Studies On In Vitro Culture And Plant Regeneration Of Unfertilized Ovary Of Pumpkin

With rich nutrition and health care, pumpkin (Cucurbita moschata) has became one of the important vegetable crops of Cucurbitaceae. Obtain inbred line to accelerate the breeding process of pumpkin is significant. In vitro, culturing haploid female nucleus to get double haploid is an important way to obtain inbred lines.Therefore, using unfertilized ovaries of pumpkin as explants in this experiment, effects of different combinations and concentrations of growth regulators, genotypes, embryo sac development stages, disinfection methods and duration of high temperature pretreatments on embryoid induction via in vitro culture were determined to establish a regeneration system of unfertilized ovaries of pumpkin. The results showed that:1、Among the 6 pumpkin varieties tested in this experiment conditions, two varieties(Xue feng mi ben and E yu nan gua) with stronger growth had higher embryoid induction rates and were more suitable to be used as experiment materials; The mediums of MS+4.0 mg·L-12,4-D+0.5 mg·L-1 NAA+1.0 mg·L-16-BA and MS+0.04 mg·L-1TDZ achieved higher induction rates of 19.8% and 20.1% respectively compared to other mediums examined, with the TDZ-induced method easier in operation; The bloom day ovary has highest embryoid induction,which was up to 20.0%; The operation of slicing and then sterilizing the ovaries picked in the bloom day decreased the cavernous callus formation effectively and increased the embryoid induction rate; Treatments with darkness and heat shock (35℃) for 5 days could help ovary turn green and induce embryoid; When moved to hormone-free MS medium, the cotyledon embryos developed to regenerated seedlings has highest rate, was 69.4%.2、According to the identification of ploidy of the 46 regenerated plants of 2014 autumn, we found that there were 7 regenerated plantlets with the chromosomal number of n=x=22 and the number of chloroplast in stomatal guard cell was 4-6, which indicated that they were haploid plants.3、Using the half closed-full open hardening-seedling for plant regeneration with 6-7 leaves for 3 days in the tissue culture works, then training seedlings under the condition of weak light after 3 days and transplanting them into greenhouse, the survival rate made up over 90%.