THE ROLE OF ENVIRONMENT DURING SEED DEVELOPMENT ON SUBSEQUENT SEED QUALITY OF COWPEA (Vigna unguiculata) By AN-CHING CHENG TANG A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1982 To my parents. ACKNOWLEDGEMENTS The author wishes to express her gratitude to Dr. Daniel J. Cantliffe, chairman of the supervisory committee, for his support, guidance and patience throughout the course of this research. She extends appreciation to Dr. C.B. Hall, Dr. T.E. Humphreys, Dr. T.A. Nell and Dr. I.K. Vasil for serving as her committee. Special thanks go to Dr. T.A. Nell for his assistance in the greenhouse experiments. Appreciation is extended to Frank Woods and Jeanne Fischer for their technical assistance and to Miss Rena Herb for her typing of this manuscript. The author would also like to thank those in the Vegetable Crop Deparment for their friendship and help during her study. Most of all, thanks go to the author's family. Without their support and encouragement this study would not have been possible. in TABLE OF CONTENTS Page ACKNOWLEDGEMENTS -j-ji LIST OF TABLES vi LIST OF FIGURES jx ABSTRACT xii INTRODUCTION 1 Chapter I. LITERATURE REVIEW 3 Influence of Light Stress on Seed Vigor of the Progeny 4 Influence of Water Stress on Seed Vigor of the Progeny 7 Influence of Temperature Stress on Seed Vigor of the Progeny 9 Influence of Nutritional Status of the Mother Plants on Seed Vigor of the Progeny 12 II. SEED YIELD AND VIGOR IN COWPEA SEEDS WHICH DEVELOPED UNDER DIFFERENT PHOTOPERIODS AND LIGHT INTENSITIES. 16 Materials and Methods 17 Results and Discussion 19" Summary 32 III. REDUCTION IN SEED YIELD AND VIGOR OF COWPEA DUE TO WATER STRESS DURING SEED DEVELOPMENT 33 Materials and Methods 34 Results and Discussion 36 Summary 58 IV REDUCTION IN SEED YIELD AND VIGOR OF COWPEA DUE TO TEMPERATURE STRESS DURING SEED DEVELOPMENT 59 Materials and Methods 60 Results and Discussion 61 Summary 87 IV Chapter Page V. CHANGES IN SEED VIGOR OF COWPEA DUE TO NUTRITIONAL TREATMENTS IMPOSED AT DIFFERENT GROWTH STAGES ... 89 Materials and Methods 90 Results and Discussion 92 Summary 113 SUMMARY AND CONCLUSIONS 115 LITERATURE CITED 121 APPENDIX 130 BIOGRAPHICAL SKETCH 145 LIST OF TABLES Table Page 1 Seed Germination and Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas after Accelerated Aging 21 2 Effect of Photoperiod during Seed Development on Seed Yields of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpea 22 3 Effect of Photoperiod during Seed Development on Subsequent Seed Germination of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas with the Standard Germination Test and the Accelerated Aging Test 24 4 Effects of Photoperiod during Seed Development on Subsequent Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas with the Standard Germination Test ... 26 5 Effect of Photoperiod during Seed Development on Subsequent Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas after Accelerated Aging 27 6 Effect of Light Intensity during Seed Development on Seed Yield Measurement of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas 28 7 Effect of Light Intensity during Seed Development on Subsequent Seed Germination and Seedling Growth of 'Texas Cream 40' and "Pinkeye Purple Hull' Cowpeas with the Standard Germination Test 30 8 Effect of Light Intensity during Seed Development on Subsequent Seed Germination and Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas after Accelerated Aging 31 9 Effect of Water Stress during Seed Development on Seed Size Distribution of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas 47 10 Embryo Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas in Media Containing Different Sucrose Concentrations 53 11 Embryo Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas in Different Culture Media 55 Table Page 12 Effect of Water Stress during Seed Development on Embryo Size at Seed Maturity and Its Growth in Culture of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas .... 56 13 Effect of Different Temperatures During Seed Development on Embryo Size at Seed Maturity and Its Growth in Culture of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas . . 77 14 Effect of Different Temperatures during Seed Development on the Percentage Changes in Weight and Nutrient Composition in the Axis and Cotyledons of 'Texas Cream 40' Cowpea during Germination 80 15 Effect of Different Temperatures during Seed Development on Nutrient Concentrations in the Axis and Cotyledons of 'Texas Cream 40' Cowpea after, 0, 1, 3 and 5 days of Germination 82 16 Effect of Different Temperatures during Seed Development on the Percentage Changes in Weight and Nutrient Composition in the Axis and Cotyledons of 'Pinkeye Purple Hull' Cowpea during Germination 83 17 Effect of Different Temperatures during Seed Development on Nutrient Concentrations in the Axis and Cotyledons of 'Pinkeye Purple Hull' Cowpea after 0, 1, 3 and 5 days of Germination 85 18 Effect of Different Temperatures during Seed Development on Changes in Weight and Nutrient Composition of Axis and Cotyledons of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpea during a 5 Day Germination Period 86 19 Nutritional Treatments Imposed at the Seedling Stage of the Parental Plant 91 20 Effect of Nutritional Treatments Imposed at the Seedling Stage of the Parent Plant on Seed Yields and Weights of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas ... 93 21 Effect of Nutritional Treatments Imposed at the Seedling Stage of the Parent Plant on Seed Germination and Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas with the Standard Germination Test 95 22 Effect of Nutritional Treatments Imposed at the Seedling Stage of Parent Plant on Seed Germination and Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas after Accelerated Aging 97 vn Table Page 23 Effect of Nutritional Treatments Imposed at the Seedling Stage of the Parent Plant on Seed Composition of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas 99 24 Effect of Nutritional Treatments Imposed at Anthesis of Parent Plant on Seed Yields of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas 103 25 Effect of Nutritional Treatments Imposed at Anthesis on the Pod Development Period and Seed Weights of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas 104 26 Effect of Nutritional Treatments Imposed at Anthesis of the Parent Plant on Seed Germination and Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas with the Standard Germination Test 105 27 Effect of Nutritional Treatments Imposed at Anthesis of the Parent Plant on the Average Days to Germination of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas with the Standard Germination Test 107 28 Effect of Nutritional Treatments Imposed at Anthesis of the Parent Plant on Seed Germination and Seedling Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas after Accelerated Aging 108 29 Effect of Nutritional Treatments Imposed at Anthesis of the Parent Plant on Seed Composition of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas 110 A-l Seed Coat Color, Seed Weight and Relative Maturity of Ten Cowpea Cultivars 130 A-2 Seed Germination and Seedling Growth of Ten Cowpea Cultivars with the Standard Germination Test 131 A-3 Seed Germination and Seedling Growth of Ten Cowpea Cultivars after Accelerated Aging at 41°C and 100% RH for 5 days . 132 A-4 Seed Coat Thickness of Hard and Nonhard 'Pinkeye Purple Hull' Seeds Which Developed under SD 133 A-5 Partitioning of Seedling Weight and Nutrient Composition during Germination of 'Texas Cream 40' Cowpea as affected by Temperatures during Seed Development 134 A-6 Partitioning of Seedling Weight and Nutrient Composition during Germination of 'Pinkeye Purple Hull' Cowpea as Affected by Temperatures during Seed Development .... 135 A-7 Effect of Developmental Temperature on Changes in Weight and Nutrient Composition in the Axis of 'Pinkeye Purple Hull' Seeds during Germination 136 viii LIST OF FIGURES Figure Page 1 Effect of water stress during seed development on seed yield measurements of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas 37 2 Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Texas Cream 40' with the standard germination test and after accelerated aging . . . . 39 3 Effect of water stress during seed development on seedling weight of the progeny of 'Texas Cream 40' with the standard germination test and after accelerated aging 40 4 Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Pinkeye Purple Hull' with the standard germination test and after accelerated aging ... 42 5 Effect of water stress during seed development on seedling weight of the progeny of 'Pinkeye Purple Hull' with the standard germination test and after accelerated aging 43 6 Final size and appearance of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas as affected by water stress during seed development 48 7 Effect of water stress during seed development on seed composition (% dry weight basis) of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas 49 8 Effect of water stress during seed development on seed composition (mg per seed) of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas 52 9 Effect of different temperatures during seed development on seed yield measurements of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas 63 10 Final size and appearance of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas as affected by temperature during seed development 64 Figure Page 11 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Texas Cream 40' cowpea with the standard germination test and after accelerated aging 66 12 Effect of different temperatures during seed development on seedling weight of the progeny of 'Texas Cream 40' cowpea with the standard germination test and after accelerated aging 67 13 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Pinkeye Purple Hull' cowpea with the standard germination test and after accelerated aging 69 14 Effect of different temperatures during seed development on seedling weight of the progeny of 'Pinkeye Purple Hull' cowpea with the standard germination test and after accelerated aging 70 15 Effect of different temperatures during seed development on seed composition {% dry weight basis) of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas .... 74 16 Effect of different temperatures during seed development on seed composition (mg per seed) of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas 75 17 Effect of nutritional treatments imposed at anthesis of the parent plant on seed composition of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas 112 A-l Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test . 137 A-2 Effect of water stress during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test 138 A-2 Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging .... 139 A- 3 Effect of water stress during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging 140 Figure page A- 5 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test 141 A-6 Effect of different temperatures during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test 142 A-7 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging . . 143 A-8 Effect of different temperatures during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging 144 XI Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF ENVIRONMENT DURING SEED DEVELOPMENT ON SUBSEQUENT SEED QUALITY OF COWPEA (Vigna unquiculata) By An-Ching Cheng Tang May, 1982 Chairman: Daniel J. Cantliffe Major Department: Vegetable Crops Seed yields of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas were significantly reduced when seeds were produced under high temper- ature, drought stress, low light intensity, or deficient levels of N or P in the growth media, but were unaffected by variation in photoperiod. Seed vigor of either cultivar was unaffected by photoperiod or light intensity. Hardseededness developed in 'Pinkeye Purple Hull' under shortdays. Drought stress of -4 or -8 bars during seed development reduced subsequent seedling axis growth of 'Texas Cream 40' compared to a nonstressed treatment. At seed maturity smaller embryo size and less nutrient accumulation, especially starch, in the cotyledons accounted for this reduction in seed vigor. Under similar conditions, 'Pinkeye Purple Hull' produced seeds as vigorous as the unstressed control. At day/night temperatures of 28°/20°C and 33°/25°C, seeds of both cultivars had higher vigor characteristics than seeds which developed at 38°/30°C or 23°/15°C. In 'Texas Cream 40', heat stress at 38°/30°C appeared to reduce the ability of seedlings to utilize food reserves effectively during germination. Reduced seed vigor of 'Pinkeye Purple Hull' at 38 /30 C was related to a shortage of reserve food, especially starch, at seed maturity. Vigor of seeds of both cultivars produced at 23 /15 C was less than seeds produced at 28°/20°C and 33°/25°C but was not as reduced as those produced at 38°/30°C. In nutritional experiments, seed vigor of 'Texas Cream 40' was unaffected by various nutritional treatments imposed from sowing to seed maturity. 'Pinkeye Purple Hull' plants grown on a deficient level of N, P or S had reduced seed vigor compared with those grown in complete Hoagland's solution. Seed vigor of this cultivar was also reduced by doubling the N level in the nutrient media. When imposed after anthesis, increased N and P levels in the nutrient media of the parent plant did not alter seed vigor of either cultivar. However, reducing N or P levels to one-quarter of those in a complete Hoagland's solution enhanced axis growth of the progenies of both cultivars. xm INTRODUCTION There is a great demand for increased research emphasis on high protein species to meet the food needs of the ever increasing world population. Although cowpea is nutritionally poor in S-containing amino acids, it is a main source of dietary protein in many tropical countries (Summerfield et aj_. , 1974). This is because cowpea normally contains 20 to 25% protein in the mature seed and is more widely adapted compared to other legumes. Low and inconsistent yields of cowpea have led to the initiation of more research to gain a better understanding of yield components for this species. Photoperiod, light intensity, temperature, soil moisture, and soil fertility have been shown to affect plant development and the final seed yield of cowpea. Rapid loss of viability during storage and extremely poor seedling establishment in the field constantly plague cowpea growers. Because cowpea is usually grown as a rainfed crop, the environment can greatly alter yields through a reduction in seed vigor. Thus, the importance of optimizing seed vigor in order to achieve higher yields is indisputable. Although genetically determined, seed vigor is often altered by external factors. This includes the environment during seed production, seed maturity, field weathering, pathogen and insect attack, processing, and storage conditions. Altering any of these factors could reduce seed vigor. Compared to other factors, environment during seed development is beyond human control in the field. Variation in 1 seed vigor due to different growing seasons or locations has been shown in several species including cowpea. For many species there is abundant literature on the influence of altered environmental conditions during seed development on subsequent seed quality. However, most of the literature for cowpea is restricted to studies on plant development, N fixation, and seed yields. Little is known about the influence of environment on subsequent seed vigor. Therefore, research concerning the role of environment during seed development on seed vigor of the progeny of cowpea was conducted. The objectives of the present experiments are (1) to investigate the influence of different photoperiods, light intensities, water stresses, temperatures, and nutritional media during cowpea seed development on subsequent seed vigor, and (2) to examine closely the portion of the seed in which vigor is affected. The results should be of benefit for the improvement of cowpea seed production practices to ultimately produce higher quality seed. CHAPTER I LITERATURE REVIEW Research on seed vigor has drawn great attention in the past several decades. An understanding of what seed vigor is and the cause of low vigor may help to improve the yields of high quality crops which may have a significant impact on economical returns to farmers. Seed vigor is defined, according to the Association of Official Seed Analysts (A.O.S.A.), as "the sum total of all those properties in seeds which, upon planting, result in rapid and uniform production of healthy seedlings under a wide range of environment including both favorable and stress conditions" (Woodstock, 1976, p. 4). Seedlings from high vigor seeds develop more rapidly and yield more than those from low vigor seeds. An accurate assessment of seed vigor cannot be made before a clear understanding of the cause of low vigor is understood. Several vigor tests have been established for certain species in the A.O.S.A. seed vigor testing handbook (Woodstock, 1976). They are the accelerated aging test, cold test, conductivity test, cool germination test, seedling growth rate test, seedling vigor classification test, and tetrazolium test. Other methods based on biochemical metabolism during germination, such as respiration and enzyme activity, have been correlated with seedling growth, but are not recommended as routine procedures for vigor testing because of variability in the tests (Woodstock, 1976). Although genetically determined, seed vigor is often modified by external factors. Several of these can affect seed vigor during seed development. These include the environment and nutritional status under which the mother plant grows, the stage of seed maturity at harvest, field deterioration, and pathogen attack in the field (Austin, 1972; Delouche, 1980). Environmental and nutritional stress can affect seed vigor early in the life of the seeds. If seeds which develop under adverse environments are low in vigor, they will be more susceptible to field deterioration, pathogen attack and improper handling during harvest and storage than seeds which develop under optimum conditions. Therefore, understanding how seed vigor will be affected by the environ- ment and nutritional status with which the seed develops is highly important. Yet, research on this area is rather limited compared with the other external factors which affect vigor. Influence of Light Stress on Seed Vigor of the Progeny Effect of photoperiod on seed vigor. Although the inductive effect of photoperiod on flower initiation has been intensively studied for many years, the influence of daylength during seed maturation on seed quality was not reported until the 1960's (Koller, 1962). The photoperiodic response of flower induction and seed quality is not always the same. For example, prickly lettuce (Lactuca scariola) which flowered only under 16 hours daylength produced high vigor seeds when the seeds developed under 8 hours daylength compared with seeds developed under 16 hours daylength (Gutterman et jiK , 1975). When seeds developed under 8 hours daylength, the seed weights of soybean (Glycine max) and restharrow (Ononis sicula) decreased, while those of lettuce (Lactuca sativa) and lamb's quarter (Chenopodium album) increased, compared with their counterparts which developed under longdays (16 to 20 hours daylength) (Roller, 1962; Wentland, 1965; Evenari et a]_. , 1966; Patterson et _al_. , 1977). A change in daylength during the last 12 days of seed ripening affected the germin- ability of 'Grand Rapids' lettuce (Gutterman, 1973). Seeds which developed under continuous light had increased tolerance to germination at high temperature both in continuous darkness and after a short light break, compared with seeds developed under 8 hours daylength. Conversely, pigweed (Amaranthus retroflexus) seeds which developed under 8 hours daylength had higher percentage of germination in the dark and at 30 C after a short illumination or low temperature (5 to 10 C) pretreatment than seeds which developed under 16 hours daylength (Kigel et al_. , 1977). Seed dormancy can also be affected by photoperiod during seed development. Both restharrow grown under 20 hours daylength and lamb's quarter grown under 16 hours daylength produced a higher proportion of dormant seeds than those under 8 hours daylength (Wentland, 1965; Evenari et al_. , 1966; Karssen, 1970; Gutterman and Evenari, 1972). The dormant seeds, which gave rise to scattered stands upon sowing, possessed water impermeable seed coats. Seedling growth following germination was not reported in the species discussed above. Purslane (Portulaca oleracea) seeds which matured under 8 hours daylength reached 50% germination 4 days earlier than those matured under 11 or 13 hours daylengths (Gutterman, 1974). The reduction in daylength from 16 to 8 hours during the last 8 days of seed maturation increased germination rate. Continuous light during seed development and maturation of red beet (Beta vulgaris) increased the proportion of empty seed balls and reduced the germinability of normally developed seeds by 12% (Heide et al_. , 1976). The progeny yield of roots was decreased by 10% compared with those grown under 8 hours daylength. Similarly, seeds of prickly lettuce which developed under 8 hours daylength were larger in size and had higher germination percentage and rate than those developed under 16 hours daylength (Gutterman et _al_. , 1975). Seedling growth, measured as hypocotyl length and cotyledon size, in seeds which developed under 8 hours daylength was 25% greater than in seeds which developed under 16 hours daylength. Progeny plants flowered earlier in the former situation. The high seed vigor of prickly lettuce which developed under 8 hours daylength was related to an increase in gibberellic acid content of the seeds. Effect of light intensity and quality on seed vigor. Little is known about the relationship of light intensity and quality during seed development to subsequent seed vigor. A reduction in light intensity to 27% of incident radiation during pigweed seed development affected its germination. Seed germination in the dark or after a low temperature pretreatment was less in longday shaded seeds than a longday control, but was higher in shortday shaded seeds than the shortday control (Kigel et a]_. , 1977). In mouse-ear cress (Arabidopsis thaliana), germination in the dark from seeds grown under cool white fluorescent lamps was 45%, under cool white plus incandescent lamps, 12%, and under incandescent lamps, 0% (McCul lough and Shropshire, 1970). The photochrome mediated system was thought to control this germination response because the incandescent lamps provided a larger proportion of light energy at wavelengths greater than 700 ym than cool white fluorescent lamps. This mechanism was not apparent in purslane because continuous red illumination during seed maturation did not improve germination in the dark (Gutterman, 1974). However, red-far red interruption during maturation under shortday affected germinability of purslane. Red interruption increased seed germination at 40°C, while far-red interruption increased germination at 25° to 30°C. Influence of Water Stress on Seed Vigor of the Progeny Rainfall during seed maturation of cotton (Gossypium hirsutum) accounted for 27% of the variability in seed vigor (Peacock and Hawkins, 1970). Higher rainfall led to progeny with better seedling growth than seeds produced in drier weather. The percentage of abnormal seedlings increased from 7% to 16% in peanut (Arachis hypogaea) when seeds developed without irrigation (Cox et _a_h , 1976). Excessive irrigation of bean (Phaseolus vulgaris) plant bearing mature seeds reduced seed vigor (Siddique and Goodwin, 1980b). The number of normal seedlings was reduced by 10% from plants watered with overhead irrigation every day, compared with those with surface watering every other day. Seed vigor of 'Moapa' alfalafa (Medicago sativa) was unaffected by water stress of up to -10 bars imposed during the reproductive stage (Walter and Jensen, 1970). Another cultivar 'Ranger' emerged earlier and had greater seedling weight and volume when seeds developed at soil moistures of -1 to -10 bars compared with -1/6 to -1/3 bars. Varietal differences in seed vigor due to drought stress during seed development were also reported for peanut (Pallas et al_., 1977). The large-seeded Virginia type 'Florigiant' was the most sensitive cultivar, the Spanish type 'Tifspan' the least and the runner type 'Florunner' intermediate. The number of mature seeds of 'Florigiant' was decreased by 34%, seed weight reduced by 20%, and germination of mature seeds was 20% less when the mother plant was grown at -15 bars soil water tension compared with those grown above -2 bars. 'Tifspan' grown at -15 bars had similar seed size and germinability as those grown above -2 bars. Lettuce seed germination was high (97-100%) regardless of water stress during seed development (Izzeldin et aj_. , 1980b). Seed wieght, however, was 40% higher when seeds developed at -5 bars than seeds which developed at -0.3 or -0.8 bars. The percentage of abnormal seedlings doubled and radicle length was 20% less in seeds which developed at -0.3 bars compared with -5 bars. A similar change in seed vigor by drought stress was reported for Indian ricegrass (Oryzopsis miliacea) (Whalley et aj_. , 1966). Seed weight and subsequent seedling growth of the progeny increased by 10% when the mother plant was grown in severe water stress (-15 bars) compared with an adequate water supply (-1 bar). 'Luther Hill' sweet corn (Zea mays) tolerated drought stress up to -5 bars during seed development without influencing seed size distribution or viability (El-Forgany and Makus, 1979). A seed vigor index, the combination of a cold test, germination rate and the accelerated aging test, in the progeny was the same regardless of water stress, but decreased significantly when -3 or -5 bars water stress were imposed during silking. Those plants exposed to water stress 3 to 6 weeks after silking produced seeds as vigorous as the control . Influence of Temperature Stress on Seed Vigor of the Progeny The influence of temperature during seed development on subsequent seedling growth was observed in spring wheat (Triticum aestivum) in the 1930's (Kostjucenko and Zarubailo, 1937). Exposure of the plants to low temperatures (<14 C) during the milk-ripe stage of seed development significantly reduced the requirement for vernalization. Progeny plants grew and developed more rapidly when seeds matured at 15.5°C instead of 26.5°C (Riddel! and Gries, 1958). Day/night temperature treatments (25°/20°C, 20°/15°C and 15°/10°C) during seed development of Italian ryegrass (Lolium multiflorum) , perennial ryegrass (Lolium perenne) and meadow fescue (Festuca pratensis) had a significant influence on subsequent seed germination and vigor (Akpan and Bean, 1977). The germination percentage was the highest in seeds which developed at 25°/20°C, especially at germination temperatures of 13° to 20°C. Seeds from the 15°/10°C treatment germin- ated 2 to 3 days slower, but produced seedling with 20 to 24% more dry weight than those which developed at higher temperatures (25°/20°C and 20 /15 C). In pearl millet (Pennisetum americanum), seed viability and field emergence were unaffected by the temperatures (33°/28°C, 30°/25°C, 27°/22°C and 21°/16°C) at which the seeds developed (Fussell and Pearson, 1980). However, grains which developed at 21°/16°C produced seedlings with 40 to 60% more hieght and dry weight than those at 33°/28°C. Similar improvements of seed vigor after maturation under low temperatures were reported for several legumes (Green et al . , 1965; 10 Harris et aj_. , 1965; Walter and Jensen, 1970; Harrison and Perry, 1973; Perry and Harrison, 1973). Soybean plants which matured in hot weather ( >32°C) produced seeds with a lower percentage of germin- ation and field emergence than those produced in cool weather (Green et aj_. , 1965). Seedling growth and seed yields in the progeny of soybean were reduced by high temperature (27°C) during the last 45 days of seed maturation compared with those produced under low temperature (21°C) (Harris et al_. , 1965). Seed germination was delayed and seedling growth was reduced in pea (Pisum sativum) when the mother plant encountered high temperatures (>35°C) during the 10 day period after the pods had started to wrinkle compared with those which developed at 30°C (Harrison and Perry, 1973; Perry and Harrison, 1973). Seed yields in the progeny were reduced when the seeds were originally matured at high temperature. In alfalfa, seedling weights and leaf numbers were greater in 'Ranger' when the seeds matured at 13°C than at 24°C (Walter and Jensen, 1970). Seed vigor of another cultivar 'Moapa' was unaffected by the temperatures at which seeds had matured. When 10 genotypes of bean were subjected to six temperature regimes (33°/28°C to 18°/13°C) from anthesis to seed maturity, all produced seeds of higher vigor at the lower temper- atures (21°/16°C and 18°/13°C) than at the higher temperatures (33°/28°C and 30°/25°C) (Siddique and Goodwin, 1980a). Resistance to mechanical injury increased in seeds which developed under low temperatures. Seed size and vigor of 'Apollo' bean decreased linearly as temperature increased from 21°/16°C to 33°/28°C during seed development and maturation (Siddique and Goodwin, 1980b). This adverse effect of high temperature on seed vigor was observed 11 even on plants with well -developed seeds at the yellow fleshy pod stage. Germination of tobacco (Nicotiana tabacum) was similar regardless of the temperatures (22°/18°C, 26°/22°C and 30°/26°C) during seed maturation (Thomas and Raper, 1975a). Rate of emergence, however, was 1 to 2 days slower and seedling growth, determined as dry weight, fresh weight and leaf size, was reduced by 17% when seeds matured at 33°/26°C compared with 22°/18°C or 26°/22°C. When a low temperature (18 /14 C) was imposed on tobacco plants during vegetative growth, seed germination of the progeny was 14% less than those grown at the higher temperatures (22°/18°C to 33°/26°C) (Thomas and Raper, 1975b). Seedling growth following germination was not reported. Contrary to the above, seeds of bracted plantain (Plantago aristata) which matured at warm temperature (27°C) germinated 1 to 2 days earlier and produced seedlings of 25% greater growth than seeds matured at cool temperature (16°C) (Stearns, 1960). The diff- erence in seedling growth persisted for 120 days. Similarly, seed germination of red beet decreased by 50% and the plants yielded 13% less when the mother plant was exposed to low temperature (12°C) during seed development compared with those exposed to higher temperatures (18° and 24°C) (Heide et _al_. , 1976). Controversial results, due to different experimental conditions, were reported for cotton (Peacock and Hawkins, 1970; Quisenberry and Gipson, 1974). Longer seedlings and higher yields were obtained when the mother plant matured at a cool minimum temperature (15°C) in the field compared with a warm temperature (21°C) (Peacock and Hawkins, 1970). When plants matured at night temperatures of 11°, 15°, 21° and 27°C 12 in the growth chamber, 20 to 70% less seedlings emerged and the resultant plants yielded 20* less cotton in the field from seeds which matured at 11° and 15°C than at 21° and 27°C (Ouisenberry and Gipson, 1974). Influence of Nutritional Status of the Mother Plants on Seed Vigor of the Progeny Seed viability of pepper (Capsicum annuum) , carrot (Daucus carota) and lettuce were similar regardless of the N levels (ranging from 23mM to 0.23 mM) in which the mother plants were grown (Harrington, 1960). Seedling growth, however, was not measured in these studies. A 10 fold increase in the level of N in the culture media of the mother plant did not affect seed germination and seedling growth of pea (Austin, 1966b), nor did N application at 141 Kg/ha to the mother plant influence subsequent progeny seed germination and root yield of carrot (Austin and Longden, 1965, 1966). In tobacco, an increase of N application from 50 to 94 Kg/ha to the mother plant reduced the time to germination and enhanced seedling uniformity of the progeny (Thomas and Raper, 1979). Seedling dry weights in the progeny of wheat and Indian ricegrass were also increased by the previous N supplement (Whalley et a]_. , 1966; Pies and Everson, 1973). A high percentage of seed germination was reported in pepper, carrot and lettuce regardless of the P levels (ImM to 0.0078mM) in the culture media of the mother plant (Harrington, 1960). Red cotyledon, a physiological disorder in lettuce, increased by 50% when the mother plant was grown under P deficiency conditions. These seeds germinated 2 to 3 days slower and plant development, as demonstrated by the formation of new leaves, accumulation of leaf 13 area, and vegetative maturity, was delayed 1 to 2 weeks compared with those grown in complete Hoagland's solution. Formation of a firm commercial lettuce head in the progeny was significantly reduced by P deficiency in the mother plant (Izzeldin et ah , 1980a). A reduction in seed vigor due to P deficiency in the mother plant was observed in pea, watercress (Rorippa nasturtium aquaticum) and carrot (Austin and Longden, 1965, 1966; Austin, 1966a, 1966b). Seed germination and emergence of these species were 95 to 100% regardless of the P levels imposed on the mother plants. Seedling weight and pea yields were 25% less from seeds grown in 0.4mM P than in 4.0mM P (Austin, 1966b). In watercress, early seedling dry weight and seed yields were 20 to 30% less in seeds produced at low P levels than those produced at high P levels (Austin, 1966a). A 10% increase in carrot root yield was related to an increase in seed P content which resulted from the P supplement (133 Kg/ha) to the mother plants (Austin and Longden, 1966) Phosphorus application to the mother plant of Indian ricegrass also improved subsequent seedling growth (Whalley et aj_. , 1966). When K from 6mM to 0.094mM or Ca from 5mM to 0.078mM was applied to the mother plants, the germination percentages of pepper, carrot and lettuce seeds were not altered (Harrington, 1960). Seed longevity, however, was significantly reduced when seeds developed on K or Ca deficient plants. A high proportion of red cotyledon developed in lettuce and the subsequent seedling growth was adversely affected when deficient levels of Ca were imposed on the mother plant (Harrington, 1960; Izzeldin et al_. , 1980a). Calcium content of the peanut seeds was reduced from 420ppm to 200ppm when the mother plant was grown without Ca supplement (Cox et al., 1976). Seeds low in Ca 14 germinated 40% less and produced seedlings with a higher incidence of abnormality, such as dark plumule and watery hypocotyl , than seeds high in Ca. Another abnormality, hollow heart, developed in peanuts when the mother plant was grown in low B soil (Harris and Brolmann, 1966). Similarly, soybeans grown in Mo deficient soil were low in seed Mo content which led to a reduction in seedling growth and seed yield (Harris et aJL , 1965). In brief, the nutritional status of the mother plant had little effect on seed germinability, but seed vigor was either reduced by nutrient deficiencies or improved by additional applications of nutrients to the mother plant. Some of the 'carry-over' effects were via changes in chemical composition (P or Ca) of the seeds, as observed in carrot, watercress and peanut (Austin, 1966a; Austin and Longden, 1966; Cox et a_l_. , 1976). An interesting relationship between seed protein content and seed vigor was reported in bean, wheat and oat (Avena sativa) (Schweizer and Ries, 1969; Ries, 1971; Ries and Everson, 1973). Supplemental N (50 to 100 Kg/ha) to the mother plant increased seed protein content in bean which was highly correlated with subsequent seedling size (r=0.95***) and yield (r=0.61**) (Ries, 1971). Protein content was increased in wheat seeds by the application of herbicide or N to the mother plant (Ries et aj_. , 1970; Ries and Everson, 1973). Progeny plants grown from high protein seeds were more advanced in morphological development and yielded more seeds than those from low protein seeds. A 21 to 42% increase in oat grain yield was related to an increase in seed protein content by herbicide applications to the mother plant (Schweizer and Ries, 1969). 15 In conclusion, seed vigor was altered by environmental and nutritional stress imposed on the mother plant. In order to understand how the environment alters seed development and subsequent seed vigor, the present research was undertaken. CHAPTER II SEED YIELD AND VIGOR IN COWPEA SEEDS WHICH DEVELOPED UNDER DIFFERENT PHOTOPERIODS AND LIGHT INTENSITIES Variation in light duration, intensity or spectral quality can affect plant development. Light duration (photoperiod) is well-known for its influence on flower initiation, while light intensity controls photosynthesis, and light quality affects photochrome mediated processes (Leopold and Kriedemann, 1975). Thus, changes in light during seed development might affect seed viability and vigor. A shortday (SD) treatment during seed development of red beet increased yield in the progeny compared with a longday (LD) treatment (Heide et _al_. , 1976). Improvements in seedling growth due to SD treatments during seed development of the mother plant were reported for purslane (Gutterman, 1974) and prickyly lettuce (Gutterman et ah, 1975). Diverse responses in seed yield of cowpeas to SD (11 hours 40 minutes) and LD (13 hours 20 minutes) treatment were reported for 61 cultivars examined (Huxley and Summerfield, 1974). A 50% reduction in light intensity decreased seed yield of 'Prime' cowpea, but increased seed weights (Summerfield et a]_. , 1976). The effect of light on seed germinability and vigor in the progeny was not reported. The objective of the present experiments was to investi- gate the influence of different photoperiods and light intensities during seed development on seed vigor of cowpea. 16 17 Materials and Methods Biological materials Ten cowpea cultivars were provided by Dr. R. L. Fery, USDA, Charleston, South Carolina (Table A-l). All seeds were propagated at the University of Florida, Horticulture Unit in Gainesville in the spring of 1979. The maturity date and seed weight were recorded at the time of harvest (Table A-l). After preliminary tests which included a standard germination test and an accelerated aging test (Tables A-2, A-3), 'Texas Cream 40' and 'Pinkeye Purple Hull' were chosen for the experiments which follow. Experiment I. Effect of photoperiod during seed development on seed vigor A greenhouse experiment was conducted in the winter of 1980 at a temperature of 24° + 3C day and 19° + 3C night under natural irradiation. 'Texas Cream 40' and 'Pinkeye Purple Hull' were grown in 8 liter pots filled with 3:1 peat:perlite mixture. All pots were watered daily to field capacity and fertilized once a week with N:P:K (2:1:2). Insecticides and fungicides were applied as needed. At anthesis, a SD treatment was initiated by covering the plants with black plastic to reduce daylength to 8 hours. One-half of these plants were subjected to a 1 hour light break (50 uE/m2/s) in the middle of the 16 hours dark period. This constituted the LD treatment. Daylength treatments were continued until all seeds were harvested. The experimental design was a split plot with four replications. Experiment II. Effect of light intensity during seed development on seed vigor 'Texas Cream 40' and 'Pinkeye Purple Hull' were grown in the greenhouse in the summer of 1980, using the same cultural procedures as Experiment I. At anthesis, plants were transferred to growth 18 chambers (Conviron E-15) where light intensities varied accordingly: 275, 475, or 775 uE/m2/s. Photoperiod and temperature were maintained constant in all chambers: 12 hours day and 12 hours night at a temperature of 25 C day and 20°C night. The experimental design was a split plot with four replications within each cultivar in the growth chamber. Evaluation of seed vigor Seed yield. In both experiments flowers were tagged at anthesis and the maturity date of individual pods was recorded. Seeds were harvested as the pods dried. The pod development period (Pod Dev. Per.), pod number per plant, seed number per pod, seed yield, and seed weight were recorded. After cleaning by hand, seeds were stored at 10°C and 50% RH. Standard germination test. Twenty-five or 50 seeds (dependent on the quantity of seeds available) were germinated on moist paper towels at 25°C (A.O.S.A., 1970). Germination, defined as radicle protrusion, was counted daily. At the end of the fifth day, the total number of germinated and abnormal seedlings were counted, hypocotyl and radicle lengths were measured, and fresh and dry weights of the axis and cotyledons were taken. An abnormal seedling was classified according to the A.O.S.A. rules for testing seeds (A.O.S.A., 1970). A formula EG.T./EG. was used to calculate the average days to germination (ADG), where G. was the number of germinated seeds at day T., and T. was the i-th day of germination (Quisenberry and Gipson, 1974). Accelerated aging test. A preliminary test was conducted with field grown cowpeas to determine the appropriate aging period 19 (Table 1). Fifty seeds were aged at 41°C and 100% RH for 0, 4 and 5 days, then germinated as in the standard germination test (Woodstock, 1976). Measurements of seedling growth were taken as above. Cold test. A cold test was verified for cowpea as recommended in the A.O.S.A. seed vigor testing handbook (Woodstock, 1976). Fifty seeds were sown in unsterilized soil, then imbibed at 10°C for seven days, and finally placed at 25°C for seven days after which germination percentage was determined. Determination of seed coat thickness. Seed coat thickness was measured in 'Pinkeye Purple Hull' seeds which developed under SD treatment. The hard and nonhard seeds were separated and dried in a 30 C oven for three days after imbibing in water for 24 hours. A cross-section was made through the hilum portion with a single edged razor. Sections of the seeds were then mounted on aluminum stubs with tube coat (G.C. Electronics Co., Rockford, Illinois), and coated with 50nm of gold-pal adium using a Hammers V sputter coater. Samples were viewed in a Hitachi scanning electron microscope, Model-450, with an accelerated voltage of 20 Kv and seed coat thickness was measured. Statistical analysis. Analysis of variance for the experimental data was performed with the aid of the Northeast Regional Data Center in Gainesville. Analysis of variance was used to determine whether the differences between mean values of the treatments were significant. Results and Discussion 'Texas Cream 40' and 'Pinkeye Purple Hull' were used because both had similar maturity dates {2h months) and seed weights (Table A-l). Seed germination and seedling growth were similar in 20 both cultivars with the standard germination test and after aging at 41°C and 100% RH for four days (Table 1). Seed vigor of 'Pinkeye Purple Hull' was unaffected by five days of accelerated aging, but that of 'Texas Cream 40' was dramatically reduced. Only 32% of the seedlings were classed as normal in 'Texas Cream 40' after this period. Germination rate and radicle lengths of these seedlings from the five day aging treatment were significantly less than those from unaged seeds. Because of the severity of the five day treatment on 'Texas Cream 40', an aging period of 4 days was used to compare seed vigor of both cultivars after the various environmental treatments. The cold test was excluded as a vigor index for cowpea because no seed of either cultivar was viable after exposure to 10°C for seven days (data not presented). Experiment I. Effect of photoperiod during seed development on seed vigor A greater number of heavier seeds of 'Pinkeye Purple Hull' led to a 50% increase in seed yield compared with 'Texas Cream 40' (Table 2). Although the period of seed development in 'Texas Cream 40' was five days longer than 'Pinkeye Purple Hull1, seed weight of the former was 40% less than that of the latter. Differences in photoperiod had no effect on seed yield, weight or the total time of pod development in these two cultivars of cowpea. Robertson et al . (1962) reported that peas which developed under 16 hours daylength after flowering produced heavier seeds than those under eight hours daylength. In this case, seed weight differences might have reflected plant growth differences due to the longer light period during seed development. Seed numbers per pod were similar in 'Pinkeye Purple Hull' under both daylength treatments, but fewer seeds per pod were produced 21 fO T3 cu <0 S- fO o: +-> x: 3 S- +-> o ■<- 22 OJ as ■a ai £ <° a) "° £ a> o ° £ -a , — fO O Qi o CD (I) a. JO ro +J OJ fO 3 XI 23 in 'Texas Cream 40' when plants developed under SD than under LD (Table 2). Huxley and Summerfield (1976) reported an increase in seed number per pod of 'K2809' cowpea under a LD (13 hours 20 minutes) compared with a SD (11 hours 40 minutes) treatment. Germinability of 'Texas Cream 40' was the same regardless of the photoperiod treatment during seed development (Table 3). However, germination was 20% less in 'Pinkeye Purple Hull' seeds which developed under SD than under LD. This difference was due to hardseededness, since germination was 100% after scarification of the seed coat. Seed coat thickness of both hard and nonhard types of 'Pinkeye Purple Hull' developed under SD were similar, 60 to 70um (Table A-4). Hardseededness might be caused by the change in seed coat structure and/or composition. Gutterman and Heydecker (1973) found that the cuticle layer in the thinner part of restharrow seed coat was well- developed and thickened in hard seeds which matured under 20 hours daylength. Werker et a_L (1979) observed that hardseeded pea species had a continuous and very hard pectinaceous layer on caps of the palisade cells. The presence of quinone in a continuous layer around the seed coat, either in the palisade cells or osteosclereids, correlated with water impermeability. Hard seeds of 'Pinkeye Purple Hull' grew as well as nonhard seeds; thus, seed vigor was not reduced. Hardseededness in cowpea which developed under SD might serve as a survival mechanism for this tropical species. Oropeza (1976) observed that rainfall combined with high temperature during seed maturation reduced germinability of 'Mognolia' cowpea by 20% within several days. Potts et al . (1978) reported that hardseededness in soybean was beneficial in preventing 24 e c i- o OJ -r- CD +-> « OJ -i- CU E S- Ol -t-J CD OJ X3 3 S- 0) X3 OO 1/1 C 0) *« •— Q. 3 O ! Cl) fO 1- > 0) a cu a.> a. cu o ^ ■O .O a) s- c "C T3 aj 5- cu « ui -o c +j fO c +j <1) GO E 3 Q. O O U ai - > i— CU r— 26 H O O <-H O CO XI 0) TJ C O i- •i- Q_ s- - (U Q.-a o c o -£= - Q. O 3 > O T- 27 ^ c o s_ a o < cnT3 C aj +J ro ■a s. QJ 3 s- a- a) a; -u s < >1^ ^ +J O c t-J a; CL> in i — Q-- O O CD >> 2 +-> O i- 28 0) •— ■— a. 3 -Q O O <— i~ r— a. o 29 reproductive stage of 'Prima' cowpea led to a 20% reduction in seed yield (Summerf ield et jfL , 1976). In this case, rapid leaf senescence and possibly a reduction in photosynthetic rate under low light intensity decreased available nutrients for seed development, which in turn reduced seed yield. Pod maturity of 'Texas Cream 40' and 'Pinkeye Purple Hull' was delayed two to three days when seeds developed under the lowest light intensity compared with seeds which developed under the higher two light intensities. Sofield et a]_. (1977] observed that the seed development period in wheat was unaffected by light intensities ranging from 8.1 to 48.4 Klux after anthesis. However, seed size decreased under shading. In 'Prima' cowpea, Summerfield et aj_. (1976) reported that seed weight increased 14% when plants developed under a 50% reduction of normal sunlight. Robertson et aj_. (1962) found that seed weight decreased when peas developed under 1000 f.c. compared with 1500 f.c. The capacity of nutrient redistribution and sink size (seed number per plant) probably contributed to the variation of seed weight in the species discussed above. Seeds of 'Texas Cream 40' germinated faster and generally developed into larger seedlings than 'Pinkeye Purple Hull' in both the standard germination test (Table 7) and the accelerated aging test (Table 8). Seed germinability and vigor of both cultivars before and after aging were unaffected by the intensity of light imposed after flowering. A decrease in radicle length and axis weights from seeds which developed under 475 uE/m2/s with the standard germination test was not thought to be of practical significance (Table 7). This was possibly related to a slight but O) ID C3 Q. 3 30 O «T -« Q - C3 O -u|<_> culo ai a :r >. o a> *r -^ ^.. 1 — a. E c E E E c 01 *-* ai i Ol e a 3. 3 u vr» uf) tr> «3 a> x: CM *r X c CT ai a_ z C CT i — a. £ 4-> +j ^ zs > s- -M c a; it! i — O <1J u 5 03 o 1 — >> +J o 4- 1 — c/> .Q C1J 03 3 .C3 i — O •r- (O C i — Q. 3 o Q. cij OJ > >1 QJ IV Q J*: c ■a CU Q. 31 o — r^ E -U o t- 32 non significant decrease in individual seed weight of seeds which developed under this light intensity. From these results, it appeared that seed vigor in two cultivars of cowpea was unaffected when the mother plants encountered a change in daylength or a reduction in light intensity during seed development. Seeds produced under different light conditions should be of high vigor although seed yields might be somewhat reduced by the lower light intensities. Summary 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas were subjected to different photoperiods and light intensities during seed development to investigate the influence of these environmental factors on seed yield and vigor. Photoperiods, shortday (8 hours day/16 hours night) versus longday (8 hours day/16 hours night with a 1 hour light break in the middle), did not influence seed yield, size or vigor of either cultivar. Hardseededness was observed in 'Pinkeye Purple Hull' when seeds developed under shortdays. Once the coat of such seeds was ruptured, germination and seedling growth were normal. Differences in seed coat thickness were not observed between hard and nonhard seeds; thus, it appeared that seed coat composition was altered. Seed yields of both cultivars decreased significantly when light intensity was reduced from 775 to 275 yE/m2/s. However, neither seed weight nor seed vigor was affected by light intensity differences imposed during seed development. CHAPTER III REDUCTION IN SEED YIELD AND VIGOR OF COWPEA DUE TO WATER STRESS DURING SEED DEVELOPMENT Water stress during seed development can affect seedling growth in the progenies of lettuce (Izzeldin et _al_. , 1980b), Indian ricegrass (Whalley et al_. , 1966), peanut (Cox et al_. , 1976), alfalfa (Walter and Jensen, 1970), and sweet corn (El-Forgany and Makus, 1979). Seed vigor was reduced in peanut and alfalfa, improved in lettuce and Indian ricegrass, and unaffected in sweet corn by drought stress. An increase in seed weight under drought conditions was related to an improvement in seed vigor of lettuce (Izzeldin et _al_. , 1980b) and Indian ricegrass (Whalley et _al_. , 1966). Decreased seed vigor of Spanish peanuts was associated with reduced ethylene production during early seed germination from plants grown under low soil moisture stress (Ketring et aj_. , 1978). When 'Florigiant' peanuts matured under a drought condition, seeds low in Ca were produced which gave rise to seedlings with a physiological disorder (Cox et al . , 1976). Seed chemical composition was altered when water stress was imposed during seed development of sorghum (Eck and Musick, 1979), wheat (Terman et al . , 1969; Campbell and Davidson, 1979) and oat (Chinnici and Peterson, 1979); however, the relation of this change in composition to seed vigor was not reported. Water stress, either a deficit or excess, adversely affected nodule growth in cowpea (Doku, 1970; Hong et a]_. , 1977). Seed yield 33 34 varied with water stress levels and the growth stage at which the water stress was imposed. Treatments that decreased the number of mature pods reduced cowpea seed yields (Hiler et _al_. , 1972; Kamara, 1976; Minchin et _aJL , 1978; Turk et al_. , 1980). The influence of water stress on progeny seed vigor was not observed. The present research was conducted to investigate the influence of water stress during seed development on subsequent seed vigor of cowpea. Since a decrease in vegetative growth by water stress might severely affect seed development and vigor, plants were grown with similar quantities of available water until flowering. Active embryo metabolism and nutrient supply from the cotyledons determine seedling growth at the early stages of plant development. Thus, the chemical composition of the seeds was analyzed to determine changes induced by water stress and its possible relationship with changes in seed vigor. Also, in order to determine if there was drought injury to the embryos themselves, embryos were cultured to eliminate the influence of cotyledons during early seedling development. Materials and Methods Experimental water stress treatments during seed development A greenhouse experiment was conducted in the summer of 1980 at a temperature of 33 + 3 C day and 25° + 3 C night under natural light intensity and photoperiod. 'Texas Cream 40' and 'Pinkeye Purple Hull' were germinated in flat trays filled with a 3:1 peat:perlite mixture. Seedlings with fully-expanded primary leaves were rinsed under tap water to remove debris on the root surfaces, then were transferred to 1-liter jars filled with complete Hoagland's 35 solution (Hoagland and Arnon, 1938). The pH of the solution was adjusted to 5.5-6.0. The jars were wrapped with aluminum foil to eliminate algae growth and the solutions were aerated with an air pump. At anthesis, polyethylene glycol-6000 (PEG-6000) was added at the rate of 160 or 250 g/Kg water to the culture solutions to reduce water potential to -4 or -8 bars, respectively (Michel and Kaufmann, 1973). A control, Hoagland's solution, was maintained for comparison. The amount of PEG-6000 was increased slowly so that water potential dropped 1 to 2 bars every 3 to 4 days. The stress conditions were maintained until seed harvest. The experiment was a completely randomized design with four replications. Evaluation of seed vigor Influence of water stress on seed yield and vigor. Seed yield measurements and seed vigor tests were conducted as previously reported (Chapter II). Influence of water stress on seed chemical composition. Mature seeds were ground in a Wiley mill to pass through a 40 mesh sieve. Total seed nitrogen (N) was determined by the micro-Kjeldahl method (A.O.A.C, 1975). Total inorganic phosphorus (P) was determined with a dry-ashed sample by the Fiske-Subbarow method (Fiske and Subbarow, 1925). Total protein was extracted with 0.5N NaOH in a homogenizer (Sorvall Omni mixer) at high speed for 3 minutes. After trichloroacetic acid precipitation, the pellet was resuspended in 0.1N NaOH and an aliquot was taken to determine protein content by Lowry's method ( Lowry et _al_. , 1951). Bovine albumin was used as a standard. Starch was analyzed by a modified method of Dekker and Richards (1971). Starch was extracted with 0.5N NaOH over a boiling water bath 36 for 15 minutes, then neutralized with 0.5N acetic acid. An aliquot was taken and hydrolyzed with amyloglucosidase (Sigma, Rhizopus genus) at 55 C for 30 minutes. The starch concentration in glucose equivalents was measured in a glucose analyzer (YSI Model 27). Embryo culture. A preliminary test was conducted to determine the optimum culture medium. Sterilization of the seeds was found to be unnecessary. Embryos were excised from the dry mature seeds, transferred to sterilized media containing 1.5% agar, 2% sucrose, and 0.1% casein hydrolysate. Petri dishes with embryos were set upright at an 80° angle. The embryos were grown at 25°C for 5 days, then the axis fresh weight, hypocotyl and radicle lengths were measured. Dry weights were determined after drying at 70°C for 24 hours. Statistical analysis. Analysis of variance was performed as previously described (Chapter II). Regression analysis was followed, if significant differences were observed, to determine the trend of changes in vigor characteristics due to water stress treatments. Lines shown in the figures were drawn from the regression equation for each measurement. Results and Discussion Influence of water stress on seed yield and vigor Seed yields of 'Pinkeye Purple Hull' decreased linearly as water stress intensified during seed development, while that of 'Texas Cream 40' decreased to half of the control (-0.8 bars) under both moderate (-4 bars) and severe (-8 bars) stress (Fig. 1A) . Reduction in seed yield was directly correlated with a decrease in pod number per plant (r=0.90***) (Fig. IB). Seed number per pod 37 60 J 48 Q. CTi ~36 Q _l Ixl £ 24 Q UJ LU " 12 0 26 22 i— •a: a! 18 t/7 Q O °- 14" I- 10 15 Ax -Texas Cream 40 \ \ Pinkeye Purple Hull 13 \ \ n -0.8 -4.0 -8.0 WATER POTENTIAL (bars) Fig. 1 Effect of water stress during seed development on seed yield measurements of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas. A. seed yield, B. pod number per plant, C. seed number per pod, D. weight of 100 seeds. 38 decreased in both cultivars under drought stress (Fig. 1C) . A similar reduction in seed yields by drought stress during seed development was reported for 'Bungundy' (Hiler et jfL , 1972), 'Temne' (Kamara, 1976), 'California No. 5', and 'Chino 3' (Turk et a]_. , 1980) cowpeas. Decreases in pod number per plant and/or seed number per pod also accounted for the reduction in seed yields of these other cowpea cultivars. When imposed after anthesis, drought stress did not influence the length of time to pod maturation of 'Texas Cream 40' and 'Pinkeye Purple Hull' (19 days under all water stress levels) or seed weight of 'Pinkeye Purple Hull' (Fig. ID). A 23% reduction in seed weight was observed in 'Texas Cream 40' when plants developed under both moderate and severe stress compared with the control (Fig. ID). Kamara (1976) reported that seed weight of 'Temne' cowpea decreased by 70% after withdrawing irrigation at the podding stage compared with a control watered to field capacity. The average seed yield of 'Pinkeye Purple Hull' was 54% more than that of 'Texas Cream 40', due to a greater number of heavier seeds produced by 'Pinkeye Purple Hull'. The percentage and rate of seed germination under optimum conditions were unaffected in 'Texas Cream 40' by drought stress imposed on the mother plant (Figs. 2A, B). Hypocotyl length was also unaffected by water stress, but radicle length was slightly reduced in seeds which developed under severe water stress (Figs. 2C, D) . The fresh and dry weights of the axis and whole seedling declined linearly as water stress intensified (Figs. 3A, B, D, E). A decrease in cotyledon weights (25 to 30%) from seeds which developed under -4 and -8 bars water stress was highly correlated (r=0.91***) with weight decrease in the mature seeds (23%) (Figs. 3C, F). 39 100 A TOTAL 80 Standard z 60 o 1 40 UJ o 20 f Aging ABNORMAL o i i B 1.8 1.6 |l.4 §1.2 1.0 L ! I -0.8 -4.0 10 I 9 8 8h _) _ >- 7 t— ' o o o. £ 6 - 5 • 1 i D ^15 " — ~~" — — - — _ — ._ u "^,fc ■•^ ""■'^ ?13 e: z LU _l Jl _l o Q < Q 7 1 . 1 1 -8.0 WATER P0TENTAIL -0.8 [bars] -4.0 -8.0 Fig. 2 Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Texas Cream 40' with the standard germination test and after accelerated aging. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 40 1200 960 720 480 240 0 +-> n 960 Q. cr, 3 720 2 480 J2 240 u. 0 960 720|- 480 240 h oL-i 130 104" 78" 52- 26 0 ;104 i I 78 52 26 0 104 -0.8 -4.0 -8.0 -0.8 WATER POTENTIAL (bars) -4.0 -8.0 Fig. 3 Effect of water stress during seed development on seedling weight of the progeny of 'Texas Cream 40' with the standard germination test and after accelerated aging. Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, E. axis, F. cotyledon. 41 The percentage of germinated and abnormal seedlings, and hypocotyl lengths of 'Texas Cream 40' after accelerated aging were unaffected by water stress (Figs. 2A, C). Germination was delayed in seeds which developed under -4 and -8 bars water stress, however (Fig. 2B). Radicle length (Fig. 2D), axis weights and seedling fresh weight decreased linearly as water stress that was imposed on the mother plant intensified (Figs. 3A, B, E). Cotyledon weight and seedling dry weight were correlated with the original seed weight (r=0.96***) (Figs. 3C, D, F). The influence of water stress on seed germination and seedling growth in the progeny of 'Texas Cream 40' were similar in both the standard germination test and the accelerated aging test (Figs. 2, 3). More abnormal seedlings arose after aging. Compared with the unstressed control, seeds which developed under -4 and -8 bars water stress germinated slower and had shorter radicle lengths after aging. This did not occur after the standard germination test, however. The overall radicle growth of aged seeds was 21% less than that of unaged seeds, while hypocotyl length was reduced by 1.3 cm after aging. Aging also led to a 10 to 15% decrease in axis weight and the corresponding increase in cotyledon weight, when compared with the standard germination test. Seed germination and seedling growth of 'Pinkeye Purple Hull' in the standard germination test were unaffected by water stress up to -8 bars (Figs. 4, 5). After accelerated aging, germination percentage and axis lengths of 'Pinkeye Purple Hull' were also unaffected by water stress (Fig. 4). Seeds which developed under the most severe water stress, however, germinated slower and had 42 100 80 ^-^ &5 hi) .?» o i— < 40 T. 0£ li I CJ3 20 0 1 .8 1 .6 ,_^ >1 03 T3| .4 C7 [ 1 «=! .2 1.0 r TOTAL A Standard — — Aging ABNORMAL B ' ' » -0.8 -4.0 -8.0 10 ! 9 - 7 i— o o o 31 5 ■ | I D —15 E o ^13 UJ ,J1 i o Is 7 1 I L_ -0.8 -4.0 -8.0 WATER POTENTIAL (bars) Fig. 4 Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Pinkeye Purple Hull' with the standard germination test and after accelerated aging. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 43 1200 960 720 480 240 0 ?960 n3 Q. o^720 E | 480 5 240 UJ a: 0 960 720 480 -Standard -Aging 240 - = — 130 104 78 52 - 26 - o 1 1 1 E ^104 c na O. ^ 78 E I52 UJ >- 26 0 i i i F 104 78 ___ 52 ~~ — — , 26 n — i 1 i -0.8 -4.0 -8.0 -0.8 WATER POTENTIAL (bars) -4.0 -8.0 Fig. 5 Effect of water stress during seed development on seedling weight of the progeny of 'Pinkeye Purple Hull' with the standard germination test and after accelerated aging. Fresh weight: A. seedling, B. axis, C. cotyledon Dry weight: D. seedling, E. axis, F. cotyledon 44 lower axis weights after aging when compared with the control (Figs. 5B, E). Therefore, severe water stress reduced seed vigor slightly but significantly. Compared with the standard germination test, aging did not alter seed germinability or the development of abnormal seedlings, but reduced hypocotyl lengths by 11%, radicle lengths by 17.5%, and axis dry weights by 8.4% in 'Pinkeye Purple Hull Overall, axis growth of 'Pinkeye Purple Hull' after aging decreased linearly as water stress levels increased; but the rate was much slower in 'Pinkeye Purple Hull' (-0.64 mg per plant per bar) than in 'Texas Cream 40' (-1.28 mg per plant per bar) (Figs. A-2, A-4), Average differences in axis dry weights between aged and unaged seeds were smaller in 'Pinkeye Purple Hull' (5%) than in 'Texas Cream 40' (12%). Thus, seed vigor of 'Texas Cream 40' was significantly reduced by moderate to severe water stress. Vigor was further reduced if seeds germinated under aging stress. Seed vigor of 'Pinkeye Purple Hull' was only reduced in seeds which developed under the most severe water stress and then germinated in an adverse condition. Compared with the control, decrease in axis dry weight of seeds which developed under -8 bars water stress was less in 'Pinkeye Purple Hull' (9%) than in 'Texas Cream 40' (19%). Thus, 'Pinkeye Purple Hull' appeared to be more tolerant to drought stress than 'Texas Cream 40' . Drought sensitive 'Texas Cream 40' produced small and low vigorous seeds under -4 and -8 bars water stress without any apparent influence on seed germinability and germination rate. Pallas et al ., (1977) reported similar decreases in seed size and normal seedling development in Virginia type peanuts by drought stress up to -15 bars 45 during seed development. The close relationship between seedling weights and seed weight in 'Texas Cream 40' (r=0.96) was also reported in lettuce by Izzeldin et a]_. , (1980b) and in Indian ricegrass by Wh alley et a]_.» (1966). Heavy seeds produced seedlings with longer radicles in lettuce and seedlings of greater length and weight in Indian ricegrass than light seeds. However, the change in seed weight by drought stress in 'Texas Cream 40' cowpea was opposite to those in lettuce and Indian ricegrass. Stresses up to -5 bars in lettuce and -15 bars in Indian ricegrass reduced seed yields of both crops, but compensatorily increased seed weight by 40% in lettuce and 9% in Indian ricegrass. In 'Texas Cream 40' cowpea, seed weight was reduced 23% under water stress conditions. Hence, drought stress during seed development did not appear to affect vigor in lettuce and Indian ricegrass to the extent it did with 'Texas Cream 40' cowpea. 'Pinkeye Purple Hull' was drought tolerant and able to produce seeds with comparable size and vigor as the control under -4 bars water stress. A similar observation was made in 'Luther Hill' sweet corn by El-Forgany and Makus (1979). Seed size and vigor index from a cold test, an accelerated aging test, and germination rate were unaffected by soil moisture depletion up to -5 bars during corn seed development. Severe drought stress (-8 bars), however, reduced seed vigor of 'Pinkeye Purple Hull' slightly after aging. Because seedling weight of cowpea was well -correlated with seed weight (r=0.96), an attempt to separate the effect of drought from the effect of seed size was made. Little variation was found in size distribution of 'Pinkeye Purple Hull' with or without water stress, while more small seeds were produced in 'Texas Cream 40' under water 46 stress (Table 9). Most seeds of 'Texas Cream 40' from drought stress treatments appeared less-filled and most had a wrinkled seed coat (Fig. 6). A further separation on seed fullness was performed with a slot sieve. The proportion of less-filled seeds increased from 1% to 13% as drought stress intensified. Since weight differences still existed after final sieving, water stress apparently reduced nutrient deposit in the developing seeds of 'Texas Cream 40'. Comparison of seed vigor with the same seed weight between water stress treatments was not feasible in this experiment. Influence of water stress on seed chemical composition Water stress during seed development significantly altered the chemical composition of mature seeds (Fig. 7). Compared with unstressed seeds, N concentration increased 13% in 'Texas Cream 40' and 7% in 'Pinkeye Purple Hull' seeds under both moderate and severe water stress (Fig. 7A) . The protein concentration of 'Texas Cream 40' increased from 17.5% to 20.5% as drought levels intensified, while that of 'Pinkeye Purple Hull' increased significantly over the control only under the -8 bars water stress (Fig. 7B). Similar increases in seed N and protein concentrations due to water stress during seed development were reported for wheat by Terman et _a_L (1969), for oat by Chinnici and Peterson (1979), and for sorghum by Eck and Musick (1979). Ries (1971) observed that seed vigor in bean was associated with seed protein concentration. Larger seedlings and higher bean yield were obtained from high protein seeds compared with low protein seeds of the same size. This relationship was also reported in wheat by Lowe and Ries (1972). However, in the present experiment with 'Texas Cream 40' cowpea drought stress led to the 47 f-H i-H O <£> C\J CVI Oi CTi (Jl CO >^ cu QJ Q -^ c TJ CI) Q. CD - 00. o o 00 o o 48 Fig. 6 Final size and appearance of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas as affected by water stress during seed development 49 Texas Cream 40 Pinkeye Purple Hull -0.8 4.0 0.65 0.61 ^.57 00.53 SO. 49 0.45 40 35 — 30 - :n o en 2 25- 20 -8.0 -0.8 -4.0 WATER POTENTIAL (bars) -8.0 Fig. 7 Effect of water stress during seed development on seed composition {% dry weight basis) of 'Texas Creatr 40' and 'Pinkeye Purple Hull ' cowpeas. A. nitrogen, B. protein, C. phosphorus, D. starch. 50 high protein but low vigor seeds regardless of seed size. Similar responses were observed in seeds of 'Pinkeye Purple Hull1 developed under severe water stress, but the reduction in vigor was less in 'Pinkeye Purple Hull' than in 'Texas Cream 40'. Seed P concentration increased linearly in 'Texas Cream 40' but remained unchanged in 'Pinkeye Purple Hull' as water stress intensified (Fig. 7C). Eck and Musick (1979) reported that the concentration of P in sorghum seeds was unaffected by drought stress up to a leaf water potential of -24 bars during seed development. Austin (1966a, 1966b) and Austin and Longen (1966) reported that seedling growth and crop yields of carrot, watercress and pea were related to seed P content. The higher the P concentration of the seed, the higher the seed vigor of the progeny. A similar relationship was not observed in the present work with 'Texas Cream 40' cowpea. In this case, seeds with higher P concentrations after water stress had reduced seedling vigor. The starch concentration of cowpea seeds increased 9^ in 'Pinkeye Purple Hull', but decreased U% in 'Texas Cream 40', under drought stress compared with unstressed seeds (Fig. 7D). Unlike seed N, protein and P concentrations, which were negatively correlated with the axis growth in both cultivars (r=-0.61**, -0.53** and -0.75** for N, protein and P, respectively), starch concentration was positively correlated with axis growth in 'Texas Cream 40' (r=0.61**)." Vegetative growth of the cowpea plants was similar before treatment in the present experiment. After imposing drough stress with PEG-6000, leaf senescence occurred and new pods were no longer 51 produced. The increases in seed N, protein and P concentrations in seeds of both cultivars under water stress might simply be the result of the decrease in the sink (seed number and weight) compared with the available nutrient source (current metabolism and vegetation) for seed development. The decrease in starch concentration of 'Texas Cream 40' seeds under water stress indicated that photosynthesis or carbohydrate metabolism might be reduced in this more drought sensitive cultivar compared with the N and P metabolism. Total nutrient quantities on a per seed basis were directly correlated with the seed weight in both cultivars (r=0.95***, 0.91***, 0.96*** and 0.97*** for N, protein, P and starch, respectively). Therefore, water stressed seeds of 'Texas Cream 40' contained less reserve food than unstressed seeds (Fig. 8). The total quantity of N decreased- from 13 to 14%, protein 10 to 12%, P 15 to 18% and starch 33%, in 'Texas Cream 40' seeds which developed under water stress compared with the control. In 'Pinkeye Purple Hull', however, total quantities of N, protein, P and starch in water stressed seeds varied to within +5% of unstressed seeds (Fig. 8). The composition of N, protein, P and starch accounted for 30 to 50% variation in seed vigor of both cultivars. This indicated that factors other than nutrient supply from the cotyledons might possibly be involved in vigor determination. Embryo culture Selection of a medium for embryo culture was conducted with media containing different sucrose concentrations (Table 10). Hypocotyl length of both cultivars decreased as the sucrose concentration increased from 2% to 6%. Radicle length and axis 52 oi 7 E 144 £ CD 0 O cc y- 5 5 4 35 "§30 CD E ~25 2E §20 h 15 •Texas Cream 40 T-OOr Pinkeye Purple HuJJ0.92 ^,0.84 -0.8 -4.0 -8.0 WATER POTENTIAL (bars Fig. 8 Effect of water stress during seed development on seed composition (mg per seed) of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas. A. nitrogen, B. protein, C. phosphorus, D. starch. 53 Table 10 Embryo Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas in Media Containing Different Sucrose Concentrations, Sucrose2 Axis length Axis wt Cultivar hypocotyl r adicle fresh dry % 0 mg/ 28.9c axis — 2.0d Texas Cream 40 0.9by 1.8b 2 1.7a 3.1a 44.3b 3.4c 4 1.2a 3.6a 49.5a 4.7b 6 1.0b 3.4a 42.9b 5.5a Pinkeye Purple Hull 0 1.0a 1.6b 36.0c 2.3d 2 1.9a 3.8a 53.0b 3.8c 4 1.8a 4.3a 57.7a 5.0b 6 1.3b 3.9a 54.0b 6.3a All media contained 0.1% casein hydrolysate and 1.5% agar. ^Values followed by the same letter within cultivar column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. 54 fresh weight reached maximum in 4% sucrose, then declined as sucrose concentration increased to 6%. Embryonic axis dry weight increased linearly as the sucrose concentration in the media increased. Because of a possible osmotic effect leading to a reduction in axis length, a medium containing 2% sucrose was used. Combinations of sucrose and casein hydrolysate in 1.5% agar media were also examined (Table 11). Embryos of both cultivars grew better, shown by increases in axis length and weight, with sucrose in the media than without. Casein hydrolysate in the presence of sucrose increased radicle length of 'Texas Cream 40' and axis weight of 'Pinkeye Purple Hull'. Therefore, both sucrose and casein hydrolysate were used in the medium for the cowpea embryo culture experiment. Embryos from 'Texas Cream 40' seeds which developed under -4 and -8 bars water stress were smaller in size and had shorter radicle lengths and reduced axis weights after 5 days culture at 25°C compared with the unstressed control (Table 12). Hypocotyl length of 'Texas Cream 40' was unaffected by water stress treatments to the mother plant. Although embryo size of 'Pinkeye Purple Hull' was unaffected by water stress treatments, and axis dry weight and hypocotyl length were similar after five days of culture, radicle lengths and axis fresh weights declined as the water stress treatment to the mother plant intensified. Anatomically, embryos of drought stressed seeds of both cultivars were the same as unstressed seeds, regardless of embryo size (data not presented) . Seed development of 'Pinkeye Purple Hull' seemed unaffected by drought stress. A decrease in embryonic axis fresh weight and radicle lengths of culture embryos of this cultivar 55 Table 11 Embryo Growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas in Different Culture Media . Med iaz Axis lenqth Axis wt Cultivar SU CH hypocotyl radicle fresh dry Texas Cream 40 0 0 0.8ay ■cm- 2.0c — mg/axis 13.9b 0.96b 2 0 1.0a 2.9b 16.3a 1.33a 0 0.1 0.8a 2.0c 12.7b 0.96b 2 0.1 1.1a 4.0a 15.7a 1.31a Pinkeye Purple Hull 0 0 0.9a 2.3b 15.6b 1.11b 2 0 1.0a 4.1a 15.4b 1.32ab 0 0.1 1.0a 2.5b 16.3b 1.17b 2 0.1 1.1a 4.3a 18.1a 1.56a SU: sucrose, CH: Casein hydrolysate. ■^Values followed by the same letter within cultivar column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. 56 co ro cm CM oo 10 co vo ro in in in ud ro CM ro ro co ld ro en ro ro ro CO o o r— dJ O 1— U ra •r- CC +J <— +-> c CU 3 i— Q cu o E +-> cr> c CU •!- +J S_ o >■> o ja o > 3 -t-> 57 indicated that severe drought stress during seed development might exert damage on the growth of the embryo. Since embryo morphology and size of 'Pinkeye Purple Hull1 were unaffected, internal injury in ultrastructure or metabolism was possible. Thus, drought stress imposed after anthesis might have adversely affected cowpea embryo development. The embryo size and growth of 'Texas Cream 40' and embryo growth of 'Pinkeye Purple Hull' were significantly reduced by water stress. Ketring et al_. (1978) reported that low soil moisture during maturation of Spanish type peanuts reduced ethylene production during early germination which resulted in reduced seedling growth. The embryo, being the major site of ethylene production (Ketring and Morgan, 1969), was possibly impaired by drought stress although it was not investigated in Ketring's work. The influence of water stress on embryo growth of the progeny of 'Texas Cream 40' in culture (Table 12) was simliar to that in the whole seed vigor test (Figs. 2, 3). In both, radicle lengths and axis weights decreased linearly, while hypocotyl lengths remained unchanged, as drought stress treatments intensified during seeds development. Apparently, axis growth of 'Texas Cream 40' was a reflection of embryo size at seed maturity and thus its potential to grow. Compared with unstressed seeds, axis growth of 'Texas Cream 40' from seeds which developed under moderate and severe stress was 6% and 14% less, respectively, in embryo culture. A further decrease in axis growth was observed in the whole seed vigor test: 14% and 19% less under moderate and severe stress, respectively, compared with the control. The decrease in seed nutrient content in 'Texas Cream 40' under water stress (Fig. 8) might have led to the 58 further reduction in axis growth in the whole seed. Since axis dry weight increase in culture was proportional to available sucrose (Table 10), the 33% decrease in available starch of 'Texas Cream 40' seeds which developed under water stress might be a major contributing factor that led to the reduction of axis growth. In conclusion, water stress during seed development reduced cowpea seed yield and vigor. The influence was more detrimental to 'Texas Cream 40' than to 'Pinkeye Purple Hull'. When developed under drought stress during seed development, 'Pinkeye Purple Hull' produced vigorous seeds although a reduction in seed yield resulted. In the same situation, adequate water supply was necessary for 'Texas Cream 40' to produce more vigorous seeds. Summary 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas were subjected to water stresses of -4 and -8 bars during seed development to investigate the influence of drought stress on subsequent seed vigor in the progeny. Seed yields of both cultivars were significantly reduced by water stress imposed after anthesis. When developed under water stress, drought sensitive 'Texas Cream 40' produced seeds which were smaller in size than the unstressed control. Decreases in embryo size and available nutrient reserves, especially starch, in the water stressed seeds accounted for the reduction in seed vigor of 'Texas Cream 40'. Drought tolerant 'Pinkeye Purple Hull' was able to produce seeds of the same size and vigor under water stress as the unstressed control. Embryo size and available nutrients in 'Pinkeye Purple Hull' seeds which developed under drought conditions were similar to the unstressed seeds. CHAPTER IV REDUCTION IN SEED YIELD AND VIGOR OF COWPEA DUE TO TEMPERATURE STRESS DURING SEED DEVELOPMENT Temperature stress during seed development can affect subsequent seed germinability, stand uniformity and early seedling growth. Seed viability of alfalfa (Walter and Jensen, 1970), lettuce (Koller, 1962), tobacco (Thomas and Raper, 1975a, 1975b), barley (Khan and Laude, 1969) and ryegrass (Wiesner and Grabe, 1972) increased when seeds matured at high temperature. Improvement of seedling growth due to low temperature during seed development was reported in pearl millet (Fussell and Pearson, 1980), pea (Perry and Harrison, 1973), soybean (Harris et aj_., 1965), bean (Siddique and Goodwin, 1980a, 1980b) and alfalfa (Walter and Jensen, 1970). However, little is known regarding how temperature affects seed vigor in the progeny. An increase in seedling weight of pearl millet was related to an increase in size of seeds which developed at low temperature (Fussell and Pearson, 1980). A physiological disorder, hollow heart, developed in pea seeds which matured at high temperature (Perry and Harrison, 1973). This abnormality led to poor seedling growth (Harrison and Perry, 1973). Night temperature can have a profound influence on cowpea reproductive ontogeny (Huxley and Summerfield, 1974, 1976). Plants flowered only at night temperatures above 19°C; the higher the temperature, the earlier the plant flowered. Seed vigor of cowpea 59 60 has been reported to be similar at temperatures of 33°/24°C and 27 /19 C during seed development (Ndunguru et _al_., 1978). In the present experiments temperatures were varied during seed development to determine their effect on seed vigor of cowpea. The composition of mature seeds was analyzed to determine changes induced by temperature stress during seed development and the possible relationship of this to seed vigor. Embryos were cultured in vitro to determine possible injury to the axis by temperature stress during seed development. Seedling growth and changes in the major storage components of starch and protein in the axis and cotyledons during germination were studied to evaluate the relationship between food utilization and seedling vigor. Materials and Methods Experimental treatments during seed development "Texas Cream 40' and 'Pinkeye Purple Hull1 cowpeas were grown in the greenhouse in the spring of 1980 using cultural practices as previously described (Chapter II). At anthesis, plants were transferred to growth chambers (Coviron E-15) in which the day/night temperatures were set at 38°/30°C, 33°/25°C, 28°/20°C and 23°/15°C. Light intensity, 775 uE/m2/s, and photoperiod, 12 hours day and 12 hours night, were the same in all chambers. The experiment was a split plot design with four replications within each cultivar in the growth chamber. Evaluation of seed vigor Influence of temperature stress on seed yield and vigor. Cowpea yield measurements and vigor tests were evaluated as previously described (Chapter II). 61 Influence of temperature stress on seed composition. Total seed N, protein, P and starch contents were analyzed as previously described (Chapter III). Embryo culture. Embryos were excised from mature seeds and cultured as previously described (Chapter III). Partitioning of seedling weight and nutrient composition during germination. Ten to 15 axes were excised from cotyledons after 0, 1, 3 and 5 days of germination. Both axis and cotyledons were weighed, then dried in a VirTis freeze-drier for two days. After dry weights were taken, each plant part was ground and assayed for amino acid and soluble carbohydrate contents. The tissues were extracted three times with 80% alcohol over a water bath at 82 C for 20 minutes. The alcohol extract was evaporated to near dryness, then diluted with distilled water. Total amino acid content was determined by the ninhydrin method (Moore and Stein, 1954; Moore, 1968) and total soluble carbohydrate by the phenol -sulfuric acid method (Dubois et al_. , 1956). Both starch and protein were dissolved in 0.5N NaOH by heating the residue of the alcohol extraction over a boiling water bath (Cruz et _al_. , 1970). After centrifugation, total protein and starch contents in the supernatant were determined as previously described (Chapter III). Statistical analysis Analyses of variance and regression were analyzed as previously described (Chapter II, III). Results and Discussion Influence of temperature stress on seed yield and vigor Seed yield of 'Texas Cream 40' decreased from 49g to 14g per plant as 62 temperature increased from 23°/15°C to 33°/25°C (Fig. 9). In 'Pinkeye Purple Hull', seed yield was comparable at temperatures of 23°/15°C and 28 /20 C, then declined from 23g to 5g per plant as temperature increased from 28°/20°C to 38°/30°C. Seed yields of both cultivars were directly correlated (r=0.91***) with pod number per plant (Fig. 9B). Stewart et al_. (1980) reported that seed yield of 'K2809' cowpea was reduced by 27% when plants were grown at 33°/24°C compared with 27 /19 C, and lower pod numbers also led to the lower seed yield. The delay of pod maturation at low temperature appeared to be related to an increase in seed weight of both 'Texas Cream 40' and 'Pinkeye Purple Hull' (Fig. 10), since these factors were highly correlated (r=0.81***) (Figs. 9C, D). Seed weights at 23°/15°C were double those at 38°/30°C. Wien and Ackah (1978) reported a similar relationship between pod development period, maturation temperature and seed weight of several cowpea cultivars. Roberts et ^1_. (1978) reported that 'K2809' cowpea had higher seed growth rate and heavier seeds when plants were grown at 27°/19°C than at 33°/24°C. Huxley and Summerfield (1976) observed that day-night temperatures affected seed development of 'K2809' cowpea differently. Seed weight was 19% less at a night temperature of 24°C compared with 19°C, but was 18% more at a day temperature of 33°C compared with 27°C. Siddique and Goodwin (1980b) reported that seed maturity in bean was delayed 11 days but that seed size increased 2.5 times when developmental temperatures decreased from 33°/28°C to 18°/13°C. A similar delay in seed development and increase in seed weight at low maturation temperature was reported in pea (Robertson et a]_. , 1962). Egli and Ward! aw (1980) reported 63 50 r +-> c ra ^40- q30 - _l LU o20 - LU LlJ OO 10 0 24 - 18 - £12 6 r 0 — Texas Cream 40 • — Pinkeye Purple Hull 23/15 28/20 33/25 38/30 50 40 >, -a — 30 LU a. >20 LU Q Q . £10 0 21 \- oo 17 Q LU LU s 13- o \ i— 3 9 - 5 J L \ \ \ 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE (°C) Fig. 9 Effect of different temperatures during seed development on seed yield measurements of 'Texas Cream 40' and 'Pinkeye Purple Hull ' cowpeas. A. seed yield, B. pod number per plant, C. pod development period, D. weight of 100 seeds. 64 tlllTllllT ••»••»>*> H 2 3 15 C 1 | 28 20 C | **Hmu* HlHWWJ H 33° 25°C 1 l~3 8 30 C 1 ^^^^^^^^^^^^^^^^H Purple ^^^^^^^^^^^^^^H Fig. 10 Final size and appearance of 'Texas Cream 40' and 'Pinkeye Purple Hull1 cowpeas as affected by temperature during seed development. 65 that the duration of soybean seed growth was unaffected by temperatures of 24°/19°C to 30°/25°C, but was 3 days less at 33°/28°C. Seed weight of mature soybean was 25% less at both 33°/28°C and 18°/13°C than at temperatures between this range. Seed germinability, abnormal seedling development, and radicle length of 'Texas Cream 40' with the standard germination test were unaffected by temperature during seed development (Fig. 11). Germination rate in the progeny was slower in seeds from the 23°/15°C treatment and hypocotyl length was shorter in seeds from the 38 /30 C treatment compared with those from 28°/20°C and 33°/25°C. Although seed weight increased at low temperature, seedling fresh weight and axis weight in the progeny of 'Texas Cream 40' were the greatest in seeds which devel- oped at 28°/20°C, followed by 23°/15°C and 33°/25°C, and the least at 38°/30°C (Figs. 12 A, B, E). Seedling dry weight and cotyledon weights were related to the original seed weight (r=0.97***) (Figs. 12 C, D, F). After accelerated aging, germinability of 'Texas Cream 40' was reduced in seeds which developed at 23 /15 C compared with seeds which developed at the higher temperatures (Fig. 11). Germination rate, normal seedling development and axis growth in the progeny were the greatest at developmental temperatures of 33°/25°C and 28°/20°C, and the least at 38°/30°C (Figs. 11, 12). Hypocotyl length was 30% less, radicle length 25% less, and axis weight 42% less in seeds which developed at 38°/30°C compared with 28°/20°C and 33°/25°C. Low temperature (23°/15°C) incurred during seed development led to a reduction in hypocotyl length by 20% and axis weight by 11% in the progeny compared with optimum temperatures. Seedling dry weight and cotyledon weight after aging were related to seed weight at maturity (r=0.97***). 66 100 80 x 60 o I 40 as 1 1 1 cs 20 0 2.0 ^1.5L gl.O 0.5U 0.0 TOTAL Aging Standard ABNORMAL ^ J I L 23/15 28/20 33/25 38/30 13 £11 3 -^20 CJ 15 w 10 O a ^ 5 en 0 \\ 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE ( C] Fig. 11 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Texas Cream 40' cowpea with the standard germination test and after accelerated aging. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 67 130 104 78 52 26 0 "SI 04 |- c Q. ^ 78 E l 52 UJ 3 S 26 B 104 78 -Aging .Standard 30 0 ?120 ro Q. 1 90 o 60 UJ « 30 0 120 90 60 30 o* 23/15 28/20 33/25 38/30 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE (°C) Fig. 12 Effect of different temperatures during seed development on seedling weight of the progeny of 'Texas Cream 40' cowpea with the standard germination test and after accelerated aging. Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, E. axis, F. cotyledon. 68 'Texas Cream 40' seeds which developed at 23°/15°C were larger in size but produced smaller seedlings before or after aging than seeds which developed at 28°/20°C. Axis growth from seeds which developed at 38°/30°C were 42% less after aging and 31% less before aging, compared with seeds which developed at temperatures of 28°/20°C and 33°/25°C. Heat stress (38°/30°C) during seed development of 'Texas Cream 40' led to the production of seeds with the lowest vigor. Cool temperature (23 /15°C) led to seeds of intermediate vigor, and temperatures in between led to seeds with the highest vigor. In 'Pinkeye Purple Hull', seed viability, abnormal seedling development and radicle length with the standard germination test were unaffected by temperature treatments imposed during seed development (Fig. 13). Seed germination in the progeny was delayed at developmental temperatures of 23°/15°C and 38°/30°C compared with 28°/20°C and 33°/25°C. Hypocotyl length was 20% less from seeds which developed at 23°/15°C than at the higher temperatures. The optimum temperatures for subsequent high seedling fresh weight and axis weights of 'Pinkeye Purple Hull' were 28°/20°C and 33°/25°C (Fig. 14). Temperatures below or above these values reduced axis growth in the progeny by 20%. Seedling dry weight and cotyledon weights were correlated with the initial seed weight (r=0.97***). After accelerated aging, germination of 'Pinkeye Purple Hull' was reduced by 20% and more abnormal seedlings were produced when seeds developed at 23°/15°C compared with 28°/20°C and 33°/25°C (Fig. 13). Seeds from 28°/20°C and 33°/25°C treatments during seed development germinated faster and had longer hypocotyls than seeds from 23°/15°C and 38°/30°C treatments. After aging, radicle 69 TOO 80 t ^ s-s 60 T' o 40 ■jz. ^ rf 20 0 2.0 -5L5 1.0 - 0.5 . 0.0 TOTAL 23/15 28/20 33/25 38/30 13 1=" ^11 nL I— 5 9 3 | 20 | 15 _l 3 10 5 - 0 23/15 23/20 33/25 38/30 DAY/NIGHT TEMPERATURE (UC) Fig. 13 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progeny of 'Pinkeye Purple Hull' cowpea with the standard germination test and after accelerated aging. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 70 130 104 78 52 26 0 ±104 $ 78 h = 52 J : 26 i j 0 104 78 52 26 B — Aging — Standard 150 120 90 60 30 0 120 90 60 30 0 120 90 60 30 23/15 28/20 33/25 38/30 J 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE (°C) Fig. 14 Effect of different temperatures during seed development on seedling weight of the progeny of 'Pinkeye Purple Hull1 cowpea with the standard germination test and after accelerated aging, Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, B. axis, C. cotyledon. 71 length, axis weight and seedling fresh weight were greater from seeds which developed at 28°/20°C than at 33°/25°C or 23°/15°C (Figs. 13, 14). These values were the least from seeds which developed at 38°/30°C. Seedling dry weight and cotyledon weight followed the same trend as initial seed weight, namely, weights increased as temperatures decreased. Axis growth of 'Pinkeye Purple Hull' seeds which developed at 23°/15°C, 33°/25°C and 38°/30°C were 80S, 91% and 60%, respectively, of those which developed at 28°/20°C after aging, compared with 80%, 100% and 80% before aging. Thus, seedling growth after aging was reduced from seeds which developed at 33°/25°C and 38°/30°C. Overall, temp- eratures of 28°/20°C and 33°/25°C led to the highest quality seeds of 'Pinkeye Purple Hull', while temperature of 38°/30°C led to the lowest quality seeds. The influence of temperature treatments during seed development on seed vigor was similar in both cowpea cultivars. Both produced small and low vigor seedlings when seeds developed at 38°/30°C. The heaviest seeds were produced in both cultivars at 23°/15°C, but axis growth in the progeny from these seeds was reduced compared with those which developed at 28°/20°C. The reduction in axis growth of both cultivars which developed at 23°/15°C was less than that from seeds which developed at 38°/30°C. Although axis growth in seeds which developed at the lower temperatures was greater in 'Texas Cream 40' than in 'Pinkeye Purple Hull' with the standard germination test (Fig. A-6) , this difference was not significant after aging (Fig. A-8). This indicated that 'Texas Cream 40' seeds which developed at low temperature appeared to be more sensitive to stress during germination than 'Pinkeye Purple Hull'. 72 Seeds with the highest vigor were produced in 'Texas Cream 40' and 'Pinkeye Purple Hull' when developed at 28°/20°C and 33°/25°C. A similar result was reported by Ndunguru et al_. (1978) for 'K2809' cowpea. Plant development, final seed yield and weight were unaffected by developmental temperatures of 33°/24°C and 27°/19°C imposed on the mother plant. Large seeds produced larger seedlings at the early stages of development than small seeds from both temperature treatments. The adverse effect that high temperature (>32°C) during seed development had on seed vigor was also observed by Green et aj_. (1965) in soybean. Compared with low temperature (21°C), high temperature (27 C) during the last 45 days of seed maturation in soybean reduced subsequent seedling growth and seed yield (Harris et aj_. , 1965). Siddique and Goodwin (1980a, 1980b) reported that normal seedling development in bean decreased linearly as temperature during seed development and maturation increased from 21°/16°C to 33°/28°C. Walter and Jensen (1970) reported that seeds with similar vigor were produced in 'Moapa' alfalfa at both 13°C and 24°C, while seedling weight and leaf number in the progeny were greater in 'Ranger' when seeds developed at 13°C instead of 24°C. If seedling weight of cowpea was taken as a vigor index, the influence of temperature during seed development on vigor would be similar to those of bean and alfalfa; for example, the lower the developmental temperature, the higher the seed vigor. However, axis weight from seeds which developed at 23°/15°C was less than that from 28°/20°C (Figs. 12, 14). Because larger seeds had a less axis weight, the use of seedling weight as a vigor index in the above case was concealed by the influence of temperature on seed size. 73 Influence of temperature stress on seed composition Temperature variation during seed development had a profound influence on the chemical composition of the seeds produced (Fig. 15). Seed N and protein concentrations of 'Texas Cream 40' were similar at 28°/20°C and 33°/25°C, 6% higher than those values at 38°/30°C, and 13.5% lower at 23°/15°C. In 'Pinkeye Purple Hull', seed N and protein concentrations decreased as temperature increased from 23°/15°C to 33°/25°C, then increased to 5.6% total N and 22% protein as temperature increased to 38°/30°C. Seed P concentration increased linearly in 'Texas Cream 40' and quadratically in 'Pinkeye Purple Hull' as developmental temperature increased from 23°/15°C to 38°/30°C. Starch concentrations, however, decreased in both cultivars as temperature increased. On a per seed basis, all these nutrients decreased as temperature increased (Fig. 16). Thus, seed size determined the total available nutrients. Robertson et a]_. (1962) reported that protein and starch synthesis in pea seeds was delayed by low maturation temperature; however, starch content on a fresh weight basis increased at low temperature due to prolonged duration for accumulation. Sofield et al . (1977) observed in wheat that although seed N and P concentrations increased at high developmental temperature (30°/25°C) the seeds produced were smaller in size than those which developed at the lower temperatures. This led to the reduced total N and P quantities in the seed (mg per seed). Similar results of low N and P contents in seeds produced at high temperature were reported by Chowdhury and Wardlaw (1978) in wheat, oat and sorghum. High production temperatures led to reduction in seed size, starch, and protein content in wheat 74 6.0 5 .5 ~5 .0 uj 4 e; o 1— 54 .5 .0 3 .5 21 9 2 17 - °- 15 - 13 ■Texas Cream 40 Pinkeye Purple _ Hull / S. J L 0.65 0.60 0.55 0.50 0.45 0.40 55 50 45 fc 401- 23/15 28/20 33/25 38/30 35 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE ( C) Fig. 15 Effect of different temperatures during seed development on seed composition [% dry weight basis) of 'Texas Cream 40' and 'Pinkeye Purple Hull ' cowpeas. A. nitrogen, B. protein, C. phosphorus, D. starch. 75 11. Or 9.5 -a cd CD 1 — en E 8.0 O 6.5 5.0 3.5 39 ai CD 33 E z: 27 21 - 15 -Texas Cream 40 1.20 — — Pinkeye Purple "g i 04 X Hull s 3 0.88 \ | 0.72 — oo £ 0.56 J 1 I L B 0.40 80 60 - -r 40 " 20 - J 1 L D - \ \ 23/15 28/20 33/25 38/30 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE ( C) Fig. 16 Effect of different temperatures during seed development on seed composition (mg per seed) of Texas Cream 40' and 'Pinkeye Purple Hull ' cowpeas. A. nitrogen, B. protein, C. phosphorus, D. starch, 76 (Spiertz, 1977) and in pearl millet (Fussell et afL, 1980). The influence of temperature on seed vigor of the progeny was not reported in these studies, however. Ries (1971) reported that larger seedlings and higher bean yields were obtained from seeds high in protein compared to those low in protein of the same size. Austin and Longden (1965, 1966) observed that higher P contents in seeds of carrot, watercress and pea led to better seed vigor. These relationships were not found in the present experiments with cowpea after temperature stress during seed development. The greatest axis growth of 'Texas Cream 40' and 'Pinkeye Purple Hull' was from seeds which developed at 28°/20°C and 33°/25°C, yet neither had excessively high N and P concentrations. Seed development at 38°/30°C led to the highest concentrations of N, protein, and P in the seeds but the poorest seedling axis growth in the progeny. After five days of germination, lack of nutrient supply probably accounted for the reduction in axis weight from seeds which developed at 38°/30°C. However, this would not explain why axis growth of seeds which developed at 23°/15°C was less than seeds which developed at 28°/20°C and 33°/25°C because seeds produced at 23 /15°C were the largest in size. Efficiency in the utilization of storage nutrients during seed germination might be impaired by low developmental temperature or embryo damage might have occurred at low temperature. Embryo culture Although seed weights of both cultivars increased as develop- mental temperatures decreased, embryos excised from these seeds were, for the most part, similar in size (Table 13). Radicle length and axis weight of both cultivars after five days culture at 25°C were related to the embryo size, although only the axis dry weight was significantly different among the various temperature treatments. 77 Table 13 Effect of Different Temperatures During Seed Development on Embryo Size at Seed Maturity and Its Growth in Culture of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas Day/Niqht temperature Wt/embryo Axis ler gth Axis fresh wt. Cultivar hypocotyl radicle dry °C mg <~m — mg/embryo— Texas 23/15 3.4az 0.99a 3 Oa 32a 3.2a Cream 40 28/20 3.3a 0.96a 3 2a 35a 3.0a 33/25 2.9b 0.97a 2 8a 28a 2.5b 38/30 3. lab 0.95a 2 8a 34a 3.0a Pinkeye 23/15 4.2a 1.11a 3 la 36a 3.5a Purple 28/20 3.5b 0.94a 2 8a 30a 3.0b Hull 33/25 4.3a 1.02a 3 Oa 36a 3.6a 38/30 3.9ab 1.27a 2 7a 38a 3.4a zValues followed by the same letter within cultivar column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. 78 Chang and Struckmeyer (1976) reported that onion (Allium cepa) produced many aborted seeds with retarded endosperm growth when seeds developed at 43 C. Similar seed abortion was observed in 'Texas Cream 40' and 'Pinkeye Purple Hull* cowpeas at 38°/30°C. These data were not included in seed yield. Embryos surviving heat stress were able to develop normally, although the shorter duration of the seed growth period and more rapid leaf senescence appeared to lead to a reduction in nutrient accumulation in the cotyledons. Compared axis growth of embryos in culture (Table 13) with that of whole seeds (Figs. 12, 14), embryo size and its relative growth obviously did not lead to the reduction in seed vigor at stress tempeatures of 38°/30°C and 23°/15°C during seed development. Therefore, utilization of food reserves during germination was probably affected by adverse seed developmental temperatures. Partitioning of seedling weight and nutrient composition during germination Generally, changes in seedling growth and utilization of storage reserves during germination were similar in 'Texas Cream 40' and 'Pinkeye Purple Hull' seeds which developed at different temperatures (Tables A-5, A-6). Seedling fresh weight increased almost linearly in both cultivars, but dry weight decreased slightly over the five day germination period. Protein content of the whole seedling decreased during germination, accompanied by an increase in amino acid content. Loss of starch in the whole seedling during germination was not accompanied by an immediate increase in soluble carbohydrate. In fact, soluble carbohydrate decreased during the first three days of germination from seeds which developed at 28°/20 C or higher. This loss of carbohydrate was assumed to be transformed to structural components and/or used for respiration. 79 The hydrolysis of storage protein and starch, and the loss of soluble carbohydrate during germination coincided with rapid radicle growth and hypocotyl elongation (Tables A-5, A-6). From this time on, axis weights increased dramatically, accompanied by a decrease in cotyledon dry weight. The protein, amino acid, and soluble carbohydrate in the axis during the first day of germination was apparently used for respiration. No starch was found in the axis throughout germination. A constantly low amino acid content and a decrease of soluble carbohydrate in the cotyledons during germination indicated that protein and starch were hydrolyzed and moved to the axis without significant accumulation of products in the cotyledons. Although cotyledon weight and nutrient reserves in 'Texas Cream 40' seeds which developed at 23°/15°C and 28°/20°C decreased at similar rates during germination, axis weight increase was slower at the third day of germination in seeds which developed at 23 /15 C than seeds which developed at 28o/20°C (Table 14). The decrease in amino acid and soluble carbohydrate in the cotyledons and the corresponding influx of these materials in the axis were also slower in the fomer compared to the latter at the third day of germination. Decreases in axis growth and movement of amino acid and soluble carbohydrate from cotyledons to the axis, as shown in the partitioning experiment with the standard germination test (Table 14), might have been further slowed down after aging. This possibly led to the low seed vigor in seeds which developed at 23°/15°C (Fig. 12). After five days of germination axis growth rates were similar in 'Texas Cream 40' seeds which developed at 33°/25°C or less, regardless of seed size at maturity (Table 14). Cotyledon weight and nutrient reserves decreased more rapidly in seeds which developed at 33 /25 C 80 Table 14 Effect of Different Temperatures during Seed Development on the Percentage Changes in Weight and Nutrient Composition in the Axis and Cotyledons of 'Texas Cream 40' Cowpea during Germination Day/Night temperature Axis ( Cotyledon Measurement 1 3 5 T "■' 3 5 °C o) «/ Fresh wt 23/15 28/20 33/25 38/30 452az 531a 501a 374a 6780b 8250a 7850a 6600b 22500a 26100a 25500a 18100b 87a 109a 107a 94 a 92a 105a 97a 84 a 44a 41a 19b 4c Dry wt 23/15 28/20 33/25 38/30 25a 32a 31a 14a 530b 660a 630a 540b 1750a 2000a 1850a 1310b -8a -3a -2a -8 a -20a -20a -26ab -32b -49a -53a -65b -69b Protein 23/15 28/20 33/25 38/30 16a -2a -2a 9a 243a 225a 218a 228a 760a 680a 670a 570a la 3a -6ab -9b -15a -16a -28b -33b -48a -53a -70b -71b Amino acid 23/15 28/20 33/25 38/30 87a 113a 86a 61a 1660b 1990a 1980a 1630b 6750b 7520ab 7670a 5170c 51a 49a 19b 29b 39a 19b -9c 15b -17a -37b -61d -48c Soluble carboydrate 23/15 28/20 33/25 38/30 -37a -35a -35a -47a 600b 750a 730a 540b 2030a 2220a 2050a 1160b -la -la -17b -4 a -35a -66b -77c -73c -76a -84b -89c -90c Starch 23/15 28/20 33/25 38/30 -11a -8a -6a la -22a -17a -21a -28a -50a -57ab -67bc -71c "Values followed by the same letter within measurement column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. 81 compared with the lower temperatures. This appeared to be related to the initial seed size (Table A-5), because changes in total quantities of weight and nutrients over the five days germination period were less in seeds which developed at 33°/25°C than at 28°/20°C (Table 18). A decrease in axis weight in seeds which developed at 33°/25°C over those which developed at 28°/20°C during germination was probably due to a smaller embryo size in the former at seed maturity (Table A-5). Similar seed and embryo sizes were obtained in 'Texas Cream 40' when seeds developed at 38°/30°C and 33°/25°C (Table A-5). However, axis growth rate decreased in seeds which developed at 38°/30°C after three days of germination (Table 14). Loss of protein and starch from the cotyledons during germination was similar in seeds which developed at both of the above temperatures (Table 14), but movement of amino acid and soluble carbohydrate to the axis was slower in seeds which developed at 38°/30°C than at 33°/25°C (Tables 14, 15). Therefore, the former seemed unable to utilize reserve food as efficiently as the latter. A lower starch concentration in the cotyledons, at seed maturity and during germination, was found in seeds which developed at 38°/30°C compared with seeds which developed at 33°/25°C (Table 15). This might partially explain the large decrease in soluble carbohydrate accumulation in the axis of heat stressed seeds after five days of germination. 'Pinkeye Purple Hull' seeds which developed at 23°/15°C and 28°/20°C had similar seed weights at maturity (Table A-6). However, changes in weight and nutrient composition were generally delayed in seeds which developed at 23°/15°C compared with seeds which developed at 28°/20°C or 33°/25°C (Table 16). The actual increases in weight. 82 CD co o Q. rC 0 O) i— Q. 01 3 > O T3 O c >> CU +j i- o < u a> ai *3- co «a- co tf)MD|v vc mois t iOio co i-KMiHM CM CM CM CM HMNW inomo <_> t-H CM CM CO O ^-^-v.-^ CO CO CO 00 CM CM CO CO «d- co **■ co r-~ ■d- o LT> ID CT> CTl CTi O I— 1 .—1 CO CO CO <— i <— i «— 1 1 — i O CO CO CM CM CM ro ro ro <* ro co u ro xi .Q CO i— ( *J- Lfi ro ro CTl CM ro ro r^ co o o l-H l-H o 1— 1 o 00 O CTi CTi I— H oo r- CM CM CO l-v. CM CM >> X) XI ye co *r X! CTl ro cn (J <_> ro ja j2 cn CTl 1 — CTi ro ro CM r~- ro ro ld r^ 1 oo° oo' i— 1 1— < r-«. CTl l-H *d" LD «d" ■>-<=> .