The in vitro culture (term that literally means in glass ), includes different techniques to multiply, among others, plant or animal material. Growing tissues plant involves different techniques to spread different plant material, including protoplasts ( cells deprived of their cell wall), cells, tissues, organs and plants complete. This type of cultivation consists of taking a portion of the plant and artificially providing a sterilized nutritious culture medium, which allows to regenerate one or many plants; aseptic conditions and being able to control the growth of the explants make this method a very important tool for the mass propagation of endangered plants, or recalcitrant plants or for cloning individuals that have desirable agronomic and ornamental characteristics (better fruits, resistant to drought, colors of flowers, etc.), where the first Experiences related to the cultivation of plant tissues date back to 1902 (Stewart & Kane, 2006).
Numerous applications of in vitro culture in plants have been: obtaining plants free of viruses , production of synthetic seeds, conservation of germplasm, obtaining secondary metabolites, production of new hybrids, genetic improvement of plants, among others.
The main applications of in vitro cultivation in orchids are conservation in germplasm; in addition, the plants produced in this way are of great interest for the reintroduction programs of native species in areas of environmental preservation (Araujo, 2004). For example, in 1922 the first successful experiment was achieved with in vitro germination of orchid seeds (Knudson, 1922). Since the Orchidiaceae family is one of the most diverse within the plant kingdom due to its wide variety of species (Giovanny Giraldo, 2012), there are rough estimates of 26,000 species of orchids worldwide, of which, Colombia houses approximately 15% (Betancur, 1988); the diversity of ecosystems present in the Colombian geography favors the ecology of the orchids giving rise to exotic and endemic species. It is currently believed that there are 4,030 species, grouped into 260 genera, distributed in the Andean (87.2%), Pacific and Amazonian (10.6%), Caribbean and Orinoquia regions that have the lowest values in terms of wealth and abundance (5% and 4%, respectively) (Andrade-C, 2011).
In addition, some species of orchids have been classified as bioindicators of the level of fragmentation or integrity of the plant communities of which they are part, especially those that have a terrestrial habit (Díaz, 2009) since they are sensitive to environmental changes, habitat destruction , pollution and pesticide applications, due to the fact that they require special conditions such as soil microbiota and the permanence of native flora for their survival (Janeckova et al., 2006) and several species of this family of plants present great ornamental and commercial importance due to the diversity of shapes, sizes and colors of their flowers; in addition, some have great industrial importance, as is Vanilla planifolia whose capsules are used in the manufacture of liquors, beverages, cosmetics and flavorings (Beltrán & Martínez, 1996) and the genera Bletia and Cyrtopodium whose pseudobulbs are used for the creation of glues (Vinccon & Cetzal, 2013); even, some species of the Orchidiaceae family have been attributed curative properties due to their fragrances through aromatherapy techniques. (Beltrán & Martínez, 1996)
However, the germination of orchids in the wild requires external agents such as fungi , especially of the species ( Rhizoctonia sp.) To obtain a high percentage of germination determining a symbiotic relationship seed-fungus (jijón-orchid meristem) This is due to the absence of endosperm in their seeds, which is a limiting factor for their development and survival in vivo (Salazar- Mercado , 2012).
Even though some species are locally abundant, the Orchidiaceae family has the highest number of threatened or endangered species, occupying the first place in the plants; Although they are found practically in all natural ecosystems and in many of them there are a large number of species, due to particularities in their life cycles, several orchids have low geographic and altitudinal distribution ranges , so they can be seriously threatened (Calderón -Sáenz 2007).
Therefore, it has been chosen to develop its in vitro culture as a tool for the conservation of orchids. In order to know a little more about this orchid conservation tool, in this work we seek to collect basic information about the in vitro culture of structures such as seeds, meristems and apices in the Orchidiaceae family.
In vitro cultivation
Plant tissue culture comprises a set of techniques widely used for in vitro propagation, its results in germination rates are higher compared to germination in natural conditions (Araújo, 2004). However, a disadvantage is the high cost of components used for the culture medium, among them, the agar (most used solidifier), since demand has raised its price (Martín-Gordo et al., 2012). It has been proven that the implementation of alternative starches or gums as gelling agents enables reduction in costs of means. In vitro tissue culture culture up to 70% compared to agar, physiologically speaking the best results are obtained in the partial substitutions that have been studied (Martín-Gordo et al., 2012; Mengesha et al., 2012).
Because the formulation of the culture medium is essential for in vitro propagation since it provides the necessary nutrients ( minerals , vitamins , growth regulators, etc.) for the development of the plant, it must be prepared with different combinations of nutrients according to the requirements of each species and part to propagate since there are different parts of which a plant can be developed by in vitro culture such as seeds, meristems (apices and buds), protoplasts and nodes (Faria et al ., 2002).
