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

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植株株龄与器官培植体对农友933番茄癒伤组织及悬浮细胞分化能力之影响

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

蔡凯桓 , 硕士　　导师：郑秋雄　　

繁体中文

番茄；植株龄期；器官培植体；悬浮细胞培养；体胚分化； tomato (Solanum lycopersicum cv. Known-You 933) ； plant age ； organ explant ； cell proliferation ； somatic embryogenesis

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摘要 │ 参考文献 (58) │ 国际计量

摘要〈TOP〉

本研究为建立具备分化能力之悬浮细胞，探讨供试植物栽培方式、植株龄期、器官培植体、癒伤组织来源及植物生长调节剂组成对癒伤组织诱导、体胚分化、悬浮细胞增殖与细胞团分化之影响。取农友933番茄种子以盆钵栽培与试管无菌培养，并以发芽後第8天、22天及43天株龄为供试植株。其中8天株龄之胚轴、胚根与22天及43天株龄之茎部上、中、下段及根段做为培植体。为比较供试植物栽培方式、植株龄期与器官培植体对癒伤组织诱导之影响。将上述培植体分别培养於含2 mg/l NAA及4 mg/l Kinetin之CS-1培养基22天，结果试管无菌培养43天株龄之癒伤组织形成率最佳，其上段、中段、下段茎部培植体之癒伤组织形成率与单位培植体癒伤组织鲜重分别为98%、96%及90%与0.314 g、0.543 g及0.207 g。为比较不同株龄各培植体来源癒伤组织之体胚分化率，将0.02 g及0.03 g鲜重癒伤组织培植体分别培养於含10 mg/l NAA或0.5 mg/l IAA及3.5 mg/l BA之CS-1固态培养基，结果其体胚分化率以43天株龄诱导之癒伤组织培养於含0.5 mg/l IAA及3.5 mg/l BA植物生长调节剂者最佳。其茎部上段、中段与下段来源癒伤组织之体胚分化率与单位培植体分化体胚数分别为82.2%、93.18%及100%与1.42个、2.93个及3.37个。比较不同株龄各培植体来源癒伤组织之悬浮细胞数、细胞增殖率，则以43天株龄中段茎部来源癒伤组织克鲜重之悬浮细胞数最多，约5.23×106 cells/g callus，且其细胞增殖率亦最佳，其初代培养、第一次继代培养及第二次继代培养之细胞增殖率分别为319%、716.7%及503.3%。比较不同株龄各培植体来源癒伤组织之悬浮细胞之细胞团分化率，结果以43天株龄下段茎部来源癒伤组织之悬浮细胞者最佳，每毫升培养基可分化出480个细胞团。上述结果显示，供试植株株龄与器官培植体对诱导具分化能力之癒伤组织及悬浮细胞影响至钜，株龄较成熟者优於幼龄者。本研究建立之最佳培养条件为：(1)体胚分化培养：试管内无菌培养43天株龄之下段茎部组织诱导之癒伤组织为培植体，培养於含0.5 mg/l IAA及3.5 mg/l BA之CS-1培养基14天可直接分化体胚，其体胚分化率约91.8%；(2)悬浮细胞继代培养与细胞团分化：诱导之悬浮细胞培养於含5 mg/l NAA之CS-1培养基，其继代培养增殖率约2.1倍至6.1倍。当悬浮细胞培养於含15 mg/l NAA之CSW-1液态培养基，则每毫升培养液中可分化出450个细胞团。

并列摘要〈TOP〉

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.

参考文献 ( 58 ) 〈TOP〉

陈季谦 (2008) 阿拉伯芥下胚轴对生长素之年龄专一性反应的分子特性分析。国立成功大学生命科学系硕士论文。98页。
连结：
陈威臣、夏奇铌 (2004) 植物组织培养之拟胚化培养。行政院农业委员会农业试验所技术服务。59: 33-36。
连结：
夏奇铌、陈威臣、曹进义。2014。利用试管内培养(in vitro)辅助育种-抗耐病筛选之探讨。农业试验所技术服务季刊 100: 17-21。
连结：
刘依昌、谢明宪、王仕贤 ( 2007) 夏季小果番茄栽培技术。台南区农业专讯 62: 3-10。
连结：
Amar, A.B., P. Cobanov, K. Boonrod, G. Krczal, S. Bouzid, A. Ghorbel, G. M. Reusal (2007) Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation. Plant cell Rep. 26: 1439-1447.
连结：

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