On-line version ISSN 0717-6643
Gayana Bot. vol.67 no.1 Concepción 2010
Gayana Bot. 67(1): 19-26, 2010 ISSN 0016-5301
Plant growth regulators optimization for in vitro cultivation of the orchid Guarianthe skinneri (Bateman) Dressier & W.E.Higgins
Optimización de reguladores de crecimiento para el cultivo in vitro de la orquídea Guarianthe skinneri (Bateman) Dressier & W.E.Higgins
Christian Yanelly Coello1, Clara Luz Miceli1, Carolina Orantes1, Luc DenDooven2 & Federico Antonio Gutiérrez3*
1Laboratorio de Cultivo de Tejidos Vegetales, Universidad de Ciencias y Artes de Chiapas, Libramiento norte. Tuxtla-Gutiérrez, México.
2Laboratory of Soil Ecology, Dept. Biotechnology and Bioengineering, Cinvestav, Av. Instituto Politécnico Nacional 2508, C.P. 07000 México D. F., México.
3Laboratorio de Biotecnología Vegetal, Instituto Tecnológico de Tuxtla-Gutiérrez, Tuxtla-Gutiérrez, México. *email@example.com
An in vitro culture procedure was developed to induce shoots and roots of Guarianthe skinneri (Orchidaceae) plantlets regenerated from seed-derived protocorms on a Murashige and Skoog (MS) médium supplemented with 6-benzyladenine (BA), indole-3-acetic acid (IAA), a-naftalenacetic acid (NAA) and gibberellic acid (GA3). A máximum of 10.6 shoots was obtained with 16.1 μM NAA, 17.1 μM IAA, 6.3 x 10-9 μM GA3 and 0.0023 μM BA. A máximum of 4.0 roots on each shoot with 5.4 μM NAA, 17.1 μM IAA, 0.001 μM AG3 and 4.6 x 10-9 μM BA. Máximum shoot and root length was obtained with a minimum of GA3, but a máximum IAA. GA3, was the main factor controlling shoot and root induction and elongation.
Keywords: 6-benzyladenine, indole-3-acetic acid, a-naftalenacetic acid, gibberellic acid, shoot induction, rooting.
Se desarrolló un procedimiento para el cultivo in vitro para inducir brotes y raíces en las plántulas de Guarianthe skinneri (Orchidaceae) regeneradas a partir de protocormos derivados de semillas en un medio de Murashige & Skoog (MS) complementado con 6-benziladenina (BA), ácido indol-3-acético (AIA), ácido a-naftalenacético (ANA) y ácido giberélico (GA3). Se obtuvo un máximo de 10,6 brotes con 16,1 μM de ANA, 17,1 μM de AIA, 6,3 x 10-9 μM de GA3 y 0,0023 μM de BA. Un máximo de 4,0 raíces en cada brote se obtuvo con 5,4 μM de ANA, 17,1 μM de AIA, 0,001 μM de AG3 y 4,6 x 10-9 μM de BA. La longitud máxima de brotes y raíces se obtuvo con la más baia concentración de GA3, y la mayor concentración de AIA. El GA3, fue el factor principal que controló la inducción y la elongación de los brotes y raíces.
Palabras clave: 6-benciladenina, ácido indol-3-acético, ácido a-naftalenacético, ácido giberélico, inducción de brotes, enraiz amiento.
