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The following points highlight the top two methods of gene transfer. The methods are: 1. Indirect Method (Agrobacterium Mediated Gene Transfer) 2. Direct Methods of Gene Transfer.
Method # 1. Indirect Method (Agrobacterium Mediated Gene Transfer):
The transformation of a plant can be carried out directly by using Agrobacterium spp. which is a common bacterium causing crown gall tumour in legumes. This bacterium carries a plasmid with T-DNA which is capable of being integrated into the host chromosome.
If a foreign gene is introduced in the plasmid of the bacteria and a plant tissue or cell suspension is grown in culture along with the bacteria, then ultimately the foreign gene can be transferred into the nucleus of the plant or more precisely, in the functional position of the host genome (Figs. 24.2 and 24.3). The two enzymes, restriction enzyme and ligase, play the most significant role in the process of transgenesis.
The Ti plasmids of A. tumefaciens have the several features for use as vector:
(i) They contain one or more T-DNA regions.
(ii) The plasmid contains a vir region.
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(iii) The plasmid has its own origin of replication.
(iv) They contain a con region enabling conjugative transfer.
(v) They have the genes for catabolism of opines (a class of amino acids/sugar conjugates).
A. Vector Strategy:
Vector strategies used in Agrobacterium mediated gene transfer to deliver the transgene into the genome of the plant are co-integrative vector strategy and binary vector strategy.
(a) Co-integrative Vector Strategy:
This is a Ti based plasmid vector developed by homologous DNA recombination between engineered plasmid (containing gene to be transferred along with selectable markers) of E. coli and resident Ti plasmid (disarmed) of A. tumefaciens. This results in integration of the entire engineered plasmid into T-DNA, e.g., pGV 3850::1103.
(b) Binary Vector Strategy:
It is a two-plasmid system in Agrobacterium tumefaciens in which one plasmid contains the virulence gene (helper plasmid) and another plasmid contains T-DNA borders carrying the selectable marker and the foreign DNA to be transferred (binary vector). Binary vector designed to replicate in both E. coli and Agrobacterium and thus can shuttle between both, hence also called shuttle vector, e.g., Helper plasmid (pAL 4404) + Binary vector (p Binl9) (Fig. 24.4).
B. The Mechanism of T-DNA Transfer and Integration:
The transfer of T-DNA and its integration into the plant genome can be divided into the following steps (Fig. 24.5).
(a) Signal Recognition by Agrobacterium:
Agrobacterium perceives signals, such as phenolics (acetosyringone) and sugars released from wounded plant cells which are involved in phytoalexin and lignin biosynthesis, these signals indicate the presence of competent plant cells.
(b) Attachment to Plant Cells:
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Attachment of Agrobacterium to plant cells is a two- step process, involving an initial attachment via a polysaccharide (the product of attR locus) which forms a mesh of cellulose fibres. Several chromosomal virulence (ChrV) genes are also involved in the attachment of bacterial cell to plant cells.
(c) Vir Gene Induction:
Vir A (a membrane linked sensor kinase) senses phenolics like acetosyringone, auto-phosphorylates and activates Vir G. Then Vir G induces expression of all other vir genes. Many sugars like glucose, galactose, xylose all enhance the vir gene induction; it requires also the participation of chromosomal vir gene.
(e) Transfer of T-DNA Out of the Bacterial Cell:
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The T-DNA/Vir D2 complex is exported from the bacterial cell by a T-pilus composed of proteins encoded by vir B operon and vir D4.
C. Basic Steps for Agrobacterium Mediated Transformation:
The steps involved are:
(a) Selection of plant tissue or explant;
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(b) Co-cultivation with Agrobacterium;
(c) Inhibition of Agrobacterium growth;
(d) Selection of transformed tissue;
(e) Regeneration from selected tissue (transgenic plant);
(f) Confirmation of transgenic plant
(a) Selection of Plant Tissue or Explant:
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Suitable plant tissue, to be used as a source of explants (which has good regeneration ability), is removed from the donor plant and sterilized (if the plant is not grown in sterile condition). The explants may be decapitated seedlings, cells, protoplasts or leaf tissue, callus, etc.
