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This is to certify that the dissertation entitled CHANGES IN LIPID, PROTEIN, MINERAL NUTRIENTS AND SEED QUALITY AT DIFFERENT TIMES OF DEVELOPMENT AND MATURATION OF CANOLA SEEDS (Brassica napus L.) presented by Sabry Gobran Elias has been accepted towards fulfillment of the requirements for fl) . D- degree in Cm? and $5 5mcad 1r Semi 9CM gfick no {dad n Magr professor Date Menu 13,. 1193 MS U it an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Michigan State , University 1 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE \ a.____J ’— ‘f‘T i MSU Is An Affirmative Action/Equal Opportunity institution c:\ci1cm.pma-p.t —-——-—'——_’—_”‘ " CHANGES IN LIPID, PROTEIN, MINERAL NUTRIENT S AND SEED QUALITY AT DIFFERENT TIMES OF DEVELOPMENT AND MATURATION OF CAN OLA SEEDS W mm; L.) By Sabry Gobran Elias A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1 993 ABSTRA£HT .CILANCHESHVlJflflnuImuyrEflfl,hflDflDRA1.NflflflUflflWTSIUWD SEED QUALITY AT DIFFERENT TIMES OF THE DEVELOPMENT AND MATURATION OF CANOLA SEEDS (5mm mg L.) By SabnyChflnanlflkm Pod and seed development and maturity of two spring and four winter canola cultivars (g; ngpgg L.) were studied over two growing seasons. Physiological and harvest maturity (PM, HM) for canola seed were determined using morphological and physiological markers. Seed quality and composition were monitored at different times of development. Appropriate tests for assessing canola seed quality were investigated. Loss of green color of canola pods and seed color changes from green to brownish green and greenish brown were the most reliable and practical methods for determining PM. Seeds at this stage are firm, but not hard and can be marked with a finger nail. The seeds reached maximum dry weight at 20.3-36% moisture. Pod color was yellow to light brown at HM and the seeds were brown or dark brown to black depending on the cultivar. The seeds were hard, with a moisture content near 10%. Significant correlation between seed dry weight and seed color during different times of development and maturation of all cultivars was found. Seed 1ipid.content reached maximum levels at PM; however, no significant changes occurred during the last 15-21 days before harvest, suggesting that the canola could be harvested two to three weeks before reaching HM without significant reduction in seed lipid content. However, in short growing seasons, this period lasted only about two weeks. The mean lipid content at HM was 40%. The ratio of saturated to unsaturated fatty acid was high (0.67) at the early stages of seed development, but reached the lowest level (0.087) 3-4 'weeks before HM where it remained without significant change until harvest. The average ratio was 8:92. Spring cultivars had higher protein content than winter cultivars, which remained constant in all cultivars during the last three weeks before harvest. The average protein content of spring cultivars at harvest was about 27%, compared to 22% for winter cultivars. Mineral content varied among cultivars and seasons with average of 0.83, 0.80, 0.40 and 0.35 % of dry weight for K, P, Ca and Mg, respectively. Traces of Fe, Mn, Zn, B, Cu and Al were also detected. The standard germination test, the cold test and the accelerated aging test were used to evaluate seed quality. Various combinations of time and temperature were used to determine the optimum conditions for each test. Seeds of all cultivars had greater germination and vigor at HM than at PM, suggesting that some physiological changes (e.g., hormonal mechanisms) may occur after PM which promote germination. The results also showed that seeds develop germination capacity ahead of vigor. I Minute this dissertation to the memory of my father who showed me an exemplary life of honesty, dedication and sacrifice; and to the memory of my brother-in-law, Fakhry Khalil, who made the graduate study opportunity available to me; and to my beloved wife Valerie and my daughter Saphia who were an inspiration to me during this study. 1ACIEKTWHJfllGhflDNTS I would like to texpress 'my sincere gratitude and appreciation to Dr. L. o. Copeland, my major professor, for his guidance, advice and support. He has shown me how to be a better scientist and, more importantly, how to be a better person. Many thanks to Drs. D. Penner, B. Knezek and I. Widders for serving on my guidance committee and for their valuable suggestions and reviews of this manuscript. Gratitude is extended to Dr. J. Ohlrogge and E. Cahoon for their advice in the lipid study, to Dr. C. Somerville, Clint Chapple and D. Reiter for providing the CC for lipid analysis and to J. Dahl and R. Cabrera for their help during the mineral nutrient study and to C. Bricker for his assistance in the protein study and to Dr. C. Cress for statistical guidance. They were always accessible. A special thanks for Matt Drake for taking pictures during the study. I am most deeply grateful to my wife Valerie for her help, patience and understanding during the entire study period. fi TIJHJECHFCIHWFENTS Page LIB! or mnns O O O O O O O O O O O O O O O O O O ........ O O O O O O O O 0 O ..... O 0 Vi 1 LI 8! or rlama O O O O I ..... O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O 0 O O x i v CHAPTER 1. Changes in lipid, protein, mineral nutrients and seed quality at different times of the development and maturation of canola aeede (aga§_iga_ngpg_ L. ). MODOflIO' 00...... ......... .00..........OOOOOOOOOOOO... 1 LITmm m1" 0 C I O O O ...... O ....... O O O O O O O O O O O O O O O O O O O O 5 Derivation Of canala O O O O O O O O O O O O I O O O I O O O O O O O O O O O O O O O O O O O 5 Plant growth times for oilseed rapes ................... 6 Pad and seed deve 1 opment O O O O O O O O I O O O O O O O O O O O I O O O O O O O O O O 7 seed maturation O O O O ...... O I O O O O O O O O O O O O O O O O O O O O O C O O O O O O 9 Factors influencing growth, development and quality of. canola seed ............................................ 11 Genetic factors ... ...... ............................. 11 Environmental factors .......... ...................... 12 Temperature and water stress ........................ 12 Fertilizer .............. ...... ...................... 13 Seeding date and rate ........ ....................... 14 Morphological and physiological indicators of physiological maturity (PM) ............................. 14 Chemical composition . ........... ....................... 16 Lipid, protein and mineral nutrients ................. 16 Variation of fatty acid composition of oilseeds ......... 18 Seed Quality . ...................... . ..... ..... ....... .. 19 Factors affecting seed quality ....................... 21 MATERIALS AND METHODS .................. .. ........... ..... 23 Planting, management and experimental design ........... 23 Sampling procedure ..................... ................ 23 Morphological and physiological assessments ............ 24 Seed lipid, protein and mineral nutrient contents ....... 25 Lipid content ..................... ................... 25 Protein content ........................... ........... 26 Mineral nutrient content ............ ................. 27 Seed quality .. ..................... ................... 28 Standard germination test ............................ 29 Cold test ........... ............ ..................... 29 Accelerated aging test ............... ................ 29 fii Second year study . .......... .... ...... . .............. .. 29 Statistical analysis ....... ..... ........ ......... ...... 30 RESULTS .0.00..00.0.000000000000000000000. ...... 0.0.0 ..... 31 Development and maturity of pod and seed ....... ...... .. 31 Pod length ........................................... 31 Pod width and fresh and dry weight ................... 39 loo-seed weight and number of seeds per pod .......... 47 Seed fresh weight, dry weight and moisture content .... 54 Morphological and physiological markers for physiological and harvest maturity ............ ..... .. 64 Nutrient accumulation in seeds .............. ......... .. 64 Lipids ............................. ..... .... ....... .. 64 Ratio of saturated to unsaturated fatty acids at different times of seed development ......... ...... 74 Protein .............................................. 83 Mineral composition ..... ............................. 91 Seed quality ........................................... 96 Germination and vigor ..... ..... ...................... 96 DISCUSSION 0.0.0.0... ................... 0.000000000000000. 105 mnmcna 0.000.000.0000000000 ..... 0.00000000000000000000 111 CHAPTER II. Evaluation of vigor tests for canola (g. ggggg) ”emu 0.0.0.0000. .............. 000.00.000.0000000000000 118 INTRODUCTION 0.00.0000... 0000000000 0 ....... 0 0000000000000 . 120 LITMTURB RHI" 0 0 0 0 0 0 0 0 0 . 00000000000000 0 0 0000000000 0 . 0 0 123 Definition of seed vigor ..... ...... ................... . 123 Manifestation of poor vigor .... ..... ..... ...... ........ 124 Seed vigor and seedling growth rate ..................... 124 Factors influencing seed vigor ....... ............. ..... 126 Genetic constitution ................................. 126 Environment during seed development and maturation .... 128 Seed storage ......................................... 129 Physiological and biochemical changes accompanying vigor loss ............................................ 130 Measurement of seed vigor . .......... ................... 131 MATERIALS AND KBTBODB ..... .......... .............. . ..... . 137 Cultivars and aging procedure ................. .. ..... .. 137 Seed quality determination ................... ........ .. 137 Accelerated aging test ...... ......... .. .......... .... 137 iv Page Standard germination test and first count germination tests ................................ 138 Cold test ........................................... 138 Cold soil test ...................................... 138 Electrical conductivity test ........................ 138 Field emergence ....................................... 139 Statistical analysis ............. ....... .............. 139 “Bars 00.000.00.0000000000000.0000.00.000.00.00...000.. 140 The standard germination test .......................... 140 The accelerated aging test ............................ 143 The first count germination test ...................... 145 The cold test ......................................... 147 The cold soil test .................................... 149 The electrical conductivity test ...................... 149 Field emergence ....................................... 151 D1 sense I on 0 0 0 0 0 0 . 0 . 0 . 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 1 5 9 cayenne I one m “GMAT I one 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 . 0 0 0 0 0 1 6 2 mmcns 0 0 0 0 0 0 0 0 0 0 ......... . 0 0 0 0 0 0 . . 0 . 0 . . 0 0 0 0 0 0 0 0 0 0 0 0 0 1 6 4 CHAPTER III. The effect of storage conditions on canola (5- seas) and quality new” 0 0 . 0 0 0 0 0 0 ....... 0 0 0 0 0 . . 0 0 0 0 0 0 0 0 . 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 168 INTRODUCTION 0 0 0 0 0 0 0 0 0 0000000000 0 ........... 0 0 0 0 0 0 0 0 0 0 0 0 0 169 LITme m1" 0 . 0 . 0 0 0 0 0 0 . 0 0 000000000000000 0 0 0 . 0 0 0 0 0 0 0 17 1 Factors affecting seed storability .................... 171 Changes in seed quality during storage ................ 174 Genetic changes ..................................... 174 Physiological changes ............... ................ 174 Biochemical changes .................. ............... 176 Measurement of seed deterioration ..................... 178 IATBRIALS AND METHODS .... ........................... .... 180 Cultivars, aging procedure and storage periods ........ 180 Determination of quality of stored seeds .............. 180 Standard germination test ........................... 181 Cold test ....... ................... ................. 181 Conductivity test .................. ................. 181 Page mars ooeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 182 Analysis of variance results ........................... 182 Tapas 00.0.0.00000....0.0000000000000000.......00.00.... 184 Liborius 0.000.000.0000000000000.000.000.0000.0.0.0.0... 185 Libravo eeeeeeeeeeeeeeeeeooeeeeeeeeeeeeeeeeeeeeeeeeeeeee 186 Discussxou eeoeeeeeeeeeeeeeeeeee eeeeeeeeeeeeee eeeeeeeeeeee 193 SM! m coucnus I one 0 0 0 0 0 0 . . 0 0 0 0 0 0 0 . 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 O 0 1 9 7 name‘s .0000.00.000.00.00.000000000000000.000.00.00...198 vi TABLE 10 11 12 13 14 LIST OF TABLES Page Analysis of variance for factors affecting pod and seed traits during different times of fruit growth of two spring canola cultivars in 1989 and 1990 ..... 34 Analysis of variance for factors affecting pod and seed traits during different times of fruit growth of four winter canola cultivars in 1989 and 1990 ..... 35 Pod length of two spring canola cultivars during different times of seed maturity in 1989 ....... ..... 36 Pod length of two spring canola cultivars during different times of seed maturity in 1990 ............ 36 Pod length of four winter canola cultivars during different times of seed maturity in 1989 ............ 37 Pod length of four winter canola cultivars during different times of seed maturity in 1990 ............ 37 Pod width of two spring canola cultivars during different times of seed maturity in 1989 ............ 41 Pod width of two spring canola cultivars during different times of seed maturity in 1990 . ..... ...... 41 Pod width of four winter canola cultivars during different times of seed maturity in 1989 ............ 42 Pod width of four winter canola cultivars during different times of seed maturity in 1990 ............ 42 Pod fresh weight of two spring canola cultivars during different times of seed maturity in 1989 ..... 43 Pod dry weight of two spring canola cultivars during different times of seed maturity in 1989 ............ 43 Pod fresh weight of two spring canola cultivars during different times of seed maturity in 1990 ..... 44 Pod dry weight of two spring canola cultivars during different times of seed maturity in 1990 ............ 44 vfi Table 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Page Pod fresh weight of four winter canola cultivars during different times of seed maturity in 1989 ..... 45 Pod dry weight of four winter canola cultivars during different times of seed maturity in 1989 ..... 45 Pod fresh weight of four winter canola cultivars during different times of seed maturity in 1990 ..... 46 Pod dry weight of four winter canola cultivars during different times of seed maturity in 1990 ..... 46 Number of seeds per pod of two spring canola cultivars during different times of maturity in 1989...49 100-seed weight of two spring canola cultivars during different times of seed maturity in 1989 ..... 49 Number of seeds per pod of two spring canola cultivars during different times of maturity in 1990...50 loo-seed weight of two spring canola cultivars during different times of seed maturity in 1990 ...... 50 Number of seeds per pod of four winter canola cultivars during different times of maturity in 1989...51 100-seed weight of four winter canola cultivars during different times of seed maturity in 1989 . ..... 51 Number of seeds per pod of four winter canola cultivars during different times of maturity in 1990...52 100-seed weight of four winter canola cultivars during different times of seed maturity in 1990 ...... 52 Seed fresh weight of two spring canola cultivars during different times of seed maturity 1989 ......... 56 Seed dry weight of two spring canola cultivars during different times of seed maturity in 1989 ...... 56 Seed moisture content of two spring canola cultivars during different times of maturity in 1989...57 Seed fresh weight of two spring canola cultivars during different times of seed maturity in 1990 ...... 57 vfii Table 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Page Seed dry weight of two spring canola cultivars during different times of seed maturity in 1990 ...... 58 Seed moisture content of two spring canola cultivars during different times of maturity in 1990...58 Seed fresh weight of four winter canola cultivars during different times of seed maturity in 1989 ...... 59 Seed dry weight of four winter canola cultivars during different times of seed maturity in 1989 ...... 59 Seed moisture content of four winter canola cultivars during different times of maturity in 1989...60 Seed fresh weight of four winter canola cultivars during different times of seed maturity in 1990 ...... 6O Seed dry weight of four winter canola cultivars during different times of seed maturity in 1990 ...... 61 Seed moisture content of four winter canola cultivars during different times of maturity in 1990...61 Lipid concentration in seed tissues of two spring canola cultivars at different times of seed maturity in1989 000..0.000.000.000000000000000.0.0.00000000000 66 Lipid concentration in seed tissues of two spring canola cultivars at different times of seed maturity inlggo 0.00.00.00.00...0.0.00.000000000000000.0.0.00. 66 Lipid concentration in seed tissues of four winter canola cultivars at different times of seed maturity in 1989..67 Lipid concentration in seed tissues of four winter canola cultivars at different times of seed maturity in 1990..67 Lipid content in seed tissues of two spring canola cultivars at different times of seed maturity in 1989..68 Lipid content in seed tissues of two spring canola cultivars at different times of seed maturity in 1990..68 Lipid content in seed tissues of four winter canola cultivars at different times of seed maturity in 1989..69 Lipid content in seed tissues of four winter canola cultivars at different times of seed maturity in 1990..69 ix Table 47 48 49 50 51 52 53 54 55 56 57 58 59 6O 61 62 Page Fatty acid profile of spring canola cultivar Topas during different times of seed maturity in 1989 ...... 76 Fatty acid profile of spring canola cultivar Westar during different times of seed maturity in 1989 ...... 76 Fatty acid profile of winter canola cultivar Cascade during different times of seed maturity in 1989 ...... 77 Fatty acid profile of winter canola cultivar Crystal during different times of seed maturity in 1989 ...... 77 Fatty acid profile of winter canola cultivar Glacier during different times of seed maturity in 1989 ...... 78 Fatty acid profile of winter canola cultivar Lirabon during different times of seed maturity in 1989 ...... 78 Fatty acid profile of spring canola cultivar Topas during different times of seed maturity in 1990 ...... 79 Fatty acid profile of spring canola cultivar Westar during different times of seed maturity in 1990 ...... 79 Fatty acid profile of winter canola cultivar Cascade during different times of seed maturity in 1990 ...... 80 Fatty acid profile of winter canola cultivar Crystal during different times of seed maturity in 1990 ...... 80 Fatty acid profile of winter canola cultivar Glacier during different times of seed maturity in 1990 ...... 81 Fatty acid profile of winter canola cultivar Lirabon during different times of seed maturity in 1990 ...... 81 Protein concentration in seed tissues of two spring canola cultivars at different times of seed maturity in 1989..86 Protein concentration in seed tissues of two spring canola cultivars at different times of seed maturity in 1990..86 Protein concentration in seed tissues of four winter can- ola cultivars at different times of maturity in 1989...87 Protein concentration in seed tissues of four winter can- ola cultivars at different times of maturity in 1990...87 Table 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 Page Protein content in seed tissues of two spring canola cultivars at different times of seed maturity in 1989..86 Protein content in seed tissues of two spring canola cultivars at different times of seed maturity in 1990..86 Protein content in seed tissues of four winter canola cultivars at different times of seed maturity in 1989..87 Protein content in seed tissues of four winter canola cultivars at different times of seed maturity in 1990..87 Mineral nutrient concentration of spring canola cultivar Topas at different times of seed maturity in 1989 .... 93 Mineral nutrient concentration of winter canola cultivar Glacier during different times of maturity in 1989 ... 93 Mineral nutrient concentration of two spring canola cultivars at different times of seed maturity in 1990..94 Mineral nutrient concentration of two winter canola cultivars at different times of seed maturity in 1990..94 Mineral nutrient concentration of two winter canola cultivars at different times of seed maturity in 1990..95 Standard germination test results of two spring canola cultivars at different times of seed maturity in 1989..98 Cold test results of two spring canola cultivars during different times of seed maturity in 1989 ...... 98 Accelerated aging test results of two spring canola cultivars at different times of seed maturity in 1989..99 Standard germination test results of two spring canola cultivars at different times of seed maturity in 1990..99 Cold test results of two spring canola cultivars during different times of seed maturity in 1990 ..... 100 Accelerated aging test results of two spring canola cultivars during different times of seed maturity inlggo...OOOOOOOOOOOOOO.IOOOOOOOOOOOOOOO0.0.........loo Standard germination test results of four winter canola cultivars at different times of seed maturity in 1989..101 xi Table 79 80 81 82 83 Page Cold test results of four winter canola cultivars during different times of seed maturity in 1989 ...... 101 Accelerated aging test results of four winter canola cultivars at different times of seed maturity in 1989..102 Standard germination test results of four winter canola cultivars at different times of seed maturity in l990..102 Cold test results of four winter canola cultivars during different times of seed.maturity in 1990 ...... 103 Accelerated aging test results of four winter canola cultivars at different times of seed maturity in 1990..103 CHAPTER II Analysis of variance for factors affecting seed quality of two winter (W) Ceres and Liborius and two spring (S) Delta and Bounty canola cultivars measured by accelerated aging, conductivity, first count tests and field emergence ...................... 141 Analysis of variance for factors affecting seed quality of two winter Ceres and Liborius and two spring Delta and Bounty canola cultivars measured.by cold and cold soil tests ................. 142 Accelerated aging test results of two winter and two spring canola cultivars aged for different periods oftime........ ...................... ................l44 First count germination test results of two spring canola cultivars aged for different.periods of time....146 Cold test results of two winter and two spring canola cultivars aged for different periods of time....148 Cold soil test results of two winter and two spring canola cultivars aged for different.periods of time....150 Electrical conductivity results of seeds of two winter and two spring canola cultivars aged for differentperiodof time 152 Field emergence means of seeds of two winter and two spring canola cultivars aged for different period of xfi Table 10 Page Simple correlation coefficients between field emergence and accelerated aging, first count germination, cold, cold soil and conductivity tests of two spring canola cultivars............................................157 Simple correlation coefficients between field emergence and accelerated aging, cold, cold soil and conductivity tests of twO‘winter canola cultivars ................. 158 CHAPTER III Analysis of variance for factors affecting seed storability of three canola cultivars measured by standard germination test, cold test and conductivity test 00.00.000.00...0.00............OOOOOOOOOOOOOO...183 The effect of storage conditions on the germination of three canola cultivars aged for different periods of timeOOOOO....OO.....O..0O..........OOOOOOOOOOOOOOOOOO189 Effect of storage conditions on cold test results of three canola cultivars aged for different periods of time........ ..... ... 000000000000 0.0.0.000000000000000190 Effect of storage conditions on conductivity test results of three canola cultivars aged for different periOdSOftime... ........ ......OOOOOOOOOOOOOOOOO... 191 Simple correlation coefficients between standard germination, cold and conductivity test results of three canola cultivars Topas, Liborius and Libravo aged for different periods of time ................... 192 )(iii Figure 10 11 1m9T(JFIHGHflRES CHAPTER I Page Pod length and width of the winter canola cultivar Crystal during different times of maturity in 1990 ... 38 Pod length and width of the spring canola cultivar Topas during different times of maturity in 1990 ..... 38 Seed per pod and loo-seed weight of the winter canola cultivar Crystal during different times of maturity inlggoO.I.I0.0......O...0..........OOOOOOOOOOOOOOOOO 53 Seed per pod and loo-seed weight of the spring canola cultivar Topas during different times of maturity 1111990 O....0.0.0.........OOOOOOOOOOOOOIOOOO ......... 53 Pericarp and seed dry weight and seed moisture content of the winter canola cultivar Crystal during different times Of maturity in 1990 0.00.00.00.00...0.00.0000... 62 Pericarp and seed dry weight and seed moisture content of the spring canola cultivar Topas during different times of maturity in 1990 .. ..... ..................... 62 Relationship between seed dry weight and seed color at different times of seed development and maturity of two spring canola cultivars in 1990 ............... 63 Relationship between seed dry weight and seed color at different times of seed development and maturity of four winter canola cultivars in 1989 ...... ........ 63 Relationship between lipid content and times of development and maturation of two spring canola cultivars in 1990 OOOOOOOOOOOOOOOOOOO O O O O O O O O O O O O O O O O 0 72 Relationship between lipid content and times of development and maturation of four winter canola cultivars in 1989 .................................... 72 Lipid and protein concentration of the winter canola cultivar Crystal at different times of development and maturity in 1990 ....... .............. ............ 73 xw Figure 12 13 14 15 16 17 18 Page Lipid and protein concentration of the spring canola cultivar Topas during different times of development and maturity in 1990 ................................. 73 Ratio of saturated to unsaturated fatty acids of the winter canola cultivar Crystal during different times of maturity in 1990 ........................... 82 Ratio of saturated to unsaturated fatty acids of the spring canola cultivar Topas during different times Ofmaturity in 1990 0............OOOOOOOOOOOO... 82 Relationship between protein content and times of development and maturity of seeds of two spring canola.cultivars:h11990 ............................. 90 Relationship between protein content and times of development and maturity of seeds of four winter canola cultivars in 1990 ............................. 90 Standard germination, cold and accelerated aging tests of winter canola cultivar Crystal during different times of maturity in 1990 104 Standard germination, cold and accelerated aging tests of spring canola cultivar Topas during different timeSOfmaturitYinlggoeeeeeeeeeeeeeeeeeeeeeeeeeeee104 CHAPTER II Relationship between accelerated aging, cold, cold soil and conductivity tests and field emergence of aged winter canola cultivar Ceres ........... ......... 155 Relationship between accelerated aging, cold, cold soil and conductivity tests and field emergence of aged winter canola.cultivar Liborius ................. 155 Relationship between accelerated aging, cold, cold soil and conductivity tests and field emergence of aged winter canola cultivar Delta .................... 156 Relationship between accelerated aging, cold, cold soil and conductivity tests and field emergence of aged winter canola cultivar Bounty ................... 156 XV gratin ‘ On“, 5 buy». 5 t quali ti dew Hit Can has HWTR£HNUCHHCH¢ Canola (rapeseed) is important for its use in the production of edible oil, its high protein content for supplementing animal diets and potentially for its high quality protein for human consumption (26,68). Canola has an indeterminate flowering pattern. Older flowers occur at the base of inflorescence while immature flowers continue to form on the newer branches, resulting in different stages of seed maturity occurring on the same plant (17, 18). If seeds are harvested before reaching HM, they tend to show varying degrees of quality and different storage potential, depending largely on the conditions during seed development and cultivar (6,25,73,78). The period available for harvesting canola is relatively short because over-ripened pods shatter easily, especially with high wind and rain (62). In drier areas of western Canada, swathing and/or chemical desiccation are used to hasten pod dry-down and even out the maturation of the seeds (67). It is also helpful in speeding up the time of harvest to permit timely planting of the next crop and to avoid unfavorable weather for maturation and harvesting and to help avoid damage from early frost. In a very weedy crop, desiccation (usually with diaquat) may be used for possible we: here a- 3'5? 4&5. ~ ‘I win. harvest at a somewhat earlier stage than full maturity. Therefore, it is important to know the quality and nutrient status of canola seeds at different times in which the crop may be harvested before reaching full maturity. Generally, seeds attain maximum dry weight, viability, lipid and protein content at PM (20,24,71,75). Thus, it is meaningful to be able to identify this stage to enable early harvest without sacrificing seed guality.~ However, it is difficult to accurately estimate the date of PM from measurements of seed dry weight or moisture content because of the variability within the raceme as it approaches PM. Therefore, a rapid method of determining PM based on visual indicators in the field is needed. Seed development and maturation are characterized by physical and chemical changes (1). Both genetic and environmental factors affect the rate and duration of fruit (pod) growth. These are affected by temperature, moisture availability, nutrient status and cultivar (5,11,12,14,15,38, 49,57,70). Important characteristics of canola seeds during this period include lipid content, fatty acid profile (saturated vs. unsaturated fatty acids), protein content, mineral composition and seed quality, e.g., viability and vigor. stand 6 Wm we. no wndit ...ort also ‘0 and he tomes ”RI 'e J9 ‘ The use of high quality seed is essential for a good stand establishment and crop productivity in any crop. Germination and vigor tests provide information to both seed producers and their customers to help predict the potential performance of a seed lot when planted under a wide range of conditions. Although the standard germination test is the most important and widely accepted quality test, vigor tests may also'be useful for determining the planting value of the seeds and helping to predict satisfactory field emergence (7,9). Most studies on seed development and changes in chemical composition of developing seeds have been conducted on crops other than canola (21,30,32,33,40,4l,50,61). Thus, an understanding of the changes in canola seed quality and composition during different development stages is needed. Most studies on chemical composition of rapeseed have focused on mature seed and defatted meal (6,11,27,37,49,63). -Few studies have been conducted on the changes in chemical composition of rapeseed during seed development and maturation (6,37,60). The objectives of this research were to: 1) Identify the physiological maturity (PM) and harvest maturity (HM) for canola using morphological and physiological markers. 2) Relate the different stages of seed development and nine} 3)? maturity to seed quality and composition (oil, protein and mineral nutrients). 3) Evaluate and develop tests for assessing the seed quality of canola. I 31:" 6 H“ 9 be ‘6 I !w\n I ‘eiv Jr! a Q IP': 0. ‘. (1’ f LIFERAJIHUEIUEVEE“V Derivation of Canola The name "canola" was coined by Canadians in 1979 to apply to rapeseed cultivars with low erucic acid and low glucosinolate and may be further described as "double low" or "double zero" rapeseed (68). All oilseed rape belongs to the mustard family, Brassicaceae and includes three species belonging to the genus Brassica:.B..napus (n=19), B. campestris (n=10) and B. junceea (n=18), known as rape (or Argentine rape), turnip rape (or Polish rape) and leaf mustard (or oriental or brown rape), respectively (26,27,68). Canola has silique type of fruit, consisting of two carpels, separated by a septum which divides the cavity into two locules and a replum between the carpel edges. Its inflorescence is a raceme. The flowers are bisexual, consisting of four sepals and four petals disposed in a cross, six stamens, four of which are longer than the other two and one pistil with two carpels and two locules. The leaves are alternate, simple and without stipules (18,26,74). ‘fd Plant Growth Stages for Oilseed Rape: In 1975, Harper and Berkenkamp (36) proposed a growth-stage key for B. napus and B. campestris, consisting of: 0, pre-emergence; 1, seedling; 2, rosette; 3, bud; 4, flower and 5, ripening. The first four represent vegetative stage and the last two reproductive stages. In 1984, Sylvester-Bradley and Makepeace (69) suggested a different key, consisting of seven principal growth stages: 0, germination and emergence; 1, leaf production; 2, stem extension; 3, flower bud development; 4, flowering; 5, pod development; and 6, seed development. Five to ten days after seeding, seedlings start to emerge above soil surface. Speed of germination depends on soil temperature and moisture, seed-soil contact, and depth of planting. The first true leaves form a rosette. The stem begins to grow and branches develop on the main stem. The plants reach their maximum leaf area at the bud stage and provide assimilates needed during flowering and pod filling. Later, the axis of the highest leaves on the stem each terminate in an inflorescence. Flowering begins at the lowest bud on the main.raceme and continues for about 2-3 weeks. Pod- fill is complete within about 35-45 days after flower initiation, depending on the cultivar and weather conditions (54,62,69). Pod and Seed Development Seed development begins with fertilization and terminates with development of maximum fresh weight. Growth occurs by cell division, elongation and differentiation in the embryonic axis, endosperm and cotyledons. Later, chloroplasts, spherosomes (oil bodies), protein bodies and starch grains within plastids form in the cotyledonary tissues, resulting in an increase in dry weight (1). Since limited literature is available on seed development of canola, a review for the patterns of pod and seed growth of different crops will also be made and compared with canola. Several researchers have studied the pattern of pod and seed development of different crops and concluded that the rate of growth and development of fruit (pods) depends on genotype and environmental conditions (4,5,8,11,32,33,38,57, 60,61,70). Early in 1936, Culpepper (21) observed that pods of snap bean attained full length 10-15 days after flowering and reached maximum diameter 25-30 days after flowering. This was confirmed by Watada and Morris (76) who found that pods of snap bean attained maximum length 13 days after flowering. Norton and Harris (60) reported that the embryo of B. napus completes cell division and formation before storage reserve deposition begins. Research.has shown that pods reach maximum weight before the seeds do. Egli (30) reported maximum dry weight of soybean pods when seeds had reached only 15-30% of their final weight. His:results support those reported by Flinn and Pate (32) who observed rapid increases in fresh weight of pods at the early stages of fruit growth of field pea, accompanied by very slow seed development. Oliker g1; 31. (61) also reported similar results in dry beans. The fact that pod growth precedes seed growth led to the observation that pods serve as either a competing sink or as a source of assimilates for developing seeds ( 33) . Oliker gt; 91. (61) suggested that pods and seeds do not act as competing sinks since nitrogen accumulation in the seeds begins only after it has ceased in the pods. Hocking and Pate (40) found that the pod of Lupinus albus contributes more to the seed than the testa to the embryo. Other researchers have suggested that pods and pod-bearing branches produce most of the assimilates needed for the growth and development of both pod and seed when almost all leaves below the pod canopy have senesced (5,8,16). Hocking (39) and Thorne (72) observed that the redistribution of pod dry matter to developing caster bean and soybean seeds occurred during late seed filling. Fraser g; 3].. (33) found significant correlation between pod length, width and final seed size in soybean and concluded that pod development is likely to influence the subsequent growth of seeds. Seed Maturation Maturation begins when the seed reaches maximum fresh weight and is terminated when they attain PM at maximum dry weight. During maturation, moisture content drops rapidly while dry weight increases slowly, as reflected by the growth of storage organs. This stage is also characterized by very low metabolic activity and near-cessation of embryonic axis growth (1). The first phase of seed development starts with the growth of the testa and endosperm (28). Temperature, moisture, cultivar and nutrient status are the most important factors that influence seed maturity (17,25,70,73) . Flinn and Pate (32) reported that 35 days after anthesis until seed maturity, the loss of dry matter from the pod in field peas was accompanied by an increase in seed dry matter. Norton and Harris (60) reported slow accumulation of metabolically active lipid during the early stages of seed development in oilseed rape. Appelgvist (6) reported similar results for seeds of many oil crops. Culpepper (21) found that seed of snap bean made up only 4% of fruit weight at 15 days after flowering but accounted for 53.8% of the total‘weight.at physiological maturity. Loewenberg (47) observed a slow 10 increase in seed dry weight of dry bean, reaching 17% of the final weight in the first three weeks. However, maximum fresh weight occurred 36 days after flowering. He also found that the seed coat accounted for 10% of seed weight at maturity. Dure (28) reported that the endosperm formed during early seed development of caster bean serves as a transient reservoir of sugar and amino acids to be utilized by the developing embryo, whereas the cotyledons serve as reservoir of stored nutrients during embryogenesis in legumes and cotton. Loewenberg (47) found that cell division in the cotyledonary tissue of beans was completed 21 days after anthesis. Hsu (41) observed the disappearance of the endosperm in bean seeds after 13-15 days after flowering. No comparable studies have been done for canola. Watada and Morris (76) reported that the growth pattern of snap bean seeds consists of two phases, with a lag in growth from about 20-22 days after flowering. A similar pattern was observed in beans by Hsu (41) who concluded that the second growth phase was associated with seed-filling of storage material and was therefore more important in determining seed size in legume than the initial growth phase during which the basic cell structure is formed. Hocking (39) also found that seed dry weight increased rapidly during the final third of fruit development in caster bean. Appelqvist (6) described three phases of lipid accumulation from petal condi lipic diffe lipi: star; 7" (1 f. H u hr‘ 5“ I 11 fall to full maturity. The first phase, which ranges from 10 to 30 days depending on the species and environmental conditions, is characterized by a very slow accumulation of lipids. The second phase, extending from 2 to 5 weeks in different oil seeds, is characterized by rapid accumulation of lipids. The third phase, ending at seed maturity, is again characterized by minor lipid accumulation. Factors Influencing Growth, Development, Production and Quality of Canola Seed Was In a genetic study of oilseed rape, Cambell and Konord ( 11) found that the heritability of growth characteristics was generally low. However, they found evidence of dominance of eaI‘ly flowering and early maturity. Heterosis was exhibited for yield and yield components and hybrids of rapeseed Sigl‘tificantly outyield their parents (11,45). Pathak g; 11. ( 63) studied the seed quality of 10 species of Brassica and three other cruciferous genus. They found that the oil of B. Calupestris var. B82 had the lowest free fatty acid content, and B. napus had the highest value of K and Ca. Mailer and wratten (49) reported that the quality of oilseed rape was s: ' . . . . lgnificantly affected by the differences among cultivars. "IF WC 1m “3 12 W Temperature and later Stress Environmental conditions affect the growth pattern of canola. The rate of development from flowering to maturity is largely controlled by temperature and water availability ( 8 ,30,39,59,7l) . Mendham gt :1. (57) reported that differences in spring temperatures from year to year have a significant effect on crop growth. They showed that each degree rise in temperature (within a range of 11 C to 17 C) caused about eight days' earlier maturity. Mailer and Wratten (49) found that seed yield, 1000-seed Weight and oil content increased significantly with an alternating temperature regime of 18 / 10 C, compared to 27/15 C. They also reported significant reduction of the same Parameters under moisture stress conditions. However, 91ncosinolates (chemical compounds which cause metabolic dj~8turbance in nonruminant animals) significantly increased with water stress but not with higher temperatures. Clarke and Silllpson (15) also reported an increase in yield of Brassica hapus with irrigation and increased seeding rates. Meza-Bosso g; g1. ( 56) found that protein content of soluble extracts decreased by 25% in seedlings of B. napus grow at 18 C compared to those grown at 0 C. They suggested h hfit specific polypeptides might be preferentially synthesized 13 at 0 C. However, Thurling (73) showed that the influence of the environment was greater on seed yield than on seed chemical composition . rcrtiliser Allen and Morgan (5) reported an increase in the growth rate and seed production of rape by the application of nitrogen. They suggested that nitrogen increases the supply of assimilates to the flower and young pods and that the increase in seed production was due to increase in number of Pods per plant and seeds per pod. However, nitrogen had little effect on seed weight. Christensen g; 51. (14) found similar results, reporting that nitrogen and phosphate fertilizer increased seed yield. Mailer and Wratten (49) showed that Yield and oil content reached a maximum at 15 ppm sulfur and that glucosinolates reached a maximum at 200 ppm. Henry and MacDonald (38) reported a near four-fold increase in yield by the combination of irrigation and nitrogen fertilizer. They showed a positive correlation betWeen application of N fertilizer and both yield and protein corl‘tent and a highly negative correlation with oil content. w‘aiss (77) suggested a minimum application of 50-75 kg/ha p2 0, at planting time. He also suggested that potassium is h§ Qessary to ensure adeqate uptake of phosphate and nitrogen. 