Plant Genetic Transformation Methods

Genetic transformation is a natural process in which a gene is transferred from one organism to another, resulting in biological trait diversity. Plant genetic transformation has become a popular strategy for transforming a gene of interest into a specific plant and obtaining the desired expression.

Plant Genetic Transformation is a way of integrating genes into the nuclear genome of a plant in order to achieve the desired expression. A selectable gene marker is necessary to transfer a gene from one plant to another and promoters are required for successful implementation of gene transfer methods for crop improvement. So a promoter is a non-coding DNA region that exists upstream of a gene’s coding sequence and is essential for that gene’s transcription. Moreover, they play a vital role in the process of plant gene expression and regulation.

Significance of Plant Genetic Transformation

Through PGT, one can find a gene and its function, comprehend features of interest, and favor breeding programmes by developing innovative and genetically diverse plant materials. Moreover, it is also important to increase the stress tolerance of a plant and the nutrient production.

So, in order to transform a gene in a plant step-by-step wet lab activities are carried out in a plant to introduce the foreign DNA (exogenous DNA) and evaluate its integration. So, PGT includes delivery of DNA into a single cell and the regeneration of complete fertile plants.

PGT Methods

On the basis of plant species, there are two methods that are followed for plant genetic transformation. They are Physical Methods and Biological Methods.

Physical Methods

The adjustment of many physical factors is required for gene transfer to a specific cell or tissue type. Essentially, these characteristics influence the particle’s effective momentum, the number of particles that strike the tissue, and the amount of usable DNA carried into the cells by the particles. So, the physical methods that can be used for gene transformation in plants are as followed-

1. Electroporation- The electric field creates holes in the plasma membrane, allowing the cell to absorb DNA. This approach has a significant cell death rate (25-50 percent survival). This technique can be applied to a wide range of species and tissue types, but it has the disadvantage of requiring a well-established protoplast regeneration system in the plant species being modified with foreign DNA.
2. Biolistic Gene Gun Technique-The term “Particle Bombardment” refers to a biolistic technique of gene transformation in plants. For the first time, in the 1980s, the biolistic approach of transformation was created and effectively used to plant cells for the first time. It entails injecting a fragment of DNA into the plant tissue that has to be transformed. The leaf tissues are plated onto a selective medium before being bombarded with 0.5 mm gold or tungsten DNA-coated projectiles using compressed helium. The plates are then incubated at 30°C to allow the transformants to grow. A plate confluent with transformants is usually the result of a single bombardment. The particles pass through the plant cell wall, and when they are given the right conditions, the majority of them can enter the nucleus. This approach is applicable to both monocotyledonous and dicotyledonous plant species. Examples are wheat and barley.
3. Laser Induced delivery of DNA- LASERs create transitory holes in the cell membrane through which DNA can enter the cytoplasm. The advantage is that the transformed cereal crops are widely cultivated however it can lead to a high rate of gene silencing.

Biological Methods

When using the gene gun for steady transformation, it is essential that a proportion must be maintained between delivery of DNA and reduction in cell damage.  Incubating the target tissue in medium with a reasonably high osmotic pressure is one way to improve cell survival. This method was first used in yeast, and it was shown to increase the rate of gene transformation in plants.

Agrobacterium tumefaciens- The use of Agrobacterium tumefaciens to optimize gene transformation has proven pivotal in the development of transgenic plants. Agrobacterium tumefaciens is a Gram-negative soil pathogen that infects wound sites in many dicotyledonous plant species spontaneously. Under normal circumstances, the infection leads to the development of crown gall tumor. Along with virulence proteins coding DNA, the bacteria transmits a segment of DNA known as transfer DNA or mobile DNA segment (T-DNA) into the nucleus of infected cells. After that, it is successfully incorporated into the host DNA.

Particle Bombardment- The desired gene is covered in a gold or tungsten particle and bombarded into the cells of the plant, as the name implies. The sequence is absorbed into the cells of the plant after being bombarded, and tissue culture techniques can be used to multiply them. In this method special equipment is required and the gene rearrangement is a high risk. Moreover, it can be used on a variety of plant tissues.

Microinjection- Microinjection is the process of manually injecting transgene at a microscopic or borderline macroscopic level using a fine glass micropipette. The transgene can be circular or linear and does not need to be physically attached for injection if it is in the shape of plasmids, phage, YAC products.

Following that, the DNA is mechanically injected into the nucleus or cytoplasm using a glass microcapillary shot pipette. The protoplasts are then trapped in low melting agar using a holding pipette and suction force while operating under a microscope. Following that, DNA is injected directly into the cytoplasm or nucleus and then the implanted cells are developed into plants in vitro. Rapeseed, tobacco, and a variety of other plants have all demonstrated to be successful in this method.

Chemical Method

Protoplast Transformation

Polyethylene glycol is used to transmit DNA directly to individual plant cells during protoplast transformation. It has several advantages, including delivery of several plasmids with high degrees of cotransformation, no binary vector is required, it is a high frequency transformation, and moreover protoplast isolation and transformation are possible in most plant species

Conclusion

The genetic transformation of cultured cells and tissues has resulted in the synthesis of a significant number of transgenic lines with agronomical useful genes and in the production of plants. Hundreds of transgenic plants have been converted with genes expressing antifungal proteins and are ready for field testing. However, it is important to realise that molecular technologies are not substitutes for traditional hybridization-based breeding, but they rather complement it.