Martina Sauerwein

Tropane alkaloids in the adventitious and hairy root cultures of solanaceous plants

Abstract Adventitious and hairy root cultures of several solanaceous plant species were established. The highest content of tropane alkaloids was obtained in the adventitious roots of Hyoscyamus albus cultured in hormone-free 1/2 MS liquid medium. On the other hand the hairy roots of H. albus transformed with A. rhizogenes strain MAFF 03-01724 grew very fast and yielded over 5 mg/100 ml flask of hyoscyamine within 22 days of culture. The alkaloid content in the hairy roots of H. niger was higher than that of its adventitious roots. 7β-Hydroxyhyoscyamine, recently isolated, was produced in the hairy roots of Scopolia tangutica and Datura innoxia, while it could not be detected in their adventitious root cultures.

Hernandulcin in hairy root cultures of Lippia dulcis.

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.

Alkaloid production in hairy roots of Hyoscyamus albus transformed with Agrobacterium rhizogenes

Abstract Hairy roots of Hyoscyamus albus were induced from the shoot cultures by transformation with A. rhizogenes strain MAFF 03-01724. The hairy root cultures were tested for growth and alkaloid production (5 tropane alkaloids simultaneously quantified by HPLC) in various media. Half-strength MS and Gamborg BS media containing 3% sucrose gave the highest alkaloid content (ca 2% dry wt), but only poor growth. On the other hand, fast growth was observed in Woody Plant medium containing 8% sucrose, while the production of tropane alkaloids was relatively low. The addition of KNO3 (1–50 mM) to Woody Plant medium (3% sucrose) enhanced the growth, and consequently the alkaloid yield in the hairy roots increased remarkably. The hairy roots cultured in the improved medium (Woody Plant medium containing 3% sucrose and 15 mM KNO3) for 28 days produced over 30 mg tropane alkaloids in one 100 ml flask (ca 0.8 mg 7β-hydroxyhyoscyamine, 1.7 mg littorine, 1.7 mg scopolamine, 7 mg 6β-hydroxyhyoscyamine and 19 mg hyoscyamine in 50 ml liquid medium).

Influence of Light and Phytohormones on Alkaloid Production in Transformed Root Cultures of Hyoscyamus albus

Summary In vitro cultures of Hyoscyamus albus were transformed with three different strains of Agrobacterium rhizogenes and subjected to various culture conditions and phytohormone concentrations. The production of 5 tropane alkaloids in the transformed as well as in the normal root cultures was examined by HPLC. Hyoscyamine was the main constituent produced by all of the different root cultures. The highest content of total tropane alkaloids was detected in hairy roots transformed with A. rhizogenes strain MAFF03-01724 cultured on WP solid medium containing 1 mg/L kinetin in combination with 1 mg/L IAA cultured in the dark. Littorine was especially abundant in these cultures. On the other hand the accumulation of the oxygenated alkaloids, such as 6β-hydroxyhyoscyamine, 7β-hydroxyhyoscyamine and scopolamine was enhanced in the roots cultured in the light. Depending on the strain of Agrobacterium rhizogenes used for transformation, hairy roots responded differently to exogenous phytohormones.

Indole alkaloids in hairy roots of Amsonia elliptica

Abstract Transformed root cultures of Amsonia elliptica were obtained after direct infection of the stem of the in vitro grown plants with Agrobacterium rhizogenes A4. The hairy roots cultured in Gamborg B5 liquid medium produced the same indole alkaloids as the normal roots, cultured in Gamborg B5 liquid medium containing 0.5 mg l−1 NAA, and the mother plant, but at a lower level. The addition of 0.5 mg l−1 NAA to the culture medium remarkably enhanced the growth, and alkaloid production, of the hairy roots.

A piperidone alkaloid from Hyoscyamus albus roots transformed with Agrobacterium rhizogenes

Abstract The new piperidone alkaloid hyalbidone [2α,2′β-(1,2-ethanediyl)bis-( N -methyl-3-piperidone)] has been isolated from the hairy roots of Hyoscyamus albus transformed with Agrobacterium rhizogenes strain MAFF 03-01724. The alkaloid was characterized by its chemical and physical data. Together with the piperidone, eight tropane alkaloids were isolated and identified from their NMR data in comparison with those of authentic samples.

Incorporation of 1-13C-acetate into tropane alkaloids by hairy root cultures of Hyoscyamus albus

Abstract Labelled acetate (C 2 H 3 13 COONa) was fed to fast-growing hairy roots of Hyoscyamus albus transformed with Agrobacterium rhizogenes MA

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.

On the role of opines in plants transformed with Agrobacterium rhizogenes: Tropane alkaloid metabolism, insect-toxicity and allelopathic properties

Summary The opines, mannopine and mikimopine, which are formed in roots after transformation with Agrobacterium rhizogenes 15834 and A. rhizogenes MAFF03-01724, were synthesized chemically and their influence on the alkaloid production in root cultures of Hyoscyamus albus was examined. In adventitious root cultures the addition of opines resulted in a rapid alkaloid degradation. Alkaloid content was restored to a «normal» level within 5 days. Alkaloid accumulation in hairy roots reacted differently: The addition of mikimopine enhanced alkaloid production whereas the addition of mannopine generally reduced the alkaloid content. Most of the different tropane alkaloids produced by the adventitious and the hairy root cultures were affected in the same way by the addition of the opines. Roots were able to take up exogenously supplied opines. The addition of opines to the culture medium replaced carbon or nitrogen sources of the media to some degree. Mikimopine and mannopine reduced growth of Manduca sexta larvae and had deterrent properties at higher concentrations. In addition, mikimopine showed allelopathic properties and retarded the germination of Lepidium sativum seeds and growth of seedlings. It is speculated that the opines serve both Agrobacteria and the infected plants, supposedly the latter in plant-plant and plant-insect interactions. Thus, Agrobacteria - plant relationships might be considered to be of rather symbiotic than pathogenic nature.

17α-O-Methylyohimbine and vallesiachotamine from roots ofAmsonia elliptica

Abstract The new yohimbane derivative 17α- O -methylyohimbine has been isolated from an extract of the roots of Amsonia elliptica and characterized from its spectral data. Vallesiachotamine, previously known as constituent of A. brevifolia , and pleiocarpamine have also been isolated and identified from their NMR data.