14 Seeding Date and Rate Christensen gt a1. (14) reported that a delay in seeding reduced oil content but had little or no effect on protein content. Degenhardt and Kondra (23) found that a delay in the seeding date for five cultivars of B. napus caused a significant decrease in days to first flower and maturity. Picatdever, increases in seeding rates (3,6 and 12 kg/ha) significantly reduced the length of seed formation and hastened maturity. Clarke and Simpson (15) found that net assimilation rate was increased by low seeding rates compared 11¢) Ihigher seeding rates in B. napus. IMendhamHgt._l. (57) showed that early sowing of winter B. ‘Iiéismus produced consistent, but not higher yield than late E3<>Vving based on larger number of pods per plant with less Seecis per pod for early sowing and fewer pods with more seeds 19‘31? pod for later sowing. Seed weight was not affected by time of sowing. They reported that sowing in early spring (mid- March) can increase yield due to an increase in the vegetative growth prior to flowering. Morphological and Physiological Indicators of Physiological Maturity (PM) Physiological maturity (PM) is defined as the time of Ilia3'<:i.mum dry weight accumulation in the seed (20,24,71) and is 1: he stage at which the seed becomes an independent biological Kl - hit (71) . This is also the point at which the seed is 15 considered to have highest quality (75). At PM, seeds no longer accumulate dry weight which remains constant from PM until HM (20,71). Since PM is an important stage in seed development, it is useful to define it with visual and physiological indicators. In legumes, loss of the green color of the pod has been found to be a practical indicator of PM. Crookston and Hill (20) found both seed shinkage and the loss of green pod color to be reliable indicators of PM of soybean seeds. Gbipki and Crookston (34) and TeKrony __e_t_ g1. (71) also reported similar conclusions. On the other hand, PM in maize and sorghum can be identified by a visible black layer in the Placental region of the kernels (13,22,29) . Watada and Morris (76) reported that moisture content (MC) can be reliable index of maturity in snap beans. This agrees with Crookston and Hill (20), and TeKrony g3; g1. (71) Who found seed moisture content of soybean at PM to be c(Jr'lsistent among cultivars. However, the range of moisture c(Dritent at which seed is expected to reach PM was found to be J:‘elatively wide for many crops. TeKrony g 11. (71) found that so)y'bean seeds reach PM at 54-62% MC, whereas Gbikpi and Crookston (34) reported that seed MC of soybeans was only 44% at PM. Carter and Poneleit (3) observed significant differences in MO at the time of black layer maturity (PM) in differ [‘25) , attair pm: is the attair 16 different corn inbreds ranging from 15.4 to 35%. Delouche (25) , however, reported that seeds of corn, sorghum and rice attain PM at moisture contents (MC) ranging only from 32-35%. Seeds attain PM at high moisture levels unsuitable for mechanical harvest and unsafe for storage. Further desiccation is therefore necessary before harvesting maturity (an) is attained (25) . TeKrony pt 31. (71) defined harvest maturity of soybean 1 as the first time seed moisture content falls below 14%. Between PM and HM seeds remain on the plant where they may be exPosed to adverse weather conditions which can greatly affect their quality. The time from PM to HM varies with crops and environmental conditions. TeKrony _e_t g1. (71) reported a range of 10-20 days, while Gbikpi and Crookston (34) found that 9-10 days were required for soybean to reach HM after PM. Chemical Composition I‘:.-P:id, Protein and Mineral Nutrients The storage reserves of oilseed rape are made up of lipids and protein in about a 2:1 ratio (6) . Pathak gt g1. ( 63) found a negative correlation between oil and protein thtent in oilseed rape. The average composition of rapeseed 1" percentage of dry matter, is 45 oil, 25 protein, 25 cal‘lbohydrate and 5 % compounds such as phytic acid, lignin and . . glucosinolate (77) . According to a study by Murphy gt g1. t 65' 0 oil can: my ‘PAQ n V& and 1 sage ‘, fin “ VII 0 5.79. 17 (59), seed proteins of B. napus consist of legumin, cruciferin, napin, and polypeptides. About 90% or more of the lipid of mature oilseed rape (B. napus) is neutral lipid, largely triacylglycerol (60, 67). The other 10% is made up of p11 osphatidyl-choline , phosphatidylethanolamine , diacylgalactosylglycerol, diacyldigalactosylglycerol and free fatty acids (60). Pathak g3; a1. (63) found that the oil content of 10 species of Brassica ranged from 48.78% in B. conpestris to 26.75% in B. alba. The protein content ranged from 32.87% in B. alba to 14.33% in B. chinensis. Oil, protein and mineral element content varied among cultivars within the Same species. They also reported the highest values of K in B. napus at 1.77%, Ca in B. carinata at 0.82%, P in B. nigra at 0 - 79% and Mg in B. hirta at 0.39%. Hocking and Pate (40) Observed considerable differences in mineral composition between different species and in the mobilization of specific IujJierals to the seeds. They suggested that selective uptake occurs from the phloem during seed development. Measurement of the lipid content of oilseeds is usually accomplished by one of two two methods. The standard method is performed by conversion of oils to methyl esters and the 8eDaration of esters by gas chromatography (GC) . The second n‘e‘thod is conducted by using near-infrared (MIR) spectroscopy Er - hlch is also useful for measuring protein content (35) . A new ref 156 £25 (3 '9; A < 18 technology using NMR (Nuclear Magnetic Resonance) is also available (55) . Fatty acids are synthesized at different sites within the cell, mostly in the plastids, cytosol and endoplasmic reticulum. Changes in the fatty acid composition occur during the different stages of seed development. Oleic acid (18:1) is desaturated to 18:2 and 18:3 and is elongated to 20:1 and 22:1. 18:0 may also be elongated to 20:0 and 22:0 (37). Lipids within spherosomes are first hydrolyzed into fatty acids by lipases which are then converted in glyoxysomes (types of microbodies) into succinate, then malate, and finally into carbohydrates via the glycoxylate pathway ( 1) . Norton and Harris (60) showed a gradual increase in lipid content of developing seed until maturity when it accounted for 45% of the dry matter. vfiriation of Patty Acid Composition of Oilseed Variation in the fatty acid content of seeds is caused by JDCD‘th genetic and environmental factors (6,12,73) . The major fatty acid (about 40%) of B. napus cultivar ‘Regina‘ is erucic aCid 22:1, followed by oleic 18:1, whereas in the cultivar ‘Oro‘ only a trace of erucic acid is present and oleic acid predominates (6) . Changes in fatty acid proportions can also be affected by the temperature during seed development. Canvin (12} 1 ‘W‘ Hmw RS 4 mile diffs shove linoi 19 (12) found that the erucic acid content of the rapeseed cv. ‘Nugget' grown at 15 C was 40%, but at 25 C, was only 25%. However, the oleic acid content at 15 C was 23% but at 25 C was 40%. Variation in fatty acid content can also be influenced by the position of the developing seeds within the inflorescence. Zimmerman and Picks (78) reported that at different locations on the plant, sunflower (H. annuus) seeds showed small but significant differences in the proportions of linoleic, palmitic and oleic acids in their oil. Seed Quality Seed quality is a collective term for the condition of seed including viability, vigor, physical appearance, genetic homogeneity, uniformity and field performance (48) . Although the standard germination test is the most common uI‘Aiversal test for seed quality, it does not always provide adequate information about the physiological and biochemical s‘tatus of the seed (9) that translate into field performance. cOnsequently, there is a need for the development of appropriate vigor tests that can be used for a more composite value of the seed. For example, vigor tests which measure various aspect of seed deterioration and/or genetic deficiency may be used to provide further information about seed bQl-formance potential (7) . 20 Generally, seeds have the ability to germinate before complete maturity (4), however maximum quality is usually not attained until physiological maturity (17,25,75) . Germination for seed testing purposes is defined as the emergence and development of the essential structures from the embryo and the ability to produce a normal plant under favorable conditions (42). However, environmental conditions in the field are usually less than optimum, thus, only the most vigorous seeds often germinate under actual field conditions. The Association of Official Seed Analyst (7) therefore, has defined seed vigor as the group of properties of seed which determine the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions. Abdul-Saki (2) considered vigor primarily a function of the rate of seedling emergence. Thus, vigor tests provide information to farmers and seed growers to help predict the potential of a seed lot when planted in a field under a broad range of conditions. Vigor tests may also be used to separate between lots similar in germination percentage and different in vigor (e.g., speed of germination and seedling growth rate). MaDonald and Wilson (52) classified vigor tests into three categories: 1) physical, to evaluate seed size, weight, density and.the internal anatomy of seeds (e.g., x-ray test); 21 2) physiological, such as germination, speed of germination, seedling growth rate, cold test and accelerated aging test; and 3) biochemical, such as tetrazolium, ATP, glutamic acid decarboxylase activity and conductivity test. rectors Affecting Seed Quality Seed vigor is influenced both by genetic and environmental conditions. Factors such as heterosis, disease resistance, susceptibility to mechanical damage, seed color, seed size and chemical composition may influence the expression of seed vigor (17,25,48,51,66) . Vigor may vary among cultivars of the same species and even among individual seeds in the same lot (24). Environmental conditions during seed development and maturation, such as temperature and water stress and lack of nutrients also influence seed quality ( 25) . Seed storage under poor conditions (e.g. , high temperature and relative humidity) accelerate seed deterioration and loss of vigor (24,25). Loss of vigor is accompanied by physiological and biochemical changes. Increase of metabolite leachates as a result of loss of membrane integrity, lipid autoxidation and enzyme degradation are associated with vigor loss (2,17,24, 66). Wahab and Burris (75) observed reduction in respiration rate and decrease in accumulation of sugars and amino acids as well as total fresh and dry matter in low quality soybean 22 seeds. Other symptoms of seed deterioration include gradual color changes and decrease in specific gravity, probably as a result of loss of food reserves, without a corresponding decrease in seed size (24). DLATEIUAliiABHIRNETEKNDS Four winter canola cultivars (B. napus), including Cascade, an early maturing variety, Lirabon,a medium maturing variety, Glacier and Crystal, late maturing varieties, and two Spring culivars, Topas and Iestar, were used in this study. Planting, Management and Bxperimental Design Seed of each cultivar’was planted at a rate of 5 lbs/acre in a completely randomized block design with four 5-row replications 20 feet long and 3 feet wide. 125 lbs/acre of Spring applied nitrogen was used for both winter and spring canola. In the first year, the winter cultivars were planted Sept. 7, 1988 and harvested on July 24, 1989. The spring cultivars were planted April 26, 1989 and harvested Aug. 26, 1989. In the second year, the winter cultivars were planted Sept. 5, 1989 and harvested July 22, 1990. The spring cultivars were planted April 26 and harvested Aug. 16, 1990. Sampling Procedure Pod sampling was started 7-10 days after plants reached 50% flowering, when 50% of the plants in a plot had at least one open flower. Twenty-five plants from each replication were randomly chosen and tagged. An area between branches 3 23 24 and 6 of each selected plant was marked. On each sampling date, about fifty randomly selected pods from the marked areas were measured for different parameters. Similarity in size and appearance from the sampling areas was considered and all pods which showed pronounced insect damage or other disorders were discarded. The sampled pods were immediately placed inside plastic ziploc bags and kept at 5 C until processing. Samples were collected at weekly intervals until complete harvesting maturity, at which time the pod color had turned brown and seed reached about 10% or less moisture content. Morphological and Physiological Assessments Ten randomly selected pods from each sample in 1989 and five in 1990 were collected and measured for different parameters after removing the pedicel. The pods were opened and the number of seeds per pod recorded. Both seeds and empty pods were placed into separate, labeled aluminum cups and weighed to determine their fresh weight. Then, they were dried in an oven at 65 C for 48 hours, cooled and weighed again to determine their dry weight and moisture content. The dry samples were then retained in glass vials for analysis of lipids, proteins and mineral nutrients. The remaining pods in each sample were carefully opened and the seeds removed and allowed to air dry in the laboratory 25 for a week. The seeds were then stored in plastic bags at 5 C for further germination and vigor evaluation. Percent seed.moisture content (MC) was determined by the following formula: wt. before drying (g) - wt. after drying (g) 8 MC II x 100 wt. before drying (g) The following measurements were taken: pod color, pod length, pod width, number of seeds per pod, pod fresh weight, pod dry weight, seed fresh weight, seed dry weight, seed color, seed moisture content and 100 seed weight (at 5% MC). The mean of 10 pods of each sample in the first year and five pods in the second year was calculated for all above measurements. Seed Lipid, Protein and Mineral Nutrient Content Lipid Content The lipid content and the fatty acid profile of seeds from each sampling date of all cultivars were assayed using the following method (58,65): 1. The dried seed samples (5% MC) from the different growth stages were ground to pass through a 40 mm mesh sieve. 2. Fifty mg of each sample were added to the following 26 chemicals to make to a total of 3 ml. 800 ul Toluene 100 ul standard (10 mg/l 17:0) 2.1 ml Trichloride reagent (BCl3/MeOH) After mixing well, the N gas was allowed to pass through for 5 seconds to prevent the oxidation of the fatty acids. The mixture was heated at 90 C for one hour at which time the color of the solution turned brownish. After 2 ml distilled water and 4 ml hexane were added, the solution*was shaken well and centrifuged at 6000 r/m for 10 minutes. The upper layer, composed of fatty acid methyl ester, was saved. Two ml of hexane were added to the bottom layer and centrifuged.for 5 minutes. The upper layer was added to the previous one. The process was repeated and the bottom layer discarded. The fatty acids were then extracted. The extraction was dried by passing nitrogen gas through it which allowed the hexane to evaporate. One ul of the methyl ester extraction was injected to HP5890 GC and the readings were recorded. Protein Content (Total Nitrogen) The protein content of seeds (at 5% MC) from each sampling date was measured using the Kjeldahl method outlined by Bremner (10). The dried seed samples from each maturity 27 stage were ground to pass through a 40 mm mesh sieve. 250 mg from each maturity stage of each cultivar was digested for 2 hours with 3 ml of concentrated H280, and 1.3 9 catalyst (a 100:10:1 mixture of K,SO,:CuSO,.5H20:Se) on digestion racks. Each sample was then diluted with 10 ml distilled water as it cooled. 15 ml of 10 N NaOH were added to each flask to completely neutralize the acid. Distillation was performed immediately and the distillate collected in a flask containing 5 ml of 2% 111,803 and methyl purple indicator. The samples were then titrated with H280, and the percent of total nitrogen calculated by the following formula: [14 x N x titration volume (ml) - titration blank (ml)] x 100 Sample weight (mg) where N is the normality of the acid and 14 is the atomic weight of nitrogen. The total nitrogen was multiplied by a factor of 6.25 to obtain the protein percent. Mineral Nutrient Content The total spectrum of mineral elements of each seed sample from each sampling date (2 replications of each) was determined using the following method (64): 28 Five hundred mg of plant tissue from each sample was weighed in a clean, numbered crucible and covered immediately. One standard (horticultural leaf material) and one blank control sample was included for every 25 samples of experimental material. The covered crucibles were ashed in a muffle furnace for five hours at 500 C. After cooling, the crucibles were transferred on trays to the hood for digestion. Twenty-five ml of digestion solution (3N HNO3 in 1000 ppm LiCl) was added to each crucible. After a one-hour incubation, the solution was filtered into labeled vials and capped. The samples were then ready for analysis using a D.C. Plasma Emission Spectrophotometer to determine K, P, Ca, Mg, Mn, Zn, Fe, Cu, Al and 8 content. 828D QUALITY Standard germination, cold and accelerated aging tests were performed to determine the viability and vigor of seed samples from different sampling dates of both winter and spring cultivars. Preliminary tests were conducted to determine the optimum temperatures and time required for the standard germination test, the cold 'test and accelerated aging test (AAT). The two winter cultivars, Glacier and Lirabon, and the spring 29 cultivar, Westar were used in these tests. The following procedure was used: Standard Germination: Four loo-seed replications of each cultivar were germinated at 20, 25, 22 and 20/30 C (16-8 hours, respectively) for 7 days. Moistened blotter papers were used as substrate. Only normal seedlings were counted. Cold Test: Four loo-seed replications of each cultivars were germinated at two temperatures, 5 and 10 C for 3, 5, 7 and 10 days, transferred to 22 C for 5 days and germinated as mentioned above. Accelerated Aging Test: The wire-mesh tray method described by McDonald and.Phaneendranath (53) was used, Seeds were aged at 36 and 42 C for periods of 48, 72 and 120 hours. Five hundred seeds from each sampling date of each cultivar were placed on 10 x 10 x 3 cm copper wire mesh tray, the tray was then placed in an 11 x 11 x 3.5 cm plastic box containing 2 cm water (about 100 ml) above the bottom of the box. Following incubation, seeds were germinated at 22 C for seven days as described above. The percent of normal seedlings was recorded. Second Year Study The same field and laboratory work was repeated in the second year for both winter and spring cultivars. 30 Statistical Analysis Analysis of variance, least significant difference, Duncan's multiple range test and simple correlation coefficient were used to analyze the data using the statistical package MSTAT (Michigan State University, East Lansing). IRESUIJS In 1989, the spring cultivars reached the flowering stage 55 and 58 days after planting for Westar and Topas, respectively. In 1990, plants reached the flowering stage 49 and 52 days after planting for Westar and Topas, respectively. In 1989, the‘winter'cultivars.reached.the flowering stage 247, 250, 252, 253 days after planting for Cascade, Lirabon, Crystal and Glacier, respectively. In 1990, the winter cultivars reached the flowering stage 245, 248, 251, 250 days after planting for Cascade, Lirabon, Crystal and Glacier, respectively. Development and Maturity of Pod and Seed Pod Length After fertilization and pod formation, pods of all cultivars in both years continued to grow in length and reached their full length after two to three weeks (Tables 3-6 and Fig. 1,2). In 1990, cultivars and date of sampling (stage of fruit growth) had a significant effect on the length of pods for both spring and winter cultivars (Tables 1 and 2). However, the interaction between cultivars and date of sampling was not significant, indicating that the pattern of 31 32 pod growth was similar in all cultivars. In 1989, only date of sampling significantly affected pod length of both spring and winter cultivars. Both.cultivars and the interaction between cultivars and date of sampling were significant for the winter but not for the spring cultivars (Tables 1 and 2). The results of spring and winter cultivars . in both seasons showed no significant differences in pod length after 21 days of pod formation until harvest, with a few exceptions (Tables 3-6 and Fig. 1,2). In 1989, the pod length of the spring cultivar Topas was 2.6 cm at the first sampling date and 4.7 cm after one week. After two weeks it had reached 6.4 cm and at harvest was 7.0 cm (Table 3). Slower growth occurred after the third week of pod formation (Tables 3-6 and Fig. 1,2). Similar patterns of pod length growth were obtained for the spring cultivars in 1990 and for the winter cultivars in both years (Table 4-6). The pod length of the winter cultivar Lirabon in 1989 was 2.4 cm at the first sampling date, 4.9 cm a week later, 7.1 cm after two weeks, 8.2 cm at the third week and 8.1 cm at harvesting time (Table 5). The pod length of the spring cultivar Westar was 3.2 cm at the first sampling date in 1990, 5.6 cm after one week, 6.6 cm at the second week, 7.3 cm at the third week and 7.4 cm at harvesting time (Table 4). 33 In 1990, the pod length of the winter cultivar Cascade ranged from 3.3 cm at the first sampling date to 6.4 cm two weeks later and was 6.9 cm at harvest (Table 6). 34 Table 1. Analysis of variance for the factors affecting pericarp and seed traits during different times of fruit growth of two spring canola cultivars in 1989 and 1990. Effect: Cultivar Time of measurement Interaction Trait (C) of fruit growth (T) (C) x (T) 89 90 89 90 89 9O Pod length ns ** ** ** ns ns Pod width ns * ** ** ns ** Pericarp fr. wt. ** ** ** ** ** ns Pericarp dry wt. * ns ** ** ** ns Seeds/pod ** ns ns ns ns ns Seed fr. wt. ** ns ** ** ns ns Seed dry wt. ns ns ** ** ns * Seed M.C. ** ns ** ** * ns loo-seed weight ** ns ** ** ** ** Lipid content ns ns ** ** ns ns Protein content ** ** ** ** * ** Germination test ** ** ** ** ** ** Cold test ** ns ** ** ** ** A. aging test ns ns ** ** ** ** *,** Indicate significance at 0.05 and 0.01 level of probability, respectively according to the F-test. ns Indicates no significance at 0.05 level of probability according to the F-test. 35 Table 2. Analysis of variance for the factors affecting pericarp and seed traits during different times of fruit growth of four winter canola cultivars in 1989 and 1990. Effect: Cultivar Time of measurement Interaction Trait (C) of fruit growth (T) (C) x (T) 89 90 89 90 89 90 Pod length ** ** ** ** ** ns pod width ns ** at it es ** Pericarp fr, wt, ** ** ** ** as ** Pericarp dry wt. ** ** ** ** es ** Seeds/pod ** ns ** ns ns ns Seed fr. wt. ** ** ** ** nS ** Seed dry wt, ** ** ** ** n3 ** Seed M.C. ns ** ** ** ns * loo—seed weight as *s ** es ** ** Lipid content ** ns ** ** ns ns Protein content ** ** ** ** ** ** Germination test ** ** ** ** ** ** Cold test *4 ** *e ** ** ** A. aging test ** ** ** es ** ** *,** Indicate significance at 0.05 and 0.01 level of probability, respectively according to the F-test. ns Indicates no significance at 0.05 level of probability according to the F-test. 36 Table 3. Pod length of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar cm 0 ** 2.6C* 2.8d 1 4.7b 4.6c 2 6.4a 6.4b 3 6.9a 7.1ab 4 7.0a 7.1ab 5 6.9a 7.2a 6 6.8a 6.9ab 7 7.0a 6.9ab * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** 7-8 is the first date of pod formation and 8-26 is the harvest time. Table 4. Pod length of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar cm 0 ** 2.9d* 3.2d‘ 1 4.6c 5.6c 2 6.3b 6.6b 3 6.9ab 7.3ab 4 7.0a 7.0ab 5 7.0ab 7.2ab 6 7.1a 7.3ab 7 6.8ab 7.4a * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** 7-1 is the first date of pod formation and 8-16 is the harvest time. 37 Table 5. Pod length of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod --—-- formation Cascade Crystal Glacier Lirabon cm 0 ** 3.2e* 1.9e 2.1e 2.4f l 5.4d 3.7d 4.5d 4.9e 2 7.2bc 6.3c 6.2c 7.1d 3 8.0a 8.4a 8.58 8.2bc 4 7.8ab 7.9ab 7.8b 8.9a 5 7.5abc 7.8ab 7.5b 7.8bcd 6 7.8ab 7.4b 7.7b 8.3b 7 7.6abc 7.7ab 7.9b 7.5cd 8 7.2bc 7.4b 7.1b 8.0bC * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** 5-31 is the first date of pod formation and 7-24 is the harvest time. Table 6. Pod length of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod -———-—— formation Cascade Crystal Glacier Lirabon cm 0 ** 3.3d* 2.5d 2.9d 2.7d 1 5.4a 4.9c 4.5C 5.2c 2 6.4b 6.lb 6.5b 6.7b 3 7.1ab 7.1a 7.1a 7.5a 4 7.0ab 7.2a 7.3a 7.3ab 5 7.1ab 6.9a 7.2a 7.6a 6 7.2a 7.0a 7.3a 7.5a 7 7.0ab 6.8a 7.4a 7.1ab 8 6.9ab 6.8a 7.0ab 7.3ab * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** 5-31 is the first date of pod formation and 7-22 is the harvest time. 38 Pod width (mm/pod) 8 ,: : ,:' 5 SiEiifSDj'sxyso-h 4 o .2- 7 E —3 8 .c 4- ,2, ..... .2 _. ’2 ".21” ' ‘ 1‘71 o *Pod length *Pod width " o 0 1 2 3 4 5 6 7 8 Time after pod formation (weeks) Figure 1. Pod length and width of winter canola cultivar Crystal during different times at maturity in 1990. 