Somatic Embryogenesis and Regeneration of Endangered Cycad Species

Home , Ceratozamia hildae, Encephalartos, Encephalartos woodii

R.E. Litz and P.A. Moon V.M. Chavez Avila Tropical Research and Education Center Jardin Botanico, Instituto de Biologia University of Florida Universidad Nacional Autonoma de Mexico 18905 SW 280 Street Apartado Postal 70-614 Homestead FL, 33031-3314 04510 Mexico DF USA Mexico

Keywords: Somatic embryo, gymnosperm, Cycadales, conservation

Abstract The Cycadales (Gymnospermae) include some of the world's most endangered and rare plant species. Many of the cycad species are known only as single specimen trees (e.g., Encephalartos woodii), as very small populations in the wild (e.g., Ceratozamia hildae) or have become extinct in the wild (e.g., Ceratozamia euryphyllidia). All cycads are dioecious, so that seed production is no longer possible with the rarest of the species. Conditions for induction of embryogenic cultures from leaves of mature phase trees of several species in the family Zamiaceae have been reported, and plants have been regenerated from somatic embryos. Embryogenic cultures of two species have been successfully cryopreserved. These strategies should contribute to the conservation of these endangered species and could lay the basis for commercial propagation of these beautiful but rare plants.

INTRODUCTION The Cycadales represent the most ancient surviving group of higher plants, having arisen during the Permian era and flourished in the Mesozoic and Jurassic periods. They have been referred to as "living fossils" (Gilbert, 1984). Norstog (1987) considered that the cycads are unique for the study of the evolution of development in higher plants. There are only three extant cycad families, the Cycadaceae, Stangeriaceae and Zamiaceae, and these contain approximately 224 species. Cycads are generally restricted to tropical and subtropical regions. Many cycad species are extremely beautiful, and have either been collected to near-extinction or no longer exist in the wild, e.g., Encephalartos woodii, etc. The cycads are dioecious plants, and some of the rarest species are known only from specimen adult plants representing a single sex. All cycad species are considered to be in some way endangered, and the international trade of cycads has been curtailed by CITES regulations. Recent revisions to cycad taxonomy, together with increased plant exploration, have resulted in the recognition of several new cycad species (Hill and Stevenson, 1999). Many of these newly described species are confined to small habitats, whose precise locations have not been defined in order to provide some degree of protection from illegal collectors. Conservation of cycad species has been based upon the protection of their native habitats in situ or ex situ as living collections. In situ conservation has not been very effective for a few reasons: 1) habitats of many cycad species have been destroyed for subsistence agriculture; 2) large scale theft of rare species; and 3) the illegal international trade in rare species. It has been suggested that cycad conservation could be more effective if improved methods for propagating various species were developed (Dehgan, 1983; 1996a,b; 1999; Dehgan and Almira, 1993; Giddy, 1996). More efficient propagation methods could improve availability of rare cycads and thereby discourage theft from their native habitats. Dehgan (1999) proposed that this could be accomplished by improving seed production by hand pollination, better seed germination, root pruning in order to enhance vegetative growth rate, and enhanced procedures for vegetative propagation. In vitro regeneration of cycads has made some significant recent advances. In vitro studies involving cycads were initiated by LaRue (1948, 1954), who described the

Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.-C. Chang and R. Drew Acta Hort 692, ISHS 2005 75 regeneration of plants from the megagametophyte (haploid) of Zamia floridana (= pumila). Much later, Norstog and Rhamstine (1962) induced embryogenic cultures from explanted zygotic embryos of the same species; however, some of the characteristic early stages of gymnosperm embryo development were not identified, i.e., the proembryo, the precotyledonary embryo, etc. As a result, this report and subsequent studies of Webb et al. (1983) and Webb and Osborne (1989) were considered to be anomalous with respect to gymnosperm embryogeny. Chavez et al. (1992a, b) revisited experimental embryogenesis of Zamia spp. and Ceratozamia spp. from explanted zygotic embryos and megagametophyte tissue, and described the early stages of embryo development from embryogenic cultures. Jager and van Staden (1996a, b) described the induction of embryogenic cultures and recovery of somatic embryos from zygotic embryos of Encephalartos cycadifolius, E. dyerianus and E. natalensis. Chavez et al. (1992c) later described the induction of embryogenic cultures from leaves of mature phase Ceratozamia mexicana, and demonstrated that the histology of somatic and zygotic embryos were similar (Chavez et al., 1995). These studies were confirmed when embryogenic cultures were induced from leaves of the mature phase of two related Ceratozamia species, C. hildae (Litz et al., 1995a, b) and C. euryphyllidia (Chavez et al., 1998). There have also been a few reports of cycad regeneration via organogenesis. Chavez et al. (1992a, b) and Chavez and Litz (1999) regenerated C. hildae, C. mexicana, Dioon edule, Z. pumila, Z. fischeri, and Z. furfuracea by organogenesis from excised zygotic embryos. Cycas revoluta was regenerated from callus derived from zygotic embryos (Rinaldi and Leva, 1995) and from cotyledons and epicotyls of 4- and 6-week- old seedlings (Rinaldi, 1999). Jager and van Staden (1996b) demonstrated the regeneration of E. dyerianus and E. natalensis from zygotic embryo explants. Osborne and van Staden (1987) and Osborne (1990) showed that Stangeria eriopus could be regenerated from root segments of young seedlings, although the frequency of regeneration appeared to be low. Organogenesis has limited use for conservation of plant genetic resources, due to the genetic instability of these cultures (Ashmore, 1997), problems associated with maintenance of these cultures for medium and long term periods and the use of (juvenile) material of unknown sex type. As part of a strategy for conserving these rapidly disappearing plant species, we have been examining how the tools of biotechnology could be utilized. Our studies have had the following objectives: 1). Development of regeneration protocols for cycads from their mature phase; 2). Development of a strategy for in vitro medium term storage of cycad; 3) Development of a long term cryoconservation method for in vitro cultures.

MATERIALS AND METHODS

Explanting and Induction Embryogenic cultures have been induced from zygotic embryos (Ceratozamia mexicana, C. hildae, Zamia pumila, Z. furfuraceae and Z. fischeri) and from the newly emerged pinnae of leaves in recent vegetative flushes of C. mexicana, C. hildae and C. euryphyllidia) (Chavez et al., 1992a-c; 1998; Litz et al., 1995a,b; 2004). Induction medium for explanted leaves consisted of B5 (Gamborg et al., 1968) major salts, MS (Murashige and Skoog, 1962) minor salts and organic components, 60 g liter-1 sucrose, 2 g liter-1 gellan gum and supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 2.4-4.7 μM kinetin. Cultures are incubated in darkness at 25oC. Generally, a 3-month exposure to induction conditions is sufficient to establish embryogenic cultures. Newly initiated embryogenic cultures are friable in appearance, but after approximately 3 months, the appearance of proembryos is evident. Precotyledonary embryos each consisting of a suspensor and an apical meristematic region develop from proembryos.

76 Maintenance Three months after explanting onto induction medium, embryogenic cultures are plated on semi solid plant growth medium consisting of B5 major salts, MS minor salts and organic components and 60 g liter-1 sucrose. Embryogenic cultures proliferate by the formation of secondary embryos from the meristematic region, although occasionally secondary embryos will develop directly from the suspensor. Establishment of embryogenic suspension cultures has not been successful.

Maturation Maturation of somatic embryos rarely occurs earlier than one year after induction and approx. 9 months following their subculture onto semi solid medium. Proliferating precotyledonary, hyperhydric embryos develop asynchronously on maintenance medium, becoming hard and white and cotyledonary. Moon et al. (2004) demonstrated that higher gelling agent concentrations result in greater synchrony of development. Vargas-Luna et al. (2004) observed that there were distinct physiological differences among somatic embryos of different stages of development. Subculture of developing somatic embryos onto freshly prepared medium is necessary at 1-2 month intervals. Cultures are incubated in darkness at 25oC.

