Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile-restorer combinations for heterosis breeding

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 ~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 T₀ 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 T₁ 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.