AN ABSTRACT OF THE THESIS OF
Getachew Boni for the degree of Doctoral Philosophy in Crop Science presented on May
24, 1996. Title: Expression and Inheritance of Tolerance to Waterlogging Stress in Wheat
(Triticum aestivum L.)
Redacted for Privacy
Abstract approved
To obtain genetic information pertaining to the inheritance of tolerance to
waterlogging in wheat, three tolerant (Prl/Sara, Ducula and Vee/Myna), and two sensitive
(Seri-82 and Kite/Glen) wheat lines were crossed in all possible combinations. F1, F2, F3,
and backcross generations obtained from these crosses were studied under field
conditions. Flooding was imposed when plants were at the first internode stage, and basins
were drained after 40 days of flooding. Data were collected on percentage leaf chlorosis
(four times at intervals of seven days starting at Zadoks' DC scale 41), heading date, plant
height, biomass, yield, and kernel weight. Leaf chlorosis was used as a measure of
tolerance to waterlogging.
The expression of tolerance to waterlogging was not influenced by the maternal
effect as no differences between reciprocal crosses for percentage leaf chlorosis were
observed. The F1 generations were intermediate for the symptom of leaf chlorosis,
indicating that resistant was additive. Broad sense heritability ranged from 58 percent to
83 percent. In an attempt to estimate the number of genes that control tolerance to
waterlogging, F3 populations for symptoms of leaf chlorosis were analyzed using chi-
square method. The segregation ratios of F3 lines indicated that
four genes control
tolerance to waterlogging stress. Selection of segregating lines at early generation for
tolerance to waterlogging stresses would be effective.
Laboratory experiments were designed to provide information about the
morphological and anatomical basis of tolerance to anaerobic conditions. In these
experiments the five parents were evaluated for the differences in root anatomy and 02
transport from shoot to root. Plants were grown either for four to five weeks under
continuously aerobic conditions, or for two to three weeks aerobically followed by two to
three weeks of anaerobic treatment in aerobic and anaerobic hydroponic culture.
Tolerant cultivars showed higher percentage of root porosity than the sensitive
cultivars. This was evident when well developed aerenchyma was observed under light
microscopy for the tolerant cultivars. Oxygen transport from the shoots to the roots was
observed for both tolerant and sensitive cultivars. The anatomical differences were not
associated with the differences observed between tolerant and sensitive cultivar in 02
transport. The differences in aerenchyma formation combined with the processes (enzyme
activities and substrate supply) controlling the glycolytic flux and respiration rate may
have accounted for the differences observed between tolerant and sensitive wheat
cultivars.
Expression and Inheritance of Tolerance to Waterlogging Stresses in Wheat
(Triticum aestivum L.)
By
Getachew Boru
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Doctor of Philosophy
Completed May 24, 1996
Commencement June, 1996
Doctoral Philosophy thesis Getachew Boru presented on May 24, 1996
APPROVED:
Redacted for Privacy
Major Professor, representin Crop Science
Redacted for Privacy
Head of the Crop and Soil Scien& Department
Redacted for Privacy
Dean of the Gradate School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorize release of my thesis to any reader upon
request.
Redacted for Privacy
Getachew Boni, Author
ACKNOWLEDGMENT
I wish to express my sincere gratitude and appreciation to my advisors Dr.
Maarten van Ginkel and Dr. Warren E. Kronstad for their excellent advice, guidance,
encouragement, and support throughout my graduate study at Oregon State University. It
was a pleasure and honor to have worked with these two outstanding plant breeders.
Sincere thanks and appreciation are extended to my graduate committee Drs. Larry
Boersma, Dal lice Mills, Patrick Hayes for providing their time and invaluable suggestions.
I would like to especially thank Dr. Larry Boersma for his invaluable suggestions on the
laboratory experimental methodologies and very constructive criticism in the preparation
of the manuscript.
I would like to extend special thanks to all members of cereal project for giving me
their unlimited assistance and cooperation. Special thanks to Mike Moor and Randy
Knight for their excellent help in the setting-up the laboratory experiment. Special thanks
also extended to Sonia Rowe, Peggy Forman, Mery Venhoven, Susan Wheeler, Debbie
Kelly, Mark Larson, and John Bassinette for giving me their advice expertise and support.
Special thanks are extended to Nan Scott for helping me with computer work
within and out of her office time.
I would like to extend sincere thanks to the staff and members of the CIMMYT
Wheat Program for their friendship and many helping in planting, maintenance, and
harvesting of my experiments during my thesis research at CIMMYT. Special thanks and
appreciation are extended to Jorge Manuel Montoya Moroyoqui, Adrian Corona robledo,
Jose Louis Martinez Hernandez, Rodrigo Rascon Gomez, Isedro Vega, Alferedo Valencia
Penaloza, Angel Gonzalez Vega, Horacio Vega Ortiz, Valentin Ramireze Jardon, Gabino
Espinoza Segura, and Gonzalo Ortiz Vega.
Sincere thanks are extended to my fellow graduate students for their friend ship
and helpful suggestions. A special thanks are given to Sala Rezigu, Maria Mendoza, Karim
Amar, Claudio Jobet, Julio Cereno, Ottoni de Sousa, Christopher Weight, and Alicia de
Blanco.
I am grateful to my wife Demekesh Weldegebrial for her love, understanding,
encouragement, and also for helping me to type this manuscript, without which this
endeavor would not have been possible.
A special thanks to United State Agency for International Development, OSU,
and CIMMYT for providing opportunity and financial support to further my education
and research at Oregon
TABLE OF CONTENTS
Page
CHAPTER I
1
INTRODUCTION
1
LITERATURE REVIEW
Effect of waterlogging on plant growth and development
Adaptation to waterlogging
Morphological adaptation
Metabolic adaptation
Genetic variability in waterlogging tolerance
3
3
5
5
7
9
OBJECTIVES
11
CHAPTER II
12
FIELD EXPERIMENTS (INHERITANCE STUDIES)
12
MATERIAL AND METHODS
12
Year I
Year II
Study 1
Study 2
Statistical analysis
RESULTS
Year I
Year II
Qualitative approach (Year II, study 1)
Quantitative approach (Year II, study 2)
Associations
12
13
13
18
18
20
20
21
25
30
32
DISCUSSION
41
CONCLUSION
43
TABLE OF CONTENTS (Continued)
Page
CHAPTER III
44
STUDY ON OXYGEN TRANSPORT AND SYMPTOMS OF
WATERLOGGING TOLERANCE IN SELECTED SPRING WHEAT
GENOTYPES
44
MATERIAL AND METHODS
44
Growth conditions
Observation and measurements
Measuring root porosity
Observing root anatomy (aerenchyma or cortical air space)
Evaluating cultivars for oxygen transport from shoot to root
Experimental assembly for measuring 02 transport
RESULTS AND DISCUSSION
44
46
46
47
48
48
53
Root porosity
Aerenchyma formation
Root respiration rate
53
55
58
GENERAL DISCUSSION
70
BIBLIOGRAPHY
72
APPENDIX
85
LIST OF FIGURES
Figure
Page
Basins were constructed immediately after planting the F3 lines and the
plants were flooded 5 to 7 cm above the soil level for 40 days starting
from the first internode stage
15
A-2
Flooded F3 lines segregating for leaf chlorosis
16
A-3
Segregating F3 lines after all the water was drained from the basins
17
A-4
Frequency distribution for F3 lines for mean percentage (%) leaf chlorosis
(data from the fourth scoring date) for the crosses Ducula x Vee/Myna 26
A-5
Frequency distribution for F3 lines for mean percentage (%) leaf chlorosis
(data from the fourth scoring date) for the crosses Ducula x Kite/Glen
26
A-6
Frequency distribution for F3 lines for mean percentage (%) leaf chlorosis
(data from the fourth scoring date) for the crosses Seri-82 x Kite/Glen
26
A-7
Postulated tolerance genes (based on leaf chlorosis data) in the five
parents and number of segregating genes identified in all crosses
29
A-8
Indirect effects from path coefficient analysis of the association of grain
yield with percent chlorosis and heading date for the cross Prl/Sara x
Seri-82
39
A-1
B-1
Hydroponic system used for growing wheat seedlings in nutrient solution
at aerobic and anaerobic conditions
45
B-2
Experimental arrangement based on the design by Veen (1977) for
measuring 02 uptake rate by wheat root from the root chamber
49
B-3
Percent porosity for the roots (expressed as percent of total root volume)
of tolerant (Ducula, Prl/Sara & Vee/Myna) and sensitive (Seri-82 &
Kite/glen) wheat cultivars under aerobic and anaerobic growing
conditions
54
B-4
Transverse sections of roots for the tolerant cultivar Ducula grown
aerobically (A) and anaerobically (B). Formation of aerenchyma in the
cortical tissue of anaerobically grown plants is indicated
56
LIST OF FIGURES (Continued)
Figure
B-5
Page
Transverse sections of aerobic (A and B) and anaerobic (C, D, E, F)
roots
57
B-6
Oxygen concentration in the root chamber as function of time measured
during depletion by the root system of anaerobically grown plants with
intact shoot and without shoot
59
B-7
Rate of oxygen depletion (in 1AM gdm-1 min-1) by the root systems of
anaerobically grown plants measured with and without shoots as function
60
of time
B-8
Rate of oxygen uptake by the root system of aerobically grown tolerant
(Ducula, Prl/Sara and Vee/Myna) and sensitive cultivars (Seri-82 and
Kite/Glen) measured with and without shoots
61
B-9
Rate of oxygen uptake by the root system of anaerobically grown tolerant
(Ducula, Prl/Sara and Vee/Myna) and sensitive cultivars (Seri-82 and
Kite/Glen) measured with and without shoots
62
B-10
Rate of oxygen depletion by the root systems of Ducula as functions of
oxygen concentration in the root chamber
63
B-11
Percent change in oxygen uptake rate {(without shoot with
shoot)/without shoot x 100 } for tolerant cultivars vs sensitive cultivars at
65
the aerobic and anaerobic treatments
LIST OF TABLES
Table
Page
A-1
Mean of heading date, percentage of surviving plants before flooding
A-2
Mean percentage leaf chlorosis (data from the fourth scoring date) for
A-3
A-4
20
parents and F3 lines
22
Means of agronomic traits for parents included in each cross grown under
waterlogged conditions (Year II; study 1)
23
Mean percentage leaf chlorosis for parents (P1, P2) , Fi (P1/P2) and Fir
reciprocals (P2/P1), under waterlogged conditions (Year II; study 2)
24
A-5
Means of agronomic traits for F3 lines grown under waterlogged conditions
(Year II; study 1)
25
A-6
Chi-square (X2) goodness of fit to ratios observed in segregating F3 lines
for percent chlorosis in response to waterlogging stress and broad sense
heritability
28
Joint scaling test for additive - dominance effects. Estimate of genetic
effect and their standard errors for percentage leaf chlorosis for Tolerant x
Sensitive crosses
31
Six parameter model. Estimate of genetic effect and their standard errors
for percentage leaf chlorosis for the Tolerant x Sensitive crosses for which
the additive - dominance model was insufficient
31
Genotypic correlations of percent chlorosis with heading date (Heading),
biomass (BM), plant height (Height), grain yield (Yield), and thousand
kernel weight (TKW) for F3 lines
34
Phenotypic correlations of percent chlorosis seed with heading date
(Heading), biomass (BM), plant height (Height), grain yield (Yield) and
thousand kernel weight (TKW) for F3 lines
35
A-7
A-8
A-9
A-10
A-11
Genotypic correlations of heading date with biomass (BM), plant height
(Height), grain yield (Yield), and thousand kernel weight (TKW) for F3
lines
36
LIST OF TABLES (cont.)
Page
Table
A-12
A-13
Phenotypic correlations of heading date with biomass (BM), plant height
(Height), grain yield (Yield), and thousand kernel weight (TKW) for F3
lines
37
Results of path coefficient analysis for the tolerant x sensitive crosses
39
LIST OF APPENDIX FIGURES
Page
Figure
A-1
Rate of oxygen depletion by the root systems of Prl/Sara as functions of
86
oxygen concentration in the root chamber
A-2
Rate of oxygen depletion by the root systems of Vee/Myna as functions
87
of oxygen concentration in the root chamber
A-3
Rate of oxygen depletion by the root systems of Seri-82 functions of
oxygen concentration in the root chamber
87
A-4
Rate of oxygen depletion by the root systems of Kite/Glen as functions
of oxygen concentration in the root chamber
88
IN DEDICATION TO
my wife, Demekesh Weldegebrial,
and our parents,
Boru Gari,
Weldegebrial Hagos,
Gemja Debela
and Gebretu Golem
EXPRESSION AND INHERITANCE OF TOLERANCE TO WATERLOGGING
STRESS IN WHEAT (Triticum aestivum L.)
CHAPTER I
INTRODUCTION
Wheat growth and distribution are restricted chiefly by limited available moisture.
However temporary, intermittent or continuous excess water due to flooding is also
common throughout parts of the wheat growing world (Ponnamperuma, 1972).
In humid, temperate regions, excessive rainfall resulting in waterlogged soils is a
factor limiting production. For example in the lower Mississippi valley in the USA, the
incidence of waterlogging is a common occurrence and it damages winter wheat at
establishment and tillering stages (Musgrave, 1994). In Britain about 40% of the cereal
growing area has waterlogging problems (Cannell et
al.
1980).
In high rainfall
environments such as the eastern and central African highlands, the duration and severity
of waterlogging may be aggravated because of the wide spread presence of heavy clay
vertisol soils. Waterlogging of soils is not restricted to areas of heavy rainfall, but also
occurs periodically in arid regions that are irrigated (Kozlowski 1984). In the irrigated
Indo-Gangetic alluvial plains of northern India, 2.5 million hectares of sodic soil planted to
wheat experience temporary waterlogging due to poor drainage (Sharma and Swamp,
1988). Direct flooding of soil by irrigation adversely affects plants by decreasing soil
aeration. But waterlogging can also be caused by indirect leakage from irrigation ditches.
