刀豆(Canavallia ensiformis)叶肉原生质体培养与植株再生

PLANTLETS REGENERATION FROM MESOPHYLL PROTOPLASTS IN SWORD BREAN (Canavallia ensiformis)

用刀豆原生质体为材料,从培养分化开始,经过无菌苗的培养、酶解、原生质体分离、原生质体培养及愈伤组织培养五个阶段,长成完整植株,整个周期为217d。本文对整个培养过程中的条件作了详细探索和讨论。

Protoplast isolated from mesophyll cells of Canavallia ensiformis was cultured in liquid culture medium D2a at a density of about 1×10~6/ml and separated about a per culture bottle(60×30×20mm^8). After 2 or 3 days, the protoplasts expanded and became elipse in shape. The regenerated cells began the first division in culture within 5~10 days, and the second division occurred 6 days later. After about 45 days a large number of calli were formed. When the callus Was 3 mm in diameter, it was transferred to differentiation media containing various combination of hor- mones and medium MS+2.4-D 3mg/L, 6BA 1mg/L, or D2a+2.4-D 2.5mg/L. 6BA 1 mg/L; PH 5.8. The calli of 5 mm in size were again transferred to differentiation media containing various combination: D2a+ZT 10mg/L. NAA 0.3 mg/L, IAA 0.5 mg/L, hard call/or embryoid formed after 25 days. Then, these were transferred to solid media: MS and D2a+6BA 8mg/L, NAA 0.3mg/L, IAA 0.3 mg/L. After about 19 days, some green spots were visible and these grew up into plantlets in 5 days. From protoplast to developed plantlets regeneration it took 217 days in total