Research On Technology Of Unpollinated Ovary Culture And The Ploidy Identification Of Embryo Sac Plants In Cucurbita Pepo L.

In order to establish a perfect technology system of creating haploid resources in cucurbita pepo L., 6 different genotype unpollinated ovaries was used as test materials of cucurbita pepo L., through culturing unpollinated ovaries to discuss the genotype, different kinds and proportion of hormone, the sampling period, the preprocessing method and exogenous additives to the impact on embryoid induction to improve the culture effect of megaspore culturing in. What’s more, the embryo sac plants were identificated through the root tip cells chromosome, leaf stomatal guard cells and flow cytometer to compare the results of three kinds of ploidy identification methods, and got an economic and effective ploidy identification technology of embryo sac plants. The main results of the study were as follows:1. The trend of embryoid induction effect was TDZ + NAA > TDZ > 6-BA + NAA, when unpollinated ovaries cultured in MS medium with different hormone. The average induction rate was P8 > P9 > P7 > P5 > P6 > P4 >P3 > P2 > P1 in different types of hormone combination of 9 kinds of medium. Thus, the optimal culture medium for unpollinated ovaries was P8 medium, which was MS+30 g·L-1 Suc+8 g·L-1 Agar+0.05 mg·L-1 TDZ+0.25 mg·L-1 NAA.2. The genotype and sampling periods of unpollinated ovaries affected the embryoid induced effect, the XH14-3 was best genotype material of induced effect among 6 genotypes material, followed by XH13-7. The embryoid induction rate was highest at 1 d sampling before flowering, the trend of induction effect was 1 d before flowering > 2 d before flowering > flowering day. Thus, flowering day was the best sampling time to culture unpollinated ovaries.3. The embryoid induction effect due to the different materials after processed different times at 4 ℃ of unpollinated ovaries. XH14-3 material with precooling 24 h at 4 ℃ could significantly increase the induction rate and decrease the vitrification rate, but had no effect on XH13-7 material.4. There was no significant effect on embryoid induction rate when unpollinated ovary slice were heat shock treatment 4 d, 5 d and 6 d at 35 ℃ after inoculated in medium, it could only ahead or behind the embryo time and increase the vitrification rate of different genotype materials embryoid.5. Adding 10, 20, 30 mg·L-1 AgNO3 to the culture medium P8 not only significantly reduced the vitrification rate, but also shorten the earliest embryo days, the earliest embryo days shorter 2~5 d than control, especially added 20 mg·L-1 AgNO3.6. Adding 20 mg·L-1 colchicine to culture medium P8 of unpollinated ovaries induced embryo, the growth of ovules was restrained after cultured 2, 4, 6 d, the embryoid induction rate decreased with the increase of culturing days of adding colchicine, and easy leaded to embryoid vitrificated. Thus, 20 mg·L-1 colchicine existed inhibition on embryoid induction of unpollinated ovaries.7. Three ploidy identification results compared with plant field characters identification of embryo sac plants, the accuracy rate was 68.75% of root tip chromosomes identification, leaf stomatal guard cells identification accuracy rate was 87.50%, flow cytometry identification accuracy rate was 100%. Study showed that could improve the accuracy of identification and reduce the cost if we appropriatiy chose leaf stomatal guard cells identified ploidy at first, and further identified by flow cytometry to the plants with uncertain ploidy of embryo sac plants from megaspore culture.