Plant tissue culture is a biotechnological technique that includes the maintenance of plants or their components under controlled environmental conditions, with the absence of associated microorganisms, heterotrophic nutrition, and plastic or glass containers (Suárez Padrón 2020Suárez Padrón, I. E. 2020. Prehistoria e historia del Cultivo de Tejidos Vegetales. En: Suárez Padrón, I. E. (ed.), Cultivo de Tejidos Vegetales. Fondo Editorial Universidad de Córdoba, pp. 13-19, ISBN: 978-958-5104-09-9.). One of the main problems that arise when trying to establish in vitro cultures is microbial contamination composed of various types of microorganisms (fungi, yeasts, bacteria, phytoplasmas, virus), which can cause the death of plant tissues, since that compete for nutrients and modify the culture medium (Mroginski et al. 2010Mroginski, L., Sansberro, P. & Flaschland, E. 2010. Parte I: Herramientas Básicas. Capítulo 1: Establecimiento de cultivos de tejidos vegetales. En: Levitus, G., Echenique, V., Rubinstein, C., Hopp, E., & Mroginski, L. (eds.), Biotecnología y mejoramiento vegetal II. Editorial Instituto Nacional de Tecnología Agropecuaria, Argentina, p. 1-15. and Nikoloff 2015Nikoloff, N. 2015. No siempre sale todo bien... En: Sharry, S. E., Adema, M. & Abedini, W. (eds), Plantas de probeta: manual para la propagación de plantas por cultivo de tejidos in vitro, Editorial de la Universidad Nacional de La Plata (EDULP), Buenos Aires, Argentina, pp. 1-102, ISBN: 978-950-34-1254-1.).
Among the substances used for the disinfection of explants are sodium hypochlorite (NaClO), calcium hypochlorite Ca(ClO)2, hydrogen peroxide (H2O2), commercial chlorine and bichloride of mercury (HgCl2), among others (Alvarado Capó 1998Alvarado Capó, Y. 1998. Contaminación microbiana en el cultivo in vitro de plantas. En: Pérez Ponce, J. N. (ed.), Propagación y mejora genética de plantas por biotecnología, Santa Clara, IBP, pp. 81-104, ISBN:959-7122-02-2.). Of these, the most used is sodium hypochlorite in plant micropropagation, due to its low cost, easy acquisition, and less phytotoxic effect on tissues (Ramírez Correa et al. 2014Ramírez Correa, L. A., Granados Moreno, J. E., & Carreño González, N. E. 2014. "Evaluación del efecto de tratamientos de desinfección con hipoclorito de sodio sobre segmentos nodales de Guadua angustifolia Kunth para el establecimiento del cultivo in vitro". RIAA, 5(1): 155-169, ISSN: 2145-6453.).
The Cenchrus purpureus Schum cv. Cuba CT-115 is widely used in Cuba due to its favorable characteristics of growth and biomass production, less resistance to cutting, more leaves, drought tolerance, low lignin content, high intake and use by the animal and less internodes distance as age advances. For these reasons, it offers better possibilities for its harvest as a biomass bank, including grazing (Herrera and Martínez 2015Herrera, R. S. & Martínez, R. O. 2015. Mejoramiento genético. En: Herrera, R. S. (ed.), Producción de biomasa de variedades y clones de Pennisetum purpureum para la ganadería, EDICA, Mayabeque, Cuba, pp.13-32, ISBN:078-959-7171-67-6. and Crespo and Martínez 2016Crespo, G. & Martínez, R. O. 2016. "Study of the chemical soil fertility in the biomass bank technology of Cenchrus purpureus Schum cv. CUBA CT-115 with different exploitation years". Cuban J. Agric. Sci. 50th Anniversary. 50(2): 497, ISSN: 2079-3472. and Fortes et al.2019Fortes, D., Herrera, R. S., García, M., Cruz, A. M. & Romero A. 2019. "Mineral composition of Cenchrus purpureus cv. Cuba CT-115, as biomass bank, after grazing". Cuban J. Agric. Sci. 53(4): 425-435, ISSN: 2079-3472.).
