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Micropropagation.-Plant Tissue Culture


Plant tissue culture broadly refers to the cultivation in vitro of all plant parts, whether a single cell, a tissue or organ under aseptic conditions. Recent progress in the field of plant tissue culture made this area as one of the most dynamic and promising experimental biology.

This new technique has enabled us to increase the knowledge in the following field of studies. Totipotency, nutrition, metabolism, division, differentiation and preservation of plant cells. Morphogenesis and plant regeneration from individual cells or tissues through the process namely organogenesis or somatic embryogenesis.

Variations generated through in vitro culture. Evolution of haploids through anther and pollen culture including ovule culture. Wide hybridization programmes through ovul e, ovary and embryo cultures to overcome both prezygotic and postzygotic sterility mechanisms Micropropagation of plant materials In vitro selection of mutants tolerant to biotic and abiotic stresses. In vitro culture and secondary metabolite biosynthesis. Plant genetic engineering through in vitro culture methods and DNA transfer technique. Thus plant cell, tissue and organ culture permeates plant biotechnology and cements together its various aspects like Physiology, Biochemistry, Genetics and Cell Biology. Like other subjects, plant cell and tissue culture has its own origin and development. The chronology of major events in this field is presented for the benefit of the new entrants into this field.

1756- Duharmel du Monceau H. L discovered callus formation from the decorticated elm tree. This very old work was foreword for the discovery of plant tissue culture.

1839- Schwann, T.H expressed the view that each living cell of a multicellular organism would be capable of developing independently if provided with proper external conditions.

1853- Trecul. A performed experiments on callus formation by decorticated trees such as Robinia, Pawlonia and Ulmus.

1878- Vochting. H obtained very luxuriant callus from Brassica rapa and proposed the polarity in development of buds from the upper portion and roots or callus and from the lower portion of a stem piece.

1885- Roux, W.Z made the first experimental step in tissue culture when he removed a fragment of the neural plate of a chick embryo and cultivated in warm salt solution.

1893- Rechinger, C described callus formation on isolated stem fragments and root slices.

1901- Morgan, T.H coined the term totipotency to describe the capability of a cell to form an individual plant.

1902- Haberlandt, G published a paper on "Experiments on the culture of isolated plant cells: In that he says "I should like to point out the fact that, in my cultures, despite the conspicuous growth of the cells which frequently occurred, cell division was never observed. It will be the problem of future culture experiments to discover the condition under which isolated cells undergo division". He clearly set forth the purposes and potentialities of cell culture after having attempted and failed in the culture of isolated plant cells. The reasons for his failure may be (i) use of three monocotyledonous genera for much of his work, (ii) culture of mature differentiated green mesophyll and pallisade tissues, (iii) contamination during culture growth.

1907- Harrison , R.G devised methods of cultivating fragments of living nerve.

1910- Carrel, A was the first scientist who demonstrated the culture of living cells outside the body of an organism.

1922- Kotte, W succeeded in cultivating small root tips of pea and maize in various nutrients. The roots developed well and their growth was maintained for long periods but no subculture was attempted.

1922- Robbins, W.J started cultivation of excised root tips and stem tips of maize under sterile conditions; however, the cultures did not survive independently.

1925- Liabach, F demonstrated the most important application of the embryoculture by crossing Linum perenae with L. austriacum to get hybrid plants from shrivelled seeds.

1934- Gautheret, R.J made preliminary attempts with liquid medium for cultivating plant tissues but failed completely. Later he cultured the explants on medium solidified with agar, and got healthy calli from the explants.

1934- White, P.R obtained indefinite survival of cultured tomato roots on subculturing in liquid medium.

1939- White, P.R., Gautheret, R.J. and Nobecourt, P simultaneously announced the possibility of cultivating plant tissues for unlimited periods.

194l- Van Overbeek, J., Conklin , M.E. and Blackeslee, A.F established importance of coconut milk for growth and development of very young Datura embryos.

1942- White, P.R. and Braun, A.C initiated studies on crown gall and tumour formation in plants.

1944- Skoog, F started his work on organogenesis in tobacco callus.

1946- Ball, E.A showed development of plantlets from sterile cultures of stem tips in Tropaeolum and Lupinus. He is considered as father of micropropagation.

1947- La Rue C.D initiated endosperm cultures of Zea mays and obtained subcultures.

1948- Skoog, F. and Tsui, C studied the chemical control of growth and bud formation in tobacco stem segments and callus cultured in vitro and suggested that callus induction and shoot initiation can be regulated by making manipulations in the culture medium.

1949- Street, H.E. and Dormer, K.J initiated work on root culture and its nutrient requirements.

1951- Morel, G. and Wetmore, R.H got successful culture from monocots, once considered as recalcitrants to the cultural conditions.

