usury :THH F2512) This is to certify that the thesis entitled Increasing Vigor and Seed Protein in Rice Grown in Indonesia presented by Harry Clair Bittenbender has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture 2’;an Kg, 4 Major professor Date [M zit (4/977 0-7639 INCREASING VIGOR AND SEED PROTEIN IN RICE GROWN IN INDONESIA By Harry Clair Bittenbender A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1977 ABSTRACT Increasing Vigor and Seed Protein in Rice Grown in Indonesia By Harry Clair Bittenbender Seed storage in the tropics is hindered by environmental factors, particularly high temperature and humidity which accelerate seed deterioration. Several hypotheses concerning increasing seed protein content in rice (Oryza sativa L.) and the effects of increased protein on seed vigor and subsequent yield were teSted under conditions of intense subsistence lowland rice culture in central Java, Indonesia. Foliar urea at 40 Kg N/ha increased protein and yield but was not superior to topdressed urea at the same rate in the wet season. During the dry season, the crop was severely damaged by grassy stunt disease and its vector the brown planthopper (Nilaparvata lugens). Foliar urea significantly increased protein content but at the expense of yield. High protein seeds of IR-S and Pelita l/l (2.ll mg protein/ seed) had higher germination following a five day storage stress than low protein seeds (l.63 mg protein/seed). A four factor field experi- ment was designed to compare two levels of genotype (IR-5 vs. Pelita l/l), seed protein content (2.ll vs. 1.63 mg protein/seed), degree of seed storage stress (none vs. five days), and planting method (trans- planting vs. direct seeding). It was also severely damaged by grassy Harry Clair Bittenbender stunt disease and brown planthopper infestation. There were no yields from the transplanted rice. Results from the direct seeded rice indi- cated that high protein seeds increased yield more for IR-S than for Pelita l/l. Storage stress had no effect on yield. The germination responses of high and low protein seed lots of IR-S and Pelita l/l rice exposed to two methods of storage stress were investigated. The LD50 for high protein seeds was 75 days, and 55 days for low protein seeds stored at 27°C and l8% moisture content. High protein seeds remained viable longer when stored at 40 C and 20% moisture content in sealed vials or over water. Seedlings from high protein seed had greater seedling vigor, and dry weight, regardless of storage stress. The stressing of whole seeds in storage indicated that seed vigor of high protein seeds appears to be in the embryo, while the endosperm is thought to be responsible for seedling vigor. To Susan, who suffered more than I. ii ACKNOWLEDGMENTS The author thanks Dr. S. K. Ries for his steadfast support during all phases of this study, and my committee, Drs. Stan Howell, L. O. Copeland, and E. H. Everson. The financial assistance for the research in Indonesia was provided by the Midwest Universities Consortium for International Activities, MUCIA. Drs. Adlowe Larson and Robert Clodius as MUCIA representatives provided strong personal support during our stay in Indonesia. The advice and support of IRs. Sudikno, Sunyoto, Sumartono, Isbandi and the field boss, Pak Asmudin, of the Department of Agronomy, Faculty of Agriculture, IR. Sutardi and Dr. Sudarmadji, Facilty of Agriculture Technology, Gadjah Mada University, Yogyakarta, Indonesia is appreciated. Special thanks goes to IR. NY. Titi Sudikno for her warm friendship and advice both at work and home. And finally, a big thank you to my wife, Susan, for her personal and professional help, my parents, Mr. and Mrs. C. K. Bittenbender, my in-laws, Dr. and Mrs. A. D. Engstrom, and our university friends. iii TABLE OF CONTENTS Page LIST OF TABLES .......................... v LIST OF FIGURES ......................... vii INTRODUCTION ........................... l LITERATURE REVIEW ........................ 2 SECTION 1: Increasing Protein Content of Rice and Its Effects on Seed Vigor and Subsequent Yield ......... l6 Abstract ...................... 17 Introduction .................... 18 Materials and Methods ................ 2l Results and Discussion ............... 25 Literature Cited .................. 34 SECTION II: Germination and Growth of Rice Seedlings Differing in Seed Protein Content under Various Storage Stress Conditions .................. 38 Abstract ...................... 39 Introduction .................... 40 Materials and Methods ................ 45 Results and Discussion ............... 47 Literature Cited .................. 56 SUMMARY ............................. 59 BIBLIOGRAPHY ........................... 61 iv LIST OF TABLES Table Page l Yield components, protein content, N efficiency, and yield after urea applications at anthesis for 'IR-S' and 'Pelita 1/l' rice grown at Yogyakarta, Indonesia in the wet season 1975-76 ................ 26 2 Yield components, protein content, N efficiency, and yield after urea applications at anthesis for 'IR-5' and 'Pelita l/l' rice grown at Yogyakarta, Indonesia in the dry season, 1976 ................. 27 3 Germination of 'IR—5' and 'Pelita 1/l' rice seeds as affected by seed protein content and a five day storage stress of 40°C and 20% moisture content ......... 29 4 Emergence of direct seeded 'IR-5' and 'Pelita l/l' rice seedlings as affected by seed protein content and a five day storage stress of 40°C and 20% moisture content at Yogyakarta, Indonesia in the dry season, 1976 ...... 30 5 Stand at maximum tillering of 'IR-S' and 'Pelita 1/1' rice as affected by seed protein content, a five day storage stress of 40°C and 20% moisture content, and planting method at Yogyakarta, Indonesia in the dry season, 1976 ...................... 31 6 Yield components and yield of direct seeded 'IR-5' and 'Pelita 1/1' rice as affected by seed protein content and a five day storage stress of 40°C and 20% moisture content grown at Yogyakarta, Indonesia in the dry season, 1976 ...................... 33 7 L050 determination by the Houston linearization and the Spearman-Karber methods of storage stressed 'IR-5' and 'Pelita l/l' rice seeds at two protein levels stored at 27°C and 18% moisture content ......... 49 8 Germination of 'IR-5' and 'Pelita l/l' rice seeds as affected by seed protein and storage stress at 40°C and 20% moisture content in airtight vials or over water .......................... 50 Table Page 9 Speed of germination of 'IR-S' and 'Pelita 1/1' rice seeds as affected by seed protein and storage stress at 40°C and 20% moisture content in airtight vials or over water .......................... 51 10 Dry weight of l9-days-old 'IR-S' and 'Pelita l/l' rice seedlings as affected by seed storage stress at 40°C and 20% moisture content in airtight vials and seed protein content . ............ . . ...... 53 ll Germination of 'IR-S' rice seeds and embryos on nutrient media as affected by seed protein content and seed storage stress ..................... 54 vi LIST OF FIGURES Figure Page 1 Mean linearized germination response of 'IR-5' and 'Pelita l/l' rice seeds as a function of days stored at 27°C and 18% moisture and protein content . . . . . . 48 vii INTRODUCTION There are several factors responsible for the food shortage that exists in the developing nations of the tropics; some of these are low productivity per hectare, high storage losses, and inadequate consumer- producer market systems. Solutions such as fertilizer-responsive pest- resistant varieties; improved storage methods, and planned-stabilized market systems are possible or at least probable, with the development of on-site technology. However, the implementation of these solutions is dependent upon high politico-cultural acceptability and high margi- nal return. This study was initiated to determine if increased protein content in rice seed (Oryza sativa L.) could prolong seed viability in tropical storage conditions and increase yields in an Indonesian paddy environ- ment. LITERATURE REVIEW Seed storage in the trepics. Reviews of seed storage in the tropics indicate that major problems center around extreme temperatures and humidity during storage (8). Thirty-five percent of storage losses of Southeast Asian paddy (unhulled rice seed) is attributed to heat and moisture. Most paddy is stored in bulk for 1-12 months at 12-16% moisture content (fresh weight basis) and 28-38°C (153). In Malaysia, granary humidity and air temperatures are higher than ambient. Tradi- tional, long-dormancy cultivars maintain viability longer than modern varieties under granary conditions (59). This phenomenon has been observed in rice seed buried in submerged soil. IR-8, a short dormancy cultivar and H4, with longer dormancy, were removed from the soil and germinated at weekly intervals for a year. IR-8's higher germination, 60%, occurred 12 weeks after burial and decreased to 10% by the end of the year. H4 had 80% germination from the 24th to the 46th week (66). In Costa Rice, bean (Phaseolus vulgaris L.) samples from farmer's seed stocks had an average of 16% moisture content and 72% germination. This was related to poor crop stands (13). Recent research on storage methods includes low cost desiccants, e.g. Ca0, CaCLZ, that are locally available (105)1 and freeze-drying of hard-to-store vegetable seeds (163). 1Personal communications with Ir Titi Sudikno, Agronomy Department, Fakultas Pertanian, Universitas Gadjah Mada, Yogyakarta, Indonesia. 2 Vigor. The concept and measurement of vigor has been the subject of several recent reviews (24,120,131). Kidd and West (73) are credited with collecting early vigor related literature and doing research on 'physiological predetermination' of seedling growth and yield. One definition of vigor states that a seed has a maximum potential from which there is a continuous decrease in potential over time, i.e., aging or deterioration (120). Vigor is conceptualized in different ways, as survival under adverse conditions or as an inherent physio- logical state. Use of the term 'vigor' can include all these situations: survival in the nonactive state (seed vigor), survival upon sowing (seed vigor), establishment of stand and healthy, normal growth (seed- ling vigor) (55). Resistance to microbial invasion is considered secondary to resistance to that environment conducive to microbial growth. Germination. An in-depth discussion of seed and seedling vigor requires an appreciation of the physical, morphological, and physiologi- cal events of germination. These events follow a general chronology: inbibition of water, hydration and activation of macromolecules, cell division and cell elongation, emergence of the embryo, and completion of nonrepetitive morphogenesis of the primary plant body (11). Mois- ture, temperature, light, atmospheric composition affect rate of progression of these events (24). Specific events mentioned here provide a background for the basis of seed vigor tests. The imbibition of water, the first step in germination, can also be the first obstacle, as in hard seed coats of legumes. The seed coat can also act as a gas diffusion barrier as in 02 uptake restriction, the principal cause of rice dormancy. This dormancy is broken by pricking the seed coat near the embryo (106). Other forms of dormancy, immature embryo or inhibitory levels of hormones, must be taken into account when evaluating vigor. Dormancy can manifest symtoms of low vigor, i.e. low germination when the seed may actually have a high germination potential after dormancy has been broken or satisfied. Cold temperature during imbibition of cold sensitive species affects membrane permeability. In cotton (Gossypium hirsutum L.), the glyoxysomal membrane becomes impermeable to succinate, resulting in feedback inhibition of isocitratase (108). The hydration and activation of enzymes in the seed brings about the hydrolysis of stored protein by proteases, carbohydrates by amylases, and lipids by lipases for translocation, synthesis, or oxidation for energy by the embryo (94). In rice, total protein (mostly glutelin) decreases rapidly, while soluble protein increases. The proteases are thought to be bound to the protein bodies in the endosperm. Many other enzymes, and the m-RNA for proteases, are pre- sent in the dry seed (112). Glutamic acid decarboxylase (GAD) decar- boxylates glutamic acid, a major amino acid in storage protein (18). In barley, (Hordeum vulgare L.), y-aminobutyric acid (GABA) is metabo- lized to succinate as a Krebs' cycle intermediate in the embryo (63), and serves a regulatory function for control of a-amylase (32). Theories and effects of seed aging. This discussion includes changes within the embryo and endosperm of monocots and within the epicotyl-hypocotyl axis and cotyledons of dicots. While it is general- ly assumed that the embryo is the site of aging, results of reciprocal embryo-endosperm transplants with aged and fresh wheat (Triticum aestivum L.) seed are inconclusive. Whether an 'aging factor' is translocated from embryo to endosperm or vice versa, or if both respond to aging simultaneously is uncertain, as the germination responses over time of storage for aged embryo or endosperm transplants and whole seeds are similar. The transplanted aged embryos had less germination than the corresponding endosperm transplants, but this could be caused by embryo sensitivity to transplantation (42). The physiological effects of seed aging can be studied by com- paring seedlots from different harvest years, or by creating populations of artificially aged seeds from the same seedlot by stressing the seeds with high humidity and/or temperature during storage. Another method for artificially aging seeds is the use of mutagenic chemicals or radiation. A classic study of the interaction of temperature and H20 content on rice seed viability during storage (130) shows that the effects are additive. Using multiple regression analysis, the log exposure period necessary to produce any germination level at a given tempera- ture and moisture content can be calculated. This approach to seed viability prediction has also been demonstrated for wheat, barley, broad beans, (Vicia faba L.) and peas (Pisum sativum L.) (131). Two theories are recognized as possible explanations for the immediate results of seed aging (52). The major theory, cytoplasmic dysfunction, includes accumulation of germination inhibitors, denatura- tion or reduced synthesis of macro-molecules like protein, lipids, and nucleic acids. The second theory views aging as one of increased chromosomal mutation. The accumulation of germination inhibitors (of viable seeds) has been noted in nonviable rice seed and hulls (31), and identified as ferulic and sinapic acids (143). This accumulation is conceptualized as a 'terminal dormancy'. Several general phenomenon during germination are associated with seed aging, respiration rate (02 uptake) is reduced with increasing age in wheat and barley (1,2,53), rice (116), and soybeans (Glycine max (L.) Merr.) (33). Increased membrane 'leakage' of sugars, amino acids, and other electrolytes is common (21,34,53). Incorporation studies using labeled amino acids (20) and glucose (1) showed different pat- terns of, and reduced amounts of, utilization in aged seeds. General constituent changes in dry or 24 hr-after-imbibed aged seeds are reduced soluble protein and increased free amino acids in rice (l7), wheat (1,84), and soybeans (61,45). Decreases in glutamic acid levels in aged dry rice or wheat are usually attributed to the formation of GABA (9), however, changes at H20 content below 12% are thought to be temperature-dependent and nonenzymatic in nature (10). A decrease in polar lipids and an increase in lysophosphatidyl choline in cucumber (Cucumis sativus L.) has been indirectly linked to free radical oxidation of membranes (76). No free radical differences were found in fresh or aged wheat (2). Enzymatic studies of aged seeds show strong correlation between respiration rate and germination, catalase, and peroxidase activity in rice (116) and reduced ATPase RNase, and lipase activity in soybeans (90). The loss of peroxidase activity in aged rice seed paralleled the disappearance of a major peroxidase electrophoretic band (139); the extractibility and activity of malic acid, glutamic acid, alcohol dehydrogenases (77), phosphatase and a-amylase activity (144). Membrane permeability changes in response to temperature extremes and solvent systems are responsible for leakage of a-amylase from bean embryos (139). Some enzymes like GAD are activated at seed moisture levels below germination levels (83,84). The use of GAD activity as a seed vigor test is discussed later. Reduced protein synthesis in aged seeds during germination (110) is usually related to reduced RNA synthesis (14). Using a poly-uracil- directed, cell-free system, the inhibition of amino acid binding to t- RHA is shown to be responsible for reduced protein synthesis (129,110). Recent work indicates that denaturation of an elongation factor (EFI) is involved in m-RNA translation and may be responsible for t-RNA amino acid binding inhibition (28). Structural changes in dry but aged seed also occur. Electron micrographs of nonviable rye (Secale cereale L.) embryos show plasma- lemma and mitochondria abnormalities as well as reduced DNA and RNA integrity (49). A unique series of experiments following the chromosomal mutation theory with lettuce (Lactuca sativa L.) has shown that seeds stored dry have an increased incidence of chromosomal aberrations as they age. Seeds stored fully imbibed but in a thermal-induced dormancy suffer no chromosomal aberrations or loss of germination (159). Later, it was shown that these seeds can repair chromosomal aberrations caused by heat or y-irradiation damage in the dry state when held in a thermal- induced-dormant-fully-imbibed state (160). Studies using y-radiation (64) and ethyleneimine, both mutagenic agents (65), demonstrated that glucose could improve the germination of treated rice. Subsequent seedling growth could not be improved by supplemental glucose, suggesting a block of a late germination event. Finally, the reduction of yield resulting from poor stand and/or growth was observed in transplanted rice (145) and direct seeded soy- beans (35). Measuring vigor. The goal of a vigor test (including viability) is to quantify the vigor differences between seedlots in order to pre- dict growth and yield potential. In recent years, two quick tests for seeds have become popular for determining viability and providing some idea of the storage history of the seed. The tetrazolium test works well for both monocots and dicots; it is based on the activity of dehydrogenases present in dry seed (95,24). The other test measures C02 evolution from GAD activity in ground seed wetted with a glutamic acid solution. Good correlation between GAD activity and reduced germi- nation in natural or artificially aged seeds has been shown for wheat (85) and rice (9,68,98). GAD activity is not effective in predicting germination percent between gresh and aged soybeans (4). The popularity of these methods is in part due to their speed and simplicity compared with other enzyme assays. Good correlations have been shown for isocitratase activity with seedling weight of cotton (134), a-amylase activity with seedling weight of rice (162), and 14C-glucose uptake and utilization with percent germination times hypocotyl length of soybeans (3). However, these assays and 02 uptake during imbibition of wheat (26) and soybean (3) have not gained wide acceptance by seed analysts because of the 'elaborate' instrumentation required. The germination test remains the standard vigor test (24). Unfortunately, the decrease in germination is one of the last observa- ble vigor phenomenon to be affected by seed aging. A more sensitive measure of vigor loss is the change in germination rate (55). Several methods for quantifying this are available, the simplest is called the speed of germination. This is calculated by summation of percent germination on day one divided by one plus the percent germination on day two divided by two and so on, until germination is complete (154). The germination curve as a function of seed age or stress during storage is sigmodial (55). This curve can be linearized by probit or probability graph paper (132) or using a simple log function (60). If only the time period or dose required to produce 50% germination (L050) is needed, (as in many dose-response bioassays), then the Spearman- Karber equation can be used (40). The advantage of linearizing the germination curve is the ease of comparing the rate of germination loss between seed lots, and the 50% germination value can still be determined easily. Another approach to evaluating vigor using germination has been to stress or artificially 'age' the seeds first and then germinate them under normal conditions. The U.S. method of artificial or accelerated aging recommends placing the seeds over free H20 (100% relative humidity) at 40°C (29). The British method usually calls for increase moisture content first (or uses freshly harvest