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Revista mexicana de ciencias agrícolas
versión impresa ISSN 2007-0934
Rev. Mex. Cienc. Agríc vol.7 no.2 Texcoco feb./mar. 2016
Articles
In vitro propagation of guava (Psidium guajava L.) from nodal segments
1Maestria en Ciencias en Biotecnología Agropecuaria-ITEL. (lilyx_one@hotmail.com).
2Instituto Tecnológico El Llano Aguascalientes, km 18 de la carretera Aguascalientes-San Luis Potosí, Aguascalientes, México. (silosespino@hotmail.com; lvalera2003@gmail.com; sbenitez@gmail.com).
The aim of this work was to develop and propose a protocol to propagate guava in vitro from nodal segments of trees in production. The plant material was collected field june 2012 to june 2013. For the cleanliness and initial culture medium of explants, three experiments were performed in the first four fungicides, two systemic evaluated: Benomyl and Carbendazim, one of contact, copper oxychloride and one natural, fractal, in different concentrations and combinations. PVP, chlorine, ethanol, tween, citric acid and ascorbic acid: In the second and different antioxidants were tested as disinfectants. In the third to the initial medium we were evaluated MS supplemented with different combinations of PVP, citric acid, ascorbic acid, silver nitrate and activated carbon. For in vitro multiplication two nodal segments with different combinations of two growth regulators were evaluated: IBA and BAP. Besides the main environmental contaminating microorganisms were identified. According to the results, the best fungicide treatment was a combination of: Benomyl 2 g L-1, Carbendazim 2 g L-1 and 1 g copper oxychloride L-1. The best treatment of antioxidants and disinfectants was PVP 0.5%, chlorine 5% and 3 drops of tween 20. Although there were no statistical differences, the best initial culture medium was: MS + PVP 0.75 g L-1 and activated carbon 2 g L-1. For in vitro multiplication, the best treatment was: one nodal segment MS + 0.5 mg L-1 BAP + 0.1 mg L-1 IBA, with outbreaks of 1.48 cm and three leaves per shoot. Aspergillus sp., and Alternaria sp. two fungal contaminants of the culture medium were identified. From the results obtained, a complete protocol for in vitro multiplication guava, from nodal segments trees in production is proposed.
Keywords: Psidium guajava L.; in vitro propagation; nodal segment; oxidation; pollution; regeneration
El objetivo de este trabajo fue desarrollar y proponer un protocolo para propagar in vitro el guayabo a partir de segmentos nodales de árboles en producción. El material vegetativo de campo se colectó de junio de 2012 a junio de 2013. Para la asepsia y medio de cultivo inicial de los explantes, se realizaron tres experimentos, en el primero se evaluaron cuatro fungicidas, dos sistémicos: Benomyl y Carbendazim, uno de contacto, oxicloruro de cobre y uno natural, fractal, en diferentes concentraciones y combinaciones. En el segundo se probaron diferentes antioxidantes y desinfectantes como: PVP, cloro, etanol, tween, ácido cítrico y ácido ascórbico. En el tercero, para el medio inicial, se evaluó MS suplementado con diferentes combinaciones de PVP, ácido cítrico, ácido ascórbico, nitrato de plata y carbón activado. Para la multiplicación in vitro se evaluaron dos segmentos nodales con diferentes combinaciones de dos reguladores del crecimiento: IBA y BAP. Además se identificaron los principales microorganismos contaminantes del medio. De acuerdo con los resultados obtenidos, el mejor tratamiento con fungicidas fue la combinación de: Benomyl 2 g L-1, Carbendazim 2 g L-1 y oxicloruro de cobre 1 g L-1. El mejor tratamiento de antioxidantes y desinfectantes fue PVP 0.5%, cloro 5% y 3 gotas de tween 20. Aunque no hubo diferencias estadísticas, el mejor medio de cultivo inicial fue: MS + PVP 0.75 g L-1 y carbón activado 2 g L-1. Para multiplicación in vitro, el mejor tratamiento fue: segmento nodal uno en MS + 0.5 mg L-1 BAP + 0.1 mg L-1 IBA, con brotes de 1.48 cm y tres hojas por brote. Se identificaron dos hongos contaminantes del medio de cultivo: Aspergillus sp. y Alternaria sp. A partir de los resultados obtenidos, se propone un protocolo completo para la multiplicación in vitro del guayabo, a partir de segmentos nodales de árboles en producción.