1952- Steward, F.C., Caplin, S.M. and Miller, F. K discovered the synergistic action of 2, 4-D and coconut milk in a culture of potato tissue.

1952- Morel, G. and Martin, C were the first to demonstrate that virus free plants can be recovered from infected plants through shoot meristem culture.

1953- Muir, W.H found out the cultural conditions favouring the isolation and growth of single cells from higher plants in vitro and established nurse culture technique.

1954- Muir, W.H. Hildebrandt A.C. and Riker. A.J obtained the first suspension cultures by transferring callus fragments to agitated liquid medium

1955- Miller, C.O., Skoog, F., Von Saltza, M. and Strong, F.M identified a cell division factor viz.,6-furfualamino purine commonly called kinetin.

1957- Skoog, F. and Miller, C.O advanced the hypothesis that shoot and root initiation in cultured callus can be regulated by particular ratios of auxins and cytokinin.

1957- Skoog, F. and Miller, C.O discovered and introduced the idea of synergistic effects of auxins and cytokinins in promoting cell division in tobacco.

1958- Steward, F.C., Mapes, M.O. and Mears, K observed the phenomenon of somatic embryogenesis in suspension culture of carrot. They also reported that cells in suspension derived from explanted roots of cultivated carrots were capable of forming unorganised cell clusters, which in turn could yield first roots, then shoots and ultimately whole plants.

1959- Reinert, J observed the somatic embryo formation from callus cultures of carrot grown on an agarified medium.

1959- Melchers, G. and Bergmann L were first to culture haploid tissues other than pollen.

1960- Cocking, E.C discovered the technique of isolation and culture of protoplasts after digesting the cell walls enzymatically and demonstrated new cell wall regeneration on protoplasts from tomato fruit locule tissue.

1960- Bergmann L was first to obtain callus by plating cells from suspension cultures on to solid medium. This plating involved mixing cells with warm sugar medium just prior to gelation in petridish (Bergmann plating technique)

1960- Morel, G discovered a technique to produce virus free progenies by meristem culture in Cymbidium.

1964- Guha, S. and Maheshwari, S.C cultured mature anthers of Datura innoxia to study the physiology of meiosis and accidentally noticed the development of embryoids from the anthers plated on basal medium supplemented with kinetin and coconut milk.

1965- Vasil, V. and Hildebrandt, A.C described rearing of a mature tobacco plant from a single cell grown initially in microculture.

1966- Torrey, J.G advanced the hypothesis that organogenesis in callus is initiated with the formation of clusters of meristematic cells called meristemoids.

1966- Stroun, M. Anker, P., Charles, P. and Le Doux L made DNA transfer in tomato under in vitro conditions.

1970- Kasha, K. J and Kao, K.N produced haploid plants of Hordeum vulgare by in vitro culturing of embryos obtained by cross Hordeum vulgare with Hordeum bulbosum in which elimination of bulbosum chromosome occurred.

1971- Takebe, I., Labib, G. and Melchers, G regenerated whole plants from isolated mesophyll protoplasts of tobacco.

1971- Bendich, A.J. and Filner, P used the cells and tissues in culture for transformation studies.

1972- Withers, L. and Cocking, E.C laid foundation for the protoplast fusion technique.

1973- Potrykus, I made the first attempt on chloroplast and nucleus transfer from Petunia hybrida into albino protoplasts of the same species.

1974- Melchers, G. and Labib, G proposed hybrids resembling the sexual hybrids by fusing protoplasts of two varieties of Nicotiana tabacum

1974- Murashige, T developed the concept of developmental stages in cultures in vitro: Stage I: Explant establishment; Stage II: Multiplication of propagule and Stage III: Rooting and hardening for planting into soil.

1975- Morel, G established cold storage of regenerated plants for a year.

1976- Mullin, R.H. and Schlegal, D.E successfully employed cold storage to maintain in vitro virus free plantlets of strawberry.

1976- Seibert, M established shoot initiation from carnation shoot apices frozen to -196xC.

1978- Zelcer, A., Aviv, D. and Galun E developed a protoplast fusion procedure called Donor - Recipient protoplast fusion to favour organelle transfer among plants.

1979- Polacco, J.C. Sparks, R.B. and Havir, E.A described the cloning of soyabean urease structural gene by the vector mediated transfer system.

1980- Gleba Y. Y.. and Hoffmann F synthesized a new plant "Arabidobrassica by fusing the protoplasts Arabidopsis and Brassica.

1981- Larkin, P.J. and Scowcroft, W.R developed the concept of somaclonal variation: A noval source of variability from cell cultures for plant improvement.

1981- Patnaik, G., Wilson, D. and Cocking, E.C regenerated a whole plant from a single free cultured tobacco protoplast.

