The following points highlight the six significant techniques of gene transfer by help of agrobacterium tumefaciens. The techniques are: 1. Regeneration of Plants from Protoplasts and Callus Tissues 2. The Ti plasmid of Agrobacterium Tumefaciens 3. Formation of Non-oncogenic (or Disarmed) Ti Plasmids 4. Chimeric Selectable Marker Genes and a few others.
Technique # 1. Regeneration of Plants from Protoplasts and Callus Tissues:
An outstanding feature of plant cells is their totipotency, that is, most plant cells retain the ability to give rise to any differentiated cell type of the mature plant. Many differentiated cells of plants are able to dedifferentiate to the embryonic state and to subsequently redifferentiate to new cell types. Thus, there exists no separation of germ line cells from somatic cells as in higher animals.
When excised tissues from mature plants are placed in the appropriate sterile tissue culture conditions (notably in the presence of the plant hormone 2, 4- dichlorophenoxyacetic acid 2, 4-D), cells in these tissue explants will often dedifferentiate and grow into highly unorganized cell masses called calli (or calluses, singular, callus).
If these undifferentiated callus cell-clumps are subsequently (i.e., within a year) transferred to growth medium favoring differentiation (medium lacking 2, 4-D hormone but containing growth hormones such as kinetin), plantlet will regenerate in case of many, but not all, species.
Moreover, with some plant species, one can regenerate plants from isolated protoplasts (i.e., single “cells” from which the walls have been removed by enzymatic digestion).
Technique # 2. The Ti Plasmid of Agrobacterium Tumefaciens:
Till date most important tool in the genetic engineering of plants has been the Ti plasmid of the soil bacterium Agrobacterium tumefaciens. This is the causative agent of crown gall disease of dicotyledenous plants. The name refers to the “galls” or tumours that often form at the crown (junction between the root and the stem) of infected plants.
Since the crown of the plant is usually located at the soil surface, this is where a plant is most likely to be wounded (e.g., due to a soil abrasion from the plant blowing in a strong wind) and infected by a soil bacterium such as A. tumefaciens. However, A. tumefaciens can infect a plant and induce a tumour at any wound site (e.g., leaf).
During the infection of a wound site by A. tumefaciens, two key events occur:
(1) The plant cells begin to divide and form tumours and
(2) They begin to synthesize an arginine derivative called an opine. The opine synthesized is either nopaline or octopine depending on the Agrobacterium tumefaciens strain involved.
These opines are catabolized and used as energy sources by the infecting bacteria. Interestingly, A. tumefaciens strains that induce the synthesis of nopaline can grow on nopaline, but not on octopine, and vice versa.
Clearly, an interesting interrelationship has evolved between A. tumefaciens strains and their plant hosts. The bacterium is able to divert the metabolic resources of the host plant to the synthesis of opines, which are of no apparent benefit to the plant, but which provide sustenance to the bacterium.
The ability of A. tumefaciens to induce crown gall disease in pants is controlled by genetic information carried on a large plasmid (about 200,000 nucleotide pairs) called the Ti plasmid for its Tumor-inducing capacity.
Two components, the T-DNA and vir region of the Ti plasmid are essential for the transformation of plant cells. During the transformation process, the T-DNA (for Transferred DNA) is excised from the Ti plasmid, transferred to a plant cell and integrated into the DNA of the plant cell.
Integration of the T-DNA occurs at random chromosomal sites; moreover, in some cases, multiple T-DNA integration events occur in the same cell. In nopaline-type Ti plasmids, the T-DNA is a 23,000-nucleotide pair segment that carries 13 known genes. In the octopine-type Ti-plasmid, there are two separate T-DNA segments.
Some of the genes on T-DNA segment of the Ti plasmid encode enzymes that catalyse the synthesis of phytohormones (the auxin indoleacetic acid I and the cytokinin isopentyl adenosine). These phytohormones are responsible for the tumorous growth of cells, in crown gall.
The T-DNA region is bordered by 25- nucleotide pair imperfect repeats, which are required in cis for T-DNA excision and transfer. The deletion of either border sequence completely blocks the transfer of T-DNA to plant cells.
The vir (for virulene) region of the Ti plasmid contains the genes required for the T-DNA transfer process. These genes are known to encode the DNA processing enzymes required for excision, transfer and integration of the T-DNA segment. An important factor in the construction of Ti gene- transfer vector is that vir genes can supply the functions needed for T-DNA transfer when located either cis or Trans to the T-DNA.
