Hairy root cultures, establishment

In this article, we demonstrate the establishment of the adventitious and the hairy root cultures of several solanaceous plant species including Datura, Duboisia, Hyoscyamus, and Scopolia etc. for the production and biosynthetic studies of tropane alkaloids. In addition, the isolation and structural elucidation of the new tropane alkaloid 7P-hydroxyhyoscyamine and the piperidone alkaloid hyalbidone from them is also presented. 

In order to estimate the productivity of a large number of clones, it is desirable to develop a simple assay technique to measure trace amount of the compound in a small amount of sample. Radioimmunoassay (RIA) [118] and HPLC [119, 120] have been carried out for the quantitative analysis of cardenolides. However RIA cannot be used in a usual laboratory, and HPLC requires much time for analysis. Therefore, the ELISA for cardenolides was established to investigate the production of cardenolides by hairy root cultures of D. lanata [116, 117]. 

Hyalbidone (182) was isolated from hairy root cultures of Hyoscyamus albus which were transformed with Agrobacterium rhizogenes. Its structure was established using MS and one-and two-dimensional H and 13C NMR [463]. Feeding [1-13C, 2-2h]-acetate to the above cultures of Hyoscyamus albus gave little incorporation of label into 182, suggesting that 182 was either not derived from tropine, or was located near the end of the biosynthetic pathway [464],... 

With a few exceptions, the characteristic problem of cultivation of plant explants in in vitro cultures is a low production of secondary metabolites by these cultures. One of the methods which can achieve an increase in the production of natural substances in in vitro cultures, is elicitation of cell cultures with biotic elicitors. For example, hairy root cultures of Cassia obtusifolia L. clones transformed with Agrobacterium rhizogenes strain 9402 were established to investigate anthraquinone production. It was found that changes of the elements in the culture medium and the addition of rare earth element Eu3+ can greatly influence the contents of free anthraquinones in the hairy root [320],... 

Figure 3. Establishment of "hairy root" cultures. (A) Datura innoxia seeding 2 weeks after infection with A. rhizogenes. (B) Detail of Hyoscyamus muticus "hairy roots".

A simple modification introduced in a stirred tank bioreactor allowed us the establishment of B. Candida hairy root cultures for scopolamine and 6P-hydroxyhyoscyamine production with increased alkaloid concentration compared to the Erlenmeyer flask cultures [28], It is worth pointing out that these results are potentially applicable to perform the rational scale-up of the process [28]. 

Li W, Koike K, Asada Y, Yoshikawa T, Nikaido T (2005) Rosmarinic acid production by Coleus forskohlii hairy root cultures. Plant Cell Tissue Organ Cult 80 151 Grzegorczyk I, Krolicka A, Wysokinska H (2006) Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid. Z Naturforsch 61 351 Tan SC (2000) Determinants of eating quality in fruits and vegetables. Proc Nutr Soc Aust 24 183... 

Establishment of the hairy root culture 2 Selection of the highly productive clones 3 Optimization of the production conditions 4 Scaling-up to bioreactors... 

In the last decades, numerous biotechnological investigations have made possible the establishment of hairy root cultures from the plant species, previously described, in order to produce terpene/terpenoids of pharmaceutically great interest. 

Jaziri M, Shimomura K, Yoshimatsu K et al (1995) Establishment of normal and transformed root cultures of Artemisia annua L. for artemisinin production. J Plant Physiol 145 175-177 Liu CZ, Wang YC, Ouyang E, Ye HC, Li GE (1999) Improvement of artemisinin accumulation in hairy root culture of Artemisia annua L. by fungal elicitor. Bioprocess Eng 20 161-164... 

In addition, hairy root culture was attempted by two groups for paclitaxel production. One of the hairy root lines, established using seedlings of T. cuspidate (Korean yew), accumulated 52.5 mg 1 paclitaxel after 2 weeks in a 20-1 bioreactor operating under MeJA induction [73]. The other hairy root culture generated from the plantlets of TaxusX media (a hybrid species between T. cuspidate and T. baccata) accumulated 210 pgg DW paclitaxel following MeJA induction [74]. 

Numerous studies on the CPT production from C. acuminate, Ophiorrhiza pumila, or N. foetida callus cell suspension, and hairy root cultures have been reported. Generally, CPT levels in in vitro plant tissue cultures are 100- to 1000-fold lower than that in planta [106,107]. To date, the highest reported yield of CPT (1.3% DW) is from optimized callus cultures that were derived from cotyledons of N. foetida [108]. The highest titers of secreted CPT (0.035 mg ml ) and the analog 9-methoxycamptothecin (0.026 mg ml ) were observed in N. foetida cell suspension culture medium [109]. Hairy root cultures induced from three Ophiorrhiza species produced CPT, in which O. pumila hairy roots accumulated the highest specific yield of CPT (0.08% DW) [110]. As in cell suspension culture, the partially secreted CPT in hairy root culture facilitated product enrichment by adding an absorbent resin [111]. Later, O. pumila hairy root culture in a 3-1 bioreactor was established with approximately 7 mgl total CPT titer including 17% secreted CPT [112]. 

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