seed which must be dried to the desired H20 content) and storage of the seed in a sealed container at a given temperature (131). Consequently, the atmospheric gas composition is different for seeds artificially aged by these two methods. The cold test is a stressed germination test utilizing the lO germination of seeds at temperatures below optimum. It works well for corn (ggg_mgy§_L.), soybeans (34), and rice (60). Another type cold test employs a thermo-gradient plate, so that germination can be easily evaluated at several temperatures (22,80). The slant board method uses a germination blotter raised at a 67° angle. Many seeds can be germi- nated at once, while allowing for proper geotropic responses by the shoot and root for daily measurement under different temperature and light conditions (71). Seedling vigor is usually evaluated as dry weight or height under normal conditions (24). Application of a stress during growth is now being used for evaluating seedling vigor for specific problems like drought (47). Enhancing vigor. This phrase is paradoxical in terms of the vigor dogma used in this discussion. Two common events are mistakenly called enhancement the germination change due to cessation of seed dormancy and post-dormant endogenous germination rhythms (90). Other reports of enhanced vigor utilize techniques which prevent deterioration (114), induce embryo repair (46,137), avoid environmental stress (8,16,109) or rapidly activate germination enzymes during imbi- bition (101,151). Electromagnetic seed treatment purportedly initi- ates a step in the sequential breaking of dormancy (92). Depending upon how vigor is defined, these techniques either enhance vigor or merely improve conditions internally or externally for maximum expres- sion of the inherent vigor of the seed. Environmental effects on seed characteristics and subsequent 3199;, Environmental effects, also called year or location effects, on yield are well known by every farmer and plant scientist. Seed ll characteristics including vigor are responsive to environmental condi- tions from the time of the parent plant to planting of its seed. Physi- cal factors like moisture content during harvest and cleaning procedures can predispose beans to mechanical damage (16). Seed damage affects wheat and pea seedling growth also (19). Other factors exhibit more subtle effects on vigor like temperature during beet (Beta vulgaris L.) seed development (54), season of coconut (Cocus nucifera L.) harvest (107), time after flowering of rice harvest (136), or simply random year-location effects in soybean (38). "Any reserve nutrient that can control the rate of seedling devel- opment is a potential vigor factor, therefore, any environmental condi- tion that influences accumulation of nutrients has potential for in- fluencing vigor in the following generation", (79). Seed size and protein content (44) are environmentally plastic and affect vigor. Large seed in terms of weight, volume, or density has been studied extensively in regards to its effects on germination, seedling size, and yield. Some workers find no seed size effect in sorghum (Sorghum bicolor (L.) Moench.) (148), soybeans and wheat (6). Others report significant yield and/or emergence increases due to large seed in rice (146,149,152,l64). The reported inconsistency of seed size effect on yield components is a function of proper size class comparisons, e.g., large vs. small or large vs. unselected seeds (74), or manner of seedling spacing, e.g., insufficient interclass competition (6,146). Seedgprotein and vigor. Much of the work on the effect of seed protein on seedling vigor, e.g. seedling growth and yield, of small grains has been done by S. K. Ries and his group at Michigan State University. Seeds with higher protein have greater seedling vigor than 12 normal protein seeds within a genotype of wheat (126) and rice (96). Comparisons between genotypes or fice showed that protein per seed but not percent protein correlated positively with seedling weight (164). The effects of protein content on yield is mixed; there are re- ported increases for wheat and oats (Avena sativa L.) (43,123,127,l35); sometimes there is no effect on wheat yield (56). The mechanism responsible for the protein effect on vigor is not clear. The gliadin protein fraction increases with increased protein. but no specific protein (7) or amino acid is more closely correlated with vigor than total seed protein (87). The site of the protein effect is the endosperm, as demonstrated by embryo culture (57,89), and embryo and endosperm transplants (89). High protein wheat seed produced larger seedlings than those from normal protein seeds under varying light intensity, temperature, and N03' levels (88), and they absorbed water and germinated faster also (86). Seed protein content does not affect rate of “03’ uptake (57; Stuurwold, 1977, M.S. Thesis, Michigan State University). It has been proposed that storage protein acts as a N source of last resort for seedling growth (Stuurwold, 1977). Increasing protein content. The seed protein content of most crop species is under both genetic and environmental control. In rice, the protein range of a given cultivar includes the average pro- tein content of high and low protein lines (44). Certain environmental factors like temperature (75), seed position within a wheat panicle (124) or pod position on the soybean stem (23) influence protein, but the practical application of these protein sources are limited by our present technology. 13 The major methods for increasing protein are breeding for consump- tion and application of supplemental nitrogen for improved vigor. The use of growth regulators has not been perfected yet. Before considering which form of N to use, or when and how to apply N, it is appropriate to first examine the physiological causes for genetically high protein (GHP) seed and the patterns of protein synthesis. During seed development, GHP rice seed has more free amino acids and RNA, higher rates of amino acid incorporation (25), and greater amino acid concentration in the culm sap (16). The leaves of GHP rice translocate a greater percentage of leaf N to the seed due to higher protease activity in the leaves (119). In GHP wheat, the leaf to seed ratio is larger, and the seed has greater amino acid incorpora- tion activity (118). Supplemental N as foliar urea is rapidly absorbed and translocated within 24 hours in sugarcane (Saccharum officinarum L.) (15), and the free amino acid content increases 60% in 12 hours in wheat (97). The mechanism for increasing protein as affected by growth regulators like simazine is more subtle. It is hypothesized that simazine stimulated nucleic acid synthesis resulting in increased protein synthesis and subsequent N03' uptake. This synthesis is dependent upon environmental conditions that favor accumulation of carbon skeletons to accept the reduced N in the form of soluble carbohydrate (12). During seed development, the enzymatic proteins, albumin and globulin, are synthesized first, followed by the storage proteins glutelin and gliadins (13,16). Applications of N increase the storage protein fraction (102,111,115,147), but the biological value of the total seed protein decreases compared to GHP seed (36). 14 There has been great interest in increasing protein content of seed craps since the availability of synthetic N fertilizers. In- creasing soybean protein is complicated by the inhibition of nodular N fixation' foliar urea has the most inhibitory effect on fixation (50,51) followed by topdressed NH4N03, NH4S04, and finally urea (155). Foliar 0.5% glucose plus any form of N stimulated N accumulation in 15 + leaves prior to pod formation (156). Using N, it is known that NH4 is absorbed faster than N03' by rice (99), but N03' stimulated greater N uptake (100). No form of N is superior for increasing rice protein (103). Timing of N application for protein vs. yield increase in rice is critical. Early or early-mid split applications increase yield (122), while early-late (panicle initiation or anthesis) split applications increase protein (27,58,81,104,136,150). Finney et al. (41) demonstrated that foliar applications of a 15% urea solution at anthesis increases protein content of wheat. These increases, due to foliar urea, have been observed many times (7,39,158), however, the usefulness of foliar urea on wheat has been challenged by Alkier (5). 15N-urea, NH4N03, and NH4SO4 applied as topdressings were superior to similar foliar applications for increasing protein and more topdressed N was recovered in the seed (1 vs. 40-50%). From his re- sults, it appears that foliar N stimulates the uptake of soil N or is absorbed after it is washed off. Foliar applications of 3% urea are recommended for rice to avoid burning, with an equal t0pdressing of urea (140) between panicle forma- tion and anthesis (12,48). Biuret contamination of urea can affect germination of corn, 15 barley, and peas at concentrations between 10 and 103 ppm (62,93). Rice growth and yield is reduced at 0.3% biuret contamination of foliar urea but had no effect at higher levels in topdressed urea (69,70). Biuret levels in foliar urea as high as 1.8% of urea had no effect on wheat yield (78). Much attention has been given to the discovery by Ries that sub- lethal levels of simazine, a s-triazine herbicide, increased plant and seed protein (125,128). Several s-triazines increased protein in wheat (114), beans (14), peas, maize, and spinach (142). Protein increases, but yield decreases, are seen in rice (27,67,96,157). Other non-triazine herbicides also increase seed protein (117). SECTION I Increasing Protein Content of Rice and Its Effect on Seed Vigor and Subsequent Yield 16 Increasing Protein Content of Rice and Its Effect on Seed Vigor and Subsequent Yield ABSTRACT Hypotheses tested the relationship of increased seed protein con- tent of rice (Oryza sativa L.) with seed vigor and subsequent yield. The research was conducted in the intense subsistence lowland rice culture area of central Java, Indonesia. Foliar urea at 40 Kg H/ha increased protein and yield but was not superior to topdressed urea at the same rate in the wet season. During the dry season, the crop was severely damaged by grassy stunt disease and an infestation of brown planthopper (Nilaparvata lggens). Foliar urea significantly increased protein content but at the expense of yield. High protein seeds of 'IR-5' and 'Pelita 1/1' (2.11 mg protein/seed) had higher germination following a five day storage stress than low protein seeds (1.63 mg protein/seed). A four factor field experiment comparing two levels of genotype, (IR-5 vs. Pelita l/l), seed protein content (2.11 vs. 1.63 mg protein/seed), degree of storage stress (none vs. five days), and planting method (transplanting vs. direct seeding) was severely damaged by grassy stunt and brown planthopper infestation. There were no yields from the transplanted rice. Results from the direct seeded rice indicated that high protein seeds increased yield more for IR-5 than for Pelita l/l. Storage stress had no effect on yield. 