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Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L. cv. Coker 310 FR)

  • Cell Biology and Morphogenesis
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Abstract

In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L. cv. Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation. We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol. Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends. A majority of these embryos (more than 75%) were β-glucuronidase (GUS)-positive. Embryos with an abnormal appearance showed a very poor conversion to plantlets. However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end. These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds. Regenerated plants tested positive for the transgene by Southern analysis. An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented.

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Abbreviations

2,4-D :

2,4-Dichlorophenoxyacetic acid

BA :

Benzylaminopurine

GA 3 :

Gibberellic acid

GUS :

β-Glucuronidase

NAA :

α-Naphthaleneacetic acid

nptII :

Neomycin phosphotransferase II

WT :

Wild type

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Acknowledgements

This investigation was supported by a research grant from the Department of Biotechnology, India. BC and SK were supported by Research Fellowships from the Council of Scientific and Industrial Research (CSIR). The technical assistance of Amarjeet Kumar Singh is acknowledged.

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Author notes

  1. G. S. Oinam

    Present address: SemiBiosys Genetics Inc, 110, 2985-23 Avenue N.E., Calgary, Calgary, AB, T1Y 7L3, Canada

Authors and Affiliations

  1. Department of Genetics, University of Delhi South Campus, Benito Juarez Road, 110021, New Delhi, India

    B. Chaudhary, S. Kumar, K. V. S. K. Prasad, G. S. Oinam, P. K. Burma & D. Pental

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  1. B. Chaudhary
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  2. S. Kumar
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  3. K. V. S. K. Prasad
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  4. G. S. Oinam
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  5. P. K. Burma
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  6. D. Pental
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Corresponding author

Correspondence to D. Pental.

Additional information

Communicated by R.J. Rose

B. Chaudhary and S. Kumar contributed equally to the investigation reported here.

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Chaudhary, B., Kumar, S., Prasad, K.V.S.K. et al. Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L. cv. Coker 310 FR). Plant Cell Rep 21, 955–960 (2003). https://doi.org/10.1007/s00299-003-0613-x

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  • DOI: https://doi.org/10.1007/s00299-003-0613-x

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