Applications apical meristem culture of stem

* Morphogenesis in the apical meristem.

Multiplication of plant mass.

* Recovery of pathogen free plants.

* Thermotherapy.

* Bibliography.

1. Morphogenesis in the meristem.

The evidence for growing tips and meristems isolated from different angiosperms date of the last century. Rechinger in 1893 tried to grow in sand, with and without attached nutrients, buds isolated from Populus nigra and Fraxinus ornus L., callus formation was observed basal and a slight expansion of the external parts, but there was no root system development (cited by Sahbde and Murashige, 1977).

Buds isolated from Ceratopteris thalictroides (Brougn, 1908) grown in peat, producing leaves that were smaller and morphologically simpler than those of the parent plants (cited by Sahbde and Murashige, 1977).

Later, Robbins (1922 th) made the first attempts to cultivate apices isolated in an artificial environment (in vitro). Cabbage apices 1 cm in diameter (Brassica oleracea var capitata), maize (Zea mays) and cotton (Gossypium herbaceum) showed limited development when grown in a medium containing inorganic salts and glucose, only a response in the apices cabbage and corn in the dark (there was development of chlorotic leaves and some roots).

Withe (1933) obtained positive results in isolated shoots growing in hanging drops half seal, wherein the apices survived a few weeks. Primordia formation was small and there was complete differentiation.

Ball (1946) conducted a trial with apex of Tropaeolum majus L. and Lupinus albus L., two morphologically and physiologically different angiosperms. The inoculum consisted of the apical meristem plus three leaf primordia and stem some underlying tissues. The apexes of both plants developed complete plants in different nutrient media. The importance of Ball is that it emerged that the perpetuation of growth and organogenesis in apical meristem of angiosperms requires underlying stems and leaf primordia.

The first positive result was reported by Wetmore and Morel (1949), and Wetmore (1954), with vascular cryptogams as Adiantum spp, spp and Seleginella Osmunda spp. The tips of these plants measured from 100 to 150 in length and were developed in Knop solution and 3% sucrose (this medo proved unsuitable for higher plants).

Ball (1960) tested the apical meristem culture of Lupinus albus L. in a medium containing amino acids, coconut milk, gibberellic acid and vitamins, noting only a small elongation of meristems. By repeating the experiment, leaving some leaf primordia at the meristems and obtained complete plants. Based on these results, Ball concludes that: a) The apical meristem exhibits a hormonal and nutritional dependence on the underlying stem and leaf primordia, and b) The angiosperm apical meristem undergoes a biochemical difference that prevents it from producing certain substances essential for growth and maintenance of a given meristem.

Murasihge Smith (1970) developed whole plants from apical meristem of Nicotiana glauca Grah., Tropaeolum majus, and Coleus blumei Beenth in a medium containing inorganic salts of Murashige and Skoog (1962), 100 mg / l of inositol, 04 mg / l of thiamine – HCl, 1 to 2 mg / l IAA, 30 g / l sucrose and 1% agar, we obtained the development of leaves at 12 days. They showed that the underlying structures were unnecessary, since complete plantlets were obtained in a medium containing only IAA exogenously administered. In an apical meristem culture of Coleus blumei, which included two or more pairs of leaf primordia, Smith (1970) confirms the observations of Ball (1960), regarding not require exogenous growth substances if the inoculum contained primordia, if the leaf primordium was not present supply was critical AIA, gibberellic acid represses the initiation of leaves and roots and adenine seems slightly reverse rebuke.

Shabd and Murashige (1977) demonstrated the dependence of hormonal sources of apical meristems underlying carnation leaves and stem tissues. This is because when the demo is inoculated in the absence of meristematic leaf primordia are expanded to include exogenous auxins and cytokinins in the medium. Jacobs (1961) mentioned that in Coleus blumei maximum auxin synthesis was observed in the second pair of leaf primordia and Shabd confirms the synthesis of auxin in the same place, but the source of cytokinins located in the first couple of expanded leaves.

