Improvement in Organogenesis and the Development of a Transformation Procedure for Cucumber and Muskmelon

A comprehensive study was carried out to optimise and improve a direct shoot organogenesis system to facilitate the transfer of reporter genes (GUSINT and nptII into cucumber (Cucumis sativus L.) and muskmelon (C. melo L.) plants. The studies carried out were: (i) a comparative study on direct and indirect shoot regeneration (ii) the improvement of a direct shoot regeneration system by using an ethylene action inhibitor, silver nitrate and an ethylene biosynthesis inhibitor, cobalt chloride (iii) identification of suitable strains of Agrobacterium tumefaciens and A. rhizogenes that are able to infect cucumber and muskmelon plants (iv) development of an intron containing gene transformation system for both cucurbit species through A. tumefaciens and (v) analysis of transformants. For the comparative study of direct and indirect shoot regeneration, four different types of explants from five elite commercial cucumber cultivars namely Spring Swallow, Suyo Cross, Suyo Long, Tasty Glory. Tasty Green and one muskmelon cultivar called Birdie were used. Explants from both species were cultured onto Murashige and Skoog medium containing different concentrations of 6-benzylaminopurine alone and in combination with either 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid. 6-benzylaminopurine alone at 1.0 or 2.0 mg/L significantly (p<0.05) induced shoot primordia from the largest number of proximal cotyledon and hypocotyl explants of all cucumber cultivars and muskmelon explants tested. However, the addition of either 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid to 6-benzylaminopurine, resulted in a reduction of the shoot primordia induction rate. Higher number of shoots induced from ex plants as well as high numbers of morphological normal shoots were obtained when explants were cultured on. medium containing 6-benzylaminopurine alone. Specific concentrations of indole-butyric acid and naphthalene acetic acid significantly (p<0.05) contributed to root initiation from the largest number of cucumber and muskmelon shoots, respectively. Dark treatment was sufficient to significantly induce root formation from the non-rooting cucumber and muskmelon shoots. The addition of either silver nitrate or cobalt chloride to the seed germination medium or shoot primordia induction medium caused a significant (p<0.05) enhancement of shoot regeneration rate from cucumber cv. SS and TG explants compared to the control. The regeneration rate was further enhanced when these two ethylene inhibitors were added to both SGM and SPI media. Furthermore, the number of shoots induced from explants of both Spring Swallow and Tasty Green cultivars was also enhanced upon the same treatment. However, muskmelon shoot induction and regeneration were reduced when the same treatment was employed