Methods of transformation

Efficient transformation methods require good control of the regeneration step through either organogenesis or somatic embryogenesis. In cucurbits, regeneration by direct or indirect organogenesis has been achieved from various plant tissues including cotyledons, hypocotyls and leaves.^3-5 The efficiency of regeneration has been found to be highly dependent on the stage of development and the growth conditions⁶ and the genotype.⁷ Regeneration of cucurbits has been obtained by somatic embryogenesis from callus or different explant tissues, but development of somatic embryos into plants still remains difficult, being highly dependent on thegenotype^8,9 and controlled by crossing.¹⁰ This renders the process of plant transformation more difficult and reduces the number of cultivars susceptible to be engineered. On the other hand, the regeneration process is known to induce endo-polyploidisation^{11, 12} leading to reduced productivity of the plants and altered shape and smaller size of the fruit, mainly in melon. Endo-polyploidisation occurs during the development of cotyledons, and at a lower rate in leaves.^12–14 Thus, the regeneration protocols must be adapted to yield low endo-polyploid plantlets.¹⁴

Currently, several methods for gene transformation including Agro bacterium mediated transformation and biolistic are available. A great number of melon, cucumber and squash genotypes have been transformed with different genes and in particular with genes of agronomic interest (Table 10.1). Transgenic plants currently tested in field conditions or commercialised were generated via Agro bacterium-mediated transformation. In cucurbits, cotyledonary explants and adventitious shoots have been widely used for generation. In these transformation systems, the NptII selectable marker gene is the most widely used allowing selection of transformed tissues in the presence of kanamycin.

Biolistic transformation has been used for the generation of transgenic cucumber and melon plants, respectively from highly embryogenic cell suspension cultures¹⁵ or embryosdeveloped on cotyledons¹⁶ leading to a high stability of the transgene. Genetic transfer via A. rhizogenes in cucumber³ or Cucurbita pepo,^{17, 18} or via the pollen-tube pathway in watermelon¹⁹ have been described but so far do not seem to have provided an efficient process in cucurbits.