Cryopreservation of oil palm (Elaeis guineensis Jacq.) polyembryoids via encapsulation–desiccation

The current report assesses the efficiency of encapsulation–desiccation protocol to cryopreserve oil palm (Elaeis guineensis Jacq.) polyembryoids. Specifically identified polyembryoids, comprising of haustorium and torpedo-shaped structures, were encapsulated [comprising 3% (w/v) sodium alginate and 100 mM CaCl2]. Calcium alginate-encapsulated and sucrose-precultured polyembryoids were subjected to different spans of desiccation in a laminar air-flow cabinet, followed by freezing in liquid nitrogen. The effect of sucrose preculture (with gradual exposure to 0.3, 0.5, 0.75 and 1 M for 7 days) and dehydration periods (0–10 h) under sterile air-flow on post-freezing survival and regrowth of encapsulated polyembryoids were studied. Cryopreserved and thawed polyembryoids (initially precultured in sucrose, followed by 9 h air-desiccated to 23.3% moisture content) displayed the highest survival percentage (73.3%) and regeneration (of shoot, root and secondary somatic embryo) on Murashige and Skoog regrowth medium containing sucrose (0.3–1 M) and 0.2 mg/l 2,4-dichlorophenoxy acetic acid. In addition, ultrastructural study using scanning electron microscopy exhibited successful revival of cryopreserved polyembryoids, owing to retention of cellular membrane stability through optimized and protected (encapsulated) desiccation. The present study thus substantiates the potential of this encapsulation–desiccation procedure in cryopreservation of oil palm polyembryoids for long-term conservation programs.

The Induction of Primary and Secondary Somatic Embryo to Support Arabica Coffee Propagation

The primary and secondary somatic embryogenesis can be used to propagate Coffea arabica L clonally. However, the success of this propagation was depended on plant growth regulator and varieties. This study aimed to examine the possibility of 2,4-D and thidiazuron application to form primary and secondary somatic embryo to support Arabica coffee clonal propagation. The study consisted of two activities (1) 2,4-D and thidiazuron Application to Induce Primary Somatic Embryogenesis of Arabica Coffee and (2) The Application of thidiazuron in Solid and Semi-Solid Media to Induce Secondary Somatic Embryos. The results indicated significant effect of varieties and plant growth regulator on fresh weight, number of torpedo and germinated embryo. However, it showed no significant effect on callus formation percentage. The best medium to induce primary somatic embryogenesis depending on variety, on the treatment of 4.52 μM 2,4 -D +18.16 μM thidiazuron was the best for AS2K and Sigarar Utang varieties, S 795 at 4.52 μM 2,4-D + 9.08 μM thidiazuron, whereas Kartika at 4.52 μM 2.4-D + 13.62 μM thidiazuron. The morphology of coffee somatic embryo was normal. Primary somatic embryo was developed indirectly, whereas the secondary somatic embryo was directly. The application of 9.08 μM thidiazuron increased the percentage and number of secondary somatic embryos, hence enhancing number of Arabica coffee planlet. Keywords : Coffea arabica L, 2,4-D, thidiazuron, semi-solid media, Indirect somatic embryogenesis

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.

Abscisic Acid Effect on Improving Horse Chestnut Secondary Somatic Embryogenesis

The effect of abscisic acid on the development of primary androgenic embryo and secondary somatic embryogenesis was investigated with the aim of improving multiplication rates and secondary somatic embryo quality in horse chestnut microspore and anther culture. The early embryo stage (globular) had a better response than late stages (heart, torpedo, and cotyledonary) in both types of cultures. Also, microspore culture had a high potential for mass secondary embryo production. The number of secondary somatic embryos was three times higher on hormone-free medium than on medium enriched with 0.01 mg·L−1 abscisic acid. However, most of the embryos on hormone-free medium had abnormal morphology. For this reason, abscisic acid was added to the media to improve embryo quality. The morphology of abscisic acid treated embryos was better than abscisic acid non-treated embryos. The optimal abscisic acid concentration for secondary somatic embryo induction and production of high-quality embryos was 0.01 mg·L−1. Overall, the effect of abscisic acid on the induction of secondary somatic embryogenesis and plant regeneration of androgenic embryos of this species may be helpful for the further synthesis of secondary metabolites in vitro and their application in the pharmaceutical industry.

Development of virus resistant transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus

Translatable and nontranslatable versions of the coat protein (cp) gene of a Papaya ringspot virus (PRSV) isolate collected in the state of Bahia, Brazil, were engineered for expression in Sunrise and Sunset Solo varieties of papaya (Carica papaya). The biolistic system was used to transform secondary somatic embryo cultures derived from immature zygotic embryos. Fifty-four transgenic lines, 26 translatable and 28 nontranslatable gene versions, were regenerated, with a transformation efficiency of 2.7%. Inoculation of cloned R0 plants with PRSV BR, PRSV HA or PRSV TH, Brazilian, Hawaiian and Thai isolates, respectively, revealed lines with mono-, double-, and triple-resistance. After molecular analysis and a preliminary agronomic evaluation, 13 R1 and R2 populations were incorporated into the papaya-breeding program at Embrapa Cassava and Tropical Fruits, in Cruz das Almas, Bahia, Brazil.