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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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Yellow endosperms from the polished grains.Variations in yellow coloration from carotenoids in transgenic double haploid rice plants showing different levels of expression. Variations in carotenoid content was found in different transgenic lines like ACSKBR-46-30 showed 3.188 µg g−1, ACSKBR-44-16 showed 3.036 µg g−1, ACSKBR-49-39 showed 2.12 µg g−1 whereas ACSKBR-64-7 showed 0.257 µg g−1.
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pone-0100212-g005: Yellow endosperms from the polished grains.Variations in yellow coloration from carotenoids in transgenic double haploid rice plants showing different levels of expression. Variations in carotenoid content was found in different transgenic lines like ACSKBR-46-30 showed 3.188 µg g−1, ACSKBR-44-16 showed 3.036 µg g−1, ACSKBR-49-39 showed 2.12 µg g−1 whereas ACSKBR-64-7 showed 0.257 µg g−1.

Mentions: Mature seeds from individual transgenic plants derived from anther cultures of the individual primary transgenic lines were polished together with seeds from non-transgenic control plants. Variations in the carotenoids levels and intensity of yellow coloration indicate the differential expression of transgene and carotenoid accumulation in the endosperms of the individual plants (Fig. 5). Spectrophotometric and HPLC analyses were used to quantify the total carotenoids and β–carotene levels (Fig. 6). The total carotenoid levels ranged from 0.257 µg g−1 to 3.188 µg g−1 in the endosperms of individual double haploid plants. Seeds from individual plants derived from the same primary transgenic plant showed different levels of carotenoids and β–carotene content.


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)

Yellow endosperms from the polished grains.Variations in yellow coloration from carotenoids in transgenic double haploid rice plants showing different levels of expression. Variations in carotenoid content was found in different transgenic lines like ACSKBR-46-30 showed 3.188 µg g−1, ACSKBR-44-16 showed 3.036 µg g−1, ACSKBR-49-39 showed 2.12 µg g−1 whereas ACSKBR-64-7 showed 0.257 µg g−1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0100212-g005: Yellow endosperms from the polished grains.Variations in yellow coloration from carotenoids in transgenic double haploid rice plants showing different levels of expression. Variations in carotenoid content was found in different transgenic lines like ACSKBR-46-30 showed 3.188 µg g−1, ACSKBR-44-16 showed 3.036 µg g−1, ACSKBR-49-39 showed 2.12 µg g−1 whereas ACSKBR-64-7 showed 0.257 µg g−1.
Mentions: Mature seeds from individual transgenic plants derived from anther cultures of the individual primary transgenic lines were polished together with seeds from non-transgenic control plants. Variations in the carotenoids levels and intensity of yellow coloration indicate the differential expression of transgene and carotenoid accumulation in the endosperms of the individual plants (Fig. 5). Spectrophotometric and HPLC analyses were used to quantify the total carotenoids and β–carotene levels (Fig. 6). The total carotenoid levels ranged from 0.257 µg g−1 to 3.188 µg g−1 in the endosperms of individual double haploid plants. Seeds from individual plants derived from the same primary transgenic plant showed different levels of carotenoids and β–carotene content.

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