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Agrobacterium-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (Cucumis melo L.)

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Abstract

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important western-shipper cantaloupe and honeydew melons, ‘F39’ and ‘150’, respectively. Different media were tested to select a shoot regeneration system for each of three elite breeding lines ‘F39’, ‘141’ and ‘TMS’. Murashige & Skoog (MS) basal medium supplemented with 1 mg l−1 benzyladenine (BA), 0.26 mg l−1 abscisic acid (ABA) and 0.8 mg l−1 indole-3-acetic acid (IAA) was used for shoot regeneration from cotyledonary explants in ‘F39’ and ‘150’. Kanamycin sensitivity as well as Timentin™ and Clavamox® were evaluated using wild-type ‘F39’ and ‘150’ cotyledons. Kanamycin concentrations of 200 and 150 mg l−1 were chosen as the threshold levels for ‘F39’ and ‘150’, respectively. No significant differences were found between Timentin™ and Clavamox® in ‘F39’; however, Clavamox® reduced the incidence of vitrification and increased the frequency of shoot elongation in ‘150’. A. tumefaciens strain EHA105, harboring pCNL56 carrying neomycin phosphotransferase II (nptII) and gusA reporter genes, was selected to establish a transformation protocol for ‘F39’ and ‘150’. Putative transformants were evaluated using β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR) and Southern blot analyses. Based on these parameters, the transformation efficiency for cantaloupe ‘F39’ was 0.3% and that for honeydew ‘150’ was 0.5%.

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Abbreviations

BA:

6-Benzyladenine

IAA:

Indole-3-acetic acid

ABA:

Abscisic acid

nptII :

Neomycin phosphotransferase II

GUS:

β-glucuronidase

MS:

Murashige and Skoog

PCR:

Polymerase chain reaction

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Acknowledgments

This research was supported by an USDA-CSREES Special Grant “Designing Foods for Health” (2006-34402-17121, 2008-34402-19195 and 2009-34402-19831) through the Vegetable and Fruit Improvement Center, Texas A&M University. We wish to thank Drs. Rebecca Grumet, Keerti Rathore, Sung Hun Park, Sylvain Marcel, Selvakumar Veluchamy, Mehdi Kabbage, Ms. Marianne Arnold, Sue Hammar, and Jungeun Kim for technical advice and help.

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Authors and Affiliations

  1. Department of Horticultural Sciences, Vegetable and Fruit Improvement Center, Texas A&M University, College Station, TX, USA

    Yan Ren, Haejeen Bang, Jean Gould, Bhimanagouda S. Patil & Kevin M. Crosby

  2. Texas AgriLife Research, Weslaco, TX, USA

    Ian S. Curtis

  3. Department of Ecosystem Science and Management, Texas A&M University, College Station, TX, USA

    Jean Gould

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  1. Yan Ren
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  2. Haejeen Bang
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  3. Ian S. Curtis
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Correspondence to Kevin M. Crosby.

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Ren, Y., Bang, H., Curtis, I.S. et al. Agrobacterium-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (Cucumis melo L.). Plant Cell Tiss Organ Cult 108, 147–158 (2012). https://doi.org/10.1007/s11240-011-0024-6

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