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Cryopreservation of embryogenic cell suspensions of Catharanthus roseus L. (G) Don.

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Abstract

An efficient cryopreservation protocol was established for embryogenic cell suspension cultures of Catharanthus roseus. This involved a vitrification-based cryopreservation method wherein embryogenic cells were exposed to a preculture/pretreatment medium prior to their immersion in liquid nitrogen. These cell suspension cultures were first initiated from friable embryogenic callus derived from hypocotyls of C. roseus on a medium containing 4.52 μM 2,4-Dichlorophenoxy acetic acid (2, 4-D). Among different sucrose (0.09–0.6 M) and sorbitol (0.2–0.6 M) levels evaluated during preculture, 0.4 M sucrose promoted highest cellular regrowth. Whereas, among pretreatments Dimethyl sulphoxide (DMSO) (5 or 10%) and glycerol (5 or 10%) at six different levels either alone or in combinations (PT 1–PT 6), the cryopreservation treatment combinations of either 0.4 M sucrose, 5% DMSO, and 5% glycerol (PT-5) or 0.4 M sucrose and 5% DMSO (PT-1) resulted in the highest frequency of viability of embryogenic cultures. However, the PT-1 treatment produced highest number of cell colonies (10.06 ± 0.55) following reculture of cryopreseved cultures. All calluses regrown in an optimized medium, containing 6.62 μM 6-benzyladenine (BA) and 5.37 μM α-naphthaleneacetic acid (NAA), resumed normal growth, and produced somatic embryos similar to those from non-frozen embryogenic cultures. These somatic embryos were converted into regenerated plantlets, and all plantlets exhibited normal morphology.

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Abbreviations

BA:

6-Benzyladenine

DMSO:

Dimethylsulfoxide

MS:

Murashige and Skoog medium

NAA:

α-Naphthaleneacetic acid

LN:

Liquid nitrogen

2,4-D:

2,4-Dichlorophenoxy acetic acid

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Acknowledgments

We wish to thank two anonymous reviewers, particularly the first for constructive suggestions on the manuscript. We are highly thankful to the Editor-in-Chief, Schuyler S. Korban, for his valuable comments and final editing.

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Authors and Affiliations

  1. Cellular Differentiation and Molecular Genetics Section, Department of Botany, Hamdard University, New Delhi, 110062, India

    Samar Fatima, A. Mujib, S.A. Nasim & Z.H. Siddiqui

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  1. Samar Fatima
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  2. A. Mujib
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  3. S.A. Nasim
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  4. Z.H. Siddiqui
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Correspondence to A. Mujib.

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Fatima, S., Mujib, A., Nasim, S. et al. Cryopreservation of embryogenic cell suspensions of Catharanthus roseus L. (G) Don.. Plant Cell Tiss Organ Cult 98, 1–9 (2009). https://doi.org/10.1007/s11240-009-9532-z

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