The following points highlight the top four methods of foreign gene transfer. The methods are: 1. Plasmid Method 2. Particle Bombardment Method 3. Direct DNA Transfer Method 4. By Micro-Injection Method.
1. Plasmid Method:
Plasmids are extra chromosomal elements which are found in the bacterial cell. They replicate independently of chromosomal DNA and are not essential for normal growth and function of a bacterium. Plasmids are used as cloning vectors in the genetic engineering. The soil-borne bacterium Agrobacterium tumefaciens is used for development of transgenic plants.
The Agrobacterium mediated genetic transformation consists of four main steps given below:
(a) Gene Cloning:
A technique of genetic engineering which is used to make several identical copies of a gene is called gene cloning. Plasmids of Agrobacterium contain tumour producing genes (Ti genes or oncogenes). The foreign gene which has to be used for genetic transformation is incorporated in place of oncogene for cloning. Thus several identical copies of the transgene are produced by self-replication of plasmids.
(b) Genetic Transformation:
The process of transfer, integration and expression of transgene (foreign gene) in the host cells is known as genetic transformation. For genetic transformation, bacteria with cloned gene are mixed (in millions) in the cell culture or protoplast culture of host plant for a day or two.
Out of several thousands of cells or protoplasts in the culture medium, only a few are transformed. In other works, plasmid DNA (foreign DNA) can enter, integrate and express in a few cells or protoplasts.
(c) Identification of Transformed Cells:
The third important step is identification of transformed cells. A marker gene is included with gene under consideration for easy identification of transformed cells. Generally, either an antibiotic resistant gene, such as kanamycin resistant gene, or a herbicide resistant gene is used for identification of transformed cells.
The plasmid (bacteria) mixed suspension culture or protoplast culture is grown in the culture medium containing lethal concentration of antibiotic (kanamycin) or herbicide. The non-transformed cells will die and only transformed cells or protoplasts will survive. In this way, transformed cells can be easily identified.
(d) Regeneration of Transformed Cells:
The final step is regeneration of transformed cells or protoplasts into whole plant. The transformed cells are transferred into culture medium for regeneration into whole plant. The regeneration of cell or protoplast into whole plant is a basic requirement of Agrobacterium mediated gene transfer system.
Agrobacterium tumefaciens has limited range of host. It can invest about 60% of Gymnosperms and dicotyledonous Agro-sperms. Hence, Agrobacterium mediated method of DNA transfer has been used in several dicotyledonous crop plants for genetic transformation.
This method has been successfully used for the development of transgenic plants in various crops such as cotton, potato, rapeseed, soybean, sugarbeet, sunflower, tobacco, tomato, pea, muskmelon, flax, cauliflower, carrot, bean, alfalfa, Brassica napus, lettuce, petunia, poplar, spinach, strawberry, apple, walnut, cucumber and other dicots.
In monocots, it has been successful in few species only such as asparagus and yam.
Agrobacterium mediated genetic transformation has three main limitations, viz:
(i) Host specificity,
(ii) Somaclonal variation, and
(iii) Slow regeneration.
These are discussed below:
i. Host Specificity:
Agrobacterium has limited host range. It cannot infest monocotyledonous crops, especially cereals. In dicots also Agrobacterium exhibits severe host specificity. This restricts its use as a transformation vector to few species only, many of them may be of limited commercial importance. Thus Agrobacterium mediated method of DNA transfer cannot be used for development of transgenic plants in many crop plants.
ii. Somaclonal Variation:
The variation which is induced in the tissue culture is called somaclonal variation. Tissue culture leads to induction of somaclonal variation. Lot of variability is generated in cell and protoplast cultures due to structural chromosomal changes. Somaclonal variation poses problems in the identification of transformed cells.
iii. Slow Regeneration:
The third problem of Agrobacterium mediated DNA transfer is slow regeneration of cell or protoplasts into whole plants. Regeneration of cells or protoplasts into whole plant takes more time than regeneration from meristems and organ cultures.
There are two main advantages of Agrobacterium mediated DNA transfer method. Firstly, this method has some control over the copy number and site of integration of transgenes which is not possible in particle bombardment method. Secondly, this is a cheaper method of genetic transformation than particle bombardment method.
2. Particle Bombardment Method:
In this method, foreign DNA is delivered into plant cells through high velocity metal particles. Particle bombardment method is also known as biolistic micro projectile bombardment, particle acceleration and micro ballistics.
