Mirosława Furmanowa

Camptothecin and 10-hydroxycamptothecin in callus and plantlets of t Camptotheca acuminata

The process of callus induction, organogenesis and plantlets regeneration of Camptotheca acuminata Decne is reported. The highest growth rate of callus was observed on MS medium with 1 mg l−1 NAA, 1 mg l−1 kinetin and 60 g l−1 sucrose. All tissues and organs developed in vitro contain camptothecin and 10-hydroxycamptothecin. The presence of 10-hydroxycamptothecin in shoots and callus of Camptotheca acuminata Decne is reported for the first time. The alkaloids were detected and identified using HPLC methods.

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoeaasarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.

Divergent pattern of Polyisoprenoid alcohols in the tissues of coluria geoides: A new electrospray lonization ms approach

Polyisoprenoid alcohols of the plant Coluria geoides were isolated and analyzed by HPLC with UV detection to determine the nature of the polyprenol and dolichol mixture in the organs studied. In roots, a family of dolichols (Dol-15 to Dol-23, with Dol-16 dominating, where Dol-n is dolichol composed of n isoprene units) was accompanied by traces of polyprenols of similar chain lengths, whereas in hairy roots grown in vitro, identical patterns with a slightly broader chain-length range were found. Conversely, in leaves and seeds polyprenols were the dominant form, and their pattern was shifted toward longer chains (maximal content of Pren-19, where Pren-n is polyprenol composed of n isoprene units). Interestingly, the pattern of dolichols in seeds and leaves (in which Dol-17 dominated) was similar to that found in roots.Structures of the dolichols and polyprenols isolated were confirmed by the application of a new HPLC/electrospray ionization-MS method, which also offers a much higher sensitivity in detection of these compounds compared to a UV detector. The highest sensitivity was obtained when the [M+Na]+ ions of polyprenols and dolichols were recorded in the selected ion monitoring mode and a small amount of sodium acetate solution was added post-column to enhance the formation of these ions in an electrospray ion source.

Antimitotic effect, G2/M accumulation, chromosomal and ultrastructure changes in meristematic cells of Allium cepa L. root tips treated with the extract from Rhodiola rosea roots

Abstract Rhodiola rosea is a Russian and Chinese medicinal plant used in medical practice as a stimulating, adaptogenic and antiarrhythmic agent. Many investigations have pointed to the anticancer and antimutagenic role of R. rosea. In spite of these therapeutic effects of R. rosea extract. little is known about the mechanism of its anticancer action, because few cytological investigations havebeen conducted on the mitotic activity, cellular cycle, structure, ultrastructure, and cytophysiology of both animal and plant cells treated with the extract. Our studies have shown that extract from R. rosea roots (containing rosavine and cinnamyl alcohol) cause the inhibition of a mitotic activity; however, cells resume their divisions after the postincubation period. During incubation in the extract, accumulation of G2/M cells was observed. Chromosomes inprophase and metaphase were shorter and thicker but the extract did not cause chromosomal aberrations or the formation of micronuclei. Ultrastructure changes after 24 h of incubation were connected with an increase in the electron density of ground cytoplasm, decrease in the number of mitochondria but with an increase of their surface. As early as after 6h postincubation, the number ofmitochondria doubled but their surface declined by over two times. After 24 h of incubation, the surface of plastids increased over twice in spite of their unchanged number. In postincubation (6h), their number doubled and the surface declined dramatically. The ultrastructure of the mitochondria was changed by the disappearance of cristae and a lowered electron density of the matrix, suggestingthat one of the mechanisms could be connected with changes in mitochondrial function.

Sample preparation for taxol and cephalomannine determination in various organs of Taxus sp.

Solid-phase extraction and preparative thin-layer chromatography were applied as sample preparation techniques for the purification of crude extracts from twigs and needles of various Taxus species as well as for the isolation of taxol and cephalomannine for further reversed phase high performance liquid chromatography analysis. Significant differences in the contents of taxanes examined were found. The preparative chromatographic methods used were compared and evaluated as routine and reproducible procedures for the rapid isolation and determination of taxol and cephalomannine in plant extracts.

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography.

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii.

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1microM of l-phenylalanine (6.2gL(-1)). Addition of 1microM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7microg L(-1)), which was not detected in the untreated control culture. Supplementation with 100microM of l-phenylalanine together with 100microM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3microg L(-1) (control untreated culture) to 568.2microg L(-1), the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8microg L(-1)) was observed when the medium was supplemented with 100microM of the precursor in combination with methyl jasmonate. Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.

Regeneration of Plants by Embryogenesis with Callus Cultures of Carum carvi L.

This report describes the regeneration of plants from callus cultures of caraway, Carum carvi L. Callus of hypocotyl origin was maintained on medium containing Murashige and Skoog salts with Nitsch and Nitsch vitamins in the presence of 0.3 mg · l(-1) 2,4-D. Formation of somatic embryos was induced with suspension cultures once 2,4-D had been removed. Embryos developed into plantlets when subcultured on solid medium supplemented with 0.5 mg · l(-1) IBA and 10.0 mg · l(-1) adenine sulphate. Regenerated plantlets were transferred to soil.

Comparison of the Effects of Extracts of Zephyranthes Robusta Baker, Haemanthamine and Lycorine on Mitosis and DNA Synthesis in Allium Cepa Roots

SUMMARYThis paper shows that extracts from Zephyranthes robusta Baker and a constituent alkaloids haemanthamine and lycorine inhibited mitosis in Allium cepa root tips.The antimitotic activity of haemanthamine, lycorine and extract of Zephyranthes robusta is expressed by the calculation of MI50. This value shows the concentration of solution which inhibited the mitotic index by 50%. This value was compared to MI50 calculated for Proresid and Oncovin.The use of the DNA labelling technique showed that haemanthamine inhibited 3H- thymidine uptake in onion roots. The extract from Zephyranthes robusta has the same effect. Combined treatment of JH-thymidine + haemanthamine and 3H-thymidine + extracts from Zephyranthes robusta caused a decrease in the quantity of the labelled nuclei and in their radioactivity. Proresid did not show the same effect.