8 5 , LSD (5%)=0.7 4 ~66- o & 5, —3 .c 4- ‘6 5 ~2 8 m 2_ LSD (5%)=o.4 A -1 0 *Pod “39$ *Pod width 0 0 1 2 3 4 5 6 7 Time alter pod formation (weeks) Figure 2. Pod length and width of spring canola cultivar Topas during different times of maturity in 1990. Pod width (mm/pod) 39 Pod lidth and Fresh and Dry Weight Cultivars, date of sampling and the interaction between them had significant effects on pod width and fresh and dry matter of the winter cultivars in both years (Table 2). In 1989, both pod fresh weight and dry matter were significantly affected by cultivar, date of sampling (stage of pod development) and the interaction between them, but pod width was only affected by date of sampling. In 1990, pod width and both fresh weight and dry matter were significantly affected by sampling date, however, the interaction between cultivar and sampling date was not significant in dry matter (Table 1). Pod width increased gradually during the first four weeks and reached maximum levels about 27-35 days after formation (Tables 7-10 and Fig. 1,2). However, this period varied with cultivar and weather conditions during the growing season. Pods reached maximum fresh and dry weight at about the same time, or one week after reaching maximum width at about 35 days (Tables 7-18). Thereafter, a significant gradual drop in pod fresh weight and dry matter occurred prior to harvest (Tables 11-18). In 1990, the spring cultivar Topas had a pod width of 1.03 mm at the first sampling date and 2.28 mm after two weeks and 3.53 mm at the third week. Maximum width was 4O attained (4.05 mm) 4 weeks after pod formation, however, at harvest time, the width was 3.65 m (Table 8 and Fig. 2). Pericarp reached maximum dry weight one week before the peak of seed dry weight (Tables 14,31 and Fig. 6). The dry weight of the pericarp dropped significantly as the seed matured However, the dry weight of the seeds did not drop significantly below peak levels (Tables 14,31 and Fig. 6). In the same year (1990), Westar reached maximum width of 4.25 mm 5 weeks after formation and was 3.73 mm wide at harvest (Table 8). Pericarp reached maximum dry weight one week before the peak of seed dry weight (Tables 14 and 31). In 1989, the winter cultivar Cascade attained maximum width of 4.0 mm 4 weeks after pod formation and was 3.3 mm at harvest. Maximum dry weight occurred one week after maximum pod width. Maximum seed dry matter occurred one week later (Tables 9,15 and 30). In 1990, the winter cultivar Lirabon reached maximum pod width (4.0 mm) 4 weeks after pod formation and maximum pericarp dry matter one week later (Tables 10 and 18). Seeds reached maximum dry matter one week after peak pod dry matter (Table 37). All spring and winter cultivars in both seasons showed a similar pattern of pod width and pericarp fresh and dry matter to those cited above (Tables 7-18). 41 Table 7. Pod width of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar mm 0 1.2e* 1.1e 1 1.6d 1.7d 2 2.7c 3.0c 3 3.8ab 3.8ab 4 3.9a 4.0a 5 3.7ab 3.9ab 6 3.6ab 3.7ab 7 3.5b 3.6b * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 8. Pod width of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar mm 0 1.03e* 1.153 1 1.94d 1.57d 2 2.28C 2.68C 3 3.53b 3.63b 4 4.05a 3.83b 5 3.80ab 4.25a 6 3.58b 3.90b 7 3.65b 3.73b * means followed by the same letter within a column are not significantly different at 0.05 probability level. 42 Table 9. Pod width of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod — formation Cascade Crystal Glacier Lirabon mm 0 1.4f* 1.0f 1.2f 1.3e 1 2.1e 1.9e 1.8e 2.0d 2 3.2d 2.7d 3.0d 3.2c 3 3.8ab 3.6b 3.7bc 3.6b 4 4.0a 4.0a 4.3a 4.1a 5 3.7ab 4.2a 4.2a 4.2a 6 3.5de 3.5b 3.8b 3.7b 7 3.6bc 3.3bc 3.4c 3.5bc 8 3.3cd 3.1c 3.5bc 3.2c * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 10. Pod width of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod . _— formation Cascade Crystal Glacier Lirabon mm 0 1.2f* 1.0f 1.2e 1.1e l 1.7e 1.8e 1.8d 1.9d 2 3.2d 3.4d 3.7c 3.30 3 3.7ab 3.9bc 3.9abc 3.6b 4 3.6abc 4.3a 4.1a 4.0a 5 3.8a 4.1ab 4.1a 3.9a 6 3.6abc 3.9bc 3.9ab 3.7ab 7 3.4bc 3.8c 3.8bc 3.5b 8 3.4cd 3.7c 3.8bc 3.3c * means followed by the same letter within a column are not significantly different at 0.05 probability level. 43 Table 11. Pericarp fresh weight of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -——————— formation Topas Westar ——— mg/pericarp -—— O ** 50f* 60e 1 ** 87f 99s 2 189d 145d 3 247C 333D 4 428a 505a 5 323b 361b 6 229cd 253c 7 1422 159d * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** Whole fruit (pericarp and seeds). Table 12. Pericarp dry weight of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -—————- formation Topas Westar —- mg /pericarp — o ** 10f* 109 1 ** 25f 27g 2 70e 47f 3 103d 119a 4 205a 235a 5 198a 212b 6 179b 184C 7 129C 138d * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** Whole fruit (pericarp and seeds). 44 Table 13. Pericarp fresh weight of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar -—— mg/pericarp ——— 0 ** 38f* 55g 1 ** 74e 92f 2 145d 173d 3 211C 288C 4 379a 401a 5 323b 361b 6 236C 269C 7 141d 136C * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** Whole fruit (pericarp and seeds). Table 14. Pericarp dry weight of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar mg/pericarp 0 ** 52* 9e 1 ** lSde 21de 2 44d 52d 3 97c 94c 4 l87ab 166b 5 205a 218a 6 156b 188ab 7 122C 115C * :gzns followed by the same letter within a column are signi 1cant1y different at 0.05 probabilit level. ** Whole fruit (pericarp and seeds). y ' 45 Table 15. Pericarp fresh weight of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -—————-— formation Cascade Crystal Glacier Lirabon mg/pericarp 0 ** 60g* 50h 45f 60e 1 ** 178e lSOf 162d 129d 2 ** 286C 314d 269C 233C 3 308bc 362C 327b 280b 4 324b 424a 412a 378a 5 359a 393b 424a 399a 6 209d 2318 261C 234C 7 l34f 138f 125e 133d 3 85g 90g 105e 109d * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** Whole fruit (pericarp and seeds). Table 16. Pericarp dry weight of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod —— formation Cascade Crystal Glacier Lirabon mg/pericarp O ** 10f* 7d 5f 10f 1 ** 32e 22d 24e 30s 2 ** 64d 69c 74d 67d 3 79cd 99b 98c 91c 4 91c 141a 134b 155a 5 145a 149a 169a 170a 6 138a 142a 161a 152b 7 109b lllb 106C 1070 8 75cd 82c 94c 97c * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** Whole fruit (pericarp and seeds). 46 Table 17. Pericarp fresh weight of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— —.—— formation Cascade Crystal Glacier Lirabon mg/pericarp 0 ** 709* 509 55g 59g 1 ** 138f 125f 133f 137f 2 ** 215d 242d 255d 250d 3 321b 358b 386C 291C 4 405a 431a 462D 351b 5 428a 444a 520a 461a 6 2890 301c 359c 333b 7 185a 242d 258d 220s 8 122f 168s 19le 156f * means followed by the same letter within a column are not significantly different at 0.05 probability level. ** Whole fruit (pericarp and seeds). Table 18. Pericarp dry weight of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod ——————— '-——————— -—————— -———-—-— formation Cascade Crystal Glacier Lirabon mg/pericarp 0 ** 12f* 10f 8f 109 1 ** 328 30s 258 33f 2 ** 64d 76d 68d 77e 3 108C 142C 129C 99d 4 152b 189ab 173b 138C 5 184a 205a 212a 228a 6 175a 199a 209a 216a 7 152b 175b 196a 181b 8 109C 148C 169b 141C * means followed by the same letter within a column are not significantly different at 0. 05 probability level. ** Whole fruit (pericarp and seeds). 47 100-seed Weight and Number of Seeds per Pod Date of sampling had no significant effect on number of seeds per pod for any cultivar in either growing season. The only exception was for the winter cultivars in 1989, in which the number of seeds per pod was significantly affected by sampling dates (Tables 1 and 2) . No significant differences in number of seeds per pod occurred between sampling dates in 1989 for Cascade (Table 23). Twenty-six seeds per pod occurred for Crystal at the last sampling date, compared to 31 seeds three weeks earlier. The largest difference occurred in Glacier, in which 25 seeds occurred at the first sampling date and 30 three weeks later (Table 23) . Generally, the number of seeds per pod was established during the first week of pod development (Tables 19,21,23,25 and Fig. 3,4). In 1989, the number of seeds per pod ranged from 23 to 25 for Topas and from 23 to 26 for Westar (Table 19). In 1990, the range in seed number was 21 to 25 for Topas, 22 to 24 for Westar (Table 21 and Fig. 4), 24 to 27 for Cascade, 25 to 30 for Crystal and 26 to 29 for Glacier (Tables 23,25 and Fig. 3,4). No significant differences occurred in number of seeds per pod between the first sampling date and at harvest for Lirabon in 1990, ranging from 26 to 29 seeds (Table 25). A gradually increase in loo-seed weight (at 5% moisture content) occurred during the first weeks of seed development, 48 reached a maximum at physiological maturity, then gradually (but not significantly) decreased until harvesting maturity (Tables 20,22,24,26 and Fig. 3,4). The weight of 100 seeds at the first sampling date (three weeks after pod formation) for Topas in 1989 was 195 mg. The biggest increase in weight occurred a week later when the weight reached 318 mg and reached a maximum of 367 mg six weeks after pod formation and was 365 mg at harvest (Table 20). In 1990, the loo-seed weight of Westar at the first sampling date (2 weeks after pod formation) was 98 mg. Again, the largest increase occurred during the second sampling week (3 weeks after pod formation) when the weight reached 217 mg. By the fourth week after pod formation, the weight had increased to 300 mg, 368 mg at PM (6 weeks after pod formation) and dropped to 355 at harvest (Table 22). loo-seed weight of Lirabon at the first sampling date (3 weeks after pod formation) in 1990 was 220 mg, at PM (6 weeks after pod formation) was 410 mg and then decreased slightly to 390 mg by the time of harvest (Table 26). 49 Table 19. Number of seeds per pod of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar seeds/pod 1 23a* 25a 2 23a 26a 3 24a 26a 4 24a 24a 5 24a 24a 6 25a 23a 7 24a 24a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 20. loo-seed weight of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar ——— mg/100 seeds+ ——— 1956* 318b 367a 373a 3653 \lO‘U‘abU 257C 336b 389a 374a 3788 * means followed by the same letter within a column are not significantly different at 0.05 probability level. + seed moisture content 5% for all samples. 50 Table 21. NMmber of seeds per pod of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar seeds/pod 1 22b 24a 2 25a 24a 3 21b 24a 4 23ab 22a 5 24ab 23a 6 23ab 24a 7 25a 24a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 22. loo-seed weight of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod ———————— formation Topas Westar -—— mg/100 seeds+ -—— 2 77e 98f 3 240d 217e 4 285C 300d 5 345b 320C 6 355b 368a 7 368a 355b * means followed by the same letter within a column are not significantly different at 0.05 probability level. + seed moisture content 5% for all samples. 51 Table 23. Number of seeds per pod of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod —— —— —— formation Cascade Crystal Glacier Lirabon seeds/pod 2 268 27b 25b 28b 3 27a 288D 27ab 318b 4 30a 29ab 29ab 31ab 5 288 318 308 328 6 26a 28ab 27ab 29ab 7 26a 27ab 26ab 29ab 8 278 26b 27ab 28b * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 24. loo-seed weight of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod —— —— —— ———-— formation Cascade Crystal Glacier Lirabon mg/lOO seeds+ 3 220d* 2708 2608 2108 4 290C 320d 310d 280d 5 330b 390C 380C 340C 6 380a 430a 420a 410a 7 370a 4208b 400b 4008b 8 3808 410b 400b 390b * means followed by the same letter within a column are not significantly different at 0.05 probability level. + seed moisture content 5% for all samples. 52 Table 25. Number of seeds per pod of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— —— —— _— formation Cascade Crystal Glacier Lirabon seeds/pod 2 25a* 28a 27a 26a 3 24a 27a 27a 29a 4 27a 25a 28a 28a 5 258 278 298 268 6 258 308 278 298 7 268 308 278 27a 8 26a 27a 26a 25a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 26. loo-seed weight of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— -— —— -— formation Cascade Crystal Glacier Lirabon mg/100 seeds+ 3 230e 260d 270e 220e 4 300d 350c 360d 320d 5 370c 400b 400c 380C 6 410a 420a 430a 4108 7 390b 430a 420ab 400ab 8 400ab 420a 410bc 390bc * means followed by the same letter within a column are not significantly different at 0.05 probability level. + seed moisture content 5% for all samples. 53 500 35 1'? :.LSD::(5%)=5 ' . 30 '§4oo-) A . m ........ -25 A ° 13 ° 0 :300- _20 if c: _ . o E :7 ‘ . . f ' . 5 f,,200-;_~ . .g - , 15 2 a o 3 +10 ‘0 E 100- 2 a) :1 M -' 1‘ . V- ‘ 5211—5 o ' *100-:seedwt. 4.3m: perils.“ ,~isi3:ir:;;§ 0 Time alter pod formation (weeks) Figure 8. Seed per pod and loo-seed weight (at moisture content 596) of winter canola cultivar Crystal at different times of development and maturity in 1990. 400 25 7; i-l - :f 3' O 300-' X j i a: ... O '5 O O E, ' . - .i .‘15 & éZOO- too-seed wt 2 g E *Seedspergpodi m E100- ”, LSD (5%):12 '5 o 7 T T i T z ‘ o 1 2 3 4 5 6 7 Time after pod formation (weeks) Figure 4. Seed per pod and loo-seed weight (at moisture content 5%) of spring canola cultivar Topas at different times of development and maturity in 1990. 54 Seed Fresh weight, Dry Weight and Moisture Content At the very early stages of fruit growth, the moisture content of both pericarp and seeds was very high, whereas both fresh and dry matter weights were very low (Tables 27-38 and Fig. 5,6). At that stage, it was difficult to separate the seeds from the pod without causing damage to the seeds. The color of seeds at the very early stages of growth was transparent. With further development, they turned to light green and then darker green until at PM the pod color was a mixture of dark green and brownish green to light brown. Although fresh weight, dry weight and moisture content of both spring and winter cultivars were influenced by seed development stage, the pattern of effect varied among cultivars and seasons (Tables 1 and 2). Both fresh weight and dry weight increased gradually following seed formation at almost the same rate until reaching a maximum at PM. Fresh weight declined thereafter as a result of water loss and reached minimum levels at harvest. However, dry weight remained without significant reduction from PM until harvest (Tables 27-38 and Fig. 5,6). The first pod sample of the spring cultivars was collected July 8, although the first seeds could not be safely separated from the pods until July 29, 1989. The fresh weight of 100 seeds of Topas was 250 mg, the dry weight was 67 55 mg and the moisture content was 73.2%. Seed fresh weight reached a maximum at 663 mg about 5 weeks after pod formation and maximum dry weight at 467 mg (PM) occurred a week later and was 454 mg at harvest (Tables 27,28) . Seed moisture content at PM was 20.3% and 10.6 at harvest (Table 29). The greatest increase in both fresh and dry weight occurred during the third and the fourth weeks after pod formation. No significant change in seed dry weight of the same cultivar occurred during the last 2 weeks of seed maturity. It was 458 mg five weeks after pod formation, 467 mg at PM (6 weeks after pod formation) and 545 mg at harvest. Similar results occurred for Westar during the same season (Tables 27-29). In 1989, seeds of the winter cultivar Cascade reached maximum fresh weight at 639 mg/ 100 seeds five weeks after pod formation and maximum dry weight of 387 mg a week later, with a moisture content of 30.6% (Tables 33-35) . Dry weight remained without significant change during the last 3 weeks (Table 34). In 1990, the spring cultivar Westar attained maximum fresh weight (672 mg) five weeks after pod formation and reached maximum dry weight (475 mg) a week later with a moisture content of 23.5% (Tables 30-32). In the same year, the winter cultivar Glacier also attained maximum fresh matter weight five weeks after pod formation and reached maximum dry weight one week later at a moisture content of 31.4% (Tables 36-38). 56 Table 27. Seed fresh weight of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar -—— mg/100 seeds —-—- 3 250C* 2546 4 508b 6428b 5 6638 7218 6 586b 652b 7 508b 538C * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 28. Seed dry weight of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -——————— formation Topas Westar -—— mg/100 seeds ——— 3 67c* 73c 4 254b 288b 5 458a 500a 6 467a 509a 7 454a 479a * means followed by the same letter within a column are not significantly different at 0.05 probability level. 57 Table 29. Seed moisture content of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -—————— formation Topas Westar % 3 73.28* 71.38 4 50.0b 55.1b 5 30.9c 30.7c 6 20.3d 21.9d 7 10.68 11.08 * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 30. Seed fresh weight of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar -——— mg/100 seeds 2 70e* 75c 3 217d 172C 4 418C 499b 5 660ab 6728 6 722a 6218b 7 555bc 526b * means followed by the same letter within a column are not significantly different at 0.05 probability level. 58 Table 31. Seed dry weight of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— formation Topas Westar -———-mg/100 seeds ——— 2 13d* 17c 3 52d 44c 4 168c 219b 5 420b 442a 6 526a 475a 7 500a 471a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 32. Seed moisture content of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— formation Topas Westar % 2 80.48* 77.38 3 76.0ab 74.4a 4 59.8b 56.1b 5 36.4c 34.2c 6 27.18 23.5Cd 7 10.0d 10.5d * means followed by the same letter within a column are not significantly different at 0.05 probability level. 59 Table 33. Seed fresh weight of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod ——————— ——-————— formation Cascade Crystal Glacier Lirabon mg/100 seeds 2 129d* 1438 1508 1028 3 396b 449C 433b 3890 4 568b 598b 6158 573b 5 6398 7118 7238 6988 6 5588 601b 598D 579b 7 447C 482Cd 493C 479C 8 393C 429d 418d 426d * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 34. Seed dry weight of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -———-—— formation Cascade Crystal Glacier Lirabon mg/100 seeds 2 32d 39d 46e 29e 3 141C 180c 169d 155d 4 271b 289b 304c 269C 5 349a 3898 399D 362D 6 387a 400a 403a 4118 7 379a 396a 399ab 400ab 8 365a 390a 374b 388b * means followed by the same letter within a column are not significantly different at 0.05 probability level. 60 Table 35. Seed moisture content of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod ———————— formation Cascade Crystal Glacier Lirabon % 2 75.28* 73.08 69.08 71.68 3 64.4b 60.0b 61.0b 60.2b 4 52.3c 51.7c 50.6c 53.1c 5 45.4d 45.30 44.80 48.10 6 30.68 33.4d 32.6d 29.0d 7 15.2f 17.88 19.18 16.58 8 7.19 9.1: 10.5: 8.9f * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 36. Seed fresh weight of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —————-—— formation Cascade Crystal Glacier Lirabon mg 3 434b0* 397d 3750d 417b 4 591b 4950 5218b 546b 5 7028 676a 689a 6328 6 582D 595D 577b0 581b 7 4730d 5140 4930 4920 8 389d 439d 414d 3990 * means followed by the same letter within a Column are not significantly different at 0.05 probability level. 61 Table 37. Seed dry weight of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— —— formation Cascade Crystal Glacier Lirabon mg/100 seeds 3 1390* 121d 1280 1320 4 261b 2390 250b 248b 5 3718 3678 389a 3398 6 3858 4188 3968 3728 7 3778 4148b 3828 3668b 8 3668 395b 3748 3518b * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 38. Seed moisture content of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Cascade Crystal Glacier Lirabon 3 68.08* 69.58 65.98 68.38 4 55.8b 51.7b 52.0b 54.6b 5 47.2b 45.7c 43.5c 46.4b 6 33.80 29.7d 31.4d 36.00 7 20.3d 19.58 22.58 25.6d 8 7.58 10.0f 9.7f 12.08 * means followed by the same letter within a column are not significantly different at 0.05 probability level. ii 8 i 5? *~ g g a? s i Seed moisture (96) 5? Pericarp and seed dry wt. (mg/pod) ILSD (5%):14 5 o I ‘T H T i I I H i. 14":1 o o 1 2 3 4 5 5 7 a Time after pod formation (weeks) Figure 5. Pericarp and seed dry weight and seed moisture content of winter canola cultivar Crystal at different times of development and maturity in 1990. re 8 100 LSD (5%) = 13 'LSD.{5%) 835-80 N ? i *Pericarpdrywt. *Seed drywt .Seed MC ,-_4o Seed moisture (96) ~20 L80 (5%) = 11 Pericarp and seed dry wt. (mg/pod) 0 1 2 3 4 5 6 7 Time after pod formation (weeks) Figure 6. Pericarp and seed dry weight and seed moisture content of spring canola cultivar Topas at different times of development and maturity in 1990. a 400 j. a" .8. -‘ .- 7:; 4 E3001. f E r: 1 as a ,2 fig "g 2°° was: a 7f 7355}:- 0100 ILight reen DjarR Brownish Light Brown to green 9 green - green brown black Seed color Figure 7. Relationship between seed dry weight and seed color §§§ Dry weight (mg/100 seeds) '3‘ I i i r i 1 US” green Dark Brownish Greenish USN Brown to at different times of seed development and maturity of two spring canola cultivars in 1990. Cascade Crystal Physiological maturity r=0.88" 9"." groan ng'Gl't brown brown .31le Seed color Figure 8. Relationship between seed dry weight and seed color at different times of seed development and maturity of four winter canola cultivars in 1989. 