Population of the country is rapidly increasing day by day. However, the most urgent need to the nation is to meet the food demand of the people. To remain self-dependent and to make them available with quality food, the biotechnologists have to boost up the gene revolution programmes for crop improvement so that the improved crops should have high yield, high amount of digestible and quality proteins, and vitamins, disease/pest resistance and drough/herbicide/salt tolerance, etc. These can be done through the transfer of beneficial genes of a prokaryotic microorganism/ eukaryotic incompatible plant in a given crop plant, alteration in metabolic pathways, and making the plants resistant to invasion of pathogens/pests and herbicides/salt stress. Methods of gene transfer in a desired eucaryotic cell are : through Agrobacterium,
through virus, co-cultivation of cells, leaf disc transformation, electroporation, etc. These are discussed in the following sub-sections.
Transgenic plants Nif gene transfer Phaseolin gene transfer sunbean plantConversion of C3 plants to C4 plants Herbicide resistant plants Insect pest resistant plants
Plant improvement through genetic transformation:
Following are some of the methods through which plants can be improved :
(i) Agrobacterium tumifaciens
mediated gene transfer. A. tumifaciens
can be used as a vector for transferring the desired genes into plant cells. There are several problems related to Ti-plasmid which must be overcome before using it as vector. Being large sized, it cannot be used as such because the hormones secreted by it interfere with the normal functioning of transformed cells. Therefore, T-DNA region, after removing from Ti-plasmid must be tailored so that it may join with foreign DNA. The nos
(nopaline synthesis ) gene of this plasmid which has promoter and termination signals and recognized by the host plant can be used for this purpose. Then it is allowed to join pBR322 resulting in production of tailored pBR322 -T-DNA plasmid. At this stage a foreign gene of desired function is inserted into the non-essential region of T-DNA. Since the tailored plasmid does not contain vir
genes required for transfer of T-DNA into the host genome. Therefore, the following two systems are being adopting for this purpose.
(a) Co-integrative system. In this system of gene transfer, the tailored plasmid containing foreign DNA is allowed to transform A. tumifaciens
cells which contain a Ti- plasmid. Consequently, A. tumifaciens
cells have two plasmids. As a result of which there are much chances of reinserting T-DNA region to tailored plasmid into Ti-plasmid via homologous recombination at the repeated sequences.
(b) Binary vector system. It is an alternative method of previous one. In this method, A. tumifaciens
containing Ti-plasmid (from which T-DNA has been removed) are transformed by tailored pBR322-T-DNA plasmid. Since the vir
gene of T-DNA lacking Ti-plasmid is still effective, it integrates with foreign DNA containing tailored plasmid and transfers into plant cells. Using these techniques many more transgenic plants have been produced so far through transformed A. tumifaciens
cells. Expression of seed storage protein of soybean in transgenic Petunia
plants has been possible only due to successful transfer of foreign DNA into it.
Co-cultivation (protoplast transformation) method. In this system the young leaves are segmented and protoplasts are isolated enzymatically by using cell wall lysing enzymes. It takes 3-10 days to protoplasts to divide and form cell walls. Protoplasts after regeneration of cell walls are mobilized to infection by A. tumifaciens
cells containing foreign DNA. These are incubated for about 3 days. After transformation of protoplasts bacterial cells of the medium are now killed and protoplasts are cultured for 16-30 days. There develop microcalli which are plated on selective medium containing kanamycin as marker. Kanr
callus grows which are then analyzed easily and recultured to develop transformed cells and finally adult plantlets within 4-6 months. In general, it is tedious and time consuming method, and takes more than 4 months to develop transgenic plants, even in plants like tobacco. Moreover, it is not suitable for producing transgenic plants in calcitrant species.
Leaf disc transformation method. In this method leaf discs of 2 mm size are punched. The discs are co-cultivated in plates with a desired strain of Agrobacterium
(5-10 x 106
cells/ml) containing the foreign DNA. Infection takes place at cut edges of leaf discs. After infection and cell transformation bacterial cells of the medium are killed. There are two methods of culturing leaf discs based on the presence of plasmids in Agrobacterium.
If the bacterium, contains Ti-plasmid with a foreign DNA the discs are transferred onto filter paper of feeder plates for 48 hour; thereafter, the filter is transferred onto shooting medium containing kanamycin as marker. After shoots develop, these are transferred onto rooting medium. Finally, the adult plantlets are transferred in soil after hardening.
Moreover, if Agrobacterium
contains Ri-plasmid the transformed cells are plated onto such medium which contains kanamycin and develops adventitious roots from cut edges of discs. The adventitious roots are cut and cultured on shooting medium to allow shoot formation. These are again put onto rooting medium for the development of roots. Transgenic plantlets are obtained after 4-6 weeks of incubation which later on are transferred in soil.
Virus mediated transformation. Among plant viruses, cauliflower mosaic virus (Ca MV) and gemini viruses are being looked for potential vectors for transferring foreign DNA into plant cells. CaMV contains double stranded DNA as genetic material, therefore, has increased the possibility of being used an efficient vector for genetic engineering purposes. Since its genome is tightly packed in protein coat, there remains the least chances to insert foreign DNA. Until size limitation problem is removed by using the helper virus, it cannot be used for transformation purposes.
Geminiviruses (for example, curly top virus and maize streak virus) are known to cause diseases on plant. They contain single stranded DNA packed in protein coat. Before using them as efficient cloning vector much is to know about the essential genes such as promoter sequence, etc.
Through electroporation : See Biotechnological Applications of Plant Cell, Tissues and Organ Cultures
Through particle bombardment: See Biotechnological Applications of Plant Cell, Tissues and Organ Cultures
Through microinjection : See Biotechnological Applications of Plant Cell, Tissues and Organ Cultures
Pollen mediated tranformation. Mature pollen grains are collected from anther and incubated at conditions with foreign DNA fragments. Pollination is done artificially. Transformation is induced. Recently, transgenic rice plants have been produced by using this technique. Success of this technique lies in the potentiality of the pollen grains. In future, the scientists look forward in producing such plants which (i)
can exhibit improved level of seed storage protein, (ii)
can fix N2
gas even if they are non-legumes, (iii)
can inhibit increased photosynthetic efficiency and (iv)
can tolerate/ resist to the pathogens, pests, chemical stress or biocides, etc. (Kumar and Kumar, 1992).