As is known, a proper balance of auxins and cytokinins in vitro induces the formation of buds and / or roots, depending on the concentration of both species and it is working, which means that the young leaves are capable of producing auxin necessary for further development of the root and when mature synthesize cytokinins which promote the formation of buds, and leaf primordia whether or lateral buds (the main source of cytokinins are the roots).

When isolating the apical meristem is damaged the region subdistal (Wardlaw, 1965), as it begins to form buds if the incision is deep and provascular damages tissue, if tissue is not damaged provascular begins to form sheets. These observations showed that the metabolites necessary for growth and development of the apical meristem could be transported through the tissue provascular (Wardlaw, 1945, 1949 and 1956).

It is recalled that the relationship auxin – cytokinin in vitro is essential for the morphogenesis of the meristem and that the current research focuses on finding the optimum concentration of both the auxin and the cytokinin, taking into account changes that have inter-response vegetables.

2. Mass multiplication of plants.

The ease of using the technique of plant tissue culture for mass propagation in vitro plant material produced by the initiation of adventitious buds, bulbs, tubers, asexual embryos or shoot growth of axillary buds and mass production of plantlets from meristem .

Commercial propagators and have standardized the method of maintaining a commercial batch of parent material in vitro, where, besides having the advantage of keeping this material, considering the large number that can be kept in a confined space with environmental conditions required and desirable quality and health. This is due to morphogenetic potential with meristems and other tissues of the plant shoot production.

There are many plant genera that are propagated by tissue culture to obtain large quantities of commercially important plants, such as Anturium andreanum, Dieffenbachia amoena Snow, D. Perfection picta, Philodendron oxycurdium, Scindapsus aureus, Syngonium podophyllum, Chrysanthemum morifolium, jamesonni Gerbera, Begonia spp, Saxifraga sarmontosa Tricolo, etc.

3. Recovery of pathogen free plants.

Many vegetable crops, particularly those that are vegetatively propagated, virus contains systematic, which affect their performance or bring down your performance. Therefore, before escape is commercially desirable to produce virus-free plants that can be clonally multiplied.

In many species this may be achieved with heat treatments of various organs in vitro, or composite plants, as well as the application of chemicals (Hollings, 1965). However, certain viruses have withstood all tests for the eradication of these media and other methods are necessary.

Currently the most successful alternative is the apical meristem culture, often combined with chemotherapy and heat treatment. When these methods are used, plants are not only released the virus, but also fungi and other pathogens.

The first crop was successfully Morel and Martin (1952), who cultivated dahlias apices infected with virus and managed to get healthy plants. Morel (1955) conducted a meristem culture with Cymbidium, Cattleya and Phajus, obtaining virus-free orchids, and in 1960 reported that it is necessary to make certain changes in the environment, as genres Phalaenosis Vlandas and do not respond favorably (cited by Lecoufle, 1969).

Other research on ornamental plants, but only mentioned the most important, by the contributions they have provided in this field.

As for vegetable and fruit species have been significant research in relation to obtaining healthy material from apical meristems, the most important were made in potato (Sussex, 1963, Ingram and Robertson, 1965; Accatino, 1966; Gregorini and Lorenzi, 1964, Mellor and Stace – Smith, 1977; Wang, 1977 and Solorzano, 1983), chile (Juo, Wahg and Chien, 1973), strawberry (Belkengren and Muller, 1962), apple (Elliott 1972; Jones, 1976; Aboot, 1976, Jones and Hopgood, 1979), grapevine (Barlass and Skene, 1978), etc.

4. Thermotherapy.

Involves the application of high temperatures to complete plants or individual parts:

Some viruses are more stable than others, and this causes many problems in their eradication, and therefore require longer periods for some and shorter for others, with different treatments. Some species are damaged by continuous high temperatures, so it is recommended alternating high and low temperatures, thus decreasing the damage to the tissues of plants.

5. Chemotherapy.

It is the application of chemicals to the culture medium, resulting in a greater likelihood of plant pathogens, when applied to different chemotherapeutics of exogenously to the culture medium, we are ensuring the production of healthy material.