For example, in Western Canada, where more than 280,000 hectares of land are irrigated,
24,000 hectares are permanently waterlogged because of seepage water from irrigation
canals (Reid, 1977). Waterlogging may also be introduced in modern mechanized farming
as the pore volume of soil is decreased due to the use of bigger and heavier machines
(Erdmann et al. 1986).
2
Donman and Housten (1967) estimated that one
half to one third of the world's acreage
of irrigated land has drainage problems. Waterlogging stress affects 12 % of all
agricultural soils in the USA (Boyer, 1982). It is estimated that about 10 million hectares
of land planted to wheat in developing countries experience medium to serious
waterlogging problems (Maarten van Ginkel, 1993, personal communication).
3
LITERATURE REVIEW
Effect of waterlogging on plant growth and development
Gas spaces in most well drained soils make up 10 to 60 percent of their volume.
Exchange of gases into and out of the soil by diffusion and convective flow is fairly rapid.
When soil is waterlogged most of the pore space becomes filled with water, while
metabolism of roots and microorganisms then consumes the available oxygen and
produces carbon dioxide (Setter and Belford, 1990). The carbon dioxide concentration
may reach as high as 50 % of the total gas in flooded acid soils and it may persist for
several weeks (Ponnamperuma, 1976). Waterlogging virtually eliminates gas-filled pores
and limits gas exchange between soil and air to molecular diffusion in the soil water within
a few hours following flooding (Ponnamperuma, 1972). Consequently the supply of
oxygen to the soil will be terminated, and gases formed by soil metabolism start to
accumulate. Diffusion of gases through water is about 10,000 times slower than through
air (Armstrong, 1979), so that the concentration of gases which are consumed, such as
oxygen, will decrease rapidly, while the concentration of gases which are produced, such
as methane, hydrogen sulfide, ethylene and carbon dioxide, will continuously increase
(Ponnamperuma, 1984).
In waterlogged soils potentially inhibitory concentrations of
carbon dioxide,
ethylene, manganese, iron and organic substances may accumulate (Gammbrell and Patric,
1978; Ponnamperuma, 1984; Russel, 1952; Russel, 1973), and the availability of soluble
and exchangeable iron and manganese increases (Armstrong, 1982; Waldern, et al., 1987).
Excessive uptake of these elements by plants may lead to toxic levels (Burke et al., 1990;
Jones, 1971, 1972; Martin, 1968; Wheeler, et al., 1985).
Denitrification and leaching caused by flooding can also lead to a loss of nitrate
from the soil solution (Patric et al., 1972; Stolzy et al., 1978) resulting in nitrogen
deficiency. Adverse effects of a high water table on nutrient uptake and crop yield was
reported by Jackson and Drew (1984).
4
Reduced concentrations of the major plant nutrients, particularly nitrogen, were found in
the leaves of wheat plants grown in waterlogged soils when compared with those grown in
well drained soils (Haung et al., 1994a). During waterlogging wheat plants translocate
nitrogen from older to younger leaves. This becomes evident by characteristic yellowing
and early senescence of the lower, older leaves (Trought and Drew, 1980b).
Poor soil aeration associated with flooding induces a number of changes in the soil
plant relations that usually adversely influence plant growth. Several factors may cause
flooding injury. However, oxygen deficiency is expected to be the most important one
(Kozlowski, 1984). Roots require oxygen for respiration and other metabolic activities.
Insufficient oxygen results in anaerobic respiration, fermenting carbohydrate into alcohol
and the production of only small amounts of energy. The limited energy produced is
usually not sufficient for normal metabolism, hence many root cells die and decay under
prolonged flooding of soils (Peter et al., 1969). Under 02 limiting stress, seeds of most
higher plants fail to germinate. In some instances seeds germinated under aerobic
conditions fail to continue growing when exposed to anaerobic conditions. Germination,
and growth of wheat and oats were found to be strongly retarded under anaerobic
conditions (Amedeo and Beevers, 1983). The effects of poor aeration in cereals are
restriction of root growth, slower rate of leaf elongation and accumulation of dry matter,
chlorosis and premature senescence of leaves, decreased tillering, wilting, sterility, and
reduced kernel weights and grain yields (Sayre et al., 1994; Troughyt and Drew, 1980;
van Ginkel et al., 1992). Waterlogging can cause retardation of root growth, the death of
root tips, and the formation of adventitious roots at the base of the stem (Setter and
Belford, 1990).
Plants that are severely injured by flooding show a rapid reduction in the rate of
photosynthesis (Musgrave, 1994). Stomatal closure appears to be mainly responsible for
the rapid decrease of photosynthesis of flooded plants (Phung and Kinipling, 1976). A sign
of reduction in stomatal conductance, photosynthesis, and water potential was observed
when the two wheat cultivars "Bayles" and "Savannah" were tested under flooded
conditions (Huang et al., 1994b).
5
Typical responses of wheat to waterlogging are early senescence, slower root
growth, cessation of seminal root growth and decreased nutrient accumulation.
This response in wheat growth can be reproduced in wheat grown in nutrient solution
continuously flushed with nitrogen gas (Trought & Drew, 1980b).
Gill et al. (1993), reported that the effect of flooding on wheat was serious at the
crown-root initiation, flowering and grain filling stage. Short term (six days) waterlogging
in wheat resulted in grain yield reductions of 39 % and 47 % in alkaline and sodic soils,
respectively (Sharma and Swamp, 1988),
37 % to 45 % in the greenhouse, and 51 % in
field experiments (Musgrave, 1994).
Adaptation to waterlogging
Plants resist the damaging effects of waterlogging by either adapting to, or
tolerating, the low-oxygen environment. Waterlogging affects the growth of many plant
species. However, tolerant species, such as Carex arenaria, Senecio aquaticus, and
Phalaris arundinacea can survive temporary or prolonged anaerobic stress in the field
(Crawford, 1982). Flooding-tolerant plants adapt to anaerobic conditions by various
mechanisms, depending on species and environmental conditions. This tolerance
is
associated with both morphological and metabolic modifications (Kozlowski, 1984).
Morphological adaptation
There are several reports of morphological changes in roots of wheat seedlings
following oxygen deficiency in the rooting medium. These changes include aerenchyma
formation, and increased seminal and nodal root production (Bradford and Shang, 1981;
Erdmann et al., 1986; Erdmann and Wiedenorth, 1988; Thomson et al., 1990; Wiedenorth
and Erdmann, 1985).
Aerenchyma are continuous gas filled channels which connect the root with the
shoot. Formation of aerenchyma was observed within five to seven days of waterlogging
in wheat (Thomson et al., 1990), within two days in sunflower (Helianthus annus), and
tomato (Lycopersicon esculentum; Kawase and Whitmoyer, 1980), and within seven days
6
in maize (Zea mays L.; Drew et al., 1997). Aerenchyma, hypertrophy of lenticles, and
adventitious roots are means of morphological adaptation to waterlogging injury (Clemens
et al., 1978; Gill, 1970; Hozumi, 1966; Jackson, 1955; Jackson and Drew, 1984; Kawase,
1981; Kozlowski, 1984; Kramer, 1951). In the absence of 02 the formation of lysigenous
(by cortical cell distractions) aerenchyma was evident in the root cortex of maize (Drew et
al., 1979), rice (Oriza sativa; Armstrong, 1971), and wheat (Huang et al., 1994; Trought
and Drew, 1980b). Internal transport of oxygen from shoot to root was correlated with
aerenchyma development in adventitious roots of the wheat cultivar "Fidel" (Prioul and
Guyot, 1985).
Formation of aerenchyma before or during waterlogging increases the oxygen
supply to the growing root tips (Armstrong, 1979). Aerenchyma reduces the diffusion
resistance within the cortex and also decreases the respiratory demand of sub-apical parts
of the roots. Thus, oxygen diffusion through aerenchyma may allow survival and growth
of roots in a soil environment essentially devoid of oxygen. Oxygen can also be diffused
from the roots to the rizosphere to detoxify toxic elements accumulated around the root
zone (Greenwood, 1967). Oxygen diffusion in bulrush (Schoenoplectus lacustris) is very
effective from the shoots to the rhizomes because of high tissue porosity (Haldmann and
Brandle, 1983). The study by Armstrong and Webb (1985) indicated that internal 02
transport is sufficient to support root extension in rice. In general, the intercellular air
system from leaves to roots is more important for grasses and sedges with hollow stems.
Species which have these structures are generally more tolerant to flooding than others
(Crawford, 1982). Ueckert et al. (1990) reported that the oxygen concentration in the root
compartment is positively and proportionally correlated with aerenchyma size in kallar
grass (Leptochloa fusca L. Kunth) and rice. Differences in tap root porosity was
correlated with differences in 02 transport from shoot to root in anaerobically grown old
roots of Rumex spp (Laan et al., 1989).
Yu et al. (1969), testing waterlogging tolerance of barley (Hordeum vulgare L.),
corn, sunflower, tomato, and wheat, found that roots grown in non-waterlogged soils have
lower porosity than those grown in fully waterlogged soils. Also it was shown that
7
waterlogging injury is less in corn, sunflower, and the wheat cultivar "Pato", which have
more porous roots than for other species.
In wheat, seminal roots of 10-day old seedlings grown on flooded sand contained
25-30 % gas space in the cortex compared with 6-8 % in aerated roots (Erdmann and
Wiedenorth, 1988). Barrett-Lennard et al. (1988) reported up to 14 % root porosity for
the anaerobically grown wheat cultivar "Gamenya".
Metabolic adaptation
Shortly after flooding, plants respond with sequential changes in metabolism and
physiological processes. Other than morphological adaptations, physiological adaptation,
such as an increase in ethylene production has been observed when plants were subjected
to waterlogging conditions. Ethylene may be a casual agent in aerenchyma production. By
introducing ethylene into a nutrient solution, Drew et al. (1979) were able to observe
induction of aerenchyma formation in the root cortex of corn. Kawase (1981b) reported
the development of cortical parenchyma in sunflowers, tomatoes and beans (Phaseoles
vulgari) after applying ethylene to their stems or roots. Mango ( Mangifera indica L. )
trees subjected to flooding, responded by producing ethylene, which in turn induced the
formation of lenticle hypertrophy (Larson et
al.,
1993). An increase in ethylene
concentration was observed when rice was submerged for one to three weeks, and this
resulted in an increase in inter-nodal length (Metraux and Kende, 1993). Crawford (1978),
indicated that the morphological explanations of flooding tolerance were inadequate and
suggested that species sensitive to flooding suffer an acceleration of glycolysis and
production of ethanol. Tolerant species avoid this acceleration of glycolysis, and also
undergo a metabolic switch from ethanol to malate production (Crawford, 1978; Joly,
1994; Sachs et al., 1980; Setter et al., 1987; Vartapeterian et al., 1987; Wignarajah et al.,
1976).
8
Plants survive flooding by different metabolic pathways which include storing
products of photosynthesis near by the demanding sink, maintaining the glucose supply,
adjusting carbon metabolism, adequately controlling glycolysis, synthesizing essential
proteins, maintaining the energy charge, and by retaining the integrity of membranes and
organelles (Davies, 1980; McManmon and Crawford, 1978; Pfister-Sieber and Brindle,
1994). Wheat seedlings responded to long term oxygen deficit by accumulating soluble
carbohydrates in their roots and shoots (Albrecht et al., 1994). An elevated sugar content
was observed in response to flooding in wheat (Albrecht et al.
,
1993 & 1994; Barret-
Lennard et al., 1988), barley (Limpinunatana and Greenway, 1979; Benjamin and
Greenway, 1979), maize (Atwell et al., 1985), and beans (Papenhuijzen, 1983).
Remobilizing the stored carbohydrate immediately after resuming aeration, may be an
adaptive mechanism to low oxygen concentration in the root zone. Leakage of fr from
the vacuole to the cytoplasm due to oxygen stress may cause cytoplasmic acidosis which
reduces the survival time of plants under flooded conditions. Regulation of cytoplasmic
pH at nearly neutral is a prerequisite for survival under flooding (Roberts et al., 1985).
Although oxygen is required for normal growth and development, after severe
waterlogging stress the restoration of the normal 02 concentration can be toxic to plant
tissue, because it generates active 02 species, such as superoxide radical (0-2), hydrogen
peroxide (H202) and the hydrogen radical (Off), which are cytotoxic to plant cells
(Scandalias, 1993). In order to survive post-anoxic injury, resistant plants may use defense
mechanisms such as use antioxidants, and enzymes to eliminate potentially harmful active
radicals (Albrecht, and Wiedenorth, 1994; Crawford, 1992, 1993; Hunter, et al
Monk et al., 1987).
1993;
9
Genetic variability in waterlogging tolerance
The implementation of improved drainage systems in the field will definitely reduce
waterlogging problems. However, the large investment needed may not be economical and
the canal systems required may sometimes be impractical. The problem may be partly
alleviated by developing genotypes which are tolerant to continuous or temporary
waterlogging conditions.
There exists large variation in the degree of tolerance to anoxia among most crops
(Pfister-Sieber and Brandle, 1994). Genetic variation and its association with evolution of
adaptation to flooding was reviewed by Davy et al. (1990). Also genetic differentiation in
photosynthesis response to flooding was reported by McGee et al. (1981), and Huang, et
al. (1994a). Varietal differences in response to flooding have been reported in wheat
(Huang, et al., 1994; Poyasa, 1984; Sayre et al., 1994; Thomson, et al., 1992; van Ginkel
et al., 1992), maize and barley (Fausy et al., 1985; Takeda and Fukuyama, 1987), and
soybean (Glysine max; Hou and Thseng, 1991).
The occurrence of aerenchyma in roots of wetland plants like rice and wild rice
(Zizania aquatica) even under well-oxygenated conditions suggests that its development
is under genetic control in these species (Jackson and Drew, 1984). The presence of one
dominant gene for submergence tolerance in rice cultivars FR13A, BKNFR(76106-16-01-0) and Kurkaruppan was reported by Setter et al. (1996).
Large differences between wheat populations in their response to flooding
treatment, both in terms of the production of tillers at the vegetative stage, as in
reproductive yield, were observed (Davies & Hillman, 1988). Genotypic differences in
flooding tolerance in wheat were expressed following a five or seven day stress (Poysa,
1984). Significant cultivar differences in wheat in response to waterlogging at both
germination and booting stage were reported by Cai (1990) and Xiang et al. (1994).
