The The Study On Transformation And Expression Of The Dual Genes Of Lyz-GFP Genes In Different Gene Acceptor In Alfalfa(longdong)

In this paper, the Alfalfa cultivar LongDong was selected as the study material.Compared to the tissue culture transformation system of LongDong alfalfa, the newtransgenic technology of agrobacterium mediated vortex oscillation infection methodwas invented. By research some factors to affect the regeneration of LongDong alfalfain the transition process, the optimal time of infection and concentration offingbacteria were determined. Lyz-GFP genes was preliminarily proved in the LongDongalfalfa by testing the marker genes green fluorescent protein (GFP) and purpose gene(Lyz) of transformation plant. It confirmed that the technology of plant meristematictissue transgenic in the living conditions is feasible and the stem tip growing pointconversion technologies was tried exploratory in the alfalfa transgenic breedingtechnology.The main research contents are as follows:1. The establishment of tissue culture transformation system of transformedLyz-GFP genes LongDong alfalfa. The hypocotyl of growth5~7d LongDong alfalfawas selected as explant to transform Lyz-GFP genes. We have studied the effect ofmany factors on the regeneration of LongDong alfalfa, such as bacteria liquidconcentration, infection, Kan concentration and antibacterial element concentrations.The results show that the optimal Kan concentration for callus stage is60mg/L andthe one for differentiation stage is70mg/L. The optimal bacteria liquid concentrationis0.5~0.6. Cef as antibacterial element is better and the optimal concentration is250mg/L.In the paper, the Vitrification phenomenon of regeneration plants indifferentiation stage was analyzed and discussed and the solution was put forward.Vitrification phenomenon of LongDong alfalfa was most effective when the followingconditions were met: light intensity is1000~1200Lx, MS culture medium contained2.0mg/L6-BA and0.5mg/LNAA, Sugar concentration is20~25g/L and agarconcentration is7g/L.2. The establishment of transformation system of agrobacterium mediated vortexoscillation infection method. The sterile LongDong alfalfa of growing2-3d out ofcotyledons was selected as material and agrobacterium-mediated bacterium fluid andsterilization quartz sands were selected as medias when Lyz-GFP genes was transform into alfalfa. The following conditions need to apply:30min was the optimal infectiontime, Cef was selected as antibacterial element and the concentration is250mg/L,screening concentration of Kan is70mg/L. Rooting of LongDong alfalfa needs1.0mg/LIBA, and the tube seedlings of LongDong alfalfa were then transplanted inmatrix of nutritional soil and vermiculite (Volume ratio is1:3).3. Fluorescence detection and polymerase chain reaction (PCR) identification oftransformational plant. The same number (50transformational plants) were tested byfluorescence detection. Three of these transformational plants by agrobacteriummediated vortex oscillation infection method have fluorescent protein expressionobviously but fluorescence expression was not detected in the transformational plantsdeveloped by the tissue culture.. The three plants were testing by PCR and one of thethree plants has one strip of750bp, so Lyz-GFP genes was transformed into the alfalfaof LongDong successfully. The transgenic technology of stem tip growing pointconversion is a new and fast.