The disinfection of apical cones of C. purpureus cv. Cuba CT-115 with sodium hypochlorite could reduce contamination by endogenous microorganisms in the in vitro establishment phase of this culture. The objective of this trial was to evaluate the effect of different concentrations of sodium hypochlorite on the disinfection of explants of Cenchrus purpureus cv. Cuba CT-115 for its in vitro establishment.
Plants of Cenchrus purpureus (Schumach) Morrone cv. Cuba CT-115 from Poaceae family, with the same regrowth age, collected in the germplasm bank of Instituto de Ciencia Animal were use as plant material. A total of 24 portions of apex were taken.
The apex were disinfected with 5 % commercial sodium hypochlorite for three minutes and rinsed twice with distilled water at the time of collection. Then they were transferred to the Laboratorio de Biotecnología de la Facultad de Agronomía de la Universidad Agraria de La Habana in bottles duly washed with commercial detergent, rinsed twice with distilled water. In the laboratory, the apex were washed with a commercial detergent under agitation for five minutes and rinsed three times with distilled water.
The sowing of the explants, apical cone, was carried out in the laminar flow properly disinfected with 70 % ethanol and an ultraviolet light lamp was placed on for 30 min before starting the sowing. The apical cones were extracted in laminar flow and disinfected with 70% ethanol for one minute. Subsequently, they were washed with sufficient sterile distilled water. Next, two disinfection variants with 3 and 5% sodium hypochlorite were tested for 10 min. For this, a total of 12 apical cones per treatment were used. Finally, they were washed with enough sterile distilled water before sowing.
After disinfection, the explants were placed in Murashige and Skoog (1962)Murashige, T. & Skoog, F. 1962. "A revised medium for rapid growth and biossays with tobacco tissue culture". Physiol Plant, 15: 473-497, Online ISSN: 1399-3054. basal culture medium, with 30 g L-1 sucrose, 1 mg L-1 of 6-BAP, and 8 g L-1 of agar. The pH was fitted to 5.7 with 0.1N sodium hydroxide and 0.1N hydrochloric acid before sterilization, which was performed in an autoclave at 121 ºC and 1.5 kg cm-2 pressure for 20 min. The explants were established in test tubes with 15 mL of culture medium. The research was carried out under controlled conditions in a growth chamber with a photoperiod of 16 h of lighting and 8 h of darkness, relative humidity of 70 ± 5%, light intensity of 45 µmol m-2 s-1 and temperature of 25 ± 2 °C for a period of eight days.
The evaluations were made visually at four and eight days after sowing. The variables number of sprouted explants, number of non-sprouted explants, number of necrotic explants, number of contaminated explants, number of explants contaminated with fungi, and number of explants contaminated with bacteria were observed.
A completely random design was used, with 12 repetitions per treatment. For data processing, an analysis of proportions comparison (Chi-square) was performed using the statistical package ComparPro 1.0 (Font et al. 2007Font, H., Noda, A., Torres, V., Herrera, M., Lizazo, D., Sarduy, L. & Rodríguez, L. 2007. Paquete estadístico ComparPro versión 1, Instituto de Ciencia Animal, Dpto Biomatemática.).
Four days after sowing the explants, there were not differences (p > 0.05) between the treatments with 3 and 5 % sodium hypochlorite (table 1), for the variables number of necrotic explants (figure 1a and 1b), number of sprouted explants and number of non-sprouted explants. After four days, there was not contamination by fungi or bacteria in the two studied treatments (figure 1c).
| Treatments Variables | Sodium hypochlorite 3 % | Sodium hypochlorite 5 % | Total | SE (±) Signif. | |||
|---|---|---|---|---|---|---|---|
| No. | % | No. | % | No. | % | ||
| Number of sprouted explants | 5 | 35.71 | 9 | 64.29 | 14 | 100 | 13.36 p = 0.2850 |
| Number of non-sprouted explants | 7 | 70.00 | 3 | 30.00 | 10 | 100 | 15.81 p = 0.2059 |
| Number of necrotic explants | 2 | 40.00 | 3 | 60.00 | 5 | 100 | 15.81 p = 0.6547 |
After eight days, there were not differences (p > 0.05) between the treatments with 3 and 5 % sodium hypochlorite (table 2), for the variables number of sprouted explants, number of non-sprouted explants, number of necrotic explants, number of contaminated explants and number of explants contaminated by bacteria (Figures 2a, 2b and 2c). In this observation, there was not fungal contamination.
