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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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Vertical sections of leaves (A) with corresponding stomatal structures (B) and pollen viability (C) of selected haploid (A1, B1, C1), dihaploid (A2, B2, C2), and tetraploid (A3, B3, C3) plants.LTB Haploids have smaller stomata and are sterile, dihaploids have normal stomata and are fertile and LTB tetraploids have bigger stomata with mostly non-fertile pollen. All photographs were taken at a similar magnification (x1800).
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pone-0100212-g002: Vertical sections of leaves (A) with corresponding stomatal structures (B) and pollen viability (C) of selected haploid (A1, B1, C1), dihaploid (A2, B2, C2), and tetraploid (A3, B3, C3) plants.LTB Haploids have smaller stomata and are sterile, dihaploids have normal stomata and are fertile and LTB tetraploids have bigger stomata with mostly non-fertile pollen. All photographs were taken at a similar magnification (x1800).

Mentions: The phenotypic variability of the anther culture-derived plants is shown in Fig. 1A and 1B. Variations in leaf anatomical features, stomatal size and pollen morphology and viability were observed in the three different types of plants [likely to be (LTB) haploid, dihaploid, and likely to be (LTB) tetraploid]. The number of mesophyll cell layers, the size of the vascular bundle (Fig. 2A), the size of stomata (Fig. 2B) and pollen characteristics (Fig. 2C) were found to differ due to changes in the ploidy level, although the number of chromosomes in plants with different phenotypes was not determined. Based on their phenotypic traits, a total of 111 doubled haploids, i.e., normal phenotype, were found to be fertile in Fig. 2C-2 (all pollen obtained from dihaploid plants seems to be fertile). By contrast, staining analyses revealed that all pollen from the smaller bushy plants was sterile (Fig. 2C-1), indicating likely to be (LTB) haploid plants. The bigger pollen from the broader-leaved plants (not shown) with bigger stomata and cells size as shown in Fig. 2A-3 & 2B-3 with mostly non-fertile pollen, likely to be (LTB) tetraploid plants (Fig. 2C-3) and with larger seed (Fig. 1B).


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)

Vertical sections of leaves (A) with corresponding stomatal structures (B) and pollen viability (C) of selected haploid (A1, B1, C1), dihaploid (A2, B2, C2), and tetraploid (A3, B3, C3) plants.LTB Haploids have smaller stomata and are sterile, dihaploids have normal stomata and are fertile and LTB tetraploids have bigger stomata with mostly non-fertile pollen. All photographs were taken at a similar magnification (x1800).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0100212-g002: Vertical sections of leaves (A) with corresponding stomatal structures (B) and pollen viability (C) of selected haploid (A1, B1, C1), dihaploid (A2, B2, C2), and tetraploid (A3, B3, C3) plants.LTB Haploids have smaller stomata and are sterile, dihaploids have normal stomata and are fertile and LTB tetraploids have bigger stomata with mostly non-fertile pollen. All photographs were taken at a similar magnification (x1800).
Mentions: The phenotypic variability of the anther culture-derived plants is shown in Fig. 1A and 1B. Variations in leaf anatomical features, stomatal size and pollen morphology and viability were observed in the three different types of plants [likely to be (LTB) haploid, dihaploid, and likely to be (LTB) tetraploid]. The number of mesophyll cell layers, the size of the vascular bundle (Fig. 2A), the size of stomata (Fig. 2B) and pollen characteristics (Fig. 2C) were found to differ due to changes in the ploidy level, although the number of chromosomes in plants with different phenotypes was not determined. Based on their phenotypic traits, a total of 111 doubled haploids, i.e., normal phenotype, were found to be fertile in Fig. 2C-2 (all pollen obtained from dihaploid plants seems to be fertile). By contrast, staining analyses revealed that all pollen from the smaller bushy plants was sterile (Fig. 2C-1), indicating likely to be (LTB) haploid plants. The bigger pollen from the broader-leaved plants (not shown) with bigger stomata and cells size as shown in Fig. 2A-3 & 2B-3 with mostly non-fertile pollen, likely to be (LTB) tetraploid plants (Fig. 2C-3) and with larger seed (Fig. 1B).

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