Principal Applications of Plant Tissue Culture in Plant Science

The principal applications of plant tissue culture in plant science are discussed under the following sub-headings:

1. Micro Propagation

2. Clonal Propagation

3. Production of Genetically Variable Plants

4. Plant Pathology and Plant Tissue Culture

5. Plant Breeding, Plant Improvement and Plant Tissue Culture

6. Production of Useful Bio-chemicals

7. Preservation of Plant Genetic Resources or Gene Conservation Banks

8. Importance of Tissue Culture in Biotechnology

Application # 1. Micro Propagation:

The regeneration of whole plant through tissue culture is popularly called “micro-propa­gation”. This is a technique where a callus mass has been initiated from a single explant taken from any living part of a donor plant and within very short time and space, a large number of plantlets can be produced from such callus tis­sue. Again, it is possible to make a large number of callus pieces from the original stock culture during sub-culturing.

Then it is possible to pro­duce hundreds of plantlets that develop on each of these callus pieces. Therefore, the most obvi­ous advantage of micro-propagation is the numer­ical one. Suspension cul­tures can also be used to exploit this numerical advantage as they produce numerous cell aggre­gates relatively rapidly, generally growing faster than callus tissues.

The numbers of plantlet pro­duction depends upon the number of shoot primordia that can be induced to form within these cell aggregates. Alternatively, if the cell suspen­sion culture happens to be embryo genic, then this propagation potential depends upon the rate at which embryoids are formed by the cell aggre­gates and the rate at which new embryo genic ag­gregates are formed in culture.

Again, the shoot tip and nodes of the regenerated plantlets de­rived from callus culture and cell suspension cul­ture can be multiplied further following organ culture method. As a result, large numbers of a plant species or variety can be propagated all the year around. The plant breeder or grower is no longer restricted by season in the production of large numbers of plants.

Application # 2. Clonal Propagation:

In vitro clonal propagation is a type of mi­cro-propagation. The cultured plants raised from tissue culture are derived asexually and also mul­tiply within culture vessel by asexual means. A sexual reproduction, on the other-hand, gives rise to plants which are genetically identical to the parent plant.

Multiplication of genetically iden­tical copies of a cultivar by asexual reproduction is called clonal propagation and a plant popu­lation derived from a single donor plant in tis­sue culture constitutes a clone. So, the vari­ability that can arise from sexual reproduction and seed formation in a crop plant is omitted.

More specifically, a single plant with desirable characters can be selected from a breeding pro­gramme and propagated so that further trials and selections can be carried out as quickly as possible. The plants with long seed dormancy can be raised faster by in vitro clonal propaga­tion than in vivo seed propagation.

The undesir­able juvenile phase associated with seed raised plants in some variety does not appear in the vegetatively propagated plants from adult mate­rial. For the orchids, in vitro clonal propagation is the only commercially viable method of micro- propagation. Clonal multiplication of cultivar is very important in horticulture and silviculture.

Application # 3. Production of Genetically Variable Plants:

In some callus culture, there is a major ten­dency of the callus tissue towards the numerical variation of chromosomes in the cells that oc­curs after a number of serial subcultures. Such chromosomal variations in culture may arise be­cause of two factors. First, the cells of various ploidy and genetic constitution of the initial ex- plant may be induced to divide and secondly, culture condition may contribute new irregulari­ties.

The chromosomal instability in the cultured cells play an important role in polyploidization of cells and genetically variable plants can be raised from such polyploidized cells by subse­quent micro-propagation. Thus, tissue culture is proving to be rich and novel sources of vari­ability with a great potential in crop improve­ment without resorting to mutation or hybridiza­tion.

Variants selected through tissue culture has been variously termed to as calliclones (from callus culture) or protoclones (from protoplast culture). Larkin and Scowcroft have proposed a general term “somaclones” for plant variants achieved from tissue cultures, irrespective of their origin.

Such variant plants may show some useful characters such as resistance to a particu­lar disease, herbicide resistance, stress tolerance etc. Such changes are valuable for crops which are normally propagated by vegetative methods. Moreover, plant breeders can exploit such vari­ants for their breeding programme.