2.1. Asepsis in the in vitro culture .
It is common to find that the prepared culture media are distributed in the containers that allow the entry of light and of a suitable size for the plant to be planted in said medium (glass jars regularly), covered with aluminum foil and then wrapped On periodic paper , lots are marked with the date and finally they are taken to the autoclave where they are sterilized with wet steam under pressure at 15 pounds for 20 minutes (Botero et al., 2008), this is because said means not only have optimal conditions for The growth of orchids, there is a myriad of fungi and bacteria that can grow with great ease.
Under natural conditions, orchids are spread through the production and dispersion of tiny seeds, but even though each orchid capsule can contain between 200,000 and 600,000 seeds depending on the genusand the species (Botero et al., 2008), only a few germinate and have the possibility of reaching adulthood, thus hindering their mass production (Cañas, 1991). The germination of orchid seeds in natural conditions presents some limitations since they lack endosperm and as a consequence of the reserve of nutrients necessary for their development, this makes the presence of a mycorrhizal fungus necessary for them to develop (Chugh et al. ., 2009, Hossain et al., 2013, Pedroza – Manrique et al., 2010, Rosa – Manzano et al., 2014). Due to little knowledgeOf the associated mycorrhizal fungi, asymbiotic germination is commonly used in the propagation of tropical orchids (Botero et al., 2008) and such artificial media became a way to conserve different species of orchids (chugh et al., 2009). , Antonio, 2012; Mahendran et al, 2012).
Knudson (1922) showed that in the absence of fungi it was possible the germination of orchid seeds, that these seeds were capable of developing in a medium rich in minerals and sugars, this technique was cataloged as seed germination asymbiotic; in general terms, the medium used for the asymbiotic germination is more complex than for the symbiotic germination, since all the organic and inorganic nutrients and the sugars must be available for the orchid in an appropriate form, since there is no intermediation of the fungus that facilitates their nutrition (Botero et al., 2008). In this sense there are several methods and means developed in order to carry out conservation through micropropagation (chugh et al., 2009, Antonio, 2012, Mahendran et al, 2012), some media that come out are Morel (1965), ½ MS (1962) , Vacin and Went (1949) and Knudson C (1946) (Rodríguez et al 2007).
The formulation of Murashige and Skoog salts (1962), which was initially developed for the growth of tobacco callus and is currently used as a basal culture medium for an important group of plants of interest for feeding and for ornamental purposes such as what are the orchids (Faria et al ., 2002).
The capsules and / or seeds must be disinfected prior to the sowing of seeds because these can present a large quantity of microorganisms that can proliferate in the medium and inhibit the growth of the seeds. Usually for the capsules a commercial detergent (twin or powder detergent) is used in solution for 20 minutes in constant agitation, followed by washing with sterile water and subsequent immersion in 0.5% hypochlorite for 10 minutes and rinsed three times with sterile water ( Pedroza – Manrique et al 2010).
In all cases the sterile seeds are grown in glass or plastic containers on a nutritious agar medium containing the sugars and minerals necessary for the seeds to germinate and grow, and a specific pH is adjusted , which according to Knudson (1951) Optimum pH range varies between 4.5 and 5.5 (Bertolini et al, 2014). However, knowledge about the best formulation of the culture medium for each species is still limited, due to the fact that a large number of factors influence the in vitro germination and growth of orchids and these are highly dependent on the species ( Araújo et al 2006).
In results obtained with species from the state of Tabasco ( Catasetum integerrimun Hook, Encyclia alata , Encyclia cordigera , Epidendrum flexuosum , Epidendrum chlocorymbos , Epidendrum stamfordianum , Myrmecophila sp ., Trichocentrum ascendens , Trichocentrum car- thagenense , Oncidium sphacelatum , Prosthechea cochleata , Nidema boothii , Trichocentrum lindenii), acceptable results have been obtained with seed germination averages of 70 to 90% using the VW (Vacin and Went), MS, and MS media at half their concentration, all of them added with 0.1% carbo ‘N activated (Mayo Mosqueda et al, 2010).
3.1. Hormones and elements for the cultivation of seeds
In many cases, the use of auxin-type plant hormones such as ANA (naphthalene acetic acid) and AIA (indole acetic acid) in combination with cytokinin-like hormones is recommended, of which the most recommended is BAP (Bencil amino purine) (Pérez et al., 2001; Botero et al., 2008).
