The following points highlight the five transformation technologies used for trans-genesis of plants. The technologies are: 1. Agrobacterium 2. Shotgun 3. Microinjection 4. DNA Uptake 5. Antisense RNA.
Transformation Technology # 1. Agrobacterium:
Agrobacterium tumefaciens a Gram-negative soil bacterium can induce crown galls on the stems of various plants. The phytopathogenic function of the bacterium is encoded in a tumour inducing plasmid called Ti plasmid, with a size of 200 kbp. The same strain of the bacterium without the plasmid cannot induce crown gall formation.
The Ti plasmid (Fig. 17.21) contains a 12-14 kbp long section, called T-DNA or transfer DNA, which naturally be transferred and integrated into plant cell nucleus. The transfer of the T-DNA single strand from the bacteria to the plant cell chromosomes proceeds by a very complicated unknown process, probably through the same way as in bacterial conjugation.
The T-DNA is integrated randomly into chromosomes and, when inserted in a gene, can eliminate its function. This can be used to identify a gene through gene tagging in a same way to gene tagging by transposons. The integrated T-DNA gains the properties of a eukaryotic gene and is inherited in a Mendelian fashion.
Due to this transformation property, the Ti plasmids are used as transformation vectors for integrating foreign genes in their functional state in a plant genome. The Ti plasmid contains 7-8 virulence (v/r) genes, which encode some virulence proteins required to transfer the tumour-inducing genes to plant chromosome.
From the T-DNA a single strand is excised by a vir-nuclease. The ends of T-DNA are demarcated by border sequences. The vir-proteins protect the T-DNA from the digestion by the plant enzymes and also facilitate the transport into the nucleus.
To use the Ti plasmid as transformation vector it needs to be modified beforehand. The auxin and cytokinin genes are removed to prevent tumour growth of the transgenic plant and the genes for opine synthesis is also removed. Thus, the T-DNA remains demarcated only by the two border regions.
The Ti plasmid is then cleaved in the T-DNA region by certain restriction enzyme and a foreign gene, excised by the same restriction enzyme, can be inserted in this cleavage site.
More recently, binary vector containing strains of Agrobacterium are used for transformation. These vectors do not contain any vir genes and are, therefore, unable to transform a plant cell.
A second helper plasmid containing the vir genes but no T-DNA is required simultaneously for transformation. Either vector alone cannot transform a plant cell. As both the vectors are required simultaneously they are called Binary vectors (Fig. 17.20).
Transformation Technology # 2. Shotgun:
A recent development in the bombardment of plant cells with micro-projectiles has made it possible to target an extremely small area of a tissue in order to transform its cells. The technique was developed in 1985. The micro-particles or micro-projectiles are small spheres of tungsten or gold (l-4pm in diameter) coated with DNA. The gene-gun mechanism is exactly similar to that of a shotgun.
In this mechanism the DNA coated particles are used to shoot into the plant cells.
The micro-projectiles are accelerated with compressed air, helium or other gases and the target materials are embryonic tissues, callus and leaves. The velocity of the projectiles may be as high as 1500 km h-1 in a vacuum chamber in order to penetrate the cell wall of the epidermis and mesophyll cells. Accurate aiming is made possible with aid of a line crossing in a microscope.
The cells in the centre of the line of fire are destroyed, whereas the peripheral cells survive. The DNA coated on the projectiles are integrated in the nuclear genome (Fig. 17.22). The transformed cells are then selected on an agar medium containing antibiotics and induced to form a callus.
By this method of gene gun different transgenic plant lines have been obtained in case of sugarcane, wheat, sorghum and other plants. In an experiment, bacterial and viral genes were shot into the onion plants, which were then found to start producing the foreign proteins.
Transformation Technology # 3. Microinjection:
Microinjection in gene technology is a procedure in which DNA is injected directly into the nucleus of a cell with the aid of a very fine needle or micropipette. In plants microinjection of DNA into the nuclei of isolated protoplasts is an efficient means of gene transfer. The injected DNA is often incorporated at random into the nuclear DNA.
If an injected transgene carries a suitable promoter sequence, the chances of its expression are increased.
The microinjection technique is advantageous due to its:
(1) Higher transformation efficiency
(2) Minimal soma-clonal variation as the egg cell is directly injected
(3) High transfer efficiency of chromosomes and organelles
In case of animals the eggs are surgically removed from the female parent and are fertilized externally with sperms. The plasmid vector carrying the desired gene is then microinjected in the male pro-nucleus of the just fertilized egg through a micropipette. The injected DNA is then integrated into the nuclear DNA. A large number of desired gene copies are injected for multiple integration.
When the injected eggs are introduced into a female mouse and allowed to develop, the gene of interest is often expressed in some of the progenies. The animals developed from the injected eggs are always genetic mosaics with some somatic cells carrying the transgene and others not, because integration of injected DNA takes place in the very early stage of embryogenesis.
Transgenic mice production through microinjection is routinely done in the different laboratories, generally for the study of mammalian gene expression. A homozygous (for the new gene) mouse line can be established through breeding. By this technique it has been possible to introduce into mice the human growth hormone gene under the control of an inducible promoter.
Some of the progeny mice developed from the injected eggs were found to grow unusually larger than the control mouse. The demerit of the technique is that its success depends on the skill. A skilled practitioner has a high success rate, but as the cells must be injected one by one, the total number that can be treated; is small.
Transformation Technology # 4. DNA Uptake:
Transgenic plant formation is also done through protoplast transformation.
The cell walls of the plant tissues are enzymatically digested to obtain the protoplasts, which are able to take up and integrate foreign DNA added in the medium into its genome in the presence of polyethylene glycol or CaCl2.This transformation is similar to the bacterial transformation. The gene of interest is linked with an antibiotic-resistant marker gene.
If the antibiotic is added in the medium, only the transformed protoplasts survive indicating the successful transformation. From the transformed protoplasts fertile plants are regenerated through tissue culture. Transgenic maize plants have been generated by protoplast transformation.
Transformation Technology # 5. Antisense RNA:
In gene technology gene expression can be regulated by the use of antisense RNA. The DNA strand, which is transcribed into normal mRNA, is referred to as the sense strand. A promoter ensures that the mRNA is copied from the correct strand. Normally complementary strand does not contain a sequence of codons that can be translated to produce a functional protein.
If a gene is positioned in wrong orientation with respect to the promoter, the opposite or complementary strand of DNA is transcribed to produce the so-called antisense RNA or complementary RNa.
Normally, mRNA occurs as a single-strand, but when antisense RNA molecules are present in the same cytosol, both will anneal to form a double-stranded RNA. The duplex RNA is not translated, instead degraded rapidly by ribonucleases. Thus the mRNA loses its ability to encode protein synthesis in presence of corresponding antisense RNA.
This antisense RNA method is being used to:
(1) Decrease or even eliminate the synthesis of unwanted enzymes during the ripening processes in fruits and vegetables
(2) Control the viral diseases.
To produce the antisense RNA of a gene of interest within a cell or an organism, the corresponding gene is first cloned, and then inserted as cDNA in reverse orientation in a suitable vector with which the plants are transformed.
The net result is the production of antisense RNA transcripts inside the transformed cells, which will hybridize with the “sense RNA” molecules to prevent or reduce the synthesis of protein gene products. For the complete inhibition of the expression of a gene multiple copies of the antisense gene, should be reintroduced inside the host cell.
An alternative method to reduce the expression of a gene is co-suppression, where an extra copy of an endogenous gene is introduced by transformation. The overexpression of the same or similar genes causes the decrease in activity of the gene of interest.