An exclusive project report on Plant Tissue Culture. This report will help you to learn about: 1. Meaning of Plant Tissue Culture 2. Basic Requirements of Plant Tissue Culture 3. Important Steps 4. Applications of Plant Tissue Culture in Crop Improvement 5. Techniques.
Contents:
- Project Report on the Meaning of Plant Tissue Culture
- Project Report on the Basic Requirements of Plant Tissue Culture
- Project Report on Important Steps in Plant Tissue Culture
- Project Report on the Applications of Plant Tissue Culture in Crop Improvement
- Project Report on the Techniques of Tissue Culture used in Plant System
Project Report # 1. Meaning of Plant Tissue Culture:
Growth of living plant tissues in a suitable culture medium (in vitro) is known as plant tissue culture. Culture medium is a nutrient medium which contains all essential micro and macro nutrients, carbohydrates, vitamins and hormones. The pH of Culture medium should be 5.5.
However, the culture medium differs from species to species. Thus a suitable medium has to be developed to meet the requirement of a plant species. Plant tissue culture includes cell culture, protoplast culture, organ culture, meristem culture etc. (Table 31.1). The organ culture includes any plant organ which has separate identity such as anther culture, ovule culture, embryo culture and bud culture.
The plant part which is used for regeneration is called explant. It may be a cell, a protoplast, a tissue, or an organ. A mass of unorganised regenerated cells in culture medium is called callus (pleural calli) and suspension of free cells of callus in a liquid medium is known as suspension culture.
The regeneration capacity or ability of a plant cell to develop into a whole plant is known as totipotency, which reveals that each cell is capable of giving rise to a complete plant.
The cell and tissue cultures lead to regeneration of complete .plant. In some species like carrot, and sandalwood somatic embryos are developed, but in several crops like wheat, rice, barley and tobacco development of both root and shoot takes place from the calli.
Project Report # 2. Basic Requirements of Plant Tissue Culture:
In plant tissue culture techniques, there are some basic requirements, viz:
(i) Aseptic conditions,
(ii) Control of temperature,
(iii) Proper culture media, and
(iv) Sub-culturing.
These are briefly described below:
i. Aseptic Conditions:
The tissue culture laboratory should have aseptic conditions. It means it should be well sterilized against pathogens. A pathogen free environment will help in maintaining good health of the callus, cell or protoplast cultures resulting in recovery of healthy plants from such cultures. The explant and glassware should be properly sterilized before their entry into the tissue culture laboratory.
ii. Control of Temperature:
Air conditioning of the tissue culture laboratory is essential. Generally, temperature between 18-25°C is used. However, this varies from species to species. High temperature adversely affects the growth of the callus.
iii. Proper Culture Medium:
Culture media have been developed by various workers for different crop species. The medium has to be modified as per the requirement of a species. The culture media developed by Murashige and Skoog (1962) and Gamberg, et al. (1968) are used with some modification in various crop species. The composition of the culture medium developed by Murashige and Skoog (1962) is given in Table 31.2.
iv. Sub-Culturing:
Transfer of tissue or callus from old culture media to fresh culture media is called sub-culturing. This is essential to maintain good health of the callus or tissues, because after some period, some nutrients are depleted in the culture media and change of media becomes essential.
Project Report # 3. Important Steps in Plant Tissue Culture Technique:
Plant tissue culture technique generally consists of four important steps, viz:
(i) Isolation of tissues,
(ii) Regeneration and callus formation in culture medium,
(iii) Embryogenesis, and
(iv) Organogenesis (Fig. 30.1).
These are briefly described below:
i. Isolation of Tissues:
Tissues for regeneration can be isolated with the help of sterilized blade from any plant part, viz., leaf, stem, apical bud, axillary bud etc. The isolated tissues are sterilized and then grown on culture medium. Tissues should be isolated from disease free portion.
ii. Regeneration and Callus Formation:
Tissues proliferate on the culture medium and give rise to a mass of unorganized cells called callus. The callus is generally of two types, viz., friable callus and compact. The friable callus can be easily manipulated for suspension culture. However, compact callus is not suitable for suspension culture.
iii. Embryogenesis:
The process of formation of somatic embryos from the callus is called embryogenesis. Sometimes, somatic embryos are not formed rather somatic buds are formed which after germination give rise to whole plant. Sub-culturing leads to healthy growth of callus and rapid embryogenesis.
iv. Organogenesis:
The process of differentiation of shoot and root from the somatic embryos is called organogenesis. Sometimes, a complete plant develops directly from the somatic bud. In such cases somatic embryos are not formed.
Usually a plant develops from somatic embryos after germination. Plants thus obtained are transferred after sometime to pot culture from the culture medium. The soil of pots should be sterilized to make it pathogen free before transplantation of regenerated plants from culture medium to pots.
