Analysis On The Genetic Diversity With AFLP And Transformation For Plbs Of Dioscorea Alata Linn

Dioscorea alata Linn., a species of yam, an efficient crop and its tuber that can be used as food and medicine. High-yield and high-starch make it as the new choice of energy plants. Because of little blossom or no blossom, low percentage of setting seeds, low germination rate, long dormant, and asexual propagation biological characteristics, it’s germplasm was degraded and it’s production was decreased. In order to improve the yield, clearly genetic background of yam is important for further research on the new breeding technology. However, the yam genetic relationship and genetic breeding is rarely reported.Here we use the AFLP method to study on the genetic diversity of yam, which contain111germplasm, of yam come from six provinces. Three different genetic background germplasms (Da-56, Da-87,Da-137) were selected as explants which containing stem segment with node. In vitro induction of protocorm-like bodies (PLBs) and regeneration system of protocorm were established, the genetic transformation system via Agrobacterium infection system was constructed and sik SAD gene were transformed. The detailed results as follows:1. Genetic diversity of111Dioscorea alata Linn, germplasm using AFLP markers.111germplasm were used with eight AFLP primer combinations that generated a total of1291fragments. A total of1286fragments showed polymorphism in the germplasm analyzed. A variety of marker attributes like polymorphism information content (PIC), marker index (MI) and resolving power (RP) values were calculated to assess the discriminatory power of eight primer combinations. The average values of PIC (0.22), MI(35.67), and RP (50.50) indicated great polymorphic and distinguishable ability of the eight AFLP primer combinations. The primer combination E-ACT/M-CTTE-AAC/CAG-M was be most informative with PIC, MI,and RP being0.22,35.67,50.50, respectively. A total of1286fragments presented polymorphism which can be summaried as great divesity among111Dioscorea alata Linn, germplasm with extremely lower similiary (0.30to0.82with average of0.58) Germplasm. At the genetic similarity coefficient level of0.54, the unweighted pair group method with arithmetic mean phylogentic anlaysis (UPGMA) and principal coordinate analysis (PCOA) further classified those gemplasm into four groups, and the germplasm serial number Da-19, Da-76and Da-137were individually separated. The clustering results indicated no close relation to the geographic location of the germplasm. Three different genetic background germplasm, Da-56, Da-87and Da-137were choosed as test materials, based on genetic diversity. 2. Established a different genetic background materials protocorm likes body (PLBs) regeneration system; PLBs were in vitro induced by stem segment with node aseptic seedling explant using an orthogonal test. The suitable concentration that induced PLBs was MS(with3×Ca2+)+6-BA1.0mg/L+NAA0.2mg/L+PVP0.1%+sucrose3.0%. Three highest PLBs induction rate was Da-56(91.67%), Da-87(93.33%) and Da-137(90%). The optimum medium for proliferation and rooting of PLBs was MS+4mg/L6-BA+80mg/L Ad+0.1%PVP+3.0%sucrose and1/2MS+0.1mg/LNAA+0.1%PVP+3%sucrose, respectively. There had no difference of induction rate and multiplication coefficient among3kinds of PLBs. After hardening off with the base material (pearlite: roseite=2:l), transplanting survival rate was up to100%.3. Dioscorea alata Linn, propagules’(PLBs) name and origin were proved. To observe the anatomy of PLB by paraffin section technique, PLBs were constituted by epidermis, meristem and large parenchyma with PLB description, which produced the adventitious bud and adventitious roots of embryogenic aggregates. The adventitious bud derived from bud primordium all of them belong to external origin of organogenesis type. Programmed cell death in the meristematic tissue exists in the adventitious bud development process. During the process, the growth point of bud separates with the scale leaves that wrapped around the bud, and then scale leaves gradually falling off. PLB endodermal cells differentiations to vascular tissue that connect adventitious bud and adventitious root, and make them formation a strong regeneration ability individual.4. Transformation system of Agrobacterium infection in PLBs was set up. Organsilicon deformer was obtained from Agrobacterium that carrying the reporter gus gene were used as a vector to study the different transformation parameters effect on the gus gene expression rate in PLB. The cutting PLBs were transfected by Agrobacterium (OD600=0.6)30minutes, and then cultured them in the PLB induce medium which contain200umol/L AS about3days. The gus gene expression rate was51.25%.5. Detection of the PLB genetic transformation system; A sik SAD gene were transfected into the Da-87PLB via170mg/L Kanamycin selection, and gained256resistance strains. After verification by PCR10transgenic plants obtained from50resistance strain. The target gene expression proved by semi-quantitative and realtime fluorescence quantitative PCR in4transgenic plants seems higher than the control group, and their comparative quantification of sik SAD gene were high distinctly compare with control, the DaT09plants had a highest gene expression level.