Direct Gene Transfer Methods and Types | Biotechnology

In this article, we will discuss about:- 1. What is Direct Gene Transfer? 2. Types of direct Gene transfer 3. Physical gene transfer methods and 4. Chemical gene transfer methods

1. What is Direct Gene Transfer?

The term direct or vector less transfer of DNA is used when the foreign DNA is directly introduced into the plant genome. Direct DNA transfer methods rely on the delivery of naked DNA into the plant cells. This is in contrast to the Agrobacterium or vector-mediated DNA transfer which may be regarded as indirect methods. Majority of the direct DNA transfer methods are simple and effective. And in fact, several transgenic plants have been developed by this approach.

2. Types of direct Gene transfer:

The direct Gene transfer can be broadly divided into three categories.

(1) Physical gene transfer methods: electroportion, particle bombardment, microinjection, liposome fusion, silicon carbide fibres.

(2) Chemical gene transfer methods: Poly­ethylene glycol (PEG)-mediated, diethyl amino ethyl (DEAE) dextran-mediated, calcium phosphate precipitation.

(3) DNA imbibition by cells/tissues/organs

(1) Physical Gene Transfer Methods:

An overview of the general scheme for the production of transgenic plants by employing physical transfer methods is depicted in Fig. 49.9. Some details of the different techniques are described.

i. Electroporation:

Electroporation basically involves the use of high field strength electrical impulses to reversibly permeabilize the cell membranes for the uptake of DNA. This technique can be used for the delivery of DNA into intact plant cells and protoplasts.

The plant material is incubated in a buffer solution containing the desired foreign/target DNA, and subjected to high voltage electrical impulses. This results in the formation of pores in the plasma membrane through which DNA enters and gets integrated into the host cell genome.

In the early years, only protoplasts were used for gene transfer by electroporation. Now a days, intact cells, callus cultures and immature embryos can be used with suitable pre- and post-electroporation treatments. Electroporation has been successfully used for the production of transgenic plants of many cereals e.g. rice, wheat, maize.

ii. Particle Bombardment (Biolistics):

Particle (or micro projectile) bombardment is the most effective method for gene transfer, and creation of transgenic plants. This method is versatile due to the fact that it can be successfully used for the DNA transfer in mammalian cells and microorganisms.

The micro projectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Biolistics is a combination of biological and ballistics. There are other names for this technique- particle gun, gene gun, bio blaster. A diagrammatic representation of micro projectile bombardment system for the transfer of genes in plants is depicted in Fig. 49.10, and briefly described below.

Micro carriers (micro projectiles), the tungsten or gold particles coated with DNA, are carried by macro carriers (macro projectiles). These macro-carriers are inserted into the apparatus and pushed downward by rupturing the disc.

The stopping plate does not permit the movement of macro carrier while the micro carriers (with DNA) are propelled at a high speed into the plant material. Here the DNA segments are released which enter the plant cells and integrate with the genome.

iii. Microinjection:

Microinjection is a direct physical method involving the mechanical insertion of the desirable DNA into a target cell. The target cell may be the one identified from intact cells, protoplasts, callus, embryos, meristems etc. Microinjection is used for the transfer of cellular organelles and for the manipulation of chromosomes.

The technique of microinjection involves the transfer of the gene through a micropipette (0.5-10.0 pm tip) into the cytoplasm/nucleus of a plant cell or protoplast. While the gene transfer is done, the recipient cells are kept immobilized in agarose embedding, and held by a suction holding pipette (Fig. 49.11).

As the process of microinjection is complete, the transformed cell is cultured and grown to develop into a transgenic plant. In fact, transgenic tobacco and Brassica napus have been developed by this approach. The major limitations of microinjection are that it is slow, expensive, and has to be performed by trained and skilled personnel.

iv. Liposome-Mediated Transformation:

Liposomes are artificially created lipid vesicles containing a phospholipid membrane. They are successfully used in mammalian cells for the delivery of proteins, drugs etc. Liposomes carrying genes can be employed to fuse with protoplasts and transfer the genes.

The efficiency of transformation increases when the process is carried out in conjunction with polyethylene glycol (PEG). Liposome-mediated transformation involves adhesion of liposomes to the protoplast surface, its fusion at the site of attachment and release of plasmids inside the cell (Fig. 49.12).

v. Silicon Carbide Fibre-Mediated Transformation:

The silicon carbide fibres (SCF) are about 0.3-0.6 pm in diameter and 10-100 pm in length. These fibres are capable of penetrating the cell wall and plasma membrane, and thus can deliver DNA into the cells. The DNA coated silicon carbide fibres are vortexed with ‘plant material (suspension culture, calluses). During the mixing, DNA adhering to the fibres enters the cells and gets stably integrated with the host genome. The silicon carbide fibres with the trade name Whiskers are available in the market.

(2) Chemical Gene Transfer Methods:

i. Polyethylene glycol (PEG)-mediated transfer:

Polyethylene glycol (PEG), in the presence of divalent cations (using Ca2+), destabilizes the plasma membrane of protoplasts and renders it permeable to naked DNA. In this way, the DNA enters nucleus of the protoplasts and gets integrated with the genome.

The procedure involves the isolation of protoplasts and their suspension, addition of plasmid DNA, followed by a slow addition of 40% PEG-4000 (w/v) dissolved in mannitol and calcium nitrate solution. As this mixture is incubated, protoplasts get transformed.

ii. Deae Dextran-Mediated transfer:

The desirable DNA can be complexed with a high molecular weight polymer diethyl amino ethyl (DEAE) dextran and transferred. The major limitation of this approach is that it does not yield stable trans-formants.

Calcium Phosphate Co- Precipitation-Mediated Transfer:

The DNA is allowed to mix with calcium chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate. When the actively dividing cells in culture are exposed to this precipitate for several hours, the cells get transformed. The success of this method is dependent on the high concentration of DNA and the protection of the complex precipitate. Addition of dimethyl sulfoxide (DMSO) increases the efficiency of transformation.

(3) Dna Imbibition By Cells/Tissues:

Some workers have seriously tried to transform cells by incubating cell suspensions, tissues, embryos and even seeds with DNA. The belief is that the DNA gets imbibed, and the cells get transformed. DNA imbibition approach has met with little or no success.