In the present study, polyembryoids of oil palm (Elaeis guineensis Jacq.) were cryopreserved with successful revival of 68 % for the first time using the droplet vitrification technique. Excised polyembryoids (3-5-mm diameter) from 3-month-old in vitro cultures were pre-cultured for 12 h in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose. The polyembryoids were osmoprotected in loading solution [10% (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose] for 30 min at room temperature and then placed on aluminium strips where they were individually drenched in chilled droplets of vitrification solution (PVS2) [30% (w/v) glycerol plus 15% (w/v) ethylene glycol (EG) plus 15% (w/v) DMSO plus 0.4 M sucrose] for 10 min. The aluminium strips were enclosed in cryovials which were then plunged quickly into liquid nitrogen and kept there for 1 h. The polyembryoids were then thawed and unloaded (using 1.2 M sucrose solution) with subsequent transfer to regeneration medium and stored in zero irradiance. Following for 10 days of storage, polyembryoids were cultured under 16 h photoperiod of 50 μmol m(-2) s(-1) photosynthetic photon flux density, at 23 ± 1 °C. Post-thaw growth recovery of 68% was recorded within 2 weeks of culture, and new shoot development was observed at 4 weeks of growth. Scanning electron microscopy revealed that successful regeneration of cryopreserved polyembryoids was related to maintenance of cellular integrity, presumably through PVS2 exposure for 10 min. The present study demonstrated that cryopreservation by droplet vitrification enhanced the regeneration percentages of oil palm in comparison with the conventional vitrification method previously reported.
Dendrobium sonia-28 is a popular orchid hybrid due to its flowering recurrence and dense inflorescences. Unfortunately, it is being decimated by fungal diseases, especially those caused by Fusarium proliferatum. In this study, selection of F. proliferatum-tolerant protocorm-like bodies (PLBs) was carried out by assessing the effects of differing concentrations of fusaric acid (FA). PLBs were cultured on Murashige and Skoog (MS) medium supplemented with 0.05 to 0.2 millimolar (mM) concentrations of FA. Higher concentrations of FA increased mortality of PLBs and reduced their growth. The survival rate for 0.05 mM FA was 20 % but only 1 % at the highest dose of 0.2 mM. Additionally, two different size ranges of PLBs were investigated, and growth increased more at lower FA concentrations for larger PLBs, whilst the growth rate of smaller PLBs was inhibited at an FA concentration of 0.2 mM. Histological examination using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses disclosed severe cell wall and organelle damage, as well as stomatal closure in PLBs treated with the high FA concentrations. Reductions in plantlet growth were much greater at the highest concentrations of FA. Some randomly amplified polymorphic DNA (RAPD) markers clearly discriminated between selected and non-selected variants of Dendrobium sonia-28, showing different banding patterns for each FA concentration and specific bands for selected and control plants.
Cytochrome P450s constitute the largest family of enzymatic proteins in plants acting on various endogenous and xenobiotic molecules. They are monooxygenases that insert one oxygen atom into inert hydrophobic molecules to make them more reactive and hydro-soluble. Besides for physiological functions, the extremely versatile cytochrome P450 biocatalysts are highly demanded in the fields of biotechnology, medicine, and phytoremediation. The nature of reactions catalyzed by P450s is irreversible, which makes these enzymes attractions in the evolution of plant metabolic pathways. P450s are prime targets in metabolic engineering approaches for improving plant defense against insects and pathogens and for production of secondary metabolites such as the anti-neoplastic drugs taxol or indole alkaloids. The emerging examples of P450 involvement in natural product synthesis in traditional medicinal plant species are becoming increasingly interesting, as they provide new alternatives to modern medicines. In view of the divergent roles of P450s, we review their classification and nomenclature, functions and evolution, role in biosynthesis of secondary metabolites, and use as tools in pharmacology.
MicroRNAs (miRNAs) play important roles in various biological processes. Our previous study showed that inhibition of MTOR with rapamycin treatment suppressed human endothelial cell tube formation, concomitant with the down-regulation of miR-107. Similarly, inhibition of Ztor by rapamycin also suppressed vascular development in zebrafish embryos. To gain a better understanding of the role of miR-107 and MTOR in vascular development, the present study sought to validate its function by over-expressing miR-107 in zebrafish embryos via microinjection with mimic miR-107 duplexes. Alkaline phosphatase (ALP) staining was used to visualise blood vessels in the zebrafish embryo, and expressions of Pten, Ztor and Rap1 were investigated by immunoblotting. Over-expression of miR-107 in zebrafish embryos inhibited the sprouting of intersegmental vessels (ISVs) with concomitant down-regulation of phosphorylated Rps6 expression, which confirmed the inhibition of Ztor signalling. As expected, pten, which is the target of miR-107, was down-regulated. Interestingly, Rap1, a small GTPase protein that is involved in intersomitic vessels sprouting during angiogenesis, was also down-regulated when miR-107 was over-expressed. Overall, our findings suggest that miR-107 and Ztor-mediated suppression of vascular development in zebrafish embryo involves Rap1.