Heikki Rosenqvist

Establishment and growth characteristics of Glycyrrhiza glabra hairy root cultures

Hairy root cultures of Glycyrrhiza glabra producing considerable amounts of phenolic compounds were successfully established by using Agrobacterium strain C58C18(pRT GUS 104). The effect of phosphate, ammonia, nitrate and ferric-EDTA concentrations of culture medium on growth and total phenolics production of the cultures were studied. By employing statistical experimental design and linear regression analysis an improved B5 medium (B50-M) could be developed. When cultivating G. glabra hairy roots in B50-M medium we were able to obtain 9 g dried roots/l in 25 days which was twice as much as when using the initial B50 medium. According to tentative analyses the cultures did not contain glycyrrhyzin, but they produced liquiritigenin and isoliquiritigenin. The production of total phenolic substances (mg g-1 dw) was higher in the improved medium resulting in significantly higher volumetric productivity (mg phenolic compounds l-1). This will further enable the extraction and identification of the phenolic compounds produced by the cultures.

The effect of temperature on hairy root cultures of Catharanthus roseus: Growth, indole alkaloid accumulation and membrane lipid composition.

Cultivation of Catharanthus roseus hairy root cultures at different temperatures was found to have an effect on growth rate and indole alkaloid content as well as lipid composition. When lowering the temperature, the roots responded by increasing the degree of unsaturation of cellular lipids, which was mainly due to an increased proportion of linolenic acid in the main lipid classes. The modifications in lipid composition were obviously necessary for the roots to retain the proper cell membrane fluidity at each temperature. Despite of changes in membrane lipids, no effect on the distribution of indole alkaloids between the roots and the medium could be detected. Instead, the level of alkaloid accumulation showed a clear increase with lowering temperature.

The effect of temperature on growth, indole alkaloid accumulation and lipid composition of Catharanthus roseus cell suspension cultures

Cell suspension cultures of Catharanthus roseus were used to study the effect of temperature on plant cell lipids and indole alkaloid accumulation. Lowering the cultivation temperature increased the total fatty acid content per cell dry weight relative to that at higher temperatures, mainly because of increased accumulation of unsaturated C18 acids. In addition, an increase in the relative proportion of phosphatidylcholine and phosphatidylethanolamine was observed. Within individual lipids, the degree of unsaturation was increased and the mean fatty acid chain length decreased with reducing temperature. These changes may be interpreted as modifying the cell membrane fluidity to keep it optimal for growth and metabolism at each temperature. In spite of membrane modifications, the indole alkaloid content of the cells or the medium was not affected by temperature change.

Interaction of abscisic acid and indole-3-acetic acid-producing fungi with Salix leaves

The molds Botrytis cinerea, Cladosporium cladosporioides, and the yeast Aureobasidium pullulans, isolated from the leaves of three short-rotation Salix clones, were found to produce indole-3-acetic acid (IAA). Abscisic acid (ABA) production was detected in B. cinerea. The contents of IAA and ABA in the leaves of the Salix clones and the amounts of fungal propagules in these leaves were also measured, in order to evaluate whether the amounts of plant growth regulators produced by the fungi would make a significant contribution to the hormonal quantities of the leaves. The content of ABA, and to a lesser degree that of IAA, showed a positive correlation with the frequency of infection by the hormone-producing organisms. The amounts of hormone-producing fungi on leaves that bore visible colonies were, however, not sufficiently high to support the claim that either the fungal production of ABA or IAA would significantly contribute to the hormonal contents of the leaves of the Salix clones. It is therefore suggested that the effect of fungal IAA production on plants is limited to the rhizosphere and that B. cinerea, which is a known pathogen, induces ABA production by the mother plant as a response to physiological stress.

Surface lipids and their distribution on willow (Salix) leaves: A combined chemical, morphological and physicochemical study

The surfaces of leaves of three willow (Salix spp.) clones with different frost tolerance and biomass productivity were investigated in order to elucidate further the previously observed relationship between high epicuticular leaf wax load, high biomass productivity and poor overwintering survival of Salix clones. Morphological studies by scanning electron microscopy (SEM) were combined with contact angle measurements, surface overall chemical analysis by X-ray photoelectron spectroscopy (XPS) and gas chromatographic (GC) analyses of extracted waxes and cutin. The GC analyses revealed that epicuticular waxes contained n-alkanes, n-alcohols, n-aldehydes, wax esters and free fatty acids. Mono- and dihydroxy hexadecanoic and monohydroxy octadecanoic acids were major cutin acids in the leaves of the three Salix clones. Overall chemical analysis showed that the Salix leaf surfaces were composed primarily of carbon (about 90%) and oxygen (about 10%). SEM showed that the leaf surfaces of the three Salix clones were partly (15-40% of the area) covered by smooth spherical particles on or in a planar matrix. By combining these chemical and morphological analyses, it was concluded that the particles observed by SEM were composed of epicuticular waxes, and the planar matrix between the particles was cutin. The high epicuticular wax load and the high amounts of n-alkanes, high levels of dihydroxy hexadecanoic acid and total C-16 acids in cutin, and the high leaf-surface coverage by the wax spheres correlated with the poor frost tolerance and high productivity of the three Salix clones in this study. Furthermore, water contact angles on the leaf surface of the frost-susceptible high-producerclone were 93 degrees, about 20 degrees higher than on the leaf surfaces of the two other clones

Epicuticular waxes of Salix species in relation to their overwintering survival and biomass productivity

Abstract The epicuticular leaf waxes of nine willow clones (one Salix myrsinifolia, four Salix dasyclados, one Salix hybrid and three Salix viminalis clones) with different overwintering survival rates and biomass productivities were analysed by combined gas chromatography-mass spectrometry. The waxes were composed of n-alkanes (C21C31), long-chain aliphatic esters (C36C58), n-alcohols (C20C30), n-aldehydes (C24C30) and saturated free fatty acids (C16C30). Two types of waxes were observed. The S. viminalis clones had n-alkanes as their major wax component and n-docosanol as their major n-alcohol, whereas the other clones contained approximately equal proportions of n-alkanes, free fatty acids, n-alcohol and n-aldehydes and had n-hexacosanol as their major n-alcohol. The total epicuticular leaf wax contents varied from 3 μg cm−2 to 24.9 μg cm−2 and epicuticular n-alkane contents from 0.7 μg cm−2 to 14.8 μg cm−2. A correlation between high wax content, high n-alkane content, two overwintering survival and high biomass productivity of the clones was observed.

