| Roses (Rosa hybrida L.) are the most widely used woody flower plants in landscapes, and because of the beautiful concurrent flowers, high adaptability to environment and good tolerance to pruning, they are widely planted in China. Roses are one of the four famous cut-flower species in the world, however, the vase life is drastically shorter compared to that of chrysanthemums and carnations. In the past years, many researchers have carried out a lot of studies on its senescence intending to prolong the vase life, but haven't completely resolved the problem. Now more and more studies showed that some rose cultivars are very similar to ethylene climacteric cut flowers: during the course of flower opening and senescence, there is a definite climacteric rise in ethylene production, ethylene treatment accelerates flower senescence and ethylene inhibitors interfere ethylene production or function and finally prolong the vase life.The major objective of this study was to breed rose individuals with prolonged vase life through inhibition of the interior ethylene production by using the antisense RNA principal and Agrobacterium tumefaciens-mediate transformation technology. For this purpose, first, an efficient in vitro propagation system and plant regeneration system were developed for a similar climacteric rose cultivar R. hybrida cv. Samantha. Factors that affecting shoot regeneration via direct organogenesis and indirect organogenesis, including genotypes, explant types, cultivation conditions and plant growth regulators, etc., were also investigated in details. Subsequently, parameters that affect the efficiency of Agrobacterium tumefaciens-mediated transformation of R. hybrida cv. Samantha were comprehensively studied and optimized by accessing gus transient expression, and a transformation protocol was established for the callus of this cultivar. Finally, on the basis of the established transformation protocol, ACC oxidase antisense gene was stably transferred to the callus of R. hybrida cv. Samantha verified by PCR test. The major results of this study were introduced separately as following:1. An in vitro micropropagation system for 4 rose cultivars was established. 4 rose cultivars with good market value were used as materials for studies on rose in vitro propagation. The results showed: the plant materials for inoculation should be collected from April to May when the shoots started to become woody and sterilizationwas relatively easy after a cold winter; buds on the stems started to sprout successively after being cultured on medium MS+0.5mg/L BA+0.004 mg/L NAA+0.1 mg/L GA3 after 2 or 3 weeks, and the sprouting rate was about 70%; shoots were micropropagated on the same medium by being cubcultured every 4 weeks, however, yellow-flowered cultivars Gold Medal and Yellow Peace needed more time for adaptation than red-flowered cultivars Samantha and Kardinal, after they adapted to the in vitro culture condition, these 4 cultivars propagated well; shoots cultured for 4 weeks on proliferation medium differentiated adventitious roots 20~30d after being transferred to rooting medium (l/2MS+0.1mg/L IB A); the key point for successful transplantation was the age and sturdiness of the in vitro rooted plants, 80% of aged and sturdy plants could survive after transplantation to soil and only a few young plants could get through because of loss of water.2. A plant regeneration system via direct organogenesis was established for R. hybrida cv. Samantha. Effects of genotypes, explant types, duration of induction and plant growth regulators on direct adventitious bud induction were investigated. The results indicated that genotypes played an important role in direct shoot regeneration of roses, adventitious buds were induced only in Samantha, not in the other 3 cultivars, though projected structures could be detected on the leaflet petioles; cultured on Induction Medium (MS+1.5mg/L TDZ+0.05mg/L NAA+lOmg/L AgNO3) for 8d in darkness and then on Shooting Medium (MS+0.5mg/L BA+O.Olmg/LNAA+ O.lmg/L GA3) under light, leaflets and compound-leaf petioles of Samantha showed the highest regeneration frequency of 51.8% and 10%, respectively; 8d of induction in the dark was shown to be optimal, longer time would cause callus formation and reduced regeneration efficiency; folded young leaflets from different positions on a compound-leaf produced a similar regeneration result which indicated that leaflet position had minor effect on regeneration; NAA was better than IBA in inducing shoots but high concentration of the auxin would lead to callus growth which was disadvantageous for shoots regeneration.3. A regeneration system via indirect organogenesis was established for/?, hybrida cv. Samantha. White pseudobulbils were initiated from in vitro grown leaflet explants of rose cultivar Samantha following an induction period of 15d on MS basal medium supplemented with 7.0~10.0mg/L 2,4-D and a subculture of 30d on MS medium with 1.5mg/LTDZ under light. The pseudobulbils were subcultured on medium with NAA, ZT and GA3xand during the process, new psedobulbils and new calluses were observed. After almost a year of continuous selection for fresh tissues, the callus that could be longtime proliferated was obtained. This callus was friable, white to yellow in color, highly proliferated and has been maintained for 16 months till now. Adventitious buds could be induced from the callus on MS medium with l.Omg/LTDZ, O.Olmg/L NAA and 0.1 mg/L GA3 in one month and elongated into shoots on MS medium with 2.0mg/L BA and O.Olmg/L NAA.4. An Agrobacterium tumefaciens-mcdiatcd transformation protocol was established for R. hybrida cv. Samantha using callus as inoculation material. First, the sensitivity of leaflets and callus to hygromycin B was tested in order to use the two established regeneration system to select and regenerate transgenic plants. Leaflets were shown to be highly sensitive to hygromycin B and when its concentration increased to 6mg/L in Shooting Medium, adventitious buds differentiation was completely inhibited. However, callus showed much higer tolerence to the antibiotic because of efficient proliferation, and growth of 90% of the calluses was inhibited when the concentration of hygromycin B reached to 70mg/L. Conditions for efficient T-DNA transformation were comprehensively studied by assessing the transient expression of GUS gene. The results showed: explant type was a key factor deciding the success of genetic transformation, leaflets were not sensitive to Agrobacterium infection, and under different conditions, no positive transient expression was detected, while callus showed a high ability of taking foreign genes, and different efficiencies were obtained under different transformation conditions; besides explant type, light condition during co-culture, salts and AS concentration in co-culture medium were also shown to be important, but the requirements for inoculation time and pH value of co-culture medium were not strict in T-DNA transfer; in some degree, increasing the levels of inoculum density, co-culture time and co-culture temperature would improve the efficiency of gus transient expression, but too low or too high levels of the 3 factors were not good; optimized transformation was performed by inoculation of callus with A. tumefaciens EHA105 at a density of OD6oo=0.5~0.8 for 20min and co-culture in darkness under 23 °C on medium with 1/2MS salts and 300MM AS for 3d. By infection according to this optimized T-DNA transformation protocol, and selection on callus maintaining medium supplemented with 70mg/L hygromycin B and 300mg/L cefotaxime, we obtained stably transformed callus according to PCR verification.5. Calluses containing ACC oxidase antisense gene were obtained. According to the established transformation system, and by using A. tumefaciens EHA105 and GV3101 containing HPT gene and ACC oxidase antisense gene, transgenic calluses of R. hybrida cv. Samantha were obtained; the growth rate of GV3101 was slower than EHA105, and a 5d' co-culture was needed for GV3101; and PCR test showed stable integration of the foreign genes in rose cells.Issues like comparison of the two regeneration systems, factors affecting Agrobacterium-mediated rose genetic transformation, the relationship between transient expression and stable transformation, transformation efficiency and the future work of this study were also discussed.
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