Maturation and Conversion of Somatic Embryos Derived from Seeds of Olive (Olea europaea L.) cv. Dahbia: Occurrence of Secondary Embryogenesis and Adventitious Bud Formation

Maturation and conversion of somatic embryos are two crucial steps that hamper the development of efficient somatic embryogenesis systems in olive. Herein, a simple and efficient protocol for the maturation and conversion of olive somatic embryos is reported. Globular somatic embryos derived from seeds of cv. Dahbia were cultured on either half-strength olive (OM) or olive cyclic embryogenesis (ECO) media, with and without plant growth regulators (PGRs). The embryos reached the cotyledonary stage in 9 weeks, but those cultured on ECO medium containing 0.1 mg·L−1 6-(dimethylallylamino)purine (2iP), 0.1 mg·L−1 6-benzyladenine (BA) and 0.05 mg·L−1 indole-3-butyric acid (IBA) exhibited the largest sizes, with an average of 4.7 mm. Somatic embryo conversion into plantlets was evaluated using different culture media (half-strength OM or one-third strength Murashige and Skoog (MS)), light conditions (light or dark) and desiccation pretreatments. The highest rate of somatic embryo conversion (45%) was observed under a 16 h photoperiod on half strength OM medium containing 0.1 mg·L−1 gibberellic acid (GA3) and 0.1 mg·L−1 1-naphthalene acetic acid (NAA). The embryos that failed to germinate showed either necrosis, cotyledon greening with no further conversion, adventitious bud formation or secondary embryogenesis. The findings of this study will be beneficial for biotechnological applications in olive.

Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L.

Turnip is a biennial crop and, consequently, the creation of pure lines for breeding is a time-consuming process. The production of pure turnip lines using doubled haploids produced in isolated microspore culture has not been sufficiently developed. The aim of the present work was to determine some key factors inducing embryogenesis in the isolated microspore culture of turnip, as well as investigating the manners of embryo development. It was shown that the acidity of the medium is an important factor in embryo production; different optimal pH levels ranging from 6.2 to 6.6 corresponded to individual genotypes. Such factors as the cold treatment of buds and the addition of activated charcoal to the nutrient medium increased the responsiveness of all genotypes studied. The turnip variety ‘Ronde witte roodkop herfst’ demonstrated a genetic disorder in the development of microspores; namely, non-separation of some microspores from tetrads. In the in vitro culture, each of the daughter microspores developed on its own. This indicates the dependence of the possibility of embryogenesis in the turnip microspore culture on the genotype. Results suggest that the initiation of secondary embryogenesis in primary embryos leads to an increase in the proportion of doubled haploid plants.

In Vitro Secondary Embryogenesis Derived from Meta-Topoline Treatment on Mass Propagation of Phalaenopsis ‘AMP 17’

Phalaenopsis ‘AMP 17’ is an important orchid commodity in Indonesia with high market demand; however, scaling up the orchid commercially is constrained by the availability and sustainability of qualified seedlings. To overcome the problem, a reliable in vitro propagation protocol, especially via secondary embryogenesis, was undertaken. In the present study, in vitro secondary embryogenesis derived from meta-topoline (mT) treatment on mass propagation of Phalaenopsis ‘AMP 17’ was successfully established. Embryos, as explant sources, were prepared by culturing meristem tips of flower stalk shoots on Murashige and Skoog (MS) medium containing 1.5 mg/L thidiazuron (TDZ) and 0.25 mg/L N6-benzylaminopurine (BAP) for ± 3 months. High secondary somatic embryo (SSE) formation up to 64.90% with 12.30 SSEs regenerated per embryo was determined on half-strength MS augmented with 0.5 mg/L BAP and 2.5 mg/L mT. The combination also stimulated the result of high multiplication rate of SSE formation, up to 10.1 fold on the third subculture, maintained low conversion rate of germinated-embryos down to 55% and improved qualified-growth of the germinated embryos. The mT treatment produced 86% survival plantlets with high qualified-performance. The system could be applied as an alternative method to step forward towards an improved propagation protocol, commercially efficient due to high productivity. Detail findings in each step were discussed.

Induction of Somatic Embryogenesis in Vulnerable Medicinal Plant Hedychium spicatum Buch-Ham ex Smith

This protocol has been developed for somatic embryogenesis in Hedychium spicatum. Simultaneously, a method has also been developed for the production of synthetic seeds by using somatic embryos. Direct somatic embryos were developed on cotyledon explants of zygotic embryos on MS supplemented with high concentration of NAA (20.0 µM). Induction of secondary embryogenesis was best in 2,4-D supplemented medium fortified with activated charcoal. Germination of somatic embryos was enhanced by using GA3. Besides this, round and semi-hard beads of somatic embryos (synthetic seeds) could be produced by using 2% Na-alginate and 100 mM calcium chloride and more than 30% germination of synthetic seeds was achieved in MS. Well acclimated plants produced via somatic embryogenesis and/or synthetic seeds were transferred to field where more than 60% survived. This simple study enabled us to obtain a number of plantlets throughout the year each cycle requiring a short period of time. Besides propagation, this study provided an ex situ method for conservation of this vulnerable Himalayan species.D. O. I.http://dx.doi.org/10.3329/ptcb.v23i2.17506Plant Tissue Cult. & Biotech. 23(2): 147-155, 2013 (December)

High Efficiency Secondary Somatic Embryogenesis inHovenia dulcisThunb. through Solid and Liquid Cultures

Embryogenic callus was obtained from mature seed explants on medium supplemented with 2,4-dichlorophenoxyacetic acid. Primary somatic embryos (SEs) can only develop into abnormal plants. Well-developed SEs could be obtained through secondary somatic embryogenesis both in solid and liquid cultures. Temperature strongly affected induction frequency of secondary embryogenesis. Relatively high temperature (30∘C) and germinated SEs explants were effective for induction of secondary somatic embryos, and low temperature (20∘C) was more suitable for further embryo development, plantlet conversion, and transplant survival. Somatic embryos formed on agar medium had larger cotyledons than those of embryos formed in liquid medium. Supplementing 0.1 mg L−16-benzyladenine (BA) was effective for plant conversion; the rate of plant conversion was 43.3% in somatic embryos from solid culture and 36.5% in embryos from liquid culture.In vitroplants were successfully acclimatized in the greenhouse. The protocol established in this study will be helpful for large-scale vegetative propagation of this medicinal tree.