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A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

  • Cell Biology and Morphogenesis
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Abstract

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25±2°C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.

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Abbreviations

AD:

adenine free base

AS:

acetosyringone

BA:

6-benzylaminopurine

CM:

coconut milk

GUS:

β-glucuronidase

IAA:

indole-3-acetic acid

IBA:

indole-3-butyric acid

MS medium:

Murashige and Skoog (1962) medium

NAA:

1-naphthaleneacetic acid

NPTII:

neomycin phosphotransferase

PGR:

plant growth regulator

SEM:

scanning electron microscopy

X-Gluc:

5-bromo-4-chloro-3-indolyl-β-d-glucuronide

ZT:

zeatin

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Acknowledgements

We thank Calvin Lemke, Bill Chissoe and Greg Strout for technical assistance, Dr. Jia Li for advice and discussion, Drs. Zengyu Wang and Yaxing Ge at The Noble Foundation, Ardmore, OK, for their kind help, and the Department of Botany & Microbiology, Vice President for Research and Graduate Student Senate, University of Oklahoma for financial support.

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Authors and Affiliations

  1. Department of Botany and Microbiology, University of Oklahoma, Norman, OK, 73019, USA

    Xiaoping Wei, Xiaoping Gou, Tong Yuan & Scott D. Russell

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  1. Xiaoping Wei
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  2. Xiaoping Gou
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  3. Tong Yuan
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  4. Scott D. Russell
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Correspondence to Scott D. Russell.

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Communicated by R. Reski

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Wei, X., Gou, X., Yuan, T. et al. A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica . Plant Cell Rep 25, 513–521 (2006). https://doi.org/10.1007/s00299-006-0114-9

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  • DOI: https://doi.org/10.1007/s00299-006-0114-9

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