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A method to overcome the waxy surface, cell wall thickening and polyphenol induced necrosis at wound sites - the major deterrents to Agrobacterium mediated transformation of bamboo, a woody monocot

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Abstract

The method is the first successful report of Agrobacterium mediated genetic transformation of the commercially important bamboo, Dendrocalamus hamiltonii. It shows how the resistance provided by the somatic embryos of this woody monocot can be overcome using a simple and effective method. The method thus standardized can be also used for the genetic transformation of other important bamboos. Identification of the factors responsible for the resistance of the somatic embryos to Agrobacterium infection was an absolute requirement for devising a successful method. Necrosis due to polyphenol oxidation, lack of differentiation due to cell wall thickening at wound sites, waxy surfaces of somatic embryos with anti-microbial properties were found to prevent Agrobacterium attachment and infection. Therefore, the somatic embryos were transformed with fresh overnight grown Agrobacterium culture containing 500 mg/l polyvinylpyrrolidone (PVP) and 0.01 % Tween-20 as surfactant followed by co-cultivation on Murashige and Skoog (MS) medium containing the vir gene inducer acetosyringone (100 μM) and 1 mg/l 6-Benzylaminopurine BAP for 2 days. Persistent GUS expression and strong positive signals in PCR, slot blot and Southern hybridization confirmed successful genetic transformation.

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Abbreviations

BAP:

6-Benzylaminopurine

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

MS medium:

Murashige and Skoog medium

NAA:

α-Napthaleneacetic acid

PCR:

Polymerase chain reaction

PPO:

Polyphenol oxidase

PVP:

Polyvinylpyrrolidone

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Acknowledgements

The authors thank the Director CSIR-IHBT for extending the requisite facilities for carrying out this work. They also acknowledge the Department of Biotechnology, Government of India and the Council of Scientific and Industrial Research (CSIR), Government of India for funding the work. Priyanka Sood acknowledges the CSIR for her Senior Research Fellowship.

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Authors and Affiliations

  1. Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, 176 061, Himachal Pradesh, India

    Priyanka Sood, Amita Bhattacharya, Sanjoy Chanda & Anil Sood

  2. Division of Hill Area Tea Sciences, CSIR-Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, 176 061, Himachal Pradesh, India

    Robin Joshi & Ashu Gulati

Authors
  1. Priyanka Sood
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  2. Amita Bhattacharya
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  3. Robin Joshi
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  4. Ashu Gulati
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  5. Sanjoy Chanda
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  6. Anil Sood
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Corresponding author

Correspondence to Amita Bhattacharya.

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IHBT Publication number: 0869

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Fig. A

Changes in cellular structures leading to cell wall thickening in treated D. hamiltonii somatic embryos after 0, 96 and 120 h of incubation on MS1 medium; (A–C) immersion, (D–F) pricking and (G–I) vacuum infiltration where (A), (D), (G): represents 0 h; (B), (E), (H): represents 96 h; and (C), (F), (I): represents 120 h. (JPEG 146 kb)

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Sood, P., Bhattacharya, A., Joshi, R. et al. A method to overcome the waxy surface, cell wall thickening and polyphenol induced necrosis at wound sites - the major deterrents to Agrobacterium mediated transformation of bamboo, a woody monocot. J. Plant Biochem. Biotechnol. 23, 69–80 (2014). https://doi.org/10.1007/s13562-013-0189-7

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