Soil and Agricultural Systems,
Southern Illinois University,
Carbondale, IL, 62901, USA
e-mail: GA4082@SIU.EDU
2- Professor Masahiko Maekawa
Research Institute for Bioresources,
Okayama University, Chuo 2-20-1, Kurashiki, 710-0046, JAPAN;
Tel: 081-86-434-1214; Fax: 081-86-434-1249
Email: mmaekawa@rib.okayama-u.ac.jp
3
10. Approved By
Name:
Dr. Mubarik Ali
Designation: Chief Executive
Approval date of PARB Board of Governors: 14.4.09
Signature with date and seal
11. PROJECT DURATION :
36 months
12. DATE OF COMMENCEMENT
15.5.2009
13. TOTAL PROJECT COST
Rs: 14.078 million
14. LOCATION OF THE PROJECT:
Centre of Agricultural Biochemistry and Biotechnology (CABB), University of
Agriculture, Faisalabad
15. BACKGROUND INFORMATION
i.
Problem to be addressed
Wheat is the most widely grown food crop, ranks first in world crop production and is
the national staple food of many countries. At least one-third of the world's population
depends on wheat as its main food. In Pakistan, wheat being the staple diet is the most
important crop and cultivated on the largest acreages in almost every part of the
country. Drought and high salinity are the most common environmental stress factors
that influence wheat growth and development and place major limits on plant
productivity in cultivated areas of Pakistan. Water availability for crops is decreasing
and demand is increasing every year due to environmental changes. Salinity is also
increasing day by day due to mismanagement and high costs of reclamation. Population
of the country is increasing at an alarming rate. Increase in area under wheat is not
possible. Research inputs for developing stress tolerant varieties through conventional
4
breeding techniques have only been modest and the situation now calls for concerted
efforts to develop technology for better production under drought and saline conditions.
This critical situation demands for increase in wheat production under existing
conditions. The responses of wheat to various abiotic stresses have been important
subjects of physiological studies and more recently, of molecular and transgenic
studies. The identification of novel genes, determination of their expression patterns in
response to the stresses, and an improved understanding of their functions in stress
adaptation have provided us the basis of an effective engineering strategies to improve
stress tolerance in crops. Salinity and drought resistant genes have been isolated and
transformed/ over expressed successfully to develop transgenic plants with improved
resistance against abiotic stress. Therefore, the aim of this proposed study is to isolate
and transform genes linked to drought and salinity in wheat to improve resistance
against these stresses.
ii. Relevance of the Project to the problem to be addressed
Drought and salinity are by far the leading environmental stresses in agriculture both in
the Pakistan and worldwide. Drought and salinity limits the agricultural production by
preventing the crop plants from expressing their full genetic potential. Understanding
the genetic basis of drought and salt resistance in wheat is fundamental to enable
breeders and molecular biologists to develop new varieties with more drought and salt
resistance characters. Drought and Salinity are complex traits controlled by multi-gene
families. Conventional breeding efforts were successful to some extent to develop
resistant cultivars but the problem was not fully solved because of its complex nature.
New developments in plant molecular biology provide us the opportunity to investigate
the mechanisms of drought and salinity resistance in a more holistic way than in the
past. Many genes have been demonstrated to respond to drought and high salt levels,
and the proteins encoded by these genes are thought to function in protecting cells from
these stresses. Plant productivity is greatly affected by environmental stresses such as
drought and salt loading. Previous findings on these stresses in rice, Arabidopsis, and
maize suggested that some cis -acting promoter elements, the dehydration response
5
element (DRE), plays an important role in regulating gene expression in response to
these stresses. Some transcription factors specifically interact with the DRE and induce
expression of stress tolerance genes. Further over- expression of the DRE in transgenic
rice plants activated the expression of many of these stress tolerance genes under
normal growing conditions and resulted in improved tolerance to drought and salt
loading and freezing tolerance. Transactivation of gdhA (glutamate dehydrogenase)
gene of E. coli has shown improved resistance against drought in maize.
In the proposed study, our plan is the transient expression of these specific transcription
factors and DRE isolated from rice and gdhA from E. coli in wheat to develop a
transgenic wheat plants resistant to drought and salt stress. Simultaneously isolation and
cloning of these elements from wheat will be conducted. Research results from wheat
can easily be used to explore particular drought and salt resistance mechanisms in other
cereals because of the high level of synteny and colinearity.
iii. Literature review preferably for the last 5 years.
Wheat (Triticum aestivum L.) is the staple food for a large part of the world population.
In Pakistan, it is grown on 8.141 million hectares with an average yield of 2.28 tones/ha
with total production of 18535 thousand tones (Economic Survey of Pakistan, 2003 Pl.
give latest reference and figures). The wheat share in cereal crops is 65.3 and 72.1 % in
terms of area and production in Pakistan (Anonymous 2004). About 1/3 area under
wheat is devoid of any supplemental irrigation. Drought and high salinity are two of the
most important environmental stresses that alter plant water status and severely limit
plant growth and development, and thus reduce crop productivity (Rabbani et al.,
2003). Drought is a complex scenario with three main components, (i) timing of
occurrence during the season, (ii) duration, and (iii) intensity. These factors vary so
widely in nature that it is very difficult to define specific plant attributes required for
crop improvement under drought stress conditions. Dehydration causes a number of
physiological and biochemical changes in plants, such as a decrease in photochemical
activities, reduction of CO2 fixation, accumulation of osmolytes and osmoprotectants,
and alteration in carbohydrate metabolism (Tabaeizadeh, 1998). Alongwith drought soil
salinity is another constraint of low yields. Despite the advanced technologies available
6
today, salinization of millions of hectares of land continues to reduce crop productivity
severely worldwide. Historically, soil salinity contributed to the decline of several
ancient civilizations. Also, high salinity (e.g., increased concentrations of Na+ and Cl¯
in the soil solution) causes osmotic/ionic stress (Hasegawa et al., 2000).
