The basic techniques of some important tissue cultures are:
(1) Meristem Culture (2) Embryo Culture (3) Anther Culture and Haploid Production (4) Protoplast Culture and Somatic Hybridization (5) Ovule Culture and (6)Micro Propagation.
Technique # 1. Meristem Culture:
(i) One can use an explant that contains pre-existing shoot meristems and produce shoots from them. Such cultures are known as meristem culture.
(ii) The explants commonly used in meristem culture are shoot tips and nodal segments.
(iii) These explants are cultured on a medium containing a cytokine (generally BAP).
(iv) The plantlets thus obtained are subjected to hardening and ultimately established in the field.
(v) Meristem culture is carried out in potato, banana, cardamom, orchids (proto-corm stage), sugarcane, strawberry, sweet potato, etc.
It is used in:
(i) Production of virus-free plants.
(ii) Germplasm conservation,
(iii) Production of transgenic plants.
(iv) Rapid clonal multiplication.
Technique # 2. Embryo Culture:
(i) Embryo culture is the technique of taking out young embryos from developing seeds and their growth on culture medium to form seedlings and then young plants.
(ii) Older embryos are more easily cultured because they are almost completely self sufficient so that they require a simple inorganic medium with a source of energy.
(iii) The immature embryos additionally require high osmotic pressure, higher potassium level, abscise acid, ammonium nitrogen and reduced oxygen tension.
(iv) Embryo culture has the following applications:
a. Embryo rescue:
It is taking out the fragile embryos from fertilized ovules of interspecific crosses before their abortion and culturing them to form viable hybrid seedlings, e.g., jute, tomato, bean, rice.
Orchid seeds lack stored food. Embryo culture helps in developing seedlings from all the seeds. The technique is also used in clonal multiplication.
c. Dormant seeds:
Inhibitors present in endosperm and other parts of seeds do not allow the embryos to grow. Embryos of such seeds can be excised and grown over culture medium to form seedlings. It eliminates the action of inhibitors and dormancy.
d. Rare plants:
The technique is useful in multiplication of some rare plants, e.g., makapuno nut.
e. Seed sterility:
It is caused by non-completion of embryo development due to early fruit ripening (e.g., Prunus, early ripening varieties) or death of embryo caused by mutations of embryo covering structures (e.g., macapuno, coconut). Young embryo of such seeds are taken out and cultured over nutrient medium where it forms the seedling.
f. Recalcitrant dormancy:
There is no known method to break the dormancy, e.g., banana, embryo culture can help to form seeding’s in such cases.
Technique # 3. Anther Culture and Haploid Production:
(i) An individual/cell having the chromosome number found in the gametes of the species is called haploid. Formation of haploid is called haploid production.
(ii) Haploids are sterile and of no direct value.
(iii) When the chromosome number of a haploid plant is doubled, the plant of normal chromosome number for particular species is obtained.
(iv) The chromosome number of these haploid plants is doubled by using colchicine’s to obtain homozygous plants.
(v) In nature, haploid plants originate from unfertilized egg cells, but in laboratory, they can be produced from both male and female gametes.
(vi) When anthers of some plants are cultured on a suitable medium to produce haploid plants, it is called anther culture.
(vii) The technique developed by Guha and Maheshwari (1961) is highly useful for immediate expression of mutations and quick formation of pure lines.
(viii) This technique was first used in India to produce haploids of Datura.
Technique # 4. Protoplast Culture and Somatic Hybridization:
(a) When a hybrid is produced by fusion of somatic cells of two varieties or species, it is known as somatic hybrid. The process of producing somatic hybrids is called somatic hybridisation.
(b) First, the cell wall of the plant cells are removed by digestion with a combination of pectinase and cellulose. The plant cells without cell wall are called protoplasts.
(c) The protoplasts of the two plants are brought in contact and made to fuse by means of electro fusion or chemicals like polyethylene glycol (PEG) and sodium nitrate.
(d) The fusion of protoplasts not only involves the fusion of their cytoplasm but also their nuclei.
(e) The fused protoplasts are allowed to grow on culture medium.
