Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Abstract

Conditions for plant regeneration from explants of tomato (Lycopersicon esculentum) cv. UC82B were studied for optimizing transformation procedure. The best regeneration rate was obtained from cotyledon explants from 8–10-d-old seedlings on a modified Murashige and Skoog medium (1962) with 0·5 mg dm⁻³ zeatin and 0·5 mg dm⁻³ indolylacetic acid. Tomato cultivars (UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum ‘CMV sel. INRA’ were studied. The cultivarUC82B and the wild Lycopersicon species showed an efficient shoot regeneration potential. Early events in the transformation of tomato cotyledons were analysed using an Agrobacterium tumefaciens strain carrying a binary vector with an nptII (pnos) gene and a reporter GUS-intron (p35S) chimeric gene. Two days after infection, GUS activity appeared specifically at the cut surface. Subepidermal cells were more susceptible to transformation than epidermal cells. When selection for kanamycin resistance was applied 2 d after inoculation, transformed cells were efficiently recovered. Preculture with feeder cells stimulated cell transformation, but reducedregeneration capacity from transformed cells. The optimal transformation rate was observed witha time of preculture of 1 and 2 d. Transformation events for two tomato cultivars (UC82B and Monalbo) occurred at the same rate as 55% of the inoculated explants developed kanamycin resistant calli. However, transformed plants were obtained at different rates of 8% and 14% for cv. Monalbo and cv. UC82B.