Overview

Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically modified organisms are called transgenic organisms.

The first-ever transgenic plant was a tobacco plant developed in 1983 that showed resistance against the tobacco mosaic virus. Since then, many transgenic plants have been developed and commercialized for improving the agricultural, ornamental, and horticultural value of a crop plant. Transgenic plants can also be used as a bioreactor for producing economically important metabolites or proteins.

Physical methods of transferring genes to plants

Many methods, such as electroporation and microprojectile bombardment, can be used to transfer a foreign gene into a plant protoplast. During electroporation, a device delivers high voltage pulses to the protoplast suspension. This increases the permeability of the protoplast allowing the foreign DNA to enter the cell and integrate into the plant genome.

In contrast, during microprojectile bombardment or the biolistic method, spherical particles made of tungsten or gold (microprojectiles) are coated with the plasmid carrying gene of interest. The coated particles are then accelerated to 300 to 600 m/s with an instrument called a particle gun. The instrument uses high-pressure helium gas to provide propelling force that helps the plasmid-coated particles to penetrate the plant cell wall. Once inside the cell, the gene of interest integrates into the plant genome. This method can be used to introduce DNA into callus cultures, meristematic tissues, immature embryos, plant cell suspensions, etc.

Applications of Transgenic plants

The recombinant DNA technology is widely used in developing pest and virus-resistant plants. The technique is also helpful in modifying the lipid, starch, and protein content of the seeds.

For example, golden rice is a genetically modified rice plant with enhanced nutritional value. This transgenic variety of rice produces β-carotene - a precursor of vitamin A, that gives it a golden color. It is produced by Agrobacterium-mediated DNA transformation, where the recombinant DNA carrying three genes encoding enzymes-phytoene synthase, phytoene desaturase, and lycopene β-cyclase is inserted into the genome of targeted rice embryos. The embryos then develop into plants that produce β-carotene. Golden rice was developed to tackle vitamin A deficiency in developing nations.

Some other transgenic plants, such as Bt cotton, have been modified by inserting a gene isolated from the bacteria Bacillus thuringiensis - a gram-positive bacteria found in soil. Such transgenic plants produce crystal or “Cry” protein that kills harmful insects, making the plant insect resistant.

Procedure

Transgenic plants are produced by introducing a foreign gene into a plant’s genome. These plants can be developed for a variety of reasons, including virus or pest resistance, higher yield, or therapeutic protein production.

To produce a transgenic plant, a gene of interest and its promoter are introduced into the plant protoplast, a plant cell without a cell wall, by incubating both in a medium containing polyethylene glycol.

The recombinant protoplast is cultivated on a sterile medium in the presence of growth regulators and nutrients.

The cells of the protoplast grow indefinitely, producing a mass of undifferentiated cells called a callus. Some cells of the callus, called totipotent cells, give rise to new shoots and roots, which can develop into a whole new plant.

Transgenic plants can also be created using the bacteria Agrobacterium tumefaciens, a gram-negative bacterium found in soil. This bacteria is a pathogen that normally causes disease in plants and contains a Ti -plasmid that can integrate into a plant cell genome.

The Ti plasmid has two regions of interest- T-DNA or transfer DNA region which can be inserted into the host cell genome and the virulence region to aid the transfer of T-DNA into the host cell.

This plasmid is modified by inserting a desired gene into the T-DNA region and a gene for antibiotic resistance to enable selective growth of plasmid-containing organisms. The recombinant plasmid is then inserted into the bacteria by applying an electric pulse to increase cell permeability and enable the uptake of the plasmid into the bacteria.

The Agrobacterium tumefaciens carrying recombinant plasmid is grown in a culture medium containing antibiotics.

Plant parts such as the cotyledonary nodes from a tomato plant are cut and incubated in the medium containing bacteria.

The wounded plant cells at the edge of the stem node release compounds that attract the bacteria. The bacteria infects the plant and releases the recombinant plasmid, which then transfers the foreign gene into the plant cell genome.

Only the transformed cells with recombinant DNA and antibiotic-resistant genes survive in the medium containing antibiotics.

The transformed cells now form a callus and develop into a new transgenic plant carrying a transgene.