Somatic Embryogenesis And Regeneration System And Genetic Transformation In Four Cultivars Of Rosa Hybrida

Roses (Rosa hybrida) are semi-evergreen woody plants in the Rosaceae family. They are one of flowers that are cultivated for the longest time and are popular in the world. They have high ornamental and commercial values for bright colors, beautiful flowers and rich fragrances, known as one of ten traditional and famous flowers in China and one of important and ornamental flowers in the world. In this study, tissue culture and rapid propagation of four cultivars of R. hybrida was established. Besides, using unexpanded leaflets of four cultivars of R. hybrida as explants, a regeneration system via somatic embryogenesis was established. Meanwhile, somatic embryos as the genetic transformation receptors in regeneration system were used for the study of genetic transformation in R. hybrida. The major results of this study were as follows:1. Establishment of tissue culture and rapid propagation in R. hybrida ’Carola’The conditions for sterilization, initiation of shoots, propagation, rooting and transfer to the field were discussed in the tissue culture of R. hvbrida’Carola’. The results showed that the contamination rate was reduced when petioles were removed during the sterilization and axillary buds grew strongly. MS+1.0mg/L6-BA+0.05mg/L NAA+30g/L Sucrose+6.5g/L Carrageenan Gum was the optimal medium for rapid propagation and time of reproduction was3.41. And the best medium for rooting was1/2MS+0.05mg/L NAA+30g/L Sucrose+6.5g/L Carrageenan Gum and average length of roots was2.52cm. At the end, the survival percentage was up to94.44%after6days acclimatization of the plantlets before transplantation.2. Establishment of somatic embryogenesis and regeneration system for R. hybridaUsing the unexpanded leaflets of four ornamental cultivars of R. hybrida’Carola’’Black Magic’、Holiday Princess’and’Monica’as explants, the factors affecting somatic embryogenesis, proliferation and plant regeneration were discussed. The results showed that:genotypes, explant types, different concentrations of plant growth regulators and cultivation conditions all played important roles in somatic embryogenesis and plant regeneration. Using the unexpanded leaflets from both tissue culture and open fields as explants, somatic embryo was not observed via direct somatic embryogenesis in four cultivars of R. hybrid. Using the unexpanded leaflets from tissue culture as explants, somatic embryo only was observed via indirect somatic embryogenesis in R. hybrida ’Carola’. The frequency of somatic embryogenesis had significant differences in different mediums. When cultured on the medium MS+2.0mg/L ZT+0.1mg/L NAA+30g/L Glucose+2.5g/L GEL, unexpanded leaflets of’Carola’showed the highest induction rate of13.33%. Somatic embryos of R. hybrida’Carola’growed vigorously and had bright colors when cultured on the medium MS+1.5mg/L ZT+0.2mg/L NAA+0.1mg/L GA3+30g/L Glucose+2.5g/L GEL as the best medium. At that moment, time of reproduction was4.02and ratio of somatic embryos was more than90%. And secondary somatic embryos were observed. When cultured on the medium MS+1.0mg/L6-BA+0.01mg/L IBA++30g/L Glucose+2.5g/L GEL, the highest frequency of plant regeneration per somatic embryo in R. hybrida’Carola’was43.33%and the number of plant regeneration per somatic embryo was1.31.Additionally, When cultured on rooting medium1/2MS+1.0mg/L NAA+30g/L Sucrose+6.5g/L Carrageenan Gum, somatic embryos like cotyledon were induced via direct somatic embryogenesis of R. hybrida ’Carola’ in root-axis after8weeks in the light. And then cultured on the medium MS+1.5mg/L ZT+0.2mg/L NAA+0.1mg/L GA3+30g/L Glucose+2.5g/L GEL, somatic embryos growed vigorously.3. Studies of genetic transformation about somatic embryos in R. hybrida’Carola’Based on the enhanced somatic embryogenesis and regeneration system, somatic embryos as the genetic transformation receptor were used for genetic transformation of R. hybrida ’Carola’. Using somatic embryos as genetic transformation receptor, an Agrobacterium-medidited transformation protocol was studied for R. hybrida’Carola’by assessing the transient expression of GUS gene. The results showed that:when infected for40min, co-cultured for3d, supplemented with80mg/L Km and300mg/L Cef, rate of GUS transient expression was extremely low. Growth state of somatic embryos were so bad that somatic embryos did not regenerate into plantlets when cultured on the medium MS+1.0mg/L6-BA+0.01mg/L IBA+30g/L Glucose+2.5g/L GEL+300mg/L Cef+80mg/L Km.