癒傷組織來源及質地與懸浮細胞繼代培養對花蓮21號水稻懸浮細胞增殖與植株分化之影響

Effects of the Source and Texture of Callus and Subculture on Proliferation and Plant Regeneration of Suspension Cell in Oryza sativa cv. Hualien No. 21

林旻潔(Min-Jie Lin)

指導教授 ： 鄭秋雄

國立屏東科技大學/農學院/植物醫學系所/碩士(2016年)

摘要

本研究為建立具備增殖能力及植株分化能力之水稻懸浮細胞，探討癒傷組織來源、癒傷組織質地、懸浮細胞重複建立培養、培養基與植物生長調節劑對懸浮細胞增殖與分化植株之影響。比較器官培植體（種子與根段）對癒傷組織形成率與質地之影響，結果其癒傷組織形成率並無明顯差異，皆可達90%以上；但其誘導之癒傷組織有異，種子來源癒傷組織皆為硬質球形癒傷組織，而根段來源之癒傷組織有鬆軟、硬質及泥狀三種癒傷組織。比較不同質地癒傷組織之懸浮細胞數，則根段來源癒傷組織優於種子，其克鮮重癒傷組織之懸浮細胞數分別為2.2×107 cell/g callus與6.5×106 cell/g callus，但其細胞增殖率並無差異，且皆無法持續增殖。為改善懸浮細胞增殖率，取根段來源鬆軟癒傷組織進行懸浮細胞重複建立培養1~6次，並分別進行繼代培養。結果顯示，重複建立培養可提高細胞增殖率。其中，第三次建立培養之懸浮細胞之增殖率最佳，其初代與繼代培養分別為262.2%（1.6倍）與733.3%（6.3倍）。根段來源之鬆軟癒傷組織與硬質癒傷組織之懸浮細胞於繼代培養過程中易分化minicalli，其中以懸浮細胞重複建立培養第二次之minicalli分化率最高，於第一次繼代及第二次繼代培養分別為每50 mL培養液約9,418個及2,758個與8,215個及2,729個minicalli。將這些minicalli進行植株分化，兩者皆可分化植株，其植株分化率約8~27.7%。上述結果顯示，癒傷組織來源及質地與懸浮細胞重複建立培養對懸浮細胞增殖能力與植株分化能
力之影響至鉅。本研究建立之最佳培養條件為：(1)懸浮細胞繼代培養以根段來源鬆軟癒傷組織為材料，於含2 mg/L 2,4-D之CS-1培養基進行重複建立培養三次所建立之懸浮細胞，再移植至CSW-1培養基，其細胞增殖倍數最高，約1.6~6.3倍。(2)分化minicalli以根來源癒傷組織進行重複建立培養兩次之懸浮細胞進行繼代培養，則每50 mL培養液約可分化出8,215個以上minicalli。(3)minicalli之植株分化以10 mg/L NAA或3 mg/L BA或2 mg/L NAA及4 mg/L Kinetin較佳。

關鍵字

水稻（Oryza sativa cv. Hualien No.21） ； 癒傷組織質地 ； 重複建立培養 ； 細胞增殖 ； minicalli ； 植株分化

並列摘要

In order to establish the suspension cells of rice that have the good abilities of proliferation and plant differentiation, we evaluated effects of the source and texture of callus, repeated establishment culture of suspension cell, medium and the plant growth regulator on proliferation and plant differentiation of suspension cell.Compared effect of organ explants (the seeds and root fragments) on formation rate and texture of callus. The two explants differ slightly in callus formation rate, both up to 90%. But varied on callus texture, all callus induced from the seed were harden nodular; callus induced from the root fragments had three kinds of callus, friable, harden nodular and sticky. In comparison of the number of suspension cell among the different texture of callus, callus induced from the root fragments better than the seeds in the number of suspension cell, which were 2.2×107 and 6.5×106 per gram callus. However, there’s no difference in cell proliferation, and both of them couldn’t continue to proliferate. Further improve the proliferation rate of suspension cell, callus induced from the root fragments proceed repeated establishment culture 1-6 times and subculture respectively. The result showed that repeated establishment culture could raise the proliferation rate of
suspension cell. Among these treatments, repeated establishment culture three times provided the best result in proliferation rate of suspension cell, which were 262.2% (1.6-folded) and 733.3% (6.3-folded) in primary culture and subculture. In the process of subculture, the suspension cells, suspended from the friable and harden nodular callus, differentiated to minicalli easily. The suspension cells repeated establishment culture twice had the best result in differentiation rate of minicalli. The minicalli number of friable callus and harden nodular callus in the first subculture and the second subculture were 9,418 and 2,758, and 8,215 and 2,729 respectively in 50 mL medium. Both of the minicalli could differentiate to plants, the plant differentiation rate about 8-27.7%. Summarize the above-mentioned, the source and texture of callus and repeated establishment culture of suspension cell had a great influence on the abilities of proliferation and plant differentiation of the suspension cells. The best culture conditions established in this study were：(1) In subculture of suspension cell, callus induced from the root fragments offered as the materials, repeated establishment cultured three times on CS-1 medium containing 2 mg/L NAA to establish the suspension cells and transferred to CSW-1 medium, the times of cell proliferation was the highest, which about 1.6-6.3 times. (2) In minicalli differentiation, the suspension cells suspended from the callus, induced from the root fragments, repeated establishment culture twice to proceed subculture, more than 8,215 minicalli differentiated in 50 mL medium. (3) In plant differentiation of minicalli, 10 mg/L NAA or 3 mg/L BA or 2 mg/L NAA and 4 mg/L Kinetin provided the best result.

並列關鍵字

rice (Oryza sativa cv. Hualien No.21) ； callus texture ； repeated establishment culture ； cell proliferation ； minicalli ； plant regeneration

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