Gene transfer methods in plants

Gene transfer methods in plants make use of a variety of target cell types, which include protoplasts or cultured cells, raeristem cells from immature embryos, shoots or flowers and pollen or zygotes. The methods can be broadly classified into two groups : (i) Agrobacterium Ti or Ri plasmid mediated gene transfer and (ii) physical delivery or DNA mediated gene transfer (DMGT).

Transfer of genes using Agrobacterium Ti and Ri plasmids. Ti plasmid of Agrobacterium tumefaciens (causing 'crown gall' disease; Fig. 42.4) and Ri plasmid of A. rhizogenes (causing 'hairy root' disease) have been effectively used as vectors for gene transfer to plant cells. The Ti plasmid (and also Ri Plasmid) has a region called vir region (having six operons vir A, B, C, D, E, G)responsible for virulence towards host, and a T-DNA region, which is transferred to the host, along with the gene intended to be transferred.

Vectors based on Ti and Ri plasmids have been designed with the following properties : (i) they do not cause the disease, since they are disarmed by removing genes causing the disease; (ii) they carry sites for insertion of foreign gene intended to be transferred and (iii) they carry selectable markers (genes which will help in selecting the transformed cells), a powerful promoter like 35S (for high level of expression of marker) derived from cauliflower mosaic virus (CaMV), and a polyadenylation signal (for adding poly A to mRNA). The most commonly used selectable marker is neomycin phosphotransferase (npt II)imparting resistance to kanamycin, so that the transformed cells will grow in a medium supplemented with kanamycin. A vector, so designed, after insertion of the foreign gene, is transferred to A. tumefaciens or A. rhizogenes, as the case may be, and then the bacterium is used for infecting the host cells to which the gene is to be transferred (Fig. 42.5).

For gene transfer, the explants commonly used include protoplasts, suspension cultured cells, callus cell clumps, thin cell layers (epidermis), tissue slices, leaf discs, and sections of roots, stems or floral tissues. These explants are incubated or cocultivated with Agrobacterium carrying the vector with the desired foreign gene. The transformed cells or colonies are selected due to the selectable marker and used for regeneration of whole plants which are expected to be transgenic in nature. These regenerated plants can again be tested for the transfer of gene, with the help of one of the several scoreable or screenable markers (also called reporter genes), which is also transferred along with the desirable gene. The most commonly used reporter genes are npt II (neomycin phosphotransferase II gene), cat (chloramphenicol acetyltransferase gene), gus (βglucuronidase gene) and lux (luciferase gene).

Transfer of genes using physical delivery methods.
Although Agrobacterium mediated gene transfer was extensively utilized for production of transgenic dicotyledonous plants, it was not successful in monocotyledonous plants, since Agrobacterium does not infect them. Therefore, for cereals and also for some dicots (e.g. legumes), where regeneration was difficult, transgenic plants could not be produced using Agrobacterium. In view of this, DNA mediated gene transfer (DMGT) was successfully tried in a wide range of plants including both monocotyledons and dicotyledons. These DMGT methods include the following :

(i) PEG (polyethylene glycol) stimulated DNA uptake involves the use of 15-25% PEG, which stimulates uptake of DNA by endocytosis without any gross damage to protoplasts. The transformed protoplasts can be selected on a selection medium,

(ii) Microinjection involves injection of DNA, using micropipettes with 0.5-10 μm diameter tip. In this technique, the recipient cells are immobilized on a solid support (cover slip or slide, etc.) or artificially bound or held by a pipette under suction (Fig. 42.1). Specially designed micromanipulators are used for this purpose,

(iii) Macroinjection of DNA, employing needles with diameters greater than cell diameter, has been tried in rye (Secale cereale). DNA was injected into the stem below the immature floral meristem, so as to reach the sporogenous tissue, leading to the production of transgenic rye plants. Unfortunately this technique was not successful when tried with other cereals, making the validity of earlier experiments doubtful,

(iv) Microprojectiles (gold or tungsten particles coated with DNA, 1-3 |im in diameter) are carried by a 'macroprojectile' (or bullet), and are accelerated into target cells, where they penetrate the cell wall, leaving DNA to be incorporated (Fig. 42.6). This technique has been successfully used for transformation in soybean, tobacco, maize, rice, wheat, oats and tall fescue. This method is now believed to be universal in its application and will be extensively utilized in 1990s for gene transfer in plants, irrespective of species and genotype,

(v) Electroporation is also used for DNA uptake into'the protoplasts, but may not be suitable, if the target cells are available in the form of multicellular explants with cell walls,

(iv) Liposome mediated gene transfer involves encasement of DNA in lipid bags, which can be made to fuse with protoplasts with the help of a peg. This technique has been used successfully for gene transfer in a number of plant species.