Polyethylene Glycol (PEG)-Mediated Chloroplast Transformation

Introduction

Polyethylene glycol (PEG)-mediated plastid transformation methods are much less expensive than biological methods. When protoplasts are the target cells for transformation, PEG-mediated transformation is usually used. So PEG method is the second most widely used method for chloroplast transformation in chloroplast engineering, and it becomes an alternative to the biolistic delivery system. Researchers achieved stable plastid transformation on Nicotiana tabacum using the PEG method as early as 1993. The PEG-mediated plastid transformation method only needs to convert the constructed plasmid (containing exogenous gene, marker gene, homologous fragment, promoter, terminator, etc.) can be transformed by co-cultivation with plant protoplasts under a certain PEG concentration. In addition, this method has the ability to form more cells with genetically transformed plastids than with biological methods. Studies have speculated that PEG can control the osmotic conditions of protoplasts, which is very helpful for the uptake of DNA constructs during transformation.

Fig. 1. An optimised procedure for transformation of O. tauri using polyethylene glycol. (Sanchez F, et al., 2019)

Services

The PEG-mediated chloroplast transformation method opens up new prospects for researchers to explore the potential of chloroplasts, and our technology in this area is quite mature. With the support of the chloroplast genetic engineering platform, Lifeasible  can easily perform various enzymatic treatments on plant cells to obtain protoplast suspensions, and utilize the polyethylene glycol method for high-quality chloroplast genetic transformation. Furthermore, our PEG-mediated chloroplast transformation method is widely used for insertion of plant resistance genes, expression of recombinant proteins for molecular agriculture and engineering of metabolic pathways.

Our service is polyethylene glycol (PEG) treatment of leaf protoplasts in the presence of transforming DNA, a strictly quality controlled enzymatic process to obtain a chloroplast suspension free of contaminants and other cellular components. This ensures that the transformation is not affected by other plant cells and only the chloroplasts are transformed. In conclusion, our goal is to achieve efficient transformation of plant chloroplast genomes based on the PEG method, ensuring that the transgene can be integrated into specific regions of the chloroplast. Here, we provide a simplified and inexpensive procedure for transforming chloroplasts in plants using an optimized protoplast culture protocol.

(1) Enzymatic treatment to remove cell walls and obtain chloroplasts from protoplast suspension.
(2) Mix chloroplasts with DNA.
(3) Add PEG solution to the mixture.
(4) Transformation of chloroplasts with DNA constructs.
(5) Selection of transformed chloroplasts based on selectable marker genes.
(6) Cultivate desired chloroplasts for recovery and regeneration.

Advantages of PEG Treatment

The key to this method is the handling and culture of protoplasts. Recovery of large numbers of active protoplasts results in large numbers of transient transformants. But you don't need to worry about these, our professional engineers will provide you with simple procedures and reliable results. This approach has been adopted by us due to its many advantages.

• Can be mixed with polyethylene glycol in the presence of DNA without causing any physical damage to cell membranes or protoplasts.
• No expensive equipment is required and the cost is minimal.
• The steps are simple and can even be performed in any laboratory setting, as long as there is a biological safety cabinet.
• With validity and the ability to reproduce expected results.
• Can produce higher conversion efficiency.

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 polyethylene glycol (PEG)-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. Sanchez F, Geffroy S, Norest M, et al. (2019) Simplified transformation of Ostreococcus tauri using polyethylene glycol[J]. Genes. 10(5): 399.
2. O'Neill C, Horvath G V, Horvath E, et al. (1993) Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems[J]. The Plant Journal. 3(5): 729-738.