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Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

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Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.

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Cleaved amplified polymorphic sequence


Doubled haploid


Expressed sequence tag


Lycopene β-cyclase


Lycopene ε-cyclase


Murashige and Tucker


Simple sequence repeats


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This research was supported by the National Basic Research Program of China (No. 2011CB100600) and the National Natural Science Foundation of China (NSFC) (Nos. 30830078 and 30921002). We thank Yangmei Nie and Jidi Xu for their help in the ploidy analysis and other technical support. We also thank Yunjiang Cheng, Zhiyong Pan and Lijun Chai for their reviewing of the manuscript.

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Correspondence to Xiuxin Deng.

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Cao, H., Biswas, M.K., Lü, Y. et al. Doubled haploid callus lines of Valencia sweet orange recovered from anther culture. Plant Cell Tiss Organ Cult 104, 415–423 (2011).

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