The vir genes are normally expressed at very low levels in A. tumefaciens cells growing in the soil. However, exposure of the bacteria to plant cells or exudates from plant cells induces increased levels of expression of the operons containing vir gene.
Surprisingly, this induction process is very slow for bacteria, taking 10 to 15 hours to reach maximum levels of expression. Recently certain phenolic compounds, for example, acetosyringone, have been purified from plant exudates and shown to act as inducers of vir operons.
Thus, the induction process can now be studied in vitro by using these purified inducer molecules. Although many details are yet not clear, the overall process of transformation of plant cells by the Ti plasmid of Agrobacterium tumefaciens is quite clear.
Technique # 3. Formation of Non-Oncogenic (or Disarmed) Ti Plasmids:
Once it had been established that the T-DNA region of the Ti plasmid of Agrobacterium tumefaciens is transferred to plant cells and becomes integrated in plants chromosomes, the potential use of A. tumefaciens in genetic engineering of plants was apparent. One could introduce foreign genes into the T-DNA region and these genes would be transferred to the plant with rest of the T-DNA segment.
In fact, this works very well. All the available data indicate that any DNA inserted anywhere between the T-DNA border sequences is transferred to plant cells and integrates in plant chromosomes with the rest of the T-DNA.
The problem is that the transformed plant cells that have received the T-DNA from a wild-type Ti plasmid contradictory with the goals of most gene-transfer experiments. One usually does not wish to genetically engineer tumor cells, and one certainly cannot study the normal functions of transferred genes in tumor cells.
Fortunately, the solution to this problem came early with the identification of the genes in the T-DNA that were responsible for tumor formation. The deletion of one or more of these genes produces a non-oncogenic or disarmed Ti plasmid. Unfortunately, the deletion of the tumor-causing genes also makes it extremely difficult to identify plant cells that have received the disarmed T-DNA.
With wild-type Ti plasmid, the recipient plant cells form tumors and are easily identified by the tumor phenotype; with disarmed Ti plasmids, the recipient plant cells continue to grow just like their neighbours that do not harbor the T-DNA. For this, marker genes are used.
Technique # 4. Chimeric Selectable Marker Genes:
A good selectable marker gene is one that will provide resistance to a drag, antibiotic, or other agent that arrest the growth of normal plant cells (i.e., plant cells not harbouring the marker gene). The selective agent should inhibit the growth of plant cells or kill them slowly.
Agents that kill cells rapidly often result in the release of phenolic compounds and other substances (from the dead cells) that are toxic to the growth of the remaining, otherwise resistant cells.
To date, three selectable marker genes have been extensively used in plant systems, they provide resistance to the antibiotics (1) kanamycin (and the related aminoglycoside G418-a more effective growth inhibitor in eukaryotes) and (2) hygromycin, and to the drag, (3) methotrexate. Of these, kanamycin and G418 have been, by far the most widely used with plants.
The Kanr gene from the E. coli transposon Tn5 has been extensively used as a selectable marker in plants; it encodes an enzyme called neomycin phosphotransferase type II (NPT II). The NPT II is one of several prokaryotic enzymes that detoxify the Kanamycin family of aminoglycoside antibiotics by phosphorylating them.
Since the promoter sequences and transcription- termination signals are different in bacteria and plants, the native Tn5 Kanr gene cannot be used directly in plants. Instead, the NPT II coding sequence must be provided with a plant promoter (5′ to the coding sequence) and plant termination and polyadenylation signals (3′ to the coding sequence).
Such constructions with prokaryotic coding sequence flanked by eukaryotic regulatory sequences are called chimeric selectable marker genes. Regulatory sequences from several different plant genes have been used to construct chimeric marker genes.
The two most frequently used promoter sequences are those from:
(i) The nopaline synthetase (nos) gene of the Ti plasmid and
(ii) The 35S transcript of cauliflower mosaic virus (CaMV). The most frequently used termination and polyadenylation sequence has been from the nopaline synthase gene of the Ti plasmid.
One widely used chimeric selectable marker gene has the structure CaMV 35S promoter/NPT II coding sequence/Ti nos termination sequence; this chimeric gene is usually symbolized 35S/NPT II/nos.
A basic aim in converting the Ti plasmid to a useful vector for plant gene transfer was to replace the tumor-inducing genes of the T-DNA with a chimeric selectable marker gene such as 35S/NPT Il/nos. This appears like a simple task, but it is not. The difficulty results from the large size (about 200 kb) of the Ti plasmid.
