Evaluation of the in vitro growth of the culture of oregano ( Origanum vulgare ) from organogenesis ’ s technology Evaluación del crecimiento in vitro del cultivo de orégano ( Origanum vulgare ) a partir de la técnica de organogénesis

Hormones of vegetable growth, microspread, oregano There was evaluated the in vitro growth of the culture of Origanum vulgare from Organogénesis’s technology. Three treatments of disinfection were applied for the explantes, with modifications in the time of dip, etc., evaluating the following variables: percentage of explantes prosperous, contaminated and oxidized. For the phase of establishment and multiplication, there were evaluated five and three means of culture Murashige and Skoog respectively, modifying the concentrations of the hormones. In agreement to the evaluation of the treatments of disinfection, one determined that the treatment one (T1), generated 100 % of explantes prosperous, opposite to the treatments other treatments. As for the means of culture Murashige and Skoog used in the phase of establishment the half number four (Ms4) possesses a significant difference in the percentage of germination, opposite to other means of culture evaluated. In the stage of multiplication the half number three (Mn3) with regard to the variable of height of the stem obtained a significant difference in relation with other evaluated means. Finally, in the phase of establishment the way Ms4 obtained the best performance in the germination of the seeds of oregano, in the stage of multiplication of the plántulas there was demonstrated a relation of 1:20, demonstrating a massive multiplication from a plant boss in the way of culture Murashige and Skoog Mn3, vitroplantas free of pathogenic.

There was evaluated the in vitro growth of the culture of Origanum vulgare from Organogénesis's technology. Three treatments of disinfection were applied for the explantes, with modifications in the time of dip, etc., evaluating the following variables: percentage of explantes prosperous, contaminated and oxidized. For the phase of establishment and multiplication, there were evaluated five and three means of culture Murashige and Skoog respectively, modifying the concentrations of the hormones. In agreement to the evaluation of the treatments of disinfection, one determined that the treatment one (T1), generated 100 % of explantes prosperous, opposite to the treatments other treatments. As for the means of culture Murashige and Skoog used in the phase of establishment the half number four (Ms4) possesses a significant difference in the percentage of germination, opposite to other means of culture evaluated. In the stage of multiplication the half number three (Mn3) with regard to the variable of height of the stem obtained a significant difference in relation with other evaluated means. Finally, in the phase of establishment the way Ms4 obtained the best performance in the germination of the seeds of oregano, in the stage of multiplication of the plántulas there was demonstrated a relation of 1:20, demonstrating a massive multiplication from a plant boss in the way of culture Murashige and Skoog Mn3, vitroplantas free of pathogenic.

Introduction
Origanum vulgare, commonly known as Oregano, is an aromatic, dicotyledonous, perennial and branched plant [1]. It is found in a temperate to temperatewarm climate, can tolerate low temperatures, grows in loamy, permeable soils, with good drainage due to its high sensitivity to root asphyxia [2]. Its propagation can be carried out by means of the seed (heterogeneous population) and by division of plants (cuttings), normally the oregano is multiplied by means of cuttings and it is advisable to renew the plantation after 3 or 4 years.
The antimicrobial potential found in the essential oils of the genus Origanum present activity against Gram-negative bacteria (Salmonella typhimurium, Escherichia coli, etc.) and Gram-positive bacteria (Listeria monocytogenes, Bacillus subtilis, etc.) in addition to their antifungal capacity against Candida albicans, Aspergillus niger, etc. Phenols, carvacrol and thymol have the highest levels of activity against Gram-negative microorganisms, with thymol being the most active. Oleoresins are homogeneous mixtures of resin and essential oils, the latter containing volatile odoriferous principles. Some of the advantages of oleoresins over raw vegetables are that they are uniform in their characteristics, have a high purity and are economically profitable [3].
The technique of tissue culture consists essentially of carrying out an aseptic isolation of a portion of the plant called explante (e.g.: protoplast, cell, tissue, organ), in a medium of defined chemical keeping the cultures free of microbial contamination. The breadth of the definition of in vitro plant tissue cultures and the many different objectives pursued can be summarized as follows: 1. Basic studies of physiology, genetics, biochemistry and related sciences; 2. Bioconservation and production of useful compounds; 3. Increase in genetic variability; 4. Obtaining pathogen-free plants; 5. Plant propagation and 6. Conservation and exchange of germplasm [4].

