Study on the yield and grain starch synthesis of NaN3-induced mutant of Tainung 67

水稻為我國最重要的栽培作物，產量和品質為水稻栽培的兩大目標。澱粉是水稻榖粒的主要成分，穀粒內澱粉合成相關酵素活性表現，與水稻穀粒發育的量與品質有關，尤其與影響米質重要因子之直鏈性澱粉合成關係更為密切。台農67號是台灣普遍栽培的一個水稻品種，為台灣之高產代表品種之一，米粒的直鏈性澱粉含量約為20﹪，但米質及二期作產量仍有改進空間。SA419為疊氮化鈉誘變台農67號的突變體，其米粒直鏈性澱粉含量為8﹪，米質不同於台農67號，本研究以該突變體與台農67號為材料，調查其農藝性狀資料，及穀粒澱粉相關酵素表現與穀粒乾重累積和品質關係之探討。 結果發現水稻穗內不同部位穎花的產量貢獻程度有差異，SA419穗內不同部位穎花的產量貢獻程度卻相同，且SA419成熟穀粒的單粒粒重分布較台農67號集中，均為其優良特性。SA419一株穗數較少，一期作產量無法超越台農67號。二期作一株穗數與台農67號差距縮小，產量即高於高產的台農67號。一株穗數較少之因素，可藉由栽培方法改進。水稻充實期SUS、Invertase、Hexokinase、AGPase、UGPase、SSS和SBE等酵素的活性表現，與澱粉的累積有關，且SA419的穀粒充實較快速和較重的粒重，乃因比台農67號有較高活性之故。SA419的穀粒含較低量的直鏈性澱粉，可能因比台農67號有較低的GBSS和SDBE活性之故。穀粒著生位置會影響穀粒生長，因品種不同而改變。以台農67號而言，轉換蔗糖為澱粉的相關酵素活性降低，尤其穀粒充實早期更加明顯，與下位二次枝梗穀粒澱粉累積比上位一次枝梗穀粒低有關連。台農67號下位二次枝梗穀粒直鏈性澱粉含量較低可能因GBSS活性較低之故，此一現象在穀粒充實早和中期尤其明顯。由於上位一次枝梗穀粒和下位二次枝梗穀粒的GBSS活性無明顯差異，SA419的澱粉和直鏈性澱粉含量在兩部位間則無明顯差異。經由NBT酵素活性原位染色，發現水稻穀粒澱粉合成相關酵素主要在胚乳細胞表現，在穀粒發育初期所有胚乳細胞均有酵素活性；隨穀粒發育，酵素活性則由中心細胞往外層細胞失去活性；穀粒發育愈快，往外層細胞失去活性的速度愈快，NBT酵素活性的原位染色結果可提供一般生化分析的參考。希望本研究結果能應用於水稻研究，作為探討及改進台灣水稻產量及品質之參考。Rice is the most important crop in Taiwan. Yield and quality are the main goal for rice cultivation. Starch is the major constitutes in rice grains, therefore, the enzyme activities engaged in starch accumulation are closely related to the rice grain dry matter accumulation and grain quality, especially to amylose accumulation. The cultivar Tainung 67, one of high yield cultivar, is widely distributed in Taiwan, but the grain quality and the yield of second crop season are always needed to be improved. Recently a mutant SA419, mutated by sodium azide from Tainung 67 which contained 20% amylose content in rice grains, was isolated. The mutant SA419 with lower amylose content(8%)in grains has different grain quality in comparison with Tainung 67. In the present study, We compared rice cultivar Tainung 67 and mutant SA419 in the agronomic characteristics, and the relationship between grain dry mater accumulation or grain quality and sugar-starch conversion enzyme activities. The results indicated that the grains located on different branches of panicle contributed different yield, but had the same contribution in mutant SA419. The mutant SA419 also had more concentration distribution of grain weight than cultivar Tainung 67. The mutant SA419, with less panicle number in the first crop, produced less yields than cultivar Tainung 67, but produced higher yield in the second crop season, the panicle number difference between two varieties decreased. The factor of growing less panicle number could be improved by cultural changes. The changes in the rate of starch accumulation correlated well with the changes in the activities of sucrose synthase, invertase. hexokinase, AGPglucose pyrophosphorylase, UDPglucose pyrophosphorylase, phosphoglucoisomerase, phosphoglucomutase, soluble starch synthase, granule-bound starch synthase, starch branching enzyme and starch debranching enzyme, during the grain filling period. The rapid grain-fill and shorter period of grain filling in mutant SA419, as compared with Tainung 67, were associated with its higher activity of starch synthesizing at the early phase of grain growth. The lower amylose content of SA419 grains (8%) in comparison with Tainung 67 grains (20%) was due possibly to their inferiority to synthesize amylose through granule bound starch synthase and starch debranching enzyme. The tested Tainung 67 and SA419 showed different starch and amylose accumulation patterns in different grain positions. In cultivar Tainung 67, the reduced activities of grain enzymes involved in starch biosynthesis, particularly during the early phase of grain filling, were related to the decreased starch accumulation for the grains located on proximal secondary branches as compared to the grains located on distal primary branches. The decreased amylose content in the grains of Tainung 67 located on proximal secondary branches was attributable to their lower granule bound starch synthase and starch debranching enzyme activities at early and mid phases of grain filling. In mutant SA419, significant changes were not observed in the activities of grain enzymes involved in sucrose to starch conversion between the grains located on distal primary and proximal secondary branches of panicle. As a result, little position effects were found in the contents of starch and amylose for these developing grains located on different branches. We found that the technology of enzymes activity in situ staining by NBT would apply to the enzyme involved to the rice grain sugar-starch conversion. The NBT in situ staining results showed that the enzyme activities expressed mainly on the endosperm. NBT staining was expressed at all the endosperm cell at the early phase of grain growth, and gradually reduced from central part of endosperm as grains growing, the faster grains growing, the faster reduction. The results of in situ enzyme activity staining could offer some suggestions on the biochemical analysis for physiological investigation.圖目錄 II 表目錄 VI 縮寫字對照表 VII 中文摘要 1 第一章 緒言 3 第二章 前人研究 5 第三章 水稻台農67號與SA419產量及產量構成要素之 差異性研究 2-1 前人研究 13 2-2 材料與方法 14 2-3 結果 16 2-4 討論 18 第四章 穀粒充實期澱粉合成之研究 2-1 前人研究 31 2-2 材料與方法 32 2-3 結果 37 2-4 討論 39 第五章 水稻穀粒澱粉合成酵素之原位（in situ）染色 2-1 前人研究 48 2-2 材料與方法 49 2-3 結果 51 2-4 討論 53 第六章 水稻穀粒著生位置與澱粉合成之關係 2-1 前人研究 65 2-2 材料與方法 66 2-3 結果 67 2-4 討論 70 第七章 綜合討論與結論 78 英文摘要 86 參考文獻 8

