Agrobacterium-mediated transformation of bottle gourd (lagenaria siceraria standl.)

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.)
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Unformatted text preview: Plant Cell Rep (2005) 23:692698 DOI 10.1007/s00299-004-0874-z G E N E T I C T R A N S F O R M A T I O N A N D H Y B R I D I Z A T I O N J.-S. Han · C. K. Kim · S. H. Park · K. D. Hirschi · I.- G. Mok Agrobacterium-mediated transformation of bottle gourd ( Lagenaria siceraria Standl.) Received: 17 February 2004 / Revised: 31 July 2004 / Accepted: 2 August 2004 / Published online: 12 October 2004 # Springer-Verlag 2004 Abstract We describe a procedure for producing trans-genic bottle gourd plants by inoculating cotyledon ex-plants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance ( bar ) gene and the b-d-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobac-terium for 68 days on co-cultivation medium supple-mented with 0.10.001 mg/l l-a-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots di-rectly emerged at the proximal end of cotyledon explants after 23 weeks of culturing on selection medium con-taining 2 mg/l dl-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l dl-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T 1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reac-tion and Southern blot analyses. Genetic segregation anal-ysis of T 1 progenies showed that transgenes were inher-ited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transforma-tion in bottle gourd. Keywords Bottle gourd ( Lagenaria siceraria Standl.) · Agrobacterium tumefaciens · gus / bar · Transformation · Ethylene inhibitor Abbreviations Acetosyringone: 3 ,5-Dimethoxy-4 hydroxyacetophenone · AVG: l-a-(2-Aminoethoxy-vinyl) glycine · BA: 6-Benzylaminopurine · GUS: b-d-Glucuronidase · IAA: Indole acetic acid · PPT: dl-Phosphinothricin Introduction Bottle gourd ( Lagenaria siceraria Standl.) has been used routinely as a source of rootstock for watermelon and other cucurbit crops in both Korea and Japan as a means to reduce the incidence of soil-borne diseases and to promote the vigor of the root system of the crop under conditions of low temperature (Lee and Oda 2003). Re-cently the potential of Lagenaria rootstock to confer re-sistance to the carmine spider mite has also been reported (Edelstein et al. 2000). Most of the damage that arises from the continuous cropping of vegetables grown for commercial purposes in the greenhouse is caused by soil-borne diseases and salinization of the soil. To overcome these problems, more than 95% of the commercially grown watermelons are grafted onto bottle gourd or squash (an interspecific hybrid between Cucurbita maxi-ma ň C. ...
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