Phase 1 Select the process of disinfection of Oregano explants in order to propagate seedlings aseptically
 Selection of the explant and mother plant: we worked with seeds of Origanum vulgare, from plants sown in the facilities of Playón Santander's SENA, which had a high content of thymol and carvacrol, important compounds for obtaining high quality essential oils.
 Explant disinfection: Three disinfection treatments were used, which differ in concentration, type of disinfectant agent and exposure times. Table I shows the established treatments.
In the three treatments 70% alcohol was applied, each one had three types of disinfectants, in which different variables were evaluated, taking into account possible negative effects on the explants.
Respuestas, 23 (2) July -December 2018, pp. 36-42, ISSN 0122-820X Evaluation of the in vitro growth of the culture of oregano (origanum vulgare) from organogenesis's technology  Selection of the explant and mother plant: we worked with seeds of Origanum vulgare, from plants sown in the facilities of Playón Santander's SENA, which had a high content of thymol and carvacrol, important compounds for obtaining high quality essential oils.
 Explant disinfection: Three disinfection treatments were used, which differ in concentration, type of disinfectant agent and exposure times. Table I shows the established treatments. In the three treatments 70% alcohol was applied, each one had three types of disinfectants, in which different variables were evaluated, taking into account possible negative effects on the explants. In order to select the best treatment, three measurement variables were stipulated (Table II). In order to select the best treatment, three measurement variables were stipulated (Table II).

Phase 2 Develop the establishment phase of selected oregano plant material (Origanum vulgare) using modified Murashige & Skoog basal medium
Five culture media were formulated from the basal medium MS (Murashige and Skoog) which varied in the content of hormones, vitamins and activated carbon. The explants were sown and incubated at a temperature of 18 °C, with a relative humidity of 65% and a photoperiod of 16 light hours. To determine the most suitable medium, measuring variables such as germination time and percentage of sprouted seeds were established.

Phase 3 Perform the multiplication phase of the explants of Oregano using modified basal medium MS (Murashige & Skoog)
The nodal segments and apical buds of the seedling were used. Cuttings of 0.5 cm between knots were made and then sowed in three culture media formulated for this phase (Mn 1, Mn 2 and Mn3).
The culture media were evaluated using five variables such as stem length, stem diameter, number of leaves, number of knots and number of branches. In the three treatments 70% alcohol was applied, each one had three types of disinfectants, in which different variables were evaluated, taking into account possible negative effects on the explants. In order to select the best treatment, three measurement variables were stipulated (Table II).

Phase 2 Develop the establishment phase of selected oregano plant material (Origanum vulgare) using modified Murashige & Skoog basal medium
Five culture media were formulated from the basal medium MS (Murashige and Skoog) which varied in the content of hormones, vitamins and activated carbon. The explants were sown and incubated at a temperature of 18 °C, with a relative humidity of 65% and a photoperiod of 16 light hours. To determine the most suitable medium, measuring variables such as germination time and percentage of sprouted seeds were established.

Statistical analysis
Each treatment was carried out in triplicate, with a total of 15 explants for each treatment, a subsequent statistical analysis will be carried out on the variants measured by means of an ANOVA.

