苹果原生质体分离培养及植株再生

Plant Regeneration from Protoplasts of Apple

以悬浮培养细胞及叶片作为分离原生质体的起始材料 ,对影响苹果原生质体分离和培养的因素进行了系统研究。优化的原生质体培养基为 :改良MT +维生素C 5mg/L +BA0 .5～ 1mg/L +2 ,4 D 0 .2mg/L +蔗糖 0 .6 5mol/L +谷氨酰胺 50 0mg/L +CH 10 0mg/L +ME50 0mg/L ;以葡萄糖代替蔗糖作渗透压调节物质效果好 ,且在培养过程中不需降压 ;适宜的低密度培养 (0 .5× 10 5/mL)可减少褐变。悬浮系原生质体培养 5～ 6d出现第 1次细胞分裂 ,15～ 2 0d形成多细胞团 ,4 0～ 50d形成肉眼可见的小愈伤组织。经培养诱导出不定芽 ,并进一步诱导生根长成完整植株。获得了平邑甜茶、M2 6 、嘎啦 3种基因型的原生质体再生植株。

s The conditions of isolation and culture of apple protoplasts from leaves and suspension cultured cells were studied systematically.Appropriate culture medium for protoplasts was modified MT supplemented with Vc,BA,2,4 D,glutamine,CH,ME and glucose.Glucose had a better effect on osmotic regulation than sucrose.Low density culture could reduce browning.Under the above culture conditions,protoplasts from suspension culture divided for the first time on five to six days,formed multi cell clusters and grew into visible mini callus.Mini callus was transferred to solidified medium for proliferation.The callus derived from protoplasts changed to compact type and could be induced to form green bud spots,grew into green and hard pieces,and subsequently adventitious shoots when transferred to the differentiation medium.The shoots rooted in rooting medium,and plantlets have regenerated from three genotypes Malus hupehensis, M 26 and Gala.