Effects of elicitation and permeabilization on hyoscyamine content in Datura stramonium hairy roots.
Several alkaloids which are low synthesized in cell suspensions are accumulated at relatively high levels in root cultures . Currently, special attention is given to the production of secondary metabolites via Hairy Roots which derived from genetic transformation with Agrobacterium rhizogenes explants [12,18,10,24]. The hairy roots are characterized by rapid growth [16,23], a good genetic stability and increased yields in secondary metabolites [17,22].
In the present study we are assigned to optimize the production of hyoscyamine by applying many strategies in relation to the culture medium, elicitation with different agents and different doses and cellular permeabilization on the hairy roots of Datura stramonium L. Our goal is to test the effect of exogenous elicitors and cell permeabilization on biomass production and accumulation of hyoscyamine in hairy root of Datura sp.
Materials and Methods
Induction of hairy roots was made according to the protocol described by Amdoun et al. . A total of 5 lines of hairy roots have been studied. These lines were maintained in culture on B5 medium in which concentrations were reduced by half and supplemented with sucrose (+ 30 g/l) and agar (7g/l). They were then transplanted, using root fragments (3 to 4 cm), every 4-6 weeks on fresh similar medium.
Culture media, selection of efficient hairy root lines and kinetics of accumulation of HS
The establishment of hairy root culture was made in B5 (Gomborg, 1970) liquid medium. Five root sections of 4 to 5 cm were cultured together in 250 ml bottles containing 50 ml of medium. The bottles were then placed in an incubator. The S9 line was selected for its biomass growth and HS content. After selecting the S9, the growth kinetics and content of HS was studied to determine the best period for the elicitation and permeabilization treatments. The 14th day of culture was chosen for this purpose.
3. Elicitation and permeabilization:
Two elicitors have been used separately, salt stress at concentrations of 1, 3 and 5 g / l NaCl, and jasmonic acid (JA: 0, 10, 100, and 1000 [micro]M). The later was combined with Tween 20 (T20: 0 , 0.5, 1.5 and 3%).
All cultures were performed in the dark at a temperature of 26 [+ or -] 1[degrees]C. Cultures in liquid media were submitted to a rotary agitation at 100 rpm.
5. Determination of hyoscyamine content:
The extraction and determination of hyoