Study On Systematic Methods Of Rapidly Obtaining Tetraploid Of Watermelon (Citrullus Vulgaris) By Biotechnique

Experiments of rapidly obtaining tetraploids were made with cultured cotyledons of 5 diploid varieties (jingxin No.l, zhengza No.5, xincheng, fengshou No.2, xinkaiza No.2) of watermelon [Citrullus lanatus (Matsum & Nakai)] by combinning plant tissue culture with chromosome engineering without supplying any inducer. In this study, the rule of regeneration, rapid identification, multiplication and grafting in tube cultured seedling and avoiding disadvantageous factors were systematically researched. The mechanism of tetraploid regenerating was also probed. The results indicated that:Aseptic germination was fundmental to the experiments. On condition that disinfecting 6-9min with 1.0-1.5% NaClo, then soaking and vernalizating 15-17h in darkness followed light-culture 3-4 days was economical, efficient and high qualitative way.There were many factors influence the rate of regeneration and tetraploid. The basal medium MS, pH5.8, day-temperature 25-30℃ and night-temperature 20-25℃, distance(0-2 mm) from cotyledon petide after germinating 2-4d and concentration of BA 2.0-2.5 mg/L were the optimum conditions, under which the average differention frequency was70.33%, multiplication coefficient was 2.46 and tetraploid frequency was 28.85%.Compared with the methods that induction of polyploid from colchicine-treated (supplemented in medium, soaking, dripping and treating cell colony, the tetraploid frequency were 9.7%,9.1%,3.85%,0, respectively) ,it was a efficient way for obtaining tetraploid.Five methods were used and compared for identifying ploidy level of tetraploids arising from tube cultured seedlings. All the tetraploids could be easily detected in 30 days by examing the chromosome number in the apical cells of the adventitious shoots, the established critical point of determination for the method of multiplication coefficient was practicabale for large-scale identification and rapidly applying to filed, high-temperature stress and low-temperature stress andnutrition stress were workable as well, moreover, tetraploid plants showed lower multiplication coefficient and better adaptation to high- and low-temperature stress and nutrition stress than their diploid counterparts, the average ploidy rates of accurate judgement were 100%, 88%, 90%, 80%, 100%, respectively.Karyotyping and RAPD showed that tetraploid arising from cotyledons culture and diploid were nothing but doubling chromosome complement, which had not gene recombination and genetic drift, It was a completely different mechanism compared with colchicine-treated.In the process of tetraploid multiplication, MS basal medium supplemented with BA 2.0mg/L and NAA0.2-0.3mg/L gave the best result in all of the varieties, forthermore, BA was more important than NAA.It was the first report that the tube-cultured seedlings of tetraploid watermelon were grafted on the fig-leaf gourd germinated 2 days, and 100% survival ratio could be reached.Several disadvantageous factors after many times maniformed were studied. Half of the NHUNOa content of MS could prevent seedlings from vitrification, yellow and death, continuous multiplication and continuous growth could resolve the crux of scale-shaped differentiation of buds, ISA 0.1mg/L and NAA 0.1mg/L applied in medium was workable to resolve the problem of taking roots.