Jen-Tsung Chen

Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis

Leaf explants of Phalaenopsis amabilis var. formosa formed clusters of somatic embryos directly from epidermal cells without an intervening callus within 20 – 30 d when cultured on 1/2-strength modified Murashige and Skoog medium supplemented with 0.1, 1 and 3 mg dm−3 TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium.

Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium

A protocol for induction of direct somatic embryogenesis, secondary embryogenesis and plant regeneration of Dendrobium cv. Chiengmai Pink was developed. Thidiazuron (TDZ) at 0.3, 1 and 3 mg dm−3 induced 5–25 % of leaf tip segments of in vitro grown plants to directly form embryos after 60 d of culture, and 1 mg dm−3 TDZ was the best treatment. Somatic embryos mostly formed from leaf surfaces near cut ends, and occasionally found on leaf tips. Higher frequency of embryogenesis was obtained in light than in darkness. During subculture, secondary embryos developed from outer cell layers of primary embryos. All combinations of NAA (0, 0.1, 1 mg dm−3) and TDZ (0, 0.3, 1, 3 mg dm−3) increased the multiplication rate of embryos. It takes about 8 months from embryo induction, plantlet formation to eventually acclimatization in greenhouse.

Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of Phalaenopsis orchids

The influence of light regime, explant position and orientation on direct embryo formation from leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were investigated to optimize the protocol for regenerating of this orchid. When explants were cultured in light, direct embryogenesis was retarded in both species. Embryos showed whitish to pale green in color and larger size than those cultured in darkness. Furthermore, light regime induced explant browning, embryo necrosis and eventually low plantlet conversion rate. Sixty days of culture in darkness is the most suitable duration for direct embryo induction. Explant orientation also significantly affected direct embryo formation, and explants placed adaxial-side-up on culture medium had higher embryogenic response than abaxial-side-up orientation. In both species, the cut end had highest embryogenic competence than other parts of the explant. Moreover, when the leaf explant was cut transversely into two segments, the leaf basal segment had higher embryogenic competence than the leaf tip segment.

Morphogenetic routes of long-term embryogenic callus culture of Areca catechu.

Early morphogenetic events and repetitive embryogenesis from callus culture of betel nut palm (Areca catechu L.) were studied using scanning electron microscopy. On Murashige and Skoog (MS) medium supplemented with 2 mg dm−3 dicamba, callus culture has capacity to form plantlets via somatic embryogenesis and to form secondary embryos for about 4 years. However, various abnormal embryos without differentiation of the leaf sheath and shoot apical meristem were observed, which showed bell-shaped and then cup-shaped or mushroom-shaped structures. These abnormal embryos contained distinctive structures, including a disk-shape interior region, surfaces with grooves and a stalk-like posterior region. During subculture, these abnormal embryos enlarged, became deformed and gradually lose their shape and then converted into nodular, compact embryogenic callus. It was also found that secondary embryos originated from interior surfaces or posterior regions of abnormal embryos, and gave rise to the next cycle of normal and abnormal embryos.

Somatic embryogenesis and plant regeneration from leaf, root and stem-derived callus cultures of Areca catechu

Plant regeneration through somatic embryogenesis of Areca catechu L. was established using leaf, root and stem segments as explants. Embryogenic callus was induced and maintained on medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,6-dichloro-2-methoxybenzoic acid (dicamba) at concentrations 2, 4, 6 and 8 mg dm−3 in darkness. Somatic embryos were found on primary callus in the presence of 2 and 4 mg dm−3 dicamba and during subculture on 2 – 8 mg dm−3 2,4-D or 2 – 4 mg dm−3 dicamba-containing media. Plantlet conversion from embryos was successfully achieved on growth regulator-free medium. The plants grew well when transplanted to containers in shaded greenhouse.

Efficient and repetitive production of leaf-derived somatic embryos of Oncidium

Oncidium cultivars gave different embryogenic responses of leaf explants when affected by auxins (2,4-D, IAA, IBA and NAA), cytokinins (2iP, BA, kinetin, TDZ and zeatin), sucrose, NaH2PO4, casein hydrolysate, peptone, and glutamine. The best embryogenic responses of cv. Sweet Sugar were at 20 g dm−3 sucrose, 85 mg dm−3 NaH2PO4 and 3 mg dm−3 kinetin, respectively. The development of somatic embryos on leaf explants of cv. Sweet Sugar was delayed for about 10 – 20 d in comparison with cv. Gower Ramsey. On growth regulator-free medium, about 40 % of leaf derived embryos of cv. Gower Ramsey were fused together in their basal parts and so called multiple-state embryos. However, under the same condition, the embryos of cv. Sweet Sugar were all in multiple-state form.