Out of 20.2 million hectares of cultivated land in Pakistan, 6.8 million hectares are
affected with salinity where as almost 15 million hectares of cultivated land is directly
or indirectly affected by drought. The availability of water in the country has been
adversely affected by 40 percent less than normal winter rains and up to 25 percent less
snowfall since 1998 and has been further compounded, particularly in the rain-fed
areas, by the continued dry spell. Moreover, almost 70% of the groundwater available
contains moderate to high concentration of salts. According to reports, Pakistan’s need
for water is increasing at the rate of 3% per year while the supply of water is decreasing
at the rate of 1% per year (Anonymous 2004). Increase in population by 1.8%, decrease
in water resources and increase in salinity is an alarming as it has created a situation of
crises in the country. The situation calls for increased production of wheat. The
production of wheat can be increased by reclamation, drainage and water management
that can minimize the extent and spread of drought and salinity, however engineering
and management costs are high. Another way is by bringing more area under
cultivation or by increasing per hectare yield. Currently, it is nearly impossible to
increase area under wheat crop due to other competing crops, restricted supply of
irrigation water etc. Therefore, the only alternative left is to increase it’s per hectare
yield by better crop management techniques and introducing high yielding varieties
along with resistance against biotic and abiotic stresses. Therefore, new strategies to
cope with salinity and drought problems are essential. It is possible to improve the
genetic tolerance of wheat crop to salinity and drought and thereby increase the
productivity of marginal lands. Efforts to breed for drought and salinity tolerance are
slow due to limited knowledge of genetics of tolerance, inadequate screening
techniques, low selection efficiency and poor understanding of these stresses and
environmental interaction. Molecular marker technology can be used to some extent to
develop new varieties with improved traits. However, the reasonable way at this stage is the
development of transgenic wheat with improved resistance against salinity and drought.
The precise
7
mechanism(s) by which plants respond to drought or high salinity remains unresolved.
However, at the molecular level, most of the changes are likely the result of alterations
in the expression of genes. Expression of a variety of genes has been demonstrated to
be induced by these stresses in a variety of plants (Ingram and Bartels, 1996; Shinozaki
and Yamaguchi-Shinozaki, 1997, 2000; Thomashow, 1999). Therefore, it is important
to identify the relevant genes and characterize their regulation in response to water
and/or salinity stress. A little progress has been made in characterizing the genetic
determinants of drought and salt resistance, because it is a complex phenomenon
comprising a number of physio-biochemical processes at both cellular and organismic
levels at different stages of plant development (Tripathy et al. 2000). The physiologic
response to drought and salinity arises out of changes in cellular gene expression. Expression of a number
of genes has been demonstrated to be induced by these stresses.
Recently, a drought, salinity and
cold responsive cis-acting element (Yamaguchi- Shinozaki and Shinozaki, 1994);
number of drought-responsive (Ingram and Bartels, 1996; Kim et al., 2000;
Nepomuceno et al., 2000; Lightfoot et al., 2007) and salinity-responsive (Moons et al.,
1997; Ramani and Apte, 1997; Wei et al., 2000) genes were cloned and characterized
from different plant species. Transcription of many of these genes (e.g., those encoding
the late-embryogenesis-abundant, LEA proteins, (Espelund et al., 1995; rd29A and
rd29B, Yamaguchi- Shinozaki and Shinozaki, 1993; OsDREB1A and OsDREB2A
(Dubouzet et al., 2003) glyceraldehyde-3-phosphate dehydrogenase, Jeong et al., 2000;
and ABA responsive element binding proteins AREB1 and AREB2, Uno et al., 2000; is
up-regulated by both drought and salinity stress. Among these, over expression of rice
OsDREB1A and OsDREB2A has resulted in increased resistance against salinity,
drought and freezing tolerance and E.coli gdhA genes has shown improved drought
resistance in maize. The object of this study is the developments of transgenic wheat
with improved resistance against drought and salinity by the transient expression of rice
OsDREB1A and OsDREB2A and E.coli gdhA genes. Isolation and cloning of these
elements from wheat will be conducted to simultaneously to determine the difference
between transient expression and over-expression.
References
Anonymous, (2002) Agricultural Statistics of Pakistan. Government of Pakistan,
8
Ministry of Food, Agriculture and Live Stock, Economic Wing, Islamabad, 83-84.
Ameziane R, Bernhard K, Lightfoot DA (2000) Expression of the Escherichia coli
glutamate dehydrogenase gene in tobacco a Evicts plant growth and development. Plant
and Soil. 221: 45–57.
Bajaj S, Targolli J, Liu LF, Ho THD, Wu R (1999) Transgenic approaches to
increase dehydration-stress tolerance in plants. Mol Breed. 5: 493–503.
Bray EA (1997) Plant responses to water deficit. Trends Plant Sci. 2: 48–54.
Cushman JC, Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr
Opin Pl Biol. 3: 117–124.
Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M,
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Trehalose accumulation in rice plants confers high tolerance levels to different abiotic
stresses. Proc Natl Acad Sci USA 99:15898–15903.
Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and
molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51: 463–
499.
Hu, T. et al., (2003) Agrobacterium-mediated large-scale transformation of wheat
(Triticum aestivum L.) using glyphosate selection. Plant Cell Rep. 21: 1010–1019.
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Annu Rev Plant Physiol Plant Mol Biol 47: 377–403.
Jeong MJ, Park SC, Kwon HB, Byun MO (2000) Isolation and characterization of
the gene encoding glyceraldehyde-3-phosphate dehydrogenase. Biochem. Biophys. Res.
Commun. 278:192–196.
Kim JY, Mahe A, Brangeon J, Prioul JL (2000) A maize vacuolar invertase, IV2, is
induced by water stress. Organ/tissue specificity and diurnal modulation of expression.
Plant Physiol. 24:71–84.
Levitt J (1980) Responses of Plants to Environmental Stress, Ed 2. Academic Press,
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Lightfoot DA, Mungur R, Ameziane R. etc (2007) Improved drought tolerance of
transgenic Zea mays plants that express the glutamate dehydrogenase gene (gdhA) of E.
coli. Euphytica 156:103–116.
Liu, Q. et al. (1998) Two transcription factors, DREB1 and DREB2, with an
EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways
in drought- and low temperature-responsive gene expression, respectively, in
Arabidopsis. Plant Cell 10:1391-1406.
Moons A, De Keyser A, Van Montagu M (1997) A group 3 LEA cDNA of rice,
responsive to abscisic acid, but not to jasmonic acid, shows variety-specific differences
in salt stress response. Gene 191: 197-204.
