Chromosome doubling of Lychnis spp. by in vitro spindle toxin treatment of nodal segments

Lychnis (Caryophyllaceae) consists of about 30 species distributed throughout the temperate regions of the Northern Hemisphere, from East Asia to Europe. Many Lychnis spp. have high ornamental value and cultivated as pot or garden plants. In the present study, in vitro chromosome doubling of several Lychnis spp. was examined in order to widen their variability in horticultural traits. Initially effect of various spindle toxin treatments [100, 500 or 1000 mg l⁻¹ colchicine (COL), 10, 20 or 50 mg l⁻¹ oryzalin (ORY), or 1, 5, 10 mg l⁻¹ amiprophos-methyl (APM)] of nodal segments of a triploid genotype of L. senno (3x) was investigated on survival of nodal segments and chromosome doubling in nodal segment-derived plantlets. Significantly higher percentage (75.0%) of surviving segments after spindle toxin treatment was obtained in 10 mg l⁻¹ ORY treatment. Flow cytometry (FCM) analysis of leaf tissues showed that 9.4–13.8% of plantlets, which were derived from 10 to 20 mg l⁻¹ ORY, or 5 mg l⁻¹ APM treatments, were hexaploid (6x) or ploidy chimera (3x + 6x, 4x + 6x, 5x + 6x, 3x + 4x + 6x). The results obtained by FCM analysis were confirmed by chromosome observation in root tip cells. Thus 10 mg l⁻¹ ORY treatment of nodal segments is suitable for in vitro chromosome doubling of triploid L. senno. Efficient chromosome doubling was also achieved in diploid L. fulgens (2x) and L. sieboldii (2x) by treating nodal segments with 10 mg l⁻¹ ORY: 68.9–88.7% of nodal segments survived after ORY treatment, and 24.7–26.5% of plantlets derived from ORY-treated nodal segments were tetraploid (4x) or ploidy chimera (2x + 4x) in both species. These results indicate that the in vitro chromosome doubling method established for triploid L. senno may be applicable to a wide range of Lychnis spp. Tetraploid L. fulgens and L. sieboldii showed a compact plant form, and had thick stems and deep green leaves compared with the diploid mother plants. On the other hand, hexaploid L. senno showed very poor growth and died before flowering.     

The Effect of Lignite and Comamonas testosteroni on Pentachlorophenol Biodegradation and Soil Ecotoxicity

Biodegradation of pentachlorophenol (PCP) in soil by autochthonous microflora and in soil bioaugmented by the bacterial strain Comamonas testosteroni CCM 7530 was studied. Subsequent addition of lignite, an abundant source of humic acids, has also been investigated as possible sorbent for PCP immobilization. Biodegradation of PCP and number of colony-forming units were determined in the three types of soil, haplic chernozem, haplic fluvisol, and haplic arenosol, freshly spiked with PCP and amended with tested sorbent. The enhancing effect of sorbent addition and bioaugmentation on PCP biodegradation depended mainly on the soil type and the initial PCP concentration. Microbial activity resulted in biotransformation of PCP into certain potentially toxic substances, probably lower chlorinated phenols that are more soluble than PCP, and therefore more toxic toward present biota. Therefore, it was necessary to monitor soil ecotoxicity during biodegradation. Addition of lignite resulted in a significant improvement of PCP biodegradation and led to a considerable decrease of soil toxicity especially in bioaugmented soils. The method could potentially serve as a promising technique in remediation technology for reducing high initial PCP content in contaminated soils.     

Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures

The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.     

Synthesis of isoacteoside, a dihydroxyphenylethyl glycoside

The total chemical synthesis of isoacteoside (1), 2-(3,4-dihydroxyphenyl)ethyl 6-O-caffeoyl-3-O-(-l-rhamnopyranosyl)--d-glucopyranoside, is described. An acteoside acetate with benzyl groups at the catechols (3: 2-(3,4-dibenzyloxyphenyl)ethyl 2,6-di-O-acetyl-4-O-[3,4-bis(O-benzyl)caffeoyl]-3-O-(-l-rhamnopyranosyl)--d-glucopyranoside) was treated with a solution of methy-lamine in methanol (MeNH₂ in MeOH) to perform both deacetylation and caffeoyl migration, affording an isoacteoside derivative with benzyl groups at the catechols4b: 2-(3,4-dibenzyloxyphenyl)ethyl 6-O-[3,4-bis(O-benzyl) caffeoyl] -3-O-(-l-rhamnopyranosyl)--d-glucopyranoside —in 34% yield. Debenzylation of4b was successfully accomplished by catalytic transfer hydrogenation using 1,4-cyclohexadiene to give the target compound isoacteoside (1) in 54% yield.¹H and¹³C nuclear magnetic resonance spectral data of the synthesized isoacteoside (1) were identical with those of the natural isoacteoside isolated fromPaulownia tomentosa (Thumb.) Steud.Part of this research was presented at the 51st Annual Meeting of the Japan Wood Research Society, Tokyo, April 2001     

Genomic in situ hybridization (GISH) analysis of intergeneric hybrids in Colchicaceae

Some intergeneric hybrids produced by the crosses among several colchicaceous ornamentals, Gloriosa superba ??Lutea?? (2n = 2x = 22), G. ??Marron Gold?? (2n = 4x = 44), G. ??Verschild?? (2n = 7x = 77), Littonia modesta Hook. (2n = 2x = 22), and Sandersonia aurantiaca Hook. (2n = 2x = 24), were subjected to genomic in situ hybridization (GISH) analysis in order to clarify their genome constitutions. Chromosome preparation was made from root tip cells of L. modesta × G. superba ??Lutea??, L. modesta × S. aurantiaca, S. aurantiaca × G. superba ??Lutea??, L. modesta × G. ??Marron Gold??, S. aurantiaca × G. ??Marron Gold?? and G. ??Verschild?? × S. aurantiaca. Total DNA of one parent was labeled with digoxigenin or biotin and used as probe, and chromosomes were counterstained with 4??-6-diamidono-2-phenylindole (DAPI). For all the nine intergeneric hybrids, chromosomes from each parent could be clearly distinguished by GISH analysis. Thus GISH analysis is a powerful tool for identifying the genome constitution of intergeneric hybrids in colchicaceous ornamentals. The results obtained by GISH analysis study may be important for further progress in breeding of colchicaceous ornamentals.     

Chemoenzymatic synthesis of aroma active 5,6-dihydro- and tetrahydropyrazines from aliphatic acyloins produced by baker's yeast

Twenty-five acyloins were generated by biotransformation of aliphatic aldehydes and 2-ketocarboxylic acids using whole cells of baker's yeast as catalyst. Six of these acyloins were synthesized and tentatively characterized for the first time. Subsequent chemical reaction with 1,2-propanediamine under mild conditions resulted in the formation of thirteen 5,6-dihydropyrazines and six tetrahydropyrazines. Their odor qualities were evaluated, and their odor thresholds were estimated. Among these pyrazine derivatives, 2-ethyl-3,5-dimethyl-5,6-dihydropyrazine (roasted, nutty, 0.002 ng/L air), 2,3-diethyl-5-methyl-5,6-dihydropyrazine (roasted, 0.004 ng/L air), and 2-ethyl-3,5-dimethyltetrahydropyrazine (bread crustlike, 1.9 ng/L air) were the most intensive-smelling aroma active compounds.