17 INTRODUCTION The concept and measurement of vigor has been the subject of several recent reviews (5,25,31). Kidd and West (19) are credited with collecting early vigor related literature and doing research on 'Physiological predetermination' of seedling growth and yield. One definition of vigor states that a seed has a maximum potential from which there is a continuous decrease in potential over time, i.e. aging or deterioration (25). Vigor is conceptualized in different ways, as survival under adverse conditions or as an inherent physiological state. Use of the term 'vigor' can include all these situations: survival in the nonactive state (seed vigor), survival upon sowing (seed vigor), establishment of stand and healthy, normal growth (seed- ling vigor) (13). The physiological effects of seed aging can be studied by compar- ing seedlots from different harvest years, or by creating populations of artificially 'aged' seeds from the same seedlot. This may be accom- plished by stressing the seeds with high humidity and/or temperature during storage. The physiological responses of naturally aged seeds are similar to artificially aged seeds. The lack of complete under- standing of the aging process, however, warrants the use of the term storage stress rather than artificial aging. A study of the interac- tion of temperature and moisture content on rice (Oryza sativa L.) seed viability during storage (30) shows that these effects are additive. 18 19 The log exposure period necessary to produce any germination level at a given temperature and moisture can be calculated using multiple regres- sion. This approach to seed viability prediction has also been demon- strated for wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), broad beans (Vicia faba L.), and peas (Pisum sativum L.) (31). Yield reduction resulting from poor stand and/or growth due to aging is observed in transplanted rice (35) and direct seeded soybeans (7). "Any reserve nutrient that can control the rate of seedling devel- opment is a potential vigor factor, therefore, any environmental condi- tion that influences accumulation of nutrients (in the seed) has potential for influencing vigor in the following generation", (21). Much of the evaluation on the effect of seed protein on seedling vigor, e.g., seedling growth and yield, of small grains has been done by Ries and his group at Michigan State University. High protein seeds, i.e., higher percent or amount per seed, have greater seedling vigor than normal protein seeds within a genotype of wheat (28) and rice (23). Comparisons between genotypes of rice showed that protein per seed, but not percent protein correlated positively with seedling weight (39). The effects of protein content on yield are mixed; there are re- ported increases for wheat and oats (Avena sativa L.) (10,27,29,32); sometimes there is no effect on wheat yield (14). Timing of N application for protein vs. yield increase in rice is critical. Early or early-mid split applications increase yield (26), while early-late (panicle initiation or anthesis) split applications increase protein (6,15,22,24,33,36). Finney et al. (9) demonstrated that foliar application of a 15% 20 urea solution at anthesis increased protein content of wheat. These increases, due to foliar urea, have been observed many times (3,8,38), however, the effectiveness of foliar urea on wheat has been challenged by Alkier (5). 15M labeled urea, NH4NO3, and NH4SO4 applied as a top- dressing were superior to foliar N for increasing protein and more topdressed N was utilized (1 vs. 40-50%). From his results, it appears that foliar N stimulates the uptake of soil N or is absorbed after it is washed off. A foliar application of 3% urea is recommended for rice to avoid leaf burning, with an equal t0pdressing of urea (34) between panicle formation and anthesis (4,12). Rice growth and yield was reduced by 0.3% biuret contamination of foliar urea but had no effect at higher levels in topdressed urea (17,18). A series of experiments was designed to test the following hypo- thesis under intense subsistence agricultural conditions in the lowland rice region of Central Java, Indonesia. Foliar urea increases protein content of rice seed more than topdressed urea when either is applied at anthesis. High protein rice seed is more resistant to aging stress than low protein seed and produces a larger yield whether the aged or unaged seeds are compared. The effect storage stress is manifested in the yield regardless of transplanting healthy seedlings grown from or direct seeding of seeds stressed during storage. MATERIALS AND METHODS Three experiments were conducted in a village 'sawah' (wet rice field complex) near the Department of Agronomy, Gadjah Mada University, Indonesia. The sawah was irrigated by a community stream which was part of the watershed system of Mt. Merapi, an active volcano, 25 Km to the north. The soil was an entisol with a pH of 5.7, 4.9% organic matter, and 0.18% N. Seeds of 'IR-5', an early IRRI (International Rice Research Institute, Los Banos, Philippines) dwarf rice of low consumer preference, and 'Pelita l/l', a dwarf Indonesian line of medium consumer preference, were provided by rice breeder, Ir. Sumartono (Department of Agronomy, Faculty of Agriculture, Gadjah Mada University, Yogyakarta, Indonesia). Net Season Protein Increase - Experiment 1. IR-5 and Pelita l/l seedlings were transplanted two seedlings/hill at a 20 x 20 cm spacing and fertilized with 45 Kg PZOS/ha as triple superphosphate on September 22, 1975. Forty-six Kg N/ha as urea was applied as 23 Kg N/ha at one month after transplanting and 23 Kg N/ha at panicle initiation. The experimental units were 10 m2 enclosed plots with individual irriga- tion inlets to maintain a water level of 10 cm. The field design was a randomized-complete block split-plot, split in space, with four blocks. Cultivars were the main plots and treatments the subplots. The treatments were control; 40 Kg N/ha t0pdressed as urea in two equal splits five days apart starting at anthesis; 40 Kg N/ha 3% foliar urea 21 22 with 0.1% Tween 20 in three equal splits three days apart starting at anthesis; and 20 Kg/ha 1.5% foliar urea with 0.1% Tween 20 applied in the same manner as the other foliar treatment. Urea used in the treat- ments was analytical grade and biuret-free. Azodrin was used to con- trol caseworm (Nymphalia diponatalis) and stem borer (Tryporyza incertulas). Weeding was done by hand. IR-5 was harvested on January 15 and Pelita 1/1 on the 22nd, 1976. Ten random hills were sampled from the center 4 m2 for yield component estimates, and the remaining 90 hills of the 4 m2 were used for yield. Estimates of panicles/mz, seeds/panicle, seed sterility, 1000 seed weight, and yield were calcu- lated using standard procedures and adjusted to 14% H20 (11). Protein content was determined as Kjeldahl N times 6.25 by a modified macro- Kjeldahl procedure (2). Percent N efficiency was calculated as treat- ment Kg N/ha as grain protein minus control Kg N/ha as grain protein divided by Kg N/ha applied as urea treatment times 100. Dry Season Protein Increase - Experiment 2. IR-5 and Pelita l/l seeds from experiment 1 were planted in flooded seedbeds and trans- planted on April 10 and harvested July 10, 1976. The experimental units were 4.84 m2 individually enclosed plots in the same field design as experiment 1. Fertilizers were 45 Kg PZOS/ha as triple superphos- phate at transplanting and 70 Kg N/ha as urea split as in experiment 1. Sevin was used to control the brown planthopper (Nilaparvata lugens). Other cultural and analytical methods were the same as experiment 1. Protein-Vigor Yield Test - Experiment 3. The protein-vigor hypo- theses were tested by a 24 factorial experiment. The factors were geno- type, (IR—5 vs. Pelita l/l), seed protein content, (high vs. low), storage stress (stressed vs. not stressed), and planting method, 23 (transplanting vs. direct seeding). Four seedlots from experiment 1, consisting of two seedlots dif- fering in protein content bu similar seed weight were selected from each cultivar. The seedlots were selected so that the protein content, mg protein/seed was similar among cultivars. The 'high' protein seeds had 2.11 mg protein/seed and the 'low' protein seeds had 1.64 and 1.62 mg protein/seed for IR-5 and Pelita l/l, respectively. Each of the four seedlots was divided, one half was subjected to storage stress, the other was not. The moisture content of the seeds to be stressed was increased to 20% H20 by placing the seeds in a bell jar over water at room temperature (approx. 