Establishment Of Plant Regeneration System And Preliminary Studies On Genetic Transformation System Of Rosa Wichuriana’basye’s Thornless’

Rosa wichuriana’Basye’s thornless’was non-recurrent blooming with white single flower and creeping stems. These are all relative traits to the current main breeding target characters such as recurrent blooming, diverse flower color, upright stems and so on. So, studies on the regeneration and transformation systems of R. wichuriana’Basye’s thornless’will pay important roles in the molecular breeding for these desirable traits of rose and will lay foundation for the function identification of related genes.In this study, tissue culture and rapid propagation of R.wichuraiana ’Basye’s thornless’was established. Besides, regeneration system of direct organogenesis via leaflets, regeneration system of indirect organogenesis and regeneration system of somatic embryogenesis were studied. Also, the direct organogenesis and indirect organogenesis regeneration systems were used to try the genetic transformation of R.wichuraiana ’Basye’s thornless’. The major results were as follows:1. Establishment of tissue culture and rapid propagation of R.wichuraiana’Basye’s thornless’Micropropagation system via axillary bud was established for R.wichuraiana ’Basye’s thornless’. The optimum medium for shoot proliferation was:MS+O.lmg/L6-BA+0.05mg/LNAA+30g/L Sucrose+6.5g/L Agar (pH5.8-6.0). The optimum medium for aseptic microshoot rooting was:1/2MS+0.05mg/L IBA+30g/L Sucrose+6.5g/L Agar (pH5.8-6.0), and the rooting rate was up to100%.2. Establishment of direct organogenesis and regeneration system for R.wichuraiana ’Basye’s thornless’Effects of ages of explants、 days in the darkness and hormones varieties and its concentrations on adventitious buds induction were studied, using leaflets of R.wichuraiana ’Basye’s thornless’as explants. The results showed that:the induction frequencies of adventitious buds were significantly different among explants with different ages and the highest induction rate was16.80%when the topmost leaves were used. Darkness also had significant effect on the induction frequencies and the highest induction rate of adventitious buds was16%when leaflets were cultured for12days in the darkness. Excised leaflets of R.wichuraiana’Basye’s thornless’were cultured in induction medium1:1/2MS+0.05mg/L NAA+1.5mg/L TDZ+100mg/L CH+10mg/L AgNO3+30g/L Glucose+6.5g/L Agar (pH5.8-6.0) for12days in the darkness, and then transplanted to the differentiation medium of MS+0.5mg/L6-BA+0.05mg/L NAA+1.Omg/L GA3+30g/L Glucose+2.5g/L GEL (pH5.8-6.0) in the light for25days, the induction frequency of adventitious buds reached highest, which was10.38%.3. Establishment of indirect organogenesis and regeneration system for R.wichuraiana’Basye’s thornless’Effects of medium component, cultural condition and medium additives on the differentiation of callus were studied, using young leaves of R.wichuraiana’Basye’s thornless’as explants was studied. The results showed that:the optimal medium for callus induction of R.wichuraiana ’Basye’s thornless’was:MS+7.0mg/L NAA+30g/L Glucose+2.5g/L GEL (pH5.8-6.0), the induction rate reaching87.50%and with good growing callus. After being cultured in differentiation medium of MS+5.0mg/L TDZ+30g/L Glucose+2.5g/L GEL (pH5.8-6.0) with10days in darkness and then with25days in light, the callus had a highest adventitious buds induction frequency of18.34%.4. Preliminary study of somatic embryogenesis and regeneration for R.wichuraiana ’Basye’s thornless’Using the young leaflets of R.wichuraiana’Basye’s thornless’as explants, the methods of direct somatic embryogenesis and indirect somatic embryogenesis were both tried. However, none somatic embryo was observed in both ways, even though pseudobulbil and regenerated plants were observed through indirect somatic embryogenesis.5. Cytological observation of the callus developing process of callus of R.wichuraiana ’Basye’s thornless’Microscopic observation of the paraffin sections of the callus during induction、 proliferation and differentiation and ultra structural observation of the ultrathin sections of differentiating callus were carried out. The observations showed that:light yellow or light green callus with close-knit had round or ellipse cells with thinner cell walls, bigger nucleolus and more grumous cytoplasm in microstructure. This type of callus was called embryogenic callus, which was easy to differentiate and what we need.6. Preliminary study of Agrobacterium tumefaciens-mediated transformation protocol for R.wichuraiana’Basye’s thornless’ On the basis of two established regeneration systems, the reporter gene GUS was tried to transform to R.wichuraiana’Basye’s thornless’by inoculating young leaves and embryogenic callus. The ratio of transient expression of GUS gene was calculated to determine conditions for efficient transformation. The results indicated that:explant types had a mostly influence on genetic transformation of R.wichuraiana ’Basye’s thomless’. Agrobacterium infection had not effect on Leaflets and no positive transient expression was detected under any treatment. On the contrary, embryogenic callus showed a high ability of taking foreign genes and efficiencies varied with different transformation conditions. Besides explants, AS concentration in co-culture medium, inoculation time, inoculum density and co-culture time were also shown to be important.