Establishment of Agrobacterium tumefaciens mediated transient transfor- mation system in young leaves of Duli pear (Pyrus betulifolia)

Abstract:【Objective】The aim of this study was to seek for the optimal conditions for transient expres- sion of the genes to a high level in the young leaves of Duli pear (Pyrus betulifolia) using Agrobacteri- um-mediated transformation strategy.【Methods】The young leaves of the tissue cultured seedlings of Du- li pear (Pyrus betulifolia) were used as the experimental materials. The pBI121 expression vector con- taining the GUS gene under the 35S promoter was firstly transformed into two different Agrobacterium tumefaciens strains GV3101 and EHA105 through the freeze-thaw method. 100 ng of pBI121 plasmid DNA was added into the competent cells DH5α under aseptic conditions, mixed gently, and let to stand for 5 minutes in an ice water bath; The mix was quickly froozen in liquid nitrogen for 5 minutes, and the centrifuge tube was quickly placed into a 37 °C water bath for 5 minutes; then 5 minutes in an ice- water bath; 800 μL of antibiotic-free LB liquid medium was added into the tube under aseptic condi- tions, and the tube was shaken at 28 °C for 2 hours to revive the bacteria; then it was centrifuge at 6000rpm for 1 minute to harvest the bacteria, about 100 μL of supernatant was left. The obtained bacte- ria was gently suspended again, an appropriate amount of bacteria liquid was taken to, spread on the LB plate containing the corresponding antibiotics. The culture was inverted in a 28 °C incubator until 2mm plaques had appeared. Colony PCR positive identification was performed using primers 35SPro-F: 5’- CTATCCTTCGCAAGACCCTTC-3’, GUS-R: 5’-ATCGCTGATGGTATCGGTGT-3’, M13F: TGTA- AAACGACGGCCAGT and M13R: CAGGAAACAGCTATGAC, then agarose gel electrophoresis was used for analysis. A 3-level orthogonal experiment was designed with 3 factors, i.e., 3 bacterial concen- trations at OD600 = 0.6, 0.8 and 1.0, 3 vacuum infiltration durations of 10 min, 20 min and 30 min, vacu- um negative pressure was set to -0.09 MPa, and co-culture time of 2 d, 4 d and 6 d, respectively. The co- cultivation medium was adjusted to pH 5.8 using 4.4 g·L-1 MS +30 g·L-1 sucrose +8 g·L-1 agar. The transformed leaves was stained with GUS staining solution containing 5-bromo-4-chloro-3-indolyl-glu- cronide (X-Gluc) ,and the expression of the gene was observed after staining. The transformation effi- ciency and the percentage of necrosis of the leaves were recorded.【Results】Using the method of dou- ble primers identification, the plant expression vector pBI121 was successfully transferred into agrobac- terium GV3101 and EHA105. The 35SPro-F and GUS-R primers was used to identify the monoclonal PCR product with a size of 1370 bp, and the M13F and M13R primers were used to identify the mono- clonal PCR product of 3085 bp. The instantaneous transformation efficiency and necrosis rate of two different Agrobacterium in Duli pear leaves were counted respectively, and it was found that high effi- ciency transformation as observed by GUS staining was achieved in young leaves of the Duli pear using both Agrobacterium strains GV3101 and EHA105. However, the transformation efficiency of these two strains were different. A 100% transformation efficiency was obtained with GV3101 where the leaves were vacuum infiltrated with the agrobacterial solution at OD600 of 0.8 for 20 min followed by co-culture for 4 days. The leaf necrosis rate was 13.09%. However, for EHA105, the highest transformation efficiency of 83.33% was achieved with two combined conditions of (1) OD600 of 0.8, the vacuum infiltra- tion time of 30 min and the coculture time of 6 d, and (2) OD600 of 1.0, the vacuum infiltration time of 10min and the co-culture time of 2 d. The leaf necrosis rates of EHA105 transformed leaves were much higher than that of GV3101 transformed leaves. However, a much lower leaf necrosis rate was obtained with EHA105 when OD600 of 1.0 to vacuum infiltrated leaves were used for 20 minutes followed by co- culture for 4 days although the transformation efficiency was lower.【Conclusion】Both Agrobacteriumstrains of GV3101 and EHA105 could transiently transform Duli pear leaves at high efficiency of 100% and 75.09%, respectively. The optimal combined conditions for transormation by strain GV3101 was es- tablished and could be valuable for further study.