Duboisia hybrid

Gritsanapan W and Griffin WJ (1992) Alkaloids and metabolism in a Duboisia hybrid. Phytochemistry 31, 471-477. 

The adventitious root cultures of Datura innoxia, Duboisia hybrid M-II-8-14 (a cross-bred between D. myoporoides and D. leichhardtii), and Scopolia tangutica were established from the axenic shoot cultures or intact plants (in the case of Duboisia) on MS solid medium containing 0.1 mg/1 NAA or 1.0 mg/1 lAA. The adventitious roots were maintained in MS liquid medium containing the same phytohormones (0.1 mg/1 NAA or 0.5 mg/1 lAA) in the dark. Addition of auxin in the culture medium has been employed for the maintenance of adventitious root cultures [14], however, the adventitious roots of H. albus and H. niger were induced and maintained in hormone-free 1/2 MS medium [15]. 

The adventitious roots of H. albus and H. niger showed the highest level of 6P- and 7p-hydroxyhyoscyamines. The amount of hyoscyamine in the adventitious roots of H. albus increased remarkably when the roots were cultured in MS or 1/2 MS medium and it became the main alkaloid. In contrast, scopolamine was the main constituent of the adventitious roots of H. albus in Gamborg B5 (B5) [16] and WP media. Thus, in Hyoscyamus species, the ratio of tropane alkaloids produced during the culture pteriod seemed to depend mainly on the culture conditions and not on the species, as described by Hashimoto et al. [17]. In adventitious roots of H. albus the production of 7P-hydroxyhyoscyamine was at its highest level when the roots were cultured in WP medium, whereas the adventitious roots of D. innoxia and S. tangutica did not produce any detectable level of 7P-hydroxyhyoscyamine. In the Duboisia hybrid a trace amount of 7p-hydroxyhyoscyamine was detected. In the adventitious roots of H. aureus, H. muticus and H. pusillus, traces of 7P-hydroxyhyoscyamine were detected (Table 4). 

Alkaloid production by adventitious root culture of Duboisia hybrid... 

Hairy roots of Datura innoxia, Duboisia hybrid M-II-8-6, Scopolia tangutica and the Hyoscyamus species were established by the co-culture method using their leaf discs with A. rhizogenes 15834. In addition, H. albus was transformed with A. rhizogenes strain MAFF 03-01724 [15]. 

The 6-hydroxyhyoscyamine which occurs in low concentration (0.005%) in Datura species is an intermediate in the in vivo conversion of hyoscyamine into hyoscine. This has now been isolated in a relatively high yield (0.5%) from a Duboisia hybrid. 

Likewise, the overexpression of H. niger h6h gene was carried out in Duboisia hybrid D. myoporoides x D. leichhardtii) hairy roots and regenerated plants derived from them. In this study, the best hairy root line produced 74.50 mg/L scopolamine, a 3-fold increment compared to wild-type lines, which paralleled the increase in h6h transcript levels, which confirmed the direct relationship between the expression level of h6h and scopolamine contents. Regarding the regenerated plants, there was no a clear scopolamine increase when compared to controls [173]. 

The effect of auxins on the growth and the alkaloid production in Duboisia hybrid root cultures... 

Fig. (37). Periodical growth and alkaloid contents in the root cultures of Duboisia hybrid cultured with 0.5 mg/lIAA(A),0.1 tng/1 4-Cl-IAA (B) and 0.01 mg/1 5,6-C12-IAA (C).

Although there are several reports on hairy root cultures of Duboisia hybrid [76, 78, 79], precise condition for hairy root induction such as bacteria density was not described. In our preliminary experiment, hairy roots of Duboisia hybrid with satisfactory growth could not be obtained from stem explants by the direct infection method previously reported [78]. Therefore, co-culture method was employed, and the influence of bacteria density and duration of co-culture on hairy root induction was investigated using leaf explants and A. rhizogenes ATCC 15834. 

Fig. (41). Effects of basal media on the growth and the alkaloid yield in the hairy roots of Duboisia hybrid. The hairy roots (ca. 30 mg fresh weight) were inoculated into various basal liquid medium and cultured at 25 °C in the dark for 5 weeks. Bars represent standard deviation of the mean, n=3. Numbers in brackets show growth index on a dry weight basis. Growth Index = final dry weight / initial dry weight...

The best alkaloid productivity (30 mg/1 scopolamine and 14 mg/1 hyoscyamine) was obtained in MS medium after 5 weeks of culture. In our previous study on production of tropane alkaloids by adventitious root cultures of the same Duboisia hybrid, 13mg/l scopolamine and 1.7 mg/1 hyoscyamine were obtained in MS medium with 0.5 mg/1 lAA after 5 weeks of culture [67, 68] (section 2.3.6). The A. rhizogenes transformation enhanced the tropane alkaloid productivity over twice the amount of scopolamine and 8 times of hyoscyamine when compared with the adventitious roots. 

Fig. (43). Periodical yields of alkaloids in hairy root cultures of Duboisia hybrid.

Fig. (45). Shoot regeneration on the adventitious and hairy root segments of Duboisia hybrid.

The isoquinoline alkaloid contents in the transformed and non-transformed plants are indicated in Table 16. Alkaloid contents in the leaves were higher than those in the roots despite transformation. Cephaeline was the major alkaloid in all parts of in vitro plants as well as the root cultures. Alkaloid contents in the transformed plants, both in the leaves and roots, were lower than those of non-transformed plants except emetine in the roots. Any advantageous effects of T-DNA integration into ipecac on the alkaloid accumulation in the regenerated plants were not observed as previously reported for Duboisia hybrid [79] and Hyoscyamus muticus [103]. 

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