Differences were observed in plant survival rate, number of tillers, plant height, rate of leaf
senescence, number of spikes per plant, and grain yield. The presence of a few genes in
grasses for anoxic tolerance was suggested by Mujer et al. (1993).
10
Van Ginkel et al. (1992), identified 14 spring wheat lines out of 1344 different
genotypes targeted for high rainfall environments which were found to be tolerant to
flooding when screened under extended waterlogged conditions in the field. They further
evaluated these promising lines from the screening trials in replicated yield trials by
flooding the plots for five weeks starting from the early tillering stage, two weeks after
emergence. Six top yielding genotypes were identified, of which four yielded more than
2 MT/ha under waterlogging stress. The check cultivar (Pato) yielded 1.6 MT/ha. These
genotypes also performed well under waterlogged stress extending over different growth
stages, from early, the middle or at the end of the crop season. (Sayre et al., 1994).
Genetic variation in response to flooding was observed when measurements were made in
terms of number of tillers, leaf chlorosis, senescence, fertility, grain yield and kernel
weight.
Research into flooding tolerance of wheat has focused mainly on the understanding
of structural, morphological, anatomical, biochemical and metabolic adaptation caused by
lack of oxygen in plant tissue, and species and varietal differences have been compared in
response to waterlogging stress. Very little attention has been given to the genetics of
waterlogging tolerance in wheat (Cao et al., 1994). The present study was conducted to
observe the nature of genetic inheritance associated with waterlogging tolerance in
selected spring wheat cultivars.
11
OBJECTIVES
The objectives of this study were:
1) To determine the nature of inheritance in the expression of waterlogging tolerance in
selected spring wheat crosses.
2) To study certain morphological characters of selected spring wheat genotypes to
waterlogging tolerance.
3) To investigate the selected wheat genotypes for their ability to transport 02 from the
shoot to the root.
Based on the above objectives, field and laboratory experiments were conducted.
12
CHAPTER II
FIELD EXPERIMENTS (INHERITANCE STUDIES)
MATERIALS AND METHODS
Three spring bread wheat advanced lines tolerant to waterlogging under conditions
in northwestern Mexico, Parula/Sara, Ducula, and Vee/Myna, one sensitive cultivar Seri-
82, and one sensitive advanced line, Kite/Glen, were received in 1992 from Dr. Maarten
van Ginkel CIMMYT-Mexico. The five parents (Appendix, Table 1.) were crossed in all
possible combinations including reciprocals (totaling 20 crosses) in the greenhouse at
Oregon State University, Corvallis. The F1, F2, and the BC (to both parents) generations
were also generated in the greenhouse.
Field experiments were conducted for two years in north-western Mexico in
Ciudad Obregon at the CIANO Agricultural Experimental Station in collaboration with
CIMMYT. The station is located at an elevation of 39 m above sea level at 27° N latitude
and 109° W longitude. The soil of the experimental station is classified as coarse sandy
clay mixed montmomorillonitic type calliciorthid, low in organic matter ( < 10 mg g"' )
with pH near neutral. The amount of rainfall during the crop cycle is about 50 mm and the
wheat crop growth is totally dependent on irrigation.
Year I
In the 1992/93 Ciudad Obregon cropping cycle, the F1, F2, BC's and the parents
were space planted in a randomized complete block design with three replications. Plots of
0.6 m x 2 m for F1's and BC's, and 1.2 m x 2 m for F2's were used. Planting was done on
13
November 30, 1993 in dry soil and ridges were constructed subsequently to form
irrigation basins. The size of the basins was 3m x 10m for F2's, and 3m x 6m for the F3's
and the BC's. Initial irrigation for germination was given, and equivalent to about 100
mm. Excess water was drained from the basins after 45 minutes. One additional normal
irrigation was given before flooding was induced. Basins were flooded 2 to 3 cm above
the soil level when the plants reached the Zadoks' et al., DC scale 14 (Zadoks, et al.,
1974). This level was maintained by frequent replenishment of the basins. All basins were
drained when plants reached the DC scale 37. Two more irrigations were given before
maturity. Due to unexpected rain certain plots could not be properly drained after the
initial irrigation. This resulted in some irregular germination, and affected plant growth
before the flooding treatment was applied.
Notes were taken on heading date, and the date when plants reached DC scale 55.
Number of plants were counted before and after flooding, and grain yield was measured
on a per-plot basis.
Year II
The second year of the study consisted of two separate sets of experiments; study
1 and study 2.
Study 1
The material for this set of experiments consisted of parents and F3 lines generated
from 100 randomly selected F2 plants/cross, from all 20 crosses. Each cross was treated as
a separate experiment, and the 100 F3 lines, the two respective parents and the three
checks (The three parents involved in the other crosses) were planted in an alpha lattice
design (15 entries per each of seven blocks) with two replications. The seven blocks were
divided into two irrigation basins for management purposes. One, 6.25 m x 9 m,
containing four blocks, and the second, 4.5 m x 9m, accommodated three blocks. Seed
rate was 110 kg/ha. Prior to planting, nitrogen and phosphorus fertilizers were applied at
14
the rate of 100 units N/ha and 40 units P205/ha, respectively. The fungicide Folicur was
applied at DC scale 60 and 75 to protect the crop against leaf rust (Puccinia recondita).
Each entry was planted in plots of three rows, lm long. The spacing between rows was 15
CM.
Seeds were planted in dry soil on 1 Dec. 1993, and the basin's outer ridges were
subsequently constructed. Initial irrigation was applied at the equivalent of 100 mm with
the excess water drained after 45 minutes. One more flood-irrigation was given at DC
scale 20. When the plants reached DC scale 31, all basins were filled with water up to 5 to
7 cm above the soil surface (Fig. A-1, and A-2), and this level was maintained for 40 days
by replenishing the basins frequently until all of the genotypes had reached at DC scale 59
(Fig. A-3). Then the water was drained from the basins. At DC scale of 71, one additional
flood-irrigation was given to allow non-stressed maturing.
Notes were taken on heading date, the date when 75 % of the plants in the plot
were at DC scale 55. Advancement of percentage foliar chlorosis was recorded four times
at intervals of seven days starting from DC scale 41. Plant height, was measured in cm
from the base of the culm to the tip of the spike on the tallest tillers excluding the awns.
Biomass was scored visually using a 1-10 scale, one being low and 10 high. Grain yield
was weighed on a plot basis and adjusted to 12 % moisture content. Kernel weight
(grams) was determined from 200 randomly sampled kernels.
Fig. A-1. Basins were constructed immediately after planting the F3 lines and the plants were
flooded 5 to 7 cm above the soil level for 40 days starting from the first internode stage.
Fig. A-2. Flooded F3 lines segregating for leaf chlorosis.
Fig. A-3. Segregating F3 lines after draining the water from the basins.
18
Study 2
A second experiment consisting of parents (P1, and P2), F1, F2, P1 x F1, P2 x F1, F3,
and reciprocal generations for each cross, was conducted to further investigate the nature
of inheritance of waterlogging tolerance by way of generation mean analysis. Seeds from
each cross were planted in a single basin of 6 m x 4.5 m size in an alpha lattice design with
three replications. Plots of three, 1 m long rows with 15 cm spacing were used for each
entry. Seeds were planted in dry soil on Dec. 7, 1993. The trial management for this study
was the same as for study 1.
Notes were taken on heading date, percentage leaf chlorosis (four times), plant
height, biomass, grain yield and kernel weight.
Statistical Analysis
Means and variances were computed to compare parents and progenies
performance using the SAS Mixed procedure ( McLean, et al., 1991; SAS Institute Inc.,
1987). The Chi-square test was used to estimate the number of genes contributing to
waterlogging tolerance. The generation mean analysis procedure outlined by Mather and
Jinks (1982) was used to study the nature of gene action. Means of all generations, pooled
additive effects, and pooled dominant effects were fitted by the weighted least square
method.
A joint scaling test (Cavalli, 1952), using information from the six generations, was
employed to detect possible allelic interactions. Genotypic and phenotypic variances and
covariances (Singh and Chaudhary, 1977) for F3 lines of each cross were estimated using
the output from the SAS multivariate analysis (MANOVA) procedure. Phenotypic (rp) and
genotypic (rg) correlations, were calculated using the following formula: rp = apx,y / (apx x
apy)-0..5 ; and rg = agx,y / (agx x agyr. Where ap,y
=
phenotypic covariance, agx, y
genotypic covariance; apx and apy are phenotypic variances and agx and a
variances (Falconer, 1989; Griffiths et al., 1993).
=
are genotypic
19
Path coefficient analysis (Singh and Chaudhary,
1977)
was used to analyze the
direct and indirect impact of chlorosis and heading date on grain yield and yield
components. Broad sense heritablity was estimated using the procedure outlined by Allard
(1960).
Area under chlorosis progress curve (AUCPC) was computed using the following
formula: AUCPC = ERxi
Madden,
1990).
procedure.
xi+i)/2)1(ti+1
(Bjarko and Line,
1988;
Campbell and
The ANOVA for the AUCPC was computed using the SAS Mixed
20
RESULTS
Year I
Unfortunately plant stand percentage was negatively affected by excess water
before emergence. The effect of flooding on plant stand was minimum after the seedlings
reached the DC scale 14 (Table A-1). Since plants were depressed even before the
flooding treatments was induced, the flooding treatment was halted at Zadoks' et al., DC
scale 37.
The first year experiment indicated that heading date was delayed for all
cultivars when the waterlogging treatment was terminated at an early growth stage. Plants
headed earlier in the second year experiment (Table A-5) because the flooding treatment
was extended up to the DC scale 59.
Table A-1. Mean of heading date, percentage' of surviving plants before and after flooding
Crosses
No Type P,
1
2
3
4
5
6
7
8
9
10
TxT
TxT
TxS
TxS
TxT
TxS
TxS
TxS
TxS
SxS
Prl/Sara
Prl/Sara
Prl/Sara
Prl/Sara
Ducula
Ducula
Ducula
Vee/Myna
Vee/Myna
Seri-82
Heading date
P2
F1
Ducula
118
Vee/Myna 120
Seri-82
117
Kite/Glen 113
Vee/Myna 114
Seri-82
112
Kite/Glen 108
Seri-82
116
Kite/Glen 109
Kite/Glen 114
% Survival before % Survival after
flooding
flooding
F2
BC1 BC2 F1
F2
BC1 BC2 F1
F2
BC1 BC2
118
119
120
117
114
121 120 67
125 121 58
53
38
79
47
44
47
68
57
75
25
59
57
54
46
72
32
95
83
82
88
93
53
75
90
92
87
72
81
83
68
98
85
93
91
84
90
98
55
86
86
111
108
117
111
113
120
122
114
115
109
117
117
116
114
118
114
110
117
110
115 109
57
64
54
30
41
61
58
41
41
56
43
43
47
39
29
29
24
54
53
66
85
85
64
64
91
65
70
66
96
69
91
52
79
86
53
75
77
93
'Percentage of plants surviving before flooding was calculated from the initial number of
planted seeds, whereas percentage survival after flooding was calculated from the number
of plants counted before flooding was imposed.
21
Year H
Prl/Sara, Ducula, and Vee/Myna were the most waterlogging tolerant parents,
measured as leaf chlorosis response and no significant difference for percent chlorosis was
observed between them. Seri-82 and Kite/Glen were the most sensitive parents (Table A-
2). This confirmed the previous classification of Ducula, Prl/Sara and Vee/Myna as
tolerant and Seri-82 and Kite/Glen as sensitive to waterlogging stress (Sayre et al., 1994;
Van Ginkel et al., 1991). Other agronomic data for the parents, as they occurred with their
respective F3's, are presented in Table 3.
Results from Study 2 indicated that the expression of tolerance to waterlogging did
not appear to be influenced by maternal effects, since no significant differences for the
percentage chlorosis could be observed between reciprocal crosses except for the cross
Vee/Myna x Seri-82 (Table A-4 ). Although significant differences between reciprocals for
the cross Vee/Myna x Seri-82 were indicated, no alternative conclusions could be drawn,
noting the response of both parents in the other F1 crosses (Table A-4). Hence F3 data of
both reciprocals from study 1 were combined per cross for further analysis, resulting in
200 families per combination. The F1 generations were intermediate between the two
parents indicating that resistance was additive. The analysis of variance for F3 family data
indicated significant differences between populations for percent chlorosis, (data from the
fourth scoring date) AUCPC, heading date, biomass, plant height, grain yield, and kernel
weight.
The F3's from the cross between the two tolerant parents, Ducula and Vee/Myna,
had the lowest mean value for percent chlorosis (18 %) and AUCPC (341), and the
highest mean value for plant height (83 cm), biomass (6.98) and grain yield (205 g) (Table
A-5). The sensitive by sensitive cross, Seri-82 x Kite/Glen showed the lowest mean value
for plant height (67 cm), biomass (4.52), grain yield (106 g), and kernel weight (25.4 g)
and the highest mean value for percent chlorosis (70 %) and AUCPC (1382). On average
however, there was no difference in heading date of the progenies from these two crosses.
Low yield in the cross Seri-82 x Kite/Glen was associated with high percentage leaf
chlorosis, low biomass and kernel weight, (Table A-3). The plot grain yield of highest
22
yielding tolerant x tolerant cross was equivalent to 4.5 T/ha, and that of the lowest
yielding sensitive x sensitive cross was 2.4 T/ha.
Table A-2. Mean percentage leaf chlorosis (data from the fourth scoring date) for parents
and F3 lines. Each cross was grown in a separate experiment alongside all five parents.
Thus 10 experiments were planted under waterlogged conditions (Year II; study 1).
Mean percent chlorosis
Crosses
No type'
Parents
Parente
F3 P12 P2 P3
P4
P5
Lsd 5% CV %
1
T x T Prl/Sara
Ducula
29 25
13
14
68
71
16
25
2
T x T Prl/Sara
Vee/Myna
22 21
15
19
71
74
12
22
3
T x S Prl/Sara
Seri-82
39
19
13
10
57
78
14
18
4
T x S Prl/Sara
Kite/Glen
41
18
11
15
55
62
17
22
5
T x T Ducula
Vee/Myna
18
23
13
9
54
87
10
19
6
T x S Ducula
Seri-82
32
15
10
11
70 64
12
18
7
T x S Ducula
Kite/Glen
40 23
10
13
72 72
16
20
8
T x S Vee/Myna
Seri-82
36
20
11
11
78 79
16
22
9
T x S Vee/Myna
Kite/Glen
41
20
11
10
68 83
16
20
10
SxS
Kite/Glen
70
21
10
10
78 74 23
19
37 20 12
12
66 74
20
Seri-82
Mean across study 1
15
I Cross type : T = tolerant; S = sensitive.
2 PI = Prl/Sara, P2 = Ducula, P3 = Vee/Myna, P4 = Seri-82, P5 = Kite/Glen.
23
Table A-3. Means of agronomic traits for parents included in each cross grown under
waterlogged conditions (Year II; study 1).
Cross
Parents
Response to CHL AUCPC Head Height BM
waterlogging (%)
1
2
3
4
5
6
7
8
9
10
Yield
ing
(cm)
(1-10) (g/plot)
TKW
(g)
P1 Prl/Sara
Tolerant
25
578
93
78
5.02
P2 Ducula
Tolerant
13
286
85
78
8.00 259
P1 Prl/Sara
Tolerant
21
524
91
80
5.40
122
P2 Vee/Myna Tolerant
19
405
91
76
5.56
148
P1 Prl/Sara
Tolerant
19
489
91
80
5.99
146
34.25
P2 Seri-82
Sensitive
56
1015
83
71
5.02
136
27.45
P1 Prl/Sara
Tolerant
18
375
90
81
6.17 136
34.60
P2 Kite/Glen Sensitive
61
1146
80
74
5.78
26.75
P1 Ducula
13
243
85
83
7.69 248
P2 Vee/Myna Tolerant
8
240
88
81
6.66 233
P1 Ducula
Tolerant
10
264
85
78
7.49 253
40.7
P2 Seri-82
Sensitive
70
1332
84
67
5.09 113
27.25
P1 Ducula
Tolerant
10
249
85
83
8.44 273
39.1
P2 Kite/Glen Sensitive
72
1501
80
64
4.05 81
21.85
P1 Vee/Myna Tolerant
10
233
90
80
6.85
-
P2 Seri-82
Sensitive
77
1515
83
61
4.56 95
P1 Vee/Myna Tolerant
10
265
90
84
7.25
P2 Kite/Glen Sensitive
83
1677
79
65
4.25 86
P1 Seri-82
Sensitive
78
1522
84
63
4.46 82
24.8
P2 Kite/Glen Sensitive
73
1418
80
61
4.52 82
23.2
Tolerant
143
117
198
229
CHL = % Chlorosis (Data from the fourth scoring date); AUCPC = Area under chlorosis
progress curve; Heading = Number of days to heading; Height = Plant height (cm); BM =
Visual score of biomass ( 1-10 Scale, 1= low, 10 = high); Yield = Grain yield (g /plot);
TKW = Weight of 1000 seeds (grams);
= no notes were not taken on TKW.
24
Table A-4. Mean percentage leaf chlorosis for parents (P1, P2) , F1 (P1/P2) and Fir
reciprocals (P2/Pi), under waterlogged conditions (Year II; study 2).
Mean percentage leaf chlorosis
Crosses
No
Type
1
P2
P1
F1
Fir
P2
1_,Sd50/0 CV
T x T Prl/Sara
Ducula
52
60
57
17
31
41
2
T x T Prl/Sara
Vee/Myna
36
22
20
7
18
41
3
T x S Prl/Sara
Seri-82
18
45
60
37
22
30
4
T x S Prl/Sara
Kite/Glen
20
65
57
82
27
27
5
T x T Ducula
Vee/Myna
10
10
10
7
7
36
6
T x S Ducula
Seri-82
12
33
25
68
17
28
7
T x S Ducula
Kite/Glen
10
27
20
88
23
33
8
T x S Vee/Myna
Seri-82
7
25
48
43
20
40
9
T x S Vee/Myna
Kite/Glen
8
33
40
88
21
29
10
SxS
Kite/Glen
62
65
47
82
30
25
P1
Seri-82
25
Table A-5. Means of agronomic traits for F3 lines grown under waterlogged conditions
(Year II; study 1).
CHL AUCPC Heading Height BM Yield
Crosses
No
Type
P1
P2
(cm)
(
(1 - 10)(g/plot)
TKW
(g)
1
T x T Prl/Sara
Ducula
29
537
88
76
5.89
154
2
T x T Prl/Sara
Vee/Myna
22
452
91
81
6.35
147
3
TxS
Prl/Sara
Seri-82
39
775
86
76
5.85
149
28.4
4
TxS
Prl/Sara
Kite/Glen
41
795
86
75
5.25
120
28.2
5
T x T Ducula
Vee/Myna
18
341
84
83
6.98 221
6
TxS
Ducula
Seri-82
32
609
84
77
6.51
205
34.7
7
TxS
Ducula
Kite/Glen
40
717
83
74
5.95
164
31.6
8
TxS
Vee /Myna
Seri-82
36
662
86
75
6.07 171
9
TxS
Vee/Myna
Kite/Glen
41
753
86
78
5.97 162
10
SxS
Seri-82
Kite/Glen
70
1382
84
67
4.52
106
25.4
CHL = % Chlorosis (Data from the fourth scoring date); AUCPC = Area under chlorosis
progress curve; Heading = Number of days to heading; Height = Plant height (cm); BM =
Visual score of biomass ( 1-10 Scale, 1= low, 10 = high); Yield = Grain yield (g /plot);
TKW = Weight of 1000 seeds (grams);
= no notes were not taken on TKW.
Qualitative Approach (Year II; study 1)
In the qualitative study the phenotypic distributions of random F2-derived F3 lines
were used. Lines were classified into tolerant, sensitive and segregating categories in
regard to percentage leaf chlorosis. Each cross consisted of 200 F3 families when
reciprocals were combined. The frequency distributions for percentage leaf chlorosis of
the F3 lines for a tolerant x tolerant, a tolerant x sensitive, and a sensitive x sensitive
crosses are shown in Figures A-4, A-5 and A-6.
26
35
30
Ducula (D) x Vee/Myna (V)
(Tolerant x Tolerant)
25
20
15
10
5
0
5
15
25
35
25
45
55
65
75
85
95
85
95
Ducula x Kite/Glen
(Tolerant x Sensitive)
20
Ducula
15
Kite/Glen
10
5
0
5
15
30
25
35
45
55
Seri-82 x Kite/Glen
(Sensitive x Sensitive)
25
65
75
A-6
Kite/Glen
20
Seri-82
15
1
10
5
EszmaJ
0
5
15
25
35
45
55
65
75
Mean percentage Chlorosis
85
95
Fig . (A-4, A-5, and A-6 from top to bottom). Frequency distribution for F3 lines for
mean percentage (%) leaf chlorosis (data from the fourth scoring date) for the crosses
Ducula x Vee/Myna, Ducula x Kite/Glen, and Seri-82 x Kite/Glen respectively. ( )
Indicates the mean of the parents.
27
Genotypes were classified for percentage chlorosis as transgressive tolerant,
tolerant, segregating, sensitive, and transgressive sensitive, relative to the range of tolerant
and sensitive parents (Table A-6). Genetic models based on the ratios of the above
classifications were fitted, and a Chi-square test for goodness of fit was used.
When the tolerant Prl/Sara was crossed with the two sensitive genotypes Seri-82
and Kite/Glen, 1:2:1 ratios of tolerant
:
segregating
:
sensitive were observed. This
indicated the presence of a major tolerance gene (Wt/) in PrUSara.
In the crosses among tolerant lines of Vee/Myna with Ducula, and Vee/Myna with
Prl/Sara, a two gene segregation ratio of 15:1 (tolerant
:
transgressive sensitive) was
observed; and noted as Wt2 and Wt3, and Wt3 and Wt4, respectively. Sensitive F3
progenies in these crosses were observed when both of the segregating loci were
homozygous recessive. For F3 progenies from the remaining third tolerant x tolerant cross
between Prl/Sara and Ducula a 13 tolerant : 3 transgressive sensitive ratio was noted. This
also indicated that two genes (Wt2 and Wt4) were segregating. From the segregation
ratios it was evident that the three tolerant parents carry different genes (Fig. A-7).
When the tolerant line Ducula was crossed with the two sensitive genotypes, Seri-
82 and Kite/Glen, three-gene segregation ratios of 34:23:7 and 19:35:10 for tolerant
:
segregating : sensitive were observed for percent chlorosis, respectively. F3 lines from the
crosses of the tolerant Vee/Myna with Seri-82 and Kite/Glen segregated 34:20:10, and
12:42:10 for tolerant : segregating : sensitive, respectively. The segregation ratios of these
four crosses are in agreement with a segregation of three genes (Wtl/wtl, Wt2/w12, and
Walwt4 and Wtl/wtl, Wt3/wt3, and Wt4/wt4, respectively). Apparently all the tolerant
parents carry one major gene (Wil) in common.
The cross between the two sensitive parents showed 1 tolerant : 15 sensitive ratio
(Table A-6). The transgressive tolerant lines observed in this cross may be contributed by
non-allelic minor genes present in both parents. It appeared that the two sensitive parents
carry a similar minor gene (Wt4Wt4). The existence of this minor gene was supported by
the ratios observed in other crosses involving Seri-82 or Kite/Glen.
In summary the segregation ratios of the F3 lines indicated that total four genes
control tolerance to waterlogging stress. The presence of Wu plus one of the other three
28
genes (Wt2/wt2, Wt31w13, Wt4Iwt4) in homozygous dominant condition appear sufficient
to obtain a significant level of tolerance. When broad sense heritabilities (H2) were
computed, estimates from 58 percent to 83 percent were observed (Table A-6).
Table A-6. Chi-square (X2) goodness of fit to ratios observed in segregating F3 lines for
percentage leaf chlorosis in response to waterlogging stress and broad sense heritability.
Parents
Type
P1
T x T Prl/Sara
No of
Ratios
P2
Observed Expected genes
Ducula
169:31
T x T Prl/Sara Vee/Myna 190:10
T x S Prl/Sara
Seri-82 48:95:57
T x S Prl/Sara Kite/Glen 50:99:51
T x T Ducula Vee/Myna 189:11
T x S Ducula
Seri-82 102:72:22
T x S Ducula Kite/Glen 53:112:35
T x S Vee/Myna Seri-82 95:72:33
T x S Vee/Myna Kite/Glen 38:127:35
S x S Seri-82 Kite/Glen
16:184
Values
X2
1.19
13:3 a
15:1 a
1:2:1 b
1:2:1 b
15:1 a
2
2
0.74
1
1.31
34:23:7 b
19:35:10 b
34:20:10 b
12:42:10b
3
1:15c
2
1
2
3
3
3
0.03
0.33
0.37
1.42
2.56
0.64
0.74
P
0.50-0.25
0.50-0.25
0.75-0.50
0.95-0.75
0.75-0.50
0.95-0.75
0.50-0.25
0.25-0.1
0.75-0.50
0.50-0.25
Tolerant gene
Postulation
P2
%
Wt1Wt4 Wt1Wt2 58
Wt1Wt4 Wt1Wt2 66
Wt1Wt4
Wt4
83
Wt1Wt4
Wt4
70
Wt1W12 Wt1Wt3 73
Wt1Wt2
Wt4
77
Wt1Wt2
Wt4
65
Wt1Wt3
Wt4
73
Wt1Wt3
Wt4
67
Wt4
Wt4
63
P1
a Tolerant parental type : transgressive sensitive.
b Tolerant parental type : segregating : sensitive parental type.
Transgressive tolerant : sensitive parental type.
T = Tolerant; S = Sensitive
X2 = Chi-squared values to test the goodness of fit.
P = Probability of a larger chi-squared values than the observed one (The goodness of fit
is accepted for P > 0.05).
H2 = Broad sense heritability.
Wt = Response to waterlogging.
H2
29
1
3
Fig. A-7. Postulated tolerance genes (based on leaf chlorosis data) in the five parents and
number of segregating genes identified in all crosses. Genes carried by individual parent
are indicated by capital letters (Wt1Wt4, Wt4, etc.), and the number of segregating genes in
the crosses are indicated by the numbers on the double arrowhead lines.
30
Quantitative approach (Year II; study 2)
A joint scaling test (Cava lli, 1952) and a six parameter model (Mather and Jinks
1982) were used to test the presence of non-allelic gene interactions, and to determine the
magnitude and significance of the different genetic components. Only the tolerant x
sensitive crosses were studied. Percentage leaf chlorosis was used as measure of tolerance.
The joint scaling test was adequate to explain the additive-dominance model for
the crosses Ducula x Kite/Glen, Vee/Myna x Seri-82 and Vee/Myna x Kite/Glen as no
significant P value for the X2 test (P > 0.05) was observed for these crosses (Table A-7).
In these crosses both the additive and dominance effects were significant for the
inheritance of tolerance to waterlogging stress. The negative value for dominant gene
effects indicated that in these crosses the level of percentage chlorosis would be decreased
relatively to the mid parent. The joint scaling test indicated that epistasis was involved in
the inheritance of tolerance to waterlogging stress in the crosses Prl/Sara x Seri-82,
Prl/Sara x Kite/Glen and Ducula x Seri-82 (P value < 0.05). Hence the six parameter
model was used to explain the genetic variability in these crosses.
In the crosses Prl/Sara x Seri-82 and Ducula x Seri-82 the additive gene effect was
significant (Table A-8) and it was the major contributor in reducing percentage leaf
chlorosis. Although the joint scaling test indicated the presence of non allelic gene
interactions the six parameter model failed to detect this interactions showing that in these
crosses the non allelic gene effects were not significant.
For the cross Prl/Sara x Kite/Glen the additive by dominant effects were significant
in reducing percentage leaf chlorosis.
The joint scale and the six parameter model indicated that the additive gene action
had the most pronounced effect on percentage chlorosis for most of the crosses. A similar
result is also obtained from the qualitative approach. Cao et al. (1994) indicated that
tolerance to waterlogging stress in wheat was mainly controlled by additive gene effects.
The presence of significant additive effects between crosses indicates that election
for tolerance to waterlogging stress could be carried out in early segregating generations.
31
Table A-7. Joint scaling test for additive dominance effects. Estimate of genetic effect
and their standard errors for percentage leaf chlorosis for Tolerant x Sensitive crosses.
Para-
P1: Prl/Sara Prl/Sara
Ducula
Ducula
Vee/Myna
Vee/Myna
meter
P2: Seri-82 Kite/Glen
Seri-82
Kite/Glen
Seri-82
Kite/Glen
m
43.9±3.3**
50.5±4.8**
38.7±2.3**
49.2±0.9**
29.5±4.4**
48.4+1.2**
a
24.9±3.3**
25.6±4.7**
30.1±2.3**
39.1±0.9**
23.6±4.3**
39.9+1.2**
d
4.1±4.9
21.5±9.9*
-8.3+2.8**
-22.4+.7*
-15+6.1*
-12.3±5.8*
X2
10.3
10.6
20.4
6.4
6.7
3.8
P
0.02-0.01
0.02-0.01
<0.01
0.05-0.1
0.05-0.1
>0.1
m = mean, a = additive, d = dominant,
X2 = Chi-square for testing the adequacy of the additive-dominance model ;
P = Probability level associated with the chi-square value of the deviations sum squares for the
model.
*, ** significant differences at P = 0.05 and at 0.01 respectively.
Table A-8. Six parameter model. Estimate of genetic effect and their standard errors for
percentage leaf chlorosis for the Tolerant x Sensitive crosses for which the additive dominance model was insufficient.
para-
P1: Prl/Sara
meter
P2: Seri-82
Prl/Sara
t value Kite/Glen
Ducula
t value Seri-82
t value
m
46.7+ 9.3**
5.02
61.7+6.0**
10.2
33.3±8.3**
4.01
a
-20+5.5**
-3.63
18.3±15.1
1.21
-40±8.9**
-4.49
d
37.5±39.5
0.95
-15.8±40.3
-0.39
-6.7±37.9
-0.18
aa
20± 41.3
0.48
-30±31.1
-0.96
-0.0±36.6
0.0
ad
-10.8±9.3
-1.16
49.2±16**
3.08
-11.7±9.3
-1.25
dd
-81.7±46.1
-1.77
45+68.9
0.65
13.3±49.3
0.27
m = mean, a = additive, d = dominant, aa = additive x additive, ad = additive x dominant,
dd = dominant x dominant effects.
** significant differences at 0.01 level
The calculated t values (Estimate of genetic effect / standard error of genetic effect) are to be
compared with 1.96 or 2.58 which are the tabulated values oft at P = 0.05 and 0.01 respectively.
.
32
Associations
Waterlogging tolerance in some genotypes may be related to an avoidance or
escape mechanism due to delayed heading. Understanding the relations between leaf
chlorosis and heading date and their impact on grain yield and yield components would
lead to a clearer understanding of the genetics of tolerance to waterlogging stress.
To investigate the effect of chlorosis and heading date on seed yield and yield
components, genotypic (rg) and phenotypic (rp) correlations were computed for F3 lines on
a plot basis.
Negative genotypic and phenotypic correlation coefficients were observed for the
association of percent chlorosis with heading date, plant height, biomass, seed yield and
kernel weight (Table A-9 and A-10). These associations are larger in magnitude for
sensitive by sensitive, and sensitive by tolerant, than for tolerant by tolerant crosses. This
is
explained by the larger variances in those former crosses. Negative genotypic
correlation between grain yield and leaf chlorosis was reported by Van Ginkel et al.
(1992). In other studies waterlogging has been shown to reduce kernel weight significantly
(Cai et al., 1994)
The trend for both genotypic and phenotypic associations of heading date with
plant height, biomass, seed yield, and kernel weight were positive, except for a few
crosses (Table A-11 and A-12). The magnitude of the genotypic correlations tended to be
larger than the phenotypic correlations for most of the traits measured (Mean of Table A-9
vs A-10, and A-11 vs A-12). Chlorosis and heading date influenced yield and yield
components in opposite directions for most of the crosses.
The negative impact on yield by increased chlorosis was larger than the positive
effect of delayed heading. The mean estimates of the correlation coefficients of chlorosis
(Table A-9 and A-10) are larger than the those of heading date (Table A-11 and A-12).
The genetic correlation between percentage leaf chlorosis and heading date was negative
and significant (Table A-9).
33
The magnitude of the mean associations of percent chlorosis with plant height,
biomass, grain yield and thousand kernel weight (rg = -0.64, -0.72, -0.69, and -0.79,
respectively; and rp = -0.53, -0.56, -0.62, and -0.72, respectively; Table A-9 and A-10) are
larger than with heading date (rg = 0.37, 0.33, 0.17, and 0.26, respectively; rp = 0.24, 0.18,
0.09, and 0.22, respectively; Table A-11 and A-12).
Table A-9. Genotypic correlations of percent chlorosis with heading date (Heading), biomass (BM),
plant height (Height), grain yield (Yield), and thousand kernel weight (TKW) for F3 lines.
Prl/Sara Prl/Sara
P2
Ducula
Prl/Sara Prl/Sara
Vee/Myna Seri-82
Kite/Glen
Ducula
Ducula
Vee/Myna Seri-82
Ducula
Vee/Myna Vee/Myna Seri-82
Kite/Glen
Seri-82
Kite/Glen
Kite/Glen
Mean
Heading -0.66**
-0.57**
-0.68**
-0.61**
-0.69**
-0.54**
-0.51**
-0.87**
-0.76**
-0.73**
-0.66**
BM
-0.79
-0.73
-0.96**
-0.58
-0.64
-0.89
-0.33
-0.80
-0.65
-0.87
-0.72
Height
-0.79**
-0.66**
-0.88**
-0.56**
-0.58**
-0.84**
-0.16**
-0.87**
-0.54
-0.55**
-0.64**
Yield
-0.43**
-0.38**
-0.81**
-0.73**
-0.51**
-0.71**
-0.77**
-0.80**
-0.88**
-0.92**
-0.69**
-0.77**
-0.67**
-0.87**
-0.75-
-0.89**
-0.79**
TKW
** P > 0.01. - = notes were not taken on TKW; (n = 200).
Table A-10. Phenotypic correlations of percent chlorosis seed with heading date (Heading), biomass (BM), plant height (Height),
grain yield (Yield) and thousand kernel weight (TKW) for F3 lines.
Pi
Prl/Sara Prl/Sara
P2
Ducula
Prl/Sara
Vee/Myna Seri-82
Ducula
Yee/Myna Vee/Myna Seri-82
Vee/Myna Seri-82
Kite/Glen
Seri-82
Prl/Sara
Ducula
Kite/Glen
Ducula
Kite/Glen
Kite/Glen Mean
Heading -0.45
-0.60
-0.54
-0.39
-0.61
-0.54**
BM
-0.49
-0.51
-0.49
-0.32
-0.54
-0.56**
Height
-0.57**
-0.56**
-0.76**
-0.51
-0.49**
-0.68**
-0.17**
-0.73
-0.44**
-0.37
-0.53**
Yield
-0.44**
-0.43**
-0.70**
-0.65**
-0.48**
-0.64
-0.66**
-0.67
-.075
-0.79**
-0.62**
TKW
-0.66
** P > 0.01. - = notes were not taken on TKW; (n = 200).
-0.81
-0.80
Table A-11. Genotypic correlations of heading date with biomass (BM), plant height (Height), grain yield (Yield),
and thousand kernel weight (TKW) for F3 lines.
P1
Prl/Sara
P2 Ducula
Prl/Sara
Prl/Sara
Pri/Sara
Ducula
Ducula
Vee/Myna Seri-82
Kite/Glen Vee/Myna Seri-82
Ducula
Vee/Myna Vee/Myna Seri-82
Kite/Glen
Seri-82
Kite/Glen Kite/Glen Mean
BM
0.26
0.12**
0.66**
0.28**
0.012
0.26**
0.22**
0.72**
0.36**
0.43**
0.33**
Height
0.45**
0.07**
0.63**
0.32**
0.33**
0.42**
0.20**
0.85**
0.31**
0.13**
0.37**
Yield
-0.19**
-0.39**
0.33**
0.22**
-0.002
-0.02**
0.05**
0.68
0.51**
0.54**
0.17**
0.21**
0.02**
0.29**
-0.04**
0.72**
0.26**
TKW
** P > 0.01.
= notes were not taken on TKW; (n = 200).
Table A-12. Phenotypic correlations of heading date with biomass (BM), plant height (Height), grain yield (Yield),
and thousand kernel weight (TKW) for F3 lines.
P1
Prl/Sara
P2 Ducula
Prl/Sara
Prl/Sara
Ducula
Vee/Myna Seri-82
Kite/Glen
Vee/Myna Seri-82
Kite/Glen Seri-82
Kite/Glen
Kite/Glen Mean
Prl/Sara
Ducula
Ducula
Vee/Myna Vee/Myna Seri-82
BM
-0.003
-0.04
0.43
0.15
-0.02
0.16
0.14
0.48
0.22
0.29
0.18
Height
0.16**
0.006
0.47**
0.19**
0.17**
0.32**
0.16**
0.56**
0.22**
0.11**
0.24**
Yield
-0.21**
-0.28**
0.21**
0.09**
-0.09**
-0.02**
0.02**
0.44**
0.344*
0.47**
0.09**
0.19**
0.008
0.26**
0.012
0.63**
0.224*
TKW
** P > 0.01.
= notes were not taken on TKW; (n = 200).
38
To investigate the direct and indirect relative impact of heading date and chlorosis on yield and
yield components, a path coefficient analysis was computed. As noted, the genotypic correlations of
chlorosis with grain yield, biomass, kernel weight and plant height were explained by the direct effect of
percent chlorosis (Table A-13). For example, for the cross Prl/Sara x Seri-82 the path coefficient analysis
showed that a large proportion of the genetic association (r = -0.81) between grain yield and percent
chlorosis was due to the direct effect (-1.09) of percent chlorosis on grain yield, whereas a large
proportion of the associations of grain yield (r = 0.33) with heading date was explained by the indirect
effect of heading date (0.75) via percentage leaf chlorosis (Fig. A-8). This may indicate that waterlogging
stress may accelerate heading date resulting in the senescence of genotypes which are not tolerant to the
stress. However, it may also indicate that later genotypes may escape some of the stress imposed by
waterlogging during the tillering, boot and early heading phase of plant development. Percentage leaf
chlorosis and heading date as independent variable in the regression analysis explain 76 % of the total
variation in grain yield (Fig. A-8). Waterlogging stress in wheat has also been shown by Cai et al. (1994)
to induce forced maturity. The large proportion of the direct effect of percent chlorosis on plant height,
biomass, yield, and kernel weight and indicates that chlorosis can be used as a selection criteria for
genotypes tolerant to waterlogging stress.
Table A-13. Results of path coefficient analysis for the tolerant x sensitive crosses. The direct and indirect effects of percent chlorosis
(% CHL) and heading date (Heading) on biomass (BM), plant height (Height), seed yield (Yield) and thousand kernel weight (TKW)
for F3 lines.
Effects
due to
BM
Height
Yield
TKW
PI: Prl/Sara
PI : Prl/Sara
P2: Seri-82
P2: Kite/Glen P2: Seri-82
P1: Ducula
PI : Ducula
PI: Vee/Myna PI: Vee/Myna PI: Prl/Sara
P2: Kite/Glen P2: Seri-82
P2: Kite/Glen P2: Ducula
PI : Prl/Sara
P1: Ducula
P1 : Seri-82
P2: Vee/Myna P2: Vee/Myna P2: Kite/Glen
direct indirect direct indirect direct indirect direct indirect direct indirect direct indirect direct indirect direct indirect direct indirect direct indirect
% CHL
-0.94 -0.02 -0.65 0.07
-1.06 0.16
-0.29 -0.04 -0.75 -0.05 -0.88 0.23
-1.09 -0.30 -0.98 0.26
-1.22 0.58
-0.29 -0.04
Heading
0.02
0.66
-0.30 0.57
0.07
-0.46 0.72
-0.83
0.07
% CHL
-0.83
-0.04 -0.58 0.02
-0.86
Heading
0.06
0.57
-0.03 0.35
% CHL
-1.09 0.28
Heading
-0.12 0.39
0.02
0.15
0.06
0.66
-0.30 0.66
-0.45 0.57
0.85
0.15
-0.08 -0.08 -0.56 -0.32 -1.22 0.68
-0.88 -0.08 -0.92 0.27
-0.68 0.09
-0.08 -0.08
-0.04 0.46
0.16
-0.95 0.22
-1.01 0.29
-0.42 0.75
-0.36 0.58
-0.55
% CHL
-1.06 0.35
-1.03 0.37
-1.00 0.13
-1.04 0.29
-1.04 0.29
Heading
-0.51 0.72
-0.61
0.63
-0.25 0.54
-0.57 0.53
-0.59 0.53
0.04
0.37
0.49
-0.89 0.93
0.13
0.45
-0.46 0.53
-0.14 0.47
0.16
-1.00 0.23
-0.89 0.09
-1.16 0.28
-0.99 0.56
-0.90 0.52
-0.98 0.47
-1.00 0.23
0.541 -0.46 0.50
-0.09 0.78
-0.37 0.88
-0.84 0.65
-0.91
0.52
-0.68 0.68
-0.46 0.51
0.04
E.g. the direct effect of % chlorosis on grain yield is -1.09 and the value 0.75 indicates the indirect effect of heading date via %
chlorosis on grain yield. The indirect effect of % chlorosis on grain yield via heading date is 0.28. As noted the direct effect of %
chlorosis on yield as well as on BM, TKW, and plant height are much grater than the direct effects of heading date.
- = notes were not taken on TKW; (n = 200).
