Effective restoration of male-sterile (barnase) lines requires overlapping and higher levels of barstar expression: A multi-generation field analysis in Brassica juncea
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We have earlier reported the deployment of an effective pollination control system for hybrid seed production in Brassica juncea based on use of the barnase-barstar system. The current study analyses the stability and performance of four male-sterile (barnase) and 34 fertility restorer (barstar) lines under containment field conditions. Line (barnase) x tester (barstar) crosses were studied for effective fertility restoration over three successive growing seasons by screening the F1 and representative F2 progeny for three major parameters, namely segregation of Basta resistance and sensitive plants, distribution of male-sterile and fertile plants (vis-à-vis segregation of the barnase and the barstar genes) and pollen viability of the restored events. A total of 29 male sterile/restorer combinations out of 53 obtained in the first growing season were stable for their restoration ability when tested for two subsequent growing seasons. Stable restorers with high pollen viability were obtained at a significantly higher frequency for one of the barnase lines, bn 3.6, out of the four lines tested. Among the barstar lines, constructs carrying two transcription units of the barstar gene provided more effective fertility restoration. Quantitative real-time PCR analysis of restored progeny obtained for all the four barnase lines showed that three barnase lines (bn 3.23, bn 3.4 and bn 3.48) had an early and much higher level of barnase expression than the line bn 3.6 indicating that the level of barnase expression in the line bn 3.6 is optimal for better and stable restoration. It was observed that a high barstar : barnase transcript ratio and an extended window of barstar gene expression are critical parameters for the development of stable male-sterile (barnase) and fertility-restorer (barstar) combinations.
KeywordsBarnase-barstar Brassica juncea Male-sterile plants Male-fertility restoration Transgene stability Expression profiles
bar gene encoding phosphinothricin Basta resistance
wild type barstar gene
codon modified barstar gene
- TA29 and A9
tapetum specific promoters from tobacco and Arabidopsis, respectively
This work was supported by research grants from the National Dairy Development Board and its subsidiaries and the Department of Biotechnology, Government of India.
- Hartley RW, Smeaton JR (1973) On the reaction between the extracellular ribonuclease of Bacillus amyloliquifaciens (Barnase) and its intracellular inhibitor (Barstar). J Biol Chem 245:5624–5626Google Scholar
- Jagannath A, Arumugam N, Gupta V, Pradhan AK, Burma PK, Pental D (2002) Development of transgenic barstar lines and identification of a male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard (Brassica juncea). Curr Sci 82:46–52Google Scholar
- Jagannath A, Bandyopadhyay P, Arumugam N, Gupta V, Burma PK, Pental D (2001) The use of a spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high frequency generation of transgenic male sterile lines in Brassica juncea L. Mol Breed 8:11–23CrossRefGoogle Scholar