Activated charcoal is also an element widely used in plant tissue culture, normally at a concentration of 0.5%, it is used to stimulate rooting in bud propagation since it can absorb the cytokinin favoring an endogenous balance / auxin / cytokinin that facilitates initiation of the roots (Cheruvatour et al., 2010)
The results given at 53 days from the sowing of the Comparettia speciosa seed , showed that 4,512% of swollen seed was with the Murashige treatment with IBA 1 ppm BA 1 ppm being this the best (Molina Cabrera, 2012)
3.2. Natural additives
Currently, lovers of orchids produce their own plants in home laboratories from asymbiotic vitro germination, and for this they use organic constitutionculture media (Silva et al., 2002). Among the natural extracts used in the cultivation of orchid seeds, you can find the homogenized banana pulp, coconut water, peptone, tryptone, yeast, casein hydrolyzate, tomato juice, pineapple juice and potato extract (Torres et al. ., 2001). These additives contain mixtures of vitamins and amino acids and some act as growth regulators (Pierik, 1989).
Various additives such as coconut complex (endosperm, water, milk ), banana pulp peptone (green or mature) have been used in tissue culture media and germination of the orchid (George, 1993). One can find reports that the use of banana pulp on the germination of Cymbidium, Dendrobium, Phalaenopsis and Paphiopedilum (Arditti and Ernst, 1993), but according to Torres and Barbosa (2001), banana pulp can promote different effects in cultivation in vitro, as a thickener and / or root growth, depending on the variety and amount of pulp used.
Coconut water, generally 3 to 15% (v / v) and the additive that has been most used for a number of species in vitro, not only to stimulate callus growth, but also to increase somatic embryogenesis formation, induce the division of pollen grains, and in their first application, in the induction of the development of immature embryos (Caldas et al., 1998). Knudson (1922) observed that sucrose, fructose and other complexes 231 Iron chelate and coconut water in in vitro germination of large Rossioglossum(Orchidaceae), chemical plant extracts, promote germination and promote the development of protocormos. The liquid endosperm of coconut seed has been used at several concentrations and several species established in vitro, including orchids, as it contains vitamins, enzymes , sugars, nitrogen sources , growth regulators and a fraction of inorganic salts (Hicks, 2007). ). The promoting effects of coconut water are due to the presence of organic compounds such as cytokinins, zeatins, kinetins and purines, which are considered promoters of plant growth(Yong et al., 2009; Hicks, 2007). Cytokinins play a fundamental role in plant organogenesis, since they induce the formation of leaves and shoots and increase the speed of seed development and germination (Werner et al., 2001, Huan et al., 2004)
Araújo et al (2006) found that the addition of 100 g L-1 banana pulp promoted greater sprout and root lengthening, fresh weight, and higher root accumulation for germinated seeds of seed hybrids in vitro Cattleya loddigesii ‘Gran’ x Cattleya loddgesii ‘ Alba ‘.
For the initiation of seedling germination and formation of the species Prosthechea vespa and S. klotzscheana , with the medium MS + Pineapple Juice, the highest percentage of seedlings formed of the species under study was found (Salazar Mercado & Cansino, 2012). From this it can be inferred that the adequate addition of organic components, such as coconut water and pineapple juice to the MS in vitro culture medium, is an important element for the germination and development of seedlings, because they are rich in energy and contain Inorganic ions, amino acids, vitamins, growth regulators and organic acids necessary for the development of orchid seeds (Kitsaki et al., 2004, Yong et al., 2009, Abbas et al., 2011).
3.3. Combined media
There are means that combine natural additives such as hormones, example of this we have:
The combination of 50 and 200 ml L-1 coconut water and 100 g L-1 banana pulp in Knudsosn C medium, showed a significant interaction for the number of roots, this interaction provides better results in number of roots and fresh mass of the outbreaks, respectively (Araújo et al 2006).
Knudson’s medium C, added with ANA (1 mg / l) and 15% coconut water, promotes germination efficiently in the species Dendrobium chrysantumWall. (Orchidiaceae) (De et al., 2006). In addition, the coconut water used in Cattleya mendelii Dombrain (Orchidiaceae) has given significant results in germination, compared to other combined culture media such as Knudson with pineapple juice, AIA and GA3 (Salazar-Mercado, 2012).
Cultivation of meristems and apices
The cultivation of meristems is an effective method for the elimination of viral infections and is the preferred material for the conservation of germplasm. The main reason why meristematic cells are resistant to viruses is that the meristem lacks vascular tissues, which is where the viruses that contaminate the plant are spread, but this is not the only reason, there are other factors: a) The apical meristem has a higher growth rate and consequently the virus “does not reach” the meristem, b) in a meristematic cell the virus has greater difficulty of coupling with the ribosomes, because these cells have a working code very defined, and in addition, c) there is a competitionby the use of some metabolites between cells and viruses. (Muñoz, 2006).