9
Nepomuceno AL, Stewart JMCD, Oosterhuis D, Turley R, Neumaier N, Farias
JRB (2000) Isolation of a cotton NADP (H) oxidase homologue induced by drought
stress. Pesqui. Agropecu. Bras. 35:1407–1416.
Oliveira IC, Brears T, Knight TJ, Clark A, Coruzzi GM (2002) Over expression of
cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration. Plant
Physiol 129:1170–1180.
Patnaik D, Dalia V and Paramjit K (2006) Agrobacterium-mediated transformation
of mature embryos of Triticum aestivum and Triticum durum Current Science, 91: 307317.
Rabbani, M. A. et al., (2003) Monitoring expression profiles of rice genes under cold,
drought, and high-salinity stresses and abscisic acid application using cDNA microarray
and RNA gel-blot analyses. Plant Physiol., 133: 1755–1767.
Ramani S, Apte SK (1997) Transient expression of multiple genes in salinity-stress
young seedlings of rice (Oryza sativa L.) cv Bura Rata Bura Rata. Biochem. Biophys.
Res. Commun. 233:663–667.
Shinozaki, K. & Yamaguchi-Shinozaki, K. (1997) Gene expression and signal
transduction in water-stress response. Plant Physiol. 115: 327-334.
Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration
and low temperature: differences and cross-talk between two stress signaling pathways.
Curr Opin Plant Biol 3: 217-223.
Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encode transcription
activators that function in drought-, high-salt- and cold-responsive gene expression
Plant J 33: 751–763.
Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene
expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410- 417.
Sun H, Huang QM, Su J (2005) Highly eVective expression of glutamine synthetase
genes GS1 and GS2 in transgenic rice plants increases nitrogen-deWciency tolerance. J
Plant Physiol Molec Biol 31:492–498.
Tabaeizadeh, Z (1998) Drought-induced responses in plant cells. Int. Rev. Cytol.
182:193–247.
Thomashow, M.F. (1994) Arabidopsis. (Eds Meyrowitz, E. & Somerville, C.) 807-834
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Tripathy JN, Zhang J, Robin S, Nguyen HT (2000) QTLs for cell-membrane
stability mapped in rice (Oryza sativa L.) under drought stress. Theor. Appl. Genet.
100: 1197–1202.
Uno T, Furihata T, Abe H, Yoshida R, Shinozaki K (2000) Arabidopsis basic
leucine zipper transcription factors involved in an abscisic acid-independent signal
transduction under drought and high salinity conditions. Proc. Natl. Acad. Sci. USA
97:11632–11637.
Wei JZ, Tirajoh A, Effendy J, Plant A (2000) Characterization of salt-induced
10
changes in gene expression in tomato (Lycopersicon esculentum) roots and the role
played by abscisic acid. Plant Sci. 159:135–148.
Xu D, Duan X, Wang B, Ho T, Wu R (1996) Expression of a late embryogenesis
abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt
stress in transgenic rice. Plant Physiol 110: 249–257.
Yamaguchi-Shinozaki, K. & Shinozaki, K (1993) Characterization of the expression
of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its
promoter in transgenic plants. Mol. Gen. Genet. 236: 331-340.
Yamaguchi-Shinozaki, K. & Shinozaki, K. (1994) A novel cis-acting element in an
Arabidopsis gene is involved in responsiveness to drought, low-temperature, or highsalt stress. Plant Cell 6: 251-264.
Zhang JX, Klueva NY, Wang Z, Wu R, Ho TH, Nguyen HT, Ho THD (2000)
Genetic engineering for abiotic stress resistance in crop plants. In Vitro Cell Dev Biol
Plant 36: 108-114.
Zhu JK (2001) Cell signalling under salt, water and cold stresses. Curr Opin Plant Biol
4:401–406.
Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant
Biol 53:247–273.
iv. Achievements and related research in hand, if any
We have arranged for drought resistant gdhA gene and efforts are under way to amplify
salt resistant genes from rice. Professor Masahiko Maekawa of Okayama University
Japan will help for the amplification of desired genes and construct making.
Professor D. A. Lightfoot has recently (2007) published his work on drought tolerance
in maize. He and his co-workers have successfully developed transgenic maize plant
with improved resistance against drought by the transactivation of E.coli gdhA gene in
maize.
Professor D. A. Lightfoot will provide the gdhA gene construct as well as the technical
know-how about their transactivation. His collaboration will be vital for the successful
completion of the project. We will work together to know how the transactivation of
gdhA works. We will also try to investigate in the use of transacting factors to analyse
plant resistance further.
Professor Masahiko Maekawa will provide help and technical assistance for making of
OsDREB1A construct in his lab. He will also provide necessary help that will be
11
needed at various occasions during the execution of the project especially in gene
amplification, developing constructs, compilation, analysis and publication of data.
15. PROJECT PLAN
a. Scientific/technical methodology (give details):
Seed collection, germination, gene amplification and construct making: Seeds of
rice cultivar Nipponbare will be provided by Professor Masahiko Maekawa of
Okayama University, Japan. Seeds will be grown under aseptic conditions to isolate
DNA. For the preparation of cDNA, rice seeds will be germinated at 25°C in the dark
and 15-day-old etiolated seedlings will be treated with 250 mM NaCl for 12 h to
activate the salinity tolerant genes.
Gene for salinity tolerance OsDREB1A will be amplified by PCR from rice by
designing primers from the already reported sequences of this gene. Race will be
conducted to amplify the ends of respective gene. Full length cDNA will be amplified
and ORF will be used to develop construct in expression vector. If we will face any
problem in the amplification of this gene, then we will commercially purchase full
length cDNA of this gene from Japan and design construct. All the above activity will
be carried in the lab of Professor Masahiko Maekawa, Okayama University Japan. He
has all the facilities in his lab and we will be able to design construct in a short time.
Gene cassette for drought resistance gene gdhA will be provided by Professor David A.
Lighthoof, The Department of Plant, Soil and Agricultural Systems, Southern Illinois
University, Carbondale, IL, 62901, USA. Gene construct will be amplified through
transforming in competent cell.
Collection of wheat gene pool and growth conditions:
Seeds of commercial wheat cultivars, Faisalabad 2008 and Chakwal 50 (recently
approved for commercial cultivation. Faisalabad 2008 will be for normal soils and
Chakwal 50 will be for rain fed areas) will be collected from Ayub Agricultural
Research Institute, Faisalabad and Barani Research Institute Chakwal respectively.
