The following points highlight the five main types of cloning. The types are: 1. Microbial Cloning 2. Gene Cloning 3. Cell Cloning 4. Plant Cloning 5. Organismal Cloning.
Type # 1. Microbial Cloning:
From very ancient times, it is known that a variety of microbes such as bacteria and yeast (fungus) are helpful in making a large number of products, e.g., lactic acid, ethanol, vinegar, cheese, etc., as also of industrially useful substances like amylase, protease and life-saving antibiotics like penicillin and tetracycline.
Cell proteins can also be produced by microbes (bacteria and fungi). Several improved strains of these microbes were developed by using mutants and other conventional techniques.
Today, gene cloning and genetic engineering techniques are generally used to improve the useful microbial strains for so many purposes:
(a) To produce useful compounds such as enzymes and vitamins on mass scale.
(b) They can be engineered to produce several pharmaceutically useful products such as human insulin (e.g., in Escherichia coli), interferon, human growth hormone and viral vaccines.
(c) Several microbial strains are used in the various industries for performing many important functions such as the removal of undesired lignin (e.g., Trametes).
(d) Microbes (e.g., heavy-metal accumulating bacteria like Pseudomonas) can be used to purify polluted environment (called bioremediation).
(e) Certain microbes such as Trichoderma (fungus) and Rhizobium (bacterium) are used for bio-control (against plant diseases and pests) and as biofertilisers respectively.
In the process of DNA cloning fragments of eukaryotic DNA are introduced into bacterial cells, which are then grown into individual colonies on a culture dish.
As a cell proliferates to form a colony, the single DNA molecule taken in by the founder of the colony is repeatedly replicated by the enzymes present in the bacterial cells such that by the end of the growth period, the number of copies of the foreign sequence has been greatly amplified.
Cloning, therefore, can be used as a technique to produce large quantities of a particular DNA sequence, but more important, it can be used as a technique to isolate, in an absolutely pure form, any specific DNA fragment among a large, heterogeneous population of DNA molecules.
Type # 2. Gene Cloning:
The object of the cloning technique is to introduce a copy of a eukaryotic DNA fragment into a recipient bacterial cell under conditions in which it will replicate autonomously from that of the host chromosome. In order to get this objective, the eukaryotic DNA must be linked to the DNA of a vector that normally replicates within a bacterial environment.
The most common vector employed in cloning procedures is the bacterial plasmid. The use of plasmids is as follows: Plasmids are nonessential extra-chromosomal DNA molecules present in many bacteria.
Like the main bacterial chromosome, they are double-stranded circles, but they are much smaller and of variable genetic constitution. Since the plasmids and chromosomal DNAs have very different physical properties, they can be readily separated from one another.
The foreign DNA to be cloned is inserted into the plasmid to form the recombinant DNA molecule. This modified plasmid is then added to a bacterial culture as naked DNA, and in the presence of CaCl2, a small percentage of the bacteria will pick up one of these molecules from the medium. Once a plasmid is taken up, it will replicate autonomously within the recipient and will be passed on to its progeny during cell division.
A wide variety of plasmids have been isolated, many of which carry genes for resistance to antibiotics. If these plasmids are added to a bacterial culture and the cells are then treated with specific antibiotics, only these bacteria which have taken up the plasmid will be able to survive.
How the DNA of the vector is combined with that form the eukaryotic genome? Recombinant DNA molecules can be formed in several ways.
Recombinant DNAs can be constructed between DNA fragments having blunt ends. This can be done by enzymatically adding a string of A- or C- bearing nucleotides to one strand of the fragment and a complementary string of T- or G- bearing nucleotides to the opposite strand of the vector, mixing the two populations, and sealing the molecules with the ligase.
Cloning Genomic Fragments:
When working with genomic DNA, one has to isolate a particular gene or genes, such as those which code for the histones, from among the thousands of unrelated sequences. The first step in this type of experiment is to fragment the DNA with restriction endonucleases.
These enzymes recognize sequences from 4 to 6 nucleotides in length. Once the DNA is fragmented, the fragments are combined with the DNA of the vector, and the diverse population of recombinant DNA molecules is subjected to the DNA cloning procedure. Since a given bacterial cell takes up only one plasmid, all the cells of a given colony will contain copies of the same recombinant DNA molecule.
Type # 3. Cell Cloning:
The bacterial cell is at one and the same time is the somatic and the germ cell. Having selected a single bacterium, one can grow large populations of cells, all identical in morphology and biochemistry. In modem times by means of refined culture techniques the cell cloning has become possible, and the pure populations of cells have been grown from a single isolated cell.
The smallest viable units of plant one can at present envisage as reproducing, growing and developing in culture is a single cell. Several workers established method which would allow the growth of isolated plant organs and tissues in vitro, in culture.
Virtually all plants are totipotent but in animals only fertilized egg and stem cells in the embryonic blastocyst are totipotent.
Type # 4. Plant Cloning:
Cellular totipotency is the capacity of mature cells showing that when freed from the plant body, they have the ability to reorganize themselves into the new individuals. Steward and his co-workers, showed this phenomenon in the carrot cultures. Here the small pieces of mature carrot root were grown in a liquid medium supplemented with coconut milk in special containers.
These cultures were shaken generally which freed all the cell clusters into the medium. Some of them developed into rooting clumps. When these were transferred to the tubes containing a semi-solid medium, they gave rise to whole plant that flowered and set seeds.
Type # 5. Organismal Cloning:
It is characteristic of vegetative reproduction that a vegetative part, when removed from the plant and placed under suitable conditions, will replace the missing parts and produce a complete individual. It has been possible to grow several types of plant organs in sterile culture including roots, shoot apices, leaves, floral parts and fruits.
The nutrient requirements for such organ culture vary considerably from species to species and according to the type of organ in question but certain general requirements can be recognized.
Intact higher plants are autotrophic, that is, they are able to synthesize all the organic substances required for their own life from water, carbon dioxide, oxygen and mineral nutrients. But since most sterile cultures are unable to carry on photosynthesis, it is clear that they will require at-least a carbon source, usually supplied in the form of a sugar as sucrose or glucose.
In addition, sterile cultures require the same mineral nutrients as the intact plant, including both macronutrients (e.g., nitrogen, phosphorus, potassium and calcium) and micronutrients or trace elements (e.g., Mg, Fe, Mn, Zn, etc.).
In addition to the requirements for a carbon source and mineral nutrients, it is found that most isolated organs have also a requirement for certain special organic substances, such as vitamins, nicotinic acid, etc.
This type of reproduction is applied extensively to horticulturally important plants. Horticulturists usually refer to it as vegetative propagation. Its practical advantage is that a single individual can give rise to a large number of plants that will be uniform because all of them have the same genetic constitution, or genotype.
Such a group of plants derived from a single one by vegetative propagation is called a clone. Now days the organismal cloning is a common practice for the development of the clones’ of horticultural as well as forest plants.
Many orchids producing beautiful flowers on cloned plants. Scientists have genetically engineered agronomically important crop plants. Rapid production of such plants can be achieved using actively dividing meristematic cells, which occur at the apices of root and shoot of the plant.
For the agriculture, disease, drought, insect pest-resistant as well as herbicide-tolerant crops have been successfully produced by gene manipulation.
Genetically Modified Food (GMF), such as vitamin A-rich rice and lysine-rich pulse seeds are now becoming popular components of human staple food.