Aging Physiology of Peanut Seed

本研究主要目的為利用人工老化方式，探討花生種子台南11號及立枝仔之 老化生理因素，並以萌爆技術來處理高活力及低活力種子花生種子，以探 討種子活力改善之可能生理因素。試驗結果顯示，人工老化種子之發芽率 ﹑發芽速度﹑發芽係數﹑幼苗長度及幼苗乾重均明顯下降，全粒種子及切 離胚軸之滲漏均隨人工老化處理增加，顯示人工老化處理已造成種子膜系 破壞，因而造成種子之生理代謝異常，因此種子之呼吸作用率及異檸檬酸 分解﹑蘋果酸合成合成均隨人工老化處理明顯下降。本研究也同時測 試脂氧化﹑超氧歧化﹑觸﹑過氧化及ascorbate peroxidase等酵 素在人工老化期間之活性變化，以了解膜系破壞的因素，結果發現除觸 及子葉內脂氧化type-I之活性外，其他各種酵素活性均隨人工老化處理 而下降，所以由脂氧化來促進脂質過氧化而造成種子老化的可能性似乎 不高，但自由基及過氧化物清除酵素活性降低，造成過氧化物及MDA的累 積，這些現象可能與老化現象有關。萌爆處理可明顯提高種子活力，此一 現象可能與種子胚軸內超氧歧化﹑觸﹑過氧化﹑ascorbate peroxidase及種子子葉內觸﹑ascorbate peroxidase等之活性，因萌爆 處理而提升有關。The basic objective of this study was to iden- tify the factors linked to accelerated seed aging in "TN.11" and "Li-Chih Tzae" peanut cultivars. The effect of solid matric priming on high vigor and low vigor peanut seeds were investigated. The results indicated that accelerated aging reduced seed vigor parameters including germination time ,germination coefficient ,seedling length and seedling dry weight. Increasing leakage in seed and axes coincided with the vigor decline,indica- ting the loss of membrane integrity through acce- lerated aging ,Moreover , sereral metabolic acti- vities seed became abberrant , with lower respi- tion and reduced isocitrate lyase and malate synthase activities were obtained in aged seeds. In this study , we aslo evaluated the activities of lipoxygenase ,superoxide dismutase ,catalase ,peroxidase and ascorbate peroxidase in peanut seeds . subjected to diffenrent aging duration in two cultivars. It appeared that most of enzyme lost their activity at accelerated aging except that the activity of cotyledon- catalase and the activity pf cotyledon-lipoxygenase type-II remaining unchanged during accelerated aging. These results seem to indicate that the decresed lipoxygenase activity was unlikely to be the factor including lipid peroxidation . Importantly ,the reduced activities of the scavening enzymes of free radical and peroxide were responsible for the stimulated lipid peroxidation observed in aged peanut seeds . Solid matric priming improved the vigor of peanut seed.This phenomenon might be linked to the increased superoxide dismutase, catalase, peroxidase, and ascorbate activities