Guarianthe skinneri (Bateman) Dressier & W.E.Higgins belongs to Orchidaceae. The ornamental use of this species has increased its demand, but its natural habitat is being destroyed by deforestation so it is now recognized as threatened under ñame Cattleya skinneri within mexican law NOM-059-ECOL-2001 (Diario Ofcial, 2002). Guarianthe skinneri (Bateman) Dressier & W.E.Higgins Micropropagation might be a useful technique to cultívate belongs to Orchidaceae. The ornamental use of this G. skinneri. It offers the possibility to produce thousands of species has increased its demand, but its natural habitat is plants of the desired clone. The development of an effcient being destroyed by deforestation so it is now recognized micropropagation protocol can play a signifcant role in the commercial cultivation of vulnerable plant species, thereby conserving them in their natural habitat (Amoo et al. 2009). Encouraging results have been obtained with other species recognized as vulnerable using micropropagation (Bopana & Saxena 2008). In micropropagation, growth regulators are very important. Gibberellic acid (GA3) has a positive effect on root formation of loblolly pine (Pinus taeda L.) when added with Índole butyric acid (IBA) and butyric acid (BA) (Tang 2001). Auxiliary shoots of loblolly pine were sub-cultured twice at a 4-week interval on woody plant basal médium (WPM) supplemented with BA, GA3, or GA3 + BA to improve shoot growth. A máximum of 93% shoot growth was obtained on WPM supplemented with 2.2 μM BA (Park et al. 2008). Micropropagation of lentils (Lens culinaris Medik.) was optimized on Murashige Skoog (MS) media (Murashige & Skoog 1962) to regenérate shoots in vitro from nodal segments. The number of shoots per explant, the number of nodes per shoot and shoot length was most affected by the concentration of GA3 and 6-benzyladenine (BA), with only small interaction effects between them (Ahmad et al. 1997). Shoots of Acacia mangium were elongated on MS médium containing 0.045 uM thidiazuron (TDZ) supplemented with 7.22 μM GA3 (Deyu & Hong 2001). For in vitro propagation of Oroxylum indicum, a forest tree, the best médium for proliferation was MS médium with 8.87μM 6-BA and 2.85uM indole-3-acetic acid (IAA). However, incorporation of 1.44 μM GA3 was necessary to increase shoots elongation (Naomita & Ravishankar 2004). The study reported here was done to determine the optimum concentrations of 6-benzyladenine (BA), indole-3-acetic acid (IAA), a-naftalenacetic acid (NAA) and gibberellic acid (GA3) to increase shoot and roots numbers in plantlets of Guarianthe skinneri grown in vitro.
MATERIALS AND METHODS
Capsules disinfection and cultivation
G. skinneri capsules were collected from the Cañón del Sumidero national park 16°47'56.5" north latitude 93°05 '28.5'' west latitude at 966 masl in Chiapas (México). Five capsules were washed with water and commercial soap for 5 min and washed 3 times with sterile distilled water. Capsules were disinfected in 70% (v/v) ethanol for 5 min, immersed in 10% (m/v) aqueous calcium hypochlorite solution for 10 min and washed three times with sterile distilled water (Fig. 1 a). Hundred and fifty assays tubes (25 cm3) containing 20 cm3 MS médium supplemented with sucrose 30 g dnr3, myo-inositol 0.1 g dm3, NaHP04 0.05 g dnr3 and solidifed with 2.5 g dm-3 phytagel, were covered with plástic. The tubes with médium were sterilized at 1.5 kg cm2 for 15 min and two disinfected seeds were placed in each tube and incubated under cool white fuorescent light (50 μamol nr2 s1) and 16/8 photoperiod at 25-27°C.
figure 1. Micropropagation of Guarianthe skinneri. (a) Capsules, (b) germination and growth of protocorms, (c) protocorms differentiation and (d) plantlets.
figura 1. Micropropagación de Guarianthe skinneri. (a) Cápsulas, (b) germinación y crecimiento de los protocormos, (c) diferenciación de los protocormos y (d) plántulas.
Seeds germinated after four weeks culturing and zygotic protocorms were obtained after another two weeks (Fig. 1 b). Shoots developed two weeks later and each shoot was transferred to 150 cm3 bottles containing 30 cm3 of the same medium (Fig. 1c, d).