Seeds will be surface sterilized and grown under controlled environment at 22/14°C
12
(day/night) under a 13/11 h (light/dark) photoperiod with 50–70 % relative air
humidity. Immature caryopses12-14 days post-anthesis will be removed from the
spikelets, surface-sterilized first with 70% ethanol for 1 min, followed by 3% sodium
hypochlorite solution for 20 min and then rinse five times with sterilized distilled
water. Immature embryos will be isolated under aseptic conditions and embryos 0.8
mm to 2.0 mm in length will be used.
For mature embryos seeds will be surface-sterilized and embryos will be excised in a
laminar flow hood by removing the endosperm part from the caryopses with a sterile
blade.
Transformation, selection and regeneration of transformants:
The gene constructs gdhA and OsDREB1A will be transformed through agrobacterium
mediated transformation as previously described by Patnaik et al (2006) and Hu, T. et
al., (2003) for mature and immature embryos respectively. Transformation, selection
and regeneration of transformants will be conducted by the protocol as described by
Patnaik et al (2006).
Screening of resistant transgenics: Transgenic plants will be exposed to salinity (12
EC for salinity and SAR30 % for sodicity) and drought stress; as already reported by
Dubouzet et al (2003). Transgenics will be subjected to drought stress at 60-70 % field
capacity and non-transformed plants of respective cultivars will be used as control.
Transcripts of transformed genes will be determined through RT-PCR.
Field trials: Seeds of the transgenic plants showing improved resistance against
salinity (above 12 EC and SAR 30 %) and drought will be multiplied under controlled
conditions. The multiplied seeds will be subjected to field trials under natural drought
and saline conditions (against the parameters already defined) at different locations as
given in the milestone table. IPR and Bio safety rules will be followed and necessary
paper work will be completed before the transformation of genes into commercial
wheat cultivars.
Molecular studies: Efforts will be made to determine how the transactivation of gdhA
13
works. We will also try to investigate in the use of transacting factors to analyse plant
resistance further. This work will be conducted in the lab of Professor David A.
Lightfoot.
14
b. Milestones:
Item
Completion
date
Scientists Involved* and
activity %age
Cost
Million
RS
Description
Achievement indicators
Risk involved
Objective
Transgenic wheat with
improved resistance
against drought and
salinity.
No risk
14.05.2012 Dr. Nisar Ahmed*
Dr. Bushra Sadia
Dr. Azeem Iqbal Khan
Dr. Faisal Saeed Awan
Mr Ahsan Iqbal
Miss Shazia Anwar
Bukhari
14.078
Output-1
Import of drought
resistant gene
Purchase of equipment
and chemicals
Seeds of the transgenic wheat
varieties showing improved
resistance against salinity (at
12 EC and SAR 30 %) and
drought (70% field capacity)
with 5% yield increase as
compared to the nontransformed versions will be
available.
Drought resistant gene gdhA
will be available
Equipment and chemicals will
be available in stock
No risk
31.08.2009 Dr. Nisar Ahmed*
0.000
Supply of
equipment and
chemicals may
be delayed for
few days
No risk.
31.10.2009 Dr. Nisar Ahmed*
5.620
31.10.2009 Dr. Nisar Ahmed*
0.460
No risk
31.12.2009 Dr. Nisar Ahmed*
0.000
Output-2
Output-3
Output-4
Germination of seeds
of rice variety
Nipponbare under
controlled conditions.
Salt stress to 10 day old
seedlings before RNA
isolation. Synthesis of
cDNA and
development of
constructs in
expression vectors.
Necessary paperwork
to apply for biosafety
Gene construct in expression
vector will be available
Application will be
submitted to
15
licence
Output-5
Activity-1
Activity-2
Activity-3
Output-6
Standardization of
agro bacterium
mediated gene
transformation
protocol
Establishment of
protocol by
agrobacterium/with GUS
marker gene
Transgenic analysis for
marker gene
Visit of foreign
Professors. Research
activities will be
discussed. In additional
the visiting professors
will deliver Lectures on
recent advancements in
this field.
Transformation of
drought and salt
resistant genes in
wheat embryos
through agrobacterium
mediated
transformation
institutional biosafety
committee
Optimized protocol will be
available
3.970
Optimized protocol will be
available
Repetition of the
experiment may
take place
31.12.2010 Dr. Nisar Ahmed*
Dr. Bushra Sadia
Dr. Azeem Iqbal Khan
Dr. Faisal Saeed Awan
Mr Ahsan Iqbal
Miss Shazia Anwar
Bukhari
30.7.2010 Dr. Nisar Ahmed* (85%)
Dr. Bushra Sadia (5%)
Dr. Faisal Saeed Awan (5%)
Mr Ahsan Iqbal (5%)
Gus gene expression in
transgenic plant.
Repetition of the 30.11.2010 Dr. Nisar Ahmed* (75%)
Dr. Bushra Sadia (5%)
experiment may
Dr. Azeem Iqbal Khan (5%)
take place
Dr. Faisal Saeed Awan (5%)
1.500
No risk
A brief report on project
activities and lectures on recent
advances on the topic.
Tentative dates
of visit may
change.
Transgenic wheat plants with
drought and salt resistant
genes will be available
Poor response of
genotypes to
gene
transformation.
Repetition of the
experiment may
take place.
1.470
Mr Ahsan Iqbal (5%)
Miss Shazia Anwar Bukhari
(5%)
31.12.2010 Dr. Nisar Ahmed* (100%)
1.000
28.2.2011
2.908
Dr. Nisar Ahmed*
Dr. Bushra Sadia
Dr. Azeem Iqbal Khan
Dr. Faisal Saeed Awan
Mr Ahsan Iqbal
Miss Shazia Anwar
Bukhari
16
Dr. Nisar Ahmed* (80%)
Dr. Bushra Sadia (5%)
Dr. Faisal Saeed Awan (5%)
Mr Ahsan Iqbal (5%)
Miss Shazia Anwar Bukhari
(5%)
31.10.2011 Dr. Nisar Ahmed* (80%)
Dr. Azeem Iqbal Khan (5%)
Dr. Faisal Saeed Awan (5%)
Mr Ahsan Iqbal (5%)
Miss Shazia Anwar Bukhari
(5%)
31.10.2011 Dr. Nisar Ahmed*
1.408
No risk
14.05.2012 Dr. Nisar Ahmed*
Dr. Azeem Iqbal Khan
Dr. Faisal Saeed Awan
Mr Ahsan Iqbal
1.120
No risk
31.03.2012 Dr. Nisar Ahmed* (100%)
0.520
14.05.2012 Dr. Nisar Ahmed* (85%)
Dr. Azeem Iqbal (5%)
Dr. Faisal Saeed Awan (5%)
Mr Ahsan Iqbal (5%)
0.600
Activity-1
Regeneration of
immature embryos after
gene transformation
Transgenic plants harbouring
drought and salt resistant genes
will be available.
