骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明，酶解新转代7～10d的淡黄色松软愈伤组织，可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2，4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1，原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后，原生质体的产率和分裂频率均提高，分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后，可以分化并获得再生植株。纸电泳检测表明，原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。

Protoplast Culture and Plantlet Regeneration from Cell Line of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi Desv

The protoplasts were isolated from calli which were induced from hairy root segments of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi. After cultured in the DPD medium supplemented with 1.5 mg.L-1 2,4-D, 0.2 mg.L-1 6-BA, 0.3 mol.L-1 mannitol, 500 mg.L~ casein hydrolysate （CH） and 2% （W/V） sucrose, the protoplasts underwent sustained divisions and formed calli. The protoplast density of 4x 105 mL-1 and （450±3） mOsm.kg-1 osmotic pressure in culture medium were proved to be appropriate for obtaining higher division frequency of protoplasts. A lot of protoplasts could be obtained by the enzymatic hydrolysis of yellowish subcultured calli after cultured on MS medium supplemented with 1.5 mg-LI NAA, 1.0 mg.L-1 6-BA, 500 mg.L1 CH and 2% （W/V） sucrose for 7-10 d. Lower temperature （4 ℃） pretreatment of subcultured calli enhanced ratios of protoplast isolation and subsequent divisions. The division frequency of protoplasts was about 50%. After transferred on the MS medium added with 1-2 mg.L- 6-BA （or KT） and 0.2 mg.L- NAA, the protoplast- derived calli differentiated and formed the regenerated plantlets. Paper electrophoresis analysis indicated that the protoplast-derived calli and regenerated plantlets still contained special product---opine in transgenic root hairs.