Establishment Of Grapevine Embryogenic Suspension System And Protoplast Isolation

Embryoid regeneration system is an ideal system to explore the whole mechanism of plant differentiation and to produce high quality plants on a large scale.Embryogenic callus can be used as a target tissue for the study of genetic transformation.In this experiment,the shoot tips of Pinot Noir(Vitis vinefera L.cv.)and Beta(Vitis riparia×V.labrusca)were selected as the material.The effects of exogenous hormone ratio,genotype,sucrose concentration,organic matter,culture medium type and culture mode of embryogenic callus induction and embryoid production were studied.The germination and preservation conditions of embryoid were studied.Based on this,embryogenic cell suspension lines were established.The isolation conditions of protoplasts were preliminarily explored with suspended cells as the material,so as to obtain a large number of high quality raw materials,and lay a good theoretical and practical basis for the improvement of grape varieties and cell engineering.The main results are as follows:1.In this study,the stem tips of Pinot Noir and Beta were used as explants to induce callus.In the study of somatic embryogenesis pathway regeneration system,the results showed that the callus induction rate,proliferation and growth status of Beta grape were higher than those of Pinot Noir.2.The suitable culture medium for stem tip callus induction of Pinot Noir and Beta Grape Plantlets was N6+6-BA 1.0~2.0 mg·L-1+2,4-D 1 mg·L-1.There are 3 stages of embryogenic callus induction.First,the callus was cultured in the medium X6+6-BA 0.5 mg·L-1+NOA 0.5 mg·L-1 for 30 days.Then,the callus was transferred to the medium X6+6-BA 1 mg·L-1+NOA 1 mg·L-1 for 30 days.Finally,the callus was transferred to the medium X6+6-BA 1 mg·L-1+NOA 0.5 mg·L-1+2,4-D 0.5 mg·L-1 for culture.3.In embryoid induction stage,the induction rate of embryoid was 70 % in culture medium N6+6-BA 3 mg·L-1+2,4-D 0.5 mg·L-1 +KT 0.1 mg·L-1.The germination of embryoid is suitable for MS medium without plant growth regulator.The suitable subculture medium for embryogenic callus was MS+6-BA 2 mg·L-1+2,4-D 0.5 mg·L-1+KT 0.1 mg·L-1,which had better preservation in dark condition.4.Embryogenic cell suspension system of Pinot Noir and Beta grape was preliminarily established in this study.In order to maintain a good state of the suspension system,a growing callus should be selected to be cultured with 4g/20 m L inoculation in the medium of B5+NAA 0.2 mg·L-1+KT 0.5 mg·L-1,the suitable subculture period is 7~10 d.5.In protoplast isolation experiment,suspension cells,callus and plantlets were used as materials.The effects of different genotypes,cell state,enzyme combination and time on protoplast isolation were studied.The results showed that the ability of enzymolysis protoplasts of different materials was suspended cells > callus > leaves,and 12 h separation effect was better in enzyme solution 1~2 %+pectinase 0.25 %+dissociation enzyme 1 %.