Duboisia Hybrid Research Articles

Alkaloid production in Duboisia hybrid hairy roots and plants overexpressing the h6h gene

In the biosynthetic pathway leading to the tropane alkaloid scopolamine, which is the 6,7β-epoxide of hyoscyamine, the enzyme hyoscyamine 6-hydroxylase (H6H; EC 1.14.11.11) catalyses two consecutive steps, hydroxylation of hyoscyamine followed by epoxide formation, to produce scopolamine. The h6h gene encoding H6H from Hyoscyamus niger, placed under the regulation of the CaMV 35S promoter, was introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the Agrobacterium rhizogenes strain LBA9402. The presence and overexpression of the transgene in the resulting kanamycin-resistant hairy roots and the regenerated plants obtained from them were confirmed by polymerase chain reaction and Northern blot analysis, respectively. The scopolamine levels in the resulting engineered hairy root lines increased up to three times compared to wild-type hairy roots, but there was no clear increase in the engineered regenerated plants. The best engineered line (H6H-5) produced 74.50 mg/l scopolamine, which to our knowledge is the highest content ever reported for hairy roots of tropane-alkaloid-producing plants containing the 35S- h6h transgene.

Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2–4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.

Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites

Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 microM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d.

Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants

Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with A4 strain of Agrobacterium rhizogenes, induced transformed roots with the capacity to produce putrescine-derived alkaloids. In general, the hairy roots obtained showed two morphologies: typical hairy roots with high capacity to produce alkaloids and callus-like roots with faster growth capacity and lower alkaloid production. The aux1 gene of A. rhizogenes was detected by PCR analyses in all roots showing callus-like morphology. However, this gene was only detected in 25–60% of the root cultures established showing typical hairy morphology. This fact suggests a significant role of aux genes in the morphology of transformed roots. Inoculation of leaf sections with A. tumefaciens strain C58 C1 carrying the pRiA4TR- (deletion of aux genes) did not produce roots with callus-like morphology.

Intergeneric co-culture of genetically transformed organs for the production of scopolamine

This work demonstrates the effectiveness of interspecies and intergenus organ co-culture for the production of plant secondary metabolites. Co-cultures of Atropa belladonna transformed roots and Duboisia hybrid shooty teratomas produced significant levels of scopolamine, whereas no scopolamine could be detected in separate root and shoot cultures. Maximum scopolamine levels found in co-cultured shoots were 4.8 mg g −1 dry weight in a 2 × 2.5-1 dual bioreactor system, and 3.7 ± 0.9 mg g −1 in dual 1-1 shake flasks. Periodic crushing of the roots in shake flasks was effective in releasing hyoscyamine, but impaired growth and overall alkaloid synthesis. Less severe root damaging in the bioreactor was more successful in improving scopolamine accumulation. Uptake of hyoscyamine by shooty teratomas was inhibited at high hyoscyamine concentrations, was reduced in the presence of ATPase inhibitors, and could be significantly enhanced by pre-incubating the shoots on medium containing hyoscyamine.

Alkaloid metabolism in callus and regenerating shoot cultures of a Duboisia hybrid

Undifferentiated callus and regenerating shoot callus on Murashige and Skoog (MS) medium was unable to metabolize scopolamine to hyoscyamine. Shoots on MS medium supplemented with hyoscyamine on extraction afforded unchanged hyoscyamine (44%). However, 16% was converted to 6-hydroxyhyoscyamine and 12% to scopolamine. Metabolism appeared unaffected by the number of shoots formed. Callus only gave very low yields of these two metabolities. Control callus and shoots on MS medium without alkaloid contained traces of valtropine and/or butropine. However, both callus and shoots on MS medium supplemented with either scopolamine or hyoscyamine showed enhanced levels of these minor alkaloids at times by a factor exceeding 500.

Organogenesis and a general procedure for plant regeneration from callus culture of a commercial duboisia hybrid (D. leichhardtii x D. myoporoides)

The Duboisia hybrid (D. leichhardtii x D. myoporoides) was regenerated from callus derived from shoot tips and young seeds on Murashige and Skoog (MS) medium containing 54μM 1-naphthalenacetic acid (NAA) and 1μM 6-benzylaminopurine (BA). Callus derived from young seeds was superior to shoot tip callus. Buds were induced from callus on MS medium supplemented with 22μM BA. The buds formed shoots when transferred to MS medium with 5 μM BA and 0.5μM NAA. Shoots were induced to form roots when placed on MS medium containing 25 μM indolebutyric acid (IBA). Plantlets so formed were transplanted and subsequently planted out in the open. Approximately 6 months were required for the full regenerative process. Trees so formed have been grown for 3 years, reached a height of 3 metres and flowered and fruited normally.

