High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos
Transgenic doubled haploid rapeseed ( Brassica napus L. cvs. Global and PF 704 ) plants were obtained from microspore-derived embryo (MDE) hypocotyls using the microprojectile bombardment. The binary vector pCAMBIA3301 containing the gus and bar genes under control of CaMV 35S promoter was used for...
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Veröffentlicht in: | Molecular biology reports 2011-02, Vol.38 (2), p.711-719 |
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Sprache: | eng |
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Zusammenfassung: | Transgenic doubled haploid rapeseed (
Brassica napus
L. cvs. Global and PF
704
) plants were obtained from microspore-derived embryo (MDE) hypocotyls using the microprojectile bombardment. The binary vector pCAMBIA3301 containing the
gus
and
bar
genes under control of CaMV 35S promoter was used for bombardment experiments. Transformed plantlets were selected and continuously maintained on selective medium containing 10 mg l
−1
phosphinothricin (PPT) and transgenic plants were obtained by selecting transformed secondary embryos. The presence, copy numbers and expression of the transgenes were confirmed by PCR, Southern blot, RT-PCR and histochemical GUS analyses. In progeny test, three out of four primary transformants for
bar
gene produced homozygous lines. The ploidy level of transformed plants was confirmed by flow cytometery analysis before colchicine treatment. All of the regenerated plants were haploid except one that was spontaneous diploid. High frequency of transgenic doubled haploid rapeseeds (about 15.55% for
bar
gene and 11.11% for
gus
gene) were considerably produced after colchicines treatment of the haploid plantlets. This result show a remarkable increase in production of transgenic doubled haploid rapeseed plants compared to previous studies. |
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ISSN: | 0301-4851 1573-4978 |
DOI: | 10.1007/s11033-010-0158-3 |