27°C). The seeds were then placed in airtight plastic bottles, one seedlot per bottle and stored at 40°C for five days. The seeds were then removed from the bottles and allowed to dry at room temperature. The eight seedlots, four stressed and four unstressed, were soaked in 0.1 N HNO3 for 16 hr to break dormancy (40), and the floating seeds discarded. The germination was determined on four 50-seed samples from each of the eight seedlots. The seeds were germinated on filter paper strips lying on glass plates with the sides of paper in water. Germination was in the dark at room temperature. Final counts were made on day seven, germination was considered to be the presence of a normal radicle and coleoptile. The fourth factor, planting method, was included to test the effect of stressed-reduced—stand vs. a perfect initial stand, i.e., transplant- ing, on subsequent protein and stress effects on yield. The planting rates of the direct seeded treatments were based on the germination results. Two seeds/hill were planted for the unstressed high or low 24 protein seed or stressed high protein, three for stressed, low protein Pelita 1/1, and five for stressed, low protein IR-5. The field design was a randomized complete block split-plot with four blocks. Cultivars and planting methods were the main plots (31.2 m2), and protein content and storage stress the subplots (7.8 m2). On April 9, the seeds were seeded directly into the plots at 20 x 20 cm spacing or broadcast onto flooded seed beds. Emergence was de- termined on April 19 as the number of seedlings divided by the number of seeds plants. Seedlings in the seedbeds were transplanted 2 seed- ~lings/hill at 20 x 20 cm spacing on May 5. Stand was determined as the number of hills/m2 divided by 25 on June 12. Fertilization and insecti— cides were the same as experiment 2, cultural and analytical methods were the same as experiment 1. The experiment was harvested August 19, 1977. RESULTS AND DISCUSSION All urea treatments in experiment 1 increased yield, seed weight, and seed protein content (Table 1). The 40 Kg N/ha treatments de- creased seed sterility in both cultivars. Urea also increased the num- ber of seeds/panicle in Pelita l/l but not IR-5. Topdressed urea was not significantly better than foliar urea in terms of increasing pro- tein or yield. (The simplicity of t0pdressing urea would make it preferable to foliar urea for increasing protein.) The N efficiency was high for both methods compared with the results of an earlier study (1). Late applications of N seldom increase yield (1,26). This in- crease in yield can be attributed to the reduced seed sterility and increased seed size, indicating that late N applications can increase yield if N is limiting yield. During the dry season, experiments 2 and 3 were severely damaged by an epiphytotic of grassy stunt disease and its vector, the brown planthopper (Nilaparvata lugens). Damage was severe throughout central and eastern Java (Seminar Hama Wereng Tanaman Padi, June 1-3, 1976, Gadjah Mada University, Yogyakarta, Indonesia). The average yield of experiment 2 was reduced 80% compared with experiment 1's average (Table 2). All urea treatments increased pro- tein over the control, but only IR-5 topdressed with 80 Kg N/ha in- creased yield over the control. Foliar urea at 40 Kg N/ha reduced 25 26 .cm>wap=u e cmgpwz mcmwz+ .2: was: copueuw—aam mug: we e;\mx 2 >3 cmuw>vu Fogucou cw.:\mx z mazes «cmeummgu Log cpmuocn vmmm we mg\mx z¢ m an e N N_ m m ARV .>.u m.o mz m.o m.o m m mz *mo.o .o.m.4 o.m mo m.~ m.om up mm mew gmwpom om m.m No ~.m e.om ep mm mew gmwpom cc m.m en q.m m.om ep mm mmu mudguao» oe m.m - m.m ~.m~ om we mmm . o p\_ eu_~ma F.m oe m.~ m.w~ ow om omm deepen om F.m mm e.w w.wm mp mm oem smFPom oe e.e _m m.m e.em up ma meu mmeeeaee oe w.m - e.~ «.mm cm em emu . o m-m~ ee\e & N ooo_\m a eczema ee\m¥ .uz Ape—pampm mpuwcma E\mmFu_:m¢ eeem eeem \meeem ~ upmr> +zucmpopmwo z seepage mucmcoqeou upmv> cowueuVFQam z Lo>wpp=u .oulmum— commmm pm; mga cw ewmmcovcH .mucmxmzao> pm czogm more .F\F upwpma. new .mimn. Low mvmmsucm um meowpouwpaae ewe: Edema uFmP» use .aocmvuwewm z .ucmpcou :_mpoga .mucmcoasou upmv> .p aneh 27 .oop mus?» cowumuwpaam mac: me m;\m¥ 2 an umvw>wu Focpcou cw e;\m¥ z magma «cospmwcu Lug cwmuoga ummm mm m;\mx 2a .L~>mupau m cvsuwz mcmmz+ .Amcmmzp eum>gmmmpwzv gmanospcepa czocn asp .copum> may can mmemmwu pcaum mmegm xa awn so>o ceased; mm: ume>+ S o: m N z N 8 E .>.u N.o mz m.o m.P m m mz +mo.o .o.m.4 o.p N ¢.N_ w.mN um um o—F seeped ow N.P v o.FP o.mN NN Nm NNF mudguaoh ow ..p m N.mp m.mN NN mu oep mudguao» on _.F . o.op m.mN Fm NN ONP . o ~\p mappmm m.p N N.m— o.oN mN cu cup seeped oe m.p e o.NP N.oN FN om mmp mmmgvaoh co m.p m N.mp ¢.oN mN Nm amp mmmgvnoh om +e.p - ¢.Fp m.mN mN RN Nmp . o mumH m;\» N a coop\m & venues m;\mx .uz xuwpwcmum mpuwcomll E\mmpu*:ma eeem eeem \meeem N 33» ozocmmuwtm z 539:. 3:3an8 Em; 533183 2 .5533 .mNmP .commmm xgu 8:» cr upmmcoucH .mugexmxmo> we czosm mops __\p upvpma. new .man. com mpmmzucm we mcoPuouwpaam «we: sauce upmwz new .xocmPquem z .pcmucou cwmuoca .mucmcoaeou opmv> .N apes» 28 yield significantly below 80 Kg N/ha in both cultivars, and increased seed sterility in Pelita 1/1. The higher protein content of the foliar vs. t0pdressed urea at 40 Kg N/ha was probably due to increased seed sterility and reduced seed size which reduced the yield. Stress- induced protein increases have been noted for factors that reduce yield like simazine on rice (6,16,23,37) or even disease. The effect of disease on protein content can be seen by comparing the controls of the wet vs. the dry season experiments (Table 1,2). The N efficiency of the urea treatments was very low, presumably due to the disease- pest stress. The germination of stressed vs. unstressed seeds showed an inter- action of protein, stress, and cultivar (Table 3). The sharp reduction in germination of stressed, low protein IR-5 seeds compared with the stressed, low protein 'Pelita l/l' and lack of a protein effect or cultivar effect on the unstressed seeds were the causes of the three way interaction. It should be noted that the response of the low pro- tein seeds to stress was not Opposite. The field emergence of the direct seeded treatments had a strong interaction of stressed vs. unstressed seeds at both protein levels regardless of cultivar (Table 4). The stressed, low protein seeds had lower percent emergence than the stressed, high protein seeds, but if the seeds were not stressed, then protein had no effect on emergence. clearly demonstrating the greater resistance of the high protein seeds to storage stress. All three way interactions were significant for percent stand at maximum tillering (Table 5). There was no consistent effect of stress or protein on the transplanted treatments, but there was for the direct 29 Table 3. Germination of 'lR-S' and ‘Pelita l/l' rice seeds as affected by seed protein content and a five day storage stress of 40°C and 20% moisture content. Cultivar Protein Germination percentage mg/seed unstressed stressed 1. IR-5 2.11 99 96 1.64 90 36 Pelita 1/1 2.11 96 94 1.62 94 80 C.V. (%) 3 +F value for the interaction of stressed vs. unstressed at two protein levels for two cultivars is significant at the 0.01 level. 30 Table 4. Emergence of direct seeded 'IR-5' and 'Pelita l/l' rice seedlings as affected by seed protein content and a five day storage stress of 40°C and 20% moisture content at Yogyakarta, Indonesia in the dry season, 1976. Cultivar Protein Emergence percentage mg/seed unstressed stressed 1. IR-5 2.11 83 75 1.64 76 17 Pelita 1/1 2.11 84 78 1.64 84 64 C.V. (%) 8 1'F value for the interaction of stressed vs. unstressed at two protein levels is significant at the 0.01 level. Table 5. 31 Stand at maximum tillering of 'IR-5' and 'Pelita l/l' rice as affected by seed protein content, a five day storage stress of 40°C and 20% moisture content, and planting method at Yogyakarta, Indonesia in the dry season, 1976. Cultivar Protein Stand percentage mg/seed Direct Seeded Transplanted Storage stress Storage stress 0 + o + IR-5 2.11 95“ 94 96 95 1.64 91 57 95 95 Pelita 1/1 2.11 98 98 92 92 96 95 86 93 C.V. (%) 9 +F values for all third order interactions significant at the 0.01 level. 32 seeded treatments. The stand of direct seeded IR-5 was reduced by stress and low protein, but Pelita 1/1 less so. When healthy seedlings were transplanted, the effect of storage stress on seedling survival was not apparent as reported earlier (35). At the time of harvest, the transplanted rice was completed destroyed by the epiphtotic, and the yield of the direct seeded rice was reduced 90% (Table 6). It would appear that the transplanting shock rendered the rice more susceptible to the disease-pest stress. The interaction of high vs. low protein seed of IR-5 and Pelita 1/1 was significant for all yield components except seed weight, which was affected by an interaction of stress, protein, and cultivar. These interactions were the result of the low protein IR-5 having fewer hills/ m2, panicles/mz, seeds/panicle and greater sterility than the high pro- tein IR-S, while there was no protein effect on these components in the responses of Pelita 1/1. Storage stress and low protein reduced seed weight of IR-S, but only high protein Pelita 1/1 was affected by stress. Yield was more responsive to high protein for IR-5 than Pelita l/l. The positive effect on yield could be a stress resistance effect or a direct yield enhancement (10.27.29.32). Unfortunately, because of the extreme yield loss due to disease- pest damage, the effect of high protein rice seed on increasing yield could not be extrapolated to yields under normal conditions. The marked effect of increased protein content on rice seed viability under stor- age stress conditions was real and its potential for direct seeded rice should not be discounted. However, genetic interaction with protein should be clarified further. 33 .Fw>m— Fo.o mg» an “emu leewcmwm mw mcm>wupzu 03“ van m_m>m_ :wwuoca 03» pm ummmmcpmca .m> ummmmgpm mo cowuumgmucw on» Low ozpm> do ._m>mp mo.o mg» we ucmuwemcmwm m? mcm>wppao oz“ com cwwuoga sop .m> cow; mo cowuumgmucw as» com mape> m+ .Amcumsp epe>ceaepwzv goaaogucmpa czogn esp copum> my? use mmemmwu pczwm xmmeem ma umxocpmmu umumpasou mnemEpmmgp umpcepamcmcp new New Lm>o omuauwc we: mucmsummcp umuumgwu do upmw>+ Ne m m_ eN mm om ARV .>.o ep.o e.eN _e em we m.m + em. m~.o e.eN we um mm m.~ o ee.p Np.o e.eN ee em MN m.m + p_.N oN.o e.mN we mu oe m.N o FF.N _\_ eucpea _P.o N.NN Ne om ee 0.0 + em. N_.o e.eN 4N mN Nm N.m o ee.p oe.o e.MN om me am o.mp + __.N me.o _.eN we ee em e.m_ o __.N m-zH ooo_\m N .uz Newpeeeem e;\h vmmm + ummm m_uwcea\mummm E\mm—uwceg E\m_~wc umom\me + o + m m +u~mw> mpcwcoaeou upmw> mmmcum cmmuogm Le>wb_:o .mnmp .commmm Age ms» cw ewmmcoucH .eacexmxmo> um czocm ucmpcou mcaumwos NON new uoov we mmmcum mmmgoum xeu m>ww a new ucmucou :_muocq ummm Na umuomeee mm mo_c ._\_ eHPFma. uce .m-mH. cmumwm “emcee mo upmwx ecu mucmcoqeoo ape?» .m apnep 10. 11. LITERATURE CITED Alkier, A. C., G. J. Racz, and R. J. Saper. 1972. Effects of foliar and soil applied nitrogen and soil nitrate-N level on the protein content of Neepawa wheat. Am. J. Soil Sci. 52: 301-304. A. 0. A. c.1975. Methods of Analysis of Association of Official Analytic Chemists, Improved Kjeldal procedure 2-049 for Nitrate- ;ree samples. P.O. Box 540, Benjamin Franklin Sta. Wash. 0. C. 0049. Ayers, G. S., V. F. Hert, and S. K. Ries. 1976. The relationship of protein fractions and individual proteins to seedling vigor in wheat. Ann. Bot. 40:563-570. Bhaskaran, V. P. and Rajut De. 1971. Foliar spray of urea for yield increase in rice. Cur. Sci. 40:91-92. Copeland, L. 0. 1976. Principles of seed science and technology. Burgess Pub. Co., Minn. MN. 369 pp. De Datta, S. K., H. N. Obcemea, and R. K. Jana. 1972. Protein con- tent of rice grainas affected by nitrogen fertilizer and some triazines and substituted ureas. Edie, 0. T., and J. S. Burris. 1971. Effects of Soybean seed vigor on field performance. Agron. J. 63:536-538. Filipev, I. 0., L. F. Zhukova, and I. N. Koutunik. 