Tissue Culture: Definition, History and Importance

In this article we will discuss about the Tissue Culture:- 1. Definition of Tissue Culture 2. History of Tissue Culture 3. Importance.

Definition of Tissue Culture:

Tissue culture is the method of ‘in vitro’ culture of plant or animal cells, tissue or organ – on nutrient medium under aseptic conditions usually in a glass container. Tissue culture is sometimes referred to as ‘sterile culture’ or ‘in vitro’ culture. By this technique living cells can be maintained outside the body of the organism for a considerable period.

According to Street (’77) tissue culture is referred to any multicellular culture with protoplasmic continuity between cells and growing on a solid medium or atta­ched to a substratum and nourished by a liquid medium.

By plant tissue culture new plants may be raised in an artificial medium from very small parts of plants, such as, shoot tip, root tip, callus, seed, embryo, pollen grain, ovule or even a single cell, whether the cultured tissue develops into a plant or grows unorganized depends on the genetic potential of the tissue and the chemical and physical environment.

According to the parts used for culture the aseptic plant culture may be of follow­ing types:

(a) If a seedling is cultured it is called plant culture.

(b) When an embryo is cultured it is known as embryo culture.

(c) If plant organs, such as, shoot tips, root tips, leaf primordia, flower primordia or immature fruits are cultured, it is called organ culture.

(d) The culture of unorganized tissues from cell proliferations of segments of plant organs is called callus culture. Cell proliferations are formed in the explant due to injury caused by excision.

(e) When a single cell or small cell aggregate in a dispersed state is cultured, it is called cell suspension culture. It is also known as cell culture.

Culture of single cell is sometimes called single cell cloning. The portion of the plant to start the culture is called an explant. Culture derived from a single explant is called a clone. In order to maintain a culture for a comparatively longer period the culture me­dium is changed from time to time.

This process will remove those harmful excretory substances which have accumulated due to metabolism. By transferring a fragment of the parent culture to a new medium subculture is done. Such a fragment is called an inoculum.

History of Tissue Culture:

In 1832 Theodor Schwann said that cells could be cultured outside the body of the organism if provided with proper external conditions. In 1835 Wilhelm Roux cultured embryonic cells of chicken in salt solution. Reichinger (1839) said that fragments thicker than 1.5 mm were capable of growth but fragments below this limit failed to grow. He did not used any nutrient in his experiment.

Arnold (1885) and Jolly (1903) observed growth and cell division of leucocyte cells of salamander in culture. In 1907 American zoologist Ross Granville Harrison successfully cultured nerve cells of frog in solidified lymph. Harrison is known as the father of tissue culture.

M.J. Burrows (1910) cultured embryonic tissue of chicken in plasma. Mammalian cells were first cultured by Alexis Carrel. By repeated sub-culturing he was able to culture the tissue for 34 years. Organ culture was first done by D.H. Fell (1929) in England. He used solidified plasma and embryonic extract as nutrient medium.

History of Plant Tissue Culture:

German botanist Gottlieb Haberlandt first attempted to culture plant tissues ‘in vitro’. He started his work in 1898. He used cells from palisade tissues of leaves, cells from pith, epidermis and epidermal hairs of various plants for culture in media -containing Knop’s solution, aspergine, peptone and sucrose.

The cultured cells sur­vived for several months but the cells failed to proliferate.

This may be due to:

(a) Use of very simple media,

(b) Culture of highly differentiated cells and

(c) Aseptic techni­ques were not used.

In similar experiments by some later workers cells remained alive for a long period but they failed to divide.

Culture of meristematic tissue was started in early 20th century. Isolated root tips were first cultured by Robbin in 1922. Working independently Kotte (’22) also made similar observations. Robbin and Maneval (’23) cultured roots and maintained the culture for 20 weeks by sub culturing.

In 1934 White first successfully cultured isolated tomato roots in a medium conta­ining sucrose, inorganic iron salts, thiamine, glycine, pyridoxine and nicotinic acid etc. Gautheret (’34) noted that cambium culture from Salix capraea, Populus nigra etc. continued to grow for few months under aseptic conditions. He later (’37, ’38) used medium supplemented with B-vitamins and IAA.

In 1937 White recognised the importance of B-vitamins for growth of root cultures. Went and Thimann (’37) discovered the importance of auxin (IAA). Nobecourt (’37, ’38) obtained some growth in culture of carrot root explants. He also noted root differentiation in tissue culture. In 1938 tumour tissues of tabacco hybrid were succe­ssfully cultured.