Activity-2
Screening of transgenic
plants against salinity
and drought under
controlled conditions
Screening results will be
available
No risk
Biosafety of transgenic
plants
Field Trials and
molecular analysis
Biosafety permission to grow
transgenics will be available
Results of the Transgenic
wheat with improved
resistance against drought and
salinity and 5% increase in
yield will be available
Data on transactivation of gdhA
and its impact on wheat for
drought tolerance will be
available.
Field trials data will be available
No risk
Activity-3
Output-7
Activity-1
Activity-3
Molecular analysis
No risk but a
changed growth
behaviour may
be observed
Evaluation of the
No risk
transgenic at farmers
fields under natural
drought and salt
conditions at UAF, Pindi
Bhattian, BARI and
Bahawalpur
Put * mark on the name of the activity incharge, if more than one scientists are involve
30.6.2011
1.500
17
Annexure- I
DETAILES OF COSTS
Budget
Code
Item of Expenditure
A. Salaries
Daily wages labour(unskilled)
Sub-Total (A)
B. Operational
Research Material & Supplies
Chemicals, Vectors, Enzymes, Medias etc.
(Annexure II)
(Million Rupees)
Research phase
Total
Year 1 Year 2 Year 3
0.050
0.050
0.075
0.075
0.200
0.200
0.325
0.325
1.600
1.200
0.700
3.500
0.200
0.000
0.025
0.000
0.100
0.010
0.040
0.000
0.050
0.020
0.050
0.020
0.350
0.030
0.115
0.020
0.125
0.050
0.030
0.050
0.050
0.020
0.010
0.050
0.070
0.050
0.050
0.070
0.020
0.010
0.010
0.150
0.070
0.050
0.100
0.030
0.020
0.185
0.270
0.150
0.150
0.220
0.070
0.040
0.040
0.020
0.020
0.010
2.250
0.050
0.020
0.020
0.010
1.770
0.100
0.020
0.040
0.010
1.440
0.190
0.060
0.080
0.030
5.460
0.460
0.185
0.050
0.100
0.040
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.460
0.185
0.050
0.100
0.040
Glass Ware/ Plastic ware etc. (Annexure II)
Fertilizer
Selfing bags/tags/labels etc
pesticides/weedicides
Small implements, pots, big iron trays for
screening against salinity
Travelling Allowance (local)
POL
Stationery
Repair of equipments/machinery
Repair of vehicles
Rent/rates/taxes/fees
Communication costs
(postage/phone/fax/internet)
Advertisement costs
Printing costs
Others (specify)
Sub-Total (B)
C. Machinery and equipment
Centrifuge Machine
Thermo mixer
Racks for different tubes (Tube stands)
Oven
Refrigerator
18
Pipetteman set
Agarose gel electrophoresis +accessories
Stirrer/Mixer
Mini spin
Cooling Bucket
Micro Vac
Dry thermo unit (Heat block)
Water purification system
Shaker
PCR Machine
pH meter
Hot plate with Magnetic Stirrer
Computers (with all accessories)
Mild Shaker
Sub-Total (C)
Overseas Travel (D)
Sub-Total (A+B+C+D)
Management Cost (25% of the cost A+B+
C+D) = E
Sub-Total (A+B+C+D+E)
Incentives for Scientists (5% of the project
cost A+B+C+D+E)
Incentive for PM (1% of the project cost
A+B+C+D+E)
Sub-Total (F)
TOTAL PROJECT COST (A+B+C+D+E+F)
0.100
0.100
0.040
0.050
0.100
0.120
0.150
0.290
0.150
0.645
0.060
0.040
0.100
0.080
2.860
0.460
5.620
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
1.000
2.845
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.520
2.160
0.100
0.100
0.040
0.050
0.100
0.120
0.150
0.290
0.150
0.645
0.060
0.040
0.100
0.080
2.860
1.980
10.625
1.405
0.711
0.540
2.656
7.025
3.556
2.700
13.281
0.351
0.178
0.135
0.664
0.070
0.036
0.027
0.133
0.421
0.214
0.162
0.797
7.446
3.770
2.862
14.078
19
18. BUDGET INSTALMENTS
Instalment
(Half Yearly)
1st
2nd
3rd
4th
5th
6th
Total
19.
(Million Rs.)
Main Institute
5.620
1.826
2.000
1.770
1.142
1.720
14.078
Total
5.620
1.826
2.000
1.770
1.142
1.720
14.078
INTERNATIONAL COLLABORATION
Name of linking international institute(s) with justification
1-Professor David A. Lightfoot
The Department of Plant, Soil and Agricultural Systems, Southern Illinois University,
Carbondale, IL, 62901, USA e-mail: GA4082@SIU.EDU
Professor D. A. Lightfoot has published his work in 2007 on drought tolerance in maize.
He and his co-workers have successfully developed transgenic maize plant with
improved resistance against drought by the transactivation of E.coli gdhA gene in maize.
He will provide the gdhA gene construct as well as the technical know-how about their
transactivation. His collaboration will be vital for the successful completion of the
project.
2-Professor Masahiko Maekawa
Crop Genome Modification Group
Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki,7100046, JAPAN; Tel: 081-86-434-1214 ; Fax: 081-86-434-1249
Email: mmaekawa@rib.okayama-u.ac.jp
Professor Masahiko Maekawa will provide any technical assistance that will be needed
at various occasions during the execution of the project especially in gene amplification,
designing construct for salinity tolerance gene in his lab, compilation, analysis and
publication of data.
