Mangrove fan palm is an endemic plant that grows well and has for a long period been related to traditional ceremonies in southern Thailand. It is an endangered plant with a slow growth and is difficult to propagate in mass production. Tissue culture technique can be used to produce a large number of plants within a short time and to preserve in vitro germplasm resulting in the plant not becoming extinct from natural habitats. Thus, the objectives of this research were to study effects of auxins and embryo preparation on somatic embryogenesis. The sterile immature zygotic embryo was cultured on MS medium with different types and concentrations of auxins. The results showed that 1 mg/l dicamba gave the best results in callus induction at 100% and callus diameter at 4.67 mm after 8 weeks of culture. For callus proliferation, callus chopped at 100 times and transferred to 0.1 mg/l dicamba containing oil palm culture medium gave the highest callus diameter at 10.33 mm after 4 weeks of culture and somatic embryos were also produced. Upon transferring somatic embryos to MS medium with 0.2 M sorbitol, a large number of various developmental stages of secondary somatic embryos were induced within 4 weeks. Therefore, the finding of this study could be highlighted that dicamba and chopping callus have an important role to induce callus and somatic embryo. This is the first report achieved on in vitro propagation of this plant and it will be beneficial for genetic conservation and biotechnological applications in the future.

Introduction. This protocol describes a method for obtaining protoplasts from banana leaves, calli and cell suspensions, and their sustainable development via somatic embryogenesis from embryogenic cell suspensions. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials, and media preparation for protoplast production and culture. Results. The first protoplasts may be seen after 30 min of incubation in enzyme maceration. With protoplasts from embryogenic cell suspension, complete development into a whole plant, through somatic embryogenesis, is observed in 12 weeks. The first cell divisions occur on feeder layers 3–8 days after protoplast plating. Proembryo formation is observed 14–21 days after initiation of protoplast culture. The transfer of derived embryo plantlets, at 8–10 weeks after protoplast plating, onto growth regulator-free medium, leads to plant rooting and elongation.

Somatic embryo quality is an important aspect of artificial or synthetic seed production and is dependent on proper morphological development and deposition of sufficient storage reserves to support early growth of the seedling. The abilities of a somatic embryo to survive desiccation and to establish a rapidly growing seedling were used as estimates of quality and were dependent on a number of factors during the elongation and maturation phases of somatic embryo development. Embryo quality was significantly enhanced when the sucrose content of the elongation and maturation media was increased to 50 g I−1 from 30 g I−1. This increased dry weight to >2 mg embryo−1 suggesting greater deposition of storage reserves. Higher sucrose concentrations were inhibitory and reduced survival after desiccation and seedling vigour. An optimum number of embryos can be supported on one Petri plate; the number was controlled by the plating density of the embryogenic cell clusters spread on the elongation medium. This optimum was dependent to some extent on the sucrose content of the medium, indicating that sucrose had a nutritive effect on embryo development and not solely an osmotic effect. Light intensity was another critical factor. Intensities > 75 μmol m−2 s−1 photosynthetic photon flux density (PPFD) at the elongation stage of development blocked globular-stage somatic embryo development. Maturing somatic embryos were far less sensitive to high light intensities and development was more rapid at 150 μmol m−2 s−1 PPFD. Light quality was manipulated by covering plates with filters that selectively transmitted certain regions of the visible spectrum. All filter treatments reduced embryo yield and quality compared with embryos developing under fullspectrum light; varying the light spectrum does not seem to be a viable method of enhancing somatic embryo quality.

In the presence of abscisic acid (ABA), contents of glucose and fructose decreased in carrot embryoids, whereas umbelliferose increased during dehydration. The acquisition of desiccation tolerance was tested by scoring germinated embryoids. It did not keep pace with the changes in soluble carbohydrate contents during development, which suggests that in these somatic embryoids di- and oligosaccharides are not the determining factors for anhydrobiosis. However, on slow dehydration, umbelliferose increased, while sucrose decreased and the monosaccharides declined completely. These changes were positively correlated with increased desiccation tolerance. A similar analysis of slowly dried, ABA-treated alfalfa embryoids showed that stachyose and sucrose increased and the monosaccharides declined with the acquisition of desiccation tolerance. These data support the contention that carbohydrates are involved in anhydrobiosis.

Among the forage legumes somatic embryogenesis has been most studied in alfalfa (Medicago sativa L.) reflecting inherent breeding problems and the high demand for seeds of this species. Current methods of somatic embryogenesis are reviewed with emphasis on each of the 3 stages involved: the acquisition of competence, the induction of somatic embryogenesis, and somatic embryo development and maturation. The role of plant growth regulators is particularly stressed. Artificial seed production, the development of reduced-generation synthetics or hybrids and genetic transformation of alfalfa are considered. Research on somatic embryogenesis and plant transformation in a wide range of other forage legumes is examined but it is suggested that the application of artificial seed technology is unlikely in forage legumes other than alfalfa.