1982- Krens, F.A., Molondijk, L. Williams G. J. and Schilperoort, R.A developed poly ethylene glycol method for the direct delivery of DNA into protoplasts.

1983- Zambryski, P. Joos, H., Genetello, C., Leemans, J. Van Montagu M. and Schell Constructed Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.

l984- Watts , J. W. and King, J. M developed a simple method for large scale electrofusion of protoplasts.

l984- Brisson, N. Paszkowski, J. Penswick, J. R. Gronenborn, B. Potrykus, I. and Hohn, T Achieved transformation in which part of the cauliflower mosaic virus genome was replaced by selectable marker.

1985- Gheysen, G. Dahese, P., Van Montaque, M. and Schell, J developed very efficient gene transfer system using natural gene transfer mechanism of Agrobacterium tumifaciens.

1985- Cocking E. C exposed plasma membrane in the tips of root hairs of wide range of crop plants. The procedure enabled whole seedlings to have the plasma membrane at the tips of their root hairs exposed to foreign DNA and other microorganisms.

1985- Tabata, M. and Fujita, Y developed the technique of elucidation of the physical and chemical factors controlling the biosynthesis of the red napthoquinone pigments by Lythospermum erythrorhizon.

1986- Crossway, A. Oakes, J.V., Irvine , J.M., Ward B. Knauf, V.C. and Shewmaker, C.K developed a direct way of transferring cloned genes into protoplasts by microinjection of DNA directly into the nucleus of tobacco mesophyll protoplasts.

1986-Hamill, J. D. Parr, A. J., Robins, R. J. and Rhodes, M.J.C established hairy root cultures of Beta vulgaris and Nicotinna rustica following infection with Agrobacterium rhizogenes and the transformed cultures synthesized their characteristic secondary products at levels comparable with those of in vitro roots from the same variety.

1986- Abdullah, R., Cocking, E.C., and Thompson, J.A demonstrated that normal green rice plants can be regenerated efficiently and reproducibly from rice protoplasts via Somatic embryogenesis.

1986- Pirrie, A. and Power, J.B produced fertile, interspecific gametosomatic triploid hybrids of tobacco by fusing protoplasts of leaf (2n) and pollen tetrad (n).

1986- Kinsara A., Patnaik, S.N., Cocking, E.C. and Power, J.B produced somatic hybrids between Lycopersicon esculentum and L. peruvianum.

1987- Terada, R., Kyozuka, J., Nishibayashi, S., and Shimamoto, K regenerated plantlets form somatic hybrid cells of Oryza sativa, and Echinochloa oryzicola.

1987- Ethlenfelt, N.K. and Helgeson, J.P produced tetroploid and hexaploid somatic hybrids from protoplast fusions between Solanum bravidens (2x, non tuber bearing species) and 2x and 4x S. tuberosum.

1987- Neuhaus, G., Spangenberg, G. Mittelsten Sheid, O and Schweiger, H.G effected gene transfer by microinjecting the DNA into the cells of microspore derived proembryos.

1987- De la Pe$a, A., Lornz, H., Schell, J developed transgenic rye plants obtained by injecting DNA into young floral tillers.

1988- Nomoru, K. and Komamine, A used single cells of carrot from a cell suspension instead of protoplasts, for microinjection and the microinjected carrot cells could divide and differentiated to embryos at a frequency of about 50 percent.

1988- Rhodes, C.A., Pierce, D.A., Mettler, I. J.. Mascarenhas, D. and Detmer J.J produced transgenic maize plant by electroporation.

1988- Toriyama, K., Arimoto, Y. Uchimiya, H, and Hinata K produced transgenie rice plant by electroporation.

1989- Shimamoto, K., Terada, R., Izawa, T. and Fujimoto, H produced fertile transgenic rice plants regenerated from transformed protoplasts.

1989- Prioli, L. M. and Sondahl, M. R recovered fertile plants from protoplasts of maize.

1990- Milanova, V. and Zagorska, N. A succeeded in overcoming hybrid incompatibility between Nicotiana africana and N. tabacum and produced cytoplasmic male sterile plants by embryoculture.

1990- Iida, A., Seki, M. Kamada, M. YHamada Y. and Morikawa delivered genes into cultured plant cells by DNA-coated gold particles accelerated by a pneumatic particle gun.

1991- Kyozuka, J. Fujimoto, H., Izawa, T. and Shimamoto- K succeeded in getting tissue specific expression of maize alcohol dehydrogenase l gene in transgenie rice plants and their progenies.

1991- Spangenberg, G., Fredyl, E., Osusky, M. Nagel, J. and Potrykus, I developed a method for the predictable transfer of partial genomes predictable transfer of partial genomes by using subprotoplasts (cytoplasts and karyoplasts).

