Agrobacterium-mediated Transformation Of Paulownia Elongata Using Anti-LFY Gene

Paulownia plant originated in China is fasting-growing tree, which produced high quality timber, but some problems such as witches broom, large crown and short trunk obstruct Paulownia production. As the development of modern science and technology, gene engineering and cell engineering provide effective methods for solution of these problems.The thesis presented here deals with the factors affecting the transformation of P. elongata mediated with Agrobacterium tumefaciens, optimum transformation conditions, original establishment of anti-LFY genetic transformation system, DNA extraction and isolation from P. elongata leaves and verification of the transformation with PCR and GUS report gene. The main results are as following:1. The expression vector pBI121 contains anti-LFY gene, and there is a position, which can be defied by HindIII. We obtain the anticipative DNA segement when the plasmid of DH5 a is digestioned by Hindlll and EcoRI. The anti-LFY gene can be examined by PCR as it is transferred into Agrobacterium tumefaciens. The very Agrobacterium tumefaciens LBA4404 is the engineering Agrobacterium for transformation of P. elongata.2. A series of factors influencing the transformation have been studied such as the selection concentration, the antibiotics against Agrobacterium tumefaciens, the pre-culture time, the co-culture time, AS concentration, infection time and the density ofAgrobacterium. The optimized system is as following. The leaves cut off its edage are used as explants. The pre-culture time is 3 days. The density of Agrobacterium is about OD600 0.35.The leaves are incubated for 10 min in the diluted culture of Agrobacterium tumefaciens LBA4404. The co-culture of the explants in baterium is for 3 days. Resistant calli or shoots are obtained about 25 days on MS media supplemented with km 50 mg L-1 and cef 500 mg L-1. The resistant shoots go on culturing on 1/2MS midium supplemented with kan 30 mg L-1. Through this process better transformation rates can be got.3. Using the leaves from plantlet of P. elongata in vitro as the material, four different methods are compared in extraction of Paulownia genomic DNA, which belongs to two kinds, CTAB and SDS. The prepared DNA samples are tested by ultraviolet spectrophotometer analysis, agarose gel electrophoresis, restriction enzyme digestion and RAPD reaction.The results show that as for the four samples, the production rate were3.5~5.2pg per ten milli-gram of fresh leaves, A260/A280=l .7-1 .8, A260/A230>2.0, the molecular weights above 23 Kb, and they are all suitable for digesting with restrictive enzyme and RAPD analysis. However, considering all the factors, the best one shall be SDS-I correspondingly.4. By the Agrobacterium-mediated transformation, the model genes (NPTII + GUD genes) and anti-LFY gene contained in the vector pBI121 are introduced into the leaves of P. elongata. According to the detection of histochemistry and PCR in the leaves of plantlet regenerated from the resistant calli, anti-LFY gene has been integrated into plant genomes. The expression rate of GUS report gene reachs to 20 percent. Anti-LFY gene is detected with PCR in six of the seven shoots which contain GUS report gene.