Project Report # 4. Applications of Plant Tissue Culture in Crop Improvement:
Plant tissue culture has several useful applications in crop improvement.
The main applications are:
(i) Generation of variability
(ii) Development of haploids,
(iii) Embryo rescue,
(iv) Somatic hybridization,
(v) Selection for disease resistance,
(vi) Selection for salinity and metal toxicity resistance,
(vii) Selection for drought resistance,
(viii) Micro propagation, and
(ix) Preservation of germplasm.
These aspects are briefly described below:
i. Generation of Variability:
The variation which is induced in the tissue culture is an important source of variability for crop improvement. Depending upon the explant, tissue culture induced variation is of three types, viz., gametoclonal, somaclonal and protoclonal.
(a) Gametoclonal variation:
The variation which is observed among the plants which are regenerated from gametic culture is called gametoclonal variation. Such variation is observed among the plants which are regenerated from anther or ovule culture.
(b) Somaclonal variation:
The variation that is observed among the plants which are regenerated from callus cultures of somatic explants such as meristems is called somaclonal variation.
(c) Protoclonal variation:
The variation which is observed among the plans which are regenerated from callus cultures of protoplast is referred to as protoclonal variation.
Main features of culture induced variation are briefly presented below:
(i) The variation is genetic in origin and hence heritable.
(ii) Somaclonal variation occurs in both sexually and asexually propagated species. But the frequency of such variation is very high in vegetatively propagated species (up to 75% in, potato and sugarcane) and low in seed propagated species (0.8-1.2% in maize).
(iii) It occurs in both oligogenic as well as polygenic characters (Table 31.3).
(iv) In majority of cases, somaclonal variation is heterozygous in origin. Rarely the variation is true breeding or homozygous as has been reported in wheat and mustard somaclones.
(v) All three types of culture variations result due to chromosomal changes such as deletion, duplication, inversions and translocations.
(vi) The frequency of variation is higher in protoclones than in somaclones and gametoclones due to stress imposed by the process of cell wall removal and its subsequent synthesis.
Somaclonal variation is useful in several ways. It helps in the:
(i) Isolation of disease resistant,
(ii) Salt tolerant,
(iii) Herbicide tolerant,
(iv) Metal toxicity tolerant, and
(v) Early maturing variants.
Disease resistant somaclones have been isolated in several crops like sugarcane, potato, tobacco and ornamental plants. In many crops, heritable somaclonal variations lead to the development of new germplasm. From such variation, mutant’s with resistance to diseases, stress conditions and good quality can be isolated after proper screening.
ii. Development of Haploids:
Haploids can be developed by tissue culture technique. Haploids are developed by anther culture. Haploids are made diploid by colchicine treatment. Haploids have been developed in more than 250 plant species by anther culture technique.
In China, one variety each in wheat [Jinghua 1] and rice [Guan 18] have been developed for commercial cultivation by tissue culture. The new varieties are better in agronomic properties than old varieties.
iii. Embryo Rescue:
Embryo culture technique helps in making the interspecific crosses successful when there is post fertilisation disharmony between the embryo and endosperm. The embryo in interspecific cross is removed before abortion and cultured in nutrient medium. Embryo rescue technique has been used for successful interspecific hybridization in genus Trifolium and Lycopersicon.
Embryo culture technique has also been used for making inter-generic hybrids successful between Triticum and Aegilops, Triticum and Secale and several other genera. In barley, isolation of ovules from interspecific crosses immediately after fertilization has helped in regeneration of barley plants in Germany.
At the International Rice Research Institute, Philippines, embryo culture technique has been successfully used for transfer of Brown Plant Hopper (BPH) resistance from wild species Oryza officinalis to the cultivated species O. sativa.
Similarly, embryo rescue technique has been used in several other crops. The main drawback of embryo culture is that it is applicable to those distant crosses where crossing is possible and problems are related to post fertilization.
iv. Somatic Hybridization:
Crossing of plants through fusion of somatic cells is known as somatic hybridization. In such hybridization, the sexual process is bypassed. The fusion of cells takes place through protoplasts. Protoplasts are naked cells or cells without cell wall.
Somatic hybridization through protoplast fusion permits hybridization between any two plants irrespective of their taxonomic relationship. In other words, it makes incompatible crosses possible.
Somatic hybridization consists of several steps, viz:
(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, and
(vi) Characterization of hybrid plants.
(i) Protoplast isolation:
The cell wall is removed by mechanical or enzymatic process. Removal of cell wall makes possible the fusion of protoplasts of diverse origin and also uptake of foreign DNA. Protoplasts are isolated either from mesophyll tissues or from suspension cultures. Culture requirements of isolated protoplasts are similar to those of single cell.
