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Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)]

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Abstract

Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301–bar was used for transformation. The two-stage selection was carried out using 4 and 250 mg l−1 BASTA® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.

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Abbreviations

CaMV 35S:

Cauliflower mosaic virus 35S promoter

gusA :

β-Glucuronidase gene

hptII:

Hygromycin phosphotransferase

MS:

Murashige and Skoog medium

nos Poly A :

Nopaline synthase terminator

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Acknowledgements

Sivabalan Karthik, is grateful to Jawaharlal Nehru Memorial Fund, New Delhi, India, for the award of Jawaharlal Nehru Scholarship (Ref no: SU-1/88/2016-17/79) for his doctoral research.

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Author notes

  1. Sivabalan Karthik and Gadamchetty Pavan have contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India

    Sivabalan Karthik, Gadamchetty Pavan, Selvam Sathish & Markandan Manickavasagam

  2. School of Bio Sciences and Technology, VIT, Vellore, 632014, Tamil Nadu, India

    Ramamoorthy Siva

  3. Department of Biotechnology, BIT Campus, Anna University, Tiruchirappalli, 620024, Tamil Nadu, India

    Periyasamy Suresh Kumar

Authors
  1. Sivabalan Karthik
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  2. Gadamchetty Pavan
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  3. Selvam Sathish
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  4. Ramamoorthy Siva
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  5. Periyasamy Suresh Kumar
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  6. Markandan Manickavasagam
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Correspondence to Markandan Manickavasagam.

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Electronic supplementary material

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13205_2018_1231_MOESM1_ESM.tif

Supplementary material 1 (TIFF 76 kb) Supplementary Fig. 1 Illustration of the T-DNA region of the binary vector pCAMBIA1301–bar

13205_2018_1231_MOESM2_ESM.tif

Supplementary material 2 (TIFF 2296 kb) Supplementary Fig. 2 Evaluation of the germination percentage of 5 diverse peanut cultivars. One hundred seeds from each cultivar were used for the germination test. Mean values of three separate trials (±) with standard errors

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Karthik, S., Pavan, G., Sathish, S. et al. Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)]. 3 Biotech 8, 202 (2018). https://doi.org/10.1007/s13205-018-1231-1

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