Adventitious roots production

Because of the obligate outcrossing nature of alfalfa, all experiments were carried out using vegetative cuttings of primary transformants. Nodal stem sections were placed in sterile, moist vermiculite for approx 14 d to stimulate adventitious root production, then transferred to a mix of sand and soil (1 1 [v/v]) in 3.8 x 21 cm plastic cone-tainers (Stuewe Sons, Corvallis, OR) and fertilized monthly with soluble 10 10 10 (N P K) fertilizer. 

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... 

Fig. 6. Alkaloid production in the hairy roots and the adventitious roots of H. albus after 4 weeks in WP liquid medium. Bars indicate the standard error.

We compared the time course of tropane alkaloid production in the adventitious and two types of hairy roots of H. albus, transformed with the different strains of A. rhizogenes [15]. In the adventitious roots of H. albus, hyoscyamine was the main alkaloid during the first 6 weeks of culture in phytohormone-free MS liquid medium. At the seventh week of culture, with the beginning of the stationary growth, scopolamine became the major constituent (Fig. 7). 

In the hairy roots, transformed with A. rhizogenes 15834, hyoscyamine was the main alkaloid only for the first two weeks of culture in phytohormone-free WP liquid medium. After that time scopolamine became the major alkaloid. The production of scopolamine increased rapidly and it became almost double the amount of hyoscyamine at the end of the culture period. The yield of 6P-hydroxyhyoscyamine and 7P-hydroxyhyoscyamine produced in these hairy roots was comparable to that in the adventitious roots (Fig.7). 

The influence of phytohormones and light on the production of tropane alkaloids in transformed roots of Hyoscyamus albus was investigated [25]. In adventitious roots cultured in the dark, the addition of lAA (up to 4 mg/1) to the culture medium only slightly affected alkaloid production (Fig. 8a). On the other hand, when the adventitious roots were cultured under light, the alkaloid content decreased with the addition of lAA. Any combination of lAA with kinetin rapidly... 

Fig. 2 Time course of tannin production of adventitious root cultures of Sanguisorba officinalis.

Effects of various media on growth and phenolic production in adventitious root cultures. 

The adventitious and hairy root cultures of S. officinalis seem to be useful not only for the production of high Mr hydrolyzable tannins such as 10 and 11 but also for biosynthetic studies. The phenolic content in the hairy roots was almost the same as that of the adventitious roots. Taking into account the rapid growth, the hairy root cultures of this plant are more valuable than its normal root cultures for the production of these phenolics. 

The cell suspension cultures of this plant also showed good growth and formation of 2 (maximum level, 0.38 % as dw). Although the contents of 2 in tissue culture cells (both adventitious root and cell suspension cultures) were comparatively lower than those in the plants of Rhubarb (R. palmatum-6.06 % as dw R. officinale-X. % % as dw) (78), taking into account the rapid growth of the root and cell cultures, these cultures seemed to be useful for the production of 2. 

From the above results, it was apparent that for the production of hydrolyzable tannins using such liquid cultures (hairy root, adventitious root and shoot) further investigations will be required. Concerning the production of flavan-3-ols contained in the roots of this plant, the hairy root culture was the most useful method of those tested. The constitution of the flavan-3-ols found in the plant and the hairy roots and shoot cultures of P. mruri arc very similar to that observed in leaves of Thea sinensis (green tea) (97). Cultures of P. mruri might also have the same pharmacological effects as those expected from tea leaves (eg. 98,99). 

Callus and adventitious roots of ipecac (Cephaelis ipecacuanha ) were induced on the leaf segments from axenic shoot culture and their capabilities for alkaloid production were investigated [15, 52]. 

The biosynthesis of the secondary metabolites seems to be related to the differentiation of the cells or plant organs such as shoots or roots [50, 51]. In the section 2, production of isoquinoline alkaloids and tropane alkaloids by adventitious root cultures is described. However, the cultivation of intact plants or their organs is in most cases either phytohormone-dependent or of poor growth, so that these systems are not very useful for industrial purposes [73]. 

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. 

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