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High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos

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Abstract

Transgenic doubled haploid rapeseed (Brassica napus L. cvs. Global and PF704) 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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Abbreviations

MDE:

Microspore-derived embryo

PPT:

Phosphinothricin

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Author information

Authors and Affiliations

  1. Crop Production and Plant Breeding Department, Faculty of Agriculture, Bu Ali Sina University, Hamedan, Iran

    M. R. Abdollahi

  2. Plant Breeding Department, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran

    A. Moieni

  3. National Institute of Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran

    A. Mousavi & A. H. Salmanian

Authors
  1. M. R. Abdollahi
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  2. A. Moieni
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  3. A. Mousavi
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  4. A. H. Salmanian
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Corresponding authors

Correspondence to A. Moieni or A. Mousavi.

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Figure 1

Molecular characterization of putative bar transformants via PCR. MW molecular weight marker 1 kb DNA ladder (Fermentas); C+, positive control (pCAMBIA3301); C−, negative control (without DNA template); NT, non-transformed plant control; T1−14, putative transformants. The expected size of the PCR product is indicated with arrow (TIFF 811 kb)

Figure 2

Molecular characterization of putative gus transformants via PCR. MW, molecular weight marker 1 kb DNA ladder (Fermentas); C+, positive control (pCAMBIA3301); C−, negative control (without DNA template); NT, non-transformed plant control; T3 and T5–T13, putative transformants, The expected size of the PCR product is indicated with arrow (TIFF 1084 kb)

Figure 3

RT-PCR results of the rapeseed haploid transgenic plants for gus, bar and 18S genes. MW, molecular weight markers 1 kb DNA ladder (Fermentas); C−, negative control (water template); NT, negative control (RNA of a non-transgenic plant); T1 and T2, RNA of two transgenic lines. C+, positive control (TIFF 988 kb)

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Abdollahi, M.R., Moieni, A., Mousavi, A. et al. High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos. Mol Biol Rep 38, 711–719 (2011). https://doi.org/10.1007/s11033-010-0158-3

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