64 Morphological and Physiological Markers for Physiological and Harvest Maturity At physiological maturity (PM), pod color was yellowish- green or beginning to turn yellow and contained seeds with colors ranging from dark green to brownish-green and light brown. Seeds were firm but not hard and could be marked with a finger nail. Moisture content averaged 20.3-36%. Seeds had attained maximum dry matter content which remained unchanged until harvest (Tables 27-38 and Fig. 5). At harvest maturity (HM), pod color was completely yellow to light brown and the seeds were brown or dark brown to black depending on the cultivar. The seed moisture content at this stage was near 10% and seeds were hard. Seeds can be rattled inside the pod at this stage (Fig. 7,8) Nutrient Accumulation in Seeds Lipids The lipid content of seed was significantly affected by the stage of development for all cultivars in both growing seasons (Tables 1 and 2). However, neither cultivars nor the interaction between them and the different seed development stages significantly affected seed lipid content of the spring cultivars in either year, showing that the pattern of lipid 65 accumulation is similar during the various times of seed growth for both spring cultivars. Both cultivars and times of seed development significantly affected lipid content of winter cultivars in 1989. In 1990, only times of seed development and maturation significantly affected the lipid content, however the interaction between cultivars and stage of growth was not significant during either year (Tables.1 and 2) . Lipid content (ng/seed) and concentration (mg/ 100 mg fresh weight) gradually increased during the first weeks of seed development, reaching a maximum at physiological maturity or shortly after, depending on cultivars and weather conditions during the.growing season (Tables 39-46 and.Fig. 9- 12) . However, no significant differences occurred in lipid content during the last three weeks before harvest for the winter cultivars in 1990 and the last 2 weeks in 1989 (Tables 45,46). Also, the lipid content of the spring cultivars did not significantly differ during the last 2 weeks before harvest in either 1989 or 1990 (Tables 43,44). Lipid concentration of winter cultivars did not significantly change the last three weeks before harvest in 1989 and the last four weeks in 1990. Also, the lipid concentration of the spring cultivars did not significantly change during the last three weeks before harvest in 1989 and the last two weeks in 1990, probably because of the relatively short period of seed development and maturity in 1990 (Tables 39-42 and Fig. 12). 66 During early stages of fruit growth, lipid concentration of the first seeds in 1989 (one week after pod formation) was as low as 3.9% and 4.5% for Westar and Topas, respectively (Table 39). The largest increase in lipid accumulation always occurred in the first four weeks after pod formation and then remain relatively stable thereafter until harvest (Tables 43- 46 and Fig. 11). The lipid content of Topas in 1989 increased from.200 ng/seed.three weeks after pod formation to 960, 1790, 1900, and 1890 ng/seed in successive weeks. Westar had a similar pattern of lipid accumulation during the same year (Table 43). The first seed sampled from Westar and Topas in 1990 had higher lipid concentration than those in 1989, at 24.8 and 23.3%, respectively, probably due to the lateness in collecting seeds during that season (Table 40). In 1990, Westar had a lipid concentration of 24.8% two weeks after pod formation which gradual but significant increased during the next two weeks. However, no increase occurred during the last two weeks. Topas showed a similar pattern of lipid concentration during the same growing season (Table 40). The lipid concentration of the first samples collected in 1990 (one week after pod formation) from the winter cultivars was 15.3%, 8.1%, 10.3% and 9.6% for Cascade, Crystal, Glacier and Lirabon, respectively (Table 42). Lipid concentration of 67 the winter cultivar Lirabon in 1989 for samples drawn 3,4 and 5 weeks after pod formation were 25.8%, 34.0% and 36.3%. However, it.did not change significantly during the last three weeks before harvest (Table 41). 68 Table 39. Lipid concentration in seed tissues of two spring canola cultivars during different times of seed development and maturity .18 1989 weeks Cultivar after pod -—————-— formation Topas Westar — % Lipid* 1 4.5d** 3.9d 2 22.90 26.30 3 27.9b 30.8b 4 36.18 39.18 5 37.2a 39.78 6 38.78 38.68 7 39.58 38.68 ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content 5% at the time of analysis for all samples. Table 40. Lipid concentration in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod ———————- formation Topas Westar % Lipid* 2~ 23.3d** 24.8d 3 31.3c 32.7c 4 33.9b 36.7b 5 39.58 40.28 6 40.48 38.98 7 40.38 40.48 ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was at the time of analysis for all samples. 69 Table 41. Lipid concentration.in.seed tissues of four'winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -—-————— formation Cascade Crystal Glacier Lirabon % Lipid* 5 3 29.8c** 31.3d 32.5c 25.8c 4 33.8b 34.50 35.1b0 34.0b 5 36.88b 36.7b0 37.88b 36.38b 6 39.78 38.28b 40.88 38.88 7 39.98 40.38 40.08 38.38 8 38.98 39.4ab 39.48 38.78 * means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content 5% at the time of analysis for all samples. Table 42. Lipid concentration in seed tissues of four‘winter canola cultivars during different times of seed development and maturity in 1990 a weeks Cultivar after pod formation Cascade Crystal Glacier Lirabon % Lipid* 1 15.3d** 8.1d 10.3f 9.6d 2 21.60 15.40 22.08 18.60 3 29.9b 27.2b 30.8d 30.4b 4 37.08 37.08 33.90d 36.98 5 38.88 39.18 41.58b 38.28 6 41.18 40.28 43.18 38.68 7 39.48 39.28 39.98b 40.98 8 38.88 38.38 37.5b0 39.78 ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content 5% at the time of analysis for all samples. 70 Table 43. Lipid content in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod ———————— -—-————- formation Topas Westar ng/seed* 3 200c** (29.9c)+ 240c (32.9b) 4 960b (37.8b) 1190b (41.38) 5 17908 (39.18b) 20908 (41.88) 6 19008 (40.7ab) 20708 (40.78) 7 18908 (41.68) 19508 (40.78) ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content 5% at the time of analysis for all samples. + Percent of lipid content/dry weight. Table 44. Lipid content in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod ———————— -———-—— formation Topas Westar ng/seed* 2 30d** (23.1c)+ 40c (23.5d) 3 170d (32.7b) 1500 (34.10) 4 6000 (35.7b) 850b (38.8b) 5 1850b (44.08) 19008 (43.08) 6 2240a (42.68) 19508 (41.18b) 7 2120a (42.48) 2000a (42.58b) ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was at the time of analysis for all samples. + Percent of lipid content/dry weight. 71 Table 45. Lipid content in seed tissues of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -—-——-—— formation Cascade Crystal Glacier Lirabon ng/seed* 3 4428**(31.38)+ 593d (32.9d) 5788 (34.2d) 4218 (27.28) 4 9648 (36.9b) 10508 (36.30) 11238 (36.98) 9638 (35.88) 5 13528 (38.78) 15038 (38.6b) 158888(39.88) 13838 (38.2b) 6 16178 (41.88) 160888(40.288)17318 (43.08) 16798 (40.98) 7 159288 (42.08) 16808 (42.48) 168088(42.18) 16138 (40.388) 8 14958 (41.08) 16178 (41.58) 15518 (41.588)15818 (40.78) * means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content 5% at the time of analysis for all samples. + Percent of lipid content/dry weight. Table 46. Lipid content in seed tissues of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod -——————— formation Cascade Crystal Glacier Lirabon ng/seed* 3 4378**(34.08)+ 3468(28.68) 4158 (32.48) 4228 (31.98) 4 10178 (39.08) 9318(38.98) 8928 (35.78) 9638 (38.88) 5 15158 (40.888) 15108(41.188) 169988(43.78) 13638 (40.288) 6 16668 (43.38) 17698(42.38) 17978 (45.48) 15118 (40.6b0) 7 . 15648 (41.588) 17088(41.38) 16048b(42.0b) 15768 (43.18) 8 14958 (40.888) 15928(40.388) 14768b(39.5b) 14678 (41.888) ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content 5% at the time of analysis for all samples. + Percent of lipid content/dry weight. ‘3 Lipid content (ng/seed) g ’4 0| ? 2,000 .4 '0: 8 I § Lipid content (ng/seed) O 72 1,500-ifjf“ ' ..... maturity .- *TOPas “II-Westar 1" h 3 4 5 6 7 Time after pod formation (weeks) .( Figure 9. Relationship between lipid content and times of development and maturity of seeds of two spring canola cultivars in 1990. l —_ Physiological ...—..— maturity *Cascade *Crystal +Glacier *Lirabon ‘ I 4 5 6 7 8 Time after pod formation (weeks) Figure 10. Relationship between lipid content and times of development and maturity of seeds of four winter canola cultivars in 1989. 78 50 180458-37};.33773711237 ’ ‘ 5. ‘5 7'3". 51; """ gW-JLSD-(Wlfifl ‘ o . if.“ '1 , a. g o 20‘ 'o ..... 3 ::;i """"" (D10 0 I ‘ *Lipid concentration 49.688266886888868::25); 1 2 3 4 5 6 7 8 Time after pod formation (weeks) Figure 11. Lipid and protein concentration of winter canola cultivar cultivar Crystal at different times of development and maturity in 1990. 50 A40 - LSD (5%)=2.2 2‘; 8 IE 30 - U! o , V , g , , :- _ O 20 _ LSD (5%)a0.8 g - 0 . B o ”10- 0 *Lipid concentration * Protein concentration 2 3 4 5 6 7 Time after pod formation (weeks) Figure 12. Lipid and protein concentration of spring canola cultivar cultivar Topas at different times of development and maturity in 1990. 74 Ratio of Saturated/Unsaturated Fatty Acids at Different Times of Seed Development and Maturity At the early stage of seed development, the ratio of saturated/unsaturated fatty acid was relatively high at 0.67, reached lowest level of 0.087 3-4 weeks before harvest, then remained steady until harvest (Tables 47-58 and Fig. 13,14). The first seed sample of Topas in 1989 had 40.64% saturated fatty acids consisting mostly of palmitic and stearic (16:0, 18:0) and 59.36% unsaturated fatty acids consisting mostly of oleic and linoleic (18:1, 18:2). By the second week the saturated fatty acid level declined to 13.25% and the unsaturated increased to 86.75% consisting mostly of monounsaturated oleic acid (18:1). By the fourth week, the saturated fatty acid level was 8.59% compared to an unsaturated fatty acid level of 91.41%, consisting of 57.27% oleic, 22.09% linoleic and 10.3% linolenic acids. The ratio remained almost constant the last 21 days before harvest (Table 47). Westar exhibited a similar distribution pattern during the same season (Table 48). In 1990, the first sample of the winter cultivar Cascade had 23.12% saturated fats (mostly palmitic) and 76.88% unsaturated (mostly oleic and linoleic) compared to a ratio of 16.6%:83.4% the second week. During the last four weeks, the saturated fatty acid level ranged between 7.91% and 8.54% and unsaturated level between 91.46% and 92.19% (Table 55). Crystal, Glacier and Lirabon had similar patterns during 75 the same year (Tables 56-58). In 1990, Topas had 15.59% saturated fats and 84.41% unsaturated fats in the first seed sample collected on July 13. At the second week, the ratio was 9.98% saturated to 90.02% unsaturated fats, compared to 7.97% and 92.21%, respectively at harvest (Table 53). The results for Westar , were similar (Table 54). In 1989, the winter cultivar Lirabon had a saturated 8 fatty acid content of 12.73% in the first sample collected June 20, dropped to 9.88% by the second week and was 8.46% a week before harvest on July 18 (Table 52). Cascade, Crystal and Glacier had similar patterns during the same season (Tables 49-51). Although the ratio of saturated to unsaturated fats in seeds of the first week samples of both spring and winter cultivars was lower in 1989 than in 1990, the results of both years had the same trend in that seeds tended to have high saturated fatty acid content during early seed development which gradually decreased thereafter until harvest maturity (Tables 47-58 and Fig. 13,14). The low ratio of saturated to unsaturated fats in seeds from the first samples taken from of both spring cultivars in 1990 and winter cultivars in 1989 was probably due to lateness in collecting the first seed samples due to the difficultly of separating the seeds from pods during earlier stages. 76 Table 47. Fatty acid profile of spring canola cultivar Topas during different times of seed development and maturity in 1989 Weeks after pod formation Fatty acid 1 2 3 4 5 6 7 % 16:0 24.70 7.55 7.23 4.85 4.80 4.54 4.81 18:0 10.35 3.43 3.13 2.21 2.24 2.15 2.30 18:1 21.09 53.40 53.67 57.27 58.08 58.57 58.96 18:2 28.71 24.61 24.32 22.09 21.38 20.75 21.20 18:3 7.10 7.47 7.72 10.30 10.20 10.12 9.53 20:0 1.05 1.09 0.97 0.68 0.71 0.67 0.61 20:1 2.46 1.27 1.77 1.57 1.66 1.61 1.56 22:0 2.66 0.61 0.71 0.44 0.60 0.89 0.39 ' 22:1 0.18 0.35 24:0 1.88 0.57 0.48 0.41 0.33 0.70 0.29 Saturated 40.64 13.25 12.52 8.59 8.68 8.95 8.40 Unsaturated 59.36 86.75 87.48 91.41 91.32 91.05 91.60 Table 48. Fatty acid profile of spring canola cultivar Westar during different times of seed development and maturity in 1989 Weeks after pod formation Fatty acid 1 2 3 4 5 6 7 % 16:0 27.06 6.53 5.87 4.68 4.81 4.63 4.88 18:0 11.25 3.35 3.25 2.28 2.27 2.14 2.33 18:1 18.59 57.40 61.50 61.65 61.75 61.50 62.19 18:2 26.92 22.40 20.06 20.85 20.12 20.52 19.87 18:3 9.01 6.69 5.77 7.59 7.56 7.91 7.54 20:0 1.81 1.03 0.96 0.69 0.34 0.33 0.42 20:1 2.10 1.38 1.58 1.46 1.55 1.51 1.55 22:0 1.60 0.58 0.52 0.53 0.76 0.78 0.40 22:1 0.13 0.23 0.21 0.36 24:0 1.66 0.51 0.49 0.27 0.61 0.47 0.46 Saturated 43.38 12.00 11.09 8.45 8.79 8.35 8.49 Unsaturated 56.62 88.00 88.91 91.55 91.21 91.65 91.51 77 Table 49. Fatty acid profile of winter canola cultivar Cascade during different times of seed development and maturity in 1989 Weeks after pod formation Fatty acid 3 4 5 6 7 8 14:0 0.07 0.06 0.06 16:0 6.40 5.21 4.95 4.84 4.78 4.75 18:0 2.54 2.27 1.93 1.91 2.00 2.03 18:1 55.49 56.46 59.15 59.97 61.11 61.81 18:2 23.36 22.74 21.36 20.41 20.20 19.77 18:3 8.66 9.56 9.35 9.35 9.04 8.80 20:0 1.24 1.19 1.04 0.93 0.82 0.73 20:1 1.32 1.50 1.42 1.56 1.33 1.34 22:0 0.54 -0.55 0.47 0.38 0.39 0.40 22:1 0.02 0.11 0.04 24:0 0.38 0.46 0.25 0.54 0.29 0.37 Saturated 11.17 9.74 8.70 8.60 8.28 8.28 Unsaturated 88.83 90.26 91.30 91.40 91.72 92.72 Table 50. Fatty acid profile of winter canola cultivar Crystal during different times of seed development and maturity in 1989 Weeks after pod formation Fatty acid 3 4 5 6 7 8 14:0 0.06 0.06 0.06 0.07 0.07 16:0 5.58 5.06 4.91 4.99 4.81 4.69 18:0 2.61 2.37 2.12 2.04 2.12 2.10 18:1 58.95 59.33 61.86 61.40 62.83 63.15 18:2 21.39 20.29 19.42 19.54 18.55 18.87 18:3 8.29 9.63 8.92 9.26 9.01 8.42 20:0 1.19 1.23 0.99 0.93 0.81 0.70 20:1 1.22 1.27 1.18 1.22 1.17 1.23 22:0 0.40 0.40 0.34 0.35 0.32 0.46 22:1 0.03 0.04 0.03 0.07 24:0 0.28 0.32 0.26 0.21 0.28 0.24 Saturated 10.12 9.44 8.62 8.58 8.41 8.26 Unsaturated 89.88 90.56 91.38 91.42 91.59 91.74 78 Table 51. Fatty acid profile of winter canola cultivar Glacier during different times of seed development and maturity in 1989 Weeks after pod formation Fatty acid 3 4 5 6 7 8 % 14:0 0.08 0.08 0.05 0.06 0.09 0.05 16:0 5.30 5.81 4.81 4.50 4.67 4.71 18:0 2.76 2.24 2.13 2.17 2.15 2.07 18:1 58.67 59.12 59.34 60.35 61.84 59.20 18:2 20.31 20.05 19.27 18.96 18.54 18.61 18:3 8.29 9.24 8.79 8.30 8.31 8.93 20:0 1.18 1.16 0.98 0.83 0.76 0.74 20:1 1.85 1.45 2.51 2.29 2.03 3.07 22:0 0.50 0.39 1.53 1.49 1.06 1.86 22:1 0.45 0.19 0.24 0.34 0.24 0.26 24:0 0.61 0.27 0.35 0.71 0.31 0.50 Saturated 10.43 9.95 9.85 9.76 9.04 9.93 Unsaturated 89.57 90.05 90.15 90.24 90.96 90.07 Table 52. Fatty acid profile of winter canola cultivar Lirabon during different times of seed development and maturity in 1989 Weeks after pod formation Fatty acid 3 4 5 6 7 8 14:0 0.08 0.03 0.06 0.05 0.05 0.08 16:0 7.21 5.48 5.13 4.98 5.10 5.09 18:0 2.85 2.54 1.98 1.95 1.94 2.15 18:1 51.08 58.39 57.41 58.33 60.21 61.15 18:2 26.41 21.73 23.28 22.17 21.46 20.54 18:3 8.52 8.49 8.94 8.38 8.50 8.10 20:0 1.22 1.06 0.99 0.84 0.77 0.76 20:1 1.23 1.45 1.34 1.69 1.33 1.41 22:0 0.68 0.47 0.45 0.99 0.39 ' 0.43 22:1 0.03 0.06 0.16 0.19 0.05 24:0 0.69 0.30 0.26 0.43 0.20 0.29 Saturated 12.73 9.88 8.87 9.24 8.46 8.80 Unsaturated 87.27 90.12 91.13 90.76 91.54 91.20 Table 53. Fatty acid profile of spring canola cultivar 79 Topas during different times of seed development and maturity in 1990 Weeks after pod formation Fatty acid 2 3 4 5 6 7 14:0 0.08 0.03 0.02 16:0 8.87 5.29 4.31 4.39 4.30 4.29 18:0 4.04 2.55 2.05 2.10 2.14 2.10 18:1 51.66 55.52 59.15 60.85 60.52 60.99 18:2 23.52 22.93 20.30 20.01 20.09 19.73 18:3 7.99 9.29 9.94 9.27 9.56 9.52 20:0 1.15 1.16 1.10 0.93 0.90 0.79 20:1 1.24 1.93 2.03 1.59 1.68 1.72 22:0 0.63 0.58 0.43 0.38 0.38 0.37 22:1 0.35 0.38 0.13 0.18 0.25 24:0 0.82 0.37 0.31 0.35 0.23 0.24 Saturated 15.59 9.98 8.20 8.15 7.97 7.79 Unsaturated 84.41 90.02 91.90 91.85 92.03 92.21 Table 54. Fatty acid profile of spring canola cultivar Westar during different times of seed development and maturity in 1990 Weeks after pod formation Fatty acid 2 3 4 5 6 7 14:0 0.11 0.03 0.08 0.06 0.03 16:0 8.03 5.08 4.41 4.36 4.72 4.40 18:0 3.88 2.46 2.14 2.16 2.26 2.18 18:1 51.94 57.55 60.08 60.88 59.87 61.29 18:2 24.25 22.37 20.33 20.19 20.67 19.59 18:3 8.03 8.79 9.39 9.19 9.01 9.18 20:0 1.15 1.10 1.05 0.92 0.82 0.76 20:1 1.33 1.57 1.59 1.51 1.74 1.69 22:0 0.68 0.53 0.42 0.39 0.40 0.38 22:1 0.05 0.12 0.19 0.07 0.20 0.24 24:0 0.55 0.40 0.40 0.25 0.25 0.26 Saturated 14.40 9.60 8.42 8.16 8.51 8.01 Unsaturated 85.60 90.40 91.58 91.84 91.49 91.99 80 Table 55. ratty acid profile of winter canola cultivar Cascade during different times of seed development and maturity in 1990 Weeks after pod formation Fatty acid 1 2 3 4 5 6 7 8 % 14:0 0.04 16:0 17.47 11.78 5.79 5.16 4.77 4.88 5.05 4.93 18:0 2.69 2.48 1.90 1.80 2.00 1.85 1.87 1.84 18:1 37.85 40.28 53.34 58.61 62.93 61.24 60.81 60.79 18:2 27.68 31.76 25.18 22.56 19.30 20.61 20.84 20.47 18:3 9.00 9.07 10.65 9.14 8.56 9.10 8.93 8.88 20:0 1.05 0.94 0.78 0.63 0.63 0.60 0.62 0.62 20:1 2.35 2.11 1.47 1.26 1.23 1.24 1.28 1.28 22:0 1.05 0.82 0.60 0.41 0.37 0.38 0.37 0.84 22:1 0.18 0.18 0.04 24:0 0.86 0.58 0.29 0.25 0.21 0.20 0.23 0.27 Saturated 23.12 16.60 9.36 8.25 7.98 7.91 8.14 8.54 Unsatura. 76.88 83.40 90.64 91.75 92.02 92.19 91.86 91.46 Table 56. Fatty acid profile of winter canola cultivar Crystal during different times of seed development and maturity in 1990 Weeks after pod formation Fatty acid 1 2 3 4 5 6 7 8 g 14:0 0.29 16:0 27.07 11.24 6.72 5.28 4.68 4.66 4.65 5.04 18:0 4.04 2.30 2.24 1.99 1.89 1.98 1.97 2.05 18:1 34.32 36.15 52.30 59.31 62.24 62.85 63.20 61.80 18:2 20.70 34.55 25.84 21.33 18.91 18.81 18.63 19.65 18:3 6.83 11.96 9.78 9.48 8.88 8.92 9.02 8.99 20:0 1.35 0.81 0.78 0.65 0.56 0.57 0.57 0.60 20:1 2.83 1.48 1.34 1.17 1.15 1.17 1.16 1.19 22:0 1.10 0.71 0.54 0.40 1.49 0.94 0.60 0.35 22:1 0.09 0.11 24:0 1.47 0.71 0.46 0.28 0.20 0.10 0.20 0.33 Saturated 35.32 15.77 10.74 8.60 8.82 8.25 7.97 8.37 Unsatura. 64.68 84.23 89.26 91.40 91.18 91.75 92.03 91.63 81 Table 57. Fatty acid profile of winter canola cultivar Glacier during different times of seed development and maturity in 1990 Weeks after pod formation Fatty acid 1 2 3 4 5 6 7 8 % 16:0 20.52 11.11 6.58 5.14 4.69 4.51 4.67 4.59 18:0 3.20 2.24 2.13 1.98 1.89 1.90 1.97 1.90 18:1 34.28 37.21 51.06 59.06 62.91 63.15 62.33 63.19 18:2 26.13 34.37 26.60 21.96 19.40 18.60 19.05 19.12 18:3 8.85 11.47 10.26 9.22 8.79 8.66 8.87 8.59 20:0 1.09 0.77 0.73 0.64 0.55 0.54 0.57 0.55 20:1 2.46 1.49 1.34 1.24 1.23 1.61 1.38 1.33 22:0 0.90 0.70 0.55 0.46 0.37 0.30 0.65 0.31 22:1 0.27 0.17 0.45 0.07 0.48 0.27 0.24 24:0 2.30 0.47 0.30 0.30 0.10 0.25 0.24 0.18 Saturated 28.01 15.29 10.29 8.52 7.60 7.50 8.10 7.53 Unsatura. 71.99 84.71 89.71 91.48 92.40 92.50 91.90 92.47 Table 58. Fatty acid profile of winter canola cultivar Lirabon during different times of seed development and maturity in 1990 Weeks after pod formation Fatty acid 1 2 3 4 5 6 7 8 % 16:0 25.37 12.88 6.34 5.21 4.97 5.06 4.95 5.10 18:0 4.14 2.83 2.22 1.91 1.95 1.97 1.98 2.01 18:1 42.15 39.15 51.57 59.54 61.15 61.68 62.06 60.93 18:2 16.36 32.88 27.32 22.53 21.36 20.86 20.59 21.23 18:3 4.59 7.77 9.22 8.28 8.34 7.78 7.90 7.95 20:0 1.62 0.97 0.81 0.67 0.61 0.62 0.61 0.63 20:1 3.67 1.79 1.42 1.23 1.21 1.25 1.22 1.24 22:0 0.35 0.86 0.60 0.53 0.33 0.59 0.52 0.70 22:1 0.19 0.09 24:0 1.75 0.68 0.41 0.10 0.08 0.19 0.17 0.21 - Saturated 33.23 18.22 10.38 8.42 7.94 8.43 8.23 8.65 Unsatura. 66.77 81.78 89.62 91.58 91.57 91.77 91.35 92.06 82 Saturated I Unsaturated 100 - ‘ '1 :4 ’ '5. $ 80 - .3: E? .V g '1‘ v _. if!" " -. ‘ : ’1‘ .- 4.1 '52:! . . _ g. 5 '3‘- «r 3g "'1 . on 60 -‘ 3h 5“.“ M ‘ c -1. in. ~:‘- . O ‘9 W: ‘t‘. 0 ;d1 :53); :h ‘ ~ ‘0 1’". ,-_ :.‘ ' r" . 9'5? 1'“ 9‘? iv? v ’3 > 1'9 35,, 1.1 5?; - g :11: -; .... .~ It” $4. LE 20 — 1,:- 2“ \ ‘ 1.} " 3" ~.“ "'i‘ ‘ :A ‘ , : :3 r 1 : 2. t» \-. - r h; o l v v r r v 1 fi— 1 2 3 4 5 6 7 8 Time after Pod formation (weeks) Figure 13. Ratio of saturated to unsaturated fatty acids of winter canola cultivar Crystal at different times of development and maturity in 1990. E] Saturated I Unsaturated 1oo — Q, 4;: t; g z t, it: gi- .-. ‘14 7‘ § 80- '. .3 ti: v , '1". e-I __‘ ’ 1“ 5“" V}: 5 so 4 33 -.’ 0 fix. 5:1 'c -‘4 .6 4O —' ‘ if; a; “z - ' '3’ .i‘l.‘ g. ‘ 2 4 )1 1.3: ~ 1,; {7 6 20 ~ m LL g; It 1.. ’ ‘ ‘ L . 3,4 0 . f . fi— 2 3 4 5 6 7 Time after Pod formation (weeks) Figure 14. Flatio of saturated to unsaturated fatty acids of spring canola cultivar Topas at different times of development and maturity in 1990. 83 Protein Protein content in seed tissues was significantly affected by times of seed development and maturation, cultivar and the interaction between them (Tables 1 and 2) . Winter cultivars had lower protein content and concentration than spring cultivars in both growing seasons (Tables 59-66 and Fig. 11,12,15,16). In 1989, the spring cultivar Topas showed no significant differences in either seed protein content or concentration during the last 4 weeks before harvest. Seed protein concentration ranged between 29.6 and 31.2% and was highest at 31.2% at PM. The protein concentration of Westar at the same period ranged.between 27.4 and 30.8% with the highest value at 30.8% and 30.6 at PM (Table 59). In 1989, seed protein concentration of the winter cultivar Cascade during the last.5‘weeks before harvest ranged between 21.8% and 25.7%, l9.5-23.7%, 19.3-22.4 and 22.0-25.6% for Crystal, Glacier and Lirabon, respectively (Table 61). Cascade had protein concentration of 21.8% three weeks after pod formation, increased to 25.7% at the fourth week, declined to 23.9% at the fifth week and was 22.1% at harvest. The protein concentration of Crystal was 23.7% at the fourth week after pod formation and 19.5% at harvest. 