In 1939 working independently three scientists, White in USA and Nobecourt and Gautheret in France cultured successfully plant callus tissue on synthetic medium continuously. Gautheret (’39) said that carrot culture required Knop’s solution supplemented with Bertholots’ salt mixture, glucose, gelatine, cysteine HC1 and IAA.

White (’39a) in culture of procambial tissue from young stem of the hybrid Nicotiana glauca × N. langsdorfii noted unlimited and undifferentiated growth. He showed that this tissue could be repeatedly subcultured. White (’39b) recorded development of leafy buds in tissue culture of the hybrid N. glauca × N.langsdorfii in nutrient medium.

Tissues from various plants were cultured subsequently. It was noted that older cultures show increasing degree of organization. The role of vitamins in plant growth was also recognized. Wetmore and Wardlaw (’51) successfully cultured shoot tips of pteridophytes (Selaginella, Equisetum and ferns).

Tissues of Sequoia semipervirens were cultured by Ball (’55). Pollens of Taxus and Ginkgo biloba were cultured by Tulecke (’59). Conifer tissues were successfully cultured by Harvey and Grasham (’69).

From tissue culture studies important information about root-shoot relationship can be obtained. Several scientists reported about the factors controlling vascular tissue differentiation from tissue culture studies.

Van Oberbeck (’41) cultured embryos of Datura on a medium supplemented with coconut milk. Importance of coconut milk and 2-4D as nutrient was recognised. The stimulatory pro­perty of coconut milk is due to the presence of zeatin.

The potent cell division factor was found to be kinetin, which is a 6 furfurylaminopurine. Cytokinin is 6-substituted amino-purine compound, which can stimulate cell division in culture of plant tissues. Monocot tissues were successfully cultured on a medium containing coconut milk.

Callus culture of Tagetus erecta and Nicotiana tabacum on liquid culture medium when agitated on a shaker produced suspension of single cells or cell aggregates (Muir ’53). Such cell suspension could be subcultured.

Studies on cell suspension cul­ture were carried out by Muir, Hildebrandt and Riker (’54), Street, Shigomura (’57), Torrey and Reinert (’61), Reinert and Markel (’62). Muir (’53) developed paper raft nurse technique for single cell culture.

In this method isolated single cells were put on a square filter paper, placed on a active nurse tissue, which supplies the required nutrients to the growing single cell. In another method cells were suspended on a han­ging drop in a micro-chamber.

Bergman (’60) working with suspension cultures of Nicotiana tabacum var. sansum and Phaseolus vulgaris var. ‘early golden rod’ developed agar plating technique of single cell cloning. In this me­thod single cell fraction was separated by filtration, mixed with warm agar and then plated in a petridish in thin layer.

Melchers and Bergmann (’59) noted that after several cultures of the haploid shoot of Antirrhinum majus there was increase in ploidy. Ball (’46) noted the possibility of regeneration of whole plant in culture of shoot tip of angiospermic plants. Wetmore and Wardlaw (’51), Morel (’60) obtained whole plants from culture of shoot apices having 1 or 2 leaf primordia. Morel (’64) used this method for culture of orchids.

A cell which can develop into a whole organism by regeneration is called a to­tipotent cell. This term was coined by Morgan in 1901. According to White (’54) if all the cells of a multicellular organism is totipotent, then such cells in isolated condi­tion regain their dividing power and can produce whole plants. In an organism this capacity remain suppressed.

It was noted that single cells are capable of producing new plants. From pollen and anther culture haploid embryos were obtained. A method of microspore culture of Nicotiana and Datura was developed by Nitsch (’74, ’77). He was able to double the chromosome number and obtained homozygous diploid plants.

From anther culture of tobacco Bourgin and Nitsch (’67), Nakata and Tanaka (’68) obtained haploid tissues and haploid embryoids.

Cocking (’60) recorded release of protoplasts from root tip cells by using fungal cellulase in 0.6M sucrose. He was able to culture isolated protoplasts, which regenerate new cell walls and produce cell colonies and ultimately plantlets.

In many plant suspension cultures cell protoplasts had been successfully released. Plant tissue culture technique is used for the study of tumour physiology.

White and Brown (’42) were able to culture bacteria free crown gall tumour. In Scorzonera hispanica Gautheret (’46) noted that the callus culture which initially required auxin, produced some proliferations which can grow in auxin deficient medium.

Such inheri­ted changes occurring in the nutritional requirements (especially involving auxin) of cells of a culture is called habituation. An auxin habituated culture does not re­quire the supply of exogenous auxin (Butcher ’77). Butcher noted (’77) that when auxin and cytokinin habituated tissues are grafted into a healthy plant, tumours are produced.