Palabras clave: Psidium guajava L.; contaminación; propagación in vitro; oxidación; regeneración; segmento nodal
Introduction
For many years the guava is grown commercially and is a species found in the tropics and subtropics over 50 countries worldwide. The main countries where guava is grown are India, China, Thailand, Pakistan, Mexico, the Philippines, Egypt and the United States. In Mexico, Michoacan and Aguascalientes are the major producing states guava export quality (SAGARPA, 2012). The genetic material of guava in Calvillo, Aguascalientes, is of Creole origin, selected by the producers, known as "China and Chinese Media". The variability observed in the guava fruit is large, as a form, fruit size, flesh color, thickness of hull, number of seeds per fruit (Padilla et al., 2007).
Orchards in Calvillo, Aguascalientes, were initially established by seed, the main problem observed in guava spread in this way is that it guarantees the quality of the product to be harvested by the variability of the offspring (Mendoza et al., 2004). The gardens have a high genetic variability, making it difficult to meet the requirements of uniformity and quality of the fruit for the export market. Control of pests and diseases is poor, which adversely affects the development of the tree (SAGARPA, Annex B, 2011).
By asexual propagation it ensures that plants are obtained, with the same characteristics of the original plants. In guava grafting, cuttings, root suckers and air layering is used, the latter being the most widely practiced. The problem with air layering is much plant material for mass propagation (García, 2009) is needed. Another type of asexual propagation is the in vitro propagation, which is the multiplication of a species from a tissue or organ under aseptic conditions, this ensures that the regenerated plants are clones of the mother plant (Perales et al., 2005).
They have conducted several studies based guava micropropagation directly or indirectly organogenesis which were developed from explants meristem and young leaves. The results of these studies showed high levels of contamination, oxidation and dependence between genotype and organogenética answer, without being able to establish in vitro guava (Portal et al., 2003). For the establishment, multiplication and rooting of woody plants such as guava various culture media, where you can highlight the Murashige and Skoog (MS) (1962) they are used. It is at this stage that truly makes micropropagation, obtained a large number of new shoots from small amounts of tissue (Perez et al., 1999).
Auxins stimulate cell elongation, the most commonly used in in vitro culture are: 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (ANA), indole acetic acid (AIA) and indole butyric acid, 3 (IBA). Cytokinins promote cell division in meristematic tissues and not one of the most used is the benzylaminopurine (BAP) (Trigiano and Gray, 2000). The regeneration step in vitro could not be carried out without the addition of growth regulators in the culture medium. Auxins stimulate cell elongation and cytokinins promote cell division in non meristematic tissues (Gutierrez et al., 1998). Pallavi and Pravesh (2012), gained 80% of sprouting with the application of 3 mg L-1 BAP and 0.1 mg L-1 of ANA. The best results were in vitro regeneration with 1 mg L-1 BAP and 160 mg L-1 adenine obtained up to 3.2 shoots per explant. Roshan et al. (2007) in his research on in vitro regeneration of guava, established explants in MS medium and obtained greater number of rooted plantlets with 2.5 mg L-1 IBA and 2.5 mg L-1 IAA. Tariq et al. (2008) in his work on in vitro regeneration of guava used to root the combination of 1.5 mg L-1 IBA and 0.5 mg L-1 ANA, obtaining up to 85% of rooted plantlets.
Other major problems are guava micropropagation tissue oxidation and contamination. More antioxidants used in the in vitro propagation of guava are L-cysteine, polyvinylpyrrolidone (PVP), ascorbic acid and citric acid, there is a close relationship between the origin of explant, the concentration of phenols and the success of in vitro establishment (Concepción et al., 2005). Tagelsir et al. (2006) overcame oxidation explants in vitro establishment stage using 1.5% activated carbon, combined with 1 mg L-1 of silver nitrate in supplemented MS medium. The in vitro establishment of woody species is largely limited by the dimming of the explants in the medium, caused by pollution.
