| Rose (Rosa hybrida L.), a perennial and evergreen woody plant in the Rosaceae family, is an important ornamental plant and has important commercial value with graceful and beautiful flowers, heavy fragrance, and long flowering period. However, the vase life of rose cut flowers is short, and the plant is susceptible to powdery mildew and black spot, which affect its ornamental and economic value. Many conventional breeding methods have been introduced to improve these traits, however, the achievements by this method are restricted. Plant genetic engineering technology provides an opportunity to overcome these restrictions. Plant genetic engineering technology can take advantage of exogenous genes to improve desirable traits without changing other excellent traits, and thus provides a new way for breeding new rose varieties.In this study, we investigated several factors affecting the induction of somatic embryogenesis in R. hybrida’Samantha’, and then using this efficient regeneration system we developed an Agrobacterium-mediated transformation system for this genotype. Meanwhile, the transcriptome genes during the interaction between Rosa multiflora and powdery mildew were full screened using second generation of high-throughput sequencing method, and important candidate genes were sorted out. The temporal and spatial expression of these genes were analysed by real-time PCR. In the meantime, taken the results of cytology research into consideration, we determined the roles of these candidate genes in powdery mildew resistance of Rosa multiflora. The major results were as follows:1. Enhancement of regeneration system via direct somatic embryogenesisUsing in vitro root, leaf and field leaf of Rosa hybrida ’Samantha’ as explants, improvement of regeneration system via direct somatic embryogenesis was studied. The results showed that explant and PGRs played important roles on somatic embryogenesis. The germination frequency of somatic embryos differed significantly on different basic media and PGRs.Somatic embryogenesis was induced from in vitro-derived leaf explants, achieving a frequency of 7.21% following 8 weeks of culture on induction medium supplemented with 3.0 mg/L 2,4-D, and the frequency of somatic embryo induction from field leaf was only 3.18%. No somatic embryo was observed while using in vitro root as explant. At the same time, PRGs affecting the induction of somatic embryogenesis in R. hybrida ’Samantha’ using the in vitro-derived leaf as explants were investigated. Our results indicated that:the highest rate of somatic embryogenesis,10.2%, was obtained on medium with 3.0 mg/L 2,4-D and 0.05 mg/L KT. For the same PGR combination,3.0 mg/L 2,4-D was significantly better than 4.0 mg/L for somatic embryogenesis. Combined with the same concentration of 2,4-D, KT was significantly better than ZT and BA, indicating that KT played a significant role in promoting somatic embryogenesis of R. hybrida’Samantha’.When cultured on 1/2MS+1.0 mg/L BA+0.01 mg/L NAA+0.1 mg/L GA3+30 g/L Glucose+3.0 g/L GEL, the highest germination rate (73.49%) of somatic embryos was observed, while cultured on MS+0.1 mg/L TDZ+0.05 mg/L BA+0.1 mg/L GA3+30 g/L Glucose+3.0 g/L GEL, germination rate of somatic embryos was only 33.07%. Compared to MS basic medium,1/2 MS medium was more advantageous to the germination of somatic embryos. BA and NAA played important roles on germination of somatic embryos, the higher concentration of BA and NAA promoted germination of somatic embryos, and was obviously better than the combination of TDZ and BA.2. Establishment of genetic transformation system in R. hybrida’Samantha’Secondary somatic embryos were used as target tissues for Agrobacterium-mediated transformation, the effects of different infection methods and selection strategies were investigated. The results showed that:Optimal infection methods for transformation was that somatic embryos were immersed in the bacterial suspension for 40 min with continuous shaking at 28℃ (180 rpm), which improved transient GUS expression efficiency (87.5%). Hygromycin selection was significantly better than kanamycin. Efficient selection was obtained when hpt as a selectable marker gene following transformation, and the transformation efficiency was 12%. By contrast, the transformation efficiency was only 6.8% when nptⅡ as a selectable marker gene. The transgenic plantlets of R. hybrida ’Samantha’ were confirmed by GUS histochemical staining, PCR and Southern blotting analyses, which indicated that GUS gene was stablely inserted into the genome of R. hybrida.3. Study on genetic transformation of R. hybrida ’Samantha’ with DcACO and AtCPC genesIntroduction of DcACO and AtCPC genes into somatic embryos of R. hybrida ’Samantha’ were studied. PCR analyses indicated that DcACO and AtCPC genes were stably inserted the genome of R. hybrida.4. Morphological and expression analysis of powdery mildew resistance related genes in Rosa multifloraDuring the interactions involving R. multiflora and P. pannosa, the results of cytological observation showed that:some differences of leaf tissue structure were obtained between susceptible and resistant plant.In susceptible plants, after 96h and 144h inoculation, infection hyphaes were observed in infected leaves, and hyphaes infected leaf tissue through stoma of lower epiderm, meanwhile, papillaes were formed on upper epiderm of susceptible leaf tissue.Using real-time PCR, the change in transcript level of 15 important defense-related genes was analyzed in a timecourse experiment. The results determined that:Chitinase maybe played an important role in the interactions involving R.multiflora and P. pannosa. Jasmonic acid and ethylene (JA/ET) signaling pathway might be triggered in the interaction between susceptible R. multiflora and P. pannosa. In the resistant R. multiflora, salicylic acid (SA) signal pathway was induced early P. pannosa. Between susceptible plants and resistant plants, key phenylpropanoid pathway genes were induced and upregulated after P. pannosa inoculation, which demonstrated that phenylpropanoid pathway and secondary metabolites play an important and active role for R. multiflora against powdery mildew infection. |
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