Phase 1.
In each of the treatments there were no significant differences due to the fact that the p-value was 0.2178. However, it can be inferred that the most favourable data in the three treatments corresponded to treatment one (T1) (Povidone iodine, alcohol at 70% and sodium hypochlorite at 1.5% during five minutes of immersion) with a contamination percentage of 0%; followed by treatment three (T3) (Povidone iodine, 70% alcohol and 1% calcium hypochlorite during three minutes of immersion), obtaining 6.6% contamination and finally treatment two (T2) (Polysorbate 80, 70% alcohol and 1.5% sodium hypochlorite during four minutes of immersion), which had the highest value in contamination, 20%. (Table V) In the contaminated explants, the fungus found in the highest percentage was the genus Penicillium with 60%, followed by Aspergillus with 30% and Cladosporium with 10%.

hase 3 Perform the multiplication phase of the explants of Oregano using modified basal edium MS (Murashige & Skoog)
he nodal segments and apical buds of the seedling were used. Cuttings of 0.5 cm between knots ere made and then sowed in three culture media formulated for this phase (Mn 1, Mn 2 and Mn3). he culture media were evaluated using five variables such as stem length, stem diameter, number f leaves, number of knots and number of branches.

Phase 3 Perform the multiplication phase of the explants of Oregano using modified basa medium MS (Murashige & Skoog)
The nodal segments and apical buds of the seedling were used. Cuttings of 0.5 cm between kno were made and then sowed in three culture media formulated for this phase (Mn 1, Mn 2 and Mn3) The culture media were evaluated using five variables such as stem length, stem diameter, numbe of leaves, number of knots and number of branches.

Statistical analysis
Each treatment was carried out in triplicate, with a total of 15 explants for each treatment, subsequent statistical analysis will be carried out on the variants measured by means of a ANOVA.

Results and discussion
Phase 1. In each of the treatments there were no significant differences due to the fact tha the p-value was 0.2178. However, it can be inferred that the most favourable data in th three treatments corresponded to treatment one (T1) (Povidone iodine, alcohol at 70% an sodium hypochlorite at 1.5% during five minutes of immersion) with a contaminatio percentage of 0%; followed by treatment three (T3) (Povidone iodine, 70% alcohol and 1% calcium hypochlorite during three minutes of immersion), obtaining 6.6% contaminatio and finally treatment two (T2) (Polysorbate 80, 70% alcohol and 1.5% sodium hypochlorit during four minutes of immersion), which had the highest value in contamination, 20% (Table V)  In the contaminated explants, the fungus found in the highest percentage was the genus Penicillium with 60%, followed by Aspergillus with 30% and Cladosporium with 10%. The fungi found as contaminants of the culture media in the phase were reported in Table VI. Heimy Jerylee Camacho-Camacho, Christian Andrei Chacín-Zambrano, Leydy Gabriela Rodríguez Source: [5] The fungi found as contaminants of the culture media in the phase were reported in Table VI. From these results, it can be seen that the treatments containing povidone-iodine obtained the best results, due to its mechanism of bactericidal and fungicidal action, as it gradually releases the iodine it contains [6]. Likewise, it is deduced that treatment number two, which contains polysorbate 80, is used as a humectant, increasing the water retention capacity, which indicates the high humidity of the explant, resulting in the presence of fungi [4].The treatments evaluated in the oxidation variable obtained 0% oxidized phenolic substances, in addition to other substances that intervene causing cellular damage and produce seeds.