An orthogonal experimental design of L9 (34) in triplicate was used to investigate the effects of GA3, BA, NAA and IAA, on number of shoots and roots, and leaf and principal root length (Table 1) (Ross 1989). The symbol L (bc) is used to represent the orthogonal array where a is the number of experimental runs, b the number of levéis for each factor or variable and c the number of factors investigated. Independent variables were different concentrations of GA (0, 2.9 and 8.7 uM), BA (0, 2.5 and 4.9 μM), NAA (0, 5.4 and 16.1 μM) and IAA (0, 5.7 and 17.1 μM). Bottles were incubated under cool white fuorescent light (50 μamol nr2 s1) and 16/8 photoperiod at 25-27 °C for two months. Number of shoots and roots, and length of shoots and roots of each plant were determined.
Table I. Orthogonal experimental design L9 (34) done in triplícate to investígate the effect of different concentrations of gibberellic acid (GA3), 6-bencyl adenine (BA), a-naftalenacetic acid (NAA) and Índole acetic acid (IAA) on shoot and root number and length in plantlets of Guarianthe skinneri grown in vitro.
Tabla I. Diseño experimental ortogonal L9 (34) realizado por triplicado para investigar el efecto de diferentes concentraciones de ácido giberélico (GA3), 6-bencil adenina (BA), ácido a-naftalenacético (ANA) y ácido indol acético (AIA) sobre el número y longitud de los brotes y raíces en plántulas de Guarianthe skinneri cultivadas in vitro.
The plantlets obtained from each treatment with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agro lite for hardening at 22±2°C under diffuse light (16/8-h photoperiod). Potted plantlets were covered with polyethylene membranes to ensure high humidity and watered daily with liquid 1/2-MS médium free of sucrose. After 3 wk, the membranes were removed and the plantlets were irrigated with tap water. The plantlets were acclimatized for 1 wk in the laboratory conditions and then transferred to a greenhouse (Fig. 1 d).
The Statistica (2000) software was used to analyze data obtained with the L9 (34) orthogonal array using a confdence limit of 5%. The linear and quadratic valúes of all factors and the interactions between them were tested. The percent contribution was calculated to determine the portion of the total variation observed in an experiment attributable to each signifcant factor and/or interaction (Ross 1989). The percent contribution is a function of the sums of squares for each signifcant factor and indicates the relative power of each and/or interaction to reduce variation. If the factor and/or interaction levéis are controlled, then the total variation can be reduced by the amount indicated by the percent contribution.
Characteristics of the plantlets were subjected to a one-way analysis of variance (ANOVA) to test for signifcant differences. The latter analyses were performed using SAS statistical package (SAS 1989).
RESULTS AND DISCUSSION
The number of shoots varied from 5.0 in treatment 8 to 10.2 in treatment 3 (Table I). The most important factor for shoot proliferation was gibberellic acid and it explained 87% of the variation found, IAA 7% and BA and NAA 3% (Table II). A NAA concentration of 16.1 μM and 17.1 μM IAA in the MS medium resulted in a máximum number of shoots, but with a GA3 concentration of only 6.3 x 109 μM and 0.0023 μM BA. These concentrations induced 3.7 more shoots in G. skinneri plantlets when compared to the overall mean (Table III). The model derived from the experimental data explained 83% of the variability (Table IV).
Tabla II. Análisis ANO VA usando todos los factores con efecto signifcativo (P<0,05) sobre el número y longitud de los brotes y raíces en plántulas de Guarianthe skinneri cultivada in vitro.
Table III. Optimal concentration and contribution of gibberellic acid (GA3), 6-bencyl adenine (BA), a-naftalenacetic acid (NAA) and Índole acetic acid (IAA) on shoot and root number and length in plantlets of Guarianthe skinneri grown in vitro.
Tabla III. Concentración óptima y contribución del ácido giberélico (GA3), 6-bencil adenina (BA), ácido a-naftalenacético (ANA) y ácido indolacético (AIA) sobre el número y longitud de los brotes y raíces en plántulas de Guarianthe skinneri cultivada in vitro.
