Propagation and Establishment of Three Endangered Mexican
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PROPAGATION AND TISSUE CULTURE HORTSCIENCE 44(5):1395–1399. 2009. A´ vila-Dı´az and Oyama (2002) also reported in vitro germination of Laelia speciosa. The present study describes the effect of Propagation and Establishment modified Knudson C medium (KCm) and Murashige and Skoog medium (MS) as well as of Three Endangered Mexican the action of different concentrations of N6- benzyladenine (BA) and a-naphthaleneacetic Orchids from Protocorms acid (NAA) on the induction of shoots and PLBs from protocorms and also the survival Martı´n Mata-Rosas1 and Vı´ctor M. Salazar-Rojas of ex vitro plantlets. Instituto de Ecologia, A.C., Unidad de Recursos Forestales, Km 2.5 carretera antigua a Coatepec 350, Xalapa, Veracruz 91070, Mexico Materials and Methods Additional index words. micropropagation, protocorm-like bodies, ex vitro establishment, Seeds of the three species were donated conservation to the Francisco Javier Clavijero Botanical Garden. Open, ripe capsules (obtained from Abstract. Protocols for in vitro propagation from protocorms of Mormodes tuxtlensis hand-pollination) were dried for 24 to 48 h at Salazar, Cuitlauzina pendula La Llave & Lex., and Lycaste skinneri (Batem. Ex. Lind.) 25 °C. The seeds were then extracted and Lind., three endangered species distributed in Mexico and highly appreciated as stored in paper envelopes inside jars over ornamentals, were developed. The effect of two different culture media, Murashige silica gel for 15 to 30 d at 4 °C before and Skoog (MS) and modified Knudson (KCm), combined with varying concentrations of experimentation. 6 N -benzyladenine (0, 2.2, 4.4, 8.9, and 13.3 mM) and a-naphthaleneacetic acid (0, 0.5 and Seed surface sterilization. The seeds were 2.7 mM), were investigated. Shoot formation and development of protocorm-like bodies placed in a filter paper envelope (Whatman were observed. For all three species, cultures in MS produced more shoots per explant No. 1, 110 mm diameter). The envelopes than those in KCm, and those shoots were longer and more robust in appearance. were submerged in sterile distilled water for Maximum number of shoots for M. tuxtlensis (1.5) and C. pendula (24.3) were obtained 30 min, then dipped in 70% (v/v) ethanol for 6 in media supplemented with 13.3 mM and 2.2 mMN-benzyladenine, respectively. Con- 1 min, and then soaked in 30% (v/v) com- versely, for L. skinneri the greatest shoot production (16.4) was achieved in medium mercial bleach solution (1.8% NaOCl) with supplemented with 2.7 mM a-naphthaleneacetic acid. Subculturing explants in MS basal two drops of Tween-80 per 100 mL (Sigma, medium allowed further development and rooting of the shoots as well as growth of St. Louis, MO) for 30 min. This was followed protocorm-like bodies. The effect of different potting mixes on ex vitro survival plantlets by four rinses with distilled sterilized water was also investigated; pine bark:oak charcoal:pumice (3:1:1) allowed the highest under aseptic conditions. The seeds were then survival rates in all three species. sown in 125-mL baby food jars containing 25 mL of KCm (Knudson, 1946) supplemented –1 –1 Mexico has 1200 of the 25,000 orchid plummeted (Jime´nez et al., 1998). Lycaste with 37.3 mgÁL Na2EDTA and 27.8 mgÁL species that have been described (Dixon skinneri (Batem. Ex. Lind.) Lind. is an FeSO47H2O (Murashige and Skoog, 1962) et al., 2003); 40% of these are endemic epiphytic, lithophytic, or sometimes terres- plus 20 gÁL–1 sucrose. species (Espejo and Lo´pez, 1998). Several trial species that occurs from Mexico to The pH of all culture media was adjusted species are endangered as a direct or indirect Guatemala and Honduras (Fig. 3A) (Jime´nez to 5.0 ± 0.1 with 0.5 N NaOH and 0.5 N HCl result of two human activities: habitat alter- et al., 1998). In recent years, this species has before adding 5.5 gÁL–1 Agargelä (Sigma, St. ation and overcollecting (Flores-Palacios not been reported from Mexico, suggesting Louis, MO) and autoclaving at 1.2 kgÁcm–2 and Valencia-Dı´az, 2007; IUCN/SSC Orchid that it may be locally extinct. and 120 °C for 15 min. All cultures were Specialist Group, 1996). One hundred Efficient in situ conservation of threatened incubated in a growth chamber at 25 ± 1 °C seventy-nine orchid species are protected by or endangered species has proven difficult to under a 16-h photoperiod provided by cool- the Mexican government and research aimed achieve; however, ex situ conservation can white fluorescent lamps (50 mmolÁm–2Ás–1). at finding new methods to conserve and complement global conservation strategies. Protocorm culture. After germination, propagate these species is a high priority. Plant tissue culture represents an excellent protocorms with a height of 2 to 3 mm were Among these are