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The efficiency of direct somatic embryogenesis (SE) induced on internode explants of the tree fern Cyathea delgadii treated with different concentrations of sucrose at various periods after their excision from donor sporophytes was investigated. Internode explants, about 1.5 mm in length, were dissected from etiolated plantlets grown in vitro and cultured on half-strength hormone-free MS medium supplemented with 10 g l−1 sucrose, under continuous darkness. The impact of four concentrations of sucrose (0.6, 0.8, 1.0, 1.2 M) applied every second day between days 0 and 10 was studied. The highest number of somatic embryos was obtained when explants were treated with 1.0 M sucrose on the 4th day (59.8 on average) or 0.8 M sucrose on the 8th day (67.6 on average) after excision. Light and scanning electron microscopy observations revealed that a nearly 22-fold increase in SE efficiency induced by sucrose treatment was associated with a change in the origin of somatic embryos from multicellular to unicellular. Application of high sucrose concentrations on the 4th day after explants excision stimulated the accumulation of starch, a valuable energy source for intensively dividing cells and developing embryos. The study's results may be useful for the in vitro mass production of plantlets of the valuable tree fern C. delgadii and helpful in enriching the knowledge of the mechanisms governing the initial phases of SE.Key message: Sucrose-induced osmotic stress changes the way of somatic embryo origin and delaying its application stimulates somatic embryogenesis of Cyathea delgadii which may be useful for in vitro mass production of plantlets. [ABSTRACT FROM AUTHOR]

This study describes for the first time a simple and quick direct somatic embryogenesis and plant regeneration protocol for turmeric ( Curcuma longa L.) using leaf base explants. Preincubation of explants was carried out on solid Murashige and Skoog (MS) medium containing 4.49 μM 2,4-dichlorophenoxy acetic acid for 20 days under continuous dark condition. Subsequently transfer of the explants for 2 weeks to MS liquid medium containing 1.32 μM 6-benzyladenine (BA) under illuminated condition resulted in the highest percentage of somatic embryo (91.1 %) induction. Secondary somatic embryos which originated from primary somatic embryos were also observed in the same medium supplemented with 2.20 and 2.64 μM BA. Thereafter, mature somatic embryos germinated readily and the maximal plantlet development (87 %) was achieved on 1/2-strength MS medium containing gibberellic acid (GA) under dark condition. Histological analysis revealed that the proembryogenic masses were initiated directly from the area near the vascular tissues of the leaf base explants. Presence of clear protoderm in the globular structure and procambial strands in the matured stage of somatic embryos confirmed that these structures were true somatic embryos. Somatic embryos were successfully acclimatized ex vitro at a survival rate of 72.9 % and they were normal phenotypes. [ABSTRACT FROM AUTHOR]

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants. [ABSTRACT FROM AUTHOR]

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4- D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4- D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4- D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide. [ABSTRACT FROM AUTHOR]

The comparison of direct and indirect somatic embryogenesis (DSE and ISE) for biolistic transformation of sugarcane by minimal expression cassettes indicated the highest transformation efficiency with ISE (2.2 independent transgenic plants per shot) and the most rapid production of transgenic plants with DSE (12 weeks from explant to plants in soil). Microprojectiles of 0.3 μm diameter produced 5 times more transgenic lines than 1 μm microprojectiles when used at the same weight basis per shot. A significant reduction of the number of hybridization signals in the Southern blot was also observed with 0.3 μm microprojectiles when compared to 1.0 μm microprojectiles. This suggests that the lower DNA carrying capacity and greater number of the smaller microprojectiles contributes to more transgenic lines with less complex transgene integration. When geneticin sulfate, was used for selection following DSE, significantly more (4.8 times) transgenic plants were produced than with paromomycin sulfate and an equal number of non-expressing plants were produced with both selection agents. In conclusion, optimization of two alternative morphogenic routes for regeneration (DSE and ISE), biolistic and selection parameters generated rooted, transgenic plants of a commercially important sugarcane cultivar with simple transgene integration and within 12 or 19 weeks of culture initiation, respectively. Reducing the complexity of the transgene integration and minimizing the time in tissue culture will likely enhance the performance of the transgenic events. [ABSTRACT FROM AUTHOR]