Embryogenesis was induced on cotyledons of immature zygotic embryos of soybean (Glycine max (L.) Merrill) placed on solid medium containing 62.5 mm glutamine, soybean seed growth medium salts and vitamins, and 40 mg I−1 2,4-dichlorophenoxyacetic acid (2,4-D) plus 175 mm maltose, or 8 mg I−1 α-naphthaleneacetic acid (NAA) plus 88 mM sucrose. Somatic embryo development was continued in liquid medium containing 0.16 mg I−1 indole-3-butyric acid and 2.64 mg I−1 abscisic acid, glutamine and salts as above, and 88–438 mM sucrose in progressively increasing steps. Germination was on solid half-strength Murashige-Skoog medium. During maturation, somatic embryos mimicked zygotic embryos in colour, protein concentration, water and solute potentials, and respiration. Protein and lipid accumulated to 329 and 86 g kg−1 dry weight in somatic embryos. Fatty acid composition was similar to that of axes of mature seeds. Before desiccation, the water and solute potentials of maturing somatic embryos declined to −1.13 and −1.99 MPa while turgor increased to 0.86 MPa. Concomitantly, a 60% reduction in activity of the cytochrome oxidase pathway of respiration occurred with somatic embryo maturation at 600 g water kg−1 fresh weight. Although small (about 8 mg per embryo), 60% of the somatic embryos formed roots and shoots during germination after maturation without drying and 30% germinated after drying to 60 g water kg−1 fresh weight. In the greenhouse, somatic plantlets grew to mature plants with seeds.

Introduction. L’olivier (Olea europaea L.) est la principale espèce fruitière exploitée au Maroc. L’embryogenèse somatique de la Picholine marocaine a été jusqu’à présent très peu étudiée malgré l’importance de cette variété au Maroc. L’objectif de nos recherches a été d’étudier les effets de divers milieux de culture sur l’induction et le développement d’embryons somatiques chez cette variété. Matériel et méthodes. Des cotylédons entiers de graines d’olivier ont été excisés après désinfection et mis en culture. L’initiation des cals embryogènes a eu lieu sur un milieu de culture de Murashige et Skoog, solidifié par de l’agar et contenant 0,5 mg de zéatine·L–1 combinée à différentes concentrations en acide naphtalène acétique (ANA) [(1, 2, 5, 10 et 40) mg·L–1], les cultures témoins étant sans régulateurs de croissance. Après 3 semaines, les cals ont été transférés dans un milieu soit liquide, soit solidifié par de l’agar ou de la gélerite. Après 6 semaines sur le milieu d’induction, les cals ont été transférés sur un milieu maintenu liquide ou solidifié par de l’agar ou de la gélerite, et contenant 0,5 mg·L–1 de zéatine, afin de favoriser le développement des embryons somatiques et, par la suite, leur régénération en plantules. Le taux de cals induits et le poids moyens des cals formés sur les différents milieux ont été mesurés après 6 semaines de culture sur le milieu d’induction. Une analyse histologique des cals embryogènes et non embryogènes a été effectuée. Résultats. L’induction des cals a significativement été influencée par la concentration en ANA. Les meilleurs résultats ont été obtenus avec 2 mg ANA·L–1 pour l’induction des cals (82,3 %) et 5 mg ANA·L–1 pour le développement de ces cals (150 mg). Le milieu liquide s’est révélé être le meilleur pour l’induction des cals et le milieu avec agar a favorisé le développement des cals et la régénération de plantules par embryogenèse somatique. L’utilisation de la gélerite n’a permis aucune morphogenèse. Les plantules régénérées et acclimatées ont présenté une croissance normale en serre. Conclusion. L’embryogenèse somatique serait exploitable pour la micropropagation de l’olivier au Maroc.

The histological and biochemical characteristics of cacao (Theobroma cacao) endosperm were determined at different stages of seed development. At various stages, the endosperm was analysed for minerals, organic acids, carbohydrates, free amino acids, abscisic acid (ABA), indole-3-acetic acid (IAA) and cytokinins. Cacao endosperm was coenocytic and became progressively cellularized from the micropylar zone. From stage I to IIz, the endosperm started to degenerate all around the embryo. Among the minerals detected, potassium and sulphate were the most abundant. Ammonium was the major source of mineral nitrogen, and glutamine was the most abundant free amino acid. Total nitrogen concentration was quite low. No sulphur-containing amino acids could be detected. Malate was present at all endosperm stages and was the most abundant organic acid. The sugars in cacao endosperm were mainly reducing sugars: glucose and fructose. ABA was only detected from stage IIz. IAA and three cytokinins [zeatin, zeatin-riboside and isopentenyl adenosine (iPA)] were present at all stages of development. Cacao endosperm resembled the endosperm of other species in (1) the abundance of organic acids and phosphate; and (2) a unique combination of sugars. However, three features seemed to be specific to cacao: (1) nitrogen was supplied mainly in the form of ammonium and amino acids, whereas, in other species, amino acids represent the predominant form; (2) there was a relatively small amount of nitrogen, and (3) a high concentration of sulphate and the absence of sulphur amino acids.

