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Direct organogenesis from cotyledonary node explants suitable for Agrobacterium-mediated transformation in peanut (Arachis hypogaea L.)

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Abstract

Efficient and genotype-independent in vitro regeneration is an essential prerequisite for incremental trait improvement in peanut (Arachis hypogaea L.) via genetic transformation. We have optimized a facile and rapid method to obtain direct shoot organogenesis from cotyledonary node (CN) explants excised from peanut seedlings germinated on cytokinin-supplemented Murashige and Skoog (MS) basal salt medium. Starting with mature embryos, shoot induction occurred in approximately 7 weeks, followed by 4 weeks for rooting of excised shoots and 3 weeks of acclimatization of regenerated plantlets in soil. The regeneration and transformation system described here is time-efficient, yielding greenhouse-acclimatized plantlets within 14 weeks, in contrast to 12–14 months required for initiating and regenerating somatic embryogenic cultures, currently the most tractable method available for peanut transformation. The highest shoot induction frequency and shoot quality was obtained with 6.66 μM 6-benzylaminopurine, followed by adequate root induction at 5.37 μM α-Naphthaleneacetic acid. New Mexican Valencia A was chosen for Agrobacterium-mediated transformation. Stable GUS expression from pWBvec10a was obtained at a transformation rate of 1.25 %. Furthermore, results from genomic PCR and Southern blot analyses showed that 14 out of 576 putative transgenic regenerants contained transgene pSag12::IPT, therefore yielding a total transformation rate of 2.43 %. The cotyledonary node-based direct regeneration system described here is time-efficient and amenable to Agrobacterium-mediated transformation, and therefore should be further explored for peanut transgenic improvement.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

BAP:

6-benzylaminopurine

CN:

Cotyledonary node

MS:

Murashige and Skoog (1962) medium

NAA:

α-naphthalene acetic acid

RIM:

Root initiation medium

SEM:

Shoot elongation medium

SIM:

Shoot initiation medium

TDZ:

Thidiazuron

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Acknowledgments

This work is supported by Florida Peanut Producers Association. We are grateful to Dr. Phat Dang at USDA ARS for sharing his acclimatization protocol for peanut regenerants. Thanks to Prof. Fredy Altpeter and Prof. Gloria Moore at University of Florida for sharing pWBvec10a, pCAMBIA2201. Prof. Stanton B. Gelvin at Purdue University is gratefully acknowledged for sharing A. tumefaciens strain EHA105.

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  1. Mukesh Jain

    Present address: Plant Pathology Department, University of Florida, Gainesville, FL, 32610-3610, USA

Authors and Affiliations

  1. Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32610-3610, USA

    Yih-Feng Hsieh, Jianping Wang & Maria Gallo

  2. Agronomy Department, University of Florida, Gainesville, FL, 32610-3610, USA

    Mukesh Jain, Jianping Wang & Maria Gallo

  3. Genetics Institute, University of Florida, Gainesville, FL, 32610-3610, USA

    Jianping Wang & Maria Gallo

  4. Present Address: Delaware Valley University, Doylestown, PA, 18901, USA

    Maria Gallo

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  1. Yih-Feng Hsieh
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Correspondence to Jianping Wang or Maria Gallo.

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Hsieh, YF., Jain, M., Wang, J. et al. Direct organogenesis from cotyledonary node explants suitable for Agrobacterium-mediated transformation in peanut (Arachis hypogaea L.). Plant Cell Tiss Organ Cult 128, 161–175 (2017). https://doi.org/10.1007/s11240-016-1095-1

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