Promotion of direct somatic embryogenesis of Oncidium by adjusting carbon sources

To further optimize a culture medium for induction of direct embryo formation of Oncidium cvs. Gower Ramsey and Sweet Sugar, five kinds of carbon sources, cellibiose, fructose, glucose, maltose and sucrose at 10, 20, 30 and 60 g dm−3 were tested in this study. Cellibiose supply had an inhibitory effect and resulted in high percentage of explant browning in both cultivars. By contrast, fructose, glucose and sucrose were all effective for direct embryo induction. In cv. Gower Ramsey, the suitable ranges of concentration were found at 30–60 g dm−3 of sucrose, 10–20 g dm−3 of glucose and 20–30 g dm−3 of fructose, respectively. The suitable ranges for cv. Sweet Sugar were at 20–60 g dm−3 of sucrose, 10–30 g dm−3 of glucose, 10–20 g dm−3 of fructose and 30–60 g dm−3 of maltose, respectively. The highest amount of embryos was obtained at 30 g dm−3 of sucrose for cv. Gower Ramsey and at 20 g dm−3 of glucose for cv. Sweet Sugar.

Cellular origin and development of secondary somatic embryos in Oncidium leaf cultures

We report here for the first time that fully differentiated stomatal guard cells have the ability to form directly secondary somatic embryos. Histological and scanning electron microscopy studies reveal that the stomata are formed on primary embryos of both Oncidium cultivars Gower Ramsey and Sweet Sugar. Secondary embryogenesis from these guard cells could be induced by several plant growth regulators (PGRs), including N6-benzyladenine, kinetin, thidiazuron, 1-aminocyclopropane-1carboxylic acid (ACC), ancymidol and 2,3,5-triiodobenzoic acid. When compared with other PGRs, ACC at concentration 1 mg dm−3 resulted in highest number of secondary embryos that were derived from guard cells. The present communication provides a model system for studying factors and mechanism affecting totipotency or embryogenetic capacity of guard cells.

Effects of Salicylic and Acetylsalicylic Acid on Direct Somatic Embryogenesis in Oncidium

The effects of salicylic and acetylsalicylic acid on direct somatic embryogenesis were investigated using leaf explants of two cultivars of Oncidium on 1/2 MS medium with or without thidiazuron. In cv Gower Ramsey, salicylic acid (1, 5, 10, 15, 20, 50 μM) either alone or in combination with thidiazuron (4.54 μM) retarded and delayed embryogenesis. In contrast, in the presence of 4.54 μM of thidiazuron, acetylsalicylic acid at 0.1 μM concentration promoted embryogenesis. In cv Sweet Sugar, all concentrations of salicylic acid with or without thidiazuron proved inhibitory on embryo induction. However, in the presence of 0.45 μM thidiazuron, 0.1 and 1 μM acetylsalicylic acid promoted embryogenesis. In addition, in the presence of 4.54 μM thidiazuron, 0.01, 0.1 and 1 μM acetylsalicylic acid promoted embryogenesis.

Factors Affecting Thidiazuron-Induced Direct Somatic Embryogenesis of Phalaenopsis aphrodite

The effects of NaH2PO4, sucrose, activated charcoal, polyvinylpyrrolidone (PVP), and strength of MS medium were studied to optimize thidiazuron (TDZ)-induced direct somatic embryogenesis from leaf explants of Phalaenopsis aphrodite subsp. formosana. The results showed that full- and quarter-strength macroelements of MS medium were not suitable for direct embryo induction from leaf explants. Thus, a half-strength macroelement and full-strength microelements of MS nutrients plus full-strength of MS vitamins, 170 mg l−1 NaH2PO4, 1 g l−1 peptone, 3 mg l−1 TDZ, and 20 g l−1 sucrose are proposed as a suitably modified medium. In addition, PVP at 0.25 g l−1 significantly promoted direct embryogenesis on the cut ends of the explants, but activated charcoal at 0.5–1 g l−1 was inhibitory.