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Prof. Ramona Maghiar
Introduction
Vegetal biotechnology represents today a fascinating field of research and production which faces a spectacular dynamic.
The occurrence of the vegetal biotechnology produced a real revolution in the process of plants’ amelioration, in this way being able to obtain – by using modern technologies – an unlimited number of plants in a shorter time compared to their multiplication by traditional culture techniques.
Theoretical approaches
The name of biotechnology (bios ═ life, tehne ═ art, handcraft) appeared for the first time in the year 1656 in French texts meaning techniques of using the mechanisms and materials. The history of cultivating vegetal tissues starts in the year 1838, when Schleiden and then in the following year Schwann, formulated, beside the theory of common structural unit of living structures that have the cell at their basis, the hypothesis of the cellular totipotentiality. This hypothesis claims that any cell owns all the genetic information it needs in order to form an organism (excerpt from Gautheret, 1959).
Haberlandt, in the year 1902, claimed that any diploid or haploid cell contains all the hereditary information in the genome, thus every vegetal cell isolated or cultivated in optimum conditions will take back its activity, will grow and will be able to multiply.
In 1904, the successful experiments carried out by Hanning on the vegetal embryos cultures proved the possibility of some plant fragments to grow on artificial media.
Between 1904 and 1932 all the attempts to cultivate vegetal explants on artificial media do not have a significant importance, moreover, Küster - in 1926 – claimed that this issue cannot be “solved” (taken from
Morel, 1948).
After the year 1930 the discovery of auxine by Went and his collaborators, of cytokinins by Skoog and his collaborators favored the first successes in the field of “in vitro” culture of cells and vegetal tissues (Gautheret, 1939; Nobécourt, 1939 and White, 1939).
Morel and Martin (1952) obtained a devirused breeding material, starting from infected plants by “in vitro” cultivation of apical caulinar meristems.
In 1957 Skoog and Miller published an article and launched the idea according to which the growing type or the morphogenetic events are determined by the quantity interactions between auxine and cytokinine, so called “hormonal balance”.
Murashige and Skoog (fig. 1), in 1962 made a culture media that bears their names with a large concentration of mineral salts, which proved to be the best for the “in vitro” growing of several types of plants.
This culture media has today a key role in plants multiplication by vitrotechniques.
The plants obtained by biotechnology are not superior to those obtained by traditional methods of sexuate multiplication, the former being clones free of viruses, sometimes being represented by genotypes more resistant to the stressful factors from the septic medium of life.
Biotechnology allowed, by its special techniques, the countries with tradition in amelioration and cultivation of ornamental plants to obtain and trade huge quantities of flowers. Thus, Holland obtained in 1995 a total of 53.8 million flower plants, from which 22 million were flower plants in pots, 11.6 million cut flowers, 9.4 million orchids, 6 million bulb flowers and 4.8 million other ornamental plants (Pierik, 1997).
The flower species, of great interest both for researchers and cultivators are the following: Nephrolepis, from which in Holland in 1995 13 million samples were obtained, Gerbera - 6 millions, Lilium – 5,1 millions, Phalaenopsis - 4,4 millions, Spatiphyllum - 3,1 millions, Saintpaulia, Ficus, Cymbidium, Aster, Alstromeria, Limonium, totalized together over 1 million samples annually (Pierik, 1997).
By traditional methods a small number of plants can be obtained, while by “in vitro” methods great quantities of plants, of higher quality and more uniform as aspect are multiplied in a short time. According to the nature of explanted tissues or cells, to the moment when they were inoculated it is recommended to choose a certain medium and conditions of culture, depending on the objective followed.
The success of “in vitro” cultivation of vegetal explants depends, in a great extent, on the nutritive blending that has to correspond to the vital necessities of the inoculated tissues in order to compensate the lack of the most important endogen factors, on which the existence of the respective cells incorporated in the plant’s body depends.
For the “in vitro” cultivation techniques of vegetal tissues and cells, some compulsory operational steps have to be (fig. 2), and they are the following:
- setting up a culture, respectively obtaining an “in vitro” culture with regenerative capacity;
- guided incubation and growth of phytoinoculi, which implies not only inducing the regenerative processes but also the morphogenesis ones;
- subcultivation (repication or transferring the cultures);
conservation of cultures;
“ex vitro” transfer of vitroplantlets and their acclimatization to the septic life regime.
