Victor M. Chavez

Somatic embryogenesis and organogenesis of Agave victoriae-reginae: Considerations for its conservation

Agave victoriae-reginae somatic embryos were produced through a callus phase from seedling stem segments cultured on MS medium. The optimal treatment was MS medium with 2.26 μM 2,4-D. Multiple shoot regeneration was induced from axillary buds from stem segments cultured on MS medium with 2.2–4.4 μM BA. Effect of MS and modified MS medium with 50% macronutrient concentration, both containing 2.2 μM BA and sucrose at the following concentrations, 20, 30, 45 and 60 g l−1, resulted in inconsistent multiple shoot formation. Shoots and somatic embryos formed by this indirect pathway could have a multicellular origin, which might lead to genetic variation. The direct development of pre-existent buds occurred on MS basal medium and increased in the presence of BA; this might be a pathway for the rescue of genotypes of endangered species. Embryos and shoots developed and grew roots on MS medium. Complete plantlets were obtained on MS basal medium. A total of 92% per cent of the plantlets survived and grew when transferred to the greenhouse. Agave micropropagation could supply the commercial plant demand, diminishing the gathering of seeds and plants of this endangered species from the wild.

In vitro morphogenesis of Ceratozamia hildae and C. mexicana from megagametophytes and zygotic embryos

Organogenesis and somatic embryogenesis were induced from megagametophyte and zygotic embryo explants of 2 cycad species, Ceratozamia hildae and C. mexicana, cultured on modified B5 medium containing kinetin (0–13.9 μM) and 2,4-d (0–9.0 μM). Organogenesis occurred from megagametophyte explants of both species on the range of media tested. Somatic embryogenesis was largely restricted to zygotic embryo explants. Somatic embryos germinated in vitro: however, rooting of adventitious shoots was unsuccessful.

Induction of embryogenic mango cultures as affected by genotype, explanting, 2,4-D and embryogenic nurse culture

The nucellus was removed from immature seeds of 4 mango genotypes, andcultured under different induction conditions. The mango genotypes includedpolyembryonic ‘Hindi’ and ‘Nam Doc Mai’ and monoembryonic ‘Lippens’ and’Tommy Atkins‘. Nucellar explants were cultured on modified B5 basal mediumunder the following inductive conditions: 1) 4.52 µM 2,4-D; 2) nogrowth regulator (control); 3) 4.52 µM 2,4-D + embryogenic ‘Parris‘nurse culture; 4) no growth regulator + embryogenic ‘Parris’ nurse culture.Induction of embryogenic competence was mediated by 4 factors: genotype,explanting, 2,4-D and the presence of a highly embryogenic nurse culture,although there was considerable difference in genotype response. ‘Hindi’ hadthe greatest embryogenic potential, followed by ‘Lippens’, ‘Tommy Atkins‘and ‘Nam Doc Mai’, respectively. Induction of embryogenic cultures of allgenotypes at low frequency occurred as a result of explanting excisednucellus onto control medium. The most effective treatment for inducingembryogenic cultures was 2,4-D + embryogenic ‘Parris’ nurse culture with’Hindi’, ‘Lippens’ and ‘Nam doc Mai’, with the exception of ‘Tommy Atkins’,in which the treatment with 2,4-D alone was most effective.

Somatic embryogenesis from leaf callus derived from mature trees of the cycad Ceratozamia hildae (Gymnospermae)

A friable and transient embryogenic callus was initiated from pinnae removed from leaves in new vegetative flushes of mature Ceratozamia hildae Landry & Wilson, a cycad. Somatic proembryos developed from the callus approximately 3 months after explanting onto plant growth medium consisting of a modified B5 formulation with 60 g l-1 sucrose, 400 mg l-1 glutamine, 100 mg l-1 arginine, 100 mg l-1 asparagine, 4.5 μM 2,4-dichlorophenoxyacetic acid with either 1.2 μM or 4.6 μM kinetin and 1.75 g l-1 gellan gum. Following subculture of somatic proembryos at this time onto medium without plant growth regulators, they continued to proliferate by a process resembling cleavage embryony or polyembryogenesis for several months. Proliferating embryogenic cultures consisted of hyperhydric somatic proembryos. Some 15 months after explanting, the somatic proembryos began to change in appearance; the suspensors became white and opaque, but were usually highly branched due to cleavage embryony. A single cotyledonary somatic embryo usually developed from the tip of each of the suspensors. Somatic embryos were primarily dicotyledonous, and less frequently monocotyledonous. Fewer than 10% of the somatic embryos appeared to be morphologically abnormal. Germination occurred in vitro whereby the coleorhiza elongated and a tap root emerged; however, plantlet recovery has not been demonstrated because the shoot axis failed to elongate.

Somatic embryogenesis and organogenesis in Zamia fischeri, Z. furfuracea and Z. pumila

Morphogenesis from cultured megagametophyte, nucellus and immature embryos of three Zamia species on modified B5 medium containing 2,4-d and kinetin was compared. Organogenesis and somatic embryogenesis occurred from the megagamethophyte and zygotic embryo explants of Zamia pumila and Z. furfuracea tissue cultures, but only from the megamametophyte of Z. fischeri. Nucellar callus of Z. pumila produced globular structures that failed to develop further. Plantlets were recovered from somatic embryos of Z. pumila.

