Pamela A. Moon

Somatic embryogenesis in mango (Mangifera indica L.)

The mango Mangifera indica L. is considered to be one of the most important fruit crops of the world (Fig. 1). Its annual production is exceeded only by Musa (bananas and plantains), citrus, grapes and apples (FAO Production Yearbook, 1990). Currently, it is the most important fruit crop of Asia. Mango is in the Anacardiaceae family, which includes several other tropical and subtropical tree species of economic importance, e.g., cashew Anacardium occidentale, pistachio Pistachia vera, and several Spondias species, including S. cytherea, S. mombin and S. purpurea. The center of diversity for the genus Mangifera is in southeast Asia, with the greatest number of species being found on the island of Borneo. According to Mukherjee (1985), there are 39 currently recognized Mangifera species. The mango is the most widely cultivated species within the genus, and has a natural distribution throughout outheast Asia. It is found in forests as far west as the Indo-Burman region (Assam). There appear to be two distinct geographical races of the species: a polyembryonic race that occurs in the tropical rainforest of southeast Asia and a monoembryonic race that is associated with India. Some taxonomists have suggested that the mango originated in India, because of the ancient association between Indian culture and religion with this fruit. It has also been noted that the word for mango used throughout Asia is derived from the Tamil “maanga”.

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.

Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos

Inhibition of mango somatic embryo growth was inducedin vitro by treatments for 4 or more weeks with abscisic acid (0–100 μM ABA) with and without high osmolarity provided by mannitol (0–10%). High osmolarity and ABA significantly affected somatic embryo length, precocious germination and the production of good quality secondary somatic embryos. High osmolarity also affected root elongation. Abscisic acid was more effective in suppressing growth and development of ≥0.5 cm-length somatic embryos than smaller somatic embryos. Development beyond the heart stage was significantly inhibited by both ABA and mannitol treatments. The recovery of good quality somatic embryos was enhanced by high levels of ABA (100 μM) with and without mannitol (0–5%). Somatic embryos that had been pulsed with ABA were partially desiccated at different relative humidities. Weight loss was affected only by relative humidity; and ABA did not enhance desiccation tolerance.

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.

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

SummaryColletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.

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.

Temporal effect of 2,4-D on induction of embryogenic nucellar cultures and somatic embryo development of 'Carabao' mango

SummaryEmbryogenic nucellar cultures were established on B5 major salts, MS minor salts and organics, 400 mg/l−1 glutamine, 60 g/l−1 sucrose, 2 g/l−1 gellan gum, and 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D). There was no clear relationship between developmental age of the nucellar explants and induction of embryogenic cultures. The temporal requirements for culture initiation and for induction of embryogenic competence from nucellar explants were determined by pulsing the cultures for 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 d. Culture initiation required a minimum 7–14 d pulse with 2,4-D, and was maximum after a 56-d pulse; however, embryogenic competence was optimum after a minimum of 28 d exposure to 2,4-D. Somatic embryogenesis occurred directly from the nucellar explants at low frequencies. Somatic embryo maturation only occurred following plating of suspensions onto semisolid medium, and was stimulated by 2.4–4.8 µM kinetin and 4.4 µM 6-benzyladenine.

Effect of abscisic acid, osmolarity and temperature onin vitro development of recalcitrant mango nucellar embryos

Development of cotyledonary-stage nucellar embryos of mango was arrestedin vitro by exposure to 750–1750 μM ABA. The enlargement and germination of nucellar embryos was inhibited for as long as 4 weeks after subculture from ABA-containing medium. Mannitol at concentrations between 7.5 and 12.5% inhibited nucellar embryo development, presumably due to osmotic effects; however, there was no residual effect after subculture of somatic embryos onto medium without mannitol. Temperatures between 22.5 and 37.5°C stimulated embryo development, whereas lower temperatures (7.5 and 15°C) delayed germination. There was no germination 1 month after somatic embryos, pulsed for 8 weeks at 7.5°C, were transferred to 22.5°C; however, after 2 months, 86% of these somatic embryos germinated. These results indicate that it is possible to induce developmental arrest in recalcitrant mango embryos with high concentrations of ABA, mannitol or low temperature (7.5°C).