Tabla IV. Modelos de regresión de las respuestas y su significancia para el número de brotes y de raíces y de la longitud de los brotes y raíces en la micropropagación de Guarianthe skinneri (P<0,05).
The physiological effect of GA3 and positive effect on plant growth is well-known as it increases the concentrations of soluble carbohydrates during seed germination and plant growth (Bialecka & Kepcznski 2007). GA3 is therefore used in the cultivation of vegetables and fruits with plant tissue cultures (Isogai et al. 2008). GA3 stimulated the germination of Amaranthus caudatus L. and the plantlets contained larger concentrations of glucose and maltose (Bialecka & Kepcznski 2007). However, the optimum concentrations required in this study were low compared to those reported in other studies. It might be that the used MS médium was different from the one used in other studies or the effect of GA3 was transient and only a small pulse was required to obtain the best results (Naor et al. 2008).
Number of roots
The number of roots varied from 1.0 in treatment 8 to 3.7 in treatment 3 (Table I). The most important factor for root proliferation was GA3 and it explained 45% of the variation, while IAA explained 30%, NAA 18% and BA 6% (Table II). A NAA concentration of 5.4 uM and IAA of 17.1 uM in the MS médium resulted in a máximum number of shoots, but with a GA3 concentration of only 0.001 uM and 4.6 109 uM BA. These concentrations induced 0.6 more roots in G. skinneri plantlets when compared to the overall mean (Table III). The model derived from the experimental data explained 79% of the variability (Table III).
These results are important because a limited root formation is a major obstacle in micropropagation and conventional propagation, and conditions during in vitro rooting might have an important effect on performance after transfer ex vitro (De Klerk 2002). Contributions of GA3 and IAA were the most important. Soon after the discovery of IAA its rhizogenic activity was reported (De Klerk et al. 1999). However, the complementary effect of IAA and GA3 on root induction has not been reported yet. Hormonal action should be sequential because many researchers recognize that rooting is not a single process, but a developmental process consisting of distinct steps, each with its own requirements (De Klerk et al. 1999).
Shoot length varied from 0.8 cm in treatment 7, 8 and 9 to 1.3 in treatments 3 and 4 (Table 1). The most important factor for shoot growth was GA3 and it explained 74% of the variation found, IAA 13%, BA 10% and NAA 3% (Table II). The overall mean shoot length was 1.0 cm and increased with 0.5 cm when grown in MS médium supplemented with 4.6 x 109 μM GA3, 0.0043 μM BA, 1.2 x 105 μM NAA and 14.92 μM IAA (Table III). The model derived from the experimental data explained 82% of the variability (Table IV).
GA, is best known for its role in elongation of axial organs, such as stems, petioles and inforescences (DeMason 2005). The effects of GA3, on shoot elongation are well documented for several plants cultivated in vitro, e.g. Cephaelis ipecacuanha (Isogai et al. 2008) and Acacia sinuate (Vengadesan et al. 2002). The effects of GA3 on shoot elongation are due to the increase in soluble carbohydrates induced by GA3 and available for metabolic processes (Bialecka & Kepcznski 2007).
Root length varied from 0.2 cm in treatment 7, 8 and 9 to 0.9 in treatment 4 (Table I). The most important factor for root growth was GA3 and it explained 55% of the variation found, IAA 24%, BA 8% and NAA 13% (Table II). The overall mean root length was 0.4 cm and increased with 0.4 cm when grown in MS medium supplemented with 0.031 μM GA3, 7.0 x 10-8 μM BA, 16.1 μM NAA and 17.02 μM IAA (Table III). The model derived from experimental data explained 74% of the variability (Table IV).