Pathogen free plants can be obtained by culturing apical’ meristem.

By late 70’s it was evident that plant tissue culture technique can be successfully used in various field of agriculture, such as, production of pathogen free culture, pro­duction of secondary products, clonal propagation, mutant culture, haploid breeding and genetic engineering.

By tissue culture, pathogen free cultures have been produced. This technique is im­portant for plant pathological investigations. Protoplasts in culture are used for virus infection and biochemical studies.

From suspension culture secondary products can be synthesised in large amount. Some of these substances are enzymes, vitamins, food flavours, sweeteners, anti-tu­mour alkaloids and insecticides. In Japan ‘in vitro’ culture has been achieved at industrial level.

Clonal propagation of orchids and several other ornamental and economic plants have been achieved by ‘in vitro’ culture. In potato clonal propagation has been achieved by culturing leaf cell protoplasts. By using mutagens in culture followed by selection disease resistant or stress resistant mutant plants have been regenerated.

By haploid breeding few cultivers were produced. Hybrids of related but sexually incompatible species have been produced by protoplast fusion. By this technique hybrid between potato and tomato has been produced. By cell fusion of isolated cells from two different species hybrid tobacco plants are produced.

Dormancy period of seeds can be shortened by excising the seeds and culturing its embryo on artificial medium (embryo culture). Abortive embryo can be grown successfully by embryo culture.

Foreign genes with desirable characters attached to a plasmid may be inserted into the naked protoplast usually by means of liposomes. The expression of introduced gene in the mature plant is still doubtful.

Importance of Tissue Culture:

Tissue culture has great importance in studies of plant morphogenesis, physio­logy, biochemistry, pathology, embryology, cytology etc. From tissue culture studies it is possible to know bow simple cells differentiate and become specialized to perform special functions. Various changes taking place in a cell can be noted from clonal culture.

Interrelationship between two cells can be studied in tissue culture. With the help of phase contrast cine-photomicrography a very clear understanding of mitosis and meiosis is possible in tissue culture. Haberlandt noted the importance of tissue culture in studying plant morphogenesis. Relationship between growth and differentiation can be well understood from such a culture.

In vegetatively propagating plants many plantlets are formed very quickly from callus culture or culture of explants. Orchids, which normally propagate very slowly, can form many plantlets very rapidly in shoot tip culture. This is also noted in car­nation.

By tissue culture method new plant variants can be obtained by isolating gene­tically unique cells. From callus cultures of tobacco, carrot, asparagus etc. new plants are formed. Such plants show genetic variability.

From studies of mutant cells, the biochemical and developmental process of an organism can be better understood. In tissue culture, mutation can be easily induced and from such mutant cells mutant plants may be produced.

In tobacco, paddy etc. from anther culture haploid plantlets are produced. By doubling the chromosome homozygous plants are obtained most rapidly. So, this process has immense importance in plant breeding.

Tissue culture technique has been successfully used in nutritional research. The effects of various mineral salts, vitamins etc. on growth may be studied in culture. Many important information about glucose metabolism, nitrogen metabolism and hormone production can be obtained from ‘in vitro’ culture.

Suspension culture under controlled conditions may be used to solve many physiological or biochemical problems and also provides a system for the production of important plant products, such as, plant alkaloids.

From cell and organ culture under controlled environmental conditions nutri­tional and metabolic processes can be studied. Some mutant cells cannot grow in a medium which does not contain a special nutrient. From this biochemical steps of a process can be determined.

Tissue culture has great significance in pathological studies. The effect of various medicine on cells infected by pathogens can be studied in tissue culture.

Culture of maize cells from plants susceptible to the race T of Helminthosporium maydis were treated with pathotoxin of the fungus. Scientists were able to obtain cells resistant to this fungus. From such cells resistant plants were also produced.

Tissue culture technique is employed in the studies of plant tumour diseases and host parasite relationship. Disease free plants can be produced by tissue culture te­chnique. Tissue culture has great importance in vaccine production. In 1949 vaccine for poliomyelitis has been produced after observing that the poliomyelitis virus can attack human cells. Later vaccines for mumps, meseales, and influenza have been pro­duced.

The process of virus attack, effect of virus on Post cells and how new viruses are produced etc. have been studied in tissue culture. The behaviour of substances, which can prevent virus attack has been studied on virus infected cells.

In tissue culture the behaviour of normal and cancer cells can be studied. It has been noted that some viruses and carcinogenic chemicals can produce cancer. Effect of radiation and chemicals on normal and cancer cells has been studied. From such studies it may be possible to know which chemical substances can destroy cancer cells.

