Mechanism Of Regeneration From Cotyledons And The Application Of Efficient Regeneration System In Watermelon (Citrullus Lanatus)

Watermelon is one of the most important horticultural crops, and widely cultivated in the world. Traditional breeding method has a slow process and low efficiency,while mordern biotechnology can provide new approaches for germplasm resources and breed improvement. In this study, we successfully applied plant tissue culture technology into watermelon with an efficient shoot organogenesis system derived from cotyledon. According which, we examined the expression pattern of WOX gene family during in vitro shoot organogenesis. Furthermore, we studied regeneration shoot grafting, test-tube plantlet micro-grafting and in vitro tetraploid induction in watermelon using the efficient shoot organogenesis system. The results were as follows.1. The cotyledons of watermelon inbred A7 were used for establishment of shoot regeneration system. The results indicated that ethyl alcohol soaking has no effect on sterilization. Proximal part of cotyledon from seedlings incubated for 5-6 days was the best explants type. The optimum shoot induction medium was MS+1.0 mg/L 6-BA+0.1 mg/L NAA with shoot induction rate of 88.33 %. The elongation medium was MS+0.2 mg/L 6-BA. The rooting medium was 1/2MS+0.5 mg/L IBA with rooting rate of 92.22%. The survival rate of transplants was about 61.67% with sustrate consisted of eaqual vermuculite and coconut bran.2. We successfuly identified 11 WOX family genes in the published watermelon genome. Phylogenetic analysis revealed close evolutionary relations in Cucurbitaceae species. The q RT-PCR analysis demonstrated active expression of 11 WOX genes in watermelon tissues. Most watermelon WOX genes highly expressed when cotyledon incubated for 14 and 28 days, respectively. Moreover, WOX genes expressed higher in proximal parts than distal parts of cotyledons when incubated for 14 days.3. Regenerated shoots over 2cm with obvious growing points have a survival rate of 71.67% after gratfing. Test-tube plantlets with 2 euphylla and 0.8 cm in length have a survival rate over 90% after micro-grafting in MS medium with 50 g/L sucrose.There was no significant difference in agronomic traits and fruit quality among grafted regenerated shoots, micro-grafted test-tube plantlets and field seedlings.4. The in vitro tetraploid induction rate was 25% on medium with 0.1% colchicine after processing for 72 h. The shoot induction rate was 62.5% with growth coefficient3.6. In tetraploid identification, the coincident rates of morphological observation and chloroplast counting method with result of FCM were 91.8% and 100%, respectively.