It is recommended the use of PVP and activated carbon added to the culture medium and that remove toxic substances (Azofeifa, 2009). Some authors avoided contamination and oxidation with explants from seedlings propagated in vitro, because the seeds are easy to disinfect for its establishment in vitro (Tariq et al., 2008, Muhammad et al., 2012). An earlier alternative to micropropagation is that plants grow under greenhouse conditions, thereby substantially reducing contamination rates (Levitus et al., 2010). In guava in vitro propagation, the nodal segments should measure up to 2 cm for easy disinfection. To remove sufficient phenolics using 100 mg L-1 of PVP in combination with 100 mg l-1 ascorbic acid.
The aim was to develop and evaluate an optimal protocol for the in vitro establishment of guava (Psidium guajava L.) using nodal segments of trees in production for the mass reproduction of genotypes of interest to producers.
Materials and methods
This research was conducted at the Laboratory of Applied Biotechnology at the Technological Institute Aguascalientes El Llano (ITEL), located at km 18 of the road AguascalientesSan Luis Potosi, the Llano, Aguascalientes, Mexico. For handling of the plant material used tweezers and scalpels; the cleanliness of the surgical material with 100% and flashover burner was performed by immersion in etanol (Trigiano and Gray 2000).
Collection and management of plant material
Collecting guava explants was performed each month from june 2012 to june 2013. As a mother guava plants were used in the production stage of an orchard located at 22° 08' 56'' north latitude and 102° 25' 20'' west longitude, in the Mezquitera, San Tadeo, Calvillo, Aguascalientes.
Asepsis and initial culture medium
In this part of the experimental work they were used explants complete, without dividing them into segments. To have an aseptic accommodation first three treatments with four fungicides, two systemic were evaluated: Benomyl and Carbendazim, one contact, copper oxychloride and one natural, fractal (seed extract citrus) in different concentrations and combinations in a design completely randomized with three treatments and seven replicates, the percentage aseptic recorded, the explants were kept under stirring for two hours, the experimental unit was a wide mouth jar of 250 ml, with each treating solution, wherein ten explants were introduced (Table 1).
Table 1 Treatments with fungicides for in vitro establishment of explants guava.
| Tratamiento | Descripción |
| 1 | Benomyl 2 g L-1, carbendazim 2 g L-1 y oxicloruro de cobre 1 g L-1 |
| 2 | Benomyl 2 g L-1 y fractal 12 ml L-1 |
| 3 | Benomyl 1 g L-1, oxicloruro de cobre 1 g L-1y y fractal 12 ml L-1 |
To prevent rust and keep alive the explants, promoting their development and establishment during washing, four treatments with different disinfectants such as antioxidants: PVP, chlorine, ethanol, tween, citric acid and ascorbic acid in a completely randomized design and also evaluated with four treatments and four replications, the experimental unit was a wide mouth jar 250 ml, the solution of each treatment, in which ten explants were introduced (Table 2). the survival rate was recorded.
Table 2 Treatment with antioxidants and disinfectants in the wash solution explants.
| Tratamiento | Descripción |
| 1 | PVP 0.5 g L-1 , cloro 5% y etanol 70% |
| 2 | Ácido cítrico 0.2%, ácido ascórbico 0.6% y cloro 15% |
| 3 | PVP0.5%, cloro 5% y 3 gotas de tween20 |
| 4 | Ácido ascórbico 0.15 g L-1, ácido cítrico 0.2 g L-1 y cloro 5% |
The explants were placed in a solution containing sterile distilled water with antioxidants and disinfectants; depending on the treatment to prevent oxidation during transfer to the laboratory.
