An efficient regeneration protocol for genetic transformation of different melon genotypes (Cucumis melo L. var. cantalupensis)

Melon is an important fruit crop, belonging to the Cucurbitaceae family. Genomic tools exist for this species and plant biotechnology can be used to introduce genes and transfer novel characteristics for breeding or functional validation of ‘candidate genes’ involved in horticultural traits. However, the two major pre-requisites for genetic transformation through Agrobacterium tumefaciens are the availability of a reliable plant regeneration system and a suitable method for transformation. Previous studies showed that in vitro manipulation of melon is difficult; the regeneration efficiency is strongly influenced by both the genotype and the medium. The aim of this work was to set up a good protocol for melon plant regeneration to be used for Agrobacterium–mediated genetic transformation. Six melon genotypes, belonging to the var. cantalupensis (the doubled-haploid lines NAD, L6, L2 and the cvs. Isabelle, Charentais-T, Vedrantais), were employed to evaluate their morphogenetic response to different hormone concentrations. Cotyledon explants from 6-days old seedlings were cultured on MS medium added with different combinations of benzylaminopurine (BAP) and indolacetic acid (IAA). The three media were identified as ‘A’ (BAP 1.0 - IAA 1.0·10-2 mg/l), ‘B’ (BAP 1.2 - IAA 1.2·10-2 mg/l) and ‘C’ (BAP 1.3 - IAA 1.3·10-2 mg/l). Each explant was scored for developing shoot buds after 30 days of culturing; the average number of shoots/explant ranked from 0.4 (L6) to 4.1 (Charentais-T), from 0.3 (L6) to 4.4 (Charentais-T) and from 0.1 (L6) to 2.4 (Vedrantais) for the medium A, B and C, respectively. Although we obtained regenerated plants for every genotypes from all the media used, there were significant differences in the frequency of regeneration. The plant regeneration rate varied from 24% (Charentais-T, Isabelle) to 85% (L6), from 20% (L2) to 66% (NAD) and from 10% (Charentais-T) to 50% (L6) for the medium A, B and C, respectively. Subsequently, all the genotypes were used for transformation experiments mediated by Agrobacterium tumefaciens. Different concentrations of kanamycin (Km) and geneticin (Gt) were added on regeneration medium B and compared for their efficiency as selection agents for the selection and regeneration of transgenic shoots after the infection. Preliminary histochemical GUS assay confirmed the transient incorporation of a selectable marker (nptII) into the genome of transgenic plants. The effective setting of this part of the protocol is still in progress.