Phase 2.
The culture media used in this phase present a statistically significant difference with a p-value of 0,0001. It can be seen that the growing medium Ms4 has the highest seedling emergence rate after eight days of sowing (Figure 1), the second medium with the highest amount of germination was the medium. Ms2, with a 66.7% and Ms3 obtained a deficient yield with 0% germination, with these results and observing the differences in the use of AG 3 and activated carbon from the five media evaluated, the Ms3 media does not contain activated carbon and the addition of AG 3 was in low quantity, however, was not the medium with the highest content of gibberylin AG 3 the one that obtained better results, for this reason we can deduce that the seeds of Origanum vulgare, germinate in a medium that contains activated carbon and a range of 1200 -3000 µl de AG 3 .
The effect that the activated carbon has on the explants is representative, according to what was exposed by [8], explains that the dose of activated oxidation, as described by [7], the application of disinfection and rinses with sterile distilled water, as a pre-treatment to explants, are very useful, because they act in the removal of both disinfectants and carbon can vary from 0,5-5,0 g/L, and it is in this range where there is stimulation of the processes of morphogenic development, the Ms3 medium was the only one that was not added activated carbon, contain activated carbon and the addition of AG 3 was in low quantity, however, was not the medium with the highest content of gibberylin AG 3 the one that obtained better results, for this reason we can deduce that the seeds of Origanum vulgare, germinate in a medium that contains activated carbon and a range of 1200 -3000 µl de AG 3 . The effect that the activated carbon has on the explants is representative, according to what was exposed by [8], explains that the dose of activated carbon can vary from 0,5-5,0 g/L, and it is in this range where there is stimulation of the processes of morphogenic development, the Ms3 medium was the only one that was not added activated carbon, thus deducing that so much the little volume applied to the activated carbon, that it was not possible to use the activated carbon. AG 3 as the non-addition of activated carbon in the culture medium would cause the non-emergence of the radicle, nor the development of the Evaluation of the in vitro growth of the culture of oregano (origanum vulgare) from organogenesis's technology ntaminated explants, the fungus found in the highest percentage was the genus m with 60%, followed by Aspergillus with 30% and Cladosporium with 10%. The nd as contaminants of the culture media in the phase were reported in se results, it can be seen that the treatments containing povidone-iodine obtained esults, due to its mechanism of bactericidal and fungicidal action, as it gradually he iodine it contains [6]. Likewise, it is deduced that treatment number two, which polysorbate 80, is used as a humectant, increasing the water retention capacity, icates the high humidity of the explant, resulting in the presence of fungi [4].The s evaluated in the oxidation variable obtained 0% oxidation, as described by [7], ation of disinfection and rinses with sterile distilled water, as a pre-treatment to are very useful, because they act in the removal of both disinfectants and oxidized substances, in addition to other substances that intervene causing cellular damage ce seeds.
The culture media used in this phase present a statistically significant difference value of 0,0001. It can be seen that the growing medium Ms4 has the highest mergence rate after eight days of sowing (Figure 1), the second medium with the mount of germination was the medium. Ms2, with a 66.7% and Ms3 obtained a yield with 0% germination, with these results and observing the differences in the 3 and activated carbon from the five media evaluated, the Ms3 media does not ntaminated explants, the fungus found in the highest percentage was the genus m with 60%, followed by Aspergillus with 30% and Cladosporium with 10%. The nd as contaminants of the culture media in the phase were reported in se results, it can be seen that the treatments containing povidone-iodine obtained esults, due to its mechanism of bactericidal and fungicidal action, as it gradually he iodine it contains [6]. Likewise, it is deduced that treatment number two, which polysorbate 80, is used as a humectant, increasing the water retention capacity, icates the high humidity of the explant, resulting in the presence of fungi [4].The s evaluated in the oxidation variable obtained 0% oxidation, as described by [7], ation of disinfection and rinses with sterile distilled water, as a pre-treatment to are very useful, because they act in the removal of both disinfectants and oxidized substances, in addition to other substances that intervene causing cellular damage ce seeds.
The culture media used in this phase present a statistically significant difference value of 0,0001. It can be seen that the growing medium Ms4 has the highest mergence rate after eight days of sowing (Figure 1), the second medium with the mount of germination was the medium. Ms2, with a 66.7% and Ms3 obtained a yield with 0% germination, with these results and observing the differences in the thus deducing that so much the little volume applied to the activated carbon, that it was not possible to use the activated carbon. AG 3 as the non-addition of activated carbon in the culture medium would cause the non-emergence of the radicle, nor the development of the plant. Because germination is linked to the use of AG 3, that to high concentrations the seeds can remain in state of dormancy, for this reason it is possible that the medium Ms5 is with a very low percentage of germination.

Phase 3. Stem length
In the length of the stem it can be seen that the middle Mn3 had the largest size in the shoots with an average of 7.6 cm, the middle Mn1 and Mn2, showed an average of 5.5 cm of 2 cm stem length respectively ( Figure 2).