(ii) Protoplast fusion:
The fusion of protoplasts consists of three main steps given as follows:
(a) Plasma membranes of two or more protoplasts come into close contact.
(b) Fusion of membranes at small localized regions making bridge between protoplasts.
(c) Extension of cytoplasmic bridges and rounding off of the protoplasts forming spherical homo or heterokaryons. After fusion, mixing of the cytoplasm of two cell occurs. There is equal cytoplasmic contribution from both parents. Thus, somatic hybridization differs from sexual hybridization in several aspects (Table 31.4).
(iii) Selection of hybrid cell:
After fusion, there is a mixture of parental types, homokaryons and heterokaryons. Union of protoplasts of the same species leads to the development of horpokaryons. Union between protoplasts of two different species gives rise to heterokaryons. The heterokaryons are selected on the basis of phenotype. The somatic hybrids have a phenotype distinct from the parental material.
(iv) Culturing of hybrid cells:
Hybrid cells are cultured in a medium highly enriched with organic compounds. Growth hormones, viz., auxins and cytokinins are always required in the nutrient or culture medium.
(v) Regeneration:
Plants are regenerated from hybrid tissues and their various characters are recorded.
Uses:
There are several uses of somatic hybridization in plant breeding.
Some potential uses are given below:
i. It makes hybridization possible between diverse species (distant crosses).
ii. It helps in the production of somatic hybrids by way of fusing genetically different protoplasts.
iii. It results in the production of allotetraploid plants in a single step.
iv. Segregation does not occur in somatic hybrids, therefore, heterosis can be conserved in such crosses.
v. It also permits transfer of organelles between parents in intra and interspecific hybrids and also permits development of transgenic hybrids.
Interspecific somatic hybrids have been developed in Datura Nicotiana, Petunia, Brassica and Sorghum. The new hybrids have diploid chromosome complements of both the parental species. Thus, protoplast fusion between two diploid species leads to the development of an amphidiploid plant.
Pomoto and Raphano-brassica are notable examples of man-made crops which have been developed with the help of protoplast fusion. However, these crops have combinations of undesirable characters.
v. Selection for Disease Resistance:
For selection of disease resistant lines, the pathogen is included in the culture medium. The callus cultures of susceptible genotypes support the growth of pathogenic fungi while resistant cells do not support as has been observed in potato and tobacco. In potato, resistant plants to wilt and root rot, caused by Fusarium oxysporium, have been isolated by tissue culture technique.
In tomato, tobacco and alfalfa, resistance to virus was obtained by infection of callus by weak virus. Tissue culture can help in reviving some of the varieties of crop plants which have become obsolete due to their susceptibility to a particular disease.
Mosaic virus is a serious problem in pulse crops like greengram, blackgram, soybean and other crops like papaya and okra. Tissue culture can help in isolation of mosaic free lines in these crops. Disease free material has been developed in potato, tobacco, tomato, sugarcane and some fruit crops through tissue culture technique.
vi. Selection for Salinity and Metal Toxicity Resistance:
Tissue culture plays an important role in the identification and isolation of salt resistant genotypes. The cell culture technique is used in breeding of salinity resistant genotypes. Millions of cells are subjected to an elevated level of saline solution in a flask.
Only resistant or tolerant cells will survive in such solution. The surviving cells are used for regeneration of salinity resistant plants. Thus, cell culture is a simple and rapid method of developing salinity resistant genotypes. Similarly, genotypes resistant to herbicides and metal toxicity can be isolated.
vii. Selection for Drought Resistance:
In the culture medium drought is induced by the use of a chemical known as PEG (Poly ethylene glycol). Drought resistance or water stress tolerant genotypes have been isolated through tissue culture technique in tomato and sorghum. Low proline content is an indicator of water stress tolerance. It also helps in isolation of water stress tolerant lines in tissue culture.
viii. Micro propagation:
Tissue culture technique is useful in rapid and mass multiplication of plant material. Micropropagation is used for this purpose. Micropropagation refers to regeneration of plants from isolated meristemetic cells or tissues or from somatic cells. It is also known as microcloning.
Micropropagation 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. Micropropagation can also be used for mass multiplication of superior hybrids as an alternative to the production of hybrid seed.
Micropropagation has several advantages as given below:
i. It ensures pathogen free health status of the propagules. Propagules are small plants developed by micropropagation.
ii. It helps in rapid multiplication of material. Large number of propagules can be obtained from a single plant by this method.
iii. The material multiplied by this technique can be maintained in a small place. The transportation of such propagules from one place to another is also easy.
iv. The micropropagation can yield results faster than conventional breeding. It is mostly used in horticulture, floriculture and forestry.
