| London plane tree (Platanus acerifolia Willd.) is widely planted and applied in the city landscape, known as "the king of street trees". However, pollen and seed hairs from the trees tend to disperse widely during the spring, polluting the environment and triggering allergic reactions. Such negative traits restrict the use of London plane tree within urban landscapes. The rapid development of plant genetic engineering, especially for sterile plant cultivation, brings about new hope for no-seed cultivated programe of P. acerifolia.The genetic improvement programe of P. acerifolia had been extensively studied in our lab, and high efficient plant regeneration system and high-efficiency Agrobacterium-mediated stable transformation of P. acerifolia had also been developed. This study thereby analysed and determined some other factors influencing Agrobacterium-mediated transformation, and developed the improved genetic transformation model for P. acerifolia. Furthermore, two different binary vectors both containing the barnase gene was introduced into the P. acerifolia by the improved Agrobacterium-mediated transformation and the transgenic plantlets corresponding to them were produced. On the other hand, Inter-Simple Sequence Repeat(ISSR) was used to evaluate the genetic stability of long-term micropropagated plantlets of P. acerifolia, providing the molecular informations about whether the somaclonal variation of the explants for genetic transformation occur for performing the genetic improvement of P. acerifolia successfully. The main results as follow:1. Some other factors influencing the Agrobacterium-mediated transformation of P. acerifolia were analysed and determined through the GUS gene transient expression pattern, including the Agrobacterium tumefacien strain,surfactant,bacterium suspension and sucrose concentration in the co-culture medium, and thereby the improved Agrobacterium-mediated transformation system was developed. The study results indicated that EHA105 Agrobacterium strain, 0.01% surfactant Tween-20 in the infection solution, MS liquid medium supplemented with 3% sucrose used as bacterium suspension, and combined with 1.5% or 3% sucrose in the co-culture medium could produce the highest results, as indicated by 100% level of frequency of GUS transient expression.2. Based on the improved Agrobacterium-mediated transformation system for P. acerifolia, the pBI-TBSBAR/barnase gene and PTBS/barnase gene were both introduced into P. acerifolia, and thereafter the number of the putative transgenic plantlets containing these gene was 4 and 5, respectively. The results that the PCR dectetion for nptâ…¡gene was sussfully amplified in all of 9 putative transgenic plantlets indicated the interest genes had incorporated into the genome of P. acerifolia. The frequency of the improved stable Agrobacterium-mediated transformation of P. acerifolia arrived up to 5%, higher than that before (3.4%).3. Twenty micropropagated plantlets were indiscriminately chosen from a clonal collection as the tested materials to evaluate the genetic stability of long-term micropropagated plantlets of P. acerifolia. Here, 16 primers out of 38 ISSR primers screened, were found to produce clear reproducible bands resulting in a total of 103 distinct bands, Of these 103 bands, 86 were monomorphic across all 20 of the plants tested and 17 showed polymorphisms (16.5% polymorphism). A total of 1,771 scorable bands were obtained from the full combination of primers and plantlets and, of these, just 51 (2.88%) were polymorphic across the plantlets.4. Based on the ISSR band data, similarity indices and UPGMA dendrogram were analysed and developed. The results showed that similarity indices between the plantlets ranged from 0.92 to 1.00, and all 20 micropropagated plants grouped together in one major cluster with a similarity level of 91%, while assessed at the more stringent similarity level of 95%, could 4 distinct clusters be distinguished. Such a high level of similarity indices and a low level of somaclonal variability as indicated by ISSR analysis was confirmed genetic stability reletively, as to long-term micropropagated plantlets of P. acerifolia in vivo culture for more than 8 years. This study thereby demonstrates that the multiplication of shoots via axillary branch propagation may be performed for a considerable period of time with only a minimal risk of spontaneous genetic variation and, thus, is suitable for generating clonal material as part of a programe for Agrobacterium-mediated genetic improvement of P. acerifolia. |
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