In the case of orchids, the extraction of the meristem is more complex, since it starts from the germination of the seed and buds in development. The process of germination begins when the seed swells and takes on a green color , which originates an undifferentiated structure in spherical shape called protocormo, this protocormo originates the complete seedling after going through five stages of development described by Seaton and Ramsay (2005) : (1) protocor formation; (2) appearance of the rhizoids; (3) appearance of the apical meristem; (4) development of the first sheet; and (5) appearance of the first true root.
After identifying the meristem, the leaves are removed, finding the apical part to recognize and extract the meristem, this is sown and the medium should be expected to be adequate to obtain a positive result (Bertolini et al, 2014). will talk later
In the case of cutting the plant material before planting, it is recommended to check the pulse, do not forget the position of the elbows to counteract the vibrations of the flow chamber, work with insulin needles if you wish since the cut is finer and small and eliminate as much as possible the leaf primordia since it will obtain better results in the elimination of viruses. (Cruz, 2011).
As previously mentioned, the cultivation of meristems provides plants free of viruses, but there are techniques that provide a greater degree of confidence to achieve these results, among which we find thermotherapy or chemotherapy in combination with the meristem culture where: thermotherapy or heat treatment consists of raising or lowering the temperature to eliminate bacteria, mycoplasmas and inactivate viruses in whole plants, organs, tissues or meristems, when viruses are excessively harmful (Salazar, 2013) and chemotherapy is the application of chemical products to culture medium, which causes a greater probability to obtain plants free of pathogens since, when different chemotherapeutic products are applied exogenously to the culture medium, we are ensuring the obtaining of healthy material (rivero, 2007).
4.1. Means used for the cultivation of meristems and apices:
Different methods of propagation by meristems have been developed depending on the type of explant, growing vegetative shoots, which should be sterilized in calcium hypochlorite or 4% sodium for 20-30 minutes. Then they are rinsed in sterile distilled water and the isolation and sowing of the meristems or buds is carried out in culture medium (Debora, 2009).
Where, Each medium used for the in vitro cultivation of plants has special nutrient conditions for optimal growth of the species, which are specific according to the part of the plant being cultivated and the response that is desired (Penacho , et al., sf).
For example, this massive propagation from meristems allows the commercialization of native and high- value orchids from a few specimens, since the in vitro multiplication of orchids is high and in short periods of time , where several different orchids have been evaluated. culture media, as in results obtained in Costa Rica it was found that for the regeneration of extracted meristems, the culture medium Murashige and Skoog (1062) supplemented with 0.5 mg / l of benzyladenine (BA) was the best culture medium , in which 100% regeneration of the meristems was obtained (Muñoz-Bustos, 1998) and in other investigations it has been found that the appropriate pH for meristem culture is 4.8 and 5.5 (also for seeds) (Arditti, 1993).
The use of coconut water is also quite common in Cattleya meristem culture , especially in the introduction stage because of its ability to stimulate the formation of proto-brain bodies in epidermal cells. and by the presence of high values of essential amino acids in the liquid endosperm of the coconut.
However, when there are cases of oxidation in the meristem culture in addition to washing the explants with Ascorbic Acid, Thiourea and L-Cysteine, in works such as the one made with C. skinneri orchids and in the hybrid C. skinneri x C. maxima , the liquid medium was used for a greater diffusion of the phenols and the use of activated carbon in semi-solid rooting medium were practical to counteract oxidation, which produced a positive response, it is also very frequent the use of liquid culture medium in agitation and It promotes cell division and keeps dissolved the polyphenols that release tissues, cellular oxygenation and the use of nutrients is greater. ( Skinneri’s thesis ).
Finally, another important aspect is the light intensities used for the cultivation of seeds, meristems or buds, ranging from darkness to 300 Lux, usually obtained with fluorescent lamps and sometimes complemented with incandescent lamps. The most suitable photoperiods for seed germination and seedling tissue culture are also suitable for the cultivation of meristems (Arditti, 1993).
As we have seen throughout the review, there are multiple methodologies and structures that can be used for the in vitro propagation of orchids such as the cultivation of seeds and meristems (apices and buds) and thus ensure their preservation. However, they are complex processes and with many unknowns in the physiology , physiognomy and responses of these plants through this type of multiplication, however, this is due to the great variety of species that this family of plants presents and the scarce efforts in their study, therefore, it is necessary to continue generating progress and studies such as those mentioned so that this frontier of knowledge diminishes.