These results are due to gibberellic acid (GA3) is a growth factor that promotes elongation, due to the increase in the rate of cell division by promoting the growth and number of shoots, since its action is specific in active growth areas as protocorms and root apices (Pierik 1990). Alvarado (2000) was reported to multiply apices of lateral shoots that developed G. skinneri protocorms. As in the present investigation, the response was favorable for G. skinneri micropropagation, because these growth regulators at low concentrations act as promoters and inducers of cell division. Although the use of GA3 in protocorms from orchids is relatively scarce, the auxins and cytokinins have been widely used in species such as Barkeria obovata, Catasetum intergerrimun, Cattleya x Esbetts, Epidendrum veroscriptum, Cuitlauzina pendula, Dendrobium sp. Anceps Laelia, Lycaste skinneri, Mormodes tuxtlensis, Oncidium tigrinum Oncidium sp. and Stanhopea tigrina. The basal medium used for in vitro cultivation was MS (Murashige & Skoog, 1962) added with vitamins and growth regulators, in order to induce the formation of shoots and increase the number of roots. Each species responds differently, so they have developed 3 to 26 shoots, the above depending on the interaction of type and level of growth regulator, using IAA (1 mg / 1) and NAA (0 mg / 1) BA (0.5 mg / 1) and 2,4-D (0.5 mg / 1), BA (0, 0.5, 2, 3 and 5 mg / 1) and NAA (0, 0.1, 0.5 mg / 1) (Hernández et al. 2001, Salazar 2003, Baltazar 2004, Tinoco 2006, Askar et al. 2007, Kalimuthu et al. 2007, Maza 2008).
It was found that GA3 was the most important factor for shoot and root induction and elongation. However, the optimal concentrations were small and larger concentrations had a negative effect on plant development. The results of this study present new evidence regarding the effect of GA3 to micropropagate G. skinneri through protocorms and this protocol could be useful for other orchids.
The research was funded by the Dirección General de Educación Superior Tecnológica, Project UR TGZ-07 Micropropagación de plantas endémicas de Chiapas y en peligro de extinción o amenazadas evaluando su variabilidad genética.
Ahmad, M., A.G. Fautrier, D.L. Mcneil, G.D. Hill & D.J. Burritt. 1997. In vitro propagation of Lens species and their FI interspecifc hybrids. Plant Cell, Tissue and Organ Culture 47(2): 169-176. [ Links ]
Alvarado-Ulloa, C. 2000. "Micropropagación de Cattleya skinneri y Cattleya skinneri x Cattleya maxima por cultivo de ápices". Informe de proyecto. Instituto Tecnológico de Costa Rica. San José, Costa Rica. 89 pp. [ Links ]
Amoo, S.O., J.F. Finnie & J. Van Staden. 2009. In vitro propagation of Huernia hystrix: an endangered medicinal and ornamental succulent. Plant Cell, Tissue and Organ Culture 96(3): 273-278. [ Links ]
Askar, S., K. Nasiruddin & M. Hug. 2007. In vitro root formation in Dendrobium orchid plantlets with IBA. Journal Agricultural Rural Development 5(1-2): 48-51. [ Links ]
Baltazar, R. 2004. Micropropagación de Oncidium tigrinum Llave & Lex (Orchidaceae) a partir de protocormos. Tesis de Licenciatura. Universidad Veracruzana. 124 pp. [ Links ]
Bialecka, B. & J. Kepcznski. 2007. Changes in concentrations of soluble carbohydrates during germination of Amaranthus caudatus L. seeds in relation to ethylene, gibberellin A3 and methyl jasmonate. Plant Growth Regulation 51(1): 21-31. [ Links ]
Bopana, N. & S. Saxena. 2008. In vitro propagation of a high value medicinal plant: Asparagus racemosus Willd. In Vitro Cellular & Developmental Biology-Plant 44 (6): 525-532. [ Links ]