Application # 4. Plant Pathology and Plant Tissue Culture:

There have been many valuable contribu­tions of plant tissue culture to problems concern­ing plant pathology. One outstanding success is the virus eradication by apical meristem culture and the second success of tissue culture in plant pathology is the result of its application to the problems of plant tumors, especially crown gall.

Virus Eradication:

In virus infected plants, the distribution of viruses in plant body is uneven. It is well known that the apical meristems are generally either free or carry a very low concentration of viruses. The apical meristem culture is the only way to obtain a clone of virus free plant which can be multiplied vegetatively under control conditions that would protect them from the chance of reinfection. The elim­ination procedure of virus can usually be im­proved by combining it with heat therapy of the host plant or the culture.

Virus eradication by apical meristem culture has enormous horticul­tural and agricultural value e.g. in the produc­tion of plants for the cut flower industry when stock plants of registered line must be main­tained in as near-perfect condition as possible. Any infection by virus that affects growth rate or physical characteristics of size and shape is obviously very serious if it afflicts these nuclear stock, for they are the basis of all propagation and breeding.

In the agricultural world, the production or yield of a crop can fall dramatically as a result of viral infection and render that particular va­riety no longer saleable or commercially viable. Tissue culture techniques could be of value in restoring the original properties of the variety, by removing the infection and so bringing it bank into the commercial market. These virus tested stocks could provide ideal material for the na­tional and international distribution of plants, either for further propagation or use as breeding material. It is hoped that these selected virus free cultures would be acceptable to quarantine authorities.

Study of Crown Gall by Plant Tissue Culture:

Smith and Townsend (1907) discovered that crown gall or plant tumor formation was induced by a bacterium, Agrobacterium tumefaciens. Braun (1941) showed that in sunflower Agrobac­terium could induce tumors not only at the in­oculated point but at a considerable distance, where secondary tumors free of bacteria are formed.

Cells of these secondary tumors could be cultivated by tissue culture technique and mul­tiplied on a medium without adding auxin and cytokinin, whereas normal tissue required aux­ins and, in some cases, cytokinins.

Crown gall tissues deprived of bacteria give rise to tumors by grafting. Kulescha (1952) established that they synthesized more auxin than normal tis­sues. Braun demonstrated that crown gall tis­sues free of bacteria contain a tumor-inducing principle (TIP) which may be a macromolecule.

The biochemistry of the crown gall problem was studied by Lidret (1957) who discovered an ami­no acid called lysopin which might be specific for the tumor. Menage and Morel (1965), Gold- mann-Menage (1970) and Morel (1971) isolated two substances of the same chemical family octopine and nopaline and concluded that these opines were not characteristic of the plant but of the bacterium that had induced the tumor.

Crown galls induced by some strains of A. tume­faciens synthesize octopin, while tumors produ­ced by other strains elaborate nopaline. Follow­ing these observations, Morel (1971) suggested that the synthesis of opines depends on the pres­ence of TIP in the tumor cells of genes coming from the bacterium. In other words, the TIP con­sists of DNA. Zwnen et al. (1974) discovered the segment of bacterial DNA which is responsible for the tumoral transformation and opine syn­thesis.

This segment belongs to a large plasmid. Only a small part (about 8%) of the plasmid is stably incorporated and replicated in plant cells. Therefore, we can say that this transferred DNA (T-DNA) contains the genetic information which promotes the tumoral transformation and gene coding for octopine or nopaline.

Finally, geneti­cists and molecular biologists were able to estab­lish the map of the crown gall plasmid. But the basic mechanism of tumoral transformation has not been clarified. It is hoped that plant tissue culture technique can throw some light on the basic mechanism of tumoral transformation.

Application # 5. Plant Breeding, Plant Improvement and Plant Tissue Culture:

The conventional breeding methods are the most widely used for crop improvement. But in certain situations, these methods have to be sup­plemented with plant tissue culture techniques either to increase their efficiency or to be able to achieve the objective which is not possible through the conventional methods.

Embryo culture is now routinely used in recovery of hybrid plants from distant crosses. Some examples are recovery of hybrids from Hordeum vulgare x Secale cereale, Triticum aes-tivum x Agropyron repens, H. vulgare x Triti­cum species. In case of Triticale, a rare hy­brid between Triticum and Secale develop viable seeds.

