Construction of Agrobacterium rhizogenes-mediated transformation system of Carya illinoinensis without dependence on tissue culture

Abstract:【Objective】Carya illinoinensis, also known as pecan, is originated from the North America.Pecan is one of the most economically valuable woody species and a high value tree for edible nut pro-duction in the world. Current pecan transformation methods are time-consuming and low efficiency. Due to the lack of simple and efficient genetic transformation system for pecan, it is urgent to develop a simple and highly efficient transformation system so that the function of genes could be studied through the transgenic system quickly and efficiently. In recent years, an Agrobacterium rhizogenes mediated transformation method has been developed, which is less time-consuming and species dependent in gen-erating transgenic plant tissue. To obtain a simple and efficient A. rhizogenes mediated transgenic sys-tem, we established a transgenic root system with pecan seedlings.【Methods】Taking the seedlings of Carya illinoinensis (Wangenh.) K. Koch‘Zhongshan’in a forest nursery as materials, the effects of strain, the concentration of bacterial liquid, treatment site, and seedling age were studied on the infec-tion of pecan. The GFP expression vector pCAMBIA-35S:GFP was used to test the transgenic-positive roots. The plasmid pCAMBIA1300-35S:GFP was transformed into A. rhizogenes K599, A. rhizogenes C58C1, or A. rhizogenes MSU440, respectively. Then, a design of experiments (L9) with four factors and three levels was carried out. The factors studied were: strain type (ST), bacterial liquid concentra-tion (BC), treatment site (TS), and seedling stage (SS). A genetic transformation system for pecan was performed and, in each of them, strain type (STK599, STC58C1 and STMSU440), bacterial liquid con-centration (BC0.4, BC0.8 and BC1.2), treatment site (TS0-1, TS1-3 and TS3-5), and seedling stage(SS0, SS2 and SS4) were measured. The seedlings were grown in an artificial climate chamber (lumi-nance 200 lx, photoperiod 12 h light, and humidity 70%±5%). Seeds were collected and sown immedi-ately on a sowing substrate for germination. After germination, the germinated seeds were sown in plas-tic pots containing cropping substrate. The vegetative stage (2-3 months after seed sowing) was selected as a transgenic receptor. All seedlings were generated via injection or a blade. The seedlings were cov-ered for a dark treatment (48 h dark) after infestation. For efficiency analysis, hairy rooting efficiency,hairy rooting number, and transformation rate were analyzed. Hairy rooting efficiency was estimated asrooting rate per explant. The hairy rooting number was counted as the root number per explant. Trans-formation rate was calculated as positively transformed rooting per explant. All statistics were recordedat 30 days post-infestation. For detection of GFP fluorescence, roots were observed with a fluorescent microscope or a handheld fluorometer. The inserted GFP sequence was verified by PCR. After confirma-tion, the untransformed hairy roots and primary roots were cut manually. Transgenic hairy roots were maintained for seedling growth.【Results】For both infestation methods, the final transgenic rate was not different between needle injection and blade scratch. For the convenience of operation, we choose a blade to scratch in the following experiments. The callus tissues formed on the infestation sites after 10-15 days post- infestation using A. rhizogenes. The hairy roots developed from the callus after 10- 15 days. The GFP fluorescence was observed both in the callus and hairy roots. According to the results of orthogonal experiments, the factor order of influencing the transformation efficiency was the seedling stage, treatment site, bacterial liquid concentration, and strain type. The optimal influential variables, in this case, were strain type for STK599, bacterial liquid concentration for BC0.8, treatment site for TS3-5, and seedling stage for SS0. The results showed that hairy rooting efficiency was 56.5%, and transgen-ic rooting rate was 78.5%, while the final transformation rate was 45.2%. The GFP fluorescence was de-tected in the whole roots of transgenic pecan plants. The inserted GFP fragment was also confirmed byPCR amplification of the GFP coding sequence. We found that it was vital to bury the infestation sites in the substate during the whole experiment. If the infestation sites were exposed to air, the hairy rootscould not emerge from the cut surface of the shoots. The transgenic-positive pecan hairy roots were ableto develop and become thicker and more lignified. Meanwhile, the original taproot growth of pecan was arrested. The seedlings grew normally without the transgenic-negative roots and taproots.【Conclusion】We successfully established an efficient and simple A. rhizogenes mediated transgenic root system for pecan, which was not dependent on the tissue culture. It was proved that high efficiency transform could be achieved if an appropriate seedling stage was chosen for the specific infestation site for Agro-bacterium-mediated transformation. Our study provides useful information for the construction of a sim-ple and high efficient transformation system for hairy root transgenic crop production. This transgenic  system is a powerful tool for gene functional characterization. This study also provides a foundation for cultivating new varieties of pecan and the improvement of agronomic characteristics by genetic engi-neering.