20
a)
Type of collaboration:
b)
We have purely research based collaboration with both the Professors. They will
provide every possible help to complete this project successfully. In addition they will
visit our lab to discuss the problems and their possible solution. They will also deliver
lectures to faculty members and post graduate students on the latest advancements and
achievements in their related fields.
c)
Scientist(s) involved
Both the professors and their labs will fully cooperate for the successful completion of
the task.
20.
INTRNATIONAL TRAVELS
I.
a)
HOST INSTITUTE
Name of scientist(s) visiting institute and number of visits;
Dr Nisar Ahmed
One visit of each lab of Professor Masahiko Maekawa and Professor David A.
Lightfoot
b)
Purpose of each visit:
About 20 days visit is planned in August, 2009 to make OsDREB1A construct
in the lab of Professor Maekawa. Professor Maekawa has well established labs with all
the facilities. He will provide all the possible help to design construct. We do not have
the sequencing facilities and private companies take 3 to 4 weeks to provide sequence
results. This slow process will waste many months in designing gene cassette. While
making construct we will have to sequence many clones after every ligation until we
get insertion of the desired gene in proper orientation in expression vector. To save
time and for smooth running and execution of the project in time, this visit has been
planned.
Professor David A Lightfoot has successfully developed transgenic maize with
improved resistance against drought. He is working on the use of transacting factors to
21
analyse plant resistance further. He is also busy in analyzing why and how the
transactivation of gdhA works. He is very much interested to use in the transacting
factors we will determine in wheat to study the plant resistance at molecular level. A
20 days visit has been planned in February, 2012 to visit the lab of Professor Lightfoot
to plan and conduct molecular analysis to determine the transactivation of gdhA gene
and to investigate in the use of transacting factors to analyse plant resistance further.
c) Name of institute(s) to be visited
i. Genetics and Biochemistry lab, the Department of Plant, Soil and Agricultural
Systems, Southern Illinois University, Carbondale, IL, 62901, USA
ii. Crop Genome Modification Group lab, Research Institute for Bioresources, Okayama
University, Chuo 2-20-1, Kurashiki, 710-0046, JAPAN
II. COLLABORATING INSTITUTE
a)
Name of scientist(s) visiting institute and number of visits
i. Professor David A. Lightfoot
Genetics and Biochemistry lab
The Department of Plant, Soil and Agricultural Systems, Southern Illinois University,
Carbondale, IL, 62901, USA e-mail: GA4082@SIU.EDU
ii. Professor Masahiko Maekawa
Crop Genome Modification Group lab
Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki,
710-0046, JAPAN; Tel: 081-86-434-1214; Fax: 081-86-434-1249
Email: mmaekawa@rib.okayama-u.ac.jp
b)
Name of institute(s) to be visited:
Each Professor will visit Centre of Agricultural Biochemistry and
Biotechnology
(CABB), University of Agriculture, Faisalabad one time during the project period.
22
iii.
Purpose of each visit:
Professor Lightfoot has planned to visit CABB in August, 2010 for one week to review
and discuss the progress of the project activities. He will also deliver lectures on the
latest advancements/achievements in his field of research especially on the mechanism
of drought resistance in plants and the transactivation of gdhA.
Professor Masahiko Maekawa is planning to visit our institute in November, 2009 for
one week to review and discuss the progress of the project activities. He will deliver
lectures on the development of epigenetically stress tolerant crops.
21.
IMPORT OF TECHNOLOGIES. NIL
We will not directly import any equipment or chemical
22.
COMMERCIALIZATION AND BENEFIT TO END USERS
i)
Method of transferring results:
Through print and electronic media and Agriculture department
ii)
Agency/company/consultants involved in adaptation and adoption.
a)
Request will be submitted to the Agriculture Extension Department, Government of
the Punjab to transfer information to the farming community.
b)
The developed material of wheat will be shared with other plant breeders of the
Punjab for use by them in their breeding programmes
iii) Expected benefits to end users.
The responses of plants to various abiotic stresses have been important subjects of
physiological studies and more recently, of molecular and transgenic studies. Growing
populations, reducing water resources and shrinking farm lands require the world to
increase food production. Changing environmental conditions, such as drought, salinity
or outbreaks of pests and diseases, also call for new and better adapted crop varieties.
The identification of novel genes related to drought and salinity tolerance, determination
of their expression patterns in response to these stresses, and an understanding of their
23
functions in stress adaptation will provide us the basis of effective engineering strategies
to improve stress tolerance.
This will help to train the manpower in the latest techniques of biotechnology/genetic
engineering and to improve their knowledge level about the mechanisms of salinity and
drought tolerance at molecular level. The skilled manpower with improved knowledge
will help to coup with similar problems in wheat or other crops.
The execution of the proposed study will help to raise the academic and research level
of the institution as more number of trained manpower with improved knowledge will
be produced.
This project will help to develop close research contacts and share latest research ideas
with the eminent scientists of the world working on salinity and drought tolerance.
Farmers are generally the target market for biotechnologist’s efforts. Development of
improved salinity and drought resistant wheat will help to increase grain yield from salt
affected drought stricken marginal lands. This will definitely impose a positive affect on
farming community in terms of income and improved social status. This will be a step
ahead to feed the increasing number of masses and we will be able to save the marginal
lands from further destruction by utilizing them for growing wheat.
22.
FINAL REPORT SUBMISSION
14.5.2012
24
Justification of the equipment
Item name
Centrifuge
Machine
Micro mixer
Racks for
different
tubes
Oven
Refrigerator
Pipette man
set
Agarose Gel
Electrophoresi
s+
Accessories
Item specification
Digital microprocessor controlled machine,
speed range at least up to 14000 rpm, time
range 0 to 30 minutes, with angle rotor capacity
24x1.5mL/2.0 mL and adaptors 0.5 mL and
0.2mL, very low noise level, operation on 220
Volts.
Ideal for genomic and plasmid DNA, RNA
extraction, bacterial culture and
immunoprecipation, for micro tubes and
microtiter plates, Dimensions: 300w x 188d x
115h mm, Weight: 5 kg, Shaking speed: 300
to 1000 r/min (low) 1000 to 2500 r/min (high),
Shaking style: Orbital
Plastic and steel tube stands for holding of
various sized tubes during experiments.