Main features of this method of DNA delivery are given below:
(a) Plant Material to be used:
In this method, regenerable tissues or organs are required for bombardment. Generally, meristems, immature embryos, embryo-genic callus and suspension culture have been used for DNA delivery in different crop plants. Isolation of protoplasts is not essential in this method.
(b) Metal Particles used for Bombardment:
Generally, gold or tungsten particles are used for bombardment. Gold particles, being dense, can penetrate into deeper cell layers than tungsten particles. Other suitable metal particles include palladium, rhodium, platinum, irridium etc. The metal should be such that it should not react with plant tissues.
(c) Technique of Delivering DNA:
The bombardment is done with the help of particle gun.
The particle gun is of two types viz:
(i) Gun powder driven device, and
(ii) Helium particle inflow gun.
The gun powder device is more cumbersome as it requires more DNA loaded particles than helium particle inflow gun. Hence, the helium particle inflow gun is in more common use than gun powder device. Minute particles (1.0-1.5 pm) of tungsten or gold are coated with the DNA of interest. The primary cloned form of DNA i.e. plasmid construction is used for coating with gold or tungsten particles.
The DNA coated particles are accelerated with such force that they should penetrate the outer cell walls of target tissues. Some of the DNA of these particles enters in the nuclei of target cells and is integrated with the DNA of the host cell resulting in transformation.
(d) Identification and Regeneration of Transformed Tissues:
The transformed tissues are identified by polymerase chain reaction (PCR) technique. Out of several tissues, a few are transformed. These transformed tissues are regenerated in culture medium into whole plant, which are further examined for stability of gene expression.
Particle acceleration method has been used for development of transgenic plants in several crops such as wheat, barley, oat, rice, corn, sugarcane, cotton peanut, soybean, common bean, papaya, sunflower, tobacco, poplar and many other crops.
In particle bombardment methods, various plant parts such as meristems, immature embryos, embroygenic callus, suspension culture, hypocotyls are used as explants for regeneration of transgenic plants in different crops (Table 32.1).
However, meristems and immature embroys are more commonly used as explants in this method. All above explants are bombarded with gold or tungsten particles to recover transgenic plants.
Particle bombardment method has some advantages over the Agrobacterium mediated method of DNA transfer.
Advantages of this method are given below:
1. No Host Specificity:
This method does not exhibit host specificity. Hence, it can be effectively used for the development of transgenic plants in various plant species. Thus, this method permits development of transgenic plants in monocots as well as dicot plants.
2. Simple Method:
Particle bombardment method is technically simpler than agrobacterium mediated DNA transfer method. In this method, there is no need of isolating protoplasts. The various plant organs such as meristems, embryos or leaves can be bombarded and transformants can be regenerated from them.
This method has two main disadvantages. The main drawback of this method is that there is no control over the copy number and site of integration of foreign gene. Secondly, the high cost of equipment’s prohibits use of this method by many researchers for DNA transfer.
3. Direct DNA Transfer Method:
Direct DNA transfer involves electroporation or chemical fusagens such as polyethyl glycol (PEG) with calcium phosphate. This method is used with protoplast. A suspension of protoplast with desired DNA is prepared. Then a high voltage current is applied through the protoplast-DNA suspension.
The electric current leads to formation of small temporary holes in the membrane of the protoplasts through which the DNA can pass. After entry into the cell, the foreign DNA gets incorporated with the host genome, resulting in genetic transformation.
This method can be used only in those crop species in which regeneration from protoplasts is possible. Rice and corn were first transformed and regenerated into whole plant by electroporation method.
4. By Micro-Injection Method:
The plasmid DNA can also be delivered into host cells by mechanical means i.e. microscopic needles also called micro-injections. This method does not have host range limitations. This method can be effectively used with different crop plants.
However, in this method also regeneration from protoplasts is a basic requirement. Unfortunately, all crops do not regenerate from protoplasts. Hence, this method also has limited applications.
Direct DNA transfer method does not require specialized equipment and is, therefore, relatively inexpensive. However, it is more tedious than ballistic approach, as it requires efficient protoplast isolation and culture procedures which restrict its application.
Currently two DNA delivery systems, viz:
(i) Agrobacterium mediated gene transfer, and
(ii) Bombardment of cells with plasmid DNA coated particles are widely used for development of transgenic (genetically engineered) individuals. Electroporation and microinjection methods are rarely used for obtaining transgenic plants. A brief comparison of three methods of foreign gene transfer is presented in Table 32.2.