Yasuki Honda

Extracellular polysaccharides produced by tuberose callus

A high yield of extracellular polysaccharide (ECP) was obtained from callus cultures of tuberose (Polianthes tuberosa), which could be separated into an unadsorbed and two acidic fractions (TPS-1, -2) by ion-exchange column chromatography. The yields of each fraction were markedly increased by the addition of 10(-5) M 2,4-dichlorophenoxyacetic acid to the medium. Of the three fractions, the amount of TPS-1 accounted for over 60% of total yield of ECP, which was a predominant polysaccharide consisting of arabinose (Ara), mannose (Man) and galactose (Gal) as major neutral monosaccharides. Judging from the patterns of electrophoresis and ultra-centrifugation, TPS-1 was identified to be homogeneous. Methylation and GC-mass spectrometry analyses of this fraction revealed the presence of 1,2,3-linked Man, 1-linked Ara, 1,3-linked Ara, 1-linked Gal and 1,3,4-linked glucuronosyl (GlcUA) residues in a molar ratio of 1.0:1.08:0.85:0.75:1.08. Based on additional analyses of the mild acid hydrolysate and the absolute configuration of the constituent monosaccharides, a possible structure for TPS-1 was a glucuronomannan possessing the unit of -->4)-beta-D-GlcUAp-(1-->2)-alpha-D-Manp-(1--> with branching at the C-3 position, where -->1)-alpha-L-Araf, -->1)-beta-D-galp, -->1)-alpha-L-Araf-(3-->1)-alpha-L-Araf or -->1)-alpha-L-Araf-(3-->1)-beta-D-Galp were attached randomly. About 35% of the GlcUA moieties were present as methyl esters. Further confirmation was made by 1H and 13C NMR spectroscopy.

Plant regeneration from patchouli protoplasts encapsulated in alginate beads

Mesophyll protoplasts were isolated from leaves of in vitro grown patchouli (Pogostemon cablin Benth.). The protoplasts were encapsulated in alginate beads, approximately 2–3×103 protoplasts per 25 μl bead. Successful colony formation was induced when the protoplast beads were inoculated into a liquid medium supplemented with 10-6 M NAA and 10-5 M BA. The frequency of colony formation was improved greatly by the inclusion of several beads per ml medium. To induce high colony formation for a single bead, it was essential to culture protoplasts in the presence of nurse beads containing actively-growing cells of the same species. Rapid regeneration of plants from protoplast-derived calluses was accomplished by a two-step culture procedure with liquid and then solid media. Gas-chromatographic analyses showed that regenerated plants produced an essential oil comprising a full-set of patchouli sesquiterpenes.

Production of polysaccharides by liquid cultures of Polianthes tuberosa cells; high production by reduction in the viscosity of culture medium

To improve the production of extracellular polysaccharides (EPS) in liquid cultures of Polianthes tuberosa (tuberose) cells, the viscosity of the culture medium was lowered by addition of mineral salts. In cultures in the medium supplemented with 30 mM CaCl2, higher production of EPS (6.5g/l) has been realized (vs. 4.6 g/l without CaCl2).

Production of polysaccharides in liquid cultures of Polianthes tuberosa cells

SummaryThe effects of components of the medium on the production of extracellular polysaccharide (EPS) by cultured cells of Polianthes tuberosa (tuberose) were studied. Optimization of media components culturing in flask resulted in increasing EPS production from 1.4 to 4.1 g/l. In particular, relatively high concentration (10\s-5M) of 2,4-dichlorophenoxyacetic acid (2,4-D) markedly stimulated the production of EPS. Based on these results, EPS production by a 30-1 jar fermenter was attempted and the final rate of Production was 4.6 g/l at 30th day of culture. The EPS consisted mainly of acidic polysaccharides with glucuronic acid, mannose, arabinose, galactose, glucose and xylose.

Biosynthesis of extracellular polysaccharides by tuberose callus

Summary Extracellular polysaccharides (ECPs) were produced in liquid cultures of tuberose (Poliantbes tuberosa L.) callus regardless of the cell growth cycle. The major component of the ECPs was called tuberose polysaccharide (TPS), which has a molecular weight (mol.wt.) of ≧ 106. Using protoplasts labelled with 14C-glucose, no 14C-TPS was found, but two 14C-labelled low molecular weight polysaccharides (L-1: 1 × 105 mol.wt., L-2: 2–3 × 104 mol.wt.) were secreted into the medium. Both L-1 and L-2 polysaccharides were also detected in the intracellular polysaccharide (ICP) fractions extracted from calluses, single cells and protoplasts. Based on the neutral sugar compositions of 14C-labelled L-1, L-2 and TPS, the L-1 polysaccharide was identified as a putative TPS precursor. The addition of brefeldin-A blocked TPS production, whereas the intracellular synthesis of its precursor was not markedly inhibited. Histochemical and electron microscopic observations showed that TPS is deposited in the intercellular spaces among the surface cells of the callus mass.