£ 83 Table 16 Effect of Different Temperatures during Seed Development on the Percentage Changes in Weight and Nutrient Composition in the Axis and Cotyledons of 'Pinkeye Purple Hull' Cowpea during Germination Da.y/Niqht temperature Axis Cotyledon Measurement 1 3 5 1 3 " b UC — % chan % yc Fresh wt 23/15 28/20 33/25 38/30 259bZ 480a 510a 530a 5000b 7340a 6260ab 5030b 17000bc 22700a 19000b 14500c lOlab 85b 82b 118a 105a 83a 84 a 94a 75a 35b 22bc 9c Dry wt 23/15 28/20 33/25 38/30 2b 14ab 25a 20ab 400b 560a 520a 450ab 1340bc 1710a 1520ab 1110c la -7a -7a 4a -11a -22b -21b -29b -37a -51b -58b -69c Protein 23/15 28/20 33/25 38/30 5a 17a 30a -la 270a 300a 320a 160b 700b 830ab 890a 440c -5a -10a -12a -7a -30a -29a -31a -35a -52a -63b -75c -84d Amino acid 23/15 28/20 33/25 38/30 40c 94b 139a 81b 960b 1550a 1520a 1020b 3970b 5870a 5380a 3510b 38b 73a 52ab 34b 50a 52a 9b 14b 5a -28b -47c -54c Soluble carbohydrate 23/15 28/20 33/25 38/30 -53a -58a -45a -45a 410b 640a 560ab 450ab 1490b 2100a 1680ab 910c 3a -13a -14a -14a -25a -60b -68b -80c -68a -82b -85bc -90c Starch 23/15 28/20 33/25 38/30 la la 4a Oa -25a -26a -25a -48b -46a -56b -63b -79c "Values followed by the same letter within measurement column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. and nutrient composition from day three to five in the axis of the former were close to those changes in the latter (Table A-7). Therefore, the reduction in axis growth of seeds from the 23°/15°C treatment was probably due to a lag in active growth in the first day of germination (Table A-7). 'Pinkeye Purple Hull' seeds which developed at 28°/20°C and 33 /25 C had similar growth rates in the axis during germination (Table 16), regardless of seed and embryo sizes at maturity (Table A-6). Hydrolysis of food reserves in the cotyledons and utilization of the breakdown products in the axis were, for the most part, at similar rates in both seeds. Reduction in growth and nutrient accumulation in the axis of 'Pinkeye Purple Hull1 seeds which developed at 38°/30°C, compared with seeds which developed at 28°/20°C and 33°/25°C, occurred after three days of germination (Table 16). As germination progressed, the highest rates (Table 16), but the least actual quantities (Table 18), of weight and storage reserve loss in the cotyledons were observed in seeds which developed at 38°/30°C. The same seeds were the smallest in size (Table A-6) and contained the least concentration of starch (Table 17). This most likely led to a reduction in the amount of carbohydrate available for axis growth. After five days of germination most of the starch was utilized in these heat stressed seeds (Table A-5), which, in turn, probably led to the low concentration of soluble carbohydrate in the axis (Table 17). Because changes in weight and nutrient composition in the axis and cotyledons during early germination were unaffected by developmental temperatures of 38°/30°C and 33°/25°C, low seed vigor at 38°/30°C was probably due to an insufficient reserve food at seed maturity. 85 (1) , — (13 F Q. C u. S- o Z3 f= n S- OJ cu > 0) tfl C1J >-, Q qj 4- .:«: O TD C OJ l/l QJ Q. >> S- on "O 3 C C ■o o > « 3 +J I— I nooooi >a-Lnonco i— i • (DiDtriCO co voino o o o o i— I i— I i— I C\J Oi O d C\J CO «-H 1— I I— I I*-, r^ r»- oo o\ o*i r^- cr» ^r LD IT) io mroro^j- *d" CO CO CO (*>. «d- «3- co a~> r-«. o~i o co oo o oo i— • oo o o OJ T3 s- o OJ o F u ra n3 (/) >i OJ 4-> (1) 1- £, 86 Table 18 Effect of Different Temperatures during Seed Development on Changes in Weight and Nutrient Composition of Axis and Cotyledons of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpea during a 5 Day Germination Period OC Fresh wt 23/15 28/20 33/25 38/30 Dry wt 23/15 28/20 33/25 38/30 Protein 23/15 28/20 33/25 38/30 Amino acid 23/15 28/20 33/25 38/30 Soluble carbohydrate 23/15 28/20 33/25 38/30 Starch 23/15 28/20 33/25 38/30 Day/Niqht Texas Cream 40 Pinkeye Purpule Hull Measurement temperature Axis Cotyledon Axis Cotyledon my mg 776 74 709 137 821 56 815 64 722 19 788 32 602 4 597 7 55.5 -74 52.9 -62 57.0 -66 58.3 -86 48.2 -59 58.8 -78 40.0 -58 41.9 -51 4.8 -10.7 5.2 -15.8 4.8 -10.7 5.5 -17.7 4.1 -10.8 5.7 -15.7 3.7 -10.6 4.0 -12.6 6.4 -0.3 5.2 -0.1 7.9 -0.7 6.4 -0.3 6.6 -0.9 6.0 -0.8 5.5 -0.7 5.6 -0.8 17.0 -11.3 14.6 -10.2 15.4 -10.1 17.0 -11.6 13.5 -10.2 16.3 -11.4 9.8 -9.4 8.0 -8.0 -40.5 -40.9 -36.9 -48.2 -29.5 -42.6 -26.4 -23.8 87 Lowe and Ries (1973) reported an influence of storage tissue on seed vigor of wheat. A greater dry weight in wheat seedlings derived from high protein seeds than from low protein seeds was attributed to the endosperm, not the embryo. Reduction in seed vigor due to high temperature during seed development was reported to be related to a decrease in seed size of pearl millet (Fussell and Pearson, 1980). However, information regarding which part of the seed contributed to the low seed vigor due to temperature stress was not reported. In the present study, low seed vigor of 'Pinkeye Purple Hull' cowpea which developed at high temperature was due to the insufficient food reserve in the cotyledons, not due to an injury to the embryo. Although cowpea is tropical in origin, high temperature stress during seed development had a detrimental effect on seed yield and vigor. Seed vigor was also reduced by low temperature during cowpea seed development, even though large seeds were produced. The adverse effect of high or low temperature on seed vigor was probably related to a reduction in nutrient build up on the seeds or to a reduction in the ability to utilize food reserves efficiently during germination. No apparent injury to the embryo was found in the seeds harvested from the different temperature treatments in this experiment. In order to produce seeds of high vigor in cowpea, low or high temperature during seed development should be avoided by altering the planting date and/or by using short season cultivars. Summary 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas were grown at day/night temperatures of 38°/30°C, 33°/25°C, 28°/20°C, and 23°/15°C during seed development to investigate the influence of temperature stress on seed vigor. Seed yields and seed weights of both cultivars decreased as temperature increased. The greatest axis growth was observed in seeds of both cultivars which developed at 33°/25°C and 28°/20°C. Both could utilize storage nutrients efficiently during germination, regardless of seed size. Seeds of both cultivars which developed at 38°/30°C had the least axis growth. Seeds of 'Texas Cream 40' which developed at 38°/30°C appeared unable to utilize storage nutrients as efficiently as seeds which developed at 28 /20 C or 33°/25°C. In 'Pinkeye Purple Hull', low seed vigor at 38°/30°C was related to a shortage of reserve food, especially starch, at seed maturity. Exposure to low temperature (23 /15 C) during seed development reduced seed vigor of both cultivars to a less extent than the high temperature (38°/30°C). In 'Texas Cream 40', 23°/15°C exposure probably caused a slower rate of utilization of storage nutrients during germination. In 'Pinkeye Purple Hull', a lag in active seedling growth and utilization of reserve food during early germination probably led to reduced vigor of seeds produced at this temperature. No apparent injury to the embryo was found by low or high temperature stress in this study. CHAPTER V CHANGES IN SEED VIGOR OF COWPEA DUE TO NUTRITIONAL TREATMENTS IMPOSED AT DIFFERENT GROWTH STAGES Seed vigor of Indian ricegrass (Whalley et a]_. , 1966), wheat (Ries et _al_. , 1970) and snapbean (Ries, 1971) was improved by N application to the parent plant, while that of carrot (Austin and Longden, 1966) and pea (Austin, 1966b) was unaffected by N deficiency in the parent plant. Reduction in seedling growth and yield of the progenies were observed in pea (Austin, 1966b), carrot (Austin and Longden, 1966), lettuce (Harrington, 1960) and watercress (Austin, 1966a) after maturing on parent plants which suffered from P deficiency. Calcium deficiency led to the development of abnormal seedlings in the progenies of lettuce (Harrington, 1960) and peanut (Cox et_al_. , 1976) Changes in seed composition due to an alteration in the nutritional status of the parent plant were reported to affect seed vigor of wheat (Lowe and Ries, 1972), snapbean (Ries, 1971), pea (Austin, 1966b), carrot (Austin and Longden, 1966) and watercress (Austin, 1966a). Cowpea yields were increased by the application of N, P or S (Ezedinma, 1965; Nangju, 1976); however, the influence of these nutritional treatments on seed vigor of the progeny was not reported. Sulfur amino acids, which are quantitatively low in cowpea seeds, and N contents of cowpea seeds were increased by additional N, P or S fertilizer (Evans et aj_- , 1977; Nangju, 1976). Increases in seed S-amino acids and N contents improved the protein quality and quantity 90 in cowpea, yet their relationship to seed vigor was, again, not reported. The following experiments were conducted to determine the influence of various nutritional treatments on seed vigor of the progeny of cowpea. The nutritional treatments were initiated at the time of sowing or after anthesis of the first flowers in order to compare the influence of nutritional treatments imposed at different growth stages on subsequent seed vigor. A possible relationship between chemical composition of the seed and seed vigor due to nutritional treatment was examined. Materials and Methods Effect of nutritional treatments imposed at the seedling stage of the parent plant on seed vigor of the progeny A sand culture experiment was conducted in the greenhouse in the fall of 1979 at a temperature of 27° +3C day and 22° +3C night under natural irradiation. 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas were seeded in 8 liter pots filled with sterilized #2 grade sand. Nutritional treatments (Table 19) were initiated immediately after sowing and were continued for the duration of the experiment. Pesticides were applied as needed. The experiment was a completely randomized design with four replications. Effect of nutritional treatments imposed at anthesis in the parent plant on seed vigor of the progeny A similar experiment was conducted in the greenhouse in the fall of 1980 at a temperature of 30° +3C day and 24° +3C night under natural irradiation except that the plants received a complete Hoagland's solution until flowering. At that time each pot was flushed twice with 10 liters of deioinized water to remove residual nutrients from the sand. Three levels of N (420ppm, 210ppm and 52.5ppm) in combination with three levels of P (62ppm, 31ppm and 7.8ppm) were 91 Table 19 Nutritional Treatments Imposed at the Seedling Stage of the Parental Plant Nutrient solution Element al cone enti "at ion variable N P S rtruvi ppm contror 210.0 31.0 64.0 420ppm N 420.0 31.0 64.0 52.5ppm N 52.5 31.0 64.0 62ppm P 210.0 62.0 64.0 7.8ppm P 210.0 7.8 64.0 128ppm S 210.0 31.0 128.0 16ppm S 210.0 31.0 16.0 The following elements were in all treatments: K 234ppm, Ca 160ppm, Mg 48ppm, Fe 0.6ppm, B 0.5ppm, Mn 0.5ppm, Zn 0.05 ppm, Cu 0.02ppm, Mo O.Olppm. ^Complete Hoagland's solution (Hoagland and Arnon, 1938). 92 applied throughout the seed development period. The experiment was a 2x3x3 factorial in a completely randomized design with four replications. Evaluation of seed vigor Effect of nutritional treatments on seed yield and vigor. Seed yield measurements and vigor tests were conducted as previously described (Chapter II) . Effect of nutritional treatments on seed composition. Total seed N, protein, P and starch were analyzed as previously described (Chapter III). The seed S-amino acids, cysteine and methionine, were determined by a microbiological method, using Leuconostoc mesenteroides (Difco, ATCC 8042) growth as an indicator of S-amino acid content (Evans et aj_. , 1976; Hannah et aK, 1977). Statistical analysis. Analysis of variance was performed as previously described (Chapter II). Results and Discussion Effect of nutritional treatments imposed at the seedling stage of the parent plant on seed vigor of the progeny Effect of nutritional treatments on seed yield and vigor. Average seed yield and weight were similar for both cultivars (Table 20). However, the influence of various nutritional treatments on seed yield and weight varied with the cultivar. Seed yield of 'Texas Cream 40" decreased by 41% at 52.5ppm N compared with the higher two N levels, while seed weight increased by 10% at 420ppm N compared with the lower two N levels (Table 20). The 7.8ppm P treatment led to a 38% decrease in seed yield of 'Texas Cream 40' compared with the higher two P levels, however, seed weight was unaffected by P treatment. Neither seed yield nor seed weight was affected by different S levels. 93 Table 20 Effect of Nutritional Treatments Imposed at the Seedling Stage of the Parent Plant on Seed Yields and Weights of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas Variable Seed yie Id Wt/100 s seds g/plant 9 Cultivar Texas Cream 40 17.1az 16.8a Pinkeye Purple Hull 17.5a 17.7a Treatment N(ppm) Texas Cream 40 Pinkeye Purple Hull 9.8d 22.3a 18.4b Texas Cream 40 16!5bc 18.1a Pinkeye Purple Hull 52.5 210. 0y 420.0 10.6b 18.1a 19.7a 14.1c 17.0b 19.0a P(ppm) 7.8 31.0y 62.0 13.1b 18.1a 18.5a 13.5c 22.3a 24.1a 15.7c 16.5bc 16.5bc 18.0ab 17.0b 18.3ab S(ppm) 16.0 64. 0y 128.0 19.9a 18.1a 19.6a 16.4bc 22.3a 17.8b 17.8ab 16.5bc 17.5ab 19.3a 17.0b 17.8b Interaction Cv x Treatment Values followed by the same letter within each variable column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. *,**Signif icant interaction at 5%, 1% level, respectively. •^Control : complete Hoagland's soluti on 94 Seed yield of 'Pinkeye Purple Hull' decreased by 17% at 420ppm N and 56% at 52.5ppm N, compared with the 210ppm N treatment (Table 20). Seed weight increased as the supply of N increased. 'Pinkeye Purple Hull1 yielded 40% less at 7.8ppm P than the higher two P levels, but seed weight was unaffected by P treatment. Deviation from 64ppm S in the nutrient solution reduced seed yields by 20 to 26%. Seed weight, however, increased by 10% at 16ppm S compared with the higher two S levels. After the standard germination test, seed germination and seedling axis growth of 'Texas Cream 40' were generally less than those of 'Pinkeye Purple Hull' (Table 21). The former germinated more rapidly than the latter, however. In 'Texas Cream 40', seed germinability and abnormal seedling development after the standard germination test were unaffected by various nutritional treatments imposed on the parent plant (Table 21). Germination rate was similar regardless of N treatment. Axis lengths and weights were less in seeds which were produced with 420ppm N compared with the lower two N levels, although the former had a heavier seed weight at seed maturity than the latter. Compared with the 31ppm P treatment, seeds from the 7.8ppm P treatment germinated more rapidly, while seeds from the 62ppm P treatment had shorter hypocotyl lengths. Radicle length and axis weight were similar regardless of P treatment. Generally different S levels in the growth media had no influence on germination rate or seedling growth of the progeny of 'Texas Cream 40'. Seed germinability and abnormal seedling development of 'Pinkeye Purple Hull' seeds after the standard germination test were unaffected by altering nutrient supply to the parent plant (Table 21). Germination rate was slower in seeds from the 52.5 or 420ppm N treatments compared with the 210ppm N treatment. Seeds which were produced at 420ppm N 95 ro o£ •<- O O) QJ CD en.*; c: QJ - O 0) c > c -c o +-> +■> s- -,— aj X3 +j !^ +-> O i CO •r- 96 had smaller axis lengths and weights compared with those which developed at the lower two N levels. Cotyledon and seedling weights were correlated with original seed weight. Phosphorus levels other than 31ppm led to slower germination rates and less axis dry weights in the progeny. Axis length and fresh weight were unaffected by P treatment. Seeds of 'Pinkeye Purple Hull' which were produced with 64ppm S germinated more rapidly than the other S levels. In spite of a higher initial seed weight, seeds from the 16ppm S treatment had less axis growth than that which occurred at the higher two S levels. After accelerated aging, germination of 'Texas Cream 40' seeds was less than 'Pinkeye Purple Hull' (Table 22). Although 'Texas Cream 40' germinated more rapidly and had longer axis lengths than 'Pinkeye Purple Hull', axis weights were similar for both cultivars. The number of viable and abnormal seedlings from aged 'Texas Cream 40' seeds were unaffected by altering various nutrients to the parent plant (Table 22). Seeds germinated at similar rates regardless of N treatment. Seedlings with shorter axis lengths but similar axis weight were produced from seeds grown with 52.5 or 42Cppm N compared with 210ppm N. Seeds produced on 7.8ppm P had more rapid germination rates compared with those from the higher two P levels. Axis growth was similar regardless of P treatment. Different S levels in the nutrient solution had no influence on germination rates in the progeny. Subsequent axis weight, however, increased as the supply of S increased. In 'Pinkeye Purple Hull', germinability, abnormal seedling develop- ment and germination rate after aging were unaffected by various nutritional treatments imposed on the parent plant (Table 22). Seeds which were produced with 52.5 or 420ppm N developed into smaller seedlings than seeds which were produced with 210ppm N. Seedling growth 97 QJ 1/1 QJ (O OO O) >) 01 O) en •i- c o> - a> aj o =t +-> <_> ai (0 i— •D * U o> x u o t— Q.- S- QJ ai dJ > QJ CD -^ CD C rO t- +-> Q. oo - CT>T3 XJ - CD O CD *a- ?££ *r in in VO CO r- 1 in ir> to o o o o o o O o O 1 JD jO * CT»ffiM 13 .O « O <£> CVJ CO C^ CM CO r-< CM co 0*> CM CM «-< CNJ © © o O OO o o © o «r o d en CO CO .O jO .0 i-- «r o C\J CNJ OJ o o o MOID 01 .O O -O *D U « iO U t CM lO VO CM «— I JC Q."— m in m nir^o in in m C 1- »— •*- 3 Z3 O O O o o o o o o* q. a. 3: in en CM «T -O -Q vi E a .o re (j .a re re .a re O O fl9 «3 l-l ^H O O ^* vO o ^- o co cm co m cm m 01 uo *r «a- *r co ^- *r «■ *y V >— o *r o o o o o o do o jQ >> a» U J3 « «3jd n re .a X) eo •— , OCSJ^D lO CM .-• NNO C i- <— «COOt en co cd COCO CO* •*- 3 3 a. o. dz o m "0 .0 -O o> >— trfflcvi lO T-l .-! C >. r- --3 3 m *r ^r a- a. — en o o\~* «3- mm*? flJ3 U CO CO P- b s mo o CM O O co © o r-. .