Specific chitinases accumulate in seeds of many species as part of their normal developmental programme. Some chitinases can also be induced in developing and germinating seeds in response to microbial attack. All known seed isoforms belong to classes I, II, IV and VII, which are encoded by Chia genes, as well as to the more divergent class III encoded by Chib genes. The study of seed-specific chitinases has contributed significantly to current knowledge of this ubiquitous protein family, including antifungal properties, structure, specificity and catalytic mechanism. Indeed, the first plant chitinase for which a three-dimensional structure was solved had been isolated from barley seeds. Moreover, the finding that a chitinase could rescue a somatic embryo mutant was the first evidence of a non-defensive function. Several lines of evidence have substantiated the biotechnological potential of chitinases to counter plant fungal disease. The recent identification of several seed and fruit chitinases as major panallergens should be taken into account when selecting the genes to be introduced into food crops.

The osmotin gene was introduced into tea [Camellia sinensis (L.) O. Kuntze] somatic embryos at the globular stage of development via microprojectile bombardment, to determine the effects of this stress-tolerance-related gene on the accumulation of storage reserves and acquisition of desiccation tolerance during embryo maturation. Changes in total soluble sugars, starch and proteins in response to osmotin were estimated in the stably transformed embryos, using standard biochemical and histochemical methods, after the globular embryos had matured to the heart stage of development. These changes also were compared with two types of controls, comprising (1) untransformed embryos and (2) embryos that were bombarded without osmotin DNA. The control embryos showed poor reserve accumulation and negligible germination but, in contrast, the embryos bombarded with the osmotin gene showed a several-fold increase in storage reserves, normal maturation and 57–63% germination. When these transformed embryos were desiccated to 15.5% moisture content, only 40% germinated, although a further increase in the accumulation of storage reserves occurred. In contrast, the control embryos became necrotic and failed to germinate. This study demonstrates the use of somatic embryogenesis as a tool for understanding seed recalcitrance in tea, and suggests that transformation with stress-tolerance genes can overcome the developmental problems associated with recalcitrant seeds.

Introduction. Jusqu'à présent, aucun protocole n'a été développé pour l'embryogenèse somatique de l'olivier Picholine, cultivar exploité dans près de 98 % des plantations d'oliviers au Maroc. Nos travaux ont donc cherché à étudier les effets de la composition du milieu de culture et de la lumière sur l'induction et le développement d'embryons somatiques chez ce cultivar. Matériel et méthodes. Après désinfection des noyaux d'olives Picholine, des cotylédons ont été mis en culture après suppression des embryons zygotiques. Ils ont été disposés en boîtes de Pétri soit entiers, soit en distinguant les parties proximales et distales. Pour la phase d'induction de cals embryogènes, quatre milieux de base [Murashige et Skoog (MS), Bourgin et Nitsch (BN), Schenk et Hildebrandt (SH) et Canas et Benbadis (OMc)], additionnés de régulateurs de croissance (zéatine et ANA), ont été testés soit à l'obscurité, soit en photopériode de 16 h. Après 6 semaines de culture sur ces milieux d'induction, les cals ont été transférés en tubes, dans des milieux de culture frais mais sans ANA et soumis à une photopériode de 16 h. À l'issue de cette phase, les plantules présentant des racines bien développées et au moins deux feuilles ont été repiquées dans des pots contenant un substrat et acclimatées en serre. Résultats. Les meilleurs résultats d'induction et de développement des embryons somatiques ont été obtenus avec le milieu MS et aucune morphogenèse n'a été observée sur le milieu OMc. L'incubation à la lumière a significativement réduit l'induction des cals embryogènes et a inhibé la régénération des plantules. Les parties proximales des cotylédons ont donné de meilleurs résultats que les parties distales. Le taux de régénération de plantules a atteint 40 % des explants mis en culture et le pourcentage de survie obtenu après leur acclimatation a atteint 94 %. Conclusion. Le protocole décrit a démontré la capacité embryogène du cultivar Picholine marocaine. Il pourra être exploité pour la propagation de cet olivier au Maroc.