Bisht Naveen C, Jagannath Arun, Burma Pradeep K, Pradhan Akshay K, Pental Deepak
Centre for Genetic Manipulation of Crop Plants, University of Delhi, South Campus, Benito Juarez Road, New Delhi, India.
Plant Cell Rep. 2007 Jun;26(6):727-33. doi: 10.1007/s00299-006-0274-7. Epub 2007 Jan 5.
We report in this study, an improved method for identifying male sterile-restorer combinations using the barnase-barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line x tester cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66-90% of fertile retransformants (restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and approximately 30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) x tester (barstar) crosses, wherein only two viable male sterile-restorer combinations were identified by screening 88 different cross-combinations. The retransformation strategy not only generated several independent restorers for a given male sterile line from a single transformation experiment but also identified potential restorers in the T(0) generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T(1) progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major limitation.
在本研究中，我们报告了一种改进的方法，用于利用巴那斯酶-巴思塔授粉控制系统鉴定雄性不育-恢复系组合，以用于作物杂种优势育种，作为传统系×测验种杂交方法的替代方法。在该策略中，用合适的巴思塔构建体对转基因雄性不育巴那斯酶系进行再转化。鉴定并筛选携带巴那斯酶和巴思塔基因的双转化体的雄性育性状态。利用该策略，使用两种不同的巴思塔构建体在芥菜型油菜中获得了66%-90%的可育再转化体（恢复事件）。对恢复事件的花粉活力和巴思塔基因的拷贝数进行了分析。约90%的恢复事件表现出高花粉活力，约30%含有巴思塔基因的单拷贝整合。这些观察结果与我们早期使用系（巴那斯酶）×测验种（巴思塔）杂交进行的研究结果有显著差异，在早期研究中，通过筛选88种不同的杂交组合仅鉴定出两个可行的雄性不育-恢复系组合。再转化策略不仅通过一次转化实验为给定的雄性不育系产生了几个独立的恢复系，而且在T(0)代本身就鉴定出了潜在的恢复系，从而显著节省了时间、成本和劳动力。具有高花粉活力的单拷贝恢复植株自交，以在T(1)代后代中分离出雄性不育（巴那斯酶）系和恢复系（巴思塔），随后可将其多样化为用于杂种优势育种的合适组合系。该策略对于那些转化频率低和/或转化方案冗长是主要限制因素的作物将特别有用。
