摘要
从青菜（Ｂｒａｓｓｉｃａ ｃｈｉｎｅｎｓｉｓＬ．）单胞中后期至二胞早期花粉分离出原生质体，用聚乙二醇法诱导其与甘蓝型油菜（Ｂ．ｎａｐｕｓＬ．）下胚轴原生质体融合。通过控制双亲原生质体的数量与比率，提高了异源融合率。融合体在离体条件下发生细胞分裂，形成愈伤组织，再生了小植株。染色体计数与酯酶同工酶酶谱分析初步证明获得了１ 株异源三倍体，２ 株异源四倍体。这是以游离花粉时期的原生质体与体细胞原生质体融合，取得“配子－体细胞杂交”
It has been reported that “gameto somatic hybridization” was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid late unicellular to early bicellular stage the pollens for 1 5—2 5 h at 25℃ in a CPW solution containing 0.8% of cellulase, 0.5% pectinase, 0.1% pectolyase, 13% mannitol, 10% glucose, 0.3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen proto plasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC prelabeled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyl homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1∶3 to 1∶6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2,4 D, 0.25 mg/L NAA, 0.5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3% sucrose and 0.4% agarose, differentiated into a few shoots. The shoots were transferred onto a half strength MS medium supplemented with 2% sucrose, 0.1—0.2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by root tip squash method revealed that one plantlet was 2n=48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n=20) and one hypocotyl protoplast of B. napus (2n=38), and the other two plantlets were 2n=58, which might be an allotetraploid o
基金
国家自然科学基金
关键词
花粉
原生质体
融合
下胚轴
芸苔属
再生植株
Pollen protoplast
Protoplast fusion
Gameto somatic hybridization
Brassica chinensis
Brassica napus
