Deep Dive Into Plant Transformation: Protoplast-Mediated, Biolistic-Mediated and Agrobacterium-Mediated Gene Transfer

Plant transformation is the process in which a foreign gene is introduced into a plant's genome. The methods researchers use for plant transformation include protoplast-mediated transformation, biolistic-mediated transformation and Agrobacterium-mediated transformation.

Plant genetic transformation is a powerful research tool for gene discovery and function to investigate genetically controlled traits of interest in agriculture and favor breeding programs by producing novel and genetically diverse plant materials.

Protoplast-mediated transformation

Protoplast-mediated transformation involves direct uptake of DNA by naked plant cells (plants without a cell wall, or protoplasts). This process provides an expression system for researchers to identify novel candidate genes.

Usually, protoplast-mediated transformation (PMT) is reported for transient expression (genes temporarily expressed - not integrated in the genome), providing an efficient way to study many genes in a short time. In this method, protoplasts are first produced through enzymatic treatments to break the cell wall. Furthermore, staining solutions like calcofluor- white and fluorescein diacetate are used to visualize the cell membrane integrity and the cell viability respectively. Then, the direct delivery of DNA to individual plant cells is performed using polyethylene glycol (PEG) or electroporation.

Figure 1. Protoplast-mediated transformation steps.

Finally, transformed protoplasts are selectively cultivated and used for further regeneration steps. An updated step by step protoplast transformation protocol can be found here.

Biolistic-mediated transformation

Biolistic transformation, also known as particle-mediated gene transfer, was first reported in 1987 as an alternative to PMT, especially for recalcitrant specie.

Biolistic transformation can be defined as the introduction of substances into intact cells and tissues through high-velocity microprojectiles. The term came from cells shot with DNA. Since the biolistic process is a direct gene transfer method like PMT, it has no immediate effect on the DNA's chromosomal integration mechanism. Thus, the integration event arising from the biolistic process will depend upon the DNA being delivered and the organism's biology to transform.

In biolistics, the process starts by mixing a DNA construct with particles of a heavy metal, usually tungsten or gold. These fine particles stick to the negatively charged DNA. The DNA-metal particles are loaded

into a gene gun, and pressurized gas like helium, provides the force. Then, some of the metal particles will penetrate the cell membranes and deliver DNA constructs ito cells. A protocol for plants using biolistics can be found here.

Agrobacterium-mediated transformation

In this method, the natural ability of the soil bacterium Agrobacterium tumefaciens to transform host plants is exploited to develop transgenic plants. An excellent video to summarize the in vivo process can be found here.

Agrobacterium naturally transfers DNA (T-DNA) located on the tumor-inducing (Ti) plasmid into the nucleus of plant cells and stably incorporates the DNA into the plant genome. Thus, Agrobacterium-mediated gene transfer (AMGT) is widely used for stable plant transformation.

The Agrobacterium-mediated transformation process involves the following:

1. The attachment of Agrobacterium to the plant cells
2. Sensing plant signals by Agrobacterium and regulation of virulence genes in bacteria following transduction of the sensed signals (where the chemical acetosyringone works as a plant signal attracting Agrobacterium).
3. Generation and transport of T-DNA and virulence proteins from the bacterial cells into plant cells.
4. Nuclear import of T-DNA and effector proteins in the plant cells.
5. T-DNA integration and expression in the plant genome. You can check a complete review for Agrobacterium-mediated transient gene expression.

The popularity of Agrobacterium-mediated transformation

When it comes to producing transgenic plants, Agrobacterium-mediated transformation is a very popular method to use for several reasons:

• Agrobacterium is naturally infectious for most dicotyledonous plants. However, Agrobacterium-transformation has been extended successfully to monocots, including rice, banana, corn, wheat, sugarcane, forage grasses, and conifers such as spruce.

• Agrobacterium can be combined with other methods (i.e., biolistic) to improve efficiency.

• A. tumefaciens is preferable to biolistic guns for stable transformation for important commercial crops (such as fruits and nuts) due mainly to its low cost in operation and the high potential in producing modifications with a low-copy number of the inserted sequence such as genes of interest (Song et al., 2019).

• Transferring DNA via Agrobacterium allows stable integration with fewer rearrangements of long DNA molecules with defined ends and the ability to generate lines free from selectable marker genes.

Agrobacterium-mediated transformation and CRISPR

A recent advantage of Agrobacterium-mediated gene transfer comes with the advent of gene-editing tools like CRISPR/Cas.

Genome editing technologies provide powerful tools for precise manipulation of targeted genome sequences, setting up unprecedented opportunities for crop breeding and functional genomics research. However, the lack of appropriate methods to deliver genome-editing reagents (for example, constructs encoding the nuclease and target-specific RNAs) is the primary barrier to CRISPR/Cas-mediated gene editing in a variety of plants.

Currently, Agrobacterium-mediated transformation is the preferred method of CRISPR/Cas reagent delivery. Studies have already reported this success in maize, wheat and tobacco. Also, compared to the biolistics approach, the Agro-infiltration systems have significantly improved, progressing from the single Ti plasmid to the binary vector. Even today, more advanced Agrobacterium vectors have been developed like the superbinary and ternary vectors systems (Zhang et al., 2020).

In a binary vector, T-DNA could be separated from the Ti plasmid and placed on shuttle vectors. In the superbinary vector, the binary vector carries additional virulence genes and enhances Agrobacterium-mediated gene transfer of recalcitrant plants.

With a ternary vector, a third plasmid—called the accessory plasmid or the virulence helper plasmid—is used to carry the virulence gene cluster. These improvements have increased transformation efficiency throughout Agrobacterium and expanded the transformability of a wide range of plant genotypes!

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