Optimize On In Vitro Gynogenesis And Plant Regeneration Of Cucurbita Pepo L

With a high nutritional value and health care function,cucurbita pepo L has became one of the important melon vegetable crops.Breeding of excellent homozygous inbred lines has high practical application value for accelerating the research progress of cucurbita pepo L.Haploids were obtained by gynecological culture technology,then double haploid homozygous lines can be obtained by artificial or natural doubling to facilitate the configuration of hybrid combinations and shorten the breeding period.Therefore,5 hybrids of cucurbita pepo L were used as test materials in this paper.MS+TDZ(0.06mg.L-1+6-BA(0.5 mg.L-1)was the induction medium,MS was the regeneration medium.In vitro,culturing the unpollinated ovaries,effects of different genotypes,sampling period,pretreatments,disnfection ways and culture methods on embryoid induction of cucurbita pepo L were explored;Ploidy identification and morphological observation were carried out on the regeneration plants,and analyze the main factors affecting the domestication and transplanting of regenerated plants,expecting to optimize the system of isolated gynecological culture and regeneration of cucurbita pepo L.The results are as follows:1.With the same inducing conditions,embryoid body formation was observed in all 5 tested cultivars,different genotypes had significant differences,and‘ZY10'had the highest induction rate.The average amount of embryos per bottle was the highest at 12 hours before flowering,which was not significantly different from 24 hours before flowering.2.Pre-incubation at 35? in dark for 5 days,the number of swelled ovules,number of green ovules,number of induced embryoids and induction rate of embryoids were significantly higher than those of the control,in addition,35? heat shock treatment also helps the ovule enlargement prominent ovary placenta tissue;Unpollinated ovary sections were treated at 4? for 0 day(CK),1 day(L24),2 days(L48)and 3 days.The results show that the low-temperature treatment of 1?2 days had little effect on the production of embryoids,but the embryo yield decreased rapidly of 3 days cold treatment,it shows that short-term low-temperature treatment has no significant effect on the induction of embryoids,but low temperature treatment for a long time is not conducive to the expansion of ovules and hinders the induction and differentiation of embryoids.3.The ethanol soaking time and sodium hypochlorite concentrations both had influence on the induction of embryoids.75%alcohol soaking for 1 min can basically kill the placental tissue and hold the ovules with high viaility;When disinfection with 1%sodium hypochlorite,we cannot completely kill microorganisms on the explant,the contamination rate of medium is high.We got the highest embryo yield and embryoid induction when it came to 3%,on average,24 embryos were produced per bottle,and embryo induction rate as high as 33.8%,with the increase of sodium hypochlorite(5%,7%and 10%),the number of embryoids and induction rate began to gradually decrease.Therefore,the concentration of 3%sodium hypochlorite is the optimum disinfection concentration for gynogenetic culture of cucurbita pepo L,which not only has the effect of sterilization,but also has little damage to ovule development.4.There are advantages and disadvantages to the two culture methods of slice culture and stripping ovule culture after pre-culture,among them,slice culture operation is relatively simple,there is no loss of ovules without stripping,the method of stripping ovules after the preculture of slices can make the ovules fully contact with the nutrient in the medium,which is helpful to promote induction of embryoids.Observed by morphology,somatic embryo mophgenesis of unfertilized ovules of cucurbita pepo L passed through globular embryo,heart embryo,torpedo embryo and cotyledonous embryo stage,which was similar to zygotic embryo;When moved to MS medium without hormone,the cotyledonary embryos have a higher regeneration rate,was 63.9%.5.Effects of root tip,tendril tip and shoot tip of cucurbita pepo L as chromosome identification materials was tested,we found that tendril tip can be used as a substitute for ploidy identification of root tip.Identification of the ploidy of 39 regeneration plants using flow cytometry,two of them are haploid,17 strains are diploid and 10 strains are tetraploid;The number of haploid chloroplasts varies from 3 to 8,diploid from 6 to 13 and tetraploid from 11 to 24,the number of chloroplasts 7 and 12 was used as the demarcation index to identify haploid,diploid and tetraploid of cucurbita pepo L.6.Selecting 4?7 leaves root more than 3 plants,loose bottle for 2 days open bottle 3 days hardening-seedling in the tissue culture house,then training seeding under the condition of outdoor shading and transplanting them into the greenhouse,the survival rate of the seedling is 96%.