| The genetic background of grape is relatively complex,the genome is highly heterozygous,and the breeding of new varieties is restricted by season and region,which makes the traditional breeding mode longer.However,there are some problems such as the limitation of harvest season,the low induction rate of embryogenic which hinder the development of somatic embryogenesis.Therefore,the optimization of somatic embryogenesis pathway and the establishment of somatic embryogenesis induction system become one of the urgent problems in grape somatic embryogenesis.The effects of melatonin and 2,8high brassinolide on somatic embryogenesis were studied in the immature buds of Vitis vinifera’Chardonnay’at different concentrations,in order to establish a stable somatic embryo regeneration system and analyze the structural differences of callus under different conditions by cell staining.Meanwhile,to break the time limit of collecting explants,the seeds of ’Chardonnay’ with different maturity were used as explants,after the primary cotyledon embryos were induced to germinate,the second cycle of somatic embryogenesis was carried out,and the cycle induction system was established to study the effect of different explants on somatic embryogenesis under the effect of melatonin,the experiment of VvHOS1 gene transformation was carried out using ’Chardonnay’ as the acceptor,which provided new possibility for genetic engineering.The main results are as follows:1.Established a somatic embryo regeneration system using ’Chardonnay’flower buds as recipient materials.The small flower buds 7～12 d before opening were used as test materials.Among them,MC、M0 on callus induction is inferior to that of 0.2,0.4,0.6,0.8,1.0 mg·L-1 Melanin and brassinolide medium;while the induction rate of MC medium was 11.71%when the proembryo was induced,M0 reached 13.44%,1.0 mg·L-1 melatonin,0.4,0.6 mg·L-1 rapeseed Somatic embryos were induced directly from the water-soaked callus by vinolactone,and the induction rates were 8.57%,6.25%and 3.81%respectively;no somatic embryos were produced under the induction of other hormone concentrations.2.Established a somatic embryo cycle induction system using ’Chardonnay’ seeds as the recipient material.Using ’Chardonnay’ grape seeds as the test material,the MC、M0 medium is not conducive to the germination of primary cotyledon embryos,0.6 mg·L-1 melatonin has the best effect on the germination of cotyledon embryos,18.33%;Cotyledons,hypocotyls and radicles in different parts of the cyclic induction study,callus induction rates were 76.18%,42.86%and 33.33%,indicating that the cotyledon induction effect is the best.And it is preliminarily determined that the concentration of 0.6 mg·L-1 melatonin is beneficial to the formation of secondary somatic embryos.3.Established an Agrobacterium-mediated transformation system using ’Chardonnay’ seeds as the recipient material.Using mature grape seeds 80 days after flowering as explants and ubiquitin ligase VvHOSl as the target gene,the Agrobacterium-mediated test was carried out.The test results showed that the exogenous plant growth regulator,antibiotic concentration,and selection methods in the medium were adjusted.,MC medium is conducive to the induction of embryogenic callus,and melatonin is conducive to the germination of primary cotyledon embryos;screening tests with different antibiotic concentrations found that the screening effect of 30mg·L-1 Kan was weak,and the acquisition rate of resistant embryos was 59.12%;70mg·L-1 Kan has the strongest screening effect,and only 35%resistant embryos exist.Fluorescent protein detection technology was used to detect and analyze the receptor material,and 17.69%of them could observe GFP green fluorescence,which preliminarily confirmed the feasibility of this approach. |
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