1973. Foliar application of urea and the improvement of quality of winter wheat grain. Vestn. Sel'skolchoz. Nauki. 10:42-46. (Russ.) Finney, K. F., J. N. Meyer, F. W. Smith, and H. C. Fryer. 1957. Effect of foliar spraying of Pawnee wheat with Urea solutions on yield, protein content, and protein quality. Agron. J. 49: 341-347. Garay, A. D. 1975. Effect of nitrogen fertilization of wheat (Triticum spp) on chemcial and biochemical composition and per- fbrmance of seeds. Diss. Abstr. Int'l. 3512-B:5740. Gomez, K. A. 1972. Techniques for field experiments with rice. IRRI, Los Banos, Philippines. 46 pp. 34 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 35 Gupta, S. K. 1971. A short review on the foliar application of nutrients on rice. 0ryza 8:55-64. Heydecker, W. 1972. Vigour IQ E. H. Roberts (ed.) Viability of Seeds. Chapman and Hall, London, U.K. p. 448. Holzman, M. 1974. The influence of nitrogenous manuring on the quality of the seed material of winter wheat, tested on the growth intensity and nutrient absorption of the seedlings. Part I: Test of performance value on shoot length and dry matter of the seedlings and on the yield. Z. Acker-u. Pflanzenbau 139:186-213. Honjyo, K. 1971. Studies on protein content in rice grain. II. Effects of the fertilization on protein content and protein pro- duction in paddy grain. Proc. Crop Sci. Soc. Japan 40:190-196. IRRI. 1972. Annual Report 1972. Los Banos, Philippines. Jain, N. K., V. Singh, and H. P. Triputhi. 1972. Effects of biuret content of urea in soil and foliar application on Dwarf Indica Rice cultivar 1R8. J. Ind. Soc. Soil Sci. 20:287-292. Jain, N. K., and A. S. Verma. 1974. Biuret content of urea. I. Effect of Dwarf Indica Rice cv. IR8 in foliar spray. Indian J. Agric. Res. 8:97-102. Kidd, F., and C. West. 1918. Physiological predetermination: The influence of the physiological condition of the seed upon the course of subsequent growth and yield. I. Effect of soaking seeds in water. Ann. App. Biol. 5:1-13. Lal, R., and R. R. Singh. 1970. Effect of Different concentrations of biuret in urea used as fbliar spray in wheat. Balwant Vidyapeeth J. Agr. Sci. Res. 12:77-79. Lang, A. 1965. Effects of some internal and external conditions on seed germination. 13 H. Ruhland (ed.) Handbuch der Pflanzenphysio- logie. 15:848. Latchanna, A., and Y. Yogeswura Ras. 1964. Protein content of high yielding varieties of rice as influence by level and time of appli- cation of nitrogen. Andhra Agric. J. 16:137-140. Miller, Milton 0., and D. S. Mikkelsen. 1970. 1969 Rice Seed Protein Studies. Rice Journal. 73:7-8. Nagarajah, S., M. M. M. Sauffer, and S. M. Hillenberg. 1975. Timing of nitrogen application, its effect on nitrogen utiliza- tion and protein content of rice. Plant and Soil 42:349-358. Pollock, B. M., and E. E. Roos. 1972. Seed and seedling vigor. In T. T. Kozlowski (ed.) Seed Biology 1:313-387. Academic PFess, NY, NY. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 36 Reddy, K. R., and W. H. Patrick. 1976. Yield and nitrogen utilization by rice as affected by method and time of applica- tion of labelled nitrogen. Agronomy J. 68:965-969. Ries, S. K. 1971. The relationship of protein content and size of bean seed with growth and yield. J. Amer. Soc. Hort. Sci. 96:557-560. , and E. H. Everson. 1973. Protein content and seed size relationships with seedling vigor of wheat cultivars. Agron. J. 65:884-886. , O. Moreno, W. F. Meggitt, C. J. Schweizer, and S. A. Ashkar. 1970. Wheat seed protein: chemical inluence on and relationship to subsequent growth and yield in Michigan and Mexico. Agron. J. 62:746-748. Roberts, E. H. 1961. The viability of rice seed in relation to temperature, moisture content, and gaseous environment. Ann. Bot. N.S. 25:381-390. 1972. Viability of seeds. Chapman and Hall, _ London. Schweizer, C. J., and S. K. Ries. Protein content of seed: In- creases and improves growth and yield. Science 165:73-75. Seetanum, W., and S. K. De DaHa. 1973. Grain yield, milling quality, and seed viability of rice as influenced by time of nitrogen application and time of harvest. Agron. J. 65:390-394. Singh, A. K., and S. Taakur. 1973. Foliar application of urea in- creases in paddy yields. Farmer Parliament 8:2126. Sittisrsung, Prasoot. 1970. Deterioration of rice (Oryzfl sativa) seed in storage and its influence on field performance (Diss. Abstr. Int'l. 31/07-B p. 3805). ' Tairu, H. 1970. Effect of fertilization on protein content in high yield rice. Proc. Crop Sci. Soc. Japan 39:200-203. Vergara, B. S., M. Miller, and E. Avelino. 1970. Effect of Simazine on protein content of rice grain (Oryza sativa L.) Agron. J. 62:269-272. Vertii, S. A., and N. A. Vozov. 1974. Significance of urea applied to foliage in improving grain quality and protecting winter wheat against the noxious stink bug. Dokl. Vses. Akad. S-Kh. Nauk. 7:14-16 (Russ.) Wu, S. T. 1975. Genetic studies on seedling growth in rice plant. II. Chemical contents and seed size relationship with seedling vigor. J. Ag. Assoc. China 91:77-82. 37 40. Xuan, Vo-tong, and V. E. Ross. 1972. Training manual for rice production. IRRI, Los Banos, Philippines, 141 pp. SECTION II Germination and Growth of Rice Seedlings Differing in Seed Protein Content Under Various Storage Stress Conditions 38 Germination and Growth of Rice Seedlings Differing in Seed Protein Content Under Various Storage Stress Conditions ABSTRACT Deterioration of seed in storage in the tropics may be due to en- vironmental factors such as high temperature and humidity. The germi- nation responses of high and low protein seed lots of 'IR-5' and 'Pelita l/l' rice (Oryza sativa L.) exposed to two methods of storage stress were investigated. High protein seeds had a LD50 of 75 days and low protein seeds 55 days when stored at 27°C and 18% moisture content. High protein seeds remained viable longer at 40°C and 20% moisture content when stored in sealed vials or over water. Seedlings from high protein seed had greater seedling vigor, regardless of storage stress. Stressing of whole seeds by storing at 40°C and 20% moisture content for 25 and 4 days, low protein and high protein seeds, respectively, indicated that seed vigor of high protein seeds appears to be in the embryo, while the endosperm is thought to be responsible for seedling vigor. 39 INTRODUCTION Reviews of seed storage in the tr0pics indicate that major problems center around extreme temperatures and humidity during storage (2). Thirty-five percent of storage losses of Southeast Asian paddy (un- hulled rice seed) (Oryza sativa L.) is attributed to heat and moisture (33). Most paddy is stored in bulk for 1-12 months at 12-16% moisture content (fresh weight basis) and 28-38°C. In Malaysia, granary humidi- ty and air temperatures are higher than ambient. Traditional, long- dormancy cultivars maintained viability (seed vigor) longer than modern varieties under granary conditions (14). Vigor is conceptualized in different ways, as survival under ad- verse conditions or as an inherent physiological state. Use of the term 'vigor' can include all these situations: survival in the non- active state (seed vigor), survival upon sowing (seed vigor), establish- ment of stand and healthy, normal growth (seedling vigor) (11). One definition of vigor states that a seed has a maximum potential from which there is a continuous decrease in potential over time (aging or deterioration) (25). Two theories are recognized as possible explanations for the re- sults of seed aging (9). The major theory, cytoplasmic dysfunction, includes accumulation of germination inhibitors, denaturation or re- duced synthesis of macro-molecules like protein, lipids, and nucleic acids. The second theory views aging as one of the increased 40 41 chromosomal mutation. Reduced protein synthesis in aged seeds during germination (24) is usually related to reduced RNA synthesis (4). Using a poly-uracil- directed, cell-free system, the inhibition of amino acid binding to t-RNA is shown to be responsible for reduced protein synthesis (24,28). Recent work indicates that denaturation of an elongation factor (EFI) is involved in m-RNA translation and may be responsible for t-RNA amino acid binding inhibition (6). A unique series of experiments following the chromosomal mutation theory with lettuce (Lactuca sativa L.) demonstrated that fully imbibed, thermal-induced-dormant seeds would remain viable for long periods of time at temperatures at which dry seed would lose viability (35). It was shown that chromosomal damage either did not occur or was repaired in these seeds. The number of chromosomal mutations was reduced in seed when placed in the thermal-induced-dormant, fully imbibed state (36). The physiological effects of seed aging can be studied by compar- ing seedlots from different harvest years, or by creating p0pulations of artificially aged seeds from the same seedlot by exposing seeds to high humidity and/or temperature. The U.S. method of artificial or accelerated aging recommends placing the seeds over free H20 (100% rela- tive humidity) at 40°C (7). The British method usually calls for in- creased moisture content first (or uses freshly harvested seed which must be dried to the desired moisture content) and storage of the seed in a sealed container at a given temperature (30). Consequently, the atmospheric gas composition is different for seeds artificially aged by these two methods. The physiological responses of artificially and 42 naturally aged seeds are similar. The lack of complete understanding of the aging process warrants the use of the term storage stress in lieu of artificial aging. A study of the interaction of temperature and moisture content on rice seed viability during storage (29) shows that the effects are additive. Using multiple regression analysis, the log exposure period necessary to produce any germination level at a given temperature and moisture content can be calculated. This approach to seed viability prediction has also been demonstrated for wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), broad beans (Vicia faba L.) and peas (Pisum sativum L.) (30). The germination test remains the standard vigor test (5). Unfortu- nately, the decrease in germination is one of the last observable vigor phenomenon to be affected by seed aging. A more sensitive measure of vigor loss is the change in germination rate (11). Several methods for quantifying this are available, the simplest is called the speed of germination. This is calculated by summation of percent germination on day one divided by one plus the percent germination on day two divided by two and so on, until germination is complete (34). The germination curve as a function of time in storage or seed age is sigmodial (11). This curve can be linearized by probit or probabi- lity graph paper (31) or by using a simple log function (15), which is a linearization of the Henderson-Hasselbalch equation (16). If only the time period required to produce 50% germination (L050) is needed, (as in many dose response bioassays), then the Spearman-Karber equation can be used (8). The advantage of linearizing the germination curve is the ease of comparing the rate of germination loss between seedlots, and the LD50 can still be determined easily. 43 "Any reserve nutrient that can control the rate of seedling devel- opment is a potential vigor factor, therefore, any environmental condi- tion that influences accumulation of nutrients has potential for in- fluencing vigor in the following generation", (17). Much of the work on the effect of seed protein on seedling vigor, e.g. seedling growth and yield, of small grains has been done by Ries and his group at Michigan State University. High protein seeds have greater seedling vigor than normal protein seeds within a genotype of wheat (27) and rice (23). Comparisons between genotypes of rice showed that protein per seed but not percent protein correlated positively with seedling weight (37). The mechanism responsible for the protein effect on seedling vigor is not clear. The gliadin protein fraction increases with increased protein, but no specific protein (1) or amino acid is more closely correlated with vigor than total seed protein (19). The site of the protein effect is the endosperm, as demonstrated by embryo culture (21, 12), endosperm protein correlations with seedling vigor (21,21), and embryo and endosperm transplants (21). High protein wheat seed pro- duced better seedlings than those from normal protein seeds under varying light intensity, temperature, and N03- levels (20), and they absorbed water and germinated faster also (18). Seed protein content does not affect rate of N03' uptake (12; Stuurwold, 1977, M.S. Thesis, Michigan State University). It has been proposed that storage protein acts as a N source of last resort for seedling growth (Stuurwold, 1977). A series of experiments were designed to investigate the protein- vigor hypothesis in terms of storage stress of rice seed. Several technical and statistical methods were used to generate storage 44 stressed seed populations and analyze the data. Finally, the hypothesis that high protein seeds resist storage stress, and not simple recover better than low protein seeds, because of high protein endosperm was tested. MATERIALS AND METHODS Rice seeds differing in protein content (Kjeldahl N x 6.25) were selected from protein increase experiments on IR-5 and Pelita l/l grown in Indonesia in the l975-76 wet season. The high protein seeds con- tained 2.11 mg protein/seed (9.11 and 8.46% protein in IR-5 and Pelita l/l), and the low protein seeds had 1.63 mg protein/seed (7.08 and 6.7% protein for IR-5 and Pelita 1/1) as brown rice at 14% moisture content. Seed size was similar within a cultivar. Unhulled seeds were stressed by placing the seeds in a bell jar over water until the desired moisture content was attained. Moisture content was determined by air drying at 130°C for 2 hr. The seeds were either returned to the bell jar or placed in air-tight, screwtop, glass vials at 27 or 40°C. The bell jar was opened daily to allow air ex- change and to remove condensation from the underside of the lid. After stressing, the seeds were air dried at room temperature for two days to approximately 10% H20. Seeds were surface sterilized for 15 min with 0.1% (w/v) HgClz. and floating seeds discarded. The seeds were germinated in covered foil pans on filter paper wickslying on a glass plate at 30/25°C in a 12/12 hr diurnal cycle. A seed was judged germinated if it produced a normal coleoptile and radicle. Germination was recorded daily for seven days and the speed of germination calculated. Seedling growth was measured on seedlings germinated and grown in 45 46 double walled, foil wrapped, 220 m1 plastic cups filled with turface and covered with 5 mm of vermiculite. The light conditions in the growth chamber was 20 and 10 uW/cm2 in the blue and red spectral re- gions, respectively (IL 150 Photometer, International Light, Newbory, MA) with a 12 hr 30/25°C photoperiod. Six to ten surfaced sterilized seeds were placed in the vermiculite, watered until the water level was at the vermiculite layer, and covered with a sponge until emergence to prevent unequal drying between cups. On day 11, seedlings were thinned to the most uniform four per cup and fertilized every fourth day with 50 ml of 3mM N03' half strength Hoagland's nutrient solution until harvest. Seedlings (roots and shoots) were harvested on day 19, the seed remnants were removed, and the plants air dried at 80°C for 12 hr. Embryo and hulled, whole seed were cultured on White's media (10) with 2% sucrose and full strength Hoagland's micro-nutrients. Germina- tion was in the dark at 30/25°C in a 12/12 hr diurnal cycle. Germina- tion was rated as the presence of a normal coleoptile and radicle on day 7. The high protein IR-5 seeds used had been stressed 5 days and the low protein seeds 2.5 days. After stressing, the seeds were air dried to approximately 10% H20, the embryos were removed with a razor blade from half of the seeds from each protein level. Embryos and whole seeds were surface sterilized and transferred to sterile media. No contamination was observed during the seven day germination period. RESULTS AND DISCUSSION Germination of high protein rice seed, averaged for IR-5 and Pelita 1/1, remained higher longer than low protein seed when stored in air tight containers at 27°C with 19 moisture content (Figure l). The correlation coefficients (r) were highly significant for the Houston linearizing function, 109 (% germination/(100-% germ)) with days aged. The LD50 was 55 days and 75 days for low and high protein seeds, respectively. A comparison of the Houston linearization and Spearman-Karber methods show close agreement for estimating the LD50 of IR-5 and Pelita 1/1 at both protein levels (Table 7). It should be noted that when only small sample sizes and few doses are possible, the LD50 can be more accurately estimated by the Spearman-Karber method than a least squares method (3). Seeds stored in air-tight glass vials at 40°C and 20% H20 lost via- bility faster than seeds stored over water at the same temperature and initial moisture content (Table 8). High protein seeds remained viable longer than low protein seeds regardless of the method of storage stress, high protein IR-5 was more resistant to stress than Pelita 1/1. The seeds stored over water exhibited an endogenous germination rhythm before the final decrease in germination (22). The speed of germination of these seeds declined with the loss of germination (Table 9). However, the speed of germination appeared to 47 48 1.2-f Lo- -95 high pronnn (18 y=218'00288x E r=0.98** m: m 0.6'4 to 39 ' V75 0 9 0.41 E ‘< ‘2; g 0 1.10.2 4 3‘ <9 25 g O 50 ,4 low protein 02‘ y=2.47'0.0446x r=0.97** 0.4- ~25 0.6‘ 20 so 40 so so 70 80 DAYS Figure 1. Mean linearized germination response of 'IR-S' and 'Pelita l/l' rice seeds as a function of days stored at 27°C and 18% moisture and protein content. 49 Table 7. L050 determination by the Houston linearization and the Spearman-Karber methods of storage stressed 'IR-5' and 'Pelita l/l' rice seeds at two protein levels stored at 27°C and 18% moisture content. Cultivar Protein 'LDSO mg/seed Houston linearization Spearman-Karber Days IR-5 2.11 78.5 76.8 1.64 54.0 53.0 Pelita 1/1 2.11 72.0 69.6 1.63 58.5 59.1 Mean 2.11 75.0 74.3 Mean 1.63 55.0 54.4 50 Table 8. Germination of 'IR-5' and ’Pelita l/l' rice seeds as affected by seed protein and storage stress at 40°C and 20% moisture content in airtight vials or over water. Cultivar Protein Germination percentage mg/seed . Days stressed Control 0 2.5 6 9.5 12 13.5 18.5 22.5 Seeds Stored in Vials IR-5 2.11 99a+ 95a 89b 86a 27a 14a - - - 1.64 96ab 94a 94a 6d 36c 06 - - - Pelita 1/1 2.11 90b 89b 93a 36d 6b 3b - - - 1.64 916 926 83c 28c 1c 06 - - - C.V. (%) 9 6 9 10 27 55 Seeds Stored over Water IR-5 2.11 99a 95a - - 71a - 77ab 89a 64a 1.64 96ab 94a - - 74a - 60c 78ab 38c Pelita 1/1 2.11 906 896 - - 77a - 87a 67bc 526 1.64 916 926 - - 58a - 67bc 51c 39c C.V. (%) 9 6 - - 9 - 8 12 8 1'Means followed by the same letter within the same day and aging method are not significantly different at the LSD P 5_0.05 level. Table 9. 51 Speed of germination of 'IR-5' and 'Pelita l/l' rice seeds as affected by seed protein and storage stress at 40°C and 20% moisture content in airtight vials or over water. Cultivar Protein Speed of Germination mg/seed Days stressed Control 0 2.5 6 9.5 12 13.5 18.5 22.5 Seeds Stored in Vials IR-5 2.11 109a+ 127a 106a 89a 16a 6a - - - 1.64 1016 116a 806 8c 26 06 - - - Pelita 1/1 2.11 81c 110a 116a 306 lb 16 - - - 1.64 84c 99a 856 246 0c 06 - - - C.V. (%) 8 17 9 15 54 90 Seeds Stored over Water IR-5 2.11 109a 127a - - 94c - 79a 109a 82a 1.64 1016 116a - - 1026 - 52c 986 486 Pelita 1/1 2.11 81c 110a - 115a - 656 88c 606 1.64 84c 99a - - 94c - 58bC 53d 32c C.V. (%) 8 17 - - 12 - 11 8 13 IMeans followed by the same letter with the same day and aging method are not significantly different at the LSD P 5_O.05 level. 