-* cm* , — en o c (-) rO cr CD > 100 in subsequent axis growth compared with seeds which were produced with 210ppm N. A 10 to 20% increase in seed N and protein from the 420ppm N treatment, however, reduced axis growth of 'Pinkeye Purple Hull1 by 22% compared with seeds produced at 210ppm N. Therefore, a decrease in seed N and protein content below some threshold level appeared to affect axis growth of 'Pinkeye Purple Hull'. The reduction in vigor characteristics of 'Pinkeye Purple Hull' seeds produced with 420ppm N cannot be explained at present. Changes in total quantities of N and protein in 'Texas Cream 40' seeds did not affect seed vigor. An increase in N concentration of carrot seeds from a 141 Kg/ha N application to the parent plant did not affect seedling growth or root yield in the progeny. The concentration of P in 'Texas Cream 40' seeds was unaffected by N treatment, but increased as the level of P in the nutrient solution increased (Table 22). The 16ppm S treatment led to the highest, 128ppm S intermediate, and 64ppm S the lowest, P concentration in 'Texas Cream 40' seeds. 'Pinkeye Purple Hull1 seeds which were produced from 210ppm N contained less P than seeds from a lower or higher N level (Table 23). As P supply increased in the growing media, seed P concentration increased. Sulfur levels other than 64ppm led to the production of seeds which contained a higher P concentration. Austin (1966a, 1966b) and Austin and Longden (1966) reported that P deficiency led to seed with a low P content in pea, carrot and watercress. Upon germination these seeds developed into smaller seedlings and yielded less than seeds produced at a normal P level. In the present study with cowpea, the total quantity of P in 'Pinkeye Purple Hull' seeds decreased by 27% when grown with 7.8ppm P. During germination a possible reduction in energy supply in these low P seeds might partially attribute to the 26% reduction in its axis growth. An 101 increase in the total quantity of P in seeds produced with 62ppm P did not improve seed vigor of 'Pinkeye Purple Hull'. Thus, a threshold level of P might exist in 'Pinkeye Purple Hull' seeds, below which seed vigor could be reduced. Seed vigor of 'Texas Cream 40', however, was the same regardless of seed P concentration resulting from different P supplies in the growth media. Starch concentration of either cultivar was unaffected by N or S treatment, but increased as P level in the nutrient solution decreased (Table 23). The change in seed starch concentration due to various P levels appeared to be too small (+5%) to play an important role in changing growth patterns during early seedling development. Seed methionine concentration increased in both cultivars as the N level in the nutrient solution increased, but was unaffected by P or S treatment (Table 23). Cysteine concentration of 'Texas Cream 40' was 14% more when seeds were produced with 420ppm N compared with the lower two N levels (Table 23). 'Texas Cream 40* seeds produced with 7.8ppm P had a lower cysteine concentration than seeds from the other P levels. A low cysteine concentration was observed in 'Texas Cream 40' seeds when they were produced with an intermediate level of S. In 'Pinkeye Purple Hull' seeds, the concentration of cysteine decreased 9% at 52.5ppm N compared with the higher two N levels, but was unaffected by P or S treatment. Dessauer and Hannah (1978) reported a 10% increase in free methionine, total methionine and total protein contents of cowpea seeds when methionine was added to the growth media. Evans et _al_. (1977) reported that methionine and cysteine contents in three cowpea cultivars increased as the level of sulfate-S in the growing media increased to 5ppm. Above 5ppm sulfate-S cysteine concentration remained unchanged. 102 Failure to increase methionine and cysteine concentrations in cowpea seeds by an application of 20 Kg/ha S was also observed by Nangju (1976). In the present study, the content of S-amino acids in seeds of 'Texas Cream 40' and 'Pinkeye Purple Hull' were unaffected by the S level given to the parent plant. This indicated that the lowest S level (16ppm) was too high to induce S deficiency in cowpea seeds. There was no apparent influence of the S-amino acid content of the seeds on subsequent seedling growth. Effect of nutritional treatments imposed at anthesis in the parent plant on seed vigor of the progeny Effect of nutritional treatments on seed yield and vigor. Average seed yields of 'Pinkeye Purple Hull' were higher than 'Texas Cream 40' (Table 24). This was probably due to more seeds per pod produced by 'Pinkeye Purple Hull'. Pod number per plant and seed weight were similar in both cultivars, while the time to pod maturation was three days longer in 'Texas Cream 40' than in 'Pinkeye Purple Hull'. This cultivar differ- ence in seed yield was not observed in the previous experiment. Seed yield, pod number per plant and seed number per pod of either cultivar were unaffected by N or P treatment imposed during seed develop- ment (Table 24). Pod maturation was one to two days earlier at the 210ppm N-31ppm P and 420ppm N-31ppm P treatments compared with the other nutritional treatments (Table 25). Seeds which developed under these two nutritional treatments, however, were heavier than those which developed under the 52.5ppm N-31ppm P treatment (Table 25). An increase in seed weight of both cultivars at the high N level (420ppm) was observed at the high P level (62ppm). Summerfield et _al_. (1976) reported that compared with a continuous N supply, withdrawal of N after flowering reduced seed yield of 'Prima' cowpea by 33%. Seed size, however, was not affected. All seeds germinated normally in both cultivars after the standard germination test (Table 26). 'Texas Cream 40' germinated more rapidly 103 Table 24 Effect of Nutritional Treatments Imposed at Anthesis of the Parent Plant on Seed Yields of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas Variable Seed yield Pod/plant Seed/pod Wt/100 seeds Pod Dev Per g/plant No. No. g day Cultivar Texas Cream 40 Pinkeye Purple Hull 14.3bz 16.4a 9.8a 10.1a 9.4b 10.2a 15.6a 16.2a 23.1a 20.0b N(ppm) 52.5 210.0 420.0 15.8a 14.7a 15.6a 10.5a 9.6a 9.9a 9.9a 10.0a 9.5a 15.3b 15.6b 16.8a 21.9a 21.4a 21.6a P(ppm) 7.8 31.0 62.0 14.5a 15.2a 16.3a 9.5b 9.7b 10.7a 9.7a 10.1a 9.7a 15.9a 15.8a 15.9a 21.8a 21.1b 21.9a Interaction Cv x N Cv x P N x P Cv x N x P NS NS NS NS NS NS NS NS NS NS NS NS NS NS * NS NS NS * NS Values followed by the same letter within variable column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. * Significant interaction at 5% level. 104 Table 25 Effect of Nutritional Treatments Imposed at Anthesis on the Pod Development Period and Seed Weights of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas N ( PPm) P (ppm) Variable 7.8 31 62 Pod Dev. Per. (days) 52.5 210.0 21.7aZ 21.9a 21.9a 20.3b 22.0a 21.9a 420.0 21.9a 21.0b 21.7a Wt/100 seed (mg) 52.5 210.0 15.7a 15.6a 15.2b 16.4ab 15.0b 14.8b 420.0 16.5a 17.5a 16.5a Values followed by the same letter within variable column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. 105 fO D. s_ Q. 3 Q_ +-> +-> c 0) oo CI) >> QJ S- +J fO -a c <+- C T- o (O E S- 1/1 - aj •(— O CO l/l ^r CI) ■a c E s- +J r0 ra c 0) "O c s <_> , — T3 to CI) c 0) O 00 ■u "O •1- c aio: i- i— C_! i— +-> J3 CI) CO -O O 106 and had a greater axis growth than 'Pinkeye Purple Hull'. Changes in N or P level in the nutrient solution during seed development had no influence on seed germinability or abnormal seedling development of either cultivar when they were germinated under optimum conditions (Table 26). Germination rate of 'Texas Cream 40' was unaffected, while that of 'Pinkeye Purple Hull' varied among nutritional treatments (Table 27). Alteration in germination rate of 'Pinkeye Purple Hull' was not related to subsequent seedling growth, however. As the level of N increased in the nutrient media during seed development of 'Texas Cream 40', hypocotyl length and axis fresh weight in the progeny decreased after the standard germination test (Table 26). Radicle length, axis dry weight and seedling fresh weight remained unchanged. In 'Pinkeye Purple Hull', seed development at the lowest N level (52.5ppm) led to the production of smaller seedlings in the progeny compared with seeds from the higher two N levels. Cotyledon weight and seedling dry weight were related to the initial seed weight of both cultivars. Varying the P supply throughout the seed development period affected seedling growth in the progeny similarly in both cultivars after the standard germination test (Table 26). Seeds which developed with 31ppm P gave rise to smaller seedlings than seeds from the other two P. levels. Cotyledon weight and seedling dry weight were the same regardless of P treatment. After accelerated aging, over 80% of the seeds germinated normally in both cultivars (Table 28). 'Texas Cream 40' germinated more rapidly and had a greater axis growth than 'Pinkeye Purple Hull'. This was similar to that found with the standard germination test. 107 Table 27 Effect of Nutritional Treatments Imposed at Anthesis of the Parent Plant on the Average Days to Germination of 'Texas Cream 40' and 'Pinkeye Purple Hull' Cowpeas with the Standard Germination Test N ( ppm) P (ppm) Cultivar 7.8 31 62 Texas Cream 40 52.5 210.0 l.laZ 1.1a 1.1a 1.2a 1.1a 1.1a 420.0 1.2a 1.1a 1.1a Pinkeye Purpl Hull e 52.5 210.0 1.6a 1.3b 1.3b 1.6a 1.2b 1.4a 420.0 1.3b 1.5ab 1.2b Values followed by the same letter within cultivar column are not significantly different at 5% level of probability according to Duncan's Multiple Range Test. 108 - CD +J o c c *d" >r- 0) O) S- E > (t! (- n ■n s_ O) -M aj ■a ^ oo c: •r- +J 1/1 1/1 •i- C S- CO +-■ O •!- g~S sss -OS- •>- r— 1— a* 01 o 0> Ol CTi Cfi O CU 3 1— 1—1 Q_ Q. 1— rO -r- -r- C 1- 0 fC rO cc QJ cu C •1— Q. £ +J ■M ^ r: > i- 0) l/l +-> c (1) ro 1 — U C QJ 71 E Q (/) O 1 — i— >> 1 — +-> O) «3 132 d) o i— o 3 O OO S- .— 1 O «3" cn+-> r- O) T3 C O) O) CO co in cr> i— i co co en ld in co Oi a. i— ns •■- •,- O) , — S- +-> - 3 cu +-> c a. 7E > 0) a> in ci) 00 .C CT> i/i i — OO +J c i/i (.; 1/1 c >>-a l/l ^ l/> o XI s- e s- o 2^ ^ s; -a o 0) o 2 * o <— >> 4-> r— ■I- 3 CD H3 3 J3 133 Table A-4 Seed Coat Thickness of Hard and Nonhard 'Pinkeye Purple Hull' Seeds Which Developed under SD Seed coat layer Hard Nonhard Cuticle Palisade Basal Total thickness - 1.6-2.3 am - — ■ 1.7-2.0 40-47 40-50 14-25 16-20 5.6-74.3 57 .7-72.0 134 c C o CD • p— F +J D. S- JCM^rH «5f>vrs CM C\i *-» ^* •o *o XI x> > in «-h «— i ••« ^h ^- CM ^> O omcrs .-i ^* ■— *-« co 10 • 00 CT* O CO xi «o x> o LO Cn UD vD CO •— t *-< KD NVOCO fO * -a xi a% •a xj « HVDO X> « X) XI co cr, co ld (O « O CC ou ^- in P** N»OS •— > CM •— i .— . m^- TT cr* en r^. in ,^ rococo O o o o o o o o O o o o rwcon xj in ^ cc m vo co (VJHfl-O OOCON co cm cm co « X> o -a o o o o "3 X) (J (J OrHtNJN - m en oo * CO VO -h _ O X) o o ^ (O .n u 0«—cm «y coatrvvo o o o o <3 XI XI U HcoiriH CM ~* <— I ^-t « X] U O CO <— t «— i CM lO C"» r-» 143a 120b 85c 77c — < CO CO CO H^mrt *r O ~h -« ft CTi CM CM ^ ^ co co in o CO co «3J3 U U *r *r ~* ^* «T CM Q% CO *0 « «Q X> COOChO ££<££ ro ^-i *r >«a- CM CM «— ■ ^H CVJPO^Hfj m cm cr* o r» tt O oi L gs CO CO CO CO in o i ro CO co co CM CM CO CO CMCMCOCO CA © lO C* f\ji-;if>ifi CNJCVJrHrt ^ CM « ro 00 co oo oo o m in r-» co .— • CM in CM CM t-H m o m o «— I CM CM CO CO CO CO CO ■— • CM CM CO ro CO co CO ICOCO CMCMCOCO CMCMCOCO CD >> J3 O CO tO «T CO m o in o "^•^ • — *-*. CO CO CO CO i— CU o CO u c ■+-> o: c a; cu E <— d) Q. 5- -i- 3 +-i CO I— >a 3 135 (OO^VOiH CM OS CTt Cft ntVJr-l ,_, occmrxj *r o m cm — i ^h o O* «r cm* cm" d ^£2 S2210 ^"' *0 J3 U "O cm ro ^r vo cm *-, ^h ^ ui ,,* _" is! v o in p-t vo vo m *— • c 1 — o ai > +J a; ro a C "O F CD c 4- S- 3 3 T3 in C (1) o s- <— 3 o >> •f- a; •r- C J- i- ft3 Q. Q- - fo.au _ m cm i > CM «— I i-HH vo vo ro r»» M in oo n cs. CVJ t— I ^h m m *r o CM CM CM CM in cm <— • r** O ro co ^-» OisO^O cm ro •— i o Oco*~fin ^ ,-" ^ ^ O rn^- c> inr*»ro( oo co vo ro w CM CM CM CVJ Orvrvrv VD r- 1 CSJ UO ro vo tt «— ro^- ,-. r^ in vo so in vo co ro en «S t— • CM CJi VO Oi o «*■ CO CnCM^-O CMCMCMCM hmh«m i— • CM CM CM j3£1 fl it) en cy> CT* un COCOfl-O C0^Hr«-.crt > VO ^ CO ^- » OOOO OOOO OOOO — - co vo en o rv. vo ( «t ro r*. csj CM CM r-» r-t ro ^r uo co cocccoro coccocm OT* VO CT» ^" CM CM 4 O CO OS CO OOOO O ocncft os asm vo •-• cc co vo vo vo m <— t | CM .—i i— t ,— i cr» o co en CM •— « CO CO CSJ ^H f-H f-l Onnco vo in ^- en en co vo ro SSrCS LOOmo mom© C ^ -^.Q ^ cm cm ro rH(Mc\jn £ S £25 ^ co m co neon© cm csj roro csjesjroro cm csj ro co » O in o in o in o t csj csi ro rHfvjfvjrn t co ro co ro co ro co i csj ro ro cm cm ro ro O m o ' cm cm r~ ' co ro < cm csj ro i i csj ro ro co'co co 1 — cij o ai o c ro 4-> Oi c OJ 0) S r— i/i ro 3 +■> o ■i- c 2 3 -a .a OJ rO 2 -O 136 Table A-7 Effect of Developmental Temperature on Changes in Weight and Nutrient Composition in the Axis of 'Pinkeye Purple Hull' Seeds during Germination Measurement Day/Night temperature Germination period (days) 0 to 1 3 to 5 ■or Dry wt 23/15 28/20 33/25 38/30 Protein 23/15 28/20 33/25 38/30 Amino acid 23/15 28/20 33/25 38/30 Soluble carbohydrate 23/15 28/20 33/25 38/30 — mg — 0 0.5 1.0 0.7 0.04 0.12 0.19 0.09 0.05 0.10 0.15 0.13 -0.52 -0.47 -0.43 -0.40 — mg — 37.2 39.3 38.6 25.0 3.23 3.53 3.68 2.57 3.94 4.67 4.33 3.94 10.7 11.8 11.0 4.1 137 100 80 A TOTAL Texas Cream 40 E 10 9 c ~60 z o < 40 s: § 20 Pinkeye Purple Hull 1— CD UJ _l 1— o o D. >- X 8 7 6 I ABNORMAL 5 i 0 -r i I B D _1.8 (0 -a b u zc h- CT zr LU _i UJ _J o < 15 13 11 9 cd — - i i i I ° 960 720 480 240 " -Texas Cream 40 -Pinkeye Purple Hull 130 104 78 52 26 0 104 +-> | 78 a. en £52 I 0 104 78 - 52 - 26 " 01 -0.8 -4.0 -8.0 "-0.8 WATER POTENTIAL (bars) -4.0 -8.0 Fig. A-2 Effect of water stress during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test. Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, E. axis, F. cotyledon. 139 00 80 ^-^ >s Z' 60 o I— < 40 7" d; cj ZU 0 1 .8 ' .6 >i 01 -r> — .4 <.n 1.2 - l.Ol— i .TOTAL. Texas Cream 40 Pinkeye Purple Hull ABNORMAL 10 "e o w g :e i— CD S 8 _i _i >- 5 7 c_> o Q. >■ = 6 5 I15 |l3 LU —1 311 a g -0.8 -4.0 -8.0 -0.8 WATER POTENTIAL (bars) -4.0 -8.0 Fig. A-3 Effect of water stress during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 140 1200 960 - 720 - 480 - 240 0 • 960 i W20 i 480 i | 240 i 0 960 720 480 " 240 - 130 •Texas Cream 40 Pinkeye Purple Hull 104 78 52 26 0 _J04 +j c (T3 Q- 78 O) E h- 52 CD LU 3 26 >- Q 0 104 f- 78 52 - 26 - -0.8 -4.0 -8.0 u-0.8 WATER POTENTIAL (bars) -4.0 -8.0 Fig. A-4 Effect of water stress during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging. Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, E. axis, F. cotyledon. 141 TOO 80 ~ 60 o S 40 LU ^ 20 0 2.0 -.1.5 T3 ~1.0 Q < 0.5 0.0 B TOTAL Texas Cream 40 Pinkeye Purple Hull ABNORMAL 13 E Jdll 21 9 LU 3 _l _l >- o £ 5 3 -20 E u ^15 o LU _i O § 5 23/15 28/20 33/25 38/30 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE (°C) Fig. A-5 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 142 130 104 78 52 26 0 +J 104 c (O a rr, /8 b h- X W 11 1 12 X 26 U~) 111 oi 1 1 0 104 J- 78 52 26 B 150 120 90 Texas Cream 40 60 Pinkeye Purple Hull I I I 30 0 120 +-> c ro Cl. 90 rr> e ' — ■ X 60 cr iii ^£ 30 >- a: Q 0 120 90 60 30 23/15 28/20 33/25 38/30 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE (°C) Fig. A-6 Effect of different temperatures during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas with the standard germination test, Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, E. axis, F. cotyledon. 143 100 80 60 40 20 0 2.0 ^1.5 1.0 0.5 I- 0.0 Texas Cream 40 Pinkeye Purple Hull 13 X I— S 9h _i _i >- 5 7 o o Q. - 5h 3 ^20 P15 CD 23/15 28/20 33/25 38/30 ujIO C-J o / / / 1 1 \ \ \ \ \ \ \^ i i D \ \ \ 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE (°C) Fig. A-7 Effect of different temperatures during seed development on seed germination, germination rate and seedling lengths of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging. A. germination percentage, B. average days to germination, C. hypocotyl length, D. radicle length. 144 130 104 78 52 26 0 ^•104 to 5 78 E £ 52 CD UJ = 26 LjJ u. 0 104 78 Texas Cream 40 Pinkeye Purple Hull 150 120 90 60 30 0 ^120 fO § 90 E l 60 ^ 30 Q 0 120 90 60 30 0 23/15 28/20 33/25 38/30 23/15 28/20 33/25 38/30 DAY/NIGHT TEMPERATURE ( C) Fig. A-8 Effect of different temperatures during seed development on seedling weights of the progenies of 'Texas Cream 40' and 'Pinkeye Purple Hull' cowpeas after accelerated aging. Fresh weight: A. seedling, B. axis, C. cotyledon. Dry weight: D. seedling, E. axis, F. cotyledon. J L — n \ \ \ \ \ X ■■ s \ \ \ \ \ \ \ — \ \ \\ - 1 f 1 1 E - *2^ V\ m \\. >\ S i i l i F - ^5\^ _ v\ \x. \ X. \ Xv - \ \ \ x _i_ i i i BIOGRAPHICAL SKETCH The author was born in Nantou, Taiwan, on June 23, 1952. She enrolled at National Taiwan University, Taipei, Taiwan, in September, 1970, from which she received her Bachelor of Science degree with a major in agronomy in June, 1974. After receiving a Master of Science degree in agronomy from National Taiwan University in June, 1976, she worked in the Plant Physiology Department of the Asian Vegetable Research and Development Center in Shanhua, Taiwan, until November, 1977. In March, 1978, she entered the Vegetable Crops Department at the University of Florida to pursue her Doctor of Philosophy degree. She is married to Shih-Tsan Tang and has a son, Chiaming. 145 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. niel J. Cantliffe, Onail Dan Professor rman of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. k A^-<1 i^t f§f\ /2 Ys^C esley B. HaPTl Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 1U Thomas E. Humphreys Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Terril A. Nell Assistant Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Indra K. Graduate Research Professor of Botany This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May, 1982 'COCf\ kt.& Dean, CoTllege of Agriculture Dean for Graduate Studies and Research UNIVERSITY OF FLORIDA 3 1262 08553 3643