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研究生:徐振權
研究生(外文):Chen-Chuan Shyu
論文名稱:番茄癒傷組織誘導、細胞培養及體胚分化之研究
論文名稱(外文):Studies on Callus Induction, Cell Culture and Somatic Embryogenesis of Lycopersicon esculentum var. Santa
指導教授:鄭秋雄
指導教授(外文):Chiu-Chsiung Cheng
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:熱帶農業研究所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:144
中文關鍵詞:番茄癒傷組織細胞培養體胚分化
外文關鍵詞:tomato (Lycopersicon esculentum var. Santa.)calluscell culturesomatic embryogenesis
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本研究之目的為研發番茄癒傷組織誘導培養技術、懸浮細胞培養技術及體胚分化培養技術,建立經濟、快速、安全之體胚分化系統,以做為無病毒種苗大量繁殖及抗性品種篩選、育種工作之參考依據。以盆栽45-50天株齡聖女番茄植株之葉肉組織及莖部組織做為供試材料,探討培養基組成、植物生長調節劑組成、氨基酸組成、維他命組成、蔗糖含量、光照度及培植體對誘導番茄癒傷組織之影響。結果以莖組織之上、中段組織及葉塊組織做為培植體,培養於含10~12 mg/l NAA、六種維他命(0.5mg/l thiamina-HCl, 0.5 mg/l pyridoxine-HCl, 0.5 mg/l nicotinic acid, 0.1mg/l biotin, 0.4 mg/l folic acid and 0.5 mg/l ascorbic acid)、兩種氨基酸(1 mg/l glutamic acid及1 mg/l aspartic acid)及3% 蔗糖 之CS-1 固體培養基中,於3200~4200 lux之光照度等培養條件下誘導培養10天不僅可獲得高產量、質地鬆軟、褐變率低之癒傷組織,且可縮短誘導培養時間。另,為獲得增殖能高、活性佳及分化能強之番茄培養細胞,探討培養基組成、植物生長調節劑組成、碳素源及含量、pH值、光照度、培養細胞來源及培養細胞密度對培養細胞增殖之影響,結果以莖部上、中段組織之培養細胞以2.5×105 cells/ml為細胞密度培養於含10 mg/l NAA、3% 蔗糖、0.03%葡萄糖、0.03%果糖之CSW-1 液體培養基(pH 6.0~6.5)中,於無光下振盪培養(100 rpm/min.)7天可達最高之增殖倍數約1.18倍,且可降低培養細胞之破裂。以此培養條件進行番茄培養細胞生長曲線之觀察,其各時期分別為0~1天為誘導期(lag phase),1~4天為對數增殖期(exponential phase),4~5天為直線生長期(linear phase),5~9天為衰退期(progressive deceleration),9~10天為穩定期(stationary phase),故番茄懸浮細胞增殖培養之繼代培養應以4~5天為單位繼代培養時間為宜。又,為建立番茄體胚分化培養技術,探討植物生長調節劑組成、蔗糖含量、培養細胞來源、細胞團誘導培養天數及光照度對單細胞分化體胚之影響,結果以莖部中段組織之培養細胞培養於含10 mg/l NAA之CSW-1液體培養基中,於無光下培養7天分化出細胞團,再將細胞團移入含1 mg/l NAA 及 5~7 mg/l BA之CSW-1培養基於無光下行繼代培養14天(每週更換新鮮培養基)之體胚分化率最佳,約14.9%。
Abstract
The purposes of this study are to develop the techniques of callus induction, suspension cell culture, embryogenesis of tomato, to establish a economical, effective and safety system for somatic embryogenesis of tomato and to serve as the references for micropropagation of virus-free seedling and the selection and breeding of disease-resistant strains. Using the stem tissue and leaf disc of 45~50-day-old of Lycopersicon esculentum var. Santa., as the materials to evaluate the effects of the compositions of medium, plant growth regulator, amino acid and vitamin, sucrose concentration, light intensity and explants on callus induction of tomato. The results indicated that the upper and middle sections of the stem and mesophyll cultured on CS-1 medium supplemented with 10~12 mg/l NAA, 6 vitamins (0.5mg/l thiamine-HCl, 0.5 mg/l pyridoxine-HCl, 0.5 mg/l nicotinic acid, 0.1mg/l biotin, 0.4 mg/l folic acid and 0.5 mg/l ascorbic acid), 2 amino acids (1 mg/l glutamic acid and 1 mg/l aspartic acid) and 3% sucrose at 3200~4200 lux light intensity for 10 days were best in callus formation, inducing a high yield, friable, low-browned of callus as well as shorten the induction time. In order to induce the vital and highly regenerable callus, the effects of the compositions of medium, sources and contents of carbon, pH values, light intensity, sources of the culture cell and density of the culture cell on the proliferation of culture cell were evaluated. The results showed that 2.5×105 cells/ml culture cells, isolated from the stem tissue of L. esculentum var. Santa., shaking at 100 rpm in CSW-1 liquid medium supplemented with 10 mg/l NAA, 3% sucrose, 0.03% glucose and 0.03% fructose (pH 6.0~6.5) in the dark for 7 days had the highest proliferation, which increased callus formation for 1.18 times and decreased the rupture of the culture cells. The tomato cells cultured under the same conditions produced, the growth curves with phases identified as the lag phase occurred in 0~1 days, the exponential phase in 1~4 days, the linear phase in 4~5 days, the progressive deceleration in 5~9 days, and the stationary phase in 9~10 days, Consequently the time requested for subculture of cells was 4~5 days. Using the technique of somatic embryogenesis of tomato, the compositions of the plant growth regulator, sucrose concentration, light intensity, sources of the culture cell and number of days requested for of the cell cluster induction on the single cells differentiate to the somatic embryos were evaluated. The culture cells, isolated from the middle section of the stem tissue, which were first cultured in CSW-1 liquid medium supplemented with 10 mg/l NAA in the dark condition for 7 days for cell differentiate and then subcultured in CSW-1 liquid medium supplemented with 1 mg/l NAA and 5~7 mg/l BA in the dark condition for 14 days (replaced with fresh medium at intervals of one week) resulted in the best somatic embryogenesis, about 14.9%.
目錄
中文摘要………………………………………………………I
英文摘要………………………………………………………IV
誌謝……………………………………………………………VII
目錄……………………………………………………………VIII
表次索引………………………………………………………IX
圖次索引………………………………………………………XII
前言……………………………………………………………1
前人研究………………………………………………………4
一、番茄癒傷組織之誘導培養……………………………4
二、番茄懸浮細胞培養……………………………………5
三、番茄植株分化…………………………………………6
材料方法………………………………………………………8
一、番茄癒傷組織誘導培養………………………………8
二、番茄懸浮細胞增殖培養………………………………16
三、番茄細胞團分化培養及體胚分化培養………………20
結果……………………………………………………………26
一、番茄癒傷組織誘導培養………………………………26
二、番茄懸浮細胞培增殖培養……………………………34
三、番茄細胞團分化培養及體胚分化培養………………39
討論……………………………………………………………47
圖表……………………………………………………………63
參考文獻………………………………………………………122
附錄……………………………………………………………135
作者簡介………………………………………………………145
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