甲基氨草磷对橡胶树悬浮细胞同步化及微核诱导的影响

Effect of APM on Synchronization and Micronucleation in Suspension Cells of Hevea brasiliensis

植物微原生质体融合(microprotoplast fusion)是将部分基因组转移而又避免染色体损伤的不对称融合技术,微原生质体成功诱导是微原生质体融合的先决条件。本研究在摸索甲基氨草磷(amiprophos-methyl,APM)对橡胶树悬浮细胞有丝分裂中期指数和染色体形态影响基础上,研究了酶解时间及酶解过程中添加APM对微原生质体产量的影响。结果发现,悬浮细胞随APM处理浓度和时间的增加,有丝分裂中期指数先升高再下降,处理12 h时达到最大值;染色体聚集程度也先增加再降低,处理16 h时染色体开始解聚。在酶解过程中添加APM和延长酶解时间会大大降低微原生质体的产量。悬浮细胞经过1μmol/L APM处理10 h后,酶解10 h,中期细胞可达到79.4%,微核化细胞指数达到7.3%,从而建立了APM诱导橡胶树悬浮细胞同步化及高效获得橡胶树微化细胞的方法。

Microprotoplast fusion is a method for undamaged transferring a partial genome to receptor cell and successful induction of microprotoplast is the prerequisite of microproplast fusion. Herein, effects of concentration and treatment time of APM （amiprophos-methyl） on MI （metaphase index） and morphology of chromosome in metaphase on production of microprotoplast were investigated as well addition of APM to enzyme solution and treatment time. In the results, with increasing of concentration and treatment time of APM, MI increased and then decreased and the peak was occurred after 12 h treatment. Chromosome condensation and aggregation was positively correlated to APM concentration was found. However, chromosome depolymerization was observed after 16 h treatment. Additionally, addition of APM to enzyme solution sharply reduced the production of microprotoplast, the micronucleus index （MNI） decreased from 7.3% to 0.77% after 10 h enzyme treatment. Extension of enzyme treatment was also reduced production of microprotoplast, MNI decreased from 7.3% to 2.3% when enzyme treatment time extended from 10 h to 14 h. So APM could efficiently synchronize metaphase cells and produce microprotoplast in Hevea brasiliensis.