Modification Of Agrobacterium-mediated Genetic Transformation And Analysis Ofβ-glucanase Overexpression In Barley

Barley (Hordeum vulgare L.) ranks fourth among the cereal crops. Genetic transformation is one of the most important methods for gene function research and genetic improvement in barley. Recent studies showed that suitable explants and transformation procedure were the most crucial factors affecting transformation efficiency. In this study, different explants and regeneration medium have been tested to establish an efficient Agrobacterium-mediated transformation system in barley. Moreover, molecular identification and biochemical analysis of35S:Glb2transgenic barley plants, which overexpress β-glucanase, have been performed. The main experimental results are as follows:1. Modification of Agrobacterium-mediated genetic transformation procedure1.1Improvement of regeneration medium for barley immature embryosThe optimal concentration of BAP (6-Benzylaminopurine) in regeneration medium BCT (Barley Callus Transition) was determined to be lmg/L by comparing the regeneration efficiency using BAP concentration gradient. A new regeneration medium B13M containing basal medium B1as main composition and1mg/L BAP was used to induce plant regeneration. Compared with original used MS medium, the regeneration efficiency of callus was increased by11times. Rooting efficiency of two medium BGC(Barley Glasstube Culture) and BRM (Barley Rooting Medium) were also compared, and BRM had a higher rooting efficiency.1.2Modification of Agrobacterium infection materialsSince immature embryos tended to die easily during the process of co-cultivation because of excessive growth of Agrobacterium, callus induced from immature embryos was infected with Agrobacterium followed by hygromycin selection and regeneration. This method decreased the death of immature embryos and increased the work efficiency.1.3Genetic transformation research by using barley mature embryos as explantsPrimary genetic transformation system by using barley mature embryos as explants was established and15regeneration transgenic plants were obtained. Although using barley mature embryos as explants have some advantages, such as not restricted by availability of explants, the induction efficiency of callus from mature embryos was much lower than that from immature embryos. Further work is still needed to optimize the transformation system using immature embryos explants.6exogenous gene expression vectors were transferred to barley and48plants were gained. All these transgenic plants were transplanted in experiment field and needed further identification and analysis.2. Molecular identification and biochemical analysis of35S:Glb2transgenic barley plantsBarley (1,3-1,4)-p-glucanase (EC3.2.1.73) have two isozyme, which named P-glucanase EI and P-glucanase EII. P-glucanase (EII), which encoded by Glb2, only expressed in aleurone layer of seeds.35S:Glb2construct was generated and transformed into barley Golden Promise in our preliminary work. Molecular identification was performed using35S:Glb2transgenic T2and T3lines. Southern blot confirmed that EII gene was inserted into the genome of barley. Line DE3had one copy of target gene, while other lines had two or more copies of target gene. Semi-qRT-PCR showed that expression levels of Glb2in transgenic lines were significantly higher than that in wild type.β-glucanase activity in seeds of35S:Glb2transgenic barley was determined. The result showed that enzyme activity in transgenic barley was increased by72-105U/kg and dramatically higher than that in wild type barley. β-glucan content was also detected both in transgenic barley and wild type barley. It showed that P-glucan content was23-107times lower in T2transgenic barley and16-34times lower in T3transgenic barley, when compared with that in wild type control. The change of glucan content during seed germination was also tested. P-glucan content was decreased to a low state at3days after germination in wild type, while glucan content in35S:Glb2transgenic barley maintained low levels during germination.Moreover, thousand grains weight and starch content in35S:Glb2transgenic barley were measured. Thousand grains weight of transgenic barley was higher than wild type barley. By contrast, no significant difference of starch content was found between transgenic barley and wild type barley. We proposed that increase of thousand grains weight in35S:Glb2lines might due to decrease of β-glucan, which resulted in decomposition of glucan to monosaccharided and oligosaccharide stored in grain, and increased cellulose synthesis to maintain stable cell wall structure.In short, overexpression of β-glucanase in barley can dramatically decrease the content of P-glucan, which provid a strategy to generate low P-glucan content barley for quality improvement in the future.