You may have heard about plants with introduced desirable traits like resistance to specific pests or improved nutritional properties, but how can these plants become like that? What is a plant transformation protocol? Why does this matter?

Plant transformation is defined as introducing exogenous DNA into plant cells, tissues or organs, employing direct or indirect means developed by molecular and cellular biology (Jenes et al 1993). 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.

What is a plant transformation protocol?

A plant transformation protocol comprises two main steps, delivery of the DNA into a single cell and regeneration into entire fertile plants. There are three well-known types of transformation methods: protoplast-mediated, biolistic-mediated, and Agrobacterium-mediated. But, how do they work? How do they differ? How broadly applicable are these methods? Below are some clues to answer those questions. In this article, I will mainly focus on the first step, "DNA delivery." You can find more information about plant regeneration in transformation protocols in the article Natural Variation in Plant Pluripotency and Regeneration.

How the protoplast-mediated transformation method works

In this method, the direct uptake of DNA by naked plant cells (plants without a cell wall, or protoplasts) provides an expression system for research in plants 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. Subsequently, the direct delivery of DNA to individual plant cells is performed using polyethylene glycol (PEG) or electroporation.

protoplast-mediated transformation process - shows the creation of protoplast cells or naket cells, adding dna into the cells and regeneration

Figure 1. Protoplast-mediated transformation steps.

What PEG and electroporation does to facilitate the DNA transfer? PEG changes the DNA conformation, thus it compacts the DNA and also allows it to associate with the cell membrane. PEG is also used as an in vitro crowding agent to mimic highly crowded cellular conditions. On the other hand, electroporation induces the micropores opening in the membrane, then DNA to cross the membrane, and the DNA then finds its way to the nucleus. Finally, transformed protoplasts are selectively cultivated and used for further regeneration steps. An updated step by step protoplast transformation protocol can be found here.

How the biolistic-mediated transformation method works

Biolistic transformation, also known as particle-mediated gene transfer, was first reported in 1987 as an alternative to the PMT, especially for recalcitrant species (species that produce recalcitrant seeds such as avocados, mangos and peaches).

The biolistic transformation can be defined as the introduction of substances into intact cells and tissues through high-velocity microprojectiles. The Iterm 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 onto a gene gun, and pressurized gas, like helium, provides the gun force. Then, some of the metal particles will penetrate the cell membranes and deliver DNA constructs to cells. A protocol for plants using biolistics can be found here.

Biolistic-mediated transformation process illustratrion showing the mixing of DNA and nano particles, a gene gun that shots DNA into cells, and the penetration of DNA into the plant cell membrane. Biolistic-mediated transformation can use intact-plant tissue.

Figure 2. Biolistic-mediated transformation steps.

How Agrobacterium-mediated transformation method works

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 transfer 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 and protocol step by step here. To learn more, check out GoldBio's article A Quick Overview of Agrobacterium for Plant Transformation.

Agrobacterium-mediated transformation process steps showing the attachment of agrobacterium to plant cells, sensing plant signals, insertion of DNA into plant cells

Figure 3. Agrobacterium-mediated transformation steps.

How to choose a plant transformation protocol: protoplast transformation vs. biolistic transformation vs. Agrobacterium-mediated transformation

All right, then the next logical questions a researcher asks are, what plant transformation protocol should I choose? What criteria should I use to prioritize one method over others? These are not easy questions; however, there are some variables we could rely on to select a protocol.

Usually, high efficiency is frequently pursued when considering transformation protocols. The efficiency is defined as the number of explants (tissue transferred into an artificial nutrient medium in tissue culture) showing transformation over the number of explants inoculated and multiplied by 100. The transformation efficiency is usually low (<30%) and depends on species, tissue, and physiological state.

Regarding a specific method, other variables should be considered. In the case of PMT the variables are PEG concentration or electroporation voltage. In biolistics the variables are particle size and particle velocity. Finally, for Agrobacterium, the strain and use of single/binary/ternary vectors should also be considered. Let's focus on some variables to guide you in selecting a transformation protocol.
  • For instance, if you are guided by transformation efficiency, then the reports say PMT is usually low, Biolistics is considered high, and Agrobacterium has both high and stable efficiency (Keshavareddy et al., 2018).
  • Suppose you are guided by the range of transformable plant species. In that case, protoplast and biolistics are unrestricted; instead, in Agrobacterium, many species, especially dicotyledonous, are more susceptible to transformation.
  • If you are guided by the type of explant, in PMT, the protoplasts (or plant cells with stripped cell walls) are the ideal explant; however, they must be produced with digestive enzymes. In the case of biolistics, intact tissues or microspores are suitable; meanwhile, Agrobacterium allows working with a broader range of tissues, including intact cells, organs, or the whole plant.

Table 1. Choosing a plant transformation protocol quick reference chart.

how to to choose a plant transformation protocol reference chart - agrobacterium-mediated transformation vs. biolistic-mediated transformation vs. protoplast-mediated transformation

Of the three methods, Agrobacterium-mediated transformation remains popular and is among the most effective! But, Why?

Why does Agrobacterium-mediated transformation remains popular?

  • Agrobacterium is naturally infectious for most dicotyledonous plants. However, Agro-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 the transformation 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 the 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 from 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).

For instance, in the 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 in its backbone outside the T-DNA region and thus enhances Agrobacterium-mediated gene transfer of recalcitrant plants.

In the ternary vector, a third plasmid—termed 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!

CRISPR picture

At GoldBio, we can provide you with high-quality reagents to perform all related to Agrobacterium-mediated gene transfer.


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