But most of the tetraploid and hexaploid wheat carry two dominant genes K_r1 and K_r2 which prevent seed development in crosses with Secale. The hybrid seeds are minute, poorly de­veloped and show very poor germination. By embryo culture, 50-70% hybrid seedlings has been obtained. Hybrid seedlings from T. aestivum x H. vulgare are not obtained. But it has been achieved by embryo culture.

When H. vulgare or T. aesiivum (used as male) is crossed with H. bulbosum (used as fe­male) the chromosome complement of H. bulbo­sum is eliminated from the developing embryo. Most of the seedling obtained from such crosses are haploid, having only one set of chromosomes either from H. vulgare or T. aestivum parent.

Embryo culture is also useful for propaga­tion of orchids; shortening the breeding cycle and overcoming seed dormancy. In meristem culture, shoot apical meristem along some surrounding tissue is grown in vitro. It is used for clonal propagation and recovery of virus free plants and is potentially useful in germplasm exchange and long-term storage of germplasm through freeze preservation.

Anther and pollen culture has a potential application in plant breeding and plant improve­ment programme for the production of haploid as well as homozygous diploid plant. All-year-round rapid clonal propagation us­ing plant tissue culture techniques has highlight­ed possibilities for new plant improvement tech­niques.

Protoplast culture and somatic hybridiza­tion is a promising line for plant breeding and plant improvement techniques. But, at present, techniques for selection and multiplication of so­matic hybrid and regeneration of hybrid plants is very limited to a few classical plant species. So it is expected that in near future it would be possible to use this technique for a wide variety of plant species.

Another most important approach is the mutation of tissue culture cells to produce a mu­tant line from which plants can be raised. Pro­duction of mutant line is highly desirable for plant breeding. Callus cells, produced either from vegetative cell or reproductive tissues, can be subjected to a range of mutagenic chemicals e.g. N-nitroso-N-methyl urea or ionizing radia­tions e.g. gamma rays.

The hope is that per­manent changes in the DNA pattern of some of the cells would be achieved by such treatment. Plants could be raised from the treated cultures and any mutant whole plants selected from the population either by physical differences or by metabolic/biochemical differences. Biochemical mutants could be selected for disease resistance, resistance to phytotoxin, improvement of nutri­tional quality, adaptation of plants to stress con­ditions e.g. saline soils and to increase the bio­synthesis of plant products used for medicinal or industrial purposes.

Application # 6. Production of Useful Bio-chemicals:

Man depends on plants for many com­pounds other than food such as medicines, pig­ments, vitamins, hormones, flavoring agents, la­tex and tanins. If most plant somatic cells are totipotent, it should be possible to take a cul­ture of cells from a plant that naturally produces a certain biochemical and cause the culture to produce that chemical under in vitro conditions. The main difficulty is that we do not yet un­derstand the regulation mechanisms that control the production of most biochemical substance and so cannot manipulate them.

Even so, a surprising number of cell cultures have been found that do produce specialized bio- chemicals found in the intact parent, including alkaloid such as nicotine, atropine, ephedrine, caffeine and codeine and their precursors and derivatives. Production of cardiac glycosides and other steroids, benzoquinones, latex, phenolics, anthocyanin’s, organic acids, anti-tumor agents, antimicrobials and various flavors and odours have also been reported.

The first patents for producing bio chemical’s commercially by large-scale plant cell cul­ture was issued in 1956. The general approach since then has been to select high product-yield­ing cell lines, preferably as suspension cultures and to enhance their efficiency either by feed­ing them inexpensive product precursors or by manipulating their biosynthetic control mecha­nisms.

Here the expertise of the microbial prod­uct industry is valuable. Large-volume auto­mated culture vessels called fermenters have been used successfully to mass produce cultured plant cells. This technology will soon be produc­ing selected pharmaceuticals and other high-cost bio-chemicals commercially.

Application # 7. Preservation of Plant Genetic Resources or Gene Conservation Banks:

The need for a programme for the conser­vation of plant genetic resources arises from the rapid changes that are occurring in modern agri­culture practice. The primitive cultivars and wild relatives of crop plants constitute a pool of genetic diversity which is invaluable for future breeding programmes. But these have already led to the replacement by new cultivars which encompass a much narrow range of genetic di­versity.

