David L. Gustine

Corollin, coronillin and coronarian: Three new 3-nitropropanoyl-d-glucopyranoses from Coronilla varia

Abstract Three new 3-nitropropanoyl- d -glucopyranoses, 2,3,6-tri(3-nitropropanoyl)-α- d -glucopyranose (corollin), 1,2,6-tri(3-nitropropanoyl)-α- d -glucopyranose (coronillin) and 2,6-di(3-nitropropanoyl)-α- d -glucopyranose (coronarian) were isolated from the aerial parts of Coronilla varia . Structural assignments were made on the basis of 220 MHz NMR spectra.

Esterification of 3-nitropropanoic acid to glucose by suspension cultures of Coronilla varia

Abstract Coronilla varia suspension cultures incorporated exogenous 3-nitropropanoic acid into 3-nitropropanoyl- D -glucopyranoses, increasing their levels 600-fold over control values. The resulting levels of 3-nitropropanoyl- D -glucopyranoses were still lower than those in C . varia plants but were high enough for isolation of hiptagin (1,2,4,6-tetra(3-nitropropanoyl)-β- D -glucopyranose), coronarian and cibarian. Structural assignments were partially made on the basis of 360 MHz 1 H NMR spectra.

Regeneration of Coronilla varia L. (crownvetch) plants from callus culture

Coronilla varia L. (crownvetch) plants were regenerated from callus cultures through somatic embryogenesis. Callus cultures were initiated using hypocotyls excised from sterile seedlings. Cultures were then transferred from a modified Gamborgs B5 medium containing 2,4-D to a medium containing no plant growth regulators (basal B5). Formation of embryos was evident in 12 of 32 callus lines after transfer of callus to BOi2Y (modified Blaydeś medium supplemented with 100 mg inositol and 2 g yeast extract/L). Basal B5 supplemented with 10 mM asparagine or 20 mM NH4Cl could be substituted for BOi2Y. Embryos subsequently transferred to basal B5 developed roots and shoots. Plants thus formed were first transferred to vermiculite and then to soil.

Retention of phytoalexin regulation in legume callus cultures

Legume callus cultures were examined to assess whether regulation of phytoalexin biosynthetic pathways is retained in cultured tissues. Callus tissue cultures ofCanavalia ensiformis (jackbean),Medicago sativa (alfalfa), and nine species ofTrifolium (clover) were established (six clover species for the first time) and maintained on modified Gamborgs B5 medium. Phytoalexins educed in cultures incubated for 48 h with an abiotic elicitor (3.15 mM HgCl2) were detected by their antifungal activity and were purified by column chromatography and high-performance liquid chromatography. Following crystallization, phytoalexins were identified by ultraviolet and proton nuclear magnetic resonance spectroscopy. None of the treated cultures yielded the same complement of phytoalexins reported for fungal-inoculated leaves of the corresponding plants. Callus from all species exceptT. pratense yielded medicarpin, the only phytoalexin reported in treated leaves of all the corresponding plants. A second phytoalexin, maackiain, was found in treatedT. pratense andT. medium calli; maackiain has been reported in fungal-inoculated leaves of those plant species as well asT. hybridum. The phytoalexins sativan and vestitol were not found in treated callus tissues even though they were reported to be present in fungal-inoculated leaves of the same species. These results suggest that (a) the pathway for medicarpin biosynthesis is of central importance for this group of legumes, (b) some phytoalexin anabolic pathways contain metabolic blocks in cells of cultured tissue, and (c) the mechanism for regulating phytoalexin accumulation in tissues is not lost in culture.

Assessment of 13c-shift parameters in di- and tri-O-(3-nitropropanoyl)-d-Glucopyranoses

Abstract Natural-abundance 13 C-n.m.r. spectra of seven di- and tri- O -(3-nitropropanoyl)-α- and -β- d -glucopyranoses are reported and discussed. Linear regression-analysis of the extracted 13 C-substituted parameters associated with esterification at various positions of the α- and β-glucose skeletons yielded two sets of substituent chemical-shift constants. The chemical shifts calculated from these generated parameters agreed well with the experimentally obtained values for each compound. Additivity of the substituent-induced shifts as a function of esterification site and substituent orientation is discussed. The structures of three previously uncharacterized di- and tri-esters were ascertained by using the parameters established.

Versatile microcomputer-controlled, automated gradient analytical high-performance liquid chromatography system☆

Abstract We have developed a computer-controlled high-performance liquid chromatography (HPLC) system which generates binary gradients for automated analyses of a large number of samples. The 64K RAM S-100 laboratory microcomputer was programmed to control Waters 6000A and M45 HPLC pumps while simultaneously gathering data from Waters 440 absorbance and 420 fluorescence detectors (2 A/D channels, ± 5 V, 12 bit resolution). The S-100 was interfaced with a Micromeretics 725 autoinjector. Data were stored on 8-in floppy disks, which have a capacity for storage of up to 128 HPLC runs. The operator establishes parameters from menu prompts; one of eight convex or concave gradients, or a linear gradient may be chosen. The software will automatically control HPLC gradient analyses of up to 64 samples, including return of columns to initial conditions before each injections. Data are collected during analyses from the detectors, stored on the disks, and may also be plotted on a printer during the actual analysis. Data may be retrieved and analyzed to prepare reports with peak retention times and peak areas.

Spatial Autocorrelation Analysis of Genetic Structure Within White Clover Populations

White clover (Trifolium repens L.) populations exhibit high genetic and clonal diversities, while existing for many decades in grazed swards at northern midlatitudes. Genetic structure might exist within rapidly changing populations and might be a factor in creating genetic diversity. Trifoliate leaf samples were taken monthly for two years from up to 37 specific stolon points in quadrats from May to September on three central Pennsylvania farm sites. Random amplified polymorphic DNA (RAPD) profiles for individuals within populations in quadrats were tested by analysis of molecular variance (AMOVA) and spatial autocorrelation. Genetic variance by quadrat population dates in the three pastures ranged from 15 to 74 % and 46 to 80% in 1997 and 1998, respectively. Significant (P < 0.05) overall spatial autocorrelation was found in 26 populations that had clones and in seven populations without clones. No significant autocorrelation was found in 27 and seven populations with and without clones, respectively. The estimated patch size did not change significantly over two growing seasons. Number of clones and patch size was less important in determining genetic structure than variable existence of spatial autocorrelation.