From tissue culture studies information about some hereditary diseases of man has been obtained. Carriers of some diseases can also be identified through tissue cul­ture technique. From leucocyte culture the cause of mongolism in man has been dis­covered. From such culture abnormal Philadelphia chromosome has been identified. This chromosome has some relation with chronic granulocytic leukomia.

When tissue transplant is done from one person to the other then sometimes there is tissue rejection. So it is necessary to match the tissue of donor and receiver before actual transplant. This can be done by culturing the mixed leucocytes of the donor and the receiver.

Distantly related species usually do not hybridize. This difficulty can be omitted by cell fusion and protoplast fusion technique. Carlson in 1972 successfully produced hybrid plants by protoplast fusion between Nicotiana glauca X N. langsdorfii. Power (’76) obtained hybrids between Petunia hybrida and P. parodii by protoplast fusion.

Kaw and Wetter (’77) produced hybrids between tobacco and soyabean by cell fusion. Thus cell fusion and protoplast fusion techni­ques have great importance in plant breeding. Tetraploid fertile Lolium and Festuca hybrids were obtained by somatic cell fusion.

Those embryos which fail to produce mature fruits normally can be cultured and from such embryo cultures plants are produced. Embryo culture also prevents seed dormancy. Cooper (’78) obtained hybrid plants between barley and rye by embryo culture.

Conservation of Germplasm:

By tissue culture plant germplasm can be stored.

This method can be success­fully used to solve various problems:

(a) Many seeds, such as, seeds of Citrus sp., Coffea sp., Hevea brasiliensis etc. retain their viability for a short period. These can be conserved by tissue culture.

(b) Vegetatively propagated plants (such as, banana, potato, sweet potato, and yam) which do not produce seeds or which are highly heterozygous, are stored as cuttings or tubers. This requires much labour charge and are expensive to propa­gate. This problem can be solved by tissue culture.

Many fruit trees of Rosaceae are propagated by budding, grafting and layer­ing. By tissue culture rapid propagation of such plants are possible.

(c) In many economic plants, such as, coconut, date plam etc. vegetative pro­pagation normally does not occur. The germplasm of such plants can be conserved by tissue culture.

(d) Many trees reproduce very slowly. By tissue culture such paints can be multiplied rapidly and many plants with parental genotypes are formed.

For conservation of germplasm the cells should be stored in such a condition which allows minimum cell division. One of the method attempted is storing of cells in liquid nitrogen having a temperature of — 196°C.

For germplasm conservation shoot tips or plantlets can be stored. Such stored materials can be used as and when required.

In tissue culture cell division can be suppressed by various methods:

(i) To the medium growth retardant may be added. The substances used are absicic acid, mannitol, sorbitol, malic hydrazide, succinic acid etc. Potato shoots are successfully stored in a medium containing malic hydrizide.

(ii) Low temperature is helpful for storage of cells in culture. Cultures of potato, sweet potato, beet, grape, apple, etc. can be stored by this method. Temperate crops (e. g. potato) are stored usually at a temperature of 0—6°C and tropical crops (e.g. sweet potato) at 15—20°C. By this method meristem culture of strawberry has been conserved for six years.

(iii) The concentration of nutrients of the culture medium may be changed. Some substances required for normal growth may be supplied at a lower concentra­tion or may not be supplied at all.

(iv) The gas composition within the culture vessel may be changed. The atmos­pheric pressure or oxygen concentration may be lowered to conserve the cells.

Production of Secondary Metabolic Products:

Some plants produce secondary metabolic products, such as, alkaloid, anti­biotic, glycoside, resin, tannin, saponin, volatile oil, etc., which are of considerable economic importance.

By cell culture various secondary metabolites (e.g. allergin) have been synthesised artificially. Cultivation of plants producing secondary metabolites can be improved significantly by tissue culture. There are certain disadvantages in the production of secondary metabolites by tissue culture.

Some of these are:

(i) In cell culture synthesis of secondary metaboli­tes occur at a lower rate than in an entire plant,

(ii) After prolonged culture ‘in vitro’ the production of secondary metabolites may decrease or even stop,

(iii) The cost of large scale production of secondary metabolites in cell culture is high. So, only very rare and expensive secondary metabolites are produced by tissue culture.

Explants from stem, leaf (e.g. Mentha) or root (Daucus carota) are taken and cul­tured on suitable medium. After callus formation explants are sub-cultured on fresh medium. In early stages of callus formation and in first few subcultures secondary metabolites are formed. But in subsequent subcultures usually the amount of secondary metabolites decreases.