Stem diameter
The medium that provides the plants with the largest diameter of the stem is the Mn3 with an average of 1 cm, the plants of the means Mn1 and Mn2 have an average of 0.6 and 0.4 cm respectively, as shown in Figure 3.

Number of sheets
The medium Mn3 obtained the highest number of leaves, with an average of 11 leaves per plant, the cultivation media Mn1 and Mn2, have favorable values, because the average is 8 and 7 leaves respectively, to relate the variables of this phase and take into account the length and diameter of the stem, it can be inferred that the medium Mn3 is the one that provides the plant with better phenotypic characteristics (Figure4).

Number of sheets
The medium Mn3 obtained the highest number of leaves, with an average of 11 le plant, the cultivation media Mn1 and Mn2, have favorable values, because the ave and 7 leaves respectively, to relate the variables of this phase and take into acc length and diameter of the stem, it can be inferred that the medium Mn3 is the provides the plant with better phenotypic characteristics (Figure4).     ts obtained in this research are similar to those recorded by [9], who indicate that a balance should be made between auxins and cytokinins that benefit the sprouting segments of oregano, as it was made in this investigation the balances were for ml of ANA and 2 ml of BAP and for Mn2, 0,2 of ANA and 1,5 ml of BAP ly and Mn3 with a content of ANA of 2 ml/L and BAP of 2 ml/L. In addition to ded by [5], where the importance of the addition of BAP, Kinetin and AG3 for maximum regeneration values is highlighted, the efficiency of the culture s also highlighted when BAP is added in a higher proportion than ANA.
were found of possible problems, including vitrification and malformations, as [10]. Currently, after several trials that have been developed for some aromatic ncluding oregano, multiplication by in vitro cultures. The development of this new In the stage of multiplication of cuttings, is obtained as a result of a ratio of 1:20 with two repiques made, phenotypically the plants have the best characteristics found among the three media of cultivation, with an average height of 7.6 cm, a number of leaves average of 11, number of knots average of 5 and a single branching, these characteristics exposed are those that provides the plant the medium Mn3.
The results obtained in this research are similar to those recorded by [9], who indicate that a hormonal balance should be made between auxins and cytokinins that benefit the sprouting of nodal segments of oregano, as it was made in this investigation the balances were for Mn1, 0,5 ml of ANA and 2 ml of BAP and for Mn2, 0,2 of ANA and 1,5 ml of BAP respectively and Mn3 with a content of ANA of 2 ml/L and BAP of 2 ml/L. In addition to that recorded by [5], where the importance of the addition of BAP, Kinetin and AG3 for obtaining maximum regeneration values is highlighted, the efficiency of the culture medium is also highlighted when BAP is added in a higher proportion than ANA.
Records were found of possible problems, including vitrification and malformations, as explained [10]. Currently, after several trials that have been developed for some aromatic species, including oregano, multiplication by in vitro cultures. The development of this new technique initially stumbled upon the appearance of physiological malformations in the regeneration stages of cloned plants from tissue cultures. However, the plants that were cultivated in this research in this phase, no problem was found, this was due to the environmental conditions offered to the explant for growth were the most optimal, starting with the photoperiod of 16 light hours at a temperature of 18 ° C and relative humidity of 65%.

Conclusions
he disinfection treatment that showed the best effect on the explants was number 1, which had Povidona iodada, 70% alcohol and sodium hypochlorite at 1.5% during five minutes of immersion, each has mechanisms of action that helps the elimination of bacteria and fungi.
In the establishment phase, the medium that obtained the greatest amount of germinated seeds was Ms4, which has 100% germination in only eight days.
In the multiplication phase, the MS culture medium that was standardized and gave the plant good characteristics, such as height, number of leaves, number of knots, etc., is Mn3.
We obtained 20 seedlings from each node and apical bud, showing that the in vitro cultivation technique is efficient in the massive propagation of plants.