As a result, there is a very real danger of future breeding being impeded by the shrink­ing genetic bases of some crops. Therefore, stor­age of this sort of irreplaceable breeding material or germplasm (gene combinations available for breeding) and establishment of a centralised gene bank are the practical ways to solve these prob­lems.

Conventionally, germplasms are stored in the form of seeds because they occupy a rela­tively small space and can be stored for many years. But there are a number of important species, particularly root and tuber crops, which are normally propagated vegetatively.

These in­clude potato, sweet potato, yam and cassava. So conventional preservation methods is not appli­cable to vegetatively propagated plants. On the other hand, the cost of maintaining a target pro­portion of the available genotype of these plant in nurseries or field is very high and there is a risk of the plants being lost as a result of disease or environmental hazards. It is now possible with modern tissue cul­ture techniques to provide a germplasm storage procedure which uniquely combines the possibil­ities of disease elimination and rapid clonal mul­tiplication.

In addition, the possibility of using liquid nitrogen freeze-storage techniques for the preser­vation of cell, tissue and apical meristem is being studied. The advantages of this technique sire that cell division and normal cellular reactions are totally arrested at the very low temperature of liquid nitrogen (-196°C), which means that there should be a high level of genetic stabil­ity and that the chemical reaction responsible for cellular damage will not occur.

The plant materials can be stored in liquid nitrogen for desirable period. This technique could be par­ticularly valuable for storage of any germplasm which needs to be maintained in a clonal form. This technique is known as cryopreservation or freeze preservation of tissue or cell.

It has al­ready been shown to be successful with a range of cell cultures e.g., carrot cell and sycamore sus­pension cells and meristem tips from a number of crop plants including asparagus, tomato and potato. Plant tissue or meristems of such plants have been successfully recovered from liquid ni­trogen and grown into normal, mature fertile plants.

Application # 8. Importance of Tissue Culture in Biotechnology:

In 1981, the European Federation of Bio­technology defined “biotechnology as the integrated use of biochemistry, microbiology and chemical engineering in order to achieve the tech­nological application of the capacities of micro­bes and cultured tissue and cells”. Some peo­ple equate it with the new field of genetic en­gineering, while others take a broader viewpoint defining it as the evaluation and use of biological agents and materials in the production of goods and service for industry, trade and commerce.

To reduce confusion we will limit our interpre­tation to the two areas most often equated with biotechnology. One of these, the genetic engi­neering of organisms, is the endeavor that in­spired the coinage of the term “biotechnology” in the 1970’s. The other area consists of recent developments in the fields of tissue and cell cul­ture, most notably those that have enabled us to fuse two different eukaryotic cells into a sin­gle cell that possesses the combined properties of both.

Cell suspension culture in liquid medium is a relatively young field of biotechnology. This technology involves the large-scale culture of iso­lated plant cell under condition which induces them to synthesize the natural secondary meta­bolites characteristic of parent plants from which they were obtained.

In recent years, the tech­nique of callus culture and cell suspension cul­ture has also been viewed, particularly from the view-point for the study of biosynthesis and metabolism of steroids and cardiac glycosides. Biotechnologists are also trying to increase the synthesis of natural compounds or new com­pounds by higher plant cells culture as a result of mixing or feeding transformable precursors in the culture medium.

The metabolic process of transformation or conversion of such added pre­cursors into the natural or new compound within the cell is known as biotransformation. Biotech­nologists are also trying to augment the synthesis of medicinally important alkaloids in culture by means of fungal elicitor.

This means that cells are cultured in a liquid medium by adding re­quired quantity of the bacterial filter sterilized extract of certain fungi. It has been observed that the fungal extract in certain cases helps to increase the synthesis of a desirable compound by the higher plant cell.

Biotechnologists are also trying to modify the genetics of the cultured cells by three ways such as:

(i) Mutagenesis and selection of cell lines in cell suspension culture,

(ii) Transplanta­tion of foreign genetic material in protoplasts by means of genetic engineering and

(iii) Somatic hybridization by the fusion of distantly related plant proplast just to widen the genetic diversity of hybrids.

In the field of mutagenesis and selection of cell lines in vitro and the exploitation of totipotency, biotechnologists are trying to improve plants, for example by producing crop spe­cies that are more resistant to draught, disease, poor soil condition, chemical pesticides and her­bicides.

