Methods to transfer genes in plants

Use of Ti plasmids of Agrobacterium for gene transfer

The transgenic plants were created using the bacterium Agrobacterium tumefaciens which causes crown gall disease and carries tumour-inducing (Ti) plasmids. Genetic engineers have exploited the discovery that any piece of foreign DNA, inserted between the left and right borders of the plasmid’s T- DNA region is transferred to one of the plant’s chromosomes, where it also becomes integrated. This natural system used for transferring DNA into plants was further improved by deleting the genes that made cells to produce more hormones, followed by adding gene for antibiotic resistance, and then attaching “sticky ends” for the insertion of foreign DNA.

The method involves the following steps:

a) Insertion of the desired foreign gene into a Ti plasmid.
b) Then the naked plant cells or protoplasts are placed into a Petri dish and covered by a nutrient solution.
c) Addition of Agrobacterium tumefaciens containing genetically engineered plasmids.
d) Incubation of all the contents for several days at 25-30oC.
e) Plating of cells on nutrient agar with an appropriate antibiotic.
f) Only the plant cells that have taken up the gene for antibiotic-resistance with it’s foreign DNA will grow on this medium.
g) Harvesting the living cells after some growth period.
h) Each cell is then grown to produce a complete plant by cultivation on nutrient media by using different plant hormones.

Agrobacterium infection method has been extensively used to transfer foreign DNA into a number of dicotlyledonous species with the exception of soybean (Glycine max). However, this technique of gene transfer was not successful in monocotyledons for reasons yet unknown most probable being the lack of wound response of monocotyledonous cells.

Protoplast fusion

Protoplast fusion is an additional technique for inducing variation in plant crops. By fusing protoplasts from different strains of species, it is possible to transfer genes from one strain to another. The protoplasts are prepared by immersing sterilized plant material in a solution of the enzyme cellulose, either from fungi or the alimentary canal of snails. One can use ethylene glycol or apply electric field to fuse the protoplasts of different species or closely related species. The technique of protoplast fusion was used in transferring genes for resistance to late blight fungus from one variety of potato to others.
However there are limitations in using this technique especially for transferring genes for salt tolerance and disease-resistance from wild rice into cultivated varieties. Research is still going on to make this technique a reliable tool to use for inducing variations in plants.

Strategies used for Protoplast Fusion

Use of Polyethylene glycol (PEG) for DNA uptake

Direct DNA uptake by protoplasts can be stimulated by chemicals like polyethylene glycol (PEG). PEG is also used to stimulate the uptake of liposomes and to improve the efficiency of electroporation. PEG at high concentration (15-25%) precipitates ionic macromolecules such as DNA and stimulate their uptake by endocytosis without any gross damage to protoplasts.

This method has been successfully used in Petunia, Nicotiana, rice, maize etc. However there are problems related to plant regeneration from protoplasts subjected to this treatment for gene transfer.

Use of chemicals like Colchicine to introduce genetic variation in plants

The flowering plants are diploid (2n) and sexual reproduction involves fertilization of a haploid egg or ovum (n) by a haploid male gamete (n), formed in a germinating pollen grain. Haploid plants (n) can be formed by growing pollen grains or anthers on solid or liquid cultures with appropriate nutrients and growth hormones. By using Colchicine, a mutagen which causes chromosomes numbers to double during cell division, polyploidy plants are obtained with entirely new homozygous features and traits. The advantage of this technique is that it takes less than half of the time taken by conventional cross-breeding to produce pure-breeding lines.

Use of Liposomes for gene transfer

Liposomes are small lipid bags, in which large number of  plasmids are enclosed. They can be infused with protoplasts using devices like PEG, and therefore have been used for gene transfer especially in plant species like tobacco, petunia, carrot etc. The method involves the following steps:

1. Adhesion of the liposomes to the protoplast surface.
2. Fusion of liposomes at the site of adhesion
3. Release of plasmids inside the cell.

 The advantages of using this method are:

1. protection of DNA/RNA from nuclease digestion
2. low cell toxicity
3. stability and storage of nucleic acids due to encapsulation in liposomes
4. applicability to a wide variety and range of cells

Agroinfection

Cereals are important as they are the major food crop for us. Under natural circumstances, Agrobacterium tumefaciens does not attack cereals therefore it cannot be used to modify the genome of these plants. However, it was observed that if the DNA of wheat dwarf virus is inserted into a Ti plasmid, the bacteria carrying Ti plasmids will attack wounded wheat plants. Similarly, bacteria carrying Ti plasmids with DNA from maize streak virus will attack wounded maize plants. This technique is called agroinfection which was first used in 1987. In this mature cereal plants are infected with plasmid-carrying bacteria. Transformed cells develop symptoms of the viral disease, and do not need to be identified by selection. The infection spreads from cell to cell until all the cells of the cereal plant have been transformed. Efforts are being made to use this technique to introduce foreign DNA into cereals.

Electroporation

This is a technique which relies on direct uptake of DNA for gene transfer with out the use of bacterial vector. In this technique of electroporation, after mixing the protoplasts with foreign DNA, an electric shock is given. This electric pulse causes pores in the cell membranes to open up which increases the amount of exogenous DNA that enters the cell. Once the current is switched off the pores reseal. A small amount of the foreign DNA becomes incorporated into the chromosomes which causes some of the protoplasts to undergo transformation. This method has been successful in maize and rice protoplasts. Instead of electric pulse, ethylene glycol can also be used to make the membrane more permeable to exogenous DNA.

Macro- and micro-injection of foreign DNA

Results obtained from the direct microinjection of foreign DNA into young embryos have been mixed with limited success. The method of macroinjection involves the use of a syringe to inject foreign DNA into the space around the young inflorescence. Success for this approach has been claimed in the transfer of a gene for resistance to the antibiotic kanamycin.

Bombardment of intact plant cells with DNA coated spheres of tungsten or gold.

This method involves the bombardment of intact plants with very small (1-4um in diameter) DNA-coated spheres of tungsten or gold. These micro-projectiles are shot from a macro-projectile, resembling a bullet with an open tip, which is itself held by a stopping plate. The propulsive force gives the micro-projectiles sufficient acceleration. This force comes from either a shotgun ‘explosion’ or an electrical discharge. This method has been successful in transforming soyabeans.

All these methods are being used to give crop plants better protection against pests and parasites and also making them resistant to harsh environmental conditions.