A Temporary Immersion System to Improve Cannabis sativa Micropropagation.

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, ß-A and ß-H, all supplemented with 2-µM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in ß-A medium supplemented with 2-µM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species.

Characterization of the galactono-1,4-lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of nitric oxide.

Pepper fruit is one of the highest vitamin C sources of plant origin for our diet. In plants, ascorbic acid is mainly synthesized through the L-galactose pathway, being the L-galactono-1,4-lactone dehydrogenase (GalLDH) the last step. Using pepper fruits, the full GalLDH gene was cloned and the protein molecular characterization accomplished. GalLDH protein sequence (586 residues) showed a 37 amino acids signal peptide at the N-terminus, characteristic of mitochondria. The hydrophobic analysis of the mature protein displayed one transmembrane helix comprising 20 amino acids at the N-terminus. By using a polyclonal antibody raised against a GalLDH internal sequence and immunoblotting analysis, a 56kDa polypeptide cross-reacted with pepper fruit samples. Using leaves, flowers, stems and fruits, the expression of GalLDH by qRT-PCR and the enzyme activity were analyzed, and results indicate that GalLDH is a key player in the physiology of pepper plants, being possibly involved in the processes which undertake the transport of ascorbate among different organs. We also report that an NO (nitric oxide)-enriched atmosphere enhanced ascorbate content in pepper fruits about 40% parallel to increased GalLDH gene expression and enzyme activity. This is the first report on the stimulating effect of NO treatment on the vitamin C concentration in plants. Accordingly, the modulation by NO of GalLDH was addressed. In vitro enzymatic assays of GalLDH were performed in the presence of SIN-1 (peroxynitrite donor) and S-nitrosoglutahione (NO donor). Combined results of in vivo NO treatment and in vitro assays showed that NO provoked the regulation of GalLDH at transcriptional and post-transcriptional levels, but not post-translational modifications through nitration or S-nitrosylation events promoted by reactive nitrogen species (RNS) took place. These results suggest that this modulation point of the ascorbate biosynthesis could be potentially used for biotechnological purposes to increase the vitamin C levels in pepper fruits.

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons.

The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage- and current-clamp whole-cell techniques. The resting potential of SCG cells varied from -47 to -80 mV (-58.3 +/- 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 +/- 0.7 mV, n = 22) and a hyperpolarisation (7.2 +/- 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 +/- 0.2 mV, n = 8). Inhibition of voltage-gated currents unmasked a voltage-independent resting conductance reversing at -50 mV. The reversal potential of the voltage-independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage-independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage-independent conductance, with voltage-activated M and h currents playing a prominent stabilising role.