Everything you need to know about plant tissue culture and somatic hybridization !
Q. 1. Define plant tissue culture.
Ans. Plant tissue culture refers to development of whole plant from regeneration of living plant tissues in the nutrient medium (in vitro) or culture medium.
Q. 2. What is culture medium?
Ans. Culture medium is a nutrient medium which contains all essential micro and macro nutrients, carbohydrate (glucose), vitamins and hormones. The pH of culture medium should be 5.5. However, the culture medium differs from species to species.
Q. 3. What is explant?
Ans. Any plant part which is used for regeneration in tissue culture technique is called explant. It may be a cell, a protoplast (naked cell), a tissue or an organ. The effective explant may differ from species to species.
Q. 4. What is totipotency?
Ans. The regeneration capacity or ability of a plant cell to develop into a whole plant is called totipotency. It reveals that each cell is capable of giving rise to a whole plant.
Q. 5. What is callus?
Ans. A mass of unorganized regenerated cells in culture medium is called callus (plural calli). In some species like carrot and sandal wood, somatic embryos are developed from the callus, while in wheat, rice, barley and tobacco, roots and shoots develop directly from calli.
Q. 6. What are basic requirements of tissue culture?
Ans. There are some basic requirements of tissue culture techniques which are listed below:
(i) A well-equipped pathogen free laboratory.
(ii) The laboratory should be air conditioned (18-25°C).
(iii) Proper culture medium.
Q. 7. Who first developed the culture medium?
Ans. The culture medium was first developed by Murashige and Skoog in 1962. This culture medium with modifications is widely used for tissue culture technique.
Q. 8. What are important steps in tissue culture techniques?
Ans. The tissue culture technique generally involves following four important steps:
(i) Isolation of tissues/organ to be used as explant.
(ii) Regeneration and callus formation in culture medium.
(iii) Formation of somatic embryos from the callus (embryogenesis).
(iv) Differentiation of root and shoot from the somatic embryos (organogenesis).
Q. 9. What is embryogenesis?
Ans. In the tissue culture technique, the process of formation of somatic embryos from the callus is called embryogenesis. Sometimes, somatic embryos are not formed from the callus and somatic buds are formed which after germination give rise to whole plant.
Q. 10. Define organogenesis.
Ans. In tissue culture technique, the process of differentiation of roots and shoot from the somatic embryos is called organogenesis. Sometimes, the whole plant develops directly from the somatic bud. In such cases somatic embryos are not formed.
Q. 11. What are different techniques of plant tissue culture?
Ans. Depending upon the plant part used as explant, plant tissue culture techniques are of following five main types:
(i) Meristem Culture
(ii) Embryo Culture
(iii) Anther Culture
(iv) Cell Culture
(v) Ovule Culture.
Q. 12. What is meristem culture?
Ans. Meristem culture refers to regeneration of whole plant in culture medium from tissues of an actively dividing plant part such as stem tip, root tip or auxiliary bud. Generally shoot apical meristem is used for regeneration. This technique has been widely used in vegetatively propagated crops such as sugarcane, potato, banana and several fruit trees and timber species.
Q. 13. What are uses of meristem culture in crop improvement?
Ans. Important applications of meristem culture in crop improvement are given below:
(i) It is used for micro propagation in banana, strawberries, citrus and some timber trees such as Delbergia sissoo.
(ii) It is used for obtaining virus free plants.
(iii) It can be used for exchange of germplasm.
(iv) The germplasm can be conserved in the form of meristems at -196°C in liquid nitrogen for long term storage.
Q. 14. What is embryo culture?
Ans. Regeneration of whole plant from an embryo in culture medium is called embryo culture. Embryos of appropriate stage are excised from the seed and are transferred to culture medium. This technique is used when there is disharmony between the embryo and endosperm. This technique is used to make distant crosses successful. The embryo in distant crosses is removed before abortion and cultured in culture medium.
Q. 15. How embryo culture is useful in crop improvement?
Ans. The main uses of embryo culture in crop improvement are given below:
(i) It is useful in making distant crosses (interspecific and intergeneric) successful.
(ii) It is useful in obtaining haploids from distant crosses.
(iii) Embryo culture is useful in orchid breeding where seeds lack stored food and are unable to propagate.