40
% Chlorosis
-1.09t
2, Indirect p
'Cat/ Effects "oo
Heading Date
g
-0.42 t
Fig. A-8. Indirect effects from path coefficient analysis of the association of grain yield
with percent chlorosis and heading date for the cross Prl/Sara x Seri-82.
rg = genotypic correlation.
r2 = variations in grain yield due to % chlorosis and heading date.
** Significant at 0.01 level.
t = Direct effects.
41
DISCUSSION
Although differences in wheat cultivars in response to waterlogging stress has been
reported (Cai, 1990; Davies and Hillman, 1988; Huang, et al., 1995, 1994 a and b; Poyasa,
1984; Sayre et al., 1994; Thomson, et al., 1992; Van Ginkel et al., 1992; Xiang et al.,
1994),
reports on the inheritance of tolerance to waterlogging in wheat are rare. The
present study indicated tolerance to waterlogging stress in the five wheat genotypes to be
conditioned by four major genes and with no maternal effect. The presence of Wtl plus
one of the three other genes provides a considerable level of tolerance. Inheritance of
tolerance to waterlogging is mainly controlled by additive gene action. This was confirmed
by both qualitative and quantitative analysis. Cao et al. (1994) also reported that the
additive gene action
is
the major determinant of the inheritance of tolerance to
waterlogging stress in wheat. Cao et al. (1995) reported that waterlogging tolerance in
common wheat was controlled by a single dominant gene. In studying the genetics of
submergence tolerance in rice, Setter et al. (in press) also reported that tolerance to
submergence in three rice cultivars was controlled by a single dominant gene.
The three tolerant wheat genotypes in this study carried different genes although
they all posses one gene (Wtl) in common. There is a possibility that these different genes
may be related to different mechanisms of tolerance to waterlogging stress. This suggests
that increased levels of tolerance to waterlogging stress could be possible by combining all
tolerant genes in one genotype.
In wheat (Bertani and Brambilla 1982; Jaaska and Jaaska, 1980; Waters et al.,
1991) as well as in barley ( Good and Crosby, 1989; Hanson et al., 1984; Wignarajah et
al., 1976 ), rice (Andreev and Vertapetian, 1994; John and Greenway 1976; Umeda and
Uchimiya, 1994; Xie and Wu, 1989;) and maize (Chow, 1984; Johnson et al., 1989; Saligo
et al., 1988; Stromer, et al., 1993 Thomson and Greenway, 1991) tolerance
to
waterlogging stress was associated with alcohol fermentation genes such as Adh (alcohol
dehydrogenase). Hart (1970, 1980) indicated the presence of triplicated alcohol
dehydrogenase genes in wheat. In the absence of 02, the expression of three Adh barley
42
genes was observed by Hanson et al, (1984). Setter et al. (in press) indicated that
submergence tolerance is highly correlated with carbohydrate supply during anoxia and
suggested that a gene for a transcription factor may be involved in the induction of
multiple genes related to the expression of submergence tolerance. Two types of genes
coding for several enzymes in response to submergence stress of rice were reported by
Umeda and Uchimiya (1994). They indicated that the transcript levels of type I genes were
higher for the submergence tolerant rice cultivar FR13A than the intolerant IR42.
Attention should be given to study the anaeorobically induced wheat genes at the
molecular level and understand the mechanism(s) which switch them off and on, with
regard to the anaerobic stress.
The significant, high and direct associations of percentage leaf chlorosis with grain
yield, biomass, kernel weight and plant height than the associations of heading date with
grain yield, biomass, kernel weight and plant height indicated that waterlogging stress may
accelerate heading date resulting in early senescence of genotypes which are not tolerant
to the stress. However, it may also indicate that later genotypes could escape some of the
stress imposed by waterlogging during the tillering, boot and early heading phase of plant
development. Waterlogging stress in wheat has recently been shown to induce forced
maturity (Cai et al., 1994).
The large proportion of the direct effect of percent chlorosis on yield, biomass,
kernel weight and plant height indicates that chlorosis can be used as a selection criterion
for genotypes tolerant to waterlogging stress.
43
CONCLUSION
Inheritance of tolerance to waterlogging stress was not significantly affected
maternally. Waterlogging tolerance in these crosses was controlled by four major genes
with few minor genes present. Tolerance to waterlogging stress is highly heritable.
Therefore selection of segregating lines at early generation for tolerance to waterlogging
stress could be effective. Percentage leaf chlorosis can be used as a major criterion for
selecting wheat genotypes tolerant to waterlogging stress under field conditions.
44
CHAPTER III
STUDY ON OXYGEN TRANSPORT AND SYMPTOMS OF WATERLOGGING
TOLERANCE IN SELECTED SPRING WHEAT GENOTYPES
MATERIAL AND METHODS
Wheat cultivars found to have different levels of tolerance to waterlogging stress
under field conditions were examined in laboratory studies for differences in the transfer
of 02 from the shoot to the root, and for differences in anatomical traits in the root which
might be associated with survival under flooding conditions. Plants used for these
experiments were grown from seed and subjected to the oxygen transport experiments
under controlled environment conditions.
Growth conditions
Three spring wheat cultivars tolerant to waterlogging (Prl/Sara, Ducula, and
Vee/Myna) and two spring wheat cultivars sensitive to waterlogging (Seri-82, and
Kite/Glen) were evaluated in a growth chamber experiment. Following germination
seedlings were transferred to a hydroponic system (Fig. B-1) and grown under aerobic and
anaerobic conditions maintained in two metal tanks, of 400 liter each. The tanks were
filled with distilled water and covered with an opaque plastic lid with holes into which the
containers holding the seedlings could be placed. The containers were made by bonding
nylon screen (3 mm openings) to one end of a 10 cm long, 3.5 cm diameter PVC pipe. A
collar was attached to the opposite end of the pipe to allow it to be suspended over the
nutrient solution (Fig. B-1). This collar rested on the lid to the metal tanks.
45
PVC pipe
Solution level
Aerobic
Gravel
Screen
Fig. B-1. Hydroponic system used for growing wheat seedlings in nutrient solution at
aerobic and anaerobic conditions. Seedlings were first raised in an aerobic culture. When
seedlings were 3 to 4 weeks old, half were transferred to the anaerobic tank. The plants
were positioned in containers made of PVC pipe. Germinated seeds were placed on the
screen attached to the lower end of each pipe. When seedlings reached the three to four
leaf stage, pea gravel was added to fill the pipe to the solution level, which was about 5
cm below the cover.
The nutrient solution was prepared as follows:
(NH4)2SO4, 1.1 mM; Ca(NO3)2.4H20, 1.1 mM; MgSO4 , 0.4 mM ; KH2PO4, 2 mM;
CuSO4.5H20, 0.4 i.tM; MnC12.4H20, 1.8 ii.M; ZnSO4.7H20, 6.4 RM; H3B03, 9 11M
and daily addition of 1 11M FeSO4.
46
Seeds were germinated on wet filter paper in the dark for 2-3 days. Two
germinated seeds were placed in each container to grow positioned on the aerated
hydroponic tank. The level of the nutrient solution was set at the level of the seed for the
aerobic treatment, and 6 cm above the seeds for the anaerobic treatment. When seedlings
were about two to three weeks old, half were transferred to the anaerobic hydroponic
culture and allowed to grow for an additional two to three weeks.
The aerobic bath was vigorously aerated with aquarium air pumps (Atlantis Force
II double outlet), whereas in the anaerobic culture 02 was removed by bubbling with N2
gas. The anaerobic solution was not mixed (stagnant). Plants in both cultures were
exposed daily to 14 hours of light at 200-235 micro mole second-1 x m2 x micro ampere
and 10 hours of darkness. Measurements and observations were made on plants grown
under aerobic and anaerobic conditions.
Observation and measurements
Measuring root porosity
The objective of this experiment was to investigate differences between tolerant
and sensitive cultivars to waterlogging stress in the formation of root air space under
anaerobic growing conditions. Plants from the same cultivars were also grown aerobically
for comparison.
Root samples (two plants per cultivar per treatment) from the aerobic and
anaerobic treatment were taken to measure root air space by the pycnometer method
(Jensen et al., 1969). The sample roots were cut into 4-6 cm pieces and placed in a
narrow-mouth pycnometer bottle (25 ml). The bottle was immediately filled with water,
capped and weighed on an analytical balance providing the weight of the bottle plus the
47
weight of the water and the weight of the roots (Q1) Then, roots were taken from the
.
bottle, blotted on paper towels and weighed (R) immediately Eq. [1].
The roots were then homogenized by mortar and pestle, and placed into the empty
pycnometer, after which the Pycnometer was again filled with water and weighed. This
provided the weight of the bottle, the water and the homogenized roots (Q2). Also, the
weight of the bottle filled with water (B) was measured. Porosity was calculated using the
formula of Jensen et al. (1969).
% Porosity =100[ (Q2 Q1 ) / (B + R )
Qi
Eq. [1]
Observing root anatomy (aerenchyma or cortical air space)
This observation was aimed at investigating differences in root anatomy between
tolerant and sensitive cultivars to waterlogging stress. Measurements were made on roots
from plants grown either for 35 days under continuously aerobic conditions, or on roots
from plants grown for 15 days aerobically followed by 20 days of anaerobic treatment.
Thus the difference to be observed would occur during 20 days of anaerobic growth. Two
to three cm segments of roots were cut from 2 cm above the root tip. The segments were
fixed in formalin acetic alcohol (FAA) for about 48 hr approximately, and dehydrated in a
series of Tertiary-Butyl alcohol (TBA) solutions. They were weakly stained by safranin
and embedded in paraffin. Transverse sections of 10 gm were cut using a rotary
microtome (model Spencer '820'). Sections were mounted and fixed on slides using
Haupt's fixative. The paraffin was then removed and sections were stained with safranin
and fast green. The presence or absence of cortical air space was observed under the light
microscope (model Ernst Letiz Wetzlar Germany) at 10x magnification. Photographs were
taken using a Pentax camera (model K1000, ASAHI OPT. CO.).
48
Evaluating cultivars for oxygen transport from shoot to root
This experiment was designed to study the ability of various wheat cultivars to
transport oxygen from the atmosphere down to the roots through their shoots when
subjected to waterlogging stress. The hypothesis was that under anaerobic growing
conditions tolerant cultivars could supply 02 more efficiently to their roots than sensitive
cultivars.
Experimental assembly for measuring 02 transport
For this study a measuring system based on a design by Veen et al. (1977) was
constructed to measure the rate of oxygen depletion from an 02 saturated standard
solution by the roots (Fig. B-2). The system consisted of a root chamber (made of
Plexiglas) filled with a standard solution (A), a small electric pump (model 1-EA-42 Little
Giant) for circulating the standard solution in the system (B), a thermostat for controlling
the temperature of the standard solution (C), an oxygen measuring chamber made with
Plexiglas (D), a dissolved oxygen meter with its 02 probe for measuring 02 concentration
(E) and a strip chart recorder for recording the output from the dissolved 02 meter (F).
49
02
eter
02 measuring chamber
02 probe
Thermostat
Chart recorder
1111- 41) =->
B
Water pump
Root chamber with standard solution
Fig. B-2. Experimental arrangement based on the design by Veen (1977) for measuring
02 uptake rate by wheat root from the root chamber. The root chamber was filled with
standard solution, saturated with 02 before plants were placed in the system. Water was
continuously circulated (indicated by arrow) from the root chamber through a chamber
containing the probe for measurement of concentration of dissolved oxygen. The circuit
also contained a thermostat to control the temperature of the solution (20° C). The
standard solution was continuously mixed with a magnetic stirrer.
Different volumes of root chamber (1.83, 1.28, or 0.9 1) were used depending on
the size of the roots. The root chamber was filled with the standard nutrient solution
saturated with oxygen (7 to 8 mg/1). Plants from the aerobic or anaerobic hydroponic
treatments were evaluated for oxygen depletion with their roots placed in the root
chamber (Fig. B-2). The roots were sealed in the root chamber by placing a rubber stopper
and modeling clay at the root-shoot junction. This prevented air from leaking into the root
chamber. The system was tested for air tightness by placing physiologically dead plants in
the system and by noting changes of the oxygen concentration in the root chamber.
50
No changes in the concentration indicated that the system was air tight. An electric pump
was used to continuously circulate the standard nutrient solution from the root chamber
through a thermostat to the chamber containing the oxygen probe. The temperature of the
nutrient solution was adjusted to 20° C.
It was assumed that oxygen generated from the photosynthesis activity may be
interfere with the measuring process. Therefore, measurements were taken in the dark to
prevent photosynthetic activity.
Measurements were first made with intact shoots. Immediately after this set of
measurements, the shoot was cut off at the root shoot junction and the remaining shoot
was sealed with the modeling clay to prevent 02 leakage through the stem into the system,
and a second set of measurements was performed. The nutrient solution was renewed after
each measurement. The oxygen depletion rates of the roots were measured with a
dissolved oxygen probe (model 860 Orion Research Incorporated Laboratory product
group). The output was recorded on a strip chart recorder (Heath Schlumberger model SR
- 2558). The time required to reach zero 02 in the solution or a final concentration varied
from 7 hours to 28 hours depending on the size of the roots and the root chamber.
The differences in 02 uptake rate between plants with an intact shoot and plants
with the shoot removed were used to estimate the 02 contributed by the shoot for root
respiration (Laan et al., 1990). The dry weight values of roots and shoots were taken at
the end of each measurement to calculate the 02 uptake rate on a dry root mass basis.
Oxygen depletion curves were constructed for each cultivar grown under aerobic and
anaerobic conditions.
51
The following hypothesis were made regarding the possible responses of
wheat plants to 02 stress conditions:
1- Substrates may play a role in the rate of oxygen consumption by the roots from the root
chamber. Substrate is defined as:- carbohydrates supplied to the roots either by the shoots
or by the roots themselves.
2- The shoots may supply the roots with substrates and oxygen from the air.
3- The shoots may not supply either oxygen or substrates to the roots. It was also
assumed that there are two sources of 02 for the roots sealed in the root chamber:
(a) from the standard solution in the root chamber and
(b) from the atmosphere through the leaves, and there are two sources of substrates to
supply the roots: (a) from the roots, and (b) from the shoots.