84 Glacier had the lowest protein concentration (19.3%) of all cultivars one week before harvest and the highest (22.4%) at the fourth week after pod formation. Lirabon had the highest protein concentration (25.6%) at the third week after pod formation and the lowest (22.0%) at the fourth week (Table 61). In 1989, the protein content of the winter cultivars did not significantly differ the last three weeks before harvest with few exceptions (Table 65). In 1990, the seed protein concentration of Topas during the last four weeks before harvest ranged between 25.0% and 27.8%. The protein concentration of the sample drawn two weeks after pod formation was 21.4% and 26.0% at harvest on Aug. 16 (Table 60 and Fig.12). Westar had similar results during the same year. No significant differences in protein content of both cultivars during the last three weeks before harvest were observed (Table 64). In 1990, the winter cultivar Cascade had the highest protein levels during the first three weeks of seed development. They were 27.6% at the first week after pod formation, 28.1% at the second week, 22.1% at the third week, and 20.7% at harvest (Table 62). Inadequate seeds of Crystal, Glacier and Lirabon were obtained on June 7 (one week after pod formation) for conducting protein analysis, however, these cultivars had the highest protein content during the first two 85 weeks of sampling (i.e., two and three weeks after pod formation). Protein concentration ranged between 27.6% and 28.9% at the second week after'pod formation, 24.2% and 24.9% at the third week and were 21.6%, 20.7% and 19.9% at harvest for Crystal, Glacier and Lirabon, respectively. Protein concentration during the last four weeks before harvest ranged between 19.2 and 20.7%, 20.5 and 21.6%, 20.3 and 22.5% and 19.8 and 20.7% for Crystal, Glacier and Lirabon, respectively (Table 62 and Fig. 11). In 1990, The protein content of the winter cultivars did not significantly differ the last three weeks before harvest (Table 66). 86 Table 59. Protein concentration in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar ———— % Protein*-——— 2 30.4ab** 27.4c 3 29.6b 28.5bC 4 30.8ab 30.8a 5 31.2a 30.6a 6 30.6ab 30.6a 7 29.9a 29.6ab ** means followed by the same letter within a column are not significantly different. at 0.05 probability level. * Seed moisture content was 5% at the time of analysis for all samples. Table 60. Protein concentration in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar ———— % Protein* -—— 2 21.4e** 23.16 3 27.8a 27.7a 4 25.5Cd 25.9b 5 25.0d 26.6b 6 26.4b 25.9b 7 26.0bc 25.8b ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was 5% at the time of analysis for all samples. 87 Table 61. Protein concentration in seed tissues of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod . . formation Cascade Crystal Glacier Lirabon % Protein* 3 21.8d** 20.2c 21.3ab 25.6a 4 25.7a 23.7a 22.4a 22.0C 5 23.9b 21.6b 21.2b 23.1bc 6 22.3Cd 23.6a 20.0C 22.4bc 7 23.1bc 20.1C 19.3C 23.0bc 8 22.1cd 19.5C 21.9ab 23.6b ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was 5% at the time of analysis for all samples. Table 62. Protein concentration in seed tissues of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Cascade Crystal Glacier Lirabon % Protein* 1 27.6a** 2 28.1a 27.6a 28.2a 28.9a 3 22.1b 24.2b 24.4b 24.9b 4 20.0Cd 22.1c 22.5C 20.7c 5 19.2d 20.8de 20.3d 20.3C 6 19.2d 20.5e 20.6d 19.8C 7 20.2c 21.6Cd 20.8d 20.60 8 20.7c 21.6cd 20.7d 19.9c ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was 5% at the time of analysis for all samples. Table 63. Protein content in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod ————-—— formation Topas Westar ng/seed* 3 209c** (31.2a)* 219d (30.0a) 4 823b (32.4a) 934c (32.4a) 5 1429a (31.2a) 1611a (32.2a) 6 1504a (32.2a) 1640a (32.2a) 7 1430a (31.5a) 1492b (32.1a) ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was 5% at the time of analysis for all samples. + Percent of lipid content/dry weight. Table 64. Protein content in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod —— _— formation Topas Westar ng/seed* 2 29e** (22.3b)+ 41c (24.1b) 3 152d (29.2a) 128c (29.1a) 4 451C (26.8a) 597b (27.3ab) 5 1105b (26.3a) 1238a (28.0a) 6 1462a (27.8a) 1295a (27.3ab) 7 1358a (27.2a) 1279a (27.2ab) ** means followed by the same letter within a column are not significantly different at 0.05 probability level. * Seed moisture content was 5% at the time of analysis for all samples. + Percent of lipid content/dry weight. 89 Table 65. Protein content in seed tissues of four winter canola cultivars during different times of seed development and maturity in 1989 weeks after pod formation Cascade Cultivar Crystal Glacier Lirabon QOUl-bu 324c**(23.0c)* 733b 878a 908a 922a 849a (27.0a) (25.2ab) (23.5bc) (24.3bc) (23.3bc) 383d ng/seed* (21.3bc) 379c (22.4ab) 4l8c (27.0a) 721C (24.9a) (22.7b) 884b 994a 838b (24. (21.2bc) 9a) 801bc(20.5c) 717b (23.6a) 623b (23.2b) 890a (22.3abc)880a (24.3b) 848a (21.0bc) 969a (23.6b) 811ab(20.3c) 968a (24.2b) 862a (23.0ab) 964a (24.8b) ** means followed by not significantly different at 0.0 * Seed moisture content was 5% at t all samples. + Percent of lipid content/dry weight. the same letter within a column are 5 probability level. he time of analysis for Table 66. Protein content in seed tissues of four winter canola cultivars during different times of seed development and maturity in 1990 weeks after pod formation Cascade Cultivar Crystal Glacier Lirabon mummbu 323c**(23.2a)* '549b 750a 778a 802a 797a (21.0b) (20.2b) (20.2b) (21.3ab) (21.8ab) 308d 556c 804b 902a 941a 898a (25. (23. (21. (21. (22. (22. 5a) 3b) 9b) 6b) 7b) 7b) ng/seed* 329c 592b 831a 859a 836a 815a (25.7a) (23.7ab) (21.4c) (21.7bc) (21.9bc) (21.8bc) 346c 540b 724a 775a 794a 735a (26.2a) (21.8b) (21.4b) (20.8b) (21.7b) (20.9b) means followed by not significantly Seed moisture cont for all samples. Percent of lipid content/dry weight. the same letter within a column are different at 0.05 probability level. ent was 5% at the time of analysis 1,600 ,,,,,,, 1,400- 31,200- f. g 5 800- 3’ E f - 13:33; ° ‘°°‘ r5323: '5 2 £5 3 40°“ .31? i O ' *Tapas *Wgstar :4:*;;j__:* , 2 3 4 5 6 7 Time after pod formation (weeks) Figure 15. Relationship between protein content and times of development and maturiy of seeds of two spring canola cultivars in 1990. 1,200 ‘6‘"000‘ =0.75** 8 M a 800— 5 l 5 600- a E .2 b O O": o 9 3 ,5 400 .3 g o .: § :1 0- 200 0 *Cascade *Crystal *Glacier +Lirabon 3 4 5 6 7 8 Time after pod formation (weeks) Figure 16. Relationship between protein content and times of development and maturiy of seeds of four winter canola cultivars in 1989. 91 Mineral Composition Composition of K, P, Ca, Mg and various micro-elements for both spring and winter culivars in 1989 and 1990 varied among cultivars and with seasons (Tables 67-71). In 1989, potassium (K) concentration of Topas ranged from 1.09% at early stage of seed formation to 0.91% at PM and 0.72% at harvest (Table 59) . Phosphorus (P) levels ranged between 0.81% and 0.92% with 0.88% at both PM and HM. Calcium and magnesium concentration were lower than for K and P. Calcium concentration ranged from 0.30% to 0.52%, with 0.39% at PM and 0.40% at harvest. Magnesium concentration ranged between 0.36 and 0.42%, with 0.38% at PM and 0.36% at harvest (Table 67). The winter cultivar Glacier had a similar pattern of mineral element concentration to that of Topas. At PM, the content of K, P, Ca and Mg was 0.97%, 0.77%, 0.42% and 0.34%, respectively, and was 0.77%, 0.77%, 0.40% and 0.31%, respectively at harvest (Table 60). Similar results were obtained in 1990 for both spring and winter cultivars (Tables 69-71). The K, P, Ca and Mg concentrations of Topas at PM were 0.90%, 0.66%, 0.32% and 0.34%, respectively, and were 0.68%, 0.69%, 0.32% and 0.34% at HM. At PM, Westar, had K, P, Ca and Mg levels of 0.90%, 0.74%, 0.34% and 0.38%, respectively, and 0.65%, 0.63%, 0.34% and 0.32% at HM (Table 69). 92 Potassium levels in 1990 for all winter cultivars ranged between 1.15% and 1.29% at PM, and 0.83% and 0.87% at HM. The range of P was 0.73 to 0.85% at PM and 0.81 to 0.82% at HM. Ca ranged between 0.36% and 0.43% at PM, and 0.34% and 0.45% at HM. Mg ranged between 0.34% and 0.35% at PM, and 0.33% and 0.37% at HM (Tables 70 and 71). In both 1989 and 1990, winter cultivars had greater K concentration than spring cultivars. Winter cultivars ranged from 0.77% to 0.87% compared to 0.65 and 0.72% for the spring cultivars. The winter cultivars also had higher P, Ca and Mg than the spring cultivars in 1990 but not in 1989. In 1990, winter cultivars had a mean concentration 0.40% and Mg 0.35% for P, Ca and Mg, respectively (Tables 70 and 71), compared to spring cultivars with levels of 0.66%, 0.33% and 0.33% (Table 69). Traces of Fe, Zn, Mn, B, Cu and Al ranged from 0.0003 to 0.006% were found in both spring and winter canola (Tables 67-71). 93 Table 67. Mineral nutrient concentration in seed tissues of spring canola cultivar Topas during different times of seed development and maturity in 1989 weeks % of dry weight after pod formation K P Ca Mg 3 1.08 0.81 0.30 0.36 4 1.09 0.87 0.52 0.42 5 0.96 0.92 0.40 0.40 6 1.00 0.85 0.42 0.41 7 0.91 0.88 0.39 0.38 8 0.72 0.88 0.40 0.36 * Fe, Zn, Mn, 8, Cu, and Al exist in very small amounts (0.0003 - 0.006%). Table 68. Mineral nutrient concentration in seed tissues of winter canola cultivar Glacier during different times of seed development and maturity in 1989 weeks % of dry weight after pod formation K P Ca Mg 3 1.18 0.97 0.48 0.38 4 1.29 0.85 0.47 0.39 5 1.06 0.79 0.45 0.35 6 0.97 0.77 0.42 0.34 7 0.92 0.81 0.35 0.33 8 0.77 0.77 0.40 0.31 * Fe, Zn, Mn, 8, Cu, and Al exist in very small amounts (0.0003 - 0.006%). 94 Table 69. Mineral nutrient concentration in seed tissues of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Topas Westar after pod formation K P Ca Mg K P Ca Mg % of dry weight 2 1.26 0.64 1.31 0.34 1.35 0.62 1.24 0.41 3 1.43 0.74 0.51 0.41 1.39 0.73 0.59 0.42 4 1.33 0.67 0.33 0.39 1.32 0.72 0.51 0.41 5 0.90 0.66 0.32 0.34 0.90 0.74 0.34 0.38 6 0.71 0.69 0.34 0.33 0.67 0.73 0.38 0.33 7 0.68 0.69 0.32 0.34 0.65 0.63 0.34 0.32 * Fe, Zn, Mn, 8, Cu, and A1 exist in very small amounts (0.0008 - 0.004%). Table 70. Mineral nutrient concentration in seed tissues of two winter canola cultivars during different times of seed development and maturity in 1990 weeks Cascade Crystal after pod formation K P Ca Mg K P Ca Mg of dry weight 3 0.91 0.72 0.56 0.29 0.76 0.77 0.50 0.30 4 1.63 0.80 0.44 0.35 1.03 0.83 0.51 0.35 5 1.57 0.80 0.45 0.35 1.17 0.80 0.46 0.35 6 1.28 0.77 0.36 0.35 1.15 0.85 0.42 0.37 7 0.86 0.79 0.33 0.34 0.83 0.80 0.42 0.34 8 0.83 0.82 0.38 0.35 0.83 0.81 0.42 0.35 * Fe, Zn, Mn, 8, Cu, and Al exist in very small amounts (0.0008 - 0.004%). 95 Table 71. Mineral nutrient concentration in seed tissues of two winter canola cultivars during different times of seed development and of maturity in 1990 weeks Glacier Lirabon after pod formation K P Ca Mg K P Ca Mg of dry weight 6-21 0.89 0.80 0.51 0.32 1.40 0.83 0.50 0.36 6-28 1.09 0.79 0.52 0.34 1.26 0.76 0.57 0.33 7-05 1.15 0.81 0.47 0.34 1.55 0.74 0.43 0.36 7-10 1.18 0.83 0.43 0.35 1.29 0.73 0.36 0.34 7-15 0.89 0.83 0.42 0.33 1.15 0.74 0.35 0.34 7-22 0.86 0.81 0.45 0.33 0.87 0.81 0.34 0.37 * Fe, Zn, Mn, 8, Cu, and Al exist in very small amounts (0.0008 - 0.004%). 96 Seed Quality Germination and Vigor Seed development stages, cultivars and the interaction between them had significant effect on seed viability and vigor of both spring and winter cultivars in both growing seasons (Table 2). However, differences between spring cultivars had no significant effect on accelerating aging results in 1989 nor cold and accelerating aging results in 1990 (Table 1). Generally, increased germination and vigor results occurred between physiological maturity and harvest maturity (Tables 72-83 and Fig. 17,18). After five weeks of pod formation in 1989, the spring cultivar Topas had a germination percent (GP) of 84, compared to 93% after six weeks and 94% at harvest. At the same period, the cold test results were 71, 81 and 93%, respectively and the accelerated aging results were 70, 84 and 92%, respectivelyu ‘Westar behaved similarly in the same year (Tables 72-74). The seed samples. of ‘winter’ cultivars evaluated for quality in 1989 were collected June 20, about three weeks after pod formation. The germination percent of Cascade after three weeks of pod formation was 30, increased to 88 by the six week (PM) and to 98 at harvest. Cold test results were 20, 72 and 95% and accelerated aging test results were 0, 56 and 96%, respectively for the same periods of time (Tables 78-80). 97 In 1990, germination results of the spring cultivar Westar were 0, 29, 64,79, 90, 93% on July 13 (second week after pod formation), 7-20, 7-27, 8-3, 8-10 and 8-16 (at harvest), respectively. Cold test results for the same period were 0, 27, 67, 75, 77 and 91%, respectively. The accelerated aging results were 0, 1, 25, 73, 83, 89%, respectively for the same sampling dates (Tables 75-77). In 1990, the winter cultivar Glacier a had relatively high germination of 61% at the third week after pod formation (June 21) and increased to 67, 77, 89, 94 and 97% on 6-28, 7- 5, 7-10 (PM), 7-15 and 7-22 (harvest time), respectively. Cold test results were 52, 70, 80, 87, 91 and 97%, respectively for the same time period. Accelerated aging results were 25, 33, 56, 75, 77 and 94%, respectively for the same periods (Tables 78-80). Further information about vigor tests for evaluating the quality of canola seeds is discussed in chapter two of this dissertation. 98 Table 72. Standard germination test results of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar ——— % germination -—— 3 26d* 37c 4 69C 80b 5 84b 84b 6 93a 91a 7 94a 93a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 73. Cold test results of two spring canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod ——————— formation Topas Westar ——- % germination ——— 3 20e* 32e 4 63d 67d 5 71C 786 6 81b 84b 7 938 92a * means followed by the same letter within a column are not significantly different at 0.05 probability level. 99 Table 74. Accelerated aging test results of two results canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Topas Westar -—— % germination ——— 3 38* 1e 4 19d 25d 5 700 73c 6 84b 83b 7 92a 89a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 75. Standard germination test results of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Topas Westar -—— % germination -—— 2 1e* 0e 3 22d 29d 4 59c 64c 5 75b 79b 6 93a 90a 7 94a 93a * means followed by the same letter within a column are not significantly different at 0.05 probability level. 100 Table 76. Cold test results of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod _— formation Topas Westar -—— % germination -—— 2 0f* 0e 3 24e 27d 4 63d 67c 5 68¢ 75b 6 82b 77b 7 91a 91a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 77. Accelerated aging test results of two spring canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod -—————- formation Topas Westar -——-% germination ——— 2 0e* . 0e 3 3e 1e 4 19d 25d 5 70c 73c 6 86b 83b 7 92a 89a * means followed by the same letter within a column are not significantly different at 0.05 probability level. 101 Table 78. Standard germination test results of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod ——————— ———————— formation Cascade Crystal Glacier Lirabon % germination 3 30d* 42d 57d 44e 4 29d 47d 60d 36f 5 69c 88c 87c 63d 6 88b 90bc 88bc 78c 7 92ab 96ab 94ab 90b 8 98a 98a 98a 97a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 79. Cold test results of four ‘winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod formation Cascade Crystal Glacier Lirabon % germination 3 20e* 41d 52e 32e 4 22e 44d 62d 3le 5 56d 75c 80c 59d 6 72c 87b 85b 72c 7 90b 93a 95a 88b 8 95a 95a 93a 93a * means followed by the same letter within a column are not significantly different at 0.05 probability level. 102 Table 80. Accelerated aging test results of four winter canola cultivars during different times of seed development and maturity in 1989 weeks Cultivar after pod -—————— formation Cascade Crystal Glacier Lirabon % germination 3 0e* 8e 5e 0f 4 10d 23d 21d 10e 5 34c 56c 66c 31d 6 56b 83b 83b 60c 7 94a 96a 90a 87b 8 96a 92a 94a 95a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 81. Standard germination test results of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod ——————- formation Cascade Crystal Glacier Lirabon % germination 3 47d* 57e 61e 60d 4 48d 62d 67d 72c 5 63c 84c 77c 74c 6 84b 91b 89b 81b 7 95a 93b 94a 95a 8 95a 98a 97a 95a * means followed by the same letter within a column are not significantly different at 0.05 probability level. 103 Table 82. Cold test results of four 'winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod -——————- formation Cascade Crystal Glacier Lirabon % germination 3 30d* 54e 52e 47e 4 32d 65d 70d 64d 5 62c 75c 80c 79c 6 82b 90b 87b 85b 7 93a 94ab 91b 95a 8 95a 95a 97a 94a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Table 83. Accelerated aging test results of four winter canola cultivars during different times of seed development and maturity in 1990 weeks Cultivar after pod formation Cascade Crystal Glacier Lirabon % germination 3 12e* 31f 25e 12d 4 17d 40e 33d 15d 5 32c 52d 56c 38c 6 79b 63c 75b 80b 7 94a 76b 77b 90a 8 97a 96a 94a 94a * means followed by the same letter within a column are not significantly different at 0.05 probability level. Gennlnation (96) Germination (96) 104 8 Aw] LSD (5%)=5 LSD (5%)=4 * S. germ. ' ; LSD (5%) =4 ' *Ccld'test * Accel. aging l i l I 3 4 5 6 7 8 Time after pod formation (weeks) Figure 17. Standard germination. cold and accelerated aging test results of winter canola cultivar Crystal at different times of development and maturity in 1990. 10° KS. germ. ' - , LSD (5%)=4 5 *Coldtest . 80- ; » - . OAccel. aging _ , v LSD (5%)=4 60 —.. 40 - 2° ‘ $0 (5%):5 o T fir l l I 2 3 4 5 6 7 Time after pod formation (weeks) Figure 18. Standard germination, cold and accelerated aging test results of spring canola cultivar Topas at different times of development and maturity in 1990. DISCUSSION Pods attained maximum length about 15-21 days after formation in all cultivars in both seasons of this study. Maximum pod width and fresh and dry matter were attained about 28-35 days after formation. This pattern of growth (e.g., attaining maximum pod width and weight after full pod length) agrees with earlier reports by Culpepper (21), Watada and.Morris (76), Hsu (41), Fraser g; _1. (33) and Mariga (50) for various other crops. Pod development of all canola cultivars used in this study was attained prior to seed development. Many researchers have reported similar pattern of growth for other crops, including Dure (28), Egli (30), Fraser gt g1, (33), Oliker (61), Loewenberg (47) and Mariga (50). Results of this study showed that canola fruit attained maximum growth rate (biomass) during the second and the third week after pod formation. As the canola seeds develop and mature, pods begin to lose dry weight and decrease in width. These results suggest that pod dry matter remobilization to the seeds occurred during seed growth and that the pod serves as a reservoir for assimilates for the developing seeds. This agrees with the concept proposed by Thorone (72) in soybean, Hocking (39) in caster bean and Hocking and Pate in legumes 105 106 (40). However, Crookston gt _1. (19) observed that pod is not an important photosynthetic source of dry matter for developing bean seeds. Egli (30) concluded that the accumulation of dry weight in soybean seeds is related to the ability of the plant to fix carbon during the filling period or the translocation of storage carbohydrates from other plant parts. The results of this study did not support the suggestion by Mariga (50) in Phaseolus vulgaris L. that a good flow of photosynthate from leaves remains necessary for full seed development even.after pods attain full size, since the canola plant loses most of the canopy by the time pod reaches full size. This view is also supported by Allen and Morgan (5), Bilsborrow'and.Norton (8) and Clarke (16) who studied.Brassica species. Number of seeds per pod was found to be determined at a very early stage of pod formation and agrees with conclusions reported by Fraser gt _1. (33) for soybean. Seed dry matter results suggest that canola could be harvested two to three weeks before reaching harvesting maturity without significant reduction in seed dry weight. However, seed moisture content at this stage is not suitable for harvesting and threshing. Thus, further desiccation is necessary . 107 Although seed lipid content reached maximum levels at PM, no significant differences in lipid content occurred during the last 15-21 days before harvest in any cultivar in either year. Therefore, the crop could be harvested about two to three weeks before reaching HM without a significant reduction in seed oil content. This period may become shorter (not more than two weeks before harvest) during short growing seasons (e.g. , due to high temperature and moisture stress). The average lipid content found in these studies was 41%. The pattern of lipid accumulation in developing seeds was similar to that reported by Appelqvist in rapeseed (6). The fatty acid composition of canola oil observed in these studies was a favorable balance for human nutrition for the following reasons: 1) oleic acid (18:1), the major fatty acid (60%), has been shown to reduce cardiovascular risk, 2) about 20% of canola oil is linoleic acid (18:2), the "essential" fatty acid needed in a small amounts for the human diet, 3) linolenic acid (18:3) is present in ideal proportions (2:1) relative to linoleic acid; it is regarded as potentially functional in reducing cardiovascular risk, and 4) stearic acid (18:0) at about 2% in canola oil, is a relatively harmless saturated fatty acid in the human diet. Palmitic acid (16:0) remains as the important cardiovascular risk factor in this group of fatty acids, however, at about 4.8%, canola oil has a notably low content relative to most other vegetable oils (3). 108 Protein results also suggest that canola could be harvested three weeks before HM without a significant decrease in seed protein content. However, other factors related to storage problems preclude that. The pattern of mineral accumulation was so varied that generalizations are difficult. However, the average content of K and P in all cultivars at harvest time was about 0.85% and 0.80%, respectively, about double the content of Ca and Mg at 0.40% and 0.35%, respectively. The seed quality test results supported the well-known fact that seeds develop germination capacity ahead of vigor. Slow air drying of seeds prior to germination testing helped to improve the performance of seeds, especially at early stages of development. This was supported by Adams and Rinne (4) who demonstrated that fast drying was detrimental to the germination of immature bean seeds. The using of high temperatures to dry seeds is also known to denature seed proteins (46). The quality of seeds of all canola cultivars used in this study was better at HM than at PM, in contrast to the general principle that seeds attain maximum quality (germination and vigor) at PM (75). This may be explained by physiological changes (e.g., hormonal mechanism) that occur after the PM which can aid in germination (43). 