(iv) It is effective in breaking long seed dormancy in Prunus, Iris and Taxus.
Q. 16. What is anther culture?
Ans. Regeneration of whole plant from anther or pollen in the culture medium is called anther culture. Anthers of appropriate stage are taken from the plant. The optimum stage may differ from species to species. This technique is used to obtain haploid plants. It has been used in crops like Brassica, Tobacco, Rice, Barley, Wheat, Tomato, Potato, Triticale etc.
Q. 17. Define cell culture.
Ans. Regeneration of whole plant from cells in culture medium is called cell culture. Protoplasts are also used for regeneration of whole plants. Cell culture technique has been used in different crop plants such as wheat, rice, barley, maize, tobacco etc.
Main uses of this technique are given below:
(i) It is useful in mass clonal production of different species.
(ii) Protoplast culture is useful in somatic hybridization and overcoming barriers of cross incompatibility in distant crosses.
(iii) Cell culture and protoplast culture are useful in the development of transgenic (genetically engineered) plants.
(iv) Embryo genic cell cultures are used for cryopreservation in liquid nitrogen at —196°C for long term preservation.
Q. 18. What is ovule culture?
Ans. Regeneration of whole plant from ovule in culture medium is called ovule culture. This is also called ovary culture. This technique is used for embryo rescue in distant crosses. Ovules are of two types, viz. fertilized and unfertilized. Haploid plants are obtained from culture of unfertilized ovules and diploid from fertilized ovules. This technique has been used to limited extent.
Q. 19. What is tissue culture induced variation?
Ans. Depending upon the explant, the tissue culture induced variation is of three types, viz.:
(i) Gametoclonal variation,
(ii) Somaclonal variation, and
(iii) Protoclonal variation.
Q. 20. What is somaclonal variation?
Ans. The variation that is observed among the plants which are regenerated from callus cultures of somatic explants such as meristems is called somaclonal variation. Such variation has been observed in sugarcane, potato, rice, wheat, maize etc.
Q. 21. What do you mean by gametoclonal variation?
Ans. The variation which is observed among the plants which are regenerated from gametic culture (anther or ovule culture) is called gametoclonal variation. Such variation has been observed in tobacco, rice and Brassica.
Q. 22. Define protoclonal variation.
Ans. The variation which is observed among the plants which are regenerated from callus culture of protoplasts is called protoclonal variation. Such variation has been observed in potato, tobacco, lucern and many other crops.
Q. 23. What are main features of tissue culture induced variation?
Ans. Main features of tissue culture induced variation are briefly presented below:
(i) The variation is genetic in origin and hence heritable.
(ii) It occurs in both sexually and asexually propagated species. However, the frequency of somaclonal variation is very high in vegetatively propagated species (upto 75% in potato and sugarcane) and low in seed propagated species (0.8-1.2% in maize).
(iii) It occurs in both oligogenic and polygenic characters.
(iv) Generally, somaclonal variation is heterozygous in origin and rarely homozygous.
(v) Tissue culture induced variation results due to structural chromosomal changes, viz. deletion, duplication, translocations and inversions.
Q. 24. How somaclonal variation is useful in crop improvement?
Ans. The somoclonal variation is useful in various ways.
It is useful in isolation of plants with:
(i) Salinity resistance
(ii) Drought resistance
(iii) Herbicide resistance
(iv) Tolerance to metal toxicity
(v) Early maturity and
(vi) Resistance to various insects and diseases.
Disease resistant somaclones have been identified in sugarcane, potato, tobacco and ornamental plants.
Q. 25. What are applications of tissue culture in crop improvement?
Ans. Main applications of tissue culture in crop improvement are:
(i) Creation of variability
(ii) Development of haploids
(iii) Embryo rescue
(iv) Somatic hybridization
(v) Development of transgenic plants
(vi) Drought resistance
(vii) Disease resistance
(viii) Salinity resistance
Q. 26. What is somatic hybridization?
Ans. Crossing of plants through fusion of somatic cells is known as somatic hybridization. In somatic hybridization, the sexual process is bypassed. The fusion of cells takes place through protoplasts. Somatic hybridization permits gene transfer between any two species irrespective of their taxonomic relationship. It overcomes the barriers of cross incompatibility and makes incompatible crosses compatible.