Fatty acids and alkanes in leaves of frost-tolerant and frost-susceptible willows

Abstract The susceptibility ofwillow ( Salix ) to injuries of spring and summer frost was studied in relation to aliphatic fatty acid and n -alkane analyses in 15 clones intended for use in short-rotation forestry. All the clones examined, originating from different countries but cultivated in the same fields in different climates, contained the same major long-chain fatty acids (12:0, 14:0, 16:0, 18:0,18: 1 cis -9, 18:2 cis -9,12, 18:3 cis -9,12,15, 20:0 and 22:0). The fatty acid contents of the leaves were ca 3% of the dry weight. Clone-specific variations in relative compositions of the fatty acids were evident but no correlation existed between the amount of unsaturation, the degree of unsaturation, the mean chain length and previously recorded frost tolerance. It is therefore concluded that the fluidity properties of membrane lipids do not reflect the tolerance of willows to frost. The concentrations of major n -alkanes also varied markedly from clone to clone. In frost-sensitive clones the amounts of n -alkanes were significantly and invariably higher. The higher n -alkane contents were also maintained when cultivation was carried out in southern Finland. Therefore, the observed variations are not temperature-induced but are of genetic origin. The use of n -alkane contents as a biochemical marker for frost tolerance is suggested for future breeding work.

Fatty acid and alkane changes in willow during frost-hardening

Abstract Frost-hardening of 10 basket willow ( Salix viminalis ) clones was studied by visual scoring of twig recuperation after controlled freezing treatments, together with measurements of total fatty acids, total n -alkanes (gas chromatography) and dry wts (% of fresh wt) of the twigs during the fall of 1994 in central Finland. The initial frost-tolerance of the clones was −5°. Hardening of the clones began at the end of September and proceeded similarly until mid October, when clonal differences became observable. The final frost-tolerances of the clones at the end of November varied from −37° to −80°, as 50% killing temperatures. Twig dry wts showed some increase prior to hardening, but an abrupt increase was observed during the first four weeks of hardening. After this, the increase in dry wts ceased. The total amount of twig n -alkanes (mg g −1 dry wt) decreased constantly prior to hardening until October. The total amounts of fatty acids (mg g −1 dry wt) decreased prior to hardening but doubled after hardening had begun. This increase in total fatty acids was accompanied by an abrupt increase in the ratio of unsaturated to saturated fatty acids. The amounts of linoleic (18:2) acid began to increase at the expense of linolenic (18:3) acid simultaneously with the beginning of hardening. No significant changes in fatty acids, n -alkanes or dry weights were observed during the late hardening stage. The differences in fatty acid profiles of the clones could not be attributed to clonal differences in frost-tolerances in the late hardening stage.

Role of root and stem lipids in cold tolerance of short-rotation willows

Abstract The possible basis for the cold tolerance of Salix species was studied by analyses of long chain n -alkanes and fatty acids in 15 clones known for their cold tolerance and productivity in long term field experiments. Clone-specific differences in the barks of stems and roots were compared to those of the leaves and evaluated against the long term field records. The barks of stems, but not those of the roots, contained significant amounts of n -alkanes. The n -alkane contents were also invariably higher in stem barks than in the corresponding leaves so that the stem to leaf ratios were nearly constant from clone to clone. However, different clones varied significantly with respect to n -alkane contents and composition in the stem barks; contents were invariably higher and richer in C 21 -C 29 n -alkanes in cold-sensitive clones than in their cold-tolerant counterparts. This phenomenon was reproduced in willows from southern and northern experimental fields suggesting that the average growth temperature made little contribution to the differences. Because no correlation existed between fatty acid composition and cold hardiness it is suggested that the surface coatings of air-exposed plant tissues rather than the fluidity properties of cellular membranes are critical in determining the cold hardiness.

Fiber fractions from processing of barley in production and conservation of a biologic control agent

Carriers are frequently used to overcome problems associated with microbial survival in soil after inoculation. Moreover, the use of carriers can prolong the shelf lives and lessen dusting of both biofungicides and biologic fertilizers. This study investigated the suitability of barley-based fiber fractions as growth media and immobilization matrices in the cultivation of a Streptomyces griseoviridis biologic control agent, as well as for the conservation of obtained biomass in dehydrated hydrogel capsules. The second main ingredient in all the examined carrier matrices was alginate. The aim was to find a hydrogel formulation suited for a production process in which all individual steps, including cultivation of the organism; downstream processing; and formulation, storage, and application of the product (i.e., biologic control agent), are carried out in the hydrogel matrix. Of the tested fractions, brewer’s spent grain was the best choice, when considering the price vs the nutrient contents as well as the storage time and ease of processing of the crude and the finished products. It seems that cereal fibers can be replenished with cereal fractions less rich in fiber but having a higher content of utilizable nutrients and, hence, better suited for the production of biomass. A high content of water-insoluble fiber favorably influenced the appearance as well as the applicability of the products.