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Recovery and characterization of homozygous lines from two sweet orange cultivars via anther culture

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Abstract

Homozygous genotypes are valuable for breeding and genomic studies in higher plants. The production of haploids and DHs through gametic embryogenesis allows a single-step development of complete homozygous lines from heterozygous parents, requiring much less time than the conventional selfing method. Here, we report the regeneration of haploid and double haploid lines of citrus species through anther culture. The anthers of seven citrus cultivars at the uninucleate stage were cultured and induced using four previously reported mediums. Ten haploid lines (2n = x = 9), six DH lines (2n = 2x = 18), two tetraploid lines (2n = 4x = 36) of ‘Early Gold’ sweet orange, and one haploid line of ‘Rohde Red’ Valencia sweet orange were obtained, as identified by ploidy, karyotype and simple sequence repeats (SSRs) analysis. All of them were confirmed to be fully homozygous by SSR analysis using 31 primer pairs that are distributed evenly on each of the chromosomes. Among them, plants regenerated from two DH lines of ‘Early Gold’ sweet orange grew vigorously in the greenhouse. To our knowledge, this is the first report on sweet orange anther culture with successful DH plant regeneration. The haploid, DH and tetraploid lines reported here hold great potential for future citrus genome resequencing in genetic studies and seedless breeding via somatic fusion.

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Abbreviations

DH:

Doubled haploid

MT:

Murashige and Tucker (1969) medium

N6 :

Germanà (2005) medium

6-BA:

6-benzyaminopurine

NAA:

Naphthalene acetic acid

IAA:

Indoi-3-acetic acid

KT:

Kinetin

2,4-D:

2, 4-dichlorophenoxyacetic acid

GA:

Gibberellic acid

TDZ:

Thidiazuron

DAPI:

4′,6-diamidino-2-phenylindole

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Acknowledgments

This research was financially supported by the Ministry of Science and Technology of China (No. 2011CB100606), the National NSF of China (Nos. 31125024, 31221062), and the Ministry of Education of China (IRT13065). The authors thank Han-Chang Zhu in Foreign Language College (HZAU) for his efforts to polish the language.

Authors contribution

SMW conducted most experiments, data analysis and wrote the manuscript. HL, HBC and CLC participated in chromosomal cytogenetic analysis. QX and XXD mined and provided some SSR markers. WWG conceived, supervised the research and revised the manuscript. All authors read and approved the final manuscript.

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  1. Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, Wuhan, 430070, China

    Shu-Ming Wang, Hong Lan, Hong-Bo Cao, Qiang Xu, Chun-Li Chen, Xiu-Xin Deng & Wen-Wu Guo

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  1. Shu-Ming Wang
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  2. Hong Lan
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Wang, SM., Lan, H., Cao, HB. et al. Recovery and characterization of homozygous lines from two sweet orange cultivars via anther culture. Plant Cell Tiss Organ Cult 123, 633–644 (2015). https://doi.org/10.1007/s11240-015-0866-4

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