52 decline more slowly as a function of germination for those seeds stored over water. The atmospheric gas composition in the air-tight vials would change as C02 increased and 02 was decreased due to respiration, while the seeds over water had a fairly constant, normal gaseous environment. Experiments in which rice was stored in different initial gaseous en- vironments, air, N2, or C02, showed little effect of the gases on via- bility (29). Storage of fully imbibed, thermal-induced-dormant lettuce (Lactuca sativa L.) seeds indicates that continuous enzymatic repair occurs which maintains viability long after seeds stored at the same temperature and low H20 contents have lost viability (35,36). The rice seeds stored over water had a final equilibrium moisture content of 21% vs. 20% for the air-tight stored seeds. The relative humidity surround- ing the seeds over water was 99-100%, perhaps some degree of enzymatic repair occurred for a short period to maintain viability. The dry weights of 19-days-old seedlings grown from the seeds stored in air-tight vials were consistently higher for high protein seeds regardless of cultivar or degree of storage stress (Table 10). The dry weights were high for seedlings grown from seeds stressed six days because one seedling was grown per cup due to low germination of the low protein IR-5 seed. The lack of an apparent decrease in dry weight due to storage stress was contrary to a previous observation on rice (32). The marked effect of high protein seed content on seedling growth was in full agreement with other studies on rice (23) and wheat (19,21,26). The test of the 'recovery from vs. resistance to storage stress' hypothesis resulted in no difference in germination between storage 53 Table 10. Dry weight of l9-days-old 'IR-5' and 'Pelita l/l' rice seed- lings as affected by seed storage stress at 40°C and 20% moisture content in airtight vials and seed protein content. Cultivar Protein Days Stressed mg/seed Control 0 2.5 61 mg/seedling IR-5 V 2.11 67a+ 80a 80a 95a 1.64 556 566 606 81ab Pelita l/l 2.11 69a 72a 74a 766 1.62 606 59b 60b 54c C.V. (%) 8 ll 9 18 1'l-‘Ieans within the same column followed by the same nificantly different at the LSD P 5_0.05 level. 10ne seedling per cup. letter are not sig- 54 Table 11. Germination of 'IR-5' rice seeds and embryos on nutrient media as affected by seed protein content and seed storage stress. Protein Germination Percentage mg/seed Embryo Whole Seed 2.11 85+ 77+ 1.64 48 48 C.V. (%) 18 +F value for the comparison of high vs. low protein is significant at the 0.01 level. 55 stress; recovery via endosperm nutrition was not a factor responsible for the protein extended seed viability in rice. The effect of high seed protein on vigor of rice appeared to be of two types. One type was seed vigor, the embryos of high protein seed were more resistant to loss of viability due to storage stress. The second type was seedling vigor. Seedlings from high protein seeds regardless of storage stress were larger, probably due to endosperm nutrition (21; Stuurwold, 1977). 10. 11. 12. 13. LITERATURE CITED Ayers, G. S., V. F. Wert, and S. K. Ries. 1976. The relationship of protein fractions and individual proteins to seedling vigour in wheat. Ann. Bot. 40:563-570. Bass, L. 1975. Seed moisture and storage. Seed Sci. & Tech. 3:743-746. Bittenbender, H. C., and G. S. Howell. 1974. Adaptation of the Spearman-Karber method for estimating the T39 of cold stressed flower buds. J. Amer. Soc. Hort. Sci. 99:1 -l90. Bray, C. M., and J. Dasgupta. l976. Ribonucleic acid synthesis and loss of viability in pea seed. Planta 132:103-108. Copeland, L. 0. 1976. Principles of seed science and technology Burgess Pub. Co., Minn., MN 369 p. Dell'Aquila, A., G. Zocchi, G. A. Lanzani, and P. 0. Leo. 1976. Different forms of EFI and viability in wheat embryos. Phyto- chemistry 15:1607-1610. Delouche, J. C., and C. C. Baskin. 1973. Accelerated ageing techniques for predicting the relative storability of seed lots. Seed Sci. & Tech. 1:427-452. Finney, D. J. 1964. Statistical methods in biological assay. 2nd Ed., Hafner Pub. Co., NY, NY p. 333. .' Harrington, J. F. 1973. Biochemical basis of seed 'bngevity. Seed Sci. & Tech. 1:453-461. Hartman, H. T., and D. E. Kester. 1968. Plant Propagation. Prentice-Hall, Englewood Cliffs, N.J. 700 p. ”EYdECker: "- 1972- Vigour In E. H. Roberts (ed.) Viability of Seeds. Chapman and Hall, Londan, U.K. p. 448. Holzman, M. 1974. The influence of nitrogenous manuring on the quality of the seed material of winter wheat tested on the growth intensity and nutrient absorption of the seedlings. Part II. Absorp- tion of nutrients. Z. Acker-und Pflanzenbau 140:11-35. Honjyo, K. 1971. Studies on protein content in rice grain. II. Effects of the fertilization on protein content and protein production in paddy grain. Proc. Crop Sci. Soc. Japan 40:190-196. 56 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 24. 26. 27. 57 Horiuchi, T. 1975. Internal microclimate and grain viability in the rice granary in West Malaysia. Jap. J. Trop. Agr. 19:1-6. Houston, 0. F. 1973. Linear function for comparing viability loss rates in stored seeds. Seed Sci. & Tech. 1:795-798. Joseph, N. R. 1958. A pH calculator based on linear transforma- tions of the Henderson-Hasselbalch Equation. Science 128:1207- 1208. Lang, A. 1965. Effects of some internal and external conditions on seed germination. Ig_W. Ruhland (ed.) Handbuch der Pflanzen- physiologie 15:848. Lopez, A., and D. F. Grabe. 1973. Effect of protein content on seed performance in wheat (Triticum aestivum L.). Proc. Assoc. Off. Seed Anal. 63:106-116. Lowe, L. B., G. S. Ayers, and S. K. Ries. 1972. Relationship of seed protein and amino acid composition to seedling vigor and yield of wheat. Agron. J. 61:608-611. , and S. K. Ries. Effects of environment on the rela- tion between seed protein and seedling vigor in wheat. Can. J. Plant Sci. 52:157-164. , and . 1973. Endosperm protein of wheat seed as a determinant of seedling growth. Plt. Physiol. 51:57-60. Luczynska, J. 1973. Activity of some enzymes from soya bean seeds ageing at various air humidities. Bull. de L'Academie de Sciences. Serie biologique 21:155-158. Miller, Milton 0., and D. S. Mikkelsen. 1970. 1969 Rice Seed Protein Studies. Rice Journal 73:7-8. Osborne, 0. J., B. E. Roberts, P. I. Payne, and S. Sen. 1974. Protein synthesis and viability in rye embryos. In_Mechanisms of Regulation of Plant Growth, Bull. 12. Royal Society of New Zealand, Wellington p. 805-812. Pollock, B. M., and E. E. Roos. 1972. Seed and seedling vigor. IQ_T. T. Kozlowski (ed.) Seed Biology. Academic Press, NY, NY 1:313-387. Ries, S. K., G. Ayers, V. Wert, and E. H. Everson. 1976. The Variation in protein, size, and seedling vigor with position of seed in heads of winter wheat cultivars. Can. J. Plt. Sci. 56: 823-827. , and E. H. Everson. 1973. Protein content and seed size relationships with seedling vigor of wheat cultivars. Agron. J. 65:884-886. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 58 Roberts, 8. E., P. I. Payne, and D. J. Osborne. 1973. Protein synthesis and the viability of rye grains. Biochem. J. 131:275-286. Roberts, E. H. 1961. The viability of rice seed in relation to temperature, moisture content, and gaseous environment. Ann. Bot. N.S. 25:381-390. 1972. Viability of Seeds. Chapman and Hall, London, U.K. 1973. Predicting the storage life of seeds. Seed Sci. & Tech. 1:499-514. Sittisroung, Prasoot. 1970. Deterioration of rice (Oryza sativa L.) seed in storage and its influence on field performance. Diss. Abstr. Int'l. 31/07-8 p. 3805. Tani, T. 1975. General status of rice storage in Southeast Asia. In Assoc. of Jap. Agr. Sci. Soc. (eds.) Rice in Asia. Univ. Tokyo PFess p.650. Throneberry, G. V., and F. G. Smith. 1955. Relation of respiratory and enzymatic activity to corn seed viability. Plt. Physiol. 30: 337-343. Villiers, T. A. 1974. Seed aging: Chromosome stability and extended viability of seeds stored fully imbibed. Plt. Physiol. 53:875-878. , and D. J. Edgcumbe. 1975. On the cause of seed deterioration in dry storage. Seed Sci.& Tech. 3:761-774. Wu, S. T. 1975. Genetic studies on seedling growth in rice plant. II. Chemical contents and seedsize relationship with seedling vigor. J. Ag. Assoc. China 91:77-82. SUMMARY AND CONCLUSION Topdressed urea, 40 Kg N/ha at anthesis, was not superior to foliar urea for increasing protein content of 'IR-S' or 'Pelita 1/1' in the wet season. During the dry season, foliar urea did increase protein more than t0pdressing, but yield was decreased by the foliar treatment and disease. This leads one to suspect a foliar urea-disease inter- action. High protein seed had higher germination after a five day storage stress than low protein seed. Storage stress reduced emergence and stand of direct-seeded, low-protein seed, but it had no effect on the stand of transplanted seedlings. The loss of the transplanted rice and a 90% yield reduction of the direct seeded rice was caused by grassy stunt disease and brown planthopper infestation. This prevented any strong conclusion about the effect of protein and seed storage stress on yield of rice. Plants grown from high protein seeds yielded more regardless of storage stress. The results from several storage stress experiments further demon- strated the increased ability of high protein seeds to maintain high levels of seed vigor. Seeds stored in air-tight vials lost viability at much faster rates than seeds stored over water, suggesting either enzymatic repair during seed stress at high humidity or production of a gaseous germination inhibitor in a closed system. Larger seedlings were produced by high protein seeds, thus 59 6O agreeing with similar findings in wheat and barley, which suggest a high protein endosperm effect. However, embryos from stressed high and low protein seeds germinated better than embryos from low protein seeds. Both germinated equally well as their whole seed counterparts on nutri- ent agar, indicating that endosperm-nourished-recovery has little effect on the viability of high protein seed. In fact, it appears that embryos from high protein seeds are more resistant to deterioration. This inexpensive, improved embryo resistance to heat and moisture stress could be an important tool in germplasm maintenance and subsis- tence level seed storage methods for rice. A more in depth understand- ing of the mechanism by which embryos from high protein seed resist storage stress would magnify the value of its application. 10. 11. BIBLIOGRAPHY Abdul-Saki, A. A. 1969. Relationship of glucose metabolism to germinability and vigor in barley and wheat seeds. Crop Sci. 9:732-737. , and J. D. Anderson. 1972. Physiological and biochemical deterioration of seeds. In__T. T. Kozlowski (ed.) 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Part V1 is designed to provide the means for including community and work-related information in the vocational education planning, assisted the secondary education system in forming the vital link between education and work. The Data Element Dictionary For Vocational Education provides a sound planning base from which new efforts and developments in vocational edu- cation can be systematically incorporated into present program activities. It brings to the program planner all the structural aspects of program design which must be dealt with for successful program implementation. Lastly, the Data Element dictionary will assist the user in the development of a total planning perspective which can be applied to the challenging problems arising out of program planning for vocational education. RSI ”11111111111111!11111111 Hillillllllllllm 3 1293 030510777