Economical gravity & mechanical convection
models designed for the budget-conscious
laboratory. Gravity and convection models
feature stainless steel interiors. Chamber
size: 280x 280 x 380 mm (11"x11"x15”) 30
Litters. Two adjustable chrome-plated wire
shelves are included. Compact desktop design
with dimension: 500 mm x 500 mm x 1180
mm. Temperature controller (600W Max) with
+/- 1o C tolerance from room temperature to
220oC. Digital temperature display for easy set
up a temperature. Power: 600 W, 220V AC.
Shipping weight: 65lbs. Shipping
dimention:20"x20"x28"
Oversized and balanced, (134A), refrigeration
system, factory sealed and pre-lubricated for
long life - holds 33°F degrees to 38°F.
Adjustable range: 1 mL, 200 µL, 20 µL and 10
µL capacity.
Horizontal, large scale analysis of samples
system, with power supply and System with
CCD- at least 2048x2048 pixels camera, 10X
zoom 20-200 mm f2.8 lens, multi-wavelength
illumination, interface with windows XP. All
compulsory and optional accessories
Justification
In the small scale
centrifugation of
research samples related
to genomics and
proteomics
For the mixing of
samples to release
DNA, RNA from
tissues and to mix Hi-Di
in samples for sequence
For handlings of tubes
of different size and
shape during research
and storage of samples.
For the culture of
different medias under
controlled temperature
conditions. Also useful
for baking of
membranes after DNA
transfer.
Storage and
Maintenance of
Samples
Measuring small
volumes in different
experiments.
In the qualitative
determination of DNA
and RNA
25
Stirrer/Mixer
Mini spin
Cooling
bucket
Micro vac
Dry thermo
unit (heat
block)
Eppendorf Thermo mixer comfort with slide
adapter for in situ hybridization and mechanical
mixing of samples. Moreover, because of the
digitally programmable temperature steps,
Thermo mixer comfort facilitates maximum
reproducibility of the experimental conditions
and requires substantially less effort. It heats,
cools and mixes samples. Temperature range is
from 13ºC to 99 ºC.
12 x 1.5 ml/2.0 ml capacity rotor, spin up to
14,000 x g, Deceleration and acceleration of
<13 s, Rotor is autoclavable at 1210C, 20 min.
ECB (+3ºC to +20ºC) Portable bench top
cooling solution with 3 types of vessel holders.,
Temperature range: +3ºC to +20ºC,
Temperature accuracy: ±1.0ºC, Temperature
display: Digital, Cooling/Heating method:
Thermoelectric
Speed 2,000 rpm, Heating On/Off, 55 degree
Celsius Vacuum Pump Oil-Free Vacuum 5HPa
Vacuum Meter 0 - 0.1 MPa (0 - 760 mmHg)
Rotor 1.5 ml x 12 places Lid Blue Acrylic
Plastic Filters Intake 0.2 micro maters Exhaust
Aerosol Filter Vacuum Release Manual
Chamber Stainless Steel Dimensions 200W x
205D x 228Hmm Net Weight 7.2 Kg Power
Requirement C110/120V,50/60Hz,3A
AC220/230/240V,50/60Hz,1.5A
Range 100° F to 1200° F (38° C to 649° C)
Accuracy ± 0.8° F at 100 to 600° F – ± 0.15%
of set point >600° F Resolution 0.1 deg (test
mode) – 0.01 deg (calibration) Set Point
Stability ± 0.15° F Stabilization Time 30 min.
max for a 1100° F change from ambient
temperature Well Uniformity ± 0.5° F
Ambient Temp. Range
Operational 32° F to 120° F Storage -67° F to
167° F Readout Units ° F or ° C Well Size 1 in.
ID x 6 in. deep Adapter Chucks 4 standard
chucks with bore diameters of 1/4, 3/8, 7/16,
and 9/16 inch Case Type Deep drawn
aluminium Case Dimensions (L x W x D) 18 in.
x 11 in. x 14 in.
Used for Enzyme
reactions,
transformation,
Denature of DNA,
cultivation of bacteria,
yeast, plasmid isolation
and hybridization
reactions
To flush samples before
use
For ligation of DNA
fragments into cloning
vectors at controlled
temperatures.
Important for the
removal of ethanol from
DNA and RNA samples
through vacuum.
To heat DNA samples
for denaturation, also
important for
transformation of
competent cells.
26
Water
purification
system
Shaker
PCR Machine
pH meter
Remote display for quality monitoring and
operation up to 3 m (10') distance
Ergonomically designed point-of-use dispense
trigger for easier water delivery and fixedvolume water dispense
The remote point-of-use dispenser for
convenient delivery of ultrapure water up to 2.5
m (8') away from your system; can be wallmounted, dispenser arm rotates 180° for total
flexibility
Foot pedal for hands-free water delivery from a
Milli-Q system or remote POU dispenser and
express 20 final filter for minimum ionic and
organic contaminant release when used for subppb analysis of volatile organics or endocrine
disrupters
Wave-PR (5 to 50 r/min, see-saw mode)
Ideal for Immuno staining, genomic DNA
extraction, filter washing in hybridization, gel
staining or destaining, Temperature range:
Tolerates 0 to +50º C in surrounding, Standard
accessories: 1 x Sticky Sheet, Dimensions:
300w x 235d x 155h mm, Weight: 5.1 kg
Thermal cycler 96 well with heated lid, thermal
block for 0.2 mL and 0.5 mL PCR tubes,
ramping speed at least 3C/s, programmable
ramp rates, gradient temperature facility,
accuracy range 0.3C or less, operating temp.
4-32C ambient, thermal range 0-105 C,
heating speed at least 3 C /s, cooling speed at
least 2 C/s, block homogeneity 0.4 C with
power failure restore function, auto-restart
option without interrupting cycles, built-in
computer and printer interface, option of
several program storage preferably with
ongoing gradient viewing, on board software
for temperature validation and calibration will
be preferred, power input according to local
power supply, with UPS (with voltage
regulation) compatible with the machine.
Digital bench model microprocessor controlled
with parallel temp. indication, pH range -1.00 to
+15, pH resolution 0.005/0.01V, mV range 999.9 to +999.9 mV, automatic temp
compensation, data memory storage.
We do not have water
purification system and
we depend on NIBGE
for this purpose. So this
system is very
important.
For shaking of different
mixers at relative speed
and room temperature,
also for washing of
membranes after
southern and northern
blotting.