1991- Sautter, C., Waldner, H. Neuhaus, G., Galli, A. Neuhaus, G. and Potrykus developed a novel method for the acceleration of microprojectiles. The method is called as microtargeting.

The history of plant tissue culture had its real beginning in 1934 when Gautheret tried to cultivate isolated cells and root tips on organised medium. The momentum from this pioneering work, a new turn in this ongoing research occurred, because of world war II. After the second world war, American plant pathologists became interested in plant tissue culture.

As Steward (1970) pointed out, the plant tissue culture technique is another "Silent Revolution in Agriculture" having very good potentials to supplement conventional breeding approaches. Its potentials and prospects are discussed in subsequent chapters. The techniques' theoretical aspects and their applicabilities are simplified and presented.



The design of facilities for tissue culture research is a key topic in the broader subject of "Plant Cell Tissue and Organ Cultue." The requirements and design for a tissue culture laboratory are mainly governed by the research programme contemplated and also the great variety of fields in which tissue culture is applied. In general, there are certain basic essential facilities which must be introduced into any laboratory where the tissue cultures are to be used.

The basic design of a tissue culture laboratory facility should have the following:

  1. Sterilization room

  2. Media preparation room

  3. Aseptic manipulation room

  4. Aseptic culture room

Sterilization Room

This should contain washer, driers, hot air ovens, and autoclaves. This room should have good ventilation. The following are the important items occupying the sterilization room.

Large sinks with resistance to alkalies and acids and plentiful water supply.

Large bench space for handling the glassware material.

Hot air ovens for sterilizing washed glassware.

Autoclaves for sterilizing media, solution, distilled water, culture vessels and inoculation equipments.

In this, the position of the sink dictates the other arrangements. It is desirable to reserve an area near the sink for all preparative work. It is also better to have the hot air ovens and autoclaves near the preparation area.

Media Preparation Room

The media preparation room should be of convenient size with work benches for cleaning and packing the materials, sinks with water supply, drying racks, storage cup boards or racks for chemicals. This room should be provided with the following items:

Freezers, refrigerators and BOD incubators for storing stock solutions, hormones and plant materials.

Storage racks for chemicals and glassware.

Hot plate-cum-magnetic stirrers for melting and mixing the media.

pH meter for adjusting pH for the media and solutions.

Double water distillation and deminieralization units.

Balances, one to weigh small quantities (preferably an electronic balance) and the other to weigh comparatively larger quantities.

Plastic carboys (l0-20 litres) to store high quality distilled water.

Wire-mesh baskets to autoclave media and culture vessels.

Glassware like glass bottles conical flasks, beaker, graduated pipettes, culture vessels etc. in different sizes.

Filter membranes and their holders to filter sterilize the solutions.

Hypodermic syringes to filter sterilize solutions.

Aseptic Manipulation Room

This facility be designed so as to have minimum of fixed furnishings with a controlled air circulatory system. All kinds of aseptic manipulations of the plant material should be done only in this room. The floor should preferably be of tiled or with polyvinyl chloride sheet for easy washing and quick drying. This room should be of fairly large size to accommodate the following equipments necessary for aseptic manipulations. The important equipments/instruments needed are:

Laminar-Air flow chamber

Spirit lamp or burners to flame instruments

Atomizer, to spray spirit in the inoculation chamber

Instrument stands, to keep sterilized instruments during aseptic manipulations

Large forceps with blunt ends, for inoculation and subculture

Forceps with fine tips

Fine needles for dissections

Scalpels and spatulas

Cork borer for excising tissue cylinders of standard sizes

Microscopes and their accessories

Haemocytometer for cell counting

Aseptic Culture Room

The culture room should be contamination free with controlled temperature-light-humidity conditions. It should have shelves to keep the cultures. Several engineering aspects should be considered in designing a culture room to have a safety and convenient electrical system to have a continuous airflow for uniform temperature regulation, arrange the shelves in a proper way to have a contamination free environment. The following primary facilities are also essential to provide optimal environmental condition in a culture room.

Provision for diffused light with intensity range of 5,000-l0,000 lux.

Air-conditioners to maintain the temperature at 24 q 2xC.

A humidifier to maintain humidity at or above 80 percent.

Automatic timers to regulate the diurnal control of illumination.

Steel racks to keep the cultures.

A shaker for the culture of cell suspensions.

Provision to maintain cultures in dark (as separate dark room or chambers).

General Facilities

The facilities so far described are essential for any tissue culture laboratory. The refinements or sophistications can be made depending on the nature of the research undertaken. The access to the scientific bulletins published by various scientific instruments/equipments and chemicals companies will give a fair idea about the needs for a tissue culture laboratory. The other best way to know about the needs of a laboratory for its layout and equipments is to visit an already established tissue culture laboratory.

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