Germination and Conversion Somatic embryos generally germinate before the apical meristem is fully differentiated. Transfer to light conditions does not occur until shoot formation has been initiated. Light (16 h photoperiod) has been provided by cool white fluorescent tubes with 40-80 μmol m-2 s-1.

Cryopreservation Studies have been carried out with Ceratozamia euryphyllidia and C. hildae to determine if cryopreservation can be utilized for conserving embryogenic cycad cultures (Stewart, 1998). Polyembryogenic cycad cultures were dehydrated on plant growth medium for 2 days and encapsulated in sodium alginate gel. The encapsulated cultures were desiccated for 6 h, and then cooled to -196oC. Viability of the cultures following their removal from liquid nitrogen was determined on the basis of vital stain and growth of the cultures.

RESULTS AND DISCUSSION Embryogenic cycad cultures derived from mature phase Ceratozamia species have been maintained as proliferating polyembryogenic cultures on basal medium for up to 13 years, without loss of embryogenic competence. Cultures induced in late 1987 continue to proliferate in the absence of auxin and cytokinin as late as autumn, 2001. The appearance of proliferating cultures varies according to species, and the morphology of polyembryogenesis is less distinct with Z. pumila and C. mexicana than it is with C. hildae (Litz et al., 1995a); the length of the suspensor and the degree of hyperhydricity vary. Proliferating precotyledonary embryos consist of multiple branching forms, and are usually hyperhydric in appearance. Proliferation of embryogenic cultures occurs by the repetitive formation of secondary embryos from the meristematic region of precotyledonary embryos, and to a lesser extent from the suspensor of precotyledonary embryos. Embryogenic cultures have been demonstrated to remain viable after cryopreservation (Stewart, 1998; Litz et al., 2004). Unlike conifer somatic embryo development, cycad somatic embryo development is not affected by increased medium osmolarity together with abscisic acid. The quality of early cotyledonary embryos can be improved by increasing the hardness of the plant growth medium (Moon et al., 2004). Somatic embryos that develop from the meristematic region are generally dicotyledonous, although Ceratozamia zygotic embryos are monocotyledonous. There has been some attempt to characterize the biochemistry during somatic embryo development (Alvarez Rodriguez et al., 2001); however, these results are

77 preliminary. The C. mexicana apical meristem remains quiescent until germination; the root primordium of early cotyledonary somatic embryos is protected by a coleorhiza. A small proportion of C. mexicana somatic embryos also develop a coleoptile-like structure (Chavez et al., 1992c). The suspensor becomes desiccated quite late during maturation, usually after germination has occurred. Somatic embryos germinate in vitro, and thereafter the apical meristem enlarges. The leaves emerge from the apex and elongate. Each leaf consists of a rachis with 4-6 pinnae or leaflets. The fronds emerge at approximately 4-6 month intervals. Plantlets have been established in potting mixture, but at low frequency.

CONCLUSIONS The ability to regenerate cycads from cell and tissue cultures has important implications for species conservation. Highly endangered species that exist only as mature non-reproducing individual plants can potentially be regenerated from somatic tissue. Initiation of embryogenic cultures from leaves is nondestructive, and does not threaten rare plants in situ. The storage of embryogenic cultures in vitro for medium term (at least 14 years) and long term (in liquid nitrogen) is possible. The regeneration protocols described could be adapted for commercially propagating certain highly endangered cycad species. International movement of in vitro material is excluded from CITES regulations, and the illegal theft of rare species for sale on the international market could be halted. Finally, it should be possible in the near future to manipulate the sex type of critically endangered cycad species using genetic transformation in order to produce sexual offspring and assure the survival of these species.

ACKNOWLEDGEMENTS Florida Agricultural Experiment Station Journal Series No. R-08544.

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