Study On Transferring Rs-afp₁ Gene And γ-tmt Gene Into Peanut(Arachis Hypogaea L.) Respectively And High Efficient System Of Genetic Transformation

In this experiment, systematic study was done on the effect of physiology condition ofexplants, genotype and medium components on plant regeneration of the transgenic plants, 6peanut cultivars were used as source of explants. The high efficient system of peanut plantregeneration was established. For the first time,the phenomenon of floral organs regeneratedfrom calli of nutritive organs were discovered, crucial technology were studied ,and the newchannel for hastening peanut breeding process, by blooming, self-crossing and fruiting intested-tuber ,were put forward. High efficient system of peanut genetic transformation wasestablished by analysis of some influencing factors of peanut genetic transformation in manyprocedures. In order to generate transgenic plants of peanut with the resistance of peanutfungus disease, Rs-afp1 gene was introduced into three peanut cultivars. Obtained transgenicplants were verified that foreign genes had been intergraded into the genome DNA of peanutby molecular detection. In order to generate transgenic plants of peanut with elevated thevitamin E activity of seeds, γ - tmt gene was introduced into two peanut cultivars. Transgenicplants were verified that purpose genes had been intergraded into the genome DNA of peanutby molecular detection.The major results studied in the paper are abstracted as follows: 1.Study on plant regenerative system in peanut tissue culture In comparison with leaflets of seeding , embryonic leaflets cultured and infected asexplants have many characteristics such as simpler for getting materials,more abundant inmaterials source and higher frequency of plant regeneration. Embryonic leaflets cultured inmedium adding BA and NAA or adding BA, KIN and IAA can regenerate by multiple budsinduction. Induction efficiency of MSB + 4.0 mg/L BA +1.0 mg/L NAA is the highest.Regeneration capabilities of Fenghua2 and Luhua11 are the best, among 6 varieties tested.Floral organs regenerated from Calli, which generated from embryonic leaflets in MSB+1mg/L BA + 0.5mg/L KIN+2mg/LIAA ,and were cultured in MSB+1～2mg/L BA.Embryonic leaflets cultured in medium adding 2,4-D can regenerate by embryogenesis 4山东农业大学博士学位论文formation. Induction efficiency of MSB + 10 mg/L 2,4-D is the highest, among the 5 mediatested. 2. Study on influencing factors of peanut genetic transformation (1) 500 mg/L Cef can efficiently inhibit tested bacterial strains, GV3101 and EHA 105 ,growing. In comparison with CK, its effect on frequency of multiple buds formation fromleaflets does not show strong difference. (2) The results of research on selection antibiotic andits suitable density for npt Ⅱ gene in peanut genetic transformation system are abstracted asfollows: 100 mg/L Kan, 300 mg/L Neo or 10 mg/L G418,can efficiently inhibit callusformation and shoots regeneration from leaflets in peanut. Frequency of leaves etiolating is100%, when leaflets of seedling were smeared with 5 mg/ml Kan solution. (3) With the sameinfection time (15mins), difference of bacterial concentration affects frequency of genetransformation, the frequency of gene transformation is higher with bacterial concentrationOD 600= 0.65. (4) Co-cultivation for 4d is favorable for transformation. (5)The leaflets with 6d seedling age give the highest rate of transformation. (6)Time and media of pre- cultivationof explants play important roles in transformation of peanut .Pre- cultivation of explants inMSB + 4.0 BA mg/Ls + NAA1mg/L for 4d can effectively increase frequency oftransformation. (7) The research of Rs-afp1 gene transformation in peanut shows that:With thesame genotype, the difference of explants kinds obviously affected transformation efficiency.The frequency of transformation of clump buds of Luhua11 is the highest, that of embryonicleaflets of it is secondly, and that of leaflets from 6d seeding age of it is the lowest. Thefrequency of transformation...