Regeneration, micropropagation and somatic embryogenesis in tomato (Lycopersicon esculentum Mill.)
thesisposted on 06.12.2017, 00:00 by Poonam Bhatia
"This PhD thesis embodies research carried out to develop procedures for direct shoot regeneration, micropropagation and somatic embryogenesis with a view to assisting both para-sexual procedures of cultivar improvement for large-scale production of the tomato cultivar Red Coat. " -- abstract. Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. Tomato is normally grown from hybrid seeds. Hybrid seeds are expensive to produce due to their reliance on manual labour. Alternative methods are therefore needed to minimise the cost of production of seedlings. Tissue culture techniques have the potential to meet these requirements. However, these techniques have not been exploited in tomato propagation programmes due to genotypic specificity oftomato for PGRs and other factors. This PhD thesis embodies research carried out to develop procedures for direct shoot regeneration, micropropagation and somatic embryogenesis with the view to assisting both para-sexual procedures of cultivar improvement and for large-scale production of the tomato cultivar Red Coat. A series of experiments has been performed to optimise explant related and physical and chemical factors that affect adventitious shoot regeneration in tomato. The role of chemical factors, such as the type and concentration of plant growth regulators, sugar concentration, mineral nutrients, medium pH, ascorbic acid, activated charcoal and casein hydrolysate on shoot regeneration was studied. Exogenous application of cytokinin was found essential for induction of shoot regeneration; zeatin at 15 JlM was found to be the most suitable cytokinin for shoot regeneration from cotyledonary explants. Tomato tissues were also tolerant to a wide range of medium pH, as most of the traits studied were unaffected by the medium pH. The addition of activated charcoal, ascorbic acid and casein hydrolysate had little effect on shoot organogenesis. Amongst the explant related factors, the type, orientation (placement of explants on the media) and the effect of wounding on the regeneration response were also examined. Amongst the three types of explants studied, cotyledons produced the best shoot response followed by hypocotyl and leaf explants. The explant orientation also affected shoot regeneration. Cotyledons placed in abaxial (lower surface facing down) orientation consistently produced better shoot regeneration response, with a greater number of shoots and taller shoots compared to those inoculated in adaxial (upper surface facing down) orientation. Wounding of cotyledonary explants increased shoot induction and shoot number. However, the shoots obtained from wounded cotyledons were abnormal. Light was not absolutely essential for shoot regeneration in tomato. Furthermore, the shoots that were regenerated in the dark were not etiolated. A maximum shoot regeneration response occurred in the explants exposed to light/dark cycles of 16/8 h compared to 24 h dark and 24 h light. The efficiency of the regeneration protocol that was established for the cultivar Red Coat was tested for nine other commercially important cultivars (Big Beef, Grosse Lisse, Improved Apollo, Pinnacle, Roma VF, Small Fry, Super Sweet, Sweet Bite, Tiny Tim). Marked genotypic variation was observed for shoot regeneration response, shoot number and shoot height. Additional experiments were performed to establish multiplication and rooting protocol for the regenerated shoots of the cultivar Red Coat. In the absence of cytokinin, the explants failed to develop lateral shoots. The cultures raised on the media containing kinetin showed better multiplication rate than those grown on the medium containing zeatin or BA. The genetic fidelity of the regenerated shoots was also established using amplified fragment length polymorphism (AFLP). The results confirmed similarity between the regenerated and seed-grown plants at the molecular level. Tissue cultured plants were compared with those raised from the seed for morphological, physiological and fruit yield and quality related characteristics. The results suggested similarities between the two types of plants except for density of glandular trichomes on the surface of regenerated shoots. Histological studies were undertaken to elucidate the ongm of shoots from cotyledonary explants. Centres of cell division occurred in pockets through the subepidermal and mesophyll regions of the cotyledon. Meristematic centres in the subepidermal regions became dominant, giving rise to shoot meristems. Development of a micropropagation protocol -another pathway of regeneration, was also investigated using the shoot apex of F1 seedlings of the cultivar Red Coat. The results indicated that the tomato shoot apex could initiate shoots with or without the presence of plant growth regulators, but the highest concentration (5 JlM) of NAA markedly reduced shoot initiation. At the initiation stage, the number of shoots produced per explant was highly dependent on a specific combination of cytokinin x auxin. At the multiplication stage, maximum multiplication (four fold) was achieved in the MS medium containing 15 JlM kinetin and 5 JlM IAA. Maximum root induction (100%) occurred in the MS medium containing 2.5 JlM IBA. The VI successful hardening of the regenerated seedlings also demonstrated the potential for application ofthis protocol in commercial propagation of tomato. The effects of light and BA on somatic embryogenesis of five commercially important hybrid cultivars of tomato cultivars (Big Beef, King Kong, Red Coat, Super Sweet, Sweet Bite) were also investigated. The presence of BA was absolutely necessary for somatic embryogenesis, and the exposure to light improved induction of somatic embryos in these cultivars. The studies presented in the thesis make a significant contribution to the understanding of the role of various factors on shoot regeneration and somatic embryogenesis in tomato. Histological studies helped reveal the origin of shoots, and molecular tests assisted in determining genetic fidelity of tissue cultured plants. Furthermore, a robust micropropagation protocol using axillary branching and somatic embryogenesis has been developed. The effectiveness of these protocols on commercial hybrid cultivars demonstrates a clear feasibility of producing high quality micro plants from a few hybrid seedlings of tomato.