In the polymerization of
the DNA extracted from
plant samples
In the determination the
pH of the unknown
samples and preparing
buffers of required pH
27
Hot plate with
magnetic
Stirrer
Computer
with
accessories
Mild Shaker
Hot plate with magnetic stirrer, heating plate
temperature at least up to 100 oC, stirring
capacity (H2O) ~15L, electronic temperature
controller, support rod, holding rod, head
clamp, voltage 230V
Intel Pentium D 945 (3.4 GHz/800 FSB/2*2MB
Cache), Intel 945 G chipset, 512 MB DDRII
400/533/667 MHz RAM, hard disk drive 80
GB, combo drive internal 48X CD+/- RW and
16X DVD, floppy disk drive, built-in internet
card, CRT monitor, mouse and keyboard, laser
printer. HP Laser printer 1320+scanner
Ideal for filter washing in hybridization, gel
staining or destaining, Dimensions: 310w x
250d x 140h mm, eight: kg, Shaking speed: 20
to 70 r/min, Shaking style: Seesaw
Routing use equipment
for making solutions in
the laboratories.
For the storage and
analysis of data,
computer is needed. For
scanning of various
photos for insertion into
data scanner is required.
For printing of results
and letters printer is
needed.
For shaking of gels after
electrophoresis, for
washing of members
after southern/northern
blotting.
28
ANNEXURE II
Tentative list of chemicals, enzymes, vectors, Medias, glass and plastic wares
1.
3.
5.
7.
9.
11.
13.
15.
17.
19.
21.
23.
25.
27.
29.
31.
33.
35.
37.
39.
41.
43.
45.
46.
47.
48.
50.
52.
54.
56.
58.
60.
62.
64.
66.
68.
70.
72.
74.
76.
78.
Abscisic Acid
Ampicillin sodium salt
Adenine sulphate
Calcium triphosphate
Carmine
Cellulase RS
Citric Acid
Colchicine
Glucose
Sucrose
Phytagel
Glycine
HEPES
Hemicellulase
Indole3-Acetic acid (IAA)
Mannose
L-Asparagine Monohydrate
Maltose monohydrate
Manitol
Orcein
Tween 80
PEG MW 6000
Pectolyse Y23
Potassium monohydrogen Phos
Protoplast isolation basal salt
Sodium Hypochlorite
Sodium Docedyl Sarcosine
Sodium Bisulfate granular
Sodium Hydroxide
Sodium Pryophosphate
Thiadiazuron
Abscisic Acid
Coconut water
MES
Citric acid
GA3
Cholecalciferol
Fructose
2-mercaptaethanol
Ethanol
Isoamylalcohol
2.
4.
6.
8.
10.
12.
14.
16.
18.
20.
22.
24.
26.
28.
30.
32.
34.
36.
38.
40.
42.
44.
46.
47.
49.
49.
51.
53.
55.
57.
59.
61.
63.
65.
67.
69.
71.
73.
75.
77.
79.
Ammonium Per sulphate
Ascorbic Acid
Bovine serum albumin
Casein hydrolysate
Cellulase R 10
Charcoal activated
Cobalt chloride hexa hydrate
DMSO
EDTA, sodium salt
Ferric chloride
Gibberellic acid
Glutamine
Hydrochloric Acid
Iodine
Indole Butyric acid (IBA)
Glutamine
Macerozyme
Manganese(II)sulfatemonohydrate
Myo-Inositol
Tween 20
PEG MW 1500
Potassium acetate
Potassium chloride
Potassium phosphate monobasic
Chloroform
Gelatin
Proline
Sodium citrate dihydrate
Sodium hypochloride
Sodium sulphite anhydrous
Triphenyl tetrazolium chloride
Zeatin Riboside
Casein hydrolysate
Fumaric acid
Malic acid
Retinoic acid
Sorbitol
CTAB
Isopropanol
Chloroform
Phenol
29
80.
Glycerol
82.
EDTA
84.
RNA isolation kit
86.
Gel elution Kit
88.
plasmid isolation kit
90.
Competent cell
92.
Plasmids
94.
Agro bacterium
96.
GUS construct
100. Yeast extract
99.
Agar
101. IPTG
103. Calf intestine phosphatase
105. Ampicilline
107. Kanamycine
109. dNTP’s
111. MS medium
113. Kinetics
115. Ethidium bromide
117. SDS
119. DNAase/ RNase
121. KOH
123. PEG
125. DNA/RNA weight marker
127. Formamide
And many others
81.
83.
85.
87.
89.
91.
93.
95.
97.
98.
100.
102.
104.
106.
108.
110.
112.
114.
116.
118.
120.
122.
124.
126.
128.
Agarose
Tris
cDNA synthesis kit
Cloning kit
Restriction enzymes
Vectors
Race amplification kit
GFP construct
Tryptone
Nacl
X-gal
X-glu
Methanol
Streptomycin
Taq polymerase
Expression vector
cytokinin
Auxin
Bromophenol blue
NaoH
CaCl2
Xylene cyanol
Diethylpyrocarbonate
Formaldehyde
Sodium citrate.
Glass and Plastic Ware
Item
Petri dishes (Plastic, sterilized in the
plastic sleeves)
Sigma baby food jars with lids
Syringe filters
Cryocanes
Liquid nitrogen resistant dewars
Carrying cart Plastic
Wash Bottle
Test Tube
Reagent Bottles
Lab. Dishes PP made autoclavable
Sterilization indicator tape
Wash bottles
Measuring beakers PP made
Item
Plastic pipettes (Sterilized, individually wrapped)
Sterilin bottles (sterilized plastic bottles with lids)
Cyotubes
cryoboxes
Dewars for long-term storage of specimens
Deep Trays
Petri Dish Glass
Conical Flask
Test Tube Racks
Magnetic Bars
Pipette containers
Screw neck Reagent bottles
Measuring cylinders PP
30
Disposable weighing Boats
Plastic scoops
Clear cling foil
Scalpel blades
Disposable Pasteur pipettes LDPE
Latex gloves
Cold resistant gloves
Glass beakers of different sizes
Pasteur pipettes glass
Falcon tubes
PCR tubes
Plastic tips
Weighing forceps plastic
Aluminium foil
Forceps stainless steel with Teflon coating
Scalpel handles steel
Skin cleanser
Heat resistant gloves
Sieve
Glass bottles of different sizes
Any many others
Eppendorf tubes
Culture tubes
Plastic pots