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Cryopreservation of Citrus anthers in the National Crop Genebank of China

  • Cryopreservation
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Abstract

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L−1 sucrose, 0.1-g L−1 boric acid, 1.0-g L−1 calcium nitrate, 0.1-g L−1 potassium nitrate, 0.3-g L−1 magnesium sulfate, and 10-g L−1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.

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Acknowledgments

This study was supported by the Utilization Special Grant from the Ministry of Agriculture (Grant number: 2016NWB036-09), the Agricultural Science and Technology Innovation Program/Crop Germplasm Resources Preservation and Sharing Innovation Team (CAAS, ASTIP), the National Key Technology R&D Program (Grant number: 2013BAD01B01), and the Crop Germplasm Resources Protection, Cooperation Research on Collecting Techniques and Practice in Crop Genebank between China and United States of America (2014DFG31860). The authors would like to acknowledge Gayle M Volk (National Center for Genetic Resources Preservation, United States Department of Agriculture, Fort Collins, Colorado, USA) for assistance in revisions of the manuscript.

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Authors and Affiliations

  1. National Crop Genebank, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Jin-Mei Zhang, Xin-Xiong Lu, Xia Xin, Guang-Kun Yin, Juan-Juan He, Bin Huang & Xiao-Ling Chen

  2. Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Bin Huang

  3. Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing, 400712, China

    Dong Jiang

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  1. Jin-Mei Zhang
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  2. Xin-Xiong Lu
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  3. Xia Xin
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  7. Dong Jiang
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Contributions

Jin-Mei Zhang designed and performed research, analyzed data, and wrote the manuscript. XX, GKY, JJH, and BH participated in the acquisition of the original data and the analysis of the data. XXL and DJ participated in the revision of the manuscript. XLC designed research and revised the manuscript. All the authors have read and approved the final manuscript.

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Correspondence to Xiao-Ling Chen.

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Editor: Barbara Reed

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Zhang, JM., Lu, XX., Xin, X. et al. Cryopreservation of Citrus anthers in the National Crop Genebank of China. In Vitro Cell.Dev.Biol.-Plant 53, 318–327 (2017). https://doi.org/10.1007/s11627-017-9848-z

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