To define the initial culture medium for the in vitro establishment of guava, four treatments with seven replicates (Table 3) were evaluated using a completely randomized design, where the experimental unit was a bottle "Gerber" with five explants. In this experiment a control was included, because without the use of fungicides all contaminated explants because they come field.
Table 3 Treatments evaluated to define the initial culture medium.
| Tratamiento | Descripción |
| 1 | MS+PVP0.75gL y carbón activa 2gL-1 |
| 2 | MS + ácido cítrico 0.1 g L-1+ ácido ascórbico 0.1 g L-1 y carbón activado 2 g L-1 |
| 3 | MS + nitrato de plata lx 10-3g L-1 y carbón activado 1.5% |
The MS medium was supplemented with 1 mg L-1 of Benomyl and 0.50 g L-1 of the antibiotic cefotaxime to keep uncontaminated explants, which it withdrew from the average over a period of 15 days, gradually decreasing concentration. Cefotaxime is reported as an antibiotic that does not harm vegetable crops (Licea-Moreno et al., 2007).
In vitro multiplication
For this part of the study the explants were divided into nodal segments one and two, each 0.5 cm long, which were placed in MS medium supplemented with 30 g L-1 sugar and 7.5 g L-1 agar. For shoot induction six treatments (Table 4), of which four were added with growth regulators and the last two treatments were evaluated witnessed no growth regulators fully used a randomized design with six treatments and seven replicates, taking Gerber experimental unit one vial of explant. The variables evaluated were shoot length and number of leaves per shoot and recorded at 40 days. The environmental conditions were incubation room at 25 ± 2 °C, photoperiod 16 h light and 8 h dark and a luminous intensity of 2 000 lux. To process data from all experiments the statistical package SAS version 8 was used, where the analysis of variance (ANOVA) and mean comparison test (Duncan 5%) was performed.
Table 4 Treatments evaluated for in vitro multiplication of guava.
| Tratamiento | Descripción |
| 1 | Segmento nodal uno y 1 mg L -1 ВАР |
| 2 | Segmento nodal dos y 1 mg L -1 ВАР |
| 3 | Segmento nodal uno y 0.5 mg L-1 ВАР + 0.1 mg L-1 IBA |
| 4 | Segmento nodal dos y 0.5 mg L-1 ВАР +0.1 mg L-1 IBA |
| 5 | Segmento nodal uno, testigo |
| 6 | Segmento nodal dos, testigo |
Regenerated shoots were rooted in vitro with 1 mg L-1 IBA, treatment and tested for several years in experiments carried out in our Institute. Acclimatization of plants was carried out gradually with the Fractal he used fungicide/bactericide natural and no pollution problems arose.
Identifying contaminating microorganisms from the culture medium
Were taken samples growth of colonies or microorganisms contaminated culture media, were isolated and cultured in potato dextrose agar culture medium (PDA). After colonies developed, identifying contaminating microorganisms based on keys, photographs and the compound microscope was performed.
Results and discussion
Asepsis and initial culture medium
Importantly worked with explants obtained from trees in production and in accordance with the collections made for a year and at different times of the day, it is concluded that the best time for planting material of plants guava field is April June as early as possible. Which coincides with that reported by Amin and Jaiswal's (1987) who found less contamination of the explants in the months april to june.
According to the results obtained with fungicides, treatment one Benomyl 2 g L-1, Carbendazim 2 g L-1 and 1 g copper oxychloride L-1 had the highest percentage of disinfection, 60%, according to test comparison of means (Table 5). According to the literature reviewed, the best results are obtained with a greater concentration of systemic and contact fungicides smaller, more so when working with plant material field, for example, Ramirez et al. (2000) used a higher concentration of systemic fungicide greater tissue penetration. Although using systemic fungicides only the results are not satisfactory, as reported Flores-Mora et al. (2009) who used up to 6 g L-1 benomyl in establishing in vitro, and only 31.67% got a clean explants. The effect of the position of nodal segments of guava is also important, as reported Shekafandeh and Khosh-Khui (2008), who used Benomyl 4 g L-1 during the 30-45 min in combination with Hg2Cl 0.2 g L- 1 2 min and passed the aseptic part.