Alkaloids and metabolism in a Duboisia hybrid

A Duboisia hybrid contained scopolamine, 6-hydroxyhyoscyamine and hyoscyamine as major alkaloids and similar minor alkaloids as the parent species, D. leichhardtii and D. myoporoides. Unlike D. myoporoides, there was almost complete absence of pyridine bases. Tritiated scopolamine was infiltrated into a 2-year-old, field grown tree via a wick method. The scopolamine administered was metabolised to aposcopolamine. Hyoscyamine, 6-hydroxy-hyoscyamine and other minor Duboisia alkaloids had specific activities that were too small for them to be direct metabolites of scopolamine. Activity was located in acidic fractions that were shown to contain aromatic protons and water soluble basic fractions and are presumed to be responsible for the activity of these other alkaloids. The metabolic process occurred within the bark; alkaloids already accumulated appeared to be removed from the metabolic process.

A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay

A hybridoma clone producing a monocional antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgG1 (k) antibody with high affinity (1.6 × 101 M −1 for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak cross-reactivity with 6β- and 7β-hydroxyhyoscyamine. The cross-reaction with l-hyoscyamine, atropine, scopine and dl-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.H81 was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pg assay −1 (0.2 ng ml −1) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid.

Seasonal Variation of Alkaloids in Thai GrownDuboisia myoporoidesandDuboisiaHybrid

Seasonal Variation of Alkaloids in Thai Grown<i>Duboisia myoporoides</i>and<i>Duboisia</i>Hybrid

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

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.

The isolation of littorine from a Duboisia hybrid.

The isolation of littorine from a Duboisia hybrid.

7β-Hydroxyhyoscyamine from Duboisia myoporoides-D. leichhardtii hybrid and Hyoscyamus albus

A new tropane alkaloid, 7β-hydroxyhyoscyamine was isolated, together with 6β-hydroxyhyoscyamine, hyoscyamine, norhyoscyamine, scopolamine and tropine, from the leaves of Duboisia hybrid M-II-8-6 (crossbred between D. myoporoides and D. leichhardtii) and the hairy roots of Hyoscyamus albus.

The Effect of a Seaweed Extract on the Alkaloid Variation in a Commercial Plantation of a Duboisia hybrid

Duboisia hybrid plants of D. myoporoides R.Br. and D. leichhardtii F. Muell. were sprayed with a dilute solution of Maxicrop®, a commercial aqueous seaweed extract, over a 15 month period. The control plants experienced the usual decline in hyoscine content between May and September. This typical decline in hyoscine did not occur in the treated plants.

Cultivation of a Duboisia Hybrid. Part A. Nutritional Requirements and Effects of Growth Regulators on Alkaloid Content

Cultivation of a Duboisia Hybrid. Part A. Nutritional Requirements and Effects of Growth Regulators on Alkaloid Content

Cultivation of a Duboisia Hybrid. Part B. Alkaloid Variation in a Commercial Plantation: Effects of Seasonal Change, Soil Fertility, and Cytokinins

Hybrid plants of Duboisia myoporoides R. Br. and D. leichhardtii F. Muell., grown in a commercial plantation, were monitored for the major alkaloids, hyoscine and hyoscyamine. In the early months after planting, the hyoscine and hyoscyamine percentages were of the same order of magnitude. Gradually hyoscine became the dominant alkaloid with a maximum of 2.2% in Spring, then decreasing with a minimum in late Autumn. Soil fertilization had no effect on alkaloid yield. After harvesting, the plants were monitored as before on regrowth material. A similar pattern was established in wdiich an increase in hyoscine was accompanied by a decrease in hyoscyamine and vice versa, thus supporting alkaloid interconversion. When plants were sprayed with a commercial seaweed extract, Maxicrop®, there -was an 18% increase in leaf yield and a 16% increase in hyoscine content as compared to that of the controls. There was no significant increase in total alkaloid content.

Organization and metabolism of exogenous hyoscyamine in tissue cultures of a Duboisia hybrid

Organization and metabolism of exogenous hyoscyamine in tissue cultures of a Duboisia hybrid