A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

CRISPR/Cas9-based Antibody Production in Plants

Nowadays demand of antibody production is increased to cure different diseases including diabetes, hepatitis and cancer. For that different types of systems are used for the expression of antibody production. But these were not improved the antibody production. Plant cells have several benefits in comparison with other eukaryotic cells if it is considered as eukaryotic expression system. As compared to the human cell or other microorganisms, the plant cell is safe and decrease the contamination of antibody production. In addition, plants perform proper post-translational modification as a eukaryotic expression system. But recently, transient expression system is used due to the safe and improve the quality and quantity of antibody production. In transient expression system agroinfiltration method are mostly used. The main issue in antibody production is purification. Because in downstream process antibody is degraded due to the physical and chemical stresses. These issues can be solved with the help of CRISPR/Cas9. Plant antibody can be tagged with the help of CRISPR/Cas9. This review encompasses the applications of CRISPR technology for producing plant-based antibodies.