芹菜与CMS胡萝卜原生质体非对称性融合的初步研究

Preliminary Study of Asymmetric Protoplast Fusion Between Celery(Apium graveolens L.) and CMS Carrot(Daucus carota L.)

以芹菜和胡萝卜瓣化型细胞质不育系为试材,研究了碘乙酰胺(IOA)和紫外线对两种原生质体的钝化作用,以及融合温度、聚乙二醇6000(PEG6000)和Ca2+浓度对融合效果的影响,并对融合再生植株进行了鉴定。结果表明,6mmol.L-1IOA处理7min以上可钝化芹菜原生质体细胞质,强度约为20μmol.m-2.s-1的紫外线持续辐射9min可使胡萝卜原生质体活力趋于0;适当低温(5℃)可提高聚合率约20%;PEG6000浓度为40%,高pH液中Ca2+浓度为0.1mmol.L-1时最适宜两种原生质体融合;融合产物液体浅层培养40d后产生的可见细胞团在不添加植物生长调节剂的MS0培养基上再生出了完整植株。用AFLP分子标记对12株再生株总DNA的鉴定结果表明,再生株多不同程度地发生了芹菜和胡萝卜基因的非对称性融合。用STS分子标记鉴定再生株的细胞质基因,瓣化型雄性不育特异引物STS4的扩增结果显示,除1号单株外其余11株均成功转入了胡萝卜瓣化型雄性不育基因。

Cytoplasmic male sterility （CMS） celery is useful for hybrid seed production. Asymmetric protoplast fusion of fertile celery and petaloid CMS carrot was carried out by polyethylene glycol （PEG） 6000 treatment in order to introduce CMS from carrot to celery. Protoplasts of celery were treated with 6 mmol·L^-1 iodoacetamide for 7 rain to passivate cytoplasm, and those of carrot were continuously irradiated with UV rays （20 μmol·m^-2·s^-1） for 9 min to kill the nucleus. Low temperature （5℃） increased aggregation rates by 20% , and 40% PEG 6000 and 0. 1 mmol·L^-1 Ca^2＋ in high Ca^2＋ high pH solution were the best for fusion of two protoplasts. Twelve somatic hybrids were obtained and plants were regenerated on MS0 medium. AFLP analysis indicated that all of them were somewhat asymmetrically fused. STS marker analysis suggested that petaloid CMS were successfully introduced into 11 regenerated plants.