De KlerK, G.J. 2002. Rooting of microcuttings: Theory and practice. In Vitro Cellular & Developmental Biology-Plant 38(5): 415-422. [ Links ]
De KlerK, G.J., W. Van del Krieken & J.C. de Jong. 1999. Review the formation of adventitious roots: New Concepts, new possibilities. In Vitro Cellular & Developmental Biology-Plant 35(3): 189-199. [ Links ]
Demason, D.A. 2005. Auxincytokinin and auxin-gibberellin interactions during morphogenesis of the compound leaves of pea (Pisum sativum). Planta 222(1):151-166. [ Links ]
Deyu, X. & Y. Hong. 2001. In vitro regeneration of Acacia mangium via organogenesis. Plant Cell, Tissue and Organ Culture 66(3): 167-173. [ Links ]
Diario oficial. 2002. Norma Oficial Mexicana NOM-059-Ecol-2001 Diario ofcial de la Federación, 6 de marzo del 2002, Segunda sección México City, México. [ Links ]
Hernández, J., O. Hernández & M. Mata. 2001. Regeneración de plántulas a partir de cultivo in vitro de mitades de protocormos de Laelia anceps Lindl. y Catasetum intergerrimum Hook. Boletín Amaranto-Asociación Mexicana de Jardines Botánicos, A. C. 14(1): 3-12. [ Links ]
Isogai, S., K. Touno & K. Shimomura. 2008. Gibberellic acid improved shoot multiplication in Cephaelis ipecacuanha. In Vitro Cellular & Developmental Biology-Plant 44(3): 216-220. [ Links ]
Kalimuthu, K., R. SenThilkumar & S. Vilayakumar. 2007. In vitro micropropagation of orchid, Oncidium sp. (Dancing Dolls). African Journal of Biotechnology 6(10): 1171-1174. [ Links ]
Maza, E. 2008. Cultivo in vitro de Barkeria obovata (C.Presl) Christenson, Orchidaceae. Tesis de Licenciatura. Facultad de Ciencias Biológicas. Universidad de Ciencias y Artes de Chiapas (UNICACH). Chiapas, México. 67 pp. [ Links ]
Murashige, T. & F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497. [ Links ]
Naomita, V. D. & R.V. Ravishankar. 2004. In vitro propagation of Oroxylum indicum Vent. a medicinally important forest tree. Journal of Forest Research 9(1):61-65. [ Links ]
Naor, V., J. Kigel, y. Ben-Tal & M. Ziv. 2008. Variation in Endogenous Gibberellins, Abscisic Acid, and Carbohydrate Content During the Growth Cycle of Colored Zantedeschia spp., a Tuberous Geophyte. Journal of Plant Growth Regulation 27(3): 211-220. [ Links ]
ParK, S.Y., y.W. Kim, H.K. Moon, H.N. Murthy, y.H. Choi & H.M. Cho. 2008. Micropropagation of Salix pseudolasiogyne from nodal explants. Plant Cell, Tissue and Organ Culture 93(3): 341-346. [ Links ]
PieriK, R.1990. Cultivo in vitro de plantas superiores. Ediciones Mundi-Prensa. Madrid, España. 326 pp. [ Links ]
Ross, P.J. 1989. Taguchi techniques for quality engineering. Loss function, orthogonal experiments, parameter and tolerance design. McGraw-Hill International Editions New York. 270-279. [ Links ]
Salazar, V.M. 2003. Micropropagación de Mormodes tuxtlensis Salazar, Cuitlauzina pendula La Llave & Lex y Lycaste skinneri (Batem. ex Lindl.) Lindl. (Orchidaceae) a partir de protocormos. Tesis de Licenciatura. Benemérita Universidad Autónoma de Puebla. México.106 pp. [ Links ]
Tang, W. 2001. In vitro regeneration of loblolly pine and random amplifed polymorphic DNA analyses of regenerated plantlets. Plant Cell Reports 20(2):163-168. [ Links ]
Tinoco, M. 2006. Adquisición de competencia de Stanhopea tigrina. Tesis de Licenciatura. Universidad Veracruzana. México. 102 pp. [ Links ]
Vengadesan, G., A. Ganapathi, S. Amutha & N. Selvaraj. 2002. In vitro propagation of Acacia species - a review. Plant Science 163(4): 663-671. [ Links ]