Different Ways To Establish And Optimize Regeneration System And Studies On Genetic Transformation In Rosa Hybrida

Roses (Rosa hybrida), with bright colors, beautiful flowers and fragrant, known as one of Chinese ten famous and traditional flowers, is a perennial and evergreen woody plant in the Rosaceae family, so it has high ornamental and commercial value. However, the vase life of rose flower is short, and the plants are susceptible to powdery mildew and black spot, which all affect its ornamental and economic value. Plant genetic engineering technology can overcome the limitations of traditional breeding methods. Efficient and stable plant regeneration system is an important prerequisite for plant genetic transformation. In this study, direct organogenesis system of R. hybrida was enhanced. Besides, using leaflets and immature zygotic embryos of different cultivars of R. hybrida as explants, a regeneration system via somatic embryogenesis was established. Meanwhile, direct organogenesis system and regeneration system via somatic embryogenesis were used for the study of genetic transformation in R. hybrida. The major results of this study were as follows:1. Enhancement of direct organogenesis system in R. hybrida.Improvement of adventitious bud induction and plant regeneration using the leaflets of R. hybrida’Samantha’,’Elizabeth’,’Black Lady’as explants was studied. The results showed that:when excised leaflets were cultured on induction medium 1/2MS+1.0mg/L TDZ+0.05mg/L NAA+100mg/L CH+10mg/L AgNO3+3% Glucose+0.25% GEL for ten days in darkness, and turn to the light, together for 20 days, then transferred the adventitious buds on multiplication medium MS+0.5mg/L BA+0.004mg/L NAA+0.1mg/L GA3+3% Sucrose+7.5g/L Agar, the frequency of adventitious buds induction and plant regeneration all reached the highest. The highest rate of adventitious buds induction of’Samantha’,’Elizabeth’and’Black Lady’was 85.04%,95.30% and 81.70%, respectively, and the highest rate of plant regeneration of’Samantha’, ’Elizabeth’and’Black Lady’was 60.09%,64.49% and 55.33%.2. A regeneration system via somatic embryogenesis was established for R. hybridaUsing the leaflets and immature zygotic embryos of different cultivars of R. hybrida as explants, the factors affecting somatic embryogenesis, proliferation and plant regeneration were studied. The results showed that:genotype, explant type, plant growth regulators and cultivation conditions all played important roles in somatic embryogenesis and plant regeneration. Using the leaflets as explants, somatic embryogenesis only was observed in R. hybrida’Samantha’; Using the immature zygotic embryos as explants, somatic embryogenesis was observed in R. hybrida’Purple Haze’,’Small yellow flowers’and Climbing rose. The frequency of somatic embryogenesis had significant differences. When cultured on the medium MS+3.0mg/L 2,4-D+3% Glucose+0.25% GEL, leaflets of’Samantha’showed the highest induction rate of 7.5%, immature zygotic embryos of’Purple Haze’cultured on the medium 1/2MS+2.0mg/L 2,4-D+3% Glucose+0.25% GEL showed the highest rate of 11.11%, the highest rate of 27.41% for Climbing rose was obtained on the medium 1/2MS+2.0mg/L 2,4-D+1.0mg/L BA+3% Glucose+0.25% GEL. The highest frequency of germination of somatic embryos in R. hybrida’Samantha’,’Purple Haze’and Climbing rose was 36.38%,86.67% and 53.14%, respectively. The highest frequency of plant regeneration per somatic embryo in R. hybrida’Samantha’,’Purple Haze’and Climbing rose was 56.24%,98.88% and 47.64%, respectively. During the processes of germination of somatic embryos and plant regeneration, germination of somatic embryos in’Samantha’only showed shoots, generated weak roots or no roots, which needed rooting. However,’Purple Haze’and Climbing rose produced roots and shoots simultaneously, then generated complete plantlets.3. Studies of genetic transformation in R. hybrida with different waysBased on the enhanced direct organogenesis system, some transgenic buds were observed by Agrobacterium-mediated transformation of leaflets using GUS as a scorable marker on the selection and induction medium 1/2MS+1.0mg/L TDZ+0.05mg/L NAA+100mg/L CH+10mg/L AgNO3+300mg/L Cef+25mg/L Km+3% Glucose+0.25% GEL, however, these buds did not regenerate into plantlets. Using somatic embryos of ’Purple Haze’as inoculation material, an Agrobacterium-mediated transformation protocol was studied for R. hybrida by assessing the transient expression of GUS gene. The results showed that:infected for 40 min, co-cultured for 3d, supplemented with 75mg/L Km and 300mg/L Cef were optimal for transgenic system.