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Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings

Abstract

The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates; no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.

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Abbreviations

NYD:

Nias Yellow Dwarf

NGD:

Nias Green Dwarf

SOD:

Sagerat Orange Dwarf

MYD:

Malayan Yellow Dwarf

TKT:

Takome Tall

WAT:

West African Tall

COGENT:

Coconut genetic resources network

GMR:

Global DNA methylation rate

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Acknowledgments

The authors gratefully thank the Australian Agency for International Development (AusAID), the Australian Centre for International Agricultural Research (ACIAR), the Graduate School Research Travel Grants (GSRTG) scheme at the University of Queensland and the French CIRAD-Desi/DRS Projects for financial support. We are grateful to Dr. Hengky Novarianto and his staff at the Indonesian Coconut and Other Palm Research Institute (ICOPRI), Manado, Indonesia for preparation of plant material. The authors thank Mr. Thierry Beule, Dr. Estelle Jaligot and Mrs. Sylvie Doulbeau, IRD, Montpellier, France for their kind help in DNA methylation analysis and Ms. Angelique Berger, CIRAD, Montpellier, France for her help in microsatellite analysis.

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Correspondence to Sisunandar.

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Sisunandar, Rival, A., Turquay, P. et al. Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings. Planta 232, 435–447 (2010). https://doi.org/10.1007/s00425-010-1186-x

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Keywords

  • DNA methylation
  • Epigenetics
  • Genetic fidelity
  • Karyotype
  • Microsatellite