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Improved cryopreservation of oil palm (Elaeis guineensis Jacq.) polyembryoids using droplet vitrification approach and assessment of genetic fidelity

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Abstract

In the present study, polyembryoids of oil palm (Elaeis guineensis Jacq.) were cryopreserved with successful revival of 68 % for the first time using the droplet vitrification technique. Excised polyembryoids (3–5-mm diameter) from 3-month-old in vitro cultures were pre-cultured for 12 h in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose. The polyembryoids were osmoprotected in loading solution [10 % (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose] for 30 min at room temperature and then placed on aluminium strips where they were individually drenched in chilled droplets of vitrification solution (PVS2) [30 % (w/v) glycerol plus 15 % (w/v) ethylene glycol (EG) plus 15 % (w/v) DMSO plus 0.4 M sucrose] for 10 min. The aluminium strips were enclosed in cryovials which were then plunged quickly into liquid nitrogen and kept there for 1 h. The polyembryoids were then thawed and unloaded (using 1.2 M sucrose solution) with subsequent transfer to regeneration medium and stored in zero irradiance. Following for 10 days of storage, polyembryoids were cultured under 16 h photoperiod of 50 μmol m−2 s−1 photosynthetic photon flux density, at 23 ± 1 °C. Post-thaw growth recovery of 68 % was recorded within 2 weeks of culture, and new shoot development was observed at 4 weeks of growth. Scanning electron microscopy revealed that successful regeneration of cryopreserved polyembryoids was related to maintenance of cellular integrity, presumably through PVS2 exposure for 10 min. The present study demonstrated that cryopreservation by droplet vitrification enhanced the regeneration percentages of oil palm in comparison with the conventional vitrification method previously reported.

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Abbreviations

ISSR:

Inter-simple sequence repeats

LN:

Liquid nitrogen

LS:

Loading solution

MC:

Moisture content

MS:

Murashige and Skoog

PPFD:

Photosynthetic photon flux density

PVS2:

Plant vitrification solution 2

RAPD:

Random amplified polymorphic DNA

SEM:

Scanning electron microscope

VS:

Vitrification solution

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Acknowledgments

The authors are grateful to Yayasan Felda, Malaysia, for the financial support through research grant, Dr. Sharifah SRSA and Dr. Siti Habsah from Felda Biotechnology Centre for the supply of cell suspension culture and Dr. Sanjoy Banerjee (Institute of Bioscience, Universiti Putra Malaysia) along with Dr. Kuttichanthran Subramaniam (Faculty of Veterinary Medicine, Universiti Putra Malaysia) for their assistance during genetic integrity study, and last but not the least, the Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia for the research facilities.

Conflict of interest

We, the authors of this article, declare that there is no conflict of interest, and we do not have any financial gain from it.

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Authors and Affiliations

  1. Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Saikat Gantait, Uma Rani Sinniah, Periasamy Suranthran & Sharrmila Rengeswari Palanyandy

  2. Felda Biotechnology Centre (FBC), 71760, Bandar Enstek, Negeri Sembilan, Malaysia

    Periasamy Suranthran

  3. School of Biological Sciences, Universiti Sains Malaysia, Minden Heights, 11800, Georgetown, Penang, Malaysia

    Sreeramanan Subramaniam

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  1. Saikat Gantait
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  2. Uma Rani Sinniah
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  3. Periasamy Suranthran
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  4. Sharrmila Rengeswari Palanyandy
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  5. Sreeramanan Subramaniam
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Correspondence to Uma Rani Sinniah.

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Handling Editor: Peter Nick

S. Gantait and U.R. Sinniah conceived the idea and designed the experiments. S. Gantait P. Suranthran and S.R. Palanyandy executed the experiments. S. Gantait, U.R. Sinniah and S. Subramaniam wrote the manuscript.

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Gantait, S., Sinniah, U.R., Suranthran, P. et al. Improved cryopreservation of oil palm (Elaeis guineensis Jacq.) polyembryoids using droplet vitrification approach and assessment of genetic fidelity. Protoplasma 252, 89–101 (2015). https://doi.org/10.1007/s00709-014-0660-x

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