玉米未成熟胚培養(二)癒合組織的生長及分化

Abstract

組織培養技術，應用於作物品種改良，其前題是必須能穫得大量之再生植株，因此建立一套具有高分化能力的培養方法極爲重要。本試驗以玉米未成熟胚爲培植體，探討影響癒合組織形成的因子。結果得知以三星期間隔繼代培養癒合組織，較能維持癒合組織的生長速率及避免癒合組織褐化。培養基中加入200mg/1水解路蛋白(casein hydrolysate)，200mg/1酵母抽出物(yeast extract)或1.38 g/1脯胺酸(L-proline)可增進癒合組織的生長，而麥芽抽出物(malt extract)則無明顯效果。在含有MS基本鹽類及3%篇糖之分化培養基中，加上NAA (a-naphthalene- acetic)/BA (6-benzylamino purine )、NAA/kinetin (6-furfurylamino purine)及GA3 (gibberellic acid )等組合之植物生長調節劑，不但不能提高癒合組織分化植株之能力，反使分化效果大大降低。高濃度auxin (超過 5mg/1 dicamba (3, 6-dichloroanisic acid) or 2 mg/1 2, 4-D (2, 4-dichlorophenoxyacetic acid)所誘導的癒合組織，分化植株之能力較差。U-67甜玉米未成熟胚癒合組織經三次（每次間隔三星期）之繼代培養後，植株誘導率爲20%，二十次繼代培養後之植株分化率仍可達10%，顯示此種癒合組織是進行細胞培養及原生質體分離、再生之極佳材料。由U-67甜玉米未成熟胚癒合組織所誘導約200棵之植株，經移植於温室中種植至成熟，並經套袋自花授粉後結實，結果發現許多體細胞突變體(somaclones)，明顯的形態變異有植株接化、早熟、敗 葉或棬葉、缺少雄穗或雌穗、缺乏葉綠素、雄穗著生於植株頂端、雌雄同穗及雄不稔等個體。 The application of tissue culture technologies on crop improvement depends on the production of a large number of regenerated plants. To meet such purpose, it is important to deveop a system which enables the cultured explants to show a high differentiation ability. In this paper, factors affecting the maintenance and differentiation of callus from immature maize embryos were studied. The results shows a time interval of three weeks between subcultures of immature embryo-derived callus was appropriate in terms of maintaining the growth rate (fresh weight increase) and preventing from browning of the callus. Fresh weight of immature embryo-derived callus of maize inbred U-67 was increased by the addition of 200 mg/l casein hydrolysate, 200 mg/l yeast extract or 1.38 g/l L-proline to the medium. Malt extract had no effect in this aspect. The addition of varying concentrations of NAA (α-naphthaieneacetic acid)/BA (6-benzylamino purine), NAA/kinetin (6-furfurylamino purine) or GA3 (gibberellic acid) to the MS regeneration medium showed reversed effect on the differentiation ability of the callus. Callus induced under high concentrations of auxins (e.g., over 5 mg/l dicamba (3,6-dichloroanisic acid) or 2 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid)) supplemented to MN-6 medium showed poor regeneration ability after transferring to MS medium for organ differentiation. Twenty percent of plant regeneration rate was recorded from immature embryo-derived callus was subcultured for three generations (3 weeks interval for each generation). The plant regeneration ability was maintained at 10% even the callus was subcultured for 20 generations. Thus we concluded that the immature embryo-derived callus could be used as a good materials for establishment of cell suspension, protoplast isolation and plant regeneration. About 200 regenerated plants from immature embryo-derived callus of U-67 were raised to maturity in the green house. Some plants were morphologically abnormal, e. g., dwarf plants with early maturity, curly or wrinkled leaves, absence of tassel and/or ear, terminal ear, development of female flowers in the tassels (tassel seed), chlorophyll deficiency and male sterility etc.