Optimization of tissue culture, regeneration and genetic transformation in tomato plants

Tomato (Lycopersicon esculentum Mill.) is one of the most important vegetable fruit crops and a model plant due to its global high consumption and various types. Furthermore, with its increasing harvest area in the world followed by higher demands for high quality products, competition on offering high quality and market-friendly products has been increased. This issue caused the plant breeders to breed and introduce new market-friendly cultivars with higher yields. Genetic engineering and the introduction of external genes to plants, is one of the new methods in plant breeding which is more quick and accurate in comparison with traditional methods. One of the most important benefits of these methods is the direct transfer of external useful genes from other far species and even genus to a specific individual plant which is almost impossible in traditional methods. The plant tissue culture is the origin and the base of genetic transformation in plants and the presence of a general, quick and useful protocol for tissue culture and regeneration of specific plant can be effective in expedition and simplifying its genetic engineering programs. This could be possible only with optimization of tissue culture of that specific plant followed by representing a general and useful protocol for its genetic transformation. Besides, tissue culture can be categorized as an important and economic method in vegetative growth of plants as the cross pollinated plants could be mass propagated in economic scale without any change in their genetic heritage. On the other hand, every economic mass propagation program also needs a general and optimized economic regeneration method. In this study, two standard tomato cultivars (Red Cherry and Early Urbana VF), were used for the optimization of tissue culture of this plant under laboratory conditions. For achieving this goal, after sterilizing and culturing the seeds of these cultivars in the growth medium, three different explants (cotyledon, hypocotyls and leaf) were isolated from derived seedlings and cultured in different regeneration media containing different types and concentrations of cytokinin plant growth regulator (BAP, Zeatin and TDZ; each in two concentrations of 0.5 and 1 mg/l). In the next step, the data regarding regeneration of these explants where analyzed using factorial experiments with three factors of cultivar, explants and cytokinin PGR based on CRD design in three replications. Finally, an optimized method for tissue culture and regeneration of this plant was developed based on the effect of each factor. In a different experiment, regarding the optimized tissue culture method, a useful method for Agrobacterium-mediated genetic transformation of this plant using GUS reporter gene was introduced. For doing this, GUS 82 reporter gene was introduced to the specified cultivars separately using pBI121 and pCAMBIA3301 binary vectors and the samples were placed on optimized regeneration media. After calculating the transformation percentage for each treatment, the effect of binary vectors, cultivars and explants in transformation efficiency of tomato plant was revealed and a suitable and efficient method for genetic modification of this plant using genetic engineering was introduced. The results showed that the cultivar Red Cherry had a good potential in production of new plants in vitro. It also indicated that BAP with 1 mg/l and Zeatin with 0.5 mg/l of concentration and leaf explants were the best cytokinin PGRs and the best explants for regeneration of this plant, respectively. Finally, cultivar Red Cherry, binary vector pCAMBIA3301 and hypocotyl explants showed the highest amount of transformation.