(b) Co-Cultivation with Agrobacterium:
The tissue or explant is cut into small pieces and placed into a culture of Agrobacterium (which contains the suitable vector containing foreign gene) for about 30 min., a process known as co-cultivation. During this period, the bacteria attach to the plant tissue, and the excess culture is blotted off and placed on medium for co-cultivation.
(c) Inhibition of Agrobacterium Growth:
The incubation of the explants with Agrobacterium is allowed to continue for 2-3 days to permit the transfer of T-DNA to the plant cells. Then the explants are removed from the medium and washed in an antibiotic solution and further transferred onto antibiotic (bacteriostatic) containing medium to inhibit the growth of Agrobacterium.
(d) Selection of Transformed Plant Cells:
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The explants are then transferred onto the selective media containing proper selective agent to encourage the growth of transformed tissue.
(e) Regeneration from the Transformed Tissue:
The selected tissue part (putatively transformed), grown on selective media, are then transferred onto the regeneration media for shoot regeneration either by organogenesis or by embryogenesis in presence of proper selective agent. The shoot apices come out, those are then transferred in rooting media to get the whole plant.
(f) Confirmation of the Putatively Transformed Plant:
The transgene expression is examined either through foreign protein expression or any phenotypic character expression. The presence of foreign DNA can be examined either through PCR or Dot Blot or Southern Blot experiment. The confirmed transgenic plants then are transferred to soil to get the next generation plant. The whole process has been depicted in Fig. 24.6.
Method # 2. Direct Methods of Gene Transfer:
1. Transfer via Electroporation:
Electric field membrane permeabilization is based on the fact that the electric pulses can open the cell membrane and allow penetration of alien DNA. Heat shock, in combination with electroporation has been used resulting in a higher efficiency of the transformation.
The method requires protoplasts, because electroporation is only efficient on cell membranes. These protoplasts, harbouring the alien DNA, have to be developed into callus and regenerated into a plant.
2. Transfer via Polyethylene Glycol (PEG):
The chemical compound polyethylene glycol (PEG) changes the pore size of the cell membranes which enhances the probability of an alien DNA molecule penetrating into the cell. Heat shock may enhance the uptake of DNA. The method is mainly applied to protoplasts. The transfer of DNA via polyethylene glycol has been used for monocots as well as dicots.
3. Transfer via Biolistics:
The principle of transfer via micro-projectile bombardment (Fig. 24.7A) is to shoot particles coated with DNA into selected tissues or cells by particle gun. The gun may be driven by either air-pressure or electric discharge. The particles may consist of either tungsten or gold carrying the DNA. Any growing plant tissue may be used for this method, but the plant material has to be regenerated via callus formation.
4. Liposome Mediated Gene Transfer:
Liposomes are small lipid bags, in which a large number of plasmids can be enclosed. Those can be fused with the protoplasts using PEG (Fig. 24.7B).
This technique offers many advantages:
(i) Protection of DNA/RNA from nuclease action;
(ii) Low cell toxicity;
(iii) Stability and storage of nucleic acids due to encapsulation in liposomes;
(iv) High degree of reproducibility; and
(v) Applicability to a wide range of cell types.
5. Transfer via Microinjection:
The method involves transfer of a very small solution of DNA into a selected cell by injection with a capillary tube or a micropipette (Fig. 24.7C). The procedure has to be performed under a microscope. Often the specially designed micromanipulator is used for microinjecting the DNA.
6. Transformation using Pollen or Pollen Tube:
There lies a possibility of transferring DNA material with the pollen tube germination method, i.e., the pollen tube with alien DNA can fertilize the egg cell giving rise to transgenic plant.
7. Silicon Carbide Whiskers:
The use of silicon carbide whiskers in transforming plant cells is very simple and reproducible. These whiskers are very cheap and can penetrate cells making pores for DNA uptake.
8. Transformation by Ultra-Sonication:
There are recent reports of transformation of cultured plant tissue when sonicated with alien DNA carrying the marker genes.
9. In Planta Transformation:
In planta transformation technology is carried out in tissues or cells of growing organs of a plant in field conditions bypassing tissue culture technique. This includes seed and embryo incubation, in planta DNA inoculation, floral meristem dipping, Agrobacterium spraying, vacuum infiltration, etc.