Table 5 Response to treatment with fungicides in aseptic explants guava.
| Tratamiento | Medias (% de asepsia) |
| 1. Benomyl 2 g L-1, carbendazim 2 g L-1 y oxicloruro de cobre 1 g L-1 | 60.0 b |
| 2. Benomyl 2 g L-1 y fractal 12 ml L-1 | 42.9 b |
| 3. Benomyl 1 g L-1, oxicloruro de cobre l gL-1 y Fractal 12ml L-1 | 8.6 a |
*Medias con la misma literal son estadísticamente iguales (Duncan, p≤ 0.05).
The best results obtained with anti-oxidants and disinfectants, were presented with three treatment, 0.5% PVP, chlorine 5% and 3 drops of Tween 20 (Table 6). Explants were kept without oxidizing up to 24 h. Treatment one received only a 10% survival possibly due to ethanol 70%, than in other tissues shows good results, but not in woody species such as guava.
Table 6 Effects antioxidant agents in the wash solution explants.
| Tratamiento | Medias (% de sobrevivencia) |
| 1.РVР0.5 g L-1, с1ога5% у еtаnоl70% | 10.0 a |
| 2. Ácido cítrico 0.2%, ácido ascórbico 0.6% y cloro 5% | 30.0 ab |
| 3. PVP 0.5%, cloro 5% y 3 gotas de tween 20 | 90.0 c |
| 4. Ácido ascórbico 0.15 g L-1, ácido cítrico 0.2 g 1-1 y cloro 5% | 50.0 b |
*Medias con la misma literal son estadísticamente iguales (Duncan, p≤ 0.05).
The best treatment obtained 90% survival of the explants; similar to those reported by Ocampo and Nunez (2007), who tested as a disinfectant, chlorine 5% with 3 drops of Tween stir for 10 min and mercury dichloride 0.05% for 2 min, the two treatments had good results, but they chose the former, because it is easy to use and is nontoxic; Importantly, it is working with young plants in a greenhouse. Liu and Yang (2011) in their work of in vitro propagation of guava from nodal segments of mature trees, washed explants to prevent oxidation with 0.5% PVP for 40 min, the supplemented MS medium with 250 mg L- 1 PVP and achieved a 53.3% survival, lower than that obtained in this study.
The three treatments for evaluation of initial culture medium showed good results, keeping oxidation and contamination free explants. chose one treatment with PVP 0.75 g L-1 Activated charcoal 2 g L-1, because the treatment was modified by the authors of this paper and numerically obtained the best results (Table 7).
Table 7 Results of the treatments evaluated for the initial culture medium.
| Tratamiento | Descripción | Medias (% de sobrevivencia) | Referencia |
| 1 | PVP 0.75 g L-1 y carbón activado 2 g L-1 | 94.82 a | Modificado (2013). |
| 2 | Ácido cítrico 0.1 g L-1, ácido ascórbico 0.1 g L-1 y carbón activado 2 g L-1 | 94 82 a | Shekafandeh y Khosh Khui (2008). |
| 3 | Nitrato de plata 1 x 10-3 g L-1 y Carbón activado 1.5% | 85.71a | Tagelsir et al. (2006). |
*Medias con la misma literal son estadísticamente iguales (Duncan, p≤ 0.05).
The three treatments evaluated for choosing the initial culture medium, showed good results by the high percentage of survival of 85.71 to 94.82%, although there were no statistically significant differences, according to the analysis of variance. Saelew and Yang (2009) obtained different results in their study on the in vitro propagation of Psidium guajava L. using apexes in MS and to prevent oxidation of the culture medium supplemented with ascorbic acid 100 mg L-1 and acid citric 150 mg L-1, most sprouting, 78.8% had it with 2 mg L-1 BAP and 0.1 mg L-1 IBA.
In vitro multiplication
According to the results of the in vitro regeneration from nodal segments of guava treatments growth regulators they were equal, only two witnesses had statistically significant differences (Table 9).
