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An Efficient Method of Agrobacterium-Mediated Genetic Transformation and Regeneration in Local Indian Cultivar of Groundnut (Arachis hypogaea) Using Grafting

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Abstract

Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG-20 using de-embryonated cotyledon explant. A high regeneration 52.69 ± 2.32 % was achieved by this method with 66.6 μM 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67 ± 3.51, was found with 20 μM BAP and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and l-cysteine concentration were optimized as 1.8, 200 μM and 50 mg L−1, respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65 ± 1.06 % regeneration, of which 3.82 ± 0.6 % explants were survived on hygromycin after selection. Finally, 14.58 ± 2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and β-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BAP:

6-Benzylaminopurine

CM:

Co-cultivation media

EM:

Elongation media

GUS:

β-Glucuronidase

HPT:

Hygromycin-phosphotransferase

IAA:

Indole-3-acetic acid

LB:

Luria-Bertani broth

MS:

Murashige and Skoog basal salt media

NAA:

α-Naphthaleneacetic acid

PGR:

Plant growth regulator

RM:

Rooting media

SLM:

Selection media

SM:

Shoot induction media

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Acknowledgments

CSIR-CSMCRI Communication No.: PRIS- 119/14. The financial support received from Council of Scientific and Industrial Research (CSIR), New Delhi [SIMPLE: BSC0109 and PMSI: BSC0117] is thankfully acknowledged. VT and AKC are thankful to CSIR for Junior and Senior Research Fellowships. Dr. Siddharth Tiwari, National Agri-Food Biotechnology Institute, Mohali, India, is acknowledged for his valuable suggestions.

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Authors and Affiliations

  1. Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G.B. Road, Bhavnagar, 364002, Gujarat, India

    Vivekanand Tiwari, Amit Kumar Chaturvedi, Avinash Mishra & Bhavanath Jha

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  1. Vivekanand Tiwari
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  2. Amit Kumar Chaturvedi
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  3. Avinash Mishra
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  4. Bhavanath Jha
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Correspondence to Avinash Mishra or Bhavanath Jha.

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Tiwari, V., Chaturvedi, A.K., Mishra, A. et al. An Efficient Method of Agrobacterium-Mediated Genetic Transformation and Regeneration in Local Indian Cultivar of Groundnut (Arachis hypogaea) Using Grafting. Appl Biochem Biotechnol 175, 436–453 (2015). https://doi.org/10.1007/s12010-014-1286-3

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