Download somatic embryogenesis and regeneration of endangered cycad

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