(f) Soon they develop their own walls when they are called somatic hybrid cells (Fig. 1.13).
(g) Symmetric somatic hybrids are hybrids obtained from somatic cell hybrid in which there has been complete fusion of both the cytoplasm’s and the nuclei of the two genetically different cells.
(h) Asymmetric somatic hybrids are somatic hybrids in which nuclear genome of one parent line is intact while the nuclear genome of the other parent line is fragmentary or partially inactivated.
(i) Cytoplasmic hybrids (cybrids) are somatic hybrids in which cytoplasm’s of the two parent cell lines have fused but the nuclear genome of only one parent line persists. The other one degenerates completely.
(j) The hybrid cells give rise to callus. Callus later on differentiates into a new plant, which is a somatic hybrid between two plants.
(k) Somatic hybrids in plants were first obtained between two species of tobacco (Nicotiana glauca and N. langsdorfit) by Carlson et al in 1972.
(l) Successful somatic hybrids have also been obtained from different species of Brassica. Petunia, and Solanum.
(m) Pomato is somatic hybrid between potato and tomato that belong to two different genera. Somatic hybrids are also produced between rice and carrot.
(n) Protoplast technology has opened up avenues for development of hybrids of even asexually reproducing plants.
(o) There is a distinct possibility of development of new crop plants, e.g., pomato.
(p) Somatic hybrids may be used for the production of useful allopolyploids.
(q) Genetic manipulations can be carried out more rapidly when plant cells are in protoplast state. New genes can be introduced (e.g., male sterility herbicide resistance). Mutations will be easier.
Technique # 5. Ovule Culture:
(a) Culturing of fertilized ovules is sometimes needed in those cases where embryos abort very early and the culture of embryo is not possible due to difficulty in excision at a very early stage. Ovules can easily be excised from the ovary and cultured on the usual basal medium.
(b) In most of the cases, ovaries are often cultured either for in vitro pollination, fertilization or embryo rescue.
(c) In case of interspecies or inter generic crosses, the ovaries are excised at the zygote stage or at the two-celled pro-embryo stage and cultured on synthetic media.
(d) Fruits can be successfully obtained by culturing ovaries on synthetic media containing coconut milk, auxin or any other specific requirement.
Technique # 6. Micro Propagation:
Tissue culture technique is useful in rapid and mass multiplication of plant material. Micro propagation is used for this purpose. Micro propagation refers to regeneration of plants from isolated meristemetic cells or tissues or from somatic cells (body cells).
It is also known as micro cloning. Micro propagation can be used for rapid multiplication of crop plants which are difficult to propagate sexually or those vegetatively propagated species in which rate of multiplication is slow. Micro propagation can also be used for mass multiplication of superior hybrids as an alternative to the production of hybrid seed.
Micro propagation has several advantages as given below:
1. It ensures pathogen-free (disease free) healthy status of propagules. Propagules are small plants developed by micro propagation.
2. It helps in rapid multiplication of material. Large number of propagules can be obtained from a single plant by this method, e.g., 106 plants/year from a single explant. Therefore, this technique is highly suitable for rapid multiplication of rare genotypes, and of plants having low multiplication rate.
3. The material multiplied by this method can be maintained in a small place. The transportation of such propagules from one place to another is also convenient.
4. Plants can be maintained in vitro also in a pathogen-free state.
5. In dioecious species (separate male and female plants), plants of one sex may be more desirable than those of the other, e.g., male Asparagus or female papaya plants. In such cases, plants of the desired sex can be selectively multiplied by micro propagation.
6. In case of forest trees, mature elite trees can be identified and rapidly cloned by this method.
7. Micro propagation can be carried out throughout the year.
8. The micro propagation yields results faster than conventional breeding. It is mostly used in horticulture, floriculture and forestry. In case of many ornamental plants, tissue culture plants give better growth, more flowers and less fall out.
The technique has been used to obtain early and disease-free lines in strawberries, banana, citrus and some timber trees. In field crops, micro propagation has been used in potato, sugarcane.
The main problems of this technology are:
(1) Soma-clonal variation, and
(2) Variation in chromosome number due to continuous sub-culturing.