Establishment Of Regeneration System And Genetic Transformation In Four Rosa Species And Cultivars

Rose is an important ornamental plant in the garden and flower market.Breeding is the key way to keep and innovate good characters.The regeneration system and genetic transformation system can provide a new method and technology platform for the study of Rosa.The difficulty of the technique lies in the genotypic dependence and the culture conditions.Therefore,in consideration of the genotype,ploidy,and character,we selected Rosa multiflora var.cathayensis(2n=2x=14),R.chinensis var.spontanea(2n=2x=14),' Samantha'(2n=4x=28),R.chinensis ' Pufu Hong '(2n=2x=14)as subject,and combined with cytological observation,to screen the optimal conditions of regeneration.Using Agrobacterium tumefaciens mediated method to transform GUS gene.the key factors in genetic transformation of four Rosa species and cultivars were discussed.The four species and cultivars had a series of relative traits such as one season flowering or continuous flowering,single or multiple petals,which also lay the founction for gene function identification.The main results are as follows:1.The optimal cultivation media for callus induction and differentiation of R.multiflora var.cathayensis,R.'Samantha',R.chinensis 'Pufu Hong' are MS+1.5 mg/L 6-BA+3.0 mg/L 2,4-D+2.0 mg/L NAA+0.1 mg/L KT+300 mg/L L-proline+30 g/L Sucrose+2.4 g/L Gel,pH=5.8-5.9,the dark induction period is 30 d.The optimal cultivation media for callus induction and differentiation of R.chinensis var.spontane is MS+1.5 mg/L 6-BA+2.0-3.0 mg/L 2,4-D+2.0 mg/L NAA+0.1 mg/L KT+300 mg/L L-proline+30 g/L Sucrose+2.4 g/L Gel,pH=5.8-5.9,the dark induction period is 30 d;The optimal cultivation media for induction and differentiation of somatic embryogenesis of Rosa multiflora var.cathayensis and R.'Samantha' is MS+4.0 mg/L 2,4-D+1.5 mg/L TDZ+0.1 mg/L KT+0.1 mg/L ZT+300 mg/L L-proline+30 g/L Sucrose+2.4 g/L Gel,pH=5.8-5.9,the dark induction period is 30 d.The optimal cultivation media for induction and differentiation of somatic embryogenesis of R.chinensis var.spontane and R.chinensis 'Pufu Hong' is MS+3.0 mg/L 2,4-D+1.5 mg/L TDZ+ 0.1 mg/L KT+0.1 mg/L ZT+300 mg/L L-proline+30 g/L Sucrose+2.4 g/L Gel,pH=5.8-5.9,the dark induction period is 30d.Efficiency of callus induction of R.chinensis 'Pufu Hong',R.multiflora var.cathayensis,R.chinensis var.spontane and R.'Samantha' are 72.5%,84%,62.5%,69%,and the efficiency of somatic embryogenesis are 22.5%,18%,12%,13.5%,seperately.2.A preliminary study on Agrobacterium tumefaciens-mediated transformation protocol for the four Rosa cultivars was conducted,and the results showed that the optimal conditions for transformation of R.chinensis 'Pufu Hong' and R.'Samantha' was bacterial concentration OD600=0.4,three days' co-cultivation,40 min's infection and acetosyringone concentration of l00?mol/L.The optimal conditions for transformation of R.multiflora var.cathayensis and R.chinensis var.spontane was bacterial concentration OD600=0.5.three days' co-cultivation,40 min's infection and acetosyringone concentration of 100?mol/L.The GUS transient expression rate of R.chinensis 'Pufu Hong',R.multiflora var.cathayensis,R.chinensis var.spontane and R.hybrida'Samantha' are 38.67%,39.33%,40%,41.33%,seperatelyThe establishment of regeneration system and genetic transformation protocol of four Rosa species and cultivars provide a technical support for gene function identification and lay a theoretical basis of molecular breeding in Rosa.