Another goal of biotechnology using plant tis­sue culture technique is to produce self-fertilizing plants that would provide their own usable ni­trogen. Plants rely on a few types of bacte­ria and cyanobacteria to fix atmospheric nitro­gen into a biologically usuable form. Geneti­cally engineered plants that contain the bacterial genes for nitrogen fixation could grow well even in nitrogen-poor soil.

Genetic engineers are also developing plants that produce their own pesticides (one bite and pest dies). In 1986, the first of such genetically engineered plants successfully passed a field test.

The value of plant protein to human diet is being improved by creating corn or beans that manufacture a complete protein, one with all the amino acids essential to the human diet.

In another effort (W. David et al., Science, Vol. 234, pp. 856, 1986) the luciferase gene from the firefly, Photinus pyrahs was used as a re­porter of gene expression by light production in transfected plant cells and transgenic plant.

A complementary DNA (cDNA) clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35 S RNA promoter) was introduced into plant protoplast cells (Dancus carota) and into plants (Nicotiana tabacum) by the use of the Agrobactenum (time- facience tumor-inducing plasmid.

In this exper­iment, stable expression of the firefly luciferase gene in plant cell and transgenic plants has been achieved. The transgenic plant incubated in luciferin also emits light like firefly. The successful introduction of animal DNA into plant genome has opened a new avenue in the field of biotech­nology. With the help of this technology produc­tion of antibody has also been possible in plant (Nature 342 No. 6245, 1989).

Summary:

The importance and application of plant cell and tissue culture in plant science are vast and varied. There have been many valuable con­tribution of plant tissue culture in the field of fundamental and applied botany. The regeneration of whole plants through tissue culture is popularly called “micro-propaga­tion”. By this method, a large number of plant species can be propagated all the year round. The plant breeder is no longer restricted by sea­son in the production of large numbers of plants.

In vitro clonal propagation is a type of mi­cro-propagation. By this method the variabil­ity that can arise from sexual reproduction and seed formation in a crop plant, can be omit­ted. The plant with long seed dormancy can be raised faster by in vitro clonal propagation. For the orchid, in vitro clonal propagation is the only commercially viable method of micro-propa­gation. Clonal multiplication of a cultivar is very important in horticulture and silviculture.

The plant tissue culture is also proving to be rich and novel sources of variability with a great potential in crop improvement without re­sorting to mutation or hybridization. Larkin and Scowcroft have proposed a general term “Soma­clones” for plant variants obtained from tissue cultures, irrespective of their origin. Such vari­ant plants may show some useful characters such as resistance to a particular disease, herbicide resistance, stress tolerance etc. Plant breeders can exploit such variants for their breeding pro­gramme.

There have been many valuable uses of plant tissue culture to solve the problems con­cerning plant pathology. One outstanding suc­cess is the virus eradication by apical meristem culture and the second success of tissue culture in plant pathology is the result of its applica­tion to the problems of plant tumors, especially crown gall.

The convention, breeding methods are most widely used for crop improvement. But in cer­tain situations, these methods have to be sup­plemented with plant tissue culture technique either to increase their efficiency or to be able to achieve the objective which is not possible through conventional methods.

Embryo culture is now routinely used to recovery of hybrid plant from distant crosses. Meristem culture, anther and pollen culture, plant protoplast culture and somatic hybridization contribute a lot of poten­tial applications in plant breeding and plant im­provement programmes. Plant tissue culture, particularly cell sus­pension culture, has been exploited for the pro­duction of useful alkaloids, cardiac glycosides and other steroids.

Preservation of plant genetic resources can be achieved by plant tissue culture. Cryopreservation of plant tissue, cell or meristem in liquid nitrogen for desirable period and the recovery of whole plant via organogenesis is also a valuable tool for the preservation of very rare germplasm and to maintain a clone for crop improvement.

Plant tissue culture is relatively a young field of biotechnology. Biotechnologists are try­ing to modify the genetics of the cultured cells by three ways—mutagenesis and selection of cell lines; transplantation of foreign genetic material in protoplast by genetic engineering, and somatic hybridization by the fusion of distantly related plant protoplasts just to widen the genetic diver­sity among the existing plants.