109 Loss of the green pod color and change to brownish green or light brown was the most reliable and practical indicator of PM. Similar visual indicators have been suggested for dry beans (50,76) and soybeans (34,71). At this stage (PM), the color of canola pods turned from green to greenish-yellow and contained seeds ranging from.dark green to greenish-brown and light brown. The seeds were firm but not hard and could be marked with a finger nail, as suggested by Oplinger gt g1. (62). At harvest maturity (HM), the pod color was completely yellow to light brown and the seed color was brown or dark brown to black, depending on the cultivar. The seed moisture content was near 10% and the seeds were hard. Canola seeds in these studies reached.PM at wide range of MC from 20.3 to 36%. Thus, it is difficult to accurately estimate the date of occurrence of PM from MC measurement alone. This contrasts with reports by Fraser gt g1. (33) that MC represents a more accurate indicator of PM in soybean than seed dry weight. Although both seed dry weight and moisture content are reported to be reliable indicators of PM for many crops, they are more difficult to measure than visual markers and need to be measured over a relatively long period of time during seed maturation to determine PM. Therefore, the change of pod color from green to greenish-yellow is more convenient and rapid method for canola. 110 The time from PM to HM varied with cultivars and with environmental conditions during seed maturity. These results are supported by other reports in literature for various species (20,71). More studies on the pod growth pattern of canola and its relationship to seed quality and composition are needed. Further studies should consider the following: 1) A shorter sampling interval of about three days to improve the sensitivity in detecting changes in dry matter, moisture content, germination and nutrient status. 2) Identify specific inflorescences within the plant (sampling unit) at flowering time instead of area to improve sampling precision and ensure that the number of tagged inflorescences are enough to run all tests needed. 3) Use more vigor tests to evaluate seed quality such as speed of germination test and conductivity test. 4) The use of different environmental conditions, such as water stress to monitor the effects on seed quality and composition at different stages of fruit development and maturation. 10. 11. RIIIIUHWCES Abdul-Saki, A. A., J. E. Baker. 1973. Are changes in cellular organelles or membranes related to vigor loss in seeds?. Seed Sci. Technol. 1: 89-125. Abdul-Saki, A" A. 1980. Biochemical aspects of seed vigor. Hort Sci. 15: 765-771. Ackman, R. G. 1990. Canola fatty acids- An ideal mixture for health, nutration and food use. In: Canola and rapeseed. (ed. F. Shahidi). Van Nostrand Reinhold Inc. New York. pp. 81-98. Adams, C. A. and R. W. Rinne. 1980. Moisture content as a controlling factor in seed development and germination. Int‘l. Rev. Cytol. 68:1-8. Allen, R., J. and D. G. Morgan. 1972. A quantitave analysis of the effects of nitrogen on the growth, development and yield of oil seed rape. J. Agric. Sci. Camb. 78: 315-324. Appelqvist, L. A. Recent Advances in the chemistry and biochemistry of plant Lipids. Galliard, T., Mercer, E. I. (eds.). London: Academic Press, 1975, Proc. Phytochem. Soc. 12, 247-286 Association of Official Seed Analysts (AOSA). 1983. Seed vigor testing handbook. Bilsborrow, P. E. and G. Norton. 1987. Physiological factors controlling the yield of oilseed rape. 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Poland. 57-62. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 115 Mariga, I. K., 1987. Seed and pod and development and effect on seed quality in dry beans. (Phaseolus valgaris L.). Ph. D. Dissertation. Department of crop and soil sciences. Michigan State University. East Lansing. McDaniel, R. G. 1973. Genetic factors influencing seed vigor: biochemistry'of heterosis. Seed ci. Technol. 1: 25- 50. McDonald, M. B. Jr., and D. 0. Wilson. 1975. A review and evaluation of seed vigor tests. Proc. Assoc. Off. Seed Anal. 65: 109-139. McDonald, M. B. Jr., and B. R. Phaneendranath. 1978. A modified accelerated aging seed vigor test for soybean. J. Seed Technol. 3: 27-37. McGregor, D. I. 1981. Pattern of flower and pod development in rapeseed. Can. J. Plant Sci. 61: 275-282. McGregor, D. I. 1990. Application of Near infrared to the analysis of oil, protein, chlorophyll, and glucosinolates in canola/rapeseed. In: Canola and rapeseed. (ed. F. Shahidi). Van Nostrland Reinhold Inc. New York. pp. 221-231. Meza-Basso, L., M. Alberdi, M. Raynal, M. Ferrero-Cadinanos and M. Delseny. 1986. Changes in protein synthesis in rapeseed (Brassica napus) seedlings during a low temperature treatment. Plant Physiol. 82: 733-738. Mendham, N. J., P. A. Shipway and R. K. Scott. 1981. The effect of delayed sowing and weather on growth, development and yield of winter oil-seed rape (Brassica napus). J. Agric. Sci., Camb. 96: 389-416. Morrison, W. R. and L. M. Smith. 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron floride-methanol. J. Lipid Res. 5: 600-608. Murphy, D. J., I. Cummins and A. S. Kang. 1989. Immunocytochemical and biochemical studies of the mobilisation of storage oil-bodies and proteins in germinating cotyledons of oilseed rape, Brassica napus. J. Sci. Food Agric. 48: 209-223. Norton, G. and J. F. Harris. 1983. Triacylglycerols in oilseed rape during seed development. 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Roberts, E. H. 1972. Cytological, genetical and metabolic changes associated with loss of viability. In‘Viability of seeds (ed., E. H. Roberts). Chapman and Hall, London. pp. 253-306. Salunkhe, D. K. and B. B. Desai. 1986. Postharvest biotechnology of oilseeds. CRC Press, Inc. Boca Raton, Florida. Shahidi, F. 1990. Rapeseed and canola: Global production and distribution. In: Canola and rapeseed. (ed. F. Shahidi). Van Nostrand Reinhold Inc. pp. 3-15. Sylvester-Bradley, R. and R. J. Makepeace. 1984. A code for stages of development in oilseed rape (Brassica napus L.). Aspec. Appl. Biol. 398-419. Tayo, T. 0. and D. G. Morgan. 1979. Factors influencing flower and pod development in oil-seed rape. J. Agric. Sci. (camb.). 92: 363-373. TeKrony, D. M., D. B. Egli, J. Balles, T. Pfeiffer and R. J. Fellows. 1979. Physiological maturity in soybean. Agron. J. 71: 771-775. Thorne, J. H. 1979. Assimilate redistribution from soybean pod walls during seed development. Agron. J. 71: 812-816. 73. 74. 75. 76. 77. 78. 117 Thurling, N. 1974. Morphophysiological determinants of yield in rapeseed. Aust. J. Agric. Res. 25: 711-721. Tittonel, E. D., J. P. Chaput, F. Letoublon and O. Bonnot. 1987. Winter oilseed rape: Certain events concerning floral initiation and the beginning of spring. Seventh International Rapeseed Congress. Poland. 727-732. Wabab, A. H. and J. S. Burris. 1971. Physiological and chemical differences in low- and high-quality soybean seeds. Proc. Ass. Off. Seed Anal. 61: 58-67. Watada , A. E. and Morris. 1967 Growth and respiration patterns of snap bean fruits. Plant Physiol. 42: 757-761. Weiss, E. A. 1983. Oilseed crops. Longman Inc., New York. pp. 161-216. Zimmerman, D. C. and Fick, G. N. 1973. Fatty acid composition of sunflower (Helianthus annuus L.): Oil as influnced by seed position. J. Am. Oil Chem. Soc. 50:273-275. CILKPTERJH EVALUATION OF VIGOR TESTS FOR CANOLA (Q. mg; L.) AJEHHLACHT Various seed vigor tests and field emergence were used to evaluate the quality of different canola (gtgggigglngggg L.) seeds lots. Two winter and two spring canola cultivars were aged to provide six sub-lots with a range of seed vigor. Various combinations of time and temperature were used to determine optimum conditions for cold, cold soil, accelerated aging, conductivity, first count germination and standard germination tests. Aging for 48 h at 42 C in wire-mesh trays followed by germination for 7 d at 22 C was the best method for the accelerated aging test. Soaking 200 uninjured, untreated seeds for 16 h at room temperature (22 C) in 50 ml of distilled water was suitable for the conductivity test. The cold soil test was most successful in separating seed lots of different quality when seeds were planted on moist blotter paper, covered with soil and pre-treated for 5 d at 5 CL before germinating at 22 C for 5 d. Incubation for 5 d at 5 C without soil provided the best pre-germination exposure for the cold test, especially for lower quality seed. Temperatures of 20, 22, 25 or alternate 20/ 30 C in the standard germination test did not significantly influence germination results. Significant correlation was found between all vigor tests and field emergence for all seed lots except for the standard germination test of the spring cultivars. HWTR£HWUCHHOWJ The use of high quality, vigorous seed is one of the most important factors in crop production. The purposes of vigor testing are to provide a fair prediction of the potential performance of a seed lot under favorable or adverse field conditions and to separate high vigor seed lots from those of low and intermediate quality (8). Although the standard germination test is the most widely accepted method for measuring seed quality, some deterioration may occur even though germination remains relatively high (17). However, it can be dependable as an index of potential field performance under favorable conditions (9,27,38,41). Vigor tests can provide more information about the biochemical, cytological and physiological quality of a seed lot, thus they can be useful in predicting which seed lots have the potential for better performance under a wide range of field conditions. Generally, seeds attain maximum vigor at physiological maturity when their moisture content is relatively high. However, they are 'usually’ harvested later ‘when. moisture content is low enough for threshing and safe storage. Between physiological and harvest maturity seeds remain on the plant where they may be exposed to adverse weather conditions that 120 121 affect their quality (15,16). Factors that influence seed vigor include: genetic variation, i.e., species and cultivar; environmental conditions during seed development and maturation; physiological, biochemical and cytological deterioration of cell membranes and organelles; and storage conditions (12,31, 32,36,37,39,47). Although vigor testing has become a common practice for crops such as corn and soybean in the United States, canola, as a new crop, has no seed vigor test in practice. The possible need for seed vigor testing of canola is caused by its indeterminate habit of flowering, resulting in seeds with different maturity levels on the plant, especially if the crop is harvested before full maturity. This results in differences in vigor among seeds within the same lot. Furthermore, as an oil-seed crop, canola has a tendency to deteriorate faster than non-oilseeds because of lipid autoxidation and the accumulation of free fatty acids. The general strategy in determining seed vigor is to measure various aspects of seed deterioration or genetic deficiency which are inversely proportional to seed vigor (8). Only limited research has been reported concerning the evaluation of vigor in canola. The objectives of this study were: 1) to evaluate different.methods for determining seed vigor of canola, 2) to 122 determine the vigor of various canola seed lots using different vigor tests and 3) to study the relationship between vigor test results and field emergence. IJITHUKTURIZRENHENV Definition of Seed Vigor Seed vigor has been defined as the properties of the seed which determine the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions (8,25). Seed quality and seed vigor are often used interchangeabelly (23,31,47). Maguire (31) reported that seed quality includes genetic homogeneity, physical appearance, uniformity, viability and field performance. Woodstock (47) pointed out that the aim of vigor testing is not to predict emergence nor performance under field conditions but to provide useful and comparative information about seed quality. He emphasized that the embryo, the part of , seed that grows and develops into new plant, the whole seed which germinates, and the entire seed lot which the farmer plants all contribute to seed vigor. Heydecker (23) cited two factors that influence the expression of seed vigor: the quality of the seed and the quality of the environment. Isely (26) considered two important aspects of vigor as: (1) susceptibility to unfavorable field conditions and (2) speed of germination and seedling growth rate. 123 124 Manifestation of Poor Vigor Heydecker (23) cited several ways that seed may demonstrate poor vigor: 1) rapid deterioration during storage, 2) narrowing of the environmental conditions under which a seed will germinate, 3) reduction in germination speed and/or uniformity, 4) greater susceptibility to mild micro-organisms, 5) production of abnormal, slow-growing seedlings and 6) low yield. He also pointed out that dormancy must not be confused with lack of vigor. Perry (37) reported that the manifestation of vigor depends on the interaction between the seed and its environment. Therefore, low vigor seeds are more sensitive to stress conditions than high vigor seeds. In spite of all that.has been known about what seed.vigor is, no single test.is yet available which.measures all aspects of vigor. Grabe (19) suggested that several tests may be needed for a complete seed vigor evaluation: e.g., a growth rate test, a stress test, a biochemical test and.a mechanical damage test. Seed Vigor and Seedling Growth Rate Seedling growth rate is a component of seed vigor. Growth relates to the increase in cell numbers (cell division), cell size (cell elongation) and differentiation. Seedling growth during germination is initiated.when.the seed is hydrated and 125 terminates when the new plant becomes a complete autotroph. The embryonic axis depletes its own food reserve first and then depends on such storage tissues as the cotyledons, endosperm, scutellum and perisperm. These tissues become active upon hydrolyzation of their food reserves and re- mobilization to the growing embryonic axes. During imbibition, metabolic activities increase rapidly. Respiration and substrates required for it (sugars and fatty acids) increase, providing ATP, the energy product of respiration, for protein synthesis which, in turn, supplies enzymes and soluble proteins required for growth processes. This is accompanied by increase in activity and quantity of the protein-synthesizing machinery (ribosomes, mRNAJs, tRNA's and polysomes), mitochondria, glyoxysomes, enzymes and coenzymes (4,12). Abdul-Baki (1) found that conversion (utilization or incorporation) of glucose-"C into "C02, polysaccharides (water-soluble sugars, hemicellulose and cellulose) and protein could be used to predict seed vigor. He also reported that glucose metabolism offers a good tool for distinguishing between dead and dormant seeds. However, it failed to distinguish between dormant and non-dormant seeds of wheat and barley. 126 Factors Influencing Seed Vigor Seed vigor is a complex subject which involves both endogenous and exogenous factors. Among the factors influencing seed vigor are genetic factors (i.e., species and cultivar), physiological, biochemical and cytological factors (i.e., deterioration of cell membranes and change in cell constituent), environmental effects (i.e., conditions during seed development and maturation), storage conditions (i.e., temperature, relative humidity, storage fungi) and mechanical injury due to harvesting, conditioning and handling (12,31, 32,36,37,39,47). e 'c st tution Factors under genetic control include hybrid vigor, hard-seededness, seed size, seed chemical composition (e.g., high erucic acid and glucosinolate in rapeseed), susceptibility to mechanical damage, seed coat color and disease resistance, all of which may influence the expression of seed vigor (15,18,20,28). McDaniel (32) reported that genetic factors act through biochemical pathways to determine the seed vigor potential. He found that the superiority of large seeds of barley hybrids can be related to amount of mitochondria and therefore to higher respiration, production of ATP, metabolic activity, 127 superiority is closely correlated with the vigor and yield potential of hybrids. Thus, larger rapeseed (B. campestris) produced more and larger fruits (pods) per plant, heavier seeds (but fewer seeds per fruit) and higher seed yield per plant than plants grown from small seeds (5). Dickson (18) reported that colored seeds of many crops have been shown to be more resistant to both disease and mechanical damage than light-colored seeds. Kneebone (28) reported that mitochondria, enzyme stability, and gibberellin activity are heritable and can be modified by appropriate breeding procedures. Delouche and Caldwell (17) showed that seeds or seedlings which are inherently weak in vigor are more susceptible to a variety of adverse conditions, including microbial attack, than more vigorous ones. Roberts (39) reported the correlation between loss of viability and accumulation of chromosome damage and gene mutation, particularly under the collective effects of temperature, moisture and oxygen stress. He also cited the association between viability and nuclear damage (DNA and mRNA) and cytoplasmic organelles such as mitochondria, plastides and endoplasmic reticulum. 128 tgvitopgent Qgting Seed Development and Maturation The success of seed production of a particular crop in one area and its failure in another is evidence of the importance of environmental influences on seed development and maturation (16). Temperature, water stress and lack of nutrients during seed development and maturation also influence the quality of the developing seed. Moisture stress during seed development results in light, shriveled seeds. Warm, humid weather is favorable for the spread of infection of’ many' fungal and. bacterial. pathogens. Rainfall during harvest season also causes a significant reduction in seed quality (22). Chirco and Harman (14) studied the effect of Alternaria brassicicola infection on the viability and vigor of Brassica oleracea (cabbage) seeds. They showed that seed infection at any stage of development causes a reduction in seed quality. They also found that Thiram treatment significantly reduced the infection but did not improve germination or field emergence. Generally, seeds attain physiological maturity (maximum dry weight) at high moisture levels which are unsafe for storage. Therefore, they are usually harvested later when they reach "harvest maturity" at which seed moisture is low enough for safe storage but high enough to minimize mechanical 129 injury. Between physiological maturity and harvest seeds are stored on the plant where they may be exposed to adverse weather conditions that further affect their quality (15,16). Seed Stotage Storage conditions greatly affect seed vigor. Unfavorable storage conditions accelerate the biochemichal processes leading to seed deterioration. As seeds deteriorate, respiration decreases as a result of impairment of mitochondrial activity and reduction in number of mitochondria. Poor storage causes damage to membrane phospholipids and reduce selective permeability of cellular membranes of deteriorated seeds. Decline in activity of pre-existing enzymes such as glutamic acid decarboxylase as well as protein synthesis are other expressions of seed deterioration. Glucose utilization may incapacitate at the early stage of germination of deteriorated seeds and biomolecules hydrolyze rapidly, resulting in increased free fatty acids, amino acids and inorganic phosphate (1,12). Storage conditions (i.e. temperature, relative humidity), type of seed, moisture content and length of storage all affect the maintenance of seed vigor during storage (15). Loss of vigor can also be due to the presence of seedborne diseases or mechanical damage of stored seeds (23). 130 Stewart and Bewley (40) reported high levels of peroxidation (autoxidation) of unsaturated fatty acids and a decrease in linoleic (18:2) and linolenic (18:3) acids in stored soybean under high temperature and humidity. None of these changes occur under high temperature and low humidity. Physiological and Biochemical Changes Accompany Vigor Loss Loss of vigor is accompanied by breakdown of membrane integrity which, in turn, increases metabolite leachates (sugars, amino acids and inorganic salts) from the seed. Lipid oxidation and enzymatic degradation may also be associated with vigor loss. Other factors which may be correlated with vigor loss include low oxygen uptake, increase in CO, production and reduction in protein and carbohydrate synthesis (1,2,3,39,47). Wahab and Burris (46) found that the accumulation of fresh and dry matter, respiration rate (oxygen uptake), seedling length and total reducing sugars and.amino acids‘were significantly greater in both the embryonic axes and the cotyledons.of high quality germinating soybean seeds than from low quality seeds. Their observations suggested that low-quality seeds mobilize and interconvert reserves from cotyledons to the embryonic axes at a slower rate than high-quality seeds. They also showed that low-quality seeds 131 exhibit lower respiration activity than high-quality seeds. Abdul-Baki (1) found a positive correlation between seed vigor and protein content. Measurement of Seed Vigor The general strategy in determining seed vigor is to measure some aspects of seed deterioration or genetic deficiency which are either indirectly or directly proportional to seed vigor (8). Isely (26) categorized vigor tests into two types: (1) direct tests which simulate field conditions (e.g. cold test) and (2) indirect tests which measure certain seed physiological.and biochemical aspects of the seed. These include such as tetrazolium and accelerated aging tests, plus tests for speed of germination.