By cutting the shoots (plants without shoots) the supply of substrates and 02 from the
shoots to the roots could be eliminated.
52
Four conditions for respiration to take place in the roots can be postulated based
on the sources of 02 and substrates.
A.
IF
THEN
If the shoot does not contribute
either 02 or substrates, and only
Then no differences in rate of oxygen uptake
If the shoot does contribute 02
but no substrates, while the
roots supply substrate and the
standard solution also supplies
Then plants without a shoot will have a
If the shoot does contribute
substrate but no 02, while the
roots also supply substrate and
Then more substrates are supplied to the
roots, and the oxygen uptake rate of roots
If the shoot does contribute 02
and substrates, while the roots
also supply substrate and the
standard solution also supplies
1) If the 02 contributed by the shoots is in
the roots supply substrate and the
standard solution supplies the
02.
B.
is expected between plants measured with
intact shoots or without the shoots.
higher rate of oxygen uptake than the plants
with an intact shoot.
02.
C.
the standard
supplies 02.
D.
02.
solution
also
with an intact shoot will be higher than the
oxygen uptake rate of roots without a shoot.
excess of that required by the substrates
transported by the shoots, then the rate of
root oxygen uptake will be lower for plants
with an intact shoot than for plants without a
shoot.
2) If the 02 contributed by the shoots is
less than that required by the substrates
transported by the shoots, then the rate of
root oxygen uptake will be higher for plants
with an intact shoot than for plants without a
shoot.
3) If the 02 contributed by the shoots is
proportionally equal to that required by the
substrates transported by the shoots, then
differences in the rate of root oxygen uptake
between plants with and without a shoot
may not be observed.
53
RESULTS AND DISCUSSION
Root Porosity
A significant difference in root porosity was observed between aerobically and
anaerobically grown plants. Porosity (root air space in percent of total root volume)
ranged from 1.40 percent to 6.07 percent and from 6.13 percent to 19.75 percent for
aerobic and anaerobic treatments respectively (Fig. B-3). The present study indicated that
percent root porosity was greater for the three tolerant genotypes (Prl/Sara, Ducula, and
Vee/Myna) than for the two sensitive genotypes (Seri-82 and Kite/Glen) when they were
grown under anaerobic conditions (Fig. B-3).
The lowest percentage of root porosity in both treatments was observed for the
sensitive variety Kite/Glen. Aerobically grown plants had less air space in their roots (Fig.
B-3). Prl/Sara and Ducula showed a 493 percent and 425 percent increase in root
porosity, respectively, when they were grown anaerobically. Huang et al. (1995) observed
185 percent and 53 percent increase in root porosity of the wheat cultivars Jackson and
Coker 9835, respectively, when they were treated anaerobically. Jackson was more
tolerant to anaerobic conditions than Coker 9835. For the wheat cultivar Gamenya root
porosity increased from 3 percent in continuously aerated solution to 12 percent in a non-
aerated nutrient solution (Thomson et al., 1990). Similarly Barrett-Lennard et al. (1988)
reported a two to three fold increase in root porosity of the anaerobically grown cultivar
Gamenya. Benjamin and Greenway (1979) observed an increase in the root porosity of
barley exposed to low oxygen concentrations.
54
The increase in root porosity of tolerant genotypes in response to waterlogging
stress could be one of the strategies for adaptation to an anaerobic environment. High root
tissue porosity increases 02 diffusion from shoots to roots (Haldemann and Brandle).
Wheat genotypes with well formed aerenchyma are more tolerant to waterlogging stress
(Huang et al., 1994a and 1994b). Differences in root porosity were reflected by
differences in 02 transport from shoot to root in anaerobically grown Rumex species
(Laan et al., 1989).
Parula/Sara
Ducula
Vee/Myna
Seri-82
Tolerant
Kite/Glen
Sensitive
Varieties
IEJ Aerobic
Anaerobic
Fig. B-3. Percent porosity for the roots (expressed as percent of total root volume) of
tolerant (Ducula, Prl/Sara & Vee/Myna) and sensitive (Seri-82 & Kite/Glen) wheat
cultivars under aerobic and anaerobic growing conditions.
55
Aerenchyma formation
Formation of aerenchyma was observed within five to seven days of
waterlogging in wheat (Thomson et al., 1990). In this experiment wheat roots were
examined for aerenchyma formation after 20 days of anaerobic treatment.
Aerenchyma development in the root cortex was examined using a light
microscopy. There was a clear difference in aerenchyma formation between roots grown
under aerobic and anaerobic treatments (Fig B-4). Aerenchyma was formed by dissolution
of the cortex tissue in the roots of anaerobically grown tolerant cultivars (Fig. B-4 and B5).
Anaerobically grown roots had a larger cortical air space, whereas aerobically grown
plants did not show formation of aerenchyma tissues. This was evident when root porosity
was measured by the pycnometer method (Fig B-3).
Differences between tolerant and sensitive genotypes in the formation of root
aerenchyma were observed when they were treated anaerobically (Fig B-5). These
differences were also suggested by the root porosity data (Fig. B-3). Tolerant genotypes
responded to 02 stress by developing more advanced aerenchyma than the sensitive
genotypes. This may indicate the importance of internal aeration to survive long term
anoxia. Formation of aerenchyma has been observed in the roots of wheat (Drew, 1991;
Erdmann et al., 1986; Erdmann and Wiedenorth, 1988; Huang et al., 1994a and 1994b;
Thomson et al., 1990, 1992; Trought and Drew, 1980b;), corn (Drew et al., 1979, 1980),
sunflower and tomato (Kawase and Robert, 1980 ) when plants were grown under low 02
concentrations. Although we were not able to measure the actual amount of 02
transported from shoots
to roots in our experiment, we assume that the tolerant
genotypes with the greater root porosity were able to supply 02 from the shoot to the
roots more effectively than the sensitive genotypes.
56
Cortex
Stele
Xylem
Aerenchyma
Stele
Xylem
Cortex
Aerenchyma
Fig B-4. Transverse sections of roots from the tolerant cultivar Ducula grown aerobically
(A) and anaerobically (B). Formation of aerenchyma in the cortical tissue of anaerobically
grown plants is indicated.
57
Aerobic
Ducula (tolerant)
Anaerobic
Seri-82 (sensitive)
Prl/Sara (tolerant)
Kite/Glen (sensitive
Fig. B-5. Transvers sections of aerobic (A and B ) and anaerobic (C, D, E, and F) roots.
Differences in aerenchyma formation between tolerant and senstive cultivars when they
were grown anaerobically is indicated. No diferences were observed in aerenchyma
formaton under aerobic treatments.
58
Root Respiration Rate
The depletion of oxygen (02) from the standard solution in the root chamber was
measured as described in the Materials and Methods section. The goal was to obtain the
02 depletion rates as a function of time. The result of an experiment for plants grown
anaerobically in which 02 was measured with and without intact shoots is presented in
Figure B-6. Measurement results were read manually from the recorder tracing.
The equation
c =a(1
1
b + ce-d` )
Eq. [2]
was fitted to the measurement results, where c is the 02 concentration (mg p'), t is time
(minutes) and, a, b, and d are parameters obtained from the fitting process. The factor e-dt
described the non-linear components of the process. The equation was selected from
several equations after model fitting. This was done by trying to fit different equations to
the measurement results using statgraphics software (Statistical Graphics Corporation).
The choice of this equation was based on the slope of the recorder output in Figure B-6.
The equation models the decrease of oxygen concentration in time. Because the roots
differ in size, the time required to measure 02 depletion from the root chamber for the
same cultivar varied in different replications. Therefore, graphs presented in Figure B-6
are plotted from a representative single observation for each cultivar to show the general
trend of 02 depletion through time. The oxygen depletion rate in .tM min-1 was obtained
by differentiation of Eq. [2], resulting in a graph as presented in Figure B-7. The oxygen
depletion rate (.1M min-1) was converted to rates in p.M gdm' min-1 by dividing the
calculated rate by the dry root mass (dm).
59
The rate was then plotted as a function of the concentration of the solution in the root
chamber by combining Figures B-6 and B-7 to obtain Figure B-8 and B-9 for aerobically
and anaerobically grown plants, respectively, measured with and without intact shoots.
Ducula
0 Prl/Sara
A-- Vee/Myna
Tolerant
6 Seri-82
0 Kite/Glen Sensitive
c_
200
400
600
800
1000
1200
Time (min)
300
0 Ducula
Anaerobic without shoot
250
Tolerant
0 Vee/Myna
6 Seri-82 Sensitive
10-- PH/Sara
6 Kite/Glen
200
00
150
0" 100
50
0
0
200
400
600
800
1000
1200
Time (min)
Fig. B-6. Oxygen concentration in the root chamber as a function of time measured
during depletion by the root system of anaerobically grown plants with and without an
intact shoot. The data points were obtained from the recorder tracing of output of the
oxygen probe at 20 minute intervals. The solid line was then obtained by fitting Eq. [2] to
the data points.
60
o
I
0 Ducula
0 Prl/Sara
I
I
Anaerobic with shoot
Tolerant
A Vee/Myna
0 Seri-82
Sensitive
0 Kite/Glen
CL
0
CNJ
r,
0
100
200
300
400
500
Time (min)
Ducula
E
7
E
0 Prl/Sara
Tolerant
0 Seri-82
Sensitive
A Vee/Myna
6
0 Kite/Glen
-c7
eg
a)
a)
4
2
(NI
0
0
0
100
I
I
200
300
1
400
500
Time (min)
Fig. B-7. Rate of oxygen depletion (in 1.1M gdm-1 min-) by the root systems of
anaerobically grown plants measured with and without shoots as a function of time. The
graphs were obtained by differentiating equation 1 which gives (dc/dt) in tM min-1 and
dividing the result by dry mass (dm) of root to give unit of 1.4.M gdm-1 min-1. The graphs
shown are the means of 5 observations.
61
c
7
0--
8
Ducula
0 Prl/Sara
Tolerant
0 Seri-82
Sensitive
A Vee/Myna
6
0 Kite/Glen
D
C7)
4
io
2
0
0
0
50
100
150
200
250
02 Conc. (W)
8
Ducula
0 Prl/Sara
7
E
Tolerant
A Vee/Myna
6
Seri-82
0 Kite/Glen
Cr)
Sensitive
4
0_
2
0
50
100
02
150
200
250
conc. (LN4)
Fig. B-8. Rate of oxygen uptake by the root system of aerobically grown, tolerant
(Ducula, Prl/Sara and Vee/Myna) and sensitive cultivars (Seri-82 and Kite/Glen)
measured with and without shoots. The graph shows the decrease in 02 uptake rate as
the concentration of 02 in the root chamber decreases.
62
8
7
Ducula
Prl/Sara
Tolerant
A-- Vee/Myna
0 Seri-82
6
0 Kite/Glen
rn
a)
Sensitive
4
co
0
2
0
50
100
02
150
200
Conc. (p.M)
0 Ducula
8
PH/Sara
A Vee/Myna
E
7
E
-0
rn
250
e-
0 Seri-82
6
Kite/Glen
Tolerant
Sensitive
4
a)
(2
2
0
0
0
50
100
150
200
250
02 Conc (viM)
Fig. B-9. Rate of oxygen uptake by the root system of anaerobically grown, tolerant
(Ducula, Prl/Sara and Vee/Myna) and sensitive cultivars (Seri-82 and Kite/Glen)
measured with and without shoots. The graph shows the decrease in 02 uptake rate as
the concentration of 02 in the root chamber decreases. The 02 uptake rate is much
higher for anaerobically grown plants compared with aerobically grown plants.
63
To evaluate the responses of individual cultivars to anaerobic and aerobic
growing conditions with respect to 02 transport from the shoots to the roots, 02
depletion by the roots was measured with and without intact shoots (Fig. B-7).
al anerobic with shoot
A anaerobic without shoot
0 aerobic with shoot
T
8
aerobic without shoot
6
4
2
0
0
50
100
150
200
250
02 Conc. (.1M)
Fig. B-10. Rate of oxygen depletion by the root systems of Ducula as a function of
oxygen concentration in the root chamber. Results show the decrease in 02 uptake rate as
the concentration decreased. There is no 02 transportation from shoot to roots when the
shoots are removed. Therefore plants without a shoot have a higher 02 uptake rate than
plants with an intact shoot. Results were obtained by combining results from graphs
shown in Figure B-6 and Figure B-7. Similar graphs were constructed for all the other
cultivars (Appendix A).
Differences in 02 uptake rates between plants with and without intact shoot were
used to evaluate and compare responses of tolerant vs sensitive cultivars, and aerobically
grown plants vs anaerobically grown plants with respect to oxygen supplied by the shoots
to the roots. This comparison could indicate the difference between tolerant and sensitive
cultivars. For this comparison the percentage change in 02 uptake rate according to the
equation:
64
A=
plant without shoot plant with shoot
100
plant without shoot
Eq. [3]
was calculated. The differences in 02 uptake rate of a plant with an intact shoot and with
the shoot removed at different 02 concentrations were used to evaluate the ability of the
plant system to provide atmospheric 02 from the shoot to the roots. The shoot plays a role
by contributing substrate to the roots during the 02 depletion process and/or by supplying
oxygen to the root during this process. By removing the shoot the supply of atmospheric
02 through the shoot to the root and substrate from the shoot to the root could be
eliminated.
Results were averaged from two observations for aerobically-grown plants and
from five observations for anaerobically-grown plants, and plotted as a function of 02
concentration in the root chamber (Fig. B-11). This presentation was chosen because it
allows the analysis of the role of the shoot on the rate of oxygen depletion by the roots.
65
() Ducula
60
Pd/Sara
40
A Vee/Myna
20
() Kite /Glen
Seri -82
Tolerant
Sensitive
0
20
40
60
80
0
100
50
150
200
250
02 Conc. (.1.M)
Anaerobically grown plants
_Af
Pri/sara
Vee/Myna
Seri-82
AO Kite/Glen
0
50
100
150
200
Tolerant
Sensitive
250
02 Conc. (p.M)
Fig. B-11. Percent change in oxygen uptake rate {(without shoot with shoot)/without
shoot x 100 } for tolerant cultivars vs sensitive cultivars under aerobic and anaerobic
conditions. Results were averaged from two observations for aerobically-grown and from
five observations for anaerobically-grown plants, and plotted as a function of 02
concentrations in the root chamber.
