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采用“供-受体”双失活融合模式,以陆地棉品系YZ-1原生质体为受体,以野生棉G.davidsonii原生质体为供体,开展不对称融合.融合前,陆地棉品系YZ-1原生质体经过0.5mmol/L碘乙酰胺(IOA)室温下处理20min,野生棉G.davidsonii原生质体经过38.7J/cm2的紫外线(UV)处理30s,能有效地抑制亲本原生质体的生长.将上述条件下分别钝化处理的双亲原生质体采用电诱导融合后进行培养,获得再生植株.对获得的杂种植株进行形态学、细胞学及分子标记检测.大部分再生植株表现出新形态,部分表现亲本中间型,少数偏向受体亲本.融合植株的染色体数目在40~73之间.随机扩增多态性DNA标记(RAPD)和简单重复序列(SSR)分析证实了再生植株的杂种真实性.而细胞质基因组的酶切扩增多态性序列(CAPS)和叶绿体SSR(cpSSR)分析结果显示,杂种的线粒体和叶绿体发生了重组.本研究是棉花中开展“供-受体”双失活融合模式的首次报道,是继对称融合和基于紫外线处理的不对称融合之后在棉花原生质体融合方面又一新的进展.
In this study, asymmetric fusion of G. davidsonii protoplasts from G. gossypii was carried out by using the “donor-acceptor” double-inactivation fusion model, using as raw material Upland cotton line YZ-1 protoplasts. Protoplasts of strain YZ-1 were treated with 0.5 mmol / L iodoacetamide (IOA) for 20 min at room temperature. After treated with 38.7 J / cm2 of UV for 30 s, the protoplasts of G. davidsonii could effectively inhibit the protoplast Growth.Parent protoplasts, which were passivated respectively under the above conditions, were cultured by electroporation, and regenerated plants were obtained.The morphological, cytological and molecular markers of the hybrid plants were detected.Most of the regenerated plants showed a new morphology , And some of them showed partial parents and intermediate parents.Furthermore, the number of chromosomes in the fusion plants was between 40 and 73. Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) analysis confirmed that hybrids of regenerated plants (CAPS) and chloroplast SSR (cpSSR) analysis of the cytoplasmic genome revealed that the hybrid mitochondria and chloroplast were recombined.In this study, "Double deactivation fusion The first report of the model is another new progress in protoplast fusion of cotton after asymmetric fusion based on symmetric fusion and UV-based treatment.