Expression And Inheritance Of Lysine-rich Protein Gene In Lettuce (Lactuca Sativa L)

Various factors influencing tissue culture of lettuce (Lactuca sativa L) were investigated and a transformation system was established with explant. Transgenic lettuce was obtained by Agrobacterium tumefaciecs-mediated transformation of leaf disc. Lysine-rich protein gene was introduced into lettuce. The results indicated that the introduced gene was integrated into the T0 and T1generation transgenic plants. An efficient gene transfer system for lettuce was established. Explants were first pre-cultured on MS medium for 1-2 days, then co-cultured on original MS medium with an Agrobacterium tumefaciens strain (LBA4404) carrying the modified pBI121 plasmid which contains lysine-rich protein gene driven by CaMV35S promoter. Explant were transferred to MS medium with Kan 100mg/L and Carb 500 mg/L ,and buds elongated on MS medium containing the same concentration Kan. All elongated shoot rooted on 1/2 MS medium then transferred shoot grew 2cm. Studies on the effects of various factors influencing transformation indicated that a serious of factors, including the age of explant, the condition of Agrobacterium tumefaciens, the pre-culture of explants, the time of transformation and co-culture, influences the rate of transformation of lettuce. The results indicated that: the optimal explants age for transformation was 2-3 days;the Agrobacterium tumefaciens in the logarithm time have the most ability for transformation;the optimal time of transformation was 20 min when the Agrobacterium tumefaciens was diluted to OD600=0.8; the optimal duration of pre-culture was 1-2 days that would improve the rate of transformation;The duration of 2-3 days co-culture might improve the rate of transformation too. The transgenic nature of regenetants was indicated by plant growth on medium containing Kan. PCR and southern blot analysis demonstrated further lysine-rich protein gene integrated into transgenic plants. Simultaneously, the result of RT-PCR detection confirmed the target gene has been expressed on the level of transcription. Data analysis demonstrated the lysine content of the T0 generation transgenic lettuce has been differently improved, and in one of the most increased by 9.5% Genetic analysis for some of the T1 generation transgenic plants by PCR displayed that the target gene can inherit into T1 generation transgenic plants. But the rate of genetic of lysine rich protein gene in the lettuce was low.