悬铃木叶肉原生质体培养再生植株

MESOPHYLL PROTOPLAST CULTURE AND PLANT REGENERATION OF ORIENTAL PLANETREE (PLAT ANUS ORIENTALIS)

从1.5—2个月龄的悬铃木(Platanus orientalis)无菌苗叶片游离得到大量的叶肉原生质体,纯化后其产量为3.7×10~6g^(-1)FW。纯化的原生质体在含0.5mg/L 2,4-D,1mg/L NAA 和0.5mg/L BA 的改良 K_(8P)液体培养基中进行薄层培养。不同培养密度的试验表明,在原生质体起始培养中,较高的密度(1×10~6/ml)有利于叶肉原生质体的分裂。培养6天后开始第一次细胞分裂,14天时分裂频率可达26.8%左右,8周内形成大量的细胞团和小愈伤组织。小愈伤组织在用 gelrite 固化的含0.2mg/L NAA 和0.5mg/L BA 的 K_8培养基上进一步生长到2—3mm 时,转到含0.1mg/L NAA 和0.25mg/L BA 的 MS 增殖培养基上,得到结构紧密的米黄色愈伤组织。这种愈伤组织转到含0.5mg/L IAA,BA 和 ZT 各为1mg/L 的 MSB分化培养基上,培养4周后开始有芽的分化,分化成苗率为28.7%。待芽伸长到3cm 时,从基部切下转到含0.5mg/L IBA 和0.1mg/L BA 的1/2MS 培养基上,即生根形成完整植株。移栽盆内后,在本所人工气候室生长良好。

Large populations of mesophyll protoplasts were released from the leaves of 1.5—2 month old sterile seedlings, with a high protoplast yield (3.7 × 10~6g^(-1) FW) after protoplast purification. The purified protoplasts were cultured in a modified K_(8p) liquid medium supplemented with 0.5 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L BA. Higher density (1×10~6/ml) in the initial culture of protoplasts is favourable to the division of cultured mesophyll protoplasts of this woody species among the densities tested. The protoplasts started to divide after 6 days of culture, and achieved 26.8% division frequency by 14 days. Sustained divisions resulted in mass production of cell colonies and small calli in 8 weeks. The calli further grew to 2—3mm on the gelrite-solidified K8 medium supplemented with 0.2 mg/L NAA and 0.5 mg/L BA. Then, they were transferred onto the MSB proliferation medium with 0.1 mg/L NAA and 0.25 mg/L BA, where compact and cream-coloured calli were formed. Shoot formation was initiated on MSB differentiation medium coaing 0.5 mg/L IAA, 1 mg/L each of BA and ZT. It was observed that the frequency of shoot formation was about 28.7%. Whole plantlets were regenerated upon transferring 3 cm shoots to 1/2MS medium with 0.5mg/L IBA and 0.1mg/L BA, from which they were already transplanted into pots and grew well in the phytotron of Shanghai Institute of Plant Physiology.