three highly sought-after option for the propagation and conservation selected and transferred to two different species: Mormodes tuxtlensis, Cuitlauzina of endangered species (Gangaprasad treatment media: 1) MS medium with 2 pendula, and Lycaste skinneri. et al., 1999; Ket et al., 2004; Rubluo et al., mgÁL–1 glycine, 100 mgÁL–1 myoinositol, Mormodes tuxtlensis Salazar is an epi- 1993). and 30 gÁL–1 sucrose; and 2) KCm. Both phytic species first described in 1988. It is Although micropropagation of orchids media were supplemented with a combina- endemic to Mexico and grows in small areas has been a recognized technique for many tion of BA (0, 2.2, 4.4, 8.9, or 13.3 mM) and of the tropical rain forest in Veracruz, decades (Arditti and Ernst, 1993), its use has NAA (0, 0.5, and 2.7 mM); three protocorms Oaxaca, and Chiapas (Fig. 1A) (Salazar, been largely confined to species and hybrids were cultured in each jar. There were with 10 1988). Cuitlauzina pendula La Llave & of the genera Phalaenopsis, Cattleya, and replicates (i.e., 30 protocorms per treatment). Lexarza is an epiphytic species endemic to Oncidium (Arditti and Ernst, 1993; Chen The induction period was 120 d. the southern and western parts of Mexico et al., 1999, 2000; Chen and Chang, 2001; After the induction period, the protocorms (Fig. 2A). Its native range has been reduced, Tokuhara and Mii, 2001; Wu et al., 2004). were subcultured every 60 d to their respec- and the size of the type population has Reports of successful propagation of wild tive basal medium without plant growth Mexican species are rare. Germination and regulators (PGRs). The number of shoots development protocols for three Mexican per protocorm, shoot height, and PLB pro- species, Cattleya aurantica, Encyclia chaca- duction were recorded. Received for publication 5 June 2008. Accepted for noensis, and Brassavola nodosa were Number of shoots per protocorm and publication 16 Apr. 2009. described by Damon et al. (2004). Santos- shoot height were analyzed using one-way We thank Pamela Moon, Philip J. Brewster, and Herna´ndez et al. (2005) reported high in vitro analysis of variance followed by a least Guillermo Angeles for the suggestions and correc- tions of the English text. Vı´ctor Salazar thanks germination rates as well as bud and significant difference test (P # 0.05). Instituto de Ecologı´a, A.C. for the grant offered to protocorm-like body (PLB) formation for Lae- Ex vitro culture. Plantlet survival was conduct his professional studies. lia albida, whereas Lee-Espinosa et al. (2007) assessed using individuals that had attained 1To whom reprint requests should be addressed; reported the germination and organogenic a height of 3 to 5 cm and were at least 10 e-mail [email protected]. proliferation of L. anceps ssp. dawsonii. months old. In the case of M. tuxtlensis, the HORTSCIENCE VOL. 44(5) AUGUST 2009 1395 plating; for L. skinneri; it began between 70 to 90 d. The germination rate approached 100% for all three species, because in the different observations under stereomicro- scopy, the number of nongerminated seeds was null; but in some cases, the density of seeds was too high making it impossible to affirm that 100% of seeds had already germinated. Mormodes tuxtlensis Organogenesis and shoot formation. Seventy-one percent of protocorms cultured in KCm grew and began to form shoots. In contrast, only 61% of protocorms cultured in MS showed any morphogenic response. Those that did not respond turned brown. Shoots, formed through direct organogen- esis from protocorms that had developed rhizoids and leaves, were 5 to 8 mm long. After 90 d, small nodules formed mainly at the base of the protocorms. These nodules started to produce leaf primordial 30 d later, especially in the KCm treatments; after the protocorms were subcultured to basal medium, the nodules consolidated into ad- Fig. 1. Plant regeneration of Mormodes tuxtlensis through direct shoot formation. (A) Flowering plant. (B) ventitious shoots (Fig. 1B). 6 Multiple shoot formation from protocorms in Murashige and Skoog medium + 13.3 mMN- Shoot formation per protocorm differed benzyladenine. (C) In vitro-rooted shoots ready for ex vitro culture. (D) Plants obtained from in vitro significantly among treatments (P # 0.0001). culture after 4 months in potting mix. Bars = 2 cm. Although not significantly different from several other treatments, the highest level of shoot formation (1.5 shoots per protocorm) was achieved in the MS medium treatment supplemented with BA (13.3 mM); 1.4 shoots per protocorm developed from explants cul- tured in MS medium BA (8.9 mM) either alone or in combination with 2.7 mM NAA. In these three treatments, 85% of protocorms showed some morphogenetic response (Table 1A). In MS medium, in most cases, shoot formation tended to occur in treatments containing at least BA (4.4 mM).