Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon)

A method for shoot regeneration from leaf explants in two cultivars of cranberry (Vaccinium macrocarpon Ait.) is described. Modified Anderson's medium supplemented with combinations of thidiazuron (TDZ) with or without 1 M NAA (-naphthaleneacetic acid) was used to optimize shoot regeneration. The effect of light or dark incubation was also determined. Maximum regeneration was obtained in the light in the presence of 10 M TDZ and 1 M NAA. While this medium was suitable for leaf explants obtained from shoot cultures, regeneration did not occur from leaves collected from greenhouse-grown plants. Elongation of the regenerated shoot tips did not occur until explants were transferred to growth regulator-free medium at which time only a minority of shoots elongated. Elongated shoots could be dissected away from leaf tissue, rooted easily, and acclimitized to ambient conditions.Abbreviations NAA -naphthaleneacetic acid - TDZ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea     

Phytochrome control of short-day-induced bud set in black cottonwood

In trees and other woody perennial plants, short days (SDs) typically induce growth cessation, the initiation of cold acclimation, the formation of a terminal bud and bud dormancy. Phytochrome control of SD-induced bud set was investigated in two northern clones of black cottonwood (Populus trichocarpa Torr. & Gray) by using night breaks with red light (R) and far-red light (FR). For both clones (BC-1 and BC-2), SD-induced bud set was prevented when R night breaks as short as 2 min were given in the middle of the night. When night breaks with 2 min of R were immediately followed by 2 min of FR, substantial reversibility of bud set was observed for BC-1 but not for BC-2. By comparing the effects of the R night breaks on bud set and the length of specific internodes, we determined that the R night breaks influenced internode elongation in two opposing ways. First, the addition of a R night break to the SD treatment prevented the cessation of internode elongation that is associated with bud set. Those internodes that would not have elongated under SDs (and would have been found within the terminal bud) elongated in the R treatment. Second, the R night breaks decreased internode length relative to the long-day (LD) control. In contrast to the clonal differences in reversibility that we observed for bud set, the decrease in internode length (i.e. the second effect of R) was R/FR reversible in both clones. Based on these results, we conclude that internode elongation is influenced by two distinct types of phytochrome-mediated response. The first response is a typical response to photpperiod, whereas the second response is a typical “end-of-day” response to light quality. Our results demonstrate that SD-induced bud set in black cottonwood is controlled by phytochrome but that clonal differences have an important influence on the R/FR reversibility of this response. The availability of an experimental system in which SD-induced bud set is R/FR reversible will be valuable for studying the physiological genetics of photoperiodism in trees.     

Improved Cultivation Systems for Isolation of the Colorado Potato Beetle Spiroplasma

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

GROWTH AND DEVELOPMENT OF REPRODUCTIVE AND VEGETATIVE TISSUES OF BOUGAINVILLEA CULTURED IN VITRO AS A FUNCTION OF CARBOHYDRATE

Inflorescence meristems and vegetative tissues, excised from noninduced Bougainvillea ‘San Diego Red’ plants, were cultured in vitro in media containing either 3% fructose, glucose or sucrose as carbon sources. Growth and development of young leaves were equivalent whether sucrose or fructose was used whereas floret initiation on inflorescence meristems was much greater when fructose or glucose was the carbon sources. Brief (1-3 days) exposure of inflorescence meristems to fructose at the beginning of culture and subsequent transfer to sucrose did not increase development over continuous culture in sucrose. Longer exposures (4-7 days) to fructose with subsequent transfer to sucrose did, however, increase the percentage of meristems developing florets, but such treatment did not increase development to the same level as those exposed to fructose for the entire period in vitro. During the first 18 days of culture, growth of meristems in sucrose was linear while that in fructose was exponential. There was no difference in carbohydrate requirements for floret initiation on meristems excised from short-day induced or noninduced plants, suggesting that induction does not enhance the ability of meristems to utilize sucrose.     

Photoperiodic responses of a northern and southern ecotype of black cottonwood

Photoperiod is an important signal controlling the onset of dormancy in perennial plants. Short days typically induce growth cessation, the initiation of cold acclimation, the formation of a terminal bud. bud dormancy and other adaptive responses. Photoperiodic ecotypes have evolved in many species with large latitudinal distributions. The photoperiodic responses of two northern (53°35′ and 53°50′N) and two southern (34°10′ and 40°32′N) genotypes of black cottonwood (Populus trichocarpa Torr. & Gray) were characterized by growing trees under a range of photoperiods in the greenhouse and growth chamber. Short days induced bud set in both ecotypes. resulting in trees with fewer leaves and less height growth than trees grown under long days. Short days also enhanced anthocyanin accumulation in the northern ecotype and decreased branching of the southernmost genotype. Two aspects of the photoperiodic response were evaluated for each trail: critical photoperiod. which was defined as the longest photoperiod that elicited a short-day response, and photoperiodic sensitivity, which was defined as the change in response per unit change in photoperiod. For each of the traits analyzed, the northern ecotype had a longer critical photoperiod and greater photoperiodic sensitivity than did the southern ecotype. The short critical photoperiod and reduced photoperiodic sensitivity of the southern ecotype resulted in a significant delay in bud set compared to that of the northern ecotype, even under a 9-h photoperiod. Typically, photoperiodic ecotypes have been characterized as having different critical photoperiods. Ecotypic differences in photoperiodic sensitivity, however, indicate that differences in the photoperiodic response curves cannot be completely described by the critical photoperiod alone. These results also suggest that the critical photoperiod. photoperiodic sensitivity and speed of bud set have a common physiological basis. Bud set occurred earlier hi the northern ecotype primarily because bud scale leaves were initiated earlier. For one of the northern genotypes, leaf primordia that were initialed under long days subsequently differentiated into bud scale leaves after the trees were transferred to a 9-h photoperiod. This demonstrates that primordia initiated under long days are not necessarily committed to becoming foliage leaves. The response to photoperiod did not differ appreciably between the greenhouse and growth chamber conditions that were tested.     

The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Metabolism of progesterone in the pregnant sheep near term: identification of 3 beta-hydroxy-5 alpha-pregnan-20-one 3-sulfate as a major metabolite.

Two pregnant ewes near term were given a single injection of progesterone-4-14C via the left jugular vein, and serial blood samples were taken from the right jugular vein at 5 min intervals over a period of 40 min. Radioactive steroids in the plasma were separated into unconjugated and conjugated fractions which were further isolated and analysed by established methods. The injected hormone was rapidly metabolized with a half-life of approximately 10 min and metabolic clearance rate about 3.5 liters min. The bulk of the metabolites was found in the sulfate fraction from which a major metabolite was identified as 3 beta-hydroxy-5 alpha-pregnan-20-one. From the unconjugated fraction, 20 alpha-hydroxy-pregn-4-en-3-one, a known minor metabolite was also isolated. No radioactive estrogens were found. It is concluded that a major portion of circulating progesterone in the pregnant ewe near term is cleared by 5 alpha-reduction of ring A, followed by sulfo-conjugation.