The Ti plasmid cannot be easily manipulated like the small cloning vectors. The large 200+ kb Ti plasmid will be cut at several sites by almost all restriction enzymes. Thus, one cannot simply open it up (cut the circular DNA molecule once) and insert the chimeric gene as one can do with most small cloning vectors.
Instead, one has to insert the chimeric gene into a smaller intermediate plasmid containing DNA sequences with homology to T-DNA sequences, put this intermediate plasmid into Agrobacterium tumefaciens cells harboring a functional Ti plasmid, and rely on genetic recombination (i.e., crossovers within the regions of homology) to insert the chimeric gene into the T-DNA segment in the resident Ti plasmid. This has been performed by at least four slightly different procedures. For the sake of brevity, only one of those approaches is discussed here.
The method of Zambryski, Van Montague, Schell and colleagues (1983) was to replace the tumor-causing genes of a nopaline Ti plasmid with DNA from the E. coli plasmid pBR322. This nononcogenic or disarmed Ti plasmid functions as an acceptor plasmid for chimeric genes or other foreign DNA sequences cloned in the small pBR322 plasmid.
The pB322 derivative of interest is introduced into A. tumefaciens by conjugative transfer from E. coli mediated by trans-acting proteins encoded by broad-host range mobilizing plasmid (conjugative plasmid) that is coresident of the E. coli donor cell.
Since plasmid pBR322 cannot replicate in A. tumefaciens (it has no origin of replication that functions in Agrobacterium), DNA sequences carried on the pBR322 plasmid will only be maintained in A. tumefaciens if they are incorporated into T-DNA plasmid via crossover events.
Recombination between two plasmids to form one larger plasmid is called cointegration; a compound plasmid produced by a single crossover fusing two smaller plasmids is called a contegrate. Cointegrate plasmids are usually unstable.
However, they can be stably maintained in cells if each of the original plasmids contains a selectable marker gene and if only one of the plasmids contains an origin of replication that is functional in the host cell.
Technique # 5. Binary Ti Vectors:
The vir genes of Agrobacterium tumefaciens can act in Trans as well as in cis to the T-DNA. This had facilitated the construction of bipartite Ti vectors in which the T-DNA segment is on one plasmid and the vir genes on a second plasmid.
The plasmid that contains the T-DNA segment, but no vir genes, is called a binary Ti vector. Binary vector plasmids are very similar to the intermediate donor plasmids used to form cointegrate plasmids.
But, binary vectors differ from the cointegrate plasmids by the presence of;
(1) T-DNA border sequence and
(2) a broad-host-range origin of replication that allows them to replicate autonomously in A. tumefaciens.
The two major advantages of binary vectors are:
(1) Their small size (usually about 8 to 12 kb), which facilitates structural manipulations in vitro (e.g., the insertion of foreign genes) and
(2) Elimination of the requirement for co-integrate formation prior to T-DNA transfer to plant cells.
The vector contains two chimeric selectable marker genes. One nos/NPT II/nos, provides resistance to kanamycin (G418); the other 35S/HPH/nos (HPH is the coding sequence for the enzyme hygromycin phosphotransferase), confers resistance to the antibiotic hygromycin.
Technique # 6. Transformation by Co-Cultivation of Tissue Explants with Agrobacterium:
Transformation is now seldom carried out by inoculation of wounded plants with Agrobacterium tumefaciens. Instead, either
(1) Tissue explants or
(2) Protoplasts are co-cultivated with A. tumefaciens cells harboring the Ti plasmid of choice and plants are regenerated from transformed callus cells formed on the cut surfaces of the tissue explants or from transformed protoplasts.
The simplest method is to co-cultivate sterile leaf-discs or root sections with A. tumefaciens for a few days and then to transfer the inoculated explants to selection/regeneration medium.
The selection/regeneration medium contains;
(1) An antibiotic (usually carbenicillin) to kill Agrobacterium and
(2) The proper antibiotic (kanamycin, G4I8, hygromycin, etc.) to select for transformed plant cells.
With two to three weeks, transformed callus tissue grows on the cut surfaces and begins to differentiate into shoots. These shoots are excised and transferred to root-inducing medium. After root-formation, the plantlets are transplanted to soil. By using the leaf disc or root section co-cultivation procedure, transgenic plants can often be obtained, within five to six weeks after co-cultivation is initiated.