Abstract
Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.
Highlights
In higher plants, haploid phase is transitory and limited to gametophytes
R Arabidopsis plants rescued with Green Fluorescent Protein (GFP)-tailswap construct were high suppression of native CENH3 genes and expressing chimeric CENH3 gene was used in crosses with untransformed line, a high frequency of aneuploids and one haploid progeny were obtained suggesting that by careful choice of transgenic events, RNAi-based haploid inducer (HI) lines could be developed in polyploid crops
NPTII gene, which transgenic status of regenerated plants and most of the plants is present in both RNAi and GFP-synthetic CENH3-tailswap were found to contain one or a few copies of transgene(s) cassettes, was used as the probe in Southern hybridization
Summary
R Technical Advances in Plant Science, Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea a section of the journal Frontiers in Plant Science. Received: 02 September 2016 chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines
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