Table 9 Results of treatments for in vitro regeneration.
| Tratamiento | Medias (Longitud de brotes en cm ) | Medias (Núm. De hojas/brote) |
| 1. Segmento nodal uno, 1 mg L-1 ВАР | 1.0429 ab | 2.143 ab |
| 2. Segmento nodal dos, 1 mg L-1 ВАР | 0.8857 ab | 2.857 ab |
| 3. Segmento nodal uno, 0.5 mg L-1 BAP + 0.1mg L-1 IBA | 1.4857 a | 3.000 a |
| 4. Segmento nodal dos, 0.5 mg L-1 BAP + 0.1 mg L-1 IBA | 0.9857 ab | 2.571 ab |
| 5. Segmento nodal uno, testigo | 0.0714 b | 0.286 b |
| 6. Segmento nodal dos, testigo | 0.100 b | 0.286 b |
*Medias con la misma literal son estadísticamente iguales (Duncan, p≤ 0.05).
The three treatment obtained outbreaks of 1.4 cm in length and three leaves per shoot. Kumar et al. (2009) in his work on in vitro guava regeneration, used nodal segments established in MS, most sprouting was 1 mg L-1 BAP and developed 2.45 shoots per explant and optimal treatment to induce root was 1 mg L-1 IBA. Ali et al. (2003) in his work on in vitro micropropagation guava, used nodal segments set in MS, got up 3.72 shoots per explants with a length of 3 cm with 2 mg L-1 BAP, reported having had better results in the formation roots using 1 mg L-1 IBA, with roots to 5.4 cm in length. Tagelsir et al. (2006) reported the treatment of 1 mg L-1 IBA as the most effective for rooting Psidium guajava L. Muhammad et al. (2012) after overcoming the stage of in vitro multiplication and induce the formation of root seedlings, gradually acclimated to place them in pots with compost.
The complete standardized protocol obtained in this study for the establishment in vitro using nodal segments of guava shown in Table 10.
Table 10 Standardized protocol for in vitro multiplication of guava from nodal segments.
Identifying contaminating microorganisms from the culture medium
Two kinds of fungi were identified Aspergillus sp., and Alternaria sp. In Aspergillus conidia are chains originating from the conidiogenous cell or phialide. In Aspergillus cells adjacent to the phialides called metulae or supporting cells they were observed. The second was identified fungus Alternaria sp., having long conidiophores with conidia primary branched chains. Branching occurs predominantly from the apex and primary conidial conidio can reach up to wice the size of the next conidio wearing a short secondary conidiophores (Barnet and Hunter, 1972). The results are consistent with those reported by Acosta et al. (2002) who in his work on epiphytic and fungal contaminants mycobiota during in vitro establishment of guava (Psidium guajava L.) identified nine genera of filamentous fungi among which Aspergillus sp., and Alternaria sp., concluded that explants can be established in vitro by using antibiotics, fungicides and antioxidants in the culture medium. Fungal contaminants in vitro establishment of Psidium guajava L. are located within the area of the nodal segments, which prevents the action of surface disinfectants used. Among the pathogenic fungi in vitro culture identified guava are Aspergillus sp., and Alternaria sp. (Ramirez et al., 2000).
Conclusions
It was developed and tested a protocol for in vitro propagation of guava proposed obtained from nodal segments of trees in the field (Table 10). In step aseptic treatment was the best combination of Benomyl 2 g L-1, Carbendazim 2 g L-1 fungicide copper oxychloride and 1 g L-1. Oxidation of plant material is exceeded, the earliest collect explants, in the months of april to june and 0.5% PVP using chlorine 5% and 3 drops of Tween 20; thus ensuring their aseptic facility. In assessing the initial culture medium there were no statistical differences, but the treatment was chosen one PVP 0.75 g L-1 and activated charcoal 2 g L-1. In the multiplication stage all growth regulator treatments were statistically equal. microscopic identification of the two contaminating fungi was performed during propagation in vitro guava, Aspergillus sp., and Alternaria sp.
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Received: November 2015; Accepted: March 2016
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