1. Transformation of plant cells
Genetic transformation involves the integration of gene into genome by means other than fusion of gametes or somatic cells. The foreign gene (termed the "transgene") is incorporated into the host plant genome and stably inherited through future generations. This plant transformation approach is being used to generate plant processing trails, unachievable by conventional plant breeding, especially in case where there is no source of the desired trait in the gene pool.
In the gene of interest, the correct regulatory sequences are incorporated i.e. promoters and terminators, and then the DNA is transferred to the plant cell
or tissue
using a suitable vector. The gene
of interest
is attached to a selectable marker which allows selection for the presence of the transgene. Confirmation
for the presence of inserted
genes
is generally tested
by resistance to a specific antibiotic
present in the medium. Once the plant tissue has been transformed, the cells containing the transgene are selected and regeneration back into whole plants is carried out. This is possible as plant cells are totipotent, which means that they contain all the genetic sequence to control the development of that cell into a normal plant. Therefore, the gene
of interest
is present in every single plant cell; however, where its
expression
is controlled by the promoter. Plant transformation can be carried out by various ways depending on the species of the plant. A major method of DNA transfer in plants is Agrobacterium mediated transformation. Agrobacterium is a natural living soil bacteria and is capable of infecting a wide range of plant species, causing crown gall diseases. It has natural transformation abilities. When A. tumefaciens infects a plant cell, it transfers a copy of its T-DNA, which is a small section of DNA carried on its Ti (Tumour inducing) plasmid. This T-DNA is flanked by two (imperfect) 25 base pair repeats. Any DNA contained within these borders will be transferred to the host cell
when used as transformation vector.
2. Different types of plant transformation vectors
Plant transformation vectors comprises of plasmids that have been purposely designed to facilitate the generation of genetically modified plants. The most commonly applicable plant transformation vectors are binary vectors which have the ability to replicate in E. coli, a common lab bacterium, as well as in Agrobacterium tumefaciens, bacterium used to insert the recombinant (customized) DNA into plants. Plant transformation vectors contain three essential elements:
• Plasmids selection (creating a custom circular strand of DNA)
• Plasmids replication (so that it can be easily worked with T-DNA)
• T-DNA region (inserting the DNA into the Agrobacterium )
2.1. Co-integrate pTi vector
The discovery that the vir genes do not need to be in the same plasmid with a T-DNA region to lead its transfer and insertion into the plant genome led to the construction of a system for plant transformation where the T-DNA region and the vir region are on separate plasmids. A co-integrative vector produced by integration of recombinant intermediate vector (IV containing the DNA inserts) in to a disarmed pTi. Transformed gene is initially cloned in E. coli for easy in cloning procedure. A suitably modified E. coli plasmid is used to initiate cloning of gene (Figure 23.1). The subsequent gene transfer in to plants is obtained by co-integrative vectors. Co-integration of the two plasmids is achieved with in Agrobacterium by homologous recombination.
Figure 23.1: Diagrammatic representation of homologous recombination between disarmed pTi and recombinant IV (intermediate vector) containing the desired DNA insert to produce a cointegrative vector. (LB & RB – left and right borders of T-DNA; neo- neomycin phosphotransferase; kan r - Kanamycin resistance; ampr- ampicillin resistance).