| Treatments Variables | Sodium hypochlorite 3 % | Sodium hypochlorite 5 % | Total | SE (±) Signif. | |||
|---|---|---|---|---|---|---|---|
| No. | % | No. | % | No. | % | ||
| Number of sprouted explants | 6 | 40.00 | 9 | 60.00 | 15 | 100 | 12.91 p = 0.4386 |
| Number of non-sprouted explants | 6 | 66.67 | 3 | 33.33 | 9 | 100 | 16.67 p = 0.3127 |
| Number of necrotic explants | 3 | 50.00 | 3 | 50.00 | 6 | 100 | 15.81 p = 0.9999 |
| Number of contaminated explants | 7 | 58.33 | 5 | 41.67 | 12 | 100 | 14.43 p = 0.5637 |
| Number of explants contaminated by bacteria | 5 | 50.00 | 5 | 50.00 | 10 | 100 | 15.81 p = 0.9999 |
The superficial disinfection of plant explants is through chemical compounds. It is not possible to recommend a general procedure that guarantees removal the microorganisms with the least possible damage to the explant. Some procedures are based on the only use of ethanol or sodium hypochlorite. The most popular method consists of a double disinfection by immersing the explants in ethanol (70 %) for 20-60 seconds, followed by 1-3 % sodium hypochlorite. Next, it should be washed with sufficient water and detergent for three to 30 min, depending on the nature of the explant. Finally, it is necessary to remove the remains of these products through several washes with sterile distilled water (Mroginski et al. 2010Mroginski, L., Sansberro, P. & Flaschland, E. 2010. Parte I: Herramientas Básicas. Capítulo 1: Establecimiento de cultivos de tejidos vegetales. En: Levitus, G., Echenique, V., Rubinstein, C., Hopp, E., & Mroginski, L. (eds.), Biotecnología y mejoramiento vegetal II. Editorial Instituto Nacional de Tecnología Agropecuaria, Argentina, p. 1-15.).
In studies of genetic improvement by in vitro tissue culture techniques, Herrera et al. (2003)Herrera, R. S., Chaplé, Z., Cruz, A. M., Romero, A. & García, M. 2003. "Obtainment of Pennisetum purpureum plantles resistant to drought and salinity. Technical note". Cuban J. Agric. Sci. 37(2): 189-191, ISSN: 2079-3472. used 10 % sodium hypochlorite for 10 min for the disinfection of apical cones of Cuba CT-115. In the consulted bibliography, there is little evidence of studies referring to the disinfection of explants of C. purpureus cv. Cuba CT-115 for its in vitro establishment. However, in sugar cane (Saccharum officinarum) several studies on this aspect are reported.
Similar results are reported to those of this study with 5 % NaClO treatment for 15 min in apical meristems of S. officinarum, variety CCSP 89-43, which showed a low percentage of contamination (Betancourt Guerrero 2017Betancourt Guerrero, J. M. 2017. Evaluación del cultivo in vitro de caña de azúcar (Saccharum officinarum) var. CCSP 89-43 a partir de meristemos apicales mediante la técnica de organogénesis. Tesis en opción al título de Microbiólogo Industrial, Universidad de Santander, Colombia, pp. 14-47.). This effect could be due to the action of sodium hypochlorite on microorganisms, since it acts to inhibit enzymatic reactions and protein denaturation (Sánchez and Sáenz 2005Sánchez, L. & Sáenz, E. 2005. "Antisépticos y desinfectantes". Dermatol. Peru, 15(2):172-174, ISSN: 1609-7203.).
The results of this study differ from the high values of necrosis that were reported when disinfecting explants of the apical leaf section and axillary buds of four varieties of S. officinarum, with 5 and 7 % NaClO (Araya Contreras 2006Araya Contreras, J. F. 2006. Establecimiento in vitro de cuatro variedades de caña de azúcar a partir de explantes foliares y yemas axilares. Tesis en opción al grado de Master en Ciencias, Zamorano, Honduras, pp. 1-22.). Similarly, in the disinfection of foliar explants of S. officinarum, variety CP 72-2086, a high percentage of necrosis was obtained in plant tissues with concentrations of 10 % sodium hypochlorite for 25 min (Martínez Vega 2005Martínez Vega, A. E. 2005. Elaboración de un procedimiento de desinfección y establecimiento in vitro de caña de azúcar, variedad CP 72-2086 a partir de yemas axilares. Tesis en opción al grado de Master en Ciencias, Zamorano, Honduras, pp. 1-30.).
Similar results to the previous, when using high concentrations of 20 and 30 % sodium hypochlorite and two immersion times (15 and 20 min), to disinfect apical cones of the sugar cane varieties ITV 92-1424, Laica 82-2220 and Q28-2. In this study good results of survival and asepsis are reported when applying 20% commercial chlorine for 20min, while the dose of 30 % NaClO was effective to control the contamination, although it caused darkness of the expalnts (Rangel-Estrada et al. 2016Rangel-Estrada, S. E., Hernández-Meneses, E. & Hernández-Arenas, M. 2016. "Micropropagation of sugarcane varieties grown in México". Rev. Fitotec. Mex, 39(3): 225 - 231, ISSN: 0187-7380.).
According to Nikoloff (2015)Nikoloff, N. 2015. No siempre sale todo bien... En: Sharry, S. E., Adema, M. & Abedini, W. (eds), Plantas de probeta: manual para la propagación de plantas por cultivo de tejidos in vitro, Editorial de la Universidad Nacional de La Plata (EDULP), Buenos Aires, Argentina, pp. 1-102, ISBN: 978-950-34-1254-1., darkening, growth inhibition, necrosis, and explant death may be associated with oxidative stress derived from the use of disinfectant agents. The explant oxidation levels are related to the increase in the concentration and exposure time of the explant to the disinfectant (Lapiz-Culqui et al. 2021Lapiz-Culqui, Y. K., Tejada-Alvarado, J. J., Meléndez-Mori, J. B., Vilca-Valqui, N. C.,Huaman-Huaman, E. y Oliva, Manuel. 2021. "Establecimiento y multiplicación in vitro de papayas de montaña: Vasconcellea chachapoyensis y Vasconcellea x Heilbornii". Bioagro 33(2): 135-142, ISSN 1316-3361.), which shows the toxic effect of NaClO on plant tissues (Causil et al. 2017Causil, V. L. A., Coronado, G. J. L., Verbel, M. L. F., Vega. J. M. F., Donado, E. K. A. & Pacheco, G. C. 2017. "Cytotoxic effect of sodium hypochlorite (NaClO) in apical cells of onion roots (Allium cepa L.) ". Rev. Colomb. Cienc. Hortic. 11 (1): 97-104, Online ISSN: 2422-3719.). It could be showed that the different responses of the explants to the use of sodium hypochlorite correspond to the tolerance level of each plant species.
The difference between the results of previous studies and those obtained in the disinfection of explants of C. purpureus cv. Cuba CT-115 can be related to the manipulation of plant material, since it is one of the most common sources of contamination. In addition, it is confirmed that the superficial disinfection of the explant is conditioned by factors such as the type, concentration and time of exposure to disinfectant agents, but also by the origin of the explant (Ticona and Triguero 2020Ticona, J. & Triguero. M. L. 2020. "Evaluación del comportamiento in vitro de dos variedades de papaya (Carica papaya L.) mediante embriogénesis somática en la Estación Experimental Sapecho". RIIARn, 7(1): 55-61, ISSN: 2518-6868.).
It is concluded that the indicators studied for the disinfection of apical cones of Cuba CT-115 with concentrations of 3 and 5 % of sodium hypochlorite did not present differences. Therefore, it is recommended to disinfect with 3 % sodium hypochlorite, which saves this substance during the procedure. It is recommended to performed studies with other concentrations of sodium hypochlorite and different immersion times in the in vitro establishment stage of this culture.