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从发根农杆菌A4转化的荒漠植物——骆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-12,4-D、0.2mg·L-16-BA、0.3mol·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4×105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-16-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。
The results of protoplast isolation from the callus of Arabidopsis thaliana transformed by A. rhizogenes A4 showed that enzymatic hydrolysis of light yellow soft callus 7 ~ 10d, Vital protoplasts. Protoplasts were cultured in the medium supplemented with 1.5 mg · L-12,4-D, 0.2 mg · L-16-BA, 0.3 mol·L-1 mannitol, 2% (w / v) sucrose and 500 mg · L -1 Protein DPD medium shallow liquid culture sustainable division. The optimal osmotic pressure of culture medium was (450 ± 3) mOsm · kg-1, and the optimal density of protoplasts was 4 × 105 mL · mL-1. Protoplast production and cleavage frequency increased with the pretreatment at low temperature (4 ℃), and the cleavage frequency was up to 50%. Callus transplants formed by protoplast division can be differentiated and regenerated plants after culturing on MS medium supplemented with 1-2 mg · L-16-BA (or KT) and 0.2 mg · L-1 NAA. Paper electrophoresis showed that the callus and the differentiated plants regenerated by protoplasts still contained the specific product of hairy root transformation line - opine.