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Genetic stability developed for β-carotene synthesis in BR29 rice line using dihaploid homozygosity.

Datta K, Sahoo G, Krishnan S, Ganguly M, Datta SK - PLoS ONE (2014)

Bottom Line: Dihaploid plants in addition to haploid and tetraploid plant were generated from anther cultures of these primary transgenic plants.In addition to anatomical features of stomata, pollen of different ploidy-plants, molecular analyses confirmed the stable integration of the genes in the anther culture-derived dihaploid plants, and the yellow color of the polished seeds indicated the accumulation of carotenoids in the endosperm.High performance liquid chromatography (HPLC) analysis of the carotenoid extract further confirmed the levels of β-carotene accumulation in the endosperms of the transgenic dihaploid rice seeds.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany, Plant Molecular Biology and Biotechnology Laboratory, University of Calcutta, Kolkata, India.

ABSTRACT
Obtaining transgenic crop lines with stable levels of carotenoids is highly desirable. We addressed this issue by employing the anther culture technique to develop dihaploid lines containing genes involved in β-carotene metabolism. First, we used Agrobacterium- mediated transformation to develop primary transgenic plants containing the β-carotene biosynthetic genes, phytoene synthase (psy) and phytoene desaturase (crtI), which were engineered for expression and accumulation in the endosperm. Transgenic plants were recovered by selecting for the expression of the phosphomannose isomerase (pmi) gene. Dihaploid plants in addition to haploid and tetraploid plant were generated from anther cultures of these primary transgenic plants. In addition to anatomical features of stomata, pollen of different ploidy-plants, molecular analyses confirmed the stable integration of the genes in the anther culture-derived dihaploid plants, and the yellow color of the polished seeds indicated the accumulation of carotenoids in the endosperm. High performance liquid chromatography (HPLC) analysis of the carotenoid extract further confirmed the levels of β-carotene accumulation in the endosperms of the transgenic dihaploid rice seeds.

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RT-PCR showing the expression of crtI mRNA in anther culture-derived plants.Expression analysis of the crtI gene in homozygous anther culture-derived transgenic plant line. RT-PCR was performed with 2 µg of RNA and products were resolved on a 1.2% TAE-agarose gel. ‘N’ represents the Negative Control, ‘P’ represents the Positive Control and ‘M’ represents the Molecular marker.
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pone-0100212-g004: RT-PCR showing the expression of crtI mRNA in anther culture-derived plants.Expression analysis of the crtI gene in homozygous anther culture-derived transgenic plant line. RT-PCR was performed with 2 µg of RNA and products were resolved on a 1.2% TAE-agarose gel. ‘N’ represents the Negative Control, ‘P’ represents the Positive Control and ‘M’ represents the Molecular marker.

Mentions: Reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of crtI in seeds, based on the presence of the expected 1.03 kb amplicon for crtI cDNA, which is absent in the non-transgenic control plants (Fig. 4).


Genetic stability developed for β-carotene synthesis in BR29 rice line using dihaploid homozygosity.

Datta K, Sahoo G, Krishnan S, Ganguly M, Datta SK - PLoS ONE (2014)

RT-PCR showing the expression of crtI mRNA in anther culture-derived plants.Expression analysis of the crtI gene in homozygous anther culture-derived transgenic plant line. RT-PCR was performed with 2 µg of RNA and products were resolved on a 1.2% TAE-agarose gel. ‘N’ represents the Negative Control, ‘P’ represents the Positive Control and ‘M’ represents the Molecular marker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061092&req=5

pone-0100212-g004: RT-PCR showing the expression of crtI mRNA in anther culture-derived plants.Expression analysis of the crtI gene in homozygous anther culture-derived transgenic plant line. RT-PCR was performed with 2 µg of RNA and products were resolved on a 1.2% TAE-agarose gel. ‘N’ represents the Negative Control, ‘P’ represents the Positive Control and ‘M’ represents the Molecular marker.
Mentions: Reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of crtI in seeds, based on the presence of the expected 1.03 kb amplicon for crtI cDNA, which is absent in the non-transgenic control plants (Fig. 4).

Bottom Line: Dihaploid plants in addition to haploid and tetraploid plant were generated from anther cultures of these primary transgenic plants.In addition to anatomical features of stomata, pollen of different ploidy-plants, molecular analyses confirmed the stable integration of the genes in the anther culture-derived dihaploid plants, and the yellow color of the polished seeds indicated the accumulation of carotenoids in the endosperm.High performance liquid chromatography (HPLC) analysis of the carotenoid extract further confirmed the levels of β-carotene accumulation in the endosperms of the transgenic dihaploid rice seeds.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany, Plant Molecular Biology and Biotechnology Laboratory, University of Calcutta, Kolkata, India.

ABSTRACT
Obtaining transgenic crop lines with stable levels of carotenoids is highly desirable. We addressed this issue by employing the anther culture technique to develop dihaploid lines containing genes involved in β-carotene metabolism. First, we used Agrobacterium- mediated transformation to develop primary transgenic plants containing the β-carotene biosynthetic genes, phytoene synthase (psy) and phytoene desaturase (crtI), which were engineered for expression and accumulation in the endosperm. Transgenic plants were recovered by selecting for the expression of the phosphomannose isomerase (pmi) gene. Dihaploid plants in addition to haploid and tetraploid plant were generated from anther cultures of these primary transgenic plants. In addition to anatomical features of stomata, pollen of different ploidy-plants, molecular analyses confirmed the stable integration of the genes in the anther culture-derived dihaploid plants, and the yellow color of the polished seeds indicated the accumulation of carotenoids in the endosperm. High performance liquid chromatography (HPLC) analysis of the carotenoid extract further confirmed the levels of β-carotene accumulation in the endosperms of the transgenic dihaploid rice seeds.

Show MeSH
Related in: MedlinePlus