Q. 27. What is protoplast?
Ans. Protoplasts are nothing but naked cells or cells without cell wall. Protoplasts are isolated from mesophyll cells and callus culture. Protoplasts can be isolated mainly by two methods i.e. mechanical method and enzymatic method.
Q. 28. What are the steps involved in somatic hybridization?
Ans. Somatic hybridization consists of several steps which are listed below:
(i) Isolation of protoplasts
(ii) Fusion of protoplasts
(iii) Selection of hybrid cells
(iv) Culture of hybrid cells
(v) Regeneration of plants from hybrid tissues
(vi) Characterization of hybrid plants.
Q. 29. What are advantages of somatic hybridization?
Ans. There are several potential uses or advantages of somatic hybridization which are given below:
(i) It permits hybridization between distantly related species and unrelated species.
(ii) It permits modification of cytoplasmic genes.
(iii) It is useful in production of somatic hybrids.
(iv) It helps in production of allotetraploid in single step.
(v) It is useful in conservation of heterosis.
(vi) It is useful in development of transgenic hybrids.
(vii) It is useful in transfer of male sterility from one species to another.
Q. 30. Who coined the term somatic hybridization?
Ans. The term somatic cell hybridization was first used by Barski et al in 1960.
Q. 31. Who first used the term protoplast?
Ans. The term protoplast was first used by Hanstein in 1880.
Q. 32. What are different types of somatic hybrids?
Ans. Based on taxonomic relationship of species involved in the hybridization, somatic hybrids are of following three types:
(i) Interspecific somatic hybrids
(ii) Intergeneric somatic hybrids
(iii) Intertribal somatic hybrids.
Based on combination of chromosomes and cytoplasm from parental species, somatic hybrids are again of three types as given below:
(i) Symmetrical hybrids
(ii) Asymmetrical hybrids, and
Q. 33. What do you mean by symmetrical hybrids?
Ans. Those somatic hybrid cells or plants that contain all chromosomes of both the species involved in the fusion are called symmetrical hybrids. Such hybrids have been obtained in tobacco, carrot, petunia, etc.
Q. 34. What are asymmetrical hybrids?
Ans. Those somatic hybrid cells or plants that contain complete chromosome complement of one species and only a part of somatic complement of another species are called asymmetrical hybrids. Such hybrids have been obtained in tobacco, potato, petunia and several other intergeneric and intertribal somatic hybrids.
Q. 35. What is cybrid?
Ans. Those somatic hybrids that involve normal protoplast of one species and nucleus-less protoplast of another species are known as cybrids. Such hybrids are useful in transfer of cytoplasmic male sterility from one species to other.
Q. 36. Who coined the term cybrid?
Ans. The term cybrid was first used by Bunn et al. in 1974.
Q. 37. What is cytoplast?
Ans. A protoplast either without nucleus or with inactive nucleus is called cytoplast.
Q. 38. What are homokaryons?
Ans. Hybrid cells involving protoplasts of the same species are called homokaryones.
Q. 39. What are heterokaryons?
Ans. Hybrid cells involving protoplasts of two different species are known as heterokaryons.
Q. 40. What are limitations of somatic hybridization?
Ans. Main limitations of somatic hybridization are listed below:
(i) It can be used in those species where regeneration of plants is possible from protoplasts.
(ii) Intergeneric and intertribal hybrids are usually sterile.
(iii) Such hybrids have many undesirable traits of wild or unrelated species.
(iv) Generally, intergeneric and intertribal hybrids exhibit high degree of sterility and cannot be backcrossed with cultivated species.
Q. 41. What is micro propagation?
Ans. Mass multiplication of plant material by regeneration of somatic tissues is called micro propagation or micro-cloning. It can be used for mass multiplication of crop plants which are difficult to propagate sexually or those vegetatively propagated species in which rate of multiplication is slow.
Q. 42. What are advantages of micro propagation?
Ans. Main advantages of micro propagation are given below:
(i) It helps in rapid multiplication of material.
(ii) It helps in isolation of pathogen free propagules.
(iii) The propagules developed by micro propagation are easy for transportation and require lesser space.
Q. 43. What are differences between somatic hybridization and sexual hybridization?
Ans. Main differences between somatic hybridization and sexual hybridization are presented below in table 22.1: