The Transformation Of 'Dahong' Sweet Orange With RolB Gene

Conventional citrus breeding program have been carried out since the end of 19^th century.The genus citrus presents limitations for improvement via conventional breeding,and are directly associated with the reproductive biology of citrus,such as nucellar polyembryony(apomixis),a high level of heterozygosity,a long juvenile period and inbreeding depression.Biotechnological techniques such as cell and tissue cultures and genetic engineering have helped breeders to overcome all kinds of obstacles.Genetic transformation has become an attractive alternative method for improving citrus and other species because it is possible to maintain cultivar integrity while adding or deleting a single trait without the limitation of genotype.However, for efficient transgenic plant production,a previously defined tissue culture system for plant regeneration associated with a genetic transformation system for gene introduction is needed.The tested variety of 'Dahong' sweet orange,which is a superior choice for fresh and juice with the excellent quality and storability,is one of the local varieties in Hunan Province.Several researches,including the transformation of horticultural crops(in particular,fruit trees) showed the following characteristics belonged to rol genes,such as shorter the length of internodes,dwarfing height and generating a large number of oblique growth of hair roots.Genetic transformation with rolB gene had a high application value in the production of 'Dahong' sweet orange.At the same time, it was worth learning for the improvements of citrus and other crops.In the research, rolB gene was transferred into the genome of 'Dahong' sweet orange mediated by Agrobacterium-tumefaciens.The main results and conclusions were as follows:1.Using epicotyl segments of in vitro germinated seedlings of 'Dahong' sweet orange,the main factors which affected the plant regeneration were investigated.It turned out that plant growth regulator had a significant influence on the induction and differentiation of adventitious buds and roots.The best concentration of 6-BA for the introduction of adventitious buds was 1.0 mg·L^-1.In this case,the induction rate of adventitious buds reached 96.96%.Supplementing with suitable concentration of IBA or NAA in rooting medium could strengthen root formation.However,excessive callus were induced simultaneously by NAA.Taking all factors into consideration,the efficiency of IBA was better than that of NAA.When the IBA concentration got to 5.0 mg·L^-1,induction rate and the average root number reached the highest,which were 95.0%and 1.49,respectively.Plantlets were translated to the matrix which was made up of sand,grass charcoal and sawdust(1:1:1).Under the condition of gradually adapting to the external environment,survival rate of plantlets got up to 100%.2.Factors influencing genetic transformation of 'Dahong' sweet orange were evaluated including the concentration of Kan and Agrobacterium-tumefaciens.Higher percentage of transformation were obtained with the medium of MS+1.0mg·L^-1 6-BA+50mg·L^-1 Km+500mg·L^-1 Cef.Resistant buds were cultured through grafting and rooting,respectively.18 resistant plants were recovered from hundreds of explants.Some regenerated shoots were identified by performing PCR,RT-PCR and Southern blot analysis.First of all,PCR confirmed that No.1,7,8,9,13 and 16 were positive plants.Subsequently,RT-PCR proved that the target gene was expressed successfully in those plants of No.7,8,9,13 and 16.At last,Southern analysis revealed that at least one copy of the rolB gene was integrated into the genome of No.9 plants.3.The morphological features of transformed and control plants were observed. In grafting experiments,there were significantly differences between control and transgenic plants in leaf area and plant height.The leaf areas and plant height of control plants were 6.14～15.48 times and 6.48～9.38 folds of transformed plants, respectively.The rooting trials showed that exogenous hormone IBA could strength the rooting ability of resistant and control buds,but resistant,buds were more sensitivity to IBA.The adventitious roots germinated from resistant buds were much earlier than that from controls'.The average root number of resistant buds(9.0 roots) without the exogenous hormone were much higher than that of controls'(1.0 root), and it was almost 3 times height of resistant buds with the IBA.The characteristics of significant dwarfing,shorter internode length which displayed by grafted 'Dahong' sweet orange transformed with rolB gene were very important to the cultivation and application in production of fruits.