Study on Spikelet Distribution Pattern of the Sodium Azide-Induced Mutants of Rice Variety Tainung 67

本研究以自水稻台農67號疊氮化鈉突變庫篩選所獲得的5個穎花分佈型態之突變品系為材料，以誘變親台農67號為對照品種，進行連續2年4期作之穎花分佈調查。結果發現，無論一次枝梗數、一次枝梗穎花數、二次枝梗數及二次枝梗穎花數之數量等性狀，突變品系均產生不同於誘變親台農67號之穎花分佈型式。一次枝梗數增加或減少最多可達3個；大部分突變品系之一次枝梗穎花數均與台農67號相似，約為5~6粒，但突變品系SA0418部分枝梗可增加至7粒。整體而言，每個突變品系在第一期作（2個年度）之二次枝梗穎花數變化曲線並無明顯變異，但第二期作之穎花分佈明顯異於一期作，且年度間表現不一致。突變品系SA04018之二次枝梗穎花數明顯減少，2001年2期作甚至減少到二次枝梗幾乎無穎花之生產，台農67號之二次枝梗穎花數以中部節位最具優勢，突變品系SA04019於一期作則以中上部節位最具優勢，對穗之乾重累積特性而言，可能為優良之突變性狀。總而言之、水稻穎花分佈會隨年度、期作及誘變處理而改變，疊氫化鈉誘變處理可增加穎花分佈之變異程度及產生優良穎花分佈型態之品系，值得研究利用。 Five mutants derived from sodium aside mutagenesis with various spikelet distribution patterns on rice panicle were investigated and compared with its mother parent, TNG67 variety, for four crop seasons. The results have shown that the significant differences were observed in primary rachis-branches number, spikelet number on the primary rachis-branch, number of the secondary rachis-branch and number of spikelet on the secondary rachis-branch as compared with the characteristics of TNG67 variety. Most mutants and TNG67 produce 5~6 spikelets on the primary rachis-branch, but mutant SA0418 produced 7 spikelets on parts of its primary rachis-branch. No distinct difference in spikelet distribution on secondary rachis-branch was observed in the first crop season for two years but it was not stable in the second crop season. Mutant SA0418 produced less spikelets in the secondary rachis-branch as compared with TNG67, and most spikelets were found in the secondary rachis-branch, especially in the secondary crop season of year 2001. In mutant, SA0419, a large number of spikelets distributed in the secondary rachis-branch above the middle nodal in the first crop season providing a good characteristic for dry matter accumulation in rice panicle. In conclusion, spikelets distribution pattern of rice may have variable expressions in different year and crop season, and can also be altered by mutagenesis. Rice mutants with variations in spikelet distribution pattern provide good characteristics for further physiological study as well as variety improvement

Screening of Rice Mutants from a Sodium Azide Derived Mutation Pool of Tainung 67 Variety: Grain Appearance and Amylose Mutants