Regeneration of Ceratozamia euryphyllidia (Cycadales, Gymnospermae) plants from embryogenic leaf cultures derived from mature-phase trees

Abstract Embryogenic cultures were induced from pinnae removed from young leaf flushes of mature-phase trees of the endangered cycad species, Ceratozamia euryphyllidia. Induction media consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l–1 glutamine, 100 mg l–1 asparagine, 100 mg l–1 arginine, 60 g l–1 sucrose, 2 g l–1 gellan gum, 4.65–13.94 μm kinetin and 4.52–9.05 μm 2,4-dichlorophenoxyacetic acid. Cultures were maintained in darkness. Embryogenic cultures were comprised of precotyledonary somatic embryos that proliferated by somatic polyembryogenesis following subculture onto medium without plant growth regulators. Somatic embryo development and maturation occurred spontaneously from proliferating cultures on medium without plant growth regulators. Somatic embryos were monocotyledonous and mature somatic embryos germinated on semisolid medium without growth regulators. Subsequent development, which included the elongation of the first leaves, occurred only after subculture onto semisolid medium without plant growth regulators containing 0.5% (wt/vol) activated charcoal and under low light intensity. The time period from explanting to plant recovery was approximately 3 years.

SOMATIC EMBRYOGENESIS FROM LEAF CALLUS OF MATURE PLANTS OF THE GYMNOSPERM CERATOZAMIA MEXICANA VAR. ROBUSTA (MIQ.) DYER (CYCADALES)

SummaryEmbryogenic callus was induced from explanted pinnae of newly emerged leaves of mature plants ofCeratozamia mexicana var. Robusta (Gymnospermae, Cycadales) on a modified B5 formulation with 1 mg·liter−1 kinetin and 1 mg·liter−1 2,4-dichlorophenoxyacetic acid. Proembryos developed on induction medium, but they were more numerous after subculture onto phytohormone-free medium, which also enabled suspensors to elongate. For nearly 1.5 yr after explanting, subsequent development of somatic embryos was not observed as suspensors dedifferentiated to form embryogenic callus on phytohormone-free medium. After this time, cotyledonary somatic embryos developed at the distal end of the suspensors. Somatic embryos have germinated on phytohormone-free medium. This is the first report of regeneration by somatic embryogenesis of a gymnosperm species from a mature tree. This technique has great potential for preservation of the highly endangered cycads.

Somatic Embryogenesis in the Cycadales

The cycads (Fig. 1) constitute remnant species of an ancient class of gymnosperms, the cycadophytes, that evolved from the free-sporing progymnosperms, which also gave rise to the coniferophytes. According to Gifford & Foster (1989), the cycadophytes have included 3 orders of plants, the extinct Cycadeoidales and Pteridospermales (seed ferns), that are known only from the fossil record, and the Cycadales, that includes the cycads. The cycadophytes are supposed to be ancestral to the Gingkoales and possibly, the Gnetales (Crane & Upchurch, 1987; Taylor & Taylor, 1993). The Cycadales are believed to have originated during the Permian era, and to have flourished during the Mesozoic period, probably peaking in distribution and success during the Jurassic period. They have been referred to as “living fossils” (Gilbert, 1984). Because of their great antiquity, the living cycads have been considered to be invaluable for the study of developmental events (Norstog, 1987). Currently, the Cycadales are comprised of only 3 families, the Cycadaceae, the Stangeriaceae (monogeneric) and the Zamiaceae.

A Biotechnology Strategy for Medium- and Long-Term Conservation of Cycads

It has been possible to regenerate a few cycad species in vitro by somatic embryogenesis, either from zygotic embryos (Ceratozamia hildae, C. mexicana, Encephalartos cycadifolius,E. dyerianus, E. natalensis, Zamia fischeri, Z. furfuracea, andZ. pumila) or from leaves of mature phase trees (C. euryphyllidia, Ceratozamia hildae, andC. mexicana). This strategy has great potential for the commercial vegetative propagation of certain highly endangered species (e.g.,C. euryphyllidia) and should indirectly protect wild populations of these species by discouraging collection in situ. Embryogenic cultures of several cycad species have grown vigorously and are highly morphogenic more than 11 years after induction. The long-term conservation of cycad genetic resources can also be addressed for species that can be regenerated by somatic embryogenesis. Preliminary studies indicate that embryogenic cultures that have been pretreated on plant growth medium containing 0.75 M sucrose for two days, encapsulated in sodium alginate, and desiccated for six hours can survive immersion in liquid nitrogen (−196°C).

Organogenesis from megagametophyte and zygotic embryo explants of the gymnosperm Dioon edule Lindley (Zamiaceae, Cycadales)

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l−1 glutamine, 100 mg l−1 arginine, 100 mg l−1 asparagine, 60 g l−1 sucrose, 8 g l−1 Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated.