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Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants

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Abstract

A protocol was developed for preservation of calli of Oryza rufipogon Griff. in liquid nitrogen (−196°C). Optimal conditions for cryoprotection, pre-freezing and post-thaw recovery of calli were identified. Cryoprotectant treatment at low temperature, pre-freezing in isopropanol bath for 1 h at −70°C, rapid thawing and proper removal of cryoprotectant were critical for post-thaw survival. Genetic fidelity of the R1 plants obtained from cryopreserved, cryoprotectant-treated and untreated calli was assessed by phenotypic and molecular characterization. Comparison of phenotypic characters with seed-derived control plants showed no significant variation in the agronomic characters, but seed physical characters showed significant reduction in all the in vitro generated plants. Molecular data generated using 26 rice simple sequence repeat markers showed 4.78–7.25% change from control. Results suggested that both callus induction and cryopreservation induced heritable variations in O. rufipogon. In addition, a combination of phenotypic and molecular characterization using an appropriate marker provided better insight into genetic fidelity of recovered plants.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

AFLP:

Amplified fragment length polymorphism

AMP:

Amplified DNA methylation polymorphism

ANOVA:

Analysis of variance

BAP:

Benzyl amino purine

LSD:

Least significance difference

NAA:

α-Napthaleneacetic acid

PCA:

Principle component analysis

PCR:

Polymerase chain reaction

PGR:

Plant growth regulator

RAF:

Randomly amplified DNA fingerprinting

RAPD:

Randomly amplified polymorphic DNA

RFLP:

Restriction fragment length polymorphism

SSR:

Simple sequence repeat

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Acknowledgments

The authors sincerely thank the director, ICAR Research Complex for NEH Region for providing facilities. We also thank the anonymous reviewers whose suggestions have helped to improve the manuscript to the present form. The work was funded by grant no. BT/PR2768/AGR/142/2001 of Department of Biotechnology, Government of India to AP.

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  1. Division of Plant Breeding, ICAR Research Complex for NEH Region, Umiam, 793103, Meghalaya, India

    P. K. Zeliang, A. Pattanayak, B. Iangrai, E. A. Khongwir & B. K. Sarma

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  1. P. K. Zeliang
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  2. A. Pattanayak
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  3. B. Iangrai
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  4. E. A. Khongwir
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Correspondence to A. Pattanayak.

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Communicated by S. Merkle.

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Zeliang, P.K., Pattanayak, A., Iangrai, B. et al. Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants. Plant Cell Rep 29, 1423–1433 (2010). https://doi.org/10.1007/s00299-010-0932-7

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