This technique has been used to obtain early and disease free lines in strawberries, banana, citrus and some timber trees. In field crops, micropropagation has been used in potato and sugarcane.
The main problems with this technique are:
(i) Somaclonal variation, and
(ii) Variation in chromosome number due to continuous sub-culturing.
ix. Preservation of Germplasm:
Preservation of germplasm in the form of tissues is an important application of tissue culture in crop improvement. The cell or tissues can be preserved in liquid nitrogen in the long term storage.
The cells or tissues are treated with dimethyl sulphoxide to protect against freezing injury. Preservation of tissues is more useful in vegetatively propagated crops which usually are unable to produce seed. Such storage would require less space.
Project Report # 5. Techniques of Tissue Culture used in Plant System:
i. Embryo Culture:
The supply of proper nutrients in artificial medium under cultural set up, can promote growth of hybrid cells and mutants which being unstable in the beginning, require proper nursing in artificial medium. In fact, the embryos in culture have been utilized for overcoming compatibility barrier.
ii. Haploid Culture:
Over and above the diploid tissues, the pollen can also be cultured for the production of haploid plants as initially done by Nitsch and Nitsch in France and Maheswari and Mukherjee in India. Such haploid plants are very useful for transferring foreign gene in the cells. This is because of the fact that, single set of genes of haploid tissue does not pose any complex problem for the expression of a foreign gene.
If the tissue would have been diploid, the problem of gene interaction as well as dominance and recessive expression might stand in the functioning of the foreign gene. Further, the haploid plant also can be cultured for production of homozygous diploid through doubling of chromosomes by colchicine treatment.
In all cases, the totipotency of plant cell, that is the capacity of regeneration of the whole plant from a single cell is the basic issue for any programme on tissue culture.
iii. Protoplast Culture:
For foreign gene transfer, however, one of the essential steps in tissue culture is the use of protoplast, devoid of any cell wall. The protoplast culture technique, where the cell walls are digested through enzymes – celluloses and macerozymes, leaving the naked protoplast with the nucleus, provides the ideal- medium for incorporation of foreign genes in the cell.
iv. Protoplast Fusion:
An offshoot of protoplast culture technique utilized in biotechnological procedure is cell fusion. This method permits the fusion of cells of widely different species to undergo fusion. This is mediated by certain agents, such as polyethylene glycol (PEG), leading to the production of hybrid cells through fusion of two nuclei.
Such cells are also known as somatic hybrid cell as they contain diploid somatic nuclei of two different species. In such cases, chromosome number also become doubly diploids due to fusion of two diploids. However, if the protoplast of two haploid plants is used in fusion, the regenerated plant can be diploid.
The cell fusion method which is now widely used, was achieved initially in species of Petunia by Cocking, Nicotiana by Carlson, as well as Solanum by Melchers. The hybrid regenerants of tomato and potato were termed as “Pomato” raised by Melchers in Germany.
v. Suspension Culture:
Moreover, just as pieces of tissues, explants of different organs can be cultured, cell suspensions following softening and suspension through specific enzymes and media, are used to yield single cell in suspension which can conveniently be cultured for regeneration.
Production of secondary metabolites and biotransformation are the important applications. Cell plating is done to isolate the mutant lines through single cell culture.
vi. Micro Propagation:
One of the important uses of tissue culture is to utilize mass propagation in vitro for conservation of endangered species, as well as species of economic and medicinal value. In view of rapid denudation of forests and other human practices in relation to industry, agriculture and excessive land use, several valuable species are on the verge of extinction.
The propagation in vitro through organogenesis or embryogenesis which utilizes only small amount of tissue, has become a powerful tool for increase of individuals. The seeds of endangered as well as other economically valuable plants can also be maintained for a long period without the loss of viability through preservation in ultra cool temperature, otherwise termed as cryopreservation.
vii. Artificial Seed:
Somatic embryoids may develop in vitro through embryogenesis. These can be preserved through the preparation of artificial seeds. The artificial seeds in which the cultured embryos are preserved by coating with sodium alginate. It can maintain the viability for a long period before being attempted for regeneration.
viii. Conservation:
Cryopreservation and artificial seeding have now become extremely important strategies for conservation. The techniques for cryopreservation of both seeds and plant organs in culture form important components of germplasm bank and seed bank as well. However, germplasm bank includes both in situ and ex situ conservation in different Biosphere Reserves and National Parks.
ix. Somaclonal Variation:
In addition to these advantages, the culturing in artificial medium may lead occasionally to the origin of abnormal cells termed as ‘somatic variants’. Such somatic variants if successfully cultured and regenerated, somatic mutants may arise.
Therefore, culturing of tissues is designed to secure mass propagation of identical individuals and also occasionally, the variants. The origin of variants thus contributes to enrichment of genetic diversity.