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Optimization of factors for efficient recovery of transgenic peanut (Arachis hypogaea L.)

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Abstract

De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing β-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes. Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics (incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at 21°C on shoot induction medium containing 100 mg/L l-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate of the grafted shoots was nearly 100% in glass-house conditions. The optimized protocol took around 3 months to generate healthy plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the study.

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Abbreviations

AS:

Acetosyringone

BAP:

6-Benzylaminopurine

DEC:

De-embryonated cotyledon

GUS:

β-Glucuronidase enzyme

hpt :

Hygromycin phosphotransferase gene

nptII :

Neomycin phosphotransferase gene

PCR:

Polymerase chain reaction

SIM:

Shoot induction medium

uidA :

β-glucuronidase gene

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Acknowledgments

The authors express their gratitude to National Botanical Research Institute, Council of Scientific and Industrial Research, Lucknow, India for providing research facilities and to the Department of Science and Technology, Government of India, for a JC Bose Fellowship to Rakesh Tuli.

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  1. Siddharth Tiwari & Rakesh Tuli

    Present address: National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Govt. of India, C-127, Industrial Area, Phase VIII, SAS Nagar, Mohali, 160071, Punjab, India

Authors and Affiliations

  1. Plant Molecular Biology and Genetic Engineering Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, India

    Siddharth Tiwari & Rakesh Tuli

  2. National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology Govt. of India, C-127, Industrial Area, Phase VIII, SAS Nagar, Mohali, 160071, Punjab, India

    Rakesh Tuli

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Correspondence to Rakesh Tuli.

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Tiwari, S., Tuli, R. Optimization of factors for efficient recovery of transgenic peanut (Arachis hypogaea L.). Plant Cell Tiss Organ Cult 109, 111–121 (2012). https://doi.org/10.1007/s11240-011-0079-4

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