The results obtained by micropropagation have an indubitable economic efficiency, because by exploiting the totipotentiality of vegetal cells, the plants’ ability to multiply is developed.
The “in vitro” multiplication and propagation methods are practicable at any vegetal species, the plants generated “in vitro” are juveniles, without any diseases (but not immunes), qualities that make them to be more vigorous in culture on the field.
By micropropagation vegetal explants can be multiplied in a fast rhythm, thus from a unique meristem of orchid, during a year 4 million samples can be obtained, precise copy of the donating plant (Murashige, 1974). This multiplication is performed by subcultures operated successively “in vitro” and using a little biologic material, using microexplants, the donating plants remaining alive.
The vitrocultures of vegetal explants are also experimental models, representing valuable instruments in the modern researches of vegetal biology.
The vitroplantlets form and develop better in the conditions of thermic regime of 25˚C ± 2˚C (Torres, 1989) and a photoperiod of 16-18 hours of light per day (Hvoslef-Eide, 1989). Besides obtaining vitroplantlets, another important aim is to perform successfully the acclimatization of neoplantlets generated “in vitro” in the conditions of the septic medium, by applying those procedures that imply a diminishing of evapo-transpiration, but also ensuring the best conditions for obtaining a better rooting in “ex vitro” regime of vitroplantlets, without increasing the costs of producing the growing material, resulted by multiplication procedures (Cachiţă and Sand, 2000).
Maintaining the in vitro culture for long periods of time has the disadvantage of appearing some physiological modifications or genetic mutations in the cells of phytoinoculi, fact that asks for the application of some specific methods of in vitro cultivation of phytoinoculi (Dandekar, 2003).
Bibliography
- Cachiţă, C.D., Sand, C., Biotehnologie vegetală. Baze teoretice şi practice. Vol.1 (Vegetal Biotechnology. Theory and Practice. Volume 1), Ed.Mira Design, Sibiu, 2000.
- Dandekar A.M., Techniques for manipulating quality and productivity traits in horticultural crops. Acta Hortic; 625, 2003.
- Gautheret, R.J., Sur la possibilite de realiser la culture indefinie des tissus de tubercules de carotte, C.R.Acad.Sci., 208, 1939.
- Gautheret, R.J., La Culture des Tissus Vegetaux, Techniques et Realizations. Editor: Masson et Cie, Paris, 1959.
- Haberlandt, G., Kulturversuche mit isolierten Pflanzenzelle, Sitzungsber, Akad. Wiss., Wienn, Moth-naturwiss, K1, t.111, Sectia I, 1902.
- Hvoslef-Eide, A.K., Colture "In Vitro“ e Micropropagazione in Ortoflorofrutticoltura. Editor: Cesena A., Cesena, 1989.
- Morel, G., Recherches sur la culture associee de parasites obligatoires et de tissus vegetaux, Ann. Epiphyt., 14, 1948.
- Morel, G., Martin, G., Guerison de dahlias atteints d’une maladie a virus, C.r.hebd.Seane, Acad. Sci., 235, Paris, 1952.
- Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiologia Plantarum, 15, 1962.
- Murashige, T., Plant propagation through tissue cultures, Ann. Rev. Plant Physiol., 25, 1974.
- Nobecourt, P., Sur la perennite et laugmentation de volume des cultures de tissus vegetaux, C.R. Soc. Biol., 130, 1939.
- Pierik, R.L., Ruibing, M.A., Developments in the micropropagation industry, in: The Netherlands Plant Tissue and Biotehnology, 25, 1997.
- Skoog, F., Miller, C.O., Chemical regulation of growth and organ formation in plant tissue cultured in vitro, Symp. Soc. Exp. Biol., 11, 1957.
- Torres, K.C., Tissue Culture Techniques for Horticultural Crops. Editor: Reinhold, V.N., New York, 1989.
- White, P.R., Potentially unlimited growth of excised plant callus in an artificial nutrient, Amer. J.Bot., 26, p. 59-64, 1939.
Iconography
| Mihai Bravu Technical College, Bucharest, Romania |
| Prof. Ramona Maghiar |
