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Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica

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Abstract

The desert shrub Leptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is an important multipurpose woody species of tropical and sub-tropical arid regions. The shrub’s excellent pharmacological properties, importance in desert afforestation and role in sand dune fixation has been elaborately studied in recent years which make it a potential candidate for genetic manipulation in the global warming scenario. We have developed an Agrobacterium-mediated transformation protocol for L. pyrotechnica using hypocotyl explants from 5 days old seedlings. The reliability of the protocol has been tested by transforming the species with gus and gfp reporter genes separately. Hypocotyl explants were sonicated for 40 s, infected with Agrobacterium suspension of OD600 0.5, co-cultivated in the dark for 2 days at 26 °C in presence of 200 μM acetosyringone. Transgenic plants were obtained after 20–22 weeks at a frequency of 14 and 11 % for gus and gfp reporter genes respectively. Transgenic plants were confirmed by PCR and Southern blots. Expression of the two reporter genes has been tested in different stages of transgenic plant development. No phenotypic differences between the wild-type and transgenic plants were noted. This method will be very helpful to introduce alien genes-of-interest for various biotechnological applications.

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Abbreviations

SAAT:

Sonication-assisted Agrobacterium-mediated transformation

GUS:

β-Glucuronidase

GFP:

Green fluorescent protein

hpt :

Hygromycin phosphotransferase

X-Gluc:

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

BA:

N6-Benzyladenine

NAA:

Naphthaleneacetic acid

ABA:

Abscisic acid

IBA:

Indole-3-butyric acid

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Acknowledgments

The authors are thankful to Ms Allison Hutmacher for her technical support. This work was supported by a grant from the UAE Ministry of Presidential Affairs.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Plant Sciences, University of California, 1 Shields Ave, Davis, CA, 95616, USA

    Indrajit Dutta, Martin Kottackal, Ellen Tumimbang, Hiromi Tajima & Eduardo Blumwald

  2. Date Palm Development Research Unit, UAE University, P.O.Box 81908, Al Ain, UAE

    Abdelouahhab Zaid

Authors
  1. Indrajit Dutta
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  2. Martin Kottackal
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  3. Ellen Tumimbang
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  4. Hiromi Tajima
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  5. Abdelouahhab Zaid
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  6. Eduardo Blumwald
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Correspondence to Eduardo Blumwald.

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Dutta, I., Kottackal, M., Tumimbang, E. et al. Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica . Plant Cell Tiss Organ Cult 112, 289–301 (2013). https://doi.org/10.1007/s11240-012-0236-4

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  • DOI: https://doi.org/10.1007/s11240-012-0236-4

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