Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l^-1 NAA, 0.1 mg l^-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l^-1 TDZ in combination with 0.1 mg l^-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.Abbreviations BA benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - NAA -naphthaleneacetic acid - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Chemical and Morphological Inter‐ and Intrapopulation Variability in Natural Populations of Gentiana pneumonanthe L.

Inter‐ and intrapopulation variability in six natural populations of the rare species Gentiana pneumonanthe was examined based on morphological and chemical data. Population size and linear morphometric parameters differed significantly among populations, but without a clear connection to habitat conditions, i. e. water supply and light availability. Leaf shape varied from ovate to lanceolate in all populations, and one population was distinctive in having the largest number of leaves of transitional shape. HPLC analyses of six secondary metabolites were performed separately for belowground parts, and aboveground vegetative and reproductive parts of individual plants (6 populations ×7 individuals ×3 plant parts, n=126) in order to examine differences at the population and individual levels. Three secoiridoids (swertiamarin (SWM), sweroside (SWZ), and gentiopicrin (GP)), one xanthone (mangiferin (MGF)), and two flavones (isoorientin (IO) and isovitexin (IV)) were detected and quantified in the analyzed samples: sweroside dominated in the aboveground reproductive part, mangiferin in the aboveground vegetative part, and gentiopicrin in the belowground part. At the population level, differences in contents of the analyzed chemicals among populations were significant only for a few metabolites. At the individual level, a pronounced organ‐dependent distribution of secondary metabolites was revealed. The results of this study contribute to a better understanding of natural variability within populations of the rare and threatened G. pneumonanthe, and provide data on the contents and within‐plant distribution of secondary metabolites, which are important as pharmacologically active compounds and may be useful for further biotechnological procedures regarding this species.     

栽培措施对龙胆产量及龙胆苦苷类成分含量的影响

为给龙胆药材生产提供栽培技术,在不同时间、以不同密度、分不同种苗等级以及在不同的前茬作物地块上移栽粗糙龙胆,秋季采用五点取样法测定产量,采用HPLC测定龙胆苦苷类成分含量。结果表明秋栽、一级种苗移栽、前茬作物为玉米,密度为125株/m²龙胆产量高,且龙胆苦苷含量都超过10.8%,但密度低龙胆有效成分含量高。秋栽、采用一级种苗移栽、轮作、移栽密度为125株/m²可达到优质高产的目标。     

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6′-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.     

Isolation and characterization of microsatellite markers for Sabatia campestris (Gentianaceae), an Illinois state‐endangered plant

Sabatia campestris is a regionally endangered prairie plant. Eight polymorphic loci have been generated for S. campestris (Gentianaceae) using non‐enriched and enriched libraries. Polymorphism was quantified from an isolated population from central Illinois yielding mean observed and expected heterozygosities of 0.723 and 0.801, respectively. The number of alleles per locus for this population ranged from three to 25 with a median of 15.5. These genetic markers will be used to determine population genetic structure of Illinois populations as well as to estimate fine‐scale gene flow rates.     

Quirks of dye nomenclature. 7. Gentian violet and other violets

The name, gentian, appeared about 1880. Immediately following its discovery in 1861, this violet dye was known as Violet de Paris or as methyl violet. Initially used as a textile dye, it was soon used to color virtually anything. The names and identity of the components, the varying modes of manufacture, analytical methods and the dye’s significant contribution to biological staining are discussed here. Finally, I discuss the dye’s declining medical use following the revelation of its toxic nature.     

The Gram Stain after More than a Century

The Gram stain, the most important stain in microbiology, was described more than a century ago. Only within the past decade, however, has an understanding of its mechanism emerged. It now seems clear that the cell wall of Gram-positive microorganisms is responsible for retention of a crystal violet:iodine complex. In Gram-negative cells, the staining procedures damage the cell surface resulting in loss of dye complexes. Gram-positive microorganisms require a relatively thick cell wall, irrespective of composition, to retain the dye. Therefore, Gramstainability is a function of the cell wall and is not related to chemistry of cell constituents. This review provides a chronology of the Gram stain and discusses its recently discovered mechanism.     

Pink root rot,a revised name of brown root rot of gentian,and the causal fungi,Pyrenochaeta gentianicola sp. nov. andP. terrestris in Japan

Cultivated gentian (Gentiana scabra var.buergeri) has been seriously damaged by pink root rot since the 1970s. Diseased plants finally collapse after wilting and stunting. More than 40 fungal genera were found to be associated with roots of mature and immature diseased plants or seeds. Among these fungi,Pyrenochaeta gentianicola sp. nov. andP. terrestris were almost always associated with the diseased plants. Their morphologies and temperature responses were compared, and their pathogenicity was also demonstrated by artificial inoculation tests.Part of this work was conducted at the National Institute of Agricultural Sciences (presently National Institute of Agro-Environmental Sciences), Tsukuba S.C., Ibaraki 305, Japan by T. Watanabe.