Study Of Key Technologies In Vitro And Tetraploid Induction Of Mini-watermelon

The excellent diploid mini-watermelon inbred line(048,MH-39-1,032,028-5-5,047-1) were used as experiment materials. Firstly, mini-watermelon regeneration system was established. Secondly, mutation effects of tetraploid were studied by using colchicine and proherbicide ORYZALIN as mutagen in vitro induction of basal parts of cotyledons. Comparing the mutagenic effects of different treatments. Thirdly, identification, transplanting of tetraploid mini-watermelon plantlets were studied. The purpose of this study is to improve mini-watermelon tetraploid induction in vitro by optimizing culture parameters. The results indicated:1. Study of key technologies in vitro mini-watermelonDry mini-watermelon seeds in water for 5-6 h; carefully stripped the shells; wrapped with the wet gauze; Rinsing with 75%alcohol for about 30s; sterilizing with 0.01% HgCl2 for 10-12min and washing with sterile distilled water for 5min (3 to 5 times); flat on the agar medium; the seeds were cultured in dark for four days and then transferred into culture room at 28℃condition; After eight days, the sound seedlings were sprouted. Different genotypes had different germination rates. The highest germination rate of five genotypes seeds was 98.5%(line 048). When the mini-watermelon seedlings age was 3-5days, that turned to light green, its basal parts of cotyledons were the best explants to induce the adventitious bud directly, and the regeneration frequency was highest. The optimized regeneration medium for the basal parts of cotyledon was MS medium supplemented with 2.0 mg/L 6-BA for mini-watermelon. Different genotypes (line048, lineMH-39-1, line032, line028-5-5, line 047-1) had different regeneration frequencies. They were 90.0%,80.0%,90.0%,87.5%, 92.5%, respectively. The optimized medium for the stretch of adventitious buds was MS medium supplemented with 0.2 mg/L KT for mini-watermelon, and for rooting was 1/2MS medium supplemented with 0.3mg/L IB A and 0.2 mg/L IAA but the concentration of Fe3+ not be changed. The rooting rate and the number of roots were 79.3%,4.5; and 80.0%,5.5, respectively. Furthermore, they were beneficial for transplant of mini-watermelon.2. Results of in vitro induction of tetraploid mini-watermelon by colchicineThe results showed that the induction frequency was different from different genotype, treating concentration and time. Tetraploid mini-watermelon can be got when treated by 200-600 mg/L colchicine. The induction frequency reached highest 30.8%(line 048), when the colchicine was 400 mg/L, treated in 36 hours. The induction frequency reached highest 20.0%(line MH-39-1), when the colchicine was 600 mg/L, treated in 36 hours. The induction frequency reached highest 30.0%(line 028-5-5), when the colchicine was 400 mg/L, treated in 24 hours.The induction frequency treated 36h (11.5%) was higher than that treated 24h (8.9%) and 48h(1.7%). Tetraploid mini-watermelon can be all got in line048, lineMH-39-1, line028-5-5. The average doubling rates were 13.4%,4.9%,5.6%, respectively.3. Results of in vitro induction of tetraploid mini-watermelon by OryzalinThe results showed that the induction frequency treated by liquid medium(14.96%)in line 032 was higher significantly than that treated by solid medium (4.0%). Moreover, the doubling rate treated by 4d (16.65%) was higher significantly than that treated byld(3.35%), 2d(13.35%),3d(14.05%),5d(0), respectively. The induction frequency reached highest 33.3%, when the Oryzalin was 50mg/L, treated in 24 hours. It's much higher than using colchicine in vitro induction of tetraploid (30.8%), and the concentration of by using Oryzalin was lower than colchicine. Thus, Oryzalin can be used to in vitro induction of tetraploid watermelon replaced colchicine. The results showed that the doubling rates of line048, line 032, line 047-1 were not significantly different.4. Identification of mini-watermelon tetraploidSeveral shortcut methods were used in seedling polyploidy identification. First of all, morphological identification (height of plantlets, diameter of stem, length of internode) was carried through then use the biological character identification (length of guard cell, width of guard cell, density of stoma). Furthermore, chloroplast counting method was carried in this paper, the accuracy of this method for identification of different ploidy plant was 90.8%. At last further polyploid identification was carried by root tip cell chromosome. Therefore, the chromosome ploidy of plants derived from the basal parts of cotyledon regeneration plants in mini-watermelon can be identified by simply counting the chloroplast number in stomatal guard cells.5. Comparing the characters of different ploidy mini-watermelon in the greenhouseThe results showed that length of leaf, width of leaf, leaf length to width ratio varied significantly among the different ploidy mini-watermelon in the greenhouse.