Agrobacterium-Mediated Chloroplast Transformation

Introduction

Agrobacterium tumefaciens is a soil plant pathogen that delivers transferred (T)-DNA from bacterial cells to host plant cells via the Type IV bacterial secretion system (T4SS). Since the first report of the use of Agrobacterium-mediated methods to obtain transgenic tobacco in 1983, this technology has developed rapidly in chloroplast transformation and has played an increasingly important role in crop quality improvement. Agrobacterium T-DNA-mediated genetic transformation method refers to the construction of exogenous target genes and selectable marker genes into the Ti plasmid of Agrobacterium, and then co-cultivation with plant tissues or organs. Finally, the desired exogenous gene is transformed into the chloroplast and expressed. Transient transformation mediated by Agrobacterium tumefaciens has been applied in chloroplast transformation in a variety of plants because of its high efficiency, convenience, and ease of operation.

Fig. 1. Overall mechanism schematic of Agrobacterium-mediated chloroplast transformation. (Risha Pratiwi, et al., 2020)

Our Services

With the advancement and innovation of molecular biology technology and the in-depth understanding of the interaction between Agrobacterium and host cells, Agrobacterium has become the most popular tool for plant chloroplast transformation. Our technology in this area is quite mature. With the support of the chloroplast genetic engineering platform, Lifeasible can easily replace oncogenes in the T-DNA regions of various types of binary vectors with any gene of interest for high-quality Agrobacterium T-DNA-mediated chloroplast transformation. Our Agrobacterium-mediated chloroplast transformation method has been widely used to improve the traits of crops, including the generation of new genetic recombination, resulting in significantly improved crop yield, ease of cultivation, tolerance to environmental stress and pest resistance, etc.

Here, we constructed a dedicated vector for Agrobacterium-mediated transformation, carrying an expression cassette for a selectable marker gene within a T-DNA border. In addition, we developed new co-culture methods to compare the efficiency and stability of Agrobacterium. Our goal is to rely on the bacterial pathogen Agrobacterium tumefaciens to efficiently deliver stable, unrearranged single-copy sequences to host plants. We provide a simplified procedure for transforming chloroplasts in plants.

(1) Attaches Agrobacterium tumefaciens to gene products within plant cells and pathogens.
(2) Transfer the above gene product to T-DNA in a tumor-inducing (Ti) plasmid.
(3) T-DNA moves towards the host cell.
(4) T-DNA integrates with the chloroplast genome by non-homologous recombination.
(5) Expression of T-DNA.
(6 Chloroplasts regenerate and proliferate further.

Advantages of Agrobacterium-Mediated Method

• Agrobacterium-mediated transformation has higher and more stable expression.
• Using a binary vector system, offers great versatility and flexibility.
• Minimal equipment and facilities are required, installation and use costs are low.
• Generally low transgene copy number.
• Minimal DNA rearrangements.
• Fewer undesired mutations-somaclonal variation.

Lifeasible has successfully constructed a chloroplast gene transformation platform, using a variety of strategies to achieve chloroplast engineering. With our team's extensive experience in the construction and transformation of chloroplast gene vectors, we are able to provide high-quality Agrobacterium-based chloroplast transformation platforms to global customers. If you are interested in our services, please do not hesitate to contact us for more information.

References

1. Hwang H H, Yu M, Lai E M. (2017) Agrobacterium-mediated plant transformation: biology and applications[J]. The Arabidopsis Book. 15.
2. Risha Pratiwi, Muhammad Imam Surya. (2020) Agrobacterium-Mediated Transformation[M]. In book: Genetic Transformation in Crops. DOI:10.5772/intechopen.91132.