In the growing tips of potato (Solanum tuberosum L.) was used malachite green as a chemotherapeutic agent, the results observed in a greater number of virus-free plants “X” (Norris, 1954, Oshima and Livingstone, 1961) obtained results like to include in the medium 2, 4 – D, obtaining a higher frequency of healthy plants.

Wade Johnstone (1974) suggest that applying high concentrations of cytokinins and auxins are promoting or encouraging acts of active growth of the host, but not the viral particle. There is not much evidence for it, but said that growth regulators reduce the concentration of the virus but not eradicated (Gohen and Walkey, 1978).

Newer chemotherapy studies suggest that incorporating into the culture medium as Kibavirin chemical metabolites (Virazole), an antiviral product, we can eradicate the virus (Mough, 1976).

Combining chemotherapy with snuff meristem culture infected with virus X in potato was achieved total eradication of the (Sherpard, 1977) and more recently vizarole was added to a concentration of 50 and 100 mg / l, being that eradicated CMV meristematic tissue of Nicotiana rustica.

As you can see, the progress achieved in the meristem culture technique, assisted with the application of thermotherapy and chemotherapy, has made great strides in the matter of application of techniques for different species.

It is noteworthy that in developed countries in recent years have set up companies engaged in a commercial propagating different species, including vegetables, staple crops, fruit trees, forest species and medicinal plants.

On the next page shows a representative diagram of how to carry out a program to produce virus free plants commercially.

To view the graph select the “Download”

The selection of the mother plant is very important because the plants will get are identical to the parent (asexual propagation). If the mother plant is sick, it would be desirable to know the specific virus present, as well as growing conditions vary, thermotherapy or simply apply meristem culture.

Inoculum size is also important, since there are meristems that measure 0.01 to 0.1 mm in diameter, and stem apices measuring 0.1 to 0.3 mm in diameter, usually the number of virus-free plantlets produced is inversely proportional to size meristem or apex grown (Walkey, 1978).

Finally, it is important to note that the medium is crucial, since it plays a vital role in the nutritional, hormonal and environmental specific species we are growing, they should be similar to those that have under natural conditions.

The in vitro process includes several covers, including:

* Stage I. Establishment of aseptic culture.

* Stage II. Propagation of healthy propagules (subcultures).

* Stage III. Rooting of plantlets obtained.

* Stage IV. Transplant and conditioning soil gases.

In Stage I are inoculated or apical meristems in culture tubes, a few days observed for growth of contaminating organisms (fungi or bacteria) culture tubes contaminated should be removed and those who remain healthy should be checked regularly to observe the development of what will be the future plant (formation of leaves, buds, stems, etc.).. This stage comprises of 4 to 6 weeks.

In stage II should be separated propagules obtained and transferred to a fresh medium for the proliferation of new outbreaks and to develop them. According to the amount of material needed, will be the subcultures should be made. It is recommended not to exceed five subcultures, as the plant vigor and quality of it can decrease. Each subculture takes 4 to 6 weeks.

In Stage III was the rooting of propagules to obtain complete plants, lasting 2 to 4 weeks.

In stage IV the texture, pH and substrate composition will be given in the specific conditions of the species concerned. The conditioning step to the greenhouse lasts 6 to 8 weeks.

The plants identified as virus-free (nuclear material) will be propagated in vitro to obtain the material foundation. These are propagated by traditional methods for plants recorded and, finally, plants and certified quality.

When regenerated plantlets have been established on the ground, the virus must be indexed several times during the first year following the acquisition, as there are certain viruses whose resurgence is delayed. Driving conditions should be careful and this material is known as nuclear material. When this material is spread by the in vitro methods can be stored for long periods at low temperatures (Nullin and Schlegel, 1976), as it is a less expensive than keeping them in special greenhouses, which is what is considered foundation material.

7. Bibliography.

M. HURTADO Daniel V / MERINO M. Maria Eugenia. Editorial Trillas. Third Edition. August 1994. Mexico. Pp 233. Pg 136 to 148.

Biotec. Lucas Emilio Alfredo Carrillo