植株株齡與器官培植體對農友933番茄癒傷組織及懸浮細胞分化能力之影響

Effect of the Age of Plant and Organ Explants on Differentiation of Callus and Suspension Cells in tomato (Solanum lycopersicum cv. Known-You 933)

蔡凱桓

番茄 ； 植株齡期 ； 器官培植體 ； 懸浮細胞培養 ； 體胚分化 ； tomato (Solanum lycopersicum cv. Known-You 933) ； plant age ； organ explant ； cell proliferation ； somatic embryogenesis

屏東科技大學植物醫學系所學位論文

2016年

碩士

鄭秋雄

繁體中文

本研究為建立具備分化能力之懸浮細胞，探討供試植物栽培方式、植株齡期、器官培植體、癒傷組織來源及植物生長調節劑組成對癒傷組織誘導、體胚分化、懸浮細胞增殖與細胞團分化之影響。取農友933番茄種子以盆缽栽培與試管無菌培養，並以發芽後第8天、22天及43天株齡為供試植株。其中8天株齡之胚軸、胚根與22天及43天株齡之莖部上、中、下段及根段做為培植體。為比較供試植物栽培方式、植株齡期與器官培植體對癒傷組織誘導之影響。將上述培植體分別培養於含2 mg/l NAA及4 mg/l Kinetin之CS-1培養基22天，結果試管無菌培養43天株齡之癒傷組織形成率最佳，其上段、中段、下段莖部培植體之癒傷組織形成率與單位培植體癒傷組織鮮重分別為98%、96%及90%與0.314 g、0.543 g及0.207 g。為比較不同株齡各培植體來源癒傷組織之體胚分化率，將0.02 g及0.03 g鮮重癒傷組織培植體分別培養於含10 mg/l NAA或0.5 mg/l IAA及3.5 mg/l BA之CS-1固態培養基，結果其體胚分化率以43天株齡誘導之癒傷組織培養於含0.5 mg/l IAA及3.5 mg/l BA植物生長調節劑者最佳。其莖部上段、中段與下段來源癒傷組織之體胚分化率與單位培植體分化體胚數分別為82.2%、93.18%及100%與1.42個、2.93個及3.37個。比較不同株齡各培植體來源癒傷組織之懸浮細胞數、細胞增殖率，則以43天株齡中段莖部來源癒傷組織克鮮重之懸浮細胞數最多，約5.23×106 cells/g callus，且其細胞增殖率亦最佳，其初代培養、第一次繼代培養及第二次繼代培養之細胞增殖率分別為319%、716.7%及503.3%。比較不同株齡各培植體來源癒傷組織之懸浮細胞之細胞團分化率，結果以43天株齡下段莖部來源癒傷組織之懸浮細胞者最佳，每毫升培養基可分化出480個細胞團。上述結果顯示，供試植株株齡與器官培植體對誘導具分化能力之癒傷組織及懸浮細胞影響至鉅，株齡較成熟者優於幼齡者。本研究建立之最佳培養條件為：(1)體胚分化培養：試管內無菌培養43天株齡之下段莖部組織誘導之癒傷組織為培植體，培養於含0.5 mg/l IAA及3.5 mg/l BA之CS-1培養基14天可直接分化體胚，其體胚分化率約91.8%；(2)懸浮細胞繼代培養與細胞團分化：誘導之懸浮細胞培養於含5 mg/l NAA之CS-1培養基，其繼代培養增殖率約2.1倍至6.1倍。當懸浮細胞培養於含15 mg/l NAA之CSW-1液態培養基，則每毫升培養液中可分化出450個細胞團。

In order to establish the suspension cells that have the good ability in differentiation, we evaluated effects of the cultivate method of test plant, plant age, organ explants, callus source and the composition of plant growth regulator on callus induction, somatic embryogenesis, suspension cell proliferation and cell cluster differentiation. The seeds, Solanum lycopersicum cv. Known-You 933, cultivated in the pot or in vitro. 8, 22, 43-day-old plants after germination offered as the test plants. Hypocotyl and radicle of 8-day-old plants, the upper, middle and basal section of the stem and the root section of 22, 43-day-old plants offered as explants. In order to compare the effect of the cultivate method of test plant, plant age and organ explants on callus induction. The above-mentioned explants cultured on CS-1 medium containing 2 mg/l NAA and 4 mg/l Kinetin for 22 days. 43-day-old plants in vitro had the best result in callus formation rate. Callus formation rate of the upper, middle and basal section of stem were 98%, 96% and 90%, and the fresh weight of callus per explant were 0.314 g, 0.543 g and 0.207 g. compared the somatic embryogenesis rate of callus induced from the explants of the different age of plant. 0.02 g and 0.03 g fresh weight of callus offered as the explants, cultured on CS-1 solid medium containing 10 mg/l NAA or 0.5 mg/l IAA and 3.5 mg/l BA respectively. The result indicated that callus induced from 43-day-old-plant cultured on 0.5 mg/l NAA and 3.5 mg/l BA was the best in somatic embryogenesis. Somatic embryogenesis rate of the upper, middle and basal section of stem were 82.2%, 93.18% and 100%; the numbers of somatic embryo per explant were 1.42, 2.93 and 3.37. In comparison of the suspension cell number and cell proliferation rate among the callus induced from each explant of the different age of plants. Callus induced from the middle fragment of 43-day-old plants showed the best result in the number of suspension cell, which about 5.23×106 cells per gram callus, the proliferation rate of cell was the best, too. The cell proliferation rate in primary culture, the first subculture and the second subculture were 319%, 716.7% and 503.3%. Compared the differentiation rate of cell cluster differentiated from the suspension cells among the callus induced from each explant of the different age of plants. The suspension cells suspended from the callus induced from the basal fragment of stem of 43-day-old plants was the best, which differentiated 480 cell clusters per milliliter medium. Summarize the above-mentioned, the results indicated that the plant age and organ explants had a great influence on induction of callus and suspension cells that had the good ability in differentiation, that is, the mature plants were better than the young. In this study, it’s found that (1) In somatic embryogenesis, callus induced from the basal fragments of stem of 43-day-old plants in vitro offered as the explants, and cultured on CS-1 medium containing 0.5 mg/l IAA and 3.5 mg/l BA for 14 days. It could induced the somatic embryos directly, which about 91.8% in the rate of somatic embryogenesis. (2) In subculture of suspension cell and differentiation of cell cluster, the suspension cells cultured in CS-1 liquid medium containing 5 mg/l NAA, the cell proliferation rate in subculture was 2.1-6.1 times. While the suspension cells cultured in CSW-1 liquid medium containing 15 mg/l NAA could differentiated 450 cell clusters per milliliter medium.

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