66
The following results were obtained:
1. The 02 uptake rate decreased for both aerobic and anaerobic roots as the 02
concentration in the root chamber decreased (Fig. B-8 and B-9).
2. The 02 uptake rate of the roots of anaerobically grown plants is much higher
than the 02 uptake rate of aerobically grown plants (Fig. B-10).
3. For anaerobically grown plants the 02 uptake rate without a shoot is higher than
the 02 uptake rate with a shoot, except for the cultivar Prl/Sara (Fig. B-10 and Appendix
A).
4. For aerobically grown cultivars, Ducula and Seri-82, the 02 uptake rate is higher
for plants without a shoot than plants with a shoot. Aerobically grown Vee/Myna showed
higher 02 uptake rate with the shoot. Small differences in 02 uptake rate were observed
for aerobically grown cultivars Prl/Sara and Kite/Glen with and without a shoot (Fig. B10, Appendix A)
5. The sensitive cultivar Kite/Glen showed a lower 02 uptake rate in anaerobic
treatments compared with the other cultivars. The sensitive cultivar Seri-82 showed a
higher 02 uptake rate than tolerant cultivars Ducula and Prl/Sara, and lower 02 uptake
rate than tolerant cultivar Vee/Myna under anaerobic growing conditions.
6. For anaerobically grown plants the percentage change in 02 uptake rate
resulting from removal of shoots was greater than zero for both tolerant and sensitive
cultivars except for the tolerant cultivar Prl/Sara. A higher negative percent change was
noted only for the tolerant cultivar Vee/Myna under anaerobic growing conditions. (Fig.
B-1 1).
The results suggest that both tolerant and sensitive cultivars were able to transport
oxygen from the shoots to the roots. The lower percent change in 02 uptake rate resulting
from shoot removal for anaerobically grown tolerant cultivar could be due to a
proportionally high supply of 02 and substrate to their roots.
67
The roots of anaerobically grown the tolerant cultivars showed a higher respiration rate
than the sensitive cultivar Kite/Glen in 02 saturated media. The response of the sensitive
cultivar Seri-82 in terms of 02 uptake rate was much similar to the tolerant cultivars. The
differences in rates of 02 uptake could have been due as well to other factors such as
differences in the processes (enzyme activities) controlling the glycolytic flux and
respiration. Survival of rice seedlings during submergence is strongly associated with
carbohydrate supplies (Setter et al., 1996 (In Press)). A higher rate of root respiration
was observed for the tolerant wheat cultivar Jackson than the sensitive Coker 9835 when
aeration was resumed after 21 days of hypoxic treatments (Huang and Johnson, 1995).
The higher respiration rates of anaerobically grown plants under the 02 saturated
standard solution compared with the respiration rate of aerobically grown plants (Fig. B-9
and B-8, respectively) may indicate that anaerobically grown plants were able to store
more carbohydrates than the aerobically grown plants. The roots of anaerobically grown
plants were able to accumulate sugar because of reduced root growth. Therefore under 02
saturated standard solutions the roots of anaerobically grown plants were able to use the
stored carbohydrates at a faster rate than aerobically grown plants for building the stressed
root tissues. As a consequence anaerobically grown plants could show a higher rate of
respiration in 02 saturated media than aerobically grown plants. This result indicates the
importance of substrate supply from both roots and shoots to roots upon return to aerobic
conditions for rapidly building root tissues. Albrechet and Wiedenorth (1994) reported
that roots of the anaerobically grown wheat cultivar Alcedo had higher 02 uptake from an
02 saturated environment than the aerobically grown control.
When a proportionally large amount of substrate is supplied to the roots in
comparison with 02
,
the 02 uptake rate of plants 'With shoot" could be higher than
`Without shoot" because more substrate is available to the roots per unit time with an
intact shoot.
68
A higher rate of 02 depletion would be expected, and therefore a lower or a negative value
of the difference in 02 uptake rate (without shoot
with shoot) would result. With more
02 than substrate being supplied from the shoots to the roots, the 02 uptake rate of plants
with shoots would decrease. A less rapid depletion of 02 in the root chamber would be
expected, and therefore a positive value of the difference (plant without shoot - plant with
shoot) would occur.
A percent change in 02 uptake rate (without shoot
with shoot) less than zero,
indicates that at a given oxygen concentration the respiration rate with the shoot intact is
higher than the corresponding rate with the shoot removed. If the leaves supplied the root
system with oxygen, then this would reduce the oxygen depletion rate, not increase it, as
observed for the aerobically grown tolerant cultivar Vee/Myna and anaerobically grown
tolerant cultivar Prl/Sara. Thus the higher respiration rate with the shoot intact can only
be due to greater substrate availability for respiration. Since the root systems are identical
in all other ways, it can be assumed that the root system is supplied with substrate from
the leaves. This supply disappears when the shoots are removed, and a lower oxygen
depletion rate results. Therefore it can be assume that the lower 02 depletion rate
represents the actual respiration rate of the root system. This represents the condition
where the rate of substrate supply greatly exceeds the rate of oxygen supply. The amount
of substrate supplied by the shoot leads to a rate of oxygen use, which is much greater
than the oxygen supply from the shoot.
The inverse of this situation occurs when the rate of respiration without shoot is
greater than the rate with shoot. For the anaerobically grown plants the rate measured
with shoot removed was always higher than the rate measured with intact shoot, resulting
in a positive value of the percent change except for the tolerant cultivar Prl/Sara. When
the shoots are removed, the respiration rate, i.e., the rate of oxygen used by the root
system, can only increase because a small amount of oxygen which was supplied by the
shoot is no longer available.
69
Therefore the percent change in 02 uptake rate (without shoot - with shoot) will be always
greater than zero, indicating the situation in which rate of substrate supplied from the
shoots is small in relation to the amount of oxygen supplied by the shoot to the root.
Accumulation of soluble carbohydrates in plants during oxygen deficiency has been
indicated by several authors (Atwell et al., 1985; Albrechet et al, 1993). Remobilization of
stored carbohydrate on re-aeration enhances the 02 uptake rate resulting in a higher
growth rate (Wiednorth and Albrecht, 1990). Pfister and Brandel (1994 ) suggested that
plants may survive long term anoxia by adequately supplying carbohydrate during long
term anaerobiosis. It could be an adaptive mechanism to survive waterlogging stress for
plants to quickly use stored carbohydrate on re-aeration (Albrechet et al., 1993).
Maintenance of glucose supply during anaerobiosis as a mechanism of prolonged survival
under oxygen deficient conditions was indicated by Davies (1980). Limpinutana and
Greenway (1979) observed an increase in sugar level of barley and rice when exposed to
low oxygen concentrations. They also indicated that roots of plants grown under low
oxygen concentrations had sufficient substrate for respiration. A significant increase in the
level of sucrose and fructose in the roots of wheat cultivars treated hypoxically was
observed by Huang and Johnson (1995). They indicated that concentrations of sucrose
and glucose in roots of the tolerant cultivar Jackson were much greater than in the
sensitive cultivar Coker 9835.
70
DISCUSSION
The results obtained in this study indicated clear differences between tolerant and
sensitive cultivars in percentage of root porosity and aerenchyma formation. Formation of
aerenchyma during waterlogging facilitates 02 supply from the aerial part of the plant to
the root tissues ( Armstrong, 1979; Crawford, 1982; Justin and Armstrong, 1987;
Thomson et al., 1990, 1992). An internal transport of oxygen from shoot to root was
correlated with aerenchyma development in adventitious roots of the wheat cultivar Fidel
(Prioul and Guyot, 1985). A significant increase in internal 02 transport in anaerobically
grown Rumex was due to aerenchyma formation in the roots (Lann et al., 1990). In
general both tolerant and sensitive cultivars were able to transport oxygen from shoot to
root. Whether or not the quantity of oxygen transported from shoots to roots was enough
to satisfy the oxygen demand of the roots during anaerobiosis could not be determined.
The tolerant cultivars showed a higher root porosity and well developed
aerenchyma than the sensitive cultivars. These anatomical differences were not associated
with the differences observed between tolerant and sensitive cultivars in 02 transport from
the shoots to the roots. The results obtained in the 02 transport study do not seem to be in
agreement with the general expectation that the plants which are tolerant of flooding are
able to supply the root system with oxygen from the leaves, and that the sensitive plants
do not have this facility.
This experiment measured the rate of 02 uptake by the intact roots of wheat plants
from 02 saturated media. The 02 in the standard solution was completely depleted by the
roots of tolerant and sensitive cultivars. Yet 02 could be transported from the atmosphere
to the roots and may not be released to the standard solution either because of root
respiration, or entrapment of 02 by the aerenchyma tissues. This could be one of the
factors which prevented detection of clear differences between tolerant and sensitive
cultivars in 02 transport from the shoot to the roots.
71
We were not able to determine the respiration rate of the roots after the 02 was depleted
from the standard solution. Contributions of substrate by the shoot and roots were not
measured.
The 02 uptake rate of the sensitive cultivar Kite/Glen is lower than the 02 uptake
rate of the tolerant cultivars Ducula, Prl/Sara and Vee/Myna, and the 02 uptake rate of the
sensitive cultivar Seri-82 was higher than the two tolerant cultivars Ducula and Prl/Sara
and lower than the tolerant cultivar Vee/Myna. Thus no clear differences were observed in
02 uptake rates between the tolerant and the sensitive cultivars.
The differences in aerenchyma formation combined with the processes (enzyme
activities and substrate supply) controlling the glycolytic flux and respiration rate may
have accounted for the differences observed between tolerant and sensitive wheat
cultivars. This suggests that
more investigation is required concerning differences in
metabolic pathways (enzymes), or differences in substrate and 02 supply, or both, between
tolerant and sensitive wheat cultivars in response to waterlogging stress.
Several mechanisms may have evolved to provide tolerate under anaerobic
growing conditions. It is beyond the scope of this study to investigate all of the possible
mechanisms of tolerance to waterlogging stress. Therefore, future experiments should be
designed to verify that the tolerant cultivars have a better 02 delivery system, as well as
substrate storing and supplying mechanism than the sensitive cultivars during anaerobic
conditions. A fruitful approach for further study would be a detailed study of the phloem
transport system and the interactions between phloem and xylem transport. Also studies
are needed to determine the relative importance of CO2 removal from anaerobic tissues,
and 02 supply for survival under anaerobic stresses. Experiments should also be designed
to investigate tolerance to post anoxic effects.
72
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85
APPENDIX
86
Table 1. Brief descriptions of the five parental lines/cultivars included in the study. Names
in brackets ( ) are abbreviations.
Genotype
Selection Number
Origin
Description
Panila/Sara
CM80918-20Y-025H-0Y-17M-0Y-P2
CIMMYT-Mexico
Waterlogging
(advanced lines)
tolerant
CIMMYT-Mexico
Waterlogging
(advanced lines)
tolerant
CIMMYT-Mexico
Waterlogging
(advanced lines)
tolerant
Mexico (CIMMYT)
Waterlogging
(Commercial cultivar.)
sensitive
CIMMYT-Mexico
Waterlogging
(advanced lines)
sensitive
(= Prl/Sara)
Ducula
CM80232-28Y-03M-0Y-1M-2Y-OM
Veery/Myna
CM73815-2M- 1 Y-03M-5Y-0B-6M-0Y-OSJ
(= Vee/Myna)
CM33027-F-15M-500Y-0M-87B-OY
Seri-82
Kite/Glenson
CM90734-20Y-OH-0Y-5M-OY
(= Kite/Glen)
8
7
0 anaerobic with shoot
A anaerobic without shoot
-0- aerobic with shoot
Prl/Sara (tolerant)
aerobic without shoot
6
4
a)
is
a)
2
n.
0
Cs/
0
0
50
100
150
200
250
02 Conc. (p.M)
Fig. A-1. Rate of oxygen depletion by the root systems of Prl/Sara as a function of oxygen
concentration in the root chamber. Results show the decrease in 02 uptake rate as the
concentration decreased. Plants without a shoot have a lower 02 uptake rate than plants
with an intact shoot. It appears the shoots contribute more substrate than 02 upon return
to aerobic conditions.
87
8
E
0 anaerobic with shoot
A anaerobic without shoot
0 aerobic with shoot
A aerobic without shoot
6
-0
cl)
4
2
0
0
0
50
100
150
200
250
02 Conc. (pA)
Fig. A-2. Rate of oxygen depletion by the root systems of Vee/Myna as functions of
oxygen concentration in the root chamber. Results revealed a decrease in 02 uptake rate
as the concentration decreased. There is no 02 transportation from the shoot to roots
when the shoot is removed. Therefore, anaerobically grown plants show a higher 02
uptake without a shoot than with an intact shoot. The reverse was true for aerobically
grown plants
8
111 anaerobic with shoot
A anaerobic without shoot
0 aerobic with shoot
6
anaerobic without shoot
4
2
0
0
50
100
150
200
250
02 Conc. (.1M)
Fig. A-3. Rate of oxygen depletion by the root systems of Seri-82 as a function of oxygen
concentration in the root chamber. Results revealed a decrease in 02 uptake rate as the
concentration decreased. There is no 02 transportation from the shoot to roots when the
shoot is removed. Therefore, plants without a shoot have a higher 02 uptake rate than
plants with an intact shoot for the anaerobic treatment.
88
8
1111 anaerobic with shoot
A anaerobic without shoot
0 aerobic with shoot
L aerobic without shoot
6
rn
4
a)
2
0
0
0
50
100
150
200
250
02 Conc. (AM)
Fig. A-4. Rate of oxygen depletion by the root systems of Kite/Glen as a function of
oxygen concentration in the root chamber. Results revealed a decrease in 02 uptake rate
as the concentration decreased. There is no 02 transportation from shoot to roots when
the shoots are removed. Therefore, plants without a shoot have a higher 02 uptake rate
than plants with intact an shoot for the anaerobic treatrtment. No difference in 02 uptake
rate was observed for the aerobically grown plants measured with and without a shoot.