The following points highlight the four steps of genetic engineering in plants. The steps are: 1. Isolation and Identification of Desired DNA/Genes 2. Cloning and Production of Identical Copies of Isolated DNA Segment 3. Introduction of Cloned DNA into Plant Cells and its Integration with Plant DNA 4. Expression of Introduced Genes in the Plants.
Steps of Genetic Engineering in Plants:
- Isolation and Identification of Desired DNA/Genes
- Cloning and Production of Identical Copies of Isolated DNA Segment
- Introduction of Cloned DNA into Plant Cells and its Integration with Plant DNA
- Expression of Introduced Genes in the Plants
Genetic Engineering: Step # 1. Isolation and Identification of Desired DNA/Gene:
Two types of DNA can be isolated:
(i) Genomic DNA.
(ii) Complementary DNA (cDNA).
(i) Genomic DNA:
Total DNA isolated from the cells is called genomic DNA. It is isolated by breaking the walls of the cells by physical or biochemical methods. Finally the DNA is separated by ultracentrifugation.
(ii) Complementary DNA (cDNA):
In this case first the mRNA is isolated and then DNA is synthesized on mRNA template by the process of reverse transcription. The base sequence of this DNA is complementary to mRNA base sequence. Hence, it is known as complementary or cDNA.
Genetic Engineering: Step # 2. Cloning and Production of Identical Copies of Isolated DNA Segment:
Fragment of DNA (genes) from any source can be multiplied or amplified more than million fold. This is known as gene cloning. It may be genomic DNA cloning or cDNA cloning. In this technique, DNA to be cloned is first inserted into a plasmid (cloning). Plasmids are small circular DNA found in bacterial cells apart from genomic DNA.
Plasmids can replicate independently. During gene cloning the plasmids are first isolated from bacterial cells. They are purified. They are then cut open with restriction endonuclease. The DNA to be cloned is then joined to plasmid DNA by another enzyme known as DNA ligase. The hybrid or chimeric or recombinant plasmid is thus produced. This plasmid is then reintroduced into bacterial cells.
The transformed bacterial cells are then grown on culture medium. As the bacteria divide, the plasmids containing foreign DNA also divide and thus large number of copies of recombinant DNA is produced.
This is referred to as gene cloning and it is one step of genetic engineering. After cloning various genes of a particular species can be stored to form gene/DNA libraries.
Genetic Engineering: Step # 3. Introduction of Cloned DNA into Plant Cell and its Integration with Plant DNA:
Transfer of cloned DNA into plants can be done by use of another vector called gene transfer vector. It is different from cloning vector because cloning vector is used for gene cloning but gene transfer vector is used for gene transfer.
In dicotyledonous plants, the soil borne pathogen, bacterium, Agrobacterium tumefaciens is used as gene transfer vector. The bacterium causes crown gall disease in many species of dicotyledonous plants. The bacterium enters the plants through wound and after infection a callus or tumour is formed near the wounded area at the juncture of root and stem.
This bacterium contains, apart from its genomic DNA, a large plasmid called Ti plasmid (Tumour inducing plasmid). The plasmid contains tumour inducing genes. When the bacterium infects plant cells, a part of the Ti Plasmid with recombinant DN A gets incorporated into DN A of the plant cell. Thus, the foreign DNA becomes part of plant DNA and is called transfer or T DNA (Fig. 24.14).
The desirable donor gene/DNA after cloning is first incorporated into Ti Plasmid of Agrobacterium. This DNA is called transfer DNA, or T DNA or recombinant DNA. The bacterium is then introduced into the plant at cut end. Bacteria enter the plant tissue and a part of its plasmid having T DNA gets integrated into plant DNA.
The cells of callus thus contain foreign DNA from donor species. These cells are known as transformed or transgenic cells. Through tissue culture technique plants can be generated from the callus containing transgenic cells (Fig. 24.15). These plants will now contain additional DNA from donor species. These plants are called transformed plants or genetically modified (GM) plants or transgenic plants.
Genetic Engineering: Step # 4. Expression of Introduced Genes in Plants:
The recombinant DNA works when the recipient cell expresses protein from recombinant genes. The introduced genes should be transcribed, processed and translated in the plant, i.e., the desirable character of donor species should be expressed in the recipient plant species.