In this article we will discuss about the Gene Transfer Techniques used in Plants:- 1. Introduction to Gene Transfer Techniques 2. Vectors of Gene Transfer.
Introduction to Gene Transfer Techniques:
The aim of plant genetic engineering is to transfer a foreign gene to a recipient species to alter the genome of the recipient and to give a new characteristic of the foreign gene. The plant with the introduced foreign gene is termed as ‘Trapsaenic’ and the process is termed as ‘Trartsgenesis’. The crop thus genetically modified is called ‘GM’ (genetically modified) crop.
In case higher plants, cells or protoplasts can be cultured and used for regeneration of whole plants. The alien gene of interest can be delivered in different systems to obtain the transgenic plants. Besides cultured cells, protoplasts, meristem cells, pollen or zygotes can also be used. So for gene transfer in plants it should be decided first which will be target cell/tissue for gene transfer experiment.
Vectors of Gene Transfer:
The common features of vectors used for transformation of eukaryotic cells include:
(i) Multiple cloning sites (unique restriction site),
(ii) Bacterial origin of replication, and
(iii) Selectable marker gene which will allow to select the proper transformed cell (Fig. 18.19).
Ti and Ri Plasmid:
The potential use of the Agrobacterium Ti plasmid (A. tumefaciens) and Ri plasmid (A. rhizogenes) as vectors arises from the ability of the bacterium to somehow transfer and stably integrate a piece of the plasmid DNA into the plant nuclear genome. The transferred DNA is known as T-DNA and carries several genes which are expressed within the plants and becomes integrated into the plant chromosomal DNA.
This integration seems to depend on the presence of two repeated sequences located at either end of T-DNA. Other genes on the Ti plasmid include those for attachment of the bacterium to plant ceil walls for transfer of T-DNA into the plant cell and for the uptake and catabolism of the appropriate opine.
The non- integrating region of the Ti plasmid which is essential for transfer and integration of the T-DNA is the vir region, which is located near to the T-DNA.
Structure of Ti plasmid: Most Ti plasmids have four regions in common (Fig. 18.19):
Region A (T-DNA):
Responsible for tumour induction; comprising of T-DNA, flanked by 25 bp repeat sequence (left and right borders).
T-DNA consists of:
(i) One region of three genes, representing ‘shooty locus’ and ‘rooty locus’, responsible for auxin and cytokinin synthesis.
(ii) Os region, responsible for synthesis of opines (octopine and nopaline).
Region B (ori):
Responsible for origin of replication.
Region C (con):
Responsible for conjugation.
Region D (vir):
Responsible for virulence, also called vir region, comprising of six operons (A, B, C, D, E and G).
Plant Viruses as Vector:
There are two groups of plant viruses which contain DNA — the cauliflower mosaic viruses which have double stranded DNA and the Gemini viruses which have single stranded DNA. The Cauliflower Mosaic Virus (CaMV) is often cited as the most likely potential vector for introducing foreign genes into plants.
CaMV has one useful feature is that the naked DNA being infective, is able to enter plant cells; inside the cells, the DNA is replicated and encapsidated within virus particles, which then invade the rest of the plant. As the CaMV DNA does not become integrated into the chromosomal DNA, so it is uncertain that it goes to all cells.