本試驗以疊氮化鈉誘變所建立之台農67號突變庫已純化之1，120個M7世代突變品系為材料，進行米粒外觀及直鏈性澱粉含量突變體之篩選，結果發現突變品系之白米率及完整米率之突變範圍分別為44~81%及0~78%。突變親台農67號之穀粒外穎及種皮為無色，米粒呈透明，透明度中等，部份米粒有腹白，極少數有心白發生。經誘變後發現有完全心白、完全腹白及不同程度之乳白粒突變體出現，部分品系透明度增加，呈晶瑩剔透狀，但也有很多品系變成似糯性之不透明色。突變後米粒直鏈性澱粉含量分佈範圍從糯性至31%，變異範圍明顯廣大，且呈連續分布。而具有相同直鏈性澱粉含量之突變品系其農藝性狀、千粒重、結實率及米粒型態也呈現相當大之變異。利用糙米橫切面碘液染色，可快速分辨糥性與非糯性突變體、乳白米與糯米突變體，然而所分離的糥性突變品系間，仍有部分直鏈性澱粉合成之差異存在。總之，疊氮化鈉誘變劑誘導水稻台農67號產生許多碾米品質、米粒外觀及直鏈性澱粉含量差異之突變體，提供同一米質多樣性變異之資源，可直接評估其利用性或作為探討澱粉合成之機制及影響米質之相關生理生化特性之材料，提供改進米質與產量之參考。 In this paper, we report the screening of grain appearance and amylose content from 1,120 mutants in M7 generation of a mutation pool derived from TNG67 rice variety induced by sodium azide mutagenesis. The results show that the range of variations in milled rice rate and head rice rate are 44-81% and 0-78%, respectively. The grains of TNG67 variety show colorless husk and seed coat, middle translucent and partial white-belly, mutants colored husks and seed coats, high translucent, all white-belly, milk-white and transparent grains are observed. The grain amylose content of mutants shows continuous distribution to 31%. Mutants with similar amylose content possess various grain characteristics in 1000-grain weight, grain filling rate and morphological shape. Grain cross-section staining with iodide could be applied to differentiate the waxy mutant from non-waxy, milk-white, and waxy-pigmented mutants. These grain mutants provide good materials for rice food processing and starch metabolism studies, and germplasms for rice variety and quality improvement

芹菜種子篩選分級之研究

芹菜種子發芽緩慢不規則，且種子成熟不一又細小，故一批種子常參雜著大小 不一及重量不相等之種子。本試驗借由種子大小篩選及種子重量風選等方式做種子分級， 以比較各級種子活力之差異，供育苗及種子生產之參考。試驗結果顯示，種子大小以中粒 種子所佔比率最高，而大粒種子有較優之發芽率、發芽速度、發芽整齊度及幼苗鮮重，中 粒種子表現次之，小粒種子表現最差。大、中、小粒種子分別經過風選後發現，白骨白葉 小粒種子4.0風力級數以下、青骨青葉中粒種子3.5風力級數以下、白骨青葉中粒種子4.0 風力級數以下，其發芽率Celery seed germination is slow and irregular. Celery seed size is very small, and seed mass is different. The objects of this experiment is to study the quality of celery seeds by sorting their size to three fractions, and blowing their mass to

(57(1):33-48)Studies on Anthocyanidin and Total Phenolic Contents of Sweet Potato

甘藷 (Ipomea batatas) 之不同品種具有豐富之色彩，其花青素組成份及抗氧化能力 值得開發研究。試驗結果得知紫紅色系之藷皮及藷肉均有花青素之合成，花青素之種類 有紫紅色的矢車菊素 (cyanidin) 及紅色的芍藥素 (peonidin)，顏色愈深花青素含量愈 高，但台農73 號藷皮及藷肉之花青素含量均比芋心甘藷低。烘乾藷皮及藷肉之花青素 含量比冷凍乾燥大幅減少，但冷凍乾燥及烘乾會比新鮮材料分別提高10 倍及3-5 倍之 抗氧化能力。花青素含量與抗氧化能力無正相關存在，而與總酚含量呈正相關，但大幅 提高花青素含量，對甘藷抗氧化能力有幫助，更可豐富甘藷色彩，增進甘藷多樣性利用， 所得結果可供新品種紫肉甘藷台農73 號之利用及未來育種方向參考。Anthocyanidin composition and antioxidant activity of sweet potato (Ipomea batatas (L.) Lam) storage roots with plentiful colors were studied. The results showed that cyanidin and peonidin were synthesized in purple-red skin and storage root, and more anthocyanidins were found in darker color ones. The skin and storage root of Tainung 73 cultivar contained less anthocyanidin than that of Purple sweet potato. 70℃ drying had less anthocyanidin content of skin and storage root than freeze drying, but freeze-drying and 70℃ drying increased antioxidant activity 10 times and 3-5 times respectively as compared with fresh materials. Antioxidant activity was positively correlated to total phenolic content, but not to anthocyanidin content. However, increased anthocyanidin content was good for enhancing antioxidant activity of sweet potato. The results could be applied as a reference for the utilization of Tainung 73 cultivar and future breeding of sweet potato