(first count) and growth rate (increase in dry weight) of seedlings. Woodstock (47) reported that seed is the only biological variable involved in indirect tests (vigor per se) regardless of weather, field conditions or soil-borne diseases, whereas direct tests (e.g., soil tests) involve the seed-environment interaction. He added that vigor cannot be completely separated from viability at the cellular level as shown in the TZ test. Likewise, seedling growth (vigor) is dependent on germination (viability) and both depend on cell elongation and division. McDonald (33) divided vigor tests into three categories: (1) physical tests which includes seed 132 characteristics such as size, weight and density and the internal development; ( 2) physiological tests such as standard germination, speed of germination, seedling growth rate; and physiological stress tests such as the cold test and accelerated aging test and (3) biochemical tests such as the tetrazolium test, ATP test, glutamic acid decarboxylase activity test and electrical conductivity test. Woodstock (47) suggested similar categorizations. A survey by AOSA has shown that the six most common vigor tests in the United States are the cold test, accelerated aging test, tetrazolium test, growth rate test, the speed of germination test and the cool test (19). Isely (26) considered the cold test to be the best vigor test for corn in the United States. The relationship between seed vigor and field performance is well studied. Anfinrud and Schneiter ( 6) found correlation between the conductivity test, accelerated aging test, germination at 10 C and seedling vigor classification with field emergence of sunflower seeds. Beighley and Hopper (9) found the standard germination test and cool test to be highly correlated with field.emergence rate index. On the other hand, TeKrony and Egli (44) found that all vigor tests overpredicted field emergence of soybean under adverse soil conditions (severe crusting and cold soil temperatures). 133 They reported that the vigor index concept offers the possibility of combining laboratory parameters relating to seed and seedling vigor. Kulik and Yaklich (29) reached a similar conclusion for soybeans and reported that no single vigor test was precise enough to predict field emergence. They differentiated between an estimate of potential field emergence and a prediction of field emergence and suggested that prediction is valid under any field condition while an estimate is valid over a wide range of field conditions. They also showed that the accelerated aging, tetrazolium and cold tests have given consistent estimates of potential field emergence. Miles and Copeland (35) found a correlation between cold, accelerated aging, conductivity, tetrazolium and 4-day count tests with field emergence in soybean. Polizotto (38) found that standard germination test had the highest correlation with field emergence followed by accelerated aging, tetrazolium and the cold test. He found no relationship between vigor and yield of soybean. Suryatmama gt g1. (41) reported that the standard germination test gave the best correlation with field emergence under optimal conditions. Under less favorable conditions, a combination of standard germination and accelerated aging tests provided the best prediction of field emergence. Under stress conditions, the conductivity test appeared to give the best single 134 estimate of vigor and field emergence. They also found very low correlation between seed vigor and crop yield under both optimal and stress conditions. Bishnoi and Delouche (10) reported that the results of the cold test and the standard germination test were closely correlated for non-deteriorated cotton seeds. They also found good correlation between field establishment and results of the cold test, accelerated aging test, 02 uptake and 3-day root length. Seed weight, ATP’content.ofIhydrated.embryos or 3-day old seedling, and 7-day seedling dry weight were found to be good for predicting field emergence of thirteen barley cultivars (13). Grabe (19) reported that both speed of germination and seedling growth rate tests have similar seed attributes. He found seedling growth rate to be more closely related to potential seedling growth rate in the field than to percent stand establishment. Woodstock (47) showed a high relationship between oxygen uptake and respiratory quotient (oxygen uptake and CO, evolution) and germination capacity. He also reported that storage microfloral respiration can mask very low rates of seed respiration. Burris and Navrall (11) studied the relationship between different methods of conducting the cold test and field emergence in 15 corn inbreeds. They concluded that the elimination of soil from the test minimizes variability 135 between laboratories and that this test is not the best predictor of field emergence under cold, wet conditions. Hampton (20) found a good correlation between the cold test (5 C for 7 days, then 20 C for 4 days) and field emergence of wheat. He found no advantage of vigor testing when soil conditions are favorable. Hepburn gt g1. (21) showed that a single probe conductivity meter was better in differentiating between dead and living seeds than a multiprobe automatic seed analyzer in peas. He reported that conversion of the data to a per gram basis gave little improvement in distinguishing between dead and live seeds. Johnson and Wax (27) reported that the cold test showed a consistently high correlation with field emergence and final stand for number of soybean cultivars. They concluded that several vigor tests (e.g., conductivity, accelerated aging, standard germination) were better correlated with field emergence in favorable seedbed environments than under unfavorable environments. Loeffler gt gl. (30) used the rolled-towel, no-soil cold test to measure the reduction in corn seed quality associated with high drying temperatures. They described it as adequate, sensitive, reproducible and simple. Tao (42) studied the causes of variation in the conductivity test for soybean seeds and concluded that conductivity increases as temperature increases, as the number 136 of cracked seeds increases, as seed moisture content decreases and with the use of tap water instead of distilled or deionized water. He found that seed size had a slight effect in one seed lot but not in another. In another corn study, his results indicated that seed treatment could interfere with the conductivity test depending on the seed treatment and the source of the treated seeds. Washing seeds with methanol eliminated the seed treatment interference (43). Joo gt g1. (24) reported the conductivity test to be a quick, useful technique in predicting corn seed emergence potential. She found a high correlation between the conductivity test, the cold test and field emergence of hybrid corn. They emphasized the importance of temperature, leaching period and the initial moisture content in conducting this test. Yaklich gt g1. ( 48) found that conductivity, radioactive leachate and protein synthesis were significantly correlated with emergence under different soil types and dates of planting. Water uptake/g seed weight also showed low but significant correlation with field emergence. However, ATP and seed weight were not significantly correlated with field emergence. NLNTERflAléiAbfl)LflEEH£HMS Cultivars and Aging Procedure Two*winter, Ceres and Liborius, and two spring, Delta and Bounty, canola seed lots from the 1991 crop were used in this study. Initial seed quality, 1000-seed weight and moisture content of each lot were determined. Six quality levels of each cultivar were created by artificially aging the seeds at 42 C for 0, 16, 24, 48, 60 and 72 hours using the "wire-mesh tray" method (34) to provide sub-lots with a range of seed vigor. Seed quality Determination Each sub-lot was evaluated for quality by the following laboratory tests: Accelerated Aging Test: A single layer of seeds from each lot was placed on the copper wire mesh tray and 100 ml of distilled water added to each of the plastic boxes and the wire-mesh trays were placed inside the plastic boxes. The boxes were sealed with tape and incubated at 42 C for periods of 0, 16, 24, 48, 60 and 72 hours. The seeds were then removed, air dried at room temperature for two days and germinated using standard germination test procedures described below. 137 138 Standard Germination Test and First-Count Germination Test: Three 100-seed.replications.of each.sub-lot of the four canola cultivars were germinated on moistened blotter papers on germination trays. The trays were placed in a sealed germination cart and kept at 22 C. First counts were made four days after planting and final counts after seven days. Normal seedlings, as described in the AOSA Rules for Testing Seeds (7) , were recorded. First-count germination was performed only for the spring cultivars. Cold Test: Three loo-seed replicates of each quality level of both winter and spring cultivars were germinated on moistened blotter papers and placed in the germination cart for standard germination tests. The carts were incubated at 5 C for periods of 3 and 5 days and then transferred to 22 C for 7 days. Only normal seedlings were counted. Cold Soil Test: The same procedures of the cold test described above were used except that the seeds were planted on moistened blotter paper and covered with soil moistened with water. Normal seedlings that.emerged above the soil level were counted. Electrical Conductivity Test: A conductivity bridge apparatus 'Model 31' manufactured by Yellow Springs Instrument Co. , Inc. Yellow Springs, Ohio 45387 was used to measure the change in conductivity caused by materials (electrolytes) leached from 139 seeds into distilled water. Three replicates of 200 uninjured, untreated seeds from each sub-lot of the winter and the spring cultivars were soaked in 50 ml of distilled water at 22 C. After 16 hours of incubation, the seeds and steep water were stirred briefly and the conductivity electrode was dipped into the flask. The dial control switch was used to set the appropriate conductivity range (e.g., 100 umhos) and conductivity readings were recorded by obtaining the maximum width of the light field using the drive knob. Field Emergence Four loo-seed replicates of each quality level of the four canola cultivars were hand planted in four-foot rows in a completely randomized design. Fifteen days after planting, the emergence of each row was counted. Statistical Analysis Analysis of variance was calculated to determine the effect of each treatment on seed quality. The association between the various test results and field emergence was determined using simple correlation coefficients. The means of the different treatments were separated using the Duncan's multiple range test or the least significant difference test (LSD). IRESULTS The Standard Germination Test The initial seed quality of Ceres, Liborius, Delta and Bounty was 98, 81, 92 and.82 percent, respectively as measured by the standard germination test (Table 3). The seed moisture content of the four cultivars was about 5.5%. The 1000-seed weight was 4.35, 4.30, 3.27 and 3.46 grams for Ceres, Liborius, Delta and Bounty, respectively. Both seed vigor and viability were significantly affected by differences between cultivars and seed quality levels within each cultivar as indicated by the accelerated aging test, conductivity test, cold test, cold-soil test, first count test (for the spring cultivars) and field emergence (Tables 1,2). Incubation period (3 days and 5 days) used for cold test and cold-soil test, significantly affected the germinability of all seed lots, except for the winter cultivars ‘which. were not affected, by the difference in prechilling period (Table 2). All vigor tests were highly correlated with field emergence and with each other for both winter and spring cultivars (Tables 9,10). However, the correlation between the standard germination test and field emergence was not significant for the spring cultivars at 0.77 (P=0.05) and was significant (0.85) at (P=0.05) in winter cultivars. 140 141 Table 1. Analysis of variance for factors affecting seed quality of two winter (W), Ceres and Liborius, and two spring (8), Delta and Bounty, canola cultivars measured by accelerated aging, conductivity, first count tests and field emergence. Effect df Accel. ag. Cond. test First count field emg W S W S W S W S Probability Vuiety (v) 1 *4 «a at at .. *1: u ** Aging period (A) 5 *4 4* ** ** -- *4 *4 4* (v) x (A) 5 *4 *4 * *4 -- ** ** ns *,** Indicate significance at 0.05 and 0.01 level of probability, respectively according to the F-test. 142 Table 2. Analysis of variance for factors affecting seed quality of two winter, Ceres and Liborius, and two spring, Delta and Bounty, canola cultivars measured by cold and cold soil tests. Cold test Cold-soil test Effect df Winter Spring Winter Spring Probability Variety (V) l ** ** ** ** Prechilling period (P) 1 ns ** ** ** (3d and 5d) (V) x (P) 1 ns ns ns ns Aging period (A) 5 ** ** ** ** (v) x (A) 5 as as we as (P) X (A) 5 ns ** ng as (v) x (p) x (A) 5 a as as as *,** Indicate significance at 0.05 and 0.01 level of probability, respectively according to the F-test. 143 The Accelerated Aging Test As the aging period increased the germinability of seeds of all cultivars decreased. Aging at 42 C for different periods of time (i.e., 0, 16, 24, 48, 60 and 72 hours) created different levels of seed quality. Cultivars varied in tolerance to high temperature (42 C) and relative humidity (near 100%). After 16 hour of aging, germination of Ceres dropped from 98 to 88% and from 81 to 69% for Liborius. The spring cultivars, however, were less affected by the aging; after aging for 16 hours, germination of Delta dropped only 3% from 92% to 89%, whereas Bounty dropped 4% from 82% to 78%. After 72 hours of aging all cultivars declined sharply except for Delta which showed better tolerance to higher temperatures and had 59% germination compared to 7, 0 and 1% for Ceres, Liborius and Bounty, respectively. After 24 hours of aging, both spring cultivars lost only 7% from their initial germination levels, whereas Ceres lost 19% and Liborius 32%. Deterioration of the winter cultivar Liborius was the most rapid of all cultivars tested (Table 3 and Fig. 1-4). Accelerated aging test results of Delta did not drop sharply, even after 72 hours of aging. Likewise, electric conductivity results for Delta did not increase as sharply as for the other cultivars (Table 7). 144 Table 3. Accelerated aging test results of two winter and two spring canola cultivars aged for different periods of time. Aging Winter Spring period (h) Ceres Liborius Delta Bounty % germination 0 98a* 81a 92a 82a 16 88b 69b 89ab 78ab 24 79C 49C 85b 75b 48 65d 5d 75C 46C 60 26e 0e 65d 39d 72 7f 0e 59e le * Means followed by the same letter within a column are not significantly different (P=0.05) according to the LSD test. 145 The correlation between this test and field emergence was also highly significant (Tables 9,10). The First-Count Germination Test The first-count germination results were consistent with the standard germination and accelerated aging results in that a gradual decline in the normal seedlings occurred as the period of aging increased. Delta germinated better than Bounty, especially at lower seed quality levels. Unaged (control) Delta seeds germinated of 65% compared to 54% for Bounty. After 48 hours of aging, Delta germinated 47% compared to 19% for Bounty. After 72 hours of aging, Delta produced 32 normal seedlings compared to none for Bounty (Table 4 and Fig. 3-4). High correlation between first-count germination results and field emergence was found (Table 9). 146 Table 4. First count germination test results of seeds of two spring canola cultivars aged for different periods of time. Aging period (h) Delta Bounty % germination 0 65a* 54a 16 54b 46b 24 49c 44b 48 47c 19c 60 42d 5d 72 32e 0e * Means followed by the same letter within a column are not significantly different (P=0.05) according the to LSD test. 147 The Cold Test The response of the cultivars, and quality levels within each cultivar to different cold test pre-treatment periods (i.e., 3 and 5 days) was varied. The germinability of unaged (control) Liborius was 72% following a three-day cold period and 75% after five days, but was 37% for the sample aged for 24 hours after a 3 day cold period and 29% after 5 days. Ceres, aged for 16 hours had 84 and 82% germination after 3 day and 5 day cold exposure, respectively. However, Ceres aged for 24 hours had 52% germination after 3 days and 56% after 5 days. Delta was the only cultivar that had a consistent pattern in which the 5 day cold period decreased germinability more than the 3-day period (Table 5 and Fig. 1-4). Generally, as the quality of the seeds decreased the effect of cold treatment became more severe. The germinability of Ceres aged for 24 hours dropped from 79% to 52% after receiving the cold treatment for three days (Table 5 and Figure 1). The cold test and field emergence results were highly correlated with each other (Tables 9,10). 148 Table 5. Cold test results of two winter and two spring canola cultivars aged for different periods of time. Aging Ceres Liborius Delta Bounty period (h) 3d* 5d 3d 5d 3d 5d 3d 5d % germination 0 95a** 94a 72a 75a 91a 88a 80a 79a 16 84b 82b 64b 64b 88ab 85ab 76b 77ab 24 52d 56c 37c 29c 86b 83b 74b 75b 48 59c 55c 6d 6d 74c 69c 37c 30c 60 5e 4d 0e 0e 60d 51d 33d 19d 72 1f ld 0e 0e 53e 44e 0e 0e * Prechilling at 5 C for periods of 3 and 5 days was used in the test. ** Means followed by the same letter within a column are not significantly different (P=0.05) according to the LSD test. 149 The Cold Soil Test The cold soil test succeeded in simulating field conditions in that results of this test and field emergence were similar for all seed lots. After a five day cold exposure, germinability of all unaged (control) lots dropped significantly. Germinability of Ceres dropped from 98% to 90%, Liborius from 81% to 56%, Delta from 92% to 85% and Bounty from 82% to 76% (Table 6 and Figures 1-4). Delta retained its higher quality than the other three cultivars. All sub-lots of all cultivars showed similar trends. Unlike the cold test, a 5-day cold exposure period in cold soil test showed a more consistent pattern over 3 days (Table 6). The correlation between the results of this test and field emergence was highly significant (Tables 9,10). The Electrical Conductivity Test The electrical conductivity test accurately reflected differences in the initial seed quality between cultivars and distinguished between different seed quality levels within each cultivar. 150 Table 6. Cold soil test results of two winter and two spring canola cultivars aged for different periods of time. Aging Ceres Liborius Delta Bounty period (h) 3d* 5d 3d 5d 3d 5d 3d 5d % germination 0 92a** 90a 64a 56a 88a 85a 76a 76a 16 79b 77b 56b 55a 85ab 84ab 74a 71b 24 52C 35d 21C 21b 82b 80b 73a 69b 48 46d 42c 3d 3c 74c 70c 37b 26c 60 4e 3e 0d 0c 61d 49d 29¢ 21d 72 1e 0e 0d 0c 53e 46d 0d 0e * Prechilling at 5 C for periods of 3 and 5 days was used in the test. ** Means followed by the same letter within a column are not significantly different (P=0.05) according to the LSD test. I 151 As the seed vigor increased, the corresponding electrical conductivity also decreased. The initial standard germination of the four cultivars was 98, 81, 92 and 82% for Ceres, Liborius, Delta and Bounty, respectively; the corresponding electrical conductivity' was 67, 83, 71 and. 80 umhos/g, respectively. After aging all cultivars for 72 hours, the conductivity readings were 241, 254, 96 and 224 umhos/g, respectively (Table 7 and Figures 1-4). The accelerated aging test results showed that the germinability of Delta did not drop sharply, even after 72 hours of aging. Likewise, the electrical conductivity did not increase sharply (96 umhos/g), as was the case for the other three cultivars (Table 7). The correlation between conductivity results and field emergence was highly significant (Tables 9,10). Field Performance The field emergence results reflected differences in the initial seed vigor between cultivars as well as differences in seed quality within each cultivar. Field emergence showed the same pattern of results found in all vigor tests performed in this study for all seed lots. Ceres, Liborius, Delta and Bounty with initial quality (germination levels) of 98, 81, 92 and 82%, respectively, had field emergence results of ‘79, 56, 73 and 65%, respectively (Table 8 and Fig. 1-4). 152 Table 7. Electrical conductivity means of seeds of two winter and two spring canola cultivars aged for different periods of time. Aging Winter Spring period (h) Ceres Liborius Delta Bounty umhos/g 0 67a* 83a 71a 80a 16 86b 88a 77a 82a 24 113C 109b 86b 94b 48 133d 172c 91bc 132c 60 233e 247d 94bC 145d 72 24le 254d 96c 224e * Means followed by the same letter within a column are not significantly different (P=0.05) according to the LSD test. 153 Standard germination test.results were not significantly correlated with field emergence for the spring cultivars but were significant at P=0.05 for the winter cultivars. Field emergence results were significantly correlated with the results of all vigor tests (Table 9,10). 154 Table 8. Field emergence means of seeds of two winter and two spring canola cultivars aged for different periods of time. Aging Winter Spring period (h) Ceres Liborius Delta Bounty emergence % 0 79a* 56a 73a 65a 16 70ab 48ab 68ab 58a 24 65b 39b 58bC 56a 48 35C 2C 51C 42b 60 26C 0C 30d 24C 72 0d 0C 26d 0d * Means followed by the same letter within a column are not significantly different (P=0.05) according to the LSD test. Germination (96) Germination (96) 155 _ 8 OAcceI. waging Q'Cold test *'C9'df§9§i must”, «mean... : ”55*? 80 . , 60‘ 40- i ..... 20- ............... -LSD(5%) '0' 36 i' .5 i— .5 + =17'¢'=1‘1 0 , , . ° ‘5 24 48 60 Aging period (hours) Conductivity (umhoslg) Figure 1. Relationship between accelerated aging. cold. cold soil and conductivity tests and field emergence of aged winter canola seed cv. Ceres. o ‘ «e- -x- 4 100- Accel. aging Cold test Cold soil ‘17 280 +Cond. test '0' Field emg. +240 .4 O 80 . 3- ' .-200 g 5 60- -160 "’ a .2 4o- ' "3"120 U L50 (5%) end its .3 -F- as -s-=17j -30 S 20 - "' =“ 0 P40 \. 0 16 24 48 60 72 Aging period (hours) Figure 2. Relationship between accelerated aging, cold. cold soil. conductivity tests and field emergence of aged winter canola seed cv. Liborius. Germination (96) Germination (96) 156 200 LSD (5%)‘0 85 i- =4 4- =4 .*83'¢-811+?5. 80 g o .c < 5 60% V ' >~ .1'.’ .2 - U 40 y g c o O T.""Cond.i.testi '4' Field emL +Fil'3tCot ' ‘ 4......7....46......agg o l '1 o O 16 24 48 60 72 Aging period (hours) Figure 3. Relationship between accelerated aging, cold, cold soil, first count and conductivity tests and field emergence of aged spring canola cv. Delta. :‘0'Accel. aging (‘3' Cold. test .*