Kayo Yoshimatsu

Phenolic constituents in tissue cultures of Phyllanthus niruri

Abstract Adventitious root, hairy root, shoot and crown gall cultures of Phyllanthus niruri were established and the production of several phenolic compounds was analysed. From hairy roots cultured in 1/2 Murashige-Skoog liquid medium, six phenolic compounds; gallic acid, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin 3- O -gallate and (−)-epigallocatechin 3- O -gallate were isolated. The production of these six phenols and the related compound (+)-catechin in tissue cultures were determined by HPLC. Roots of the mother plant cultivated under hydroponic conditions contained almost the same phenolic compounds as those in root cultures. On the other hand, leaves and stems of the cultivated plant contained hydrolysable tannins such as geraniin, corilagin and galloylglucose which were similarly observed in the plant collected in Peru.

Traits of Panax ginseng hairy roots after cold storage and cryopreservation.

SummaryPanax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.

Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

SummaryTransformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 μg/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 μg morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 μg morphine equivalents/g fr. wt.) by ELISA.

Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli

Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l−1 from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.

Genetic transformation of foxglove (Digitalis purpurea) by chimeric foreign genes and production of cardioactive glycosides

The chimeric neo and gus genes on a mini Ti vector are efficiently transferred into the genome of fox glove (Digitalis purpurea L.) using a binary vector system based on a rootinducing Ri plasmid, pRi15834. The transgenic state of established transformed roots was confirmed by Southern blot analysis and by detection of agropine and mannopine. The expression of the chimeric genes controlled by the promoters from TR 1′–2′ genes, nos gene and cauliflower mosaic virus 35S RNA was demonstrated by enzymatic and histochemical assays of neomycin phosphotransferase II and ß-glucuronidase. Enzyme-linked immunosorbent assay (ELISA) was carried out using polyclonal antibody reactable against digitoxin to investigate the production of cardenolides. The results of ELISA indicated that the cardioactive glycosides were highly produced in the green transformed hairy roots.

Immunodetection of artemisinin in Artemisia annua cultivated in hydroponic conditions

Abstract A highly specific and sensitive ELISA method was developed for the detection and semi-quantitative determination of artemisinin and its structurally related compounds in crude extracts of Artemisia annua. The antibodies were raised in rabbits using a 10-succinyldihydroartemisinin-BSA conjugate as immunogen. The peroxide linkage in the artemisinin molecule was critical in determining the antibody specificity. The working range of the assay was from 0.02 to 10 ng per assay. The cross-reacting material in crude plant extracts was evaluated by chromatographic methods combined with the immunoassay method. The distribution of artemisinin equivalents in five-week-old A. annua plants cultivated in hydroponic conditions was investigated. The highest artemisinin equivalent content (1.12% dry wt) was found in the leaves of the upper parts of the plant.

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.

Plant regeneration on cultured root segments of Cephaelis ipecacuanha A. Richard

SummarySimple one step micropropagation system for Cephaelis ipecacuanha A. Richard was developed using root cultures grown in vitro. Adventitious shoots were directly formed on the cut end of root segments without callus formation, on phytohormone-free B5 solid medium in the dark. When the shoots attached with root segments were further cultured under 16 h light / 8 h dark, they developed into plantlets, which could be transplanted to soil. The regenerated plants grew well in a greenhouse with showing normal appearance and accumulated alkaloids. The influence of auxin on adventitious shoot formation was also investigated.

IAA derivative induced tropane alkaloid production in root cultures of a Duboisia hybrid.

Abstract Effects of the IAA derivatives, 4-chloro-IAA and 5,6-dichloro-IAA, on cultured roots were compared with those of IAA, NAA and 2,4-D in root cultures of a Duboisia myoporoides-D. leichhardtii hybrid. The roots grew well in MS liquid medium containing 0.5 mg l−1 IAA, 0.1 mg l−1 4-chloro-IAA, 0.01 mg l−1 5,6-dichloro-IAA or 0.5 mg l−1 NAA, and produced much more scopolamine (0.56–1.15 mg) than hyoscyamine (0.13–0.27 mg) in a 100 ml Erlenmeyer flask after four weeks of culture. Time course of growth of roots and alkaloid production in the presence of IAA derivatives were compared with those of IAA. Alkaloids were also detected in the medium, in particular, after nine weeks ca 50% of the total scopolamine was found in the cultured medium when 0.1 mg l−1 4-chloro-IAA or 0.01 mg l−1 5,6-dichloro-IAA was used.

Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard

Rapid shoot proliferation was established by adventitious shoot formation on internodal segments. Cross sections of the shoot initiation area were observed microscopically and adventitious shoots were studied under the scanning electron microscope. Shoots were directly formed on the epidermis of internodal segments in vitro without callusing, but not on that of nodal segments with axillary buds. The use of media containing 0.01 – 0.1 mg/l 6-benzyladenine or 0.1 mg /l kinetin and culture under 16 h light increased the number of shoots per segment. The shoots thus obtained were rooted on phytohormone-free Woody Plant or Gamborg B5 solid medium, and were then transferred to soil. When potted, these grew well in a greenhouse. The emetic alkaloid content of adventitious shoots and regenerated plants was determined by HPLC. In vitro shoots cultured in Woody Plant liquid medium supplemented with 0.01 – 0.1 mg/l 6-benzyladenine contained 0.04 – 0.07 % dry wt. emetine and 0.4 – 0.5 % dry wt. cephaeline. One-year old regenerated plants cultivated in a greenhouse demonstrated the same alkaloid content (roots contained 0.82 % dry wt. emetine and 2.16 % dry wt. cephaeline) as the parental plant.