Establishment And Optimization Of Agrobacterium Rhizogenes-Induced Root Transgenic System For Rosa Roxburghii Tratt

Rosa roxburghii Tratt.,belonging to the Rosaceae family and Rosa genus fruit tree,has not yet achieved a breakthrough in its regeneration system,which makes it temporarily impossible to verify gene function through stable genetic transformation.Therefore,attempting to use Agrobacterium rhizogenes to induce the production of hairy roots in R.roxburghii for genetic transformation is of great significance for elucidating and verifying the gene functions specifically expressed in key biological process of the root system;At the same time,it can lay a technical foundation for obtaining complete transgenic plants through root culture in the future.In this paper,using the A.rhizogenes K599 as the strain material and the enhanced green fluorescent protein（EGFP）gene as the reporter gene,the effects of A.rhizogenes on the rooting of R.roxburghii were analyzed through five infection methods,namely,air-layering,cutting,direct injecting,root-cutting and leaf infection.For the first time,we established a root transgenic system in R.roxburghii,further optimizing the concentration of bacterial solution,infection site,infection period and infection method.On this basis,the system was successfully carried out to achieve the overexpression of two previously screened genes,RrZIP20 in zinc/iron-regulated transporter-like protein and RrCML13 in Calmodulin-like,which laid a technical foundation for more genes functional verification and acquisition of complete R.roxburghii transgenic plants.Finally,the main findings are as follows:1.Establishment of the root transgenic system in R.roxburghii.A feasible and stable root transgenic system in R.roxburghii was successfully constructed by direct injecting,root-cutting and leaf infection methods.The specific operations were as below:（1）Direct injecting method:The robust seedlings were selected.Then,a disposable sterile syringe with the K599 infection solution was used to puncture the hypocotyl of the seedling and inject about 0.1 m L K599 infection solution into the stem,making it hung a large drop of infection solution on the injection site.After that,the wound was required to bury in the soil.（2）Root-cutting method:The robust seedlings were selected.A sterile scissor was used to cut the original root of the seedling so that the remaining hypocotyl was used as the explant.The apical portion of the hypocotyl was cut diagonally in the K599 broth.Then,the slant cut of the residual hypocotyl was inoculated on the plate grown on K599 and coated by the bacterial mass.Finally,the inoculated explant was directly planted into a wet sterile vermiculite.（3）Leaf infection method:It was the same as root-cutting method.Only the young and healthy leaves were replaced by the seedlings,and the infection was carried out after the petiole was removed.All of the infected explants were cultured under high humidity conditions.After about 8 days,callus appeared at the infected site and gradually expanded;After around 3 weeks,hairy roots similar to adventitious roots emerged from the callus.In addition,using air layering and cutting methods,it was found that K599 could promote rooting and the optimal infection concentration OD₆₀₀ was 0.4 and 0.6 respectively.2.Optimization of the root transgenic system in R.roxburghii.Using the direct injecting method,the optimal infection concentration was 0.4,the seedling age was 5 days,the injection site was 0.5-1.0 cm from the original root;Using the root-cutting method,the optimal infection concentration was 0.4,the seedling age was 5 days,the root-cutting site was 0.5 cm of the residual hypocotyl.The final root transgenic rate of direct injecting,root-cutting and leaf infection method could reach 7%,28%and 38%,respectively.And there was significant difference between them.The results indicated that the leaf infection method was more conducive to the infection of A.rhizogenes,thus inducing more transgenic roots of R.roxburghii.3.Validation of functional genes by the root transgenic system in R.roxburghii.Based on the aforemationed root transgenic system in R.roxburghii,overexpression validation was conducted on two functional genes,RrZIP20 and and RrCML13,respectively.It was found that:（1）20 members of the RrZIPs family were identified based on the whole R.roxburghii genome.Overexpression of RrZIP20 gene significantly increased the calcium content in the root and the zinc content in the leaves and roots,but decreased the iron content in the root,indicating that overexpression of the gene in the root could promote the absorption and transportation of calcium and zinc in the root,and also could transport calcium and zinc from the roots to the leaves for storage;（2）The L-Ascorbic acid（As A）content and the transcription level of the key biosynthesis gene RrGGP2（GDP-L-galactose phosphatase）in the roots of overexpressing RrCML13 were also upregulated,and there was a significant positive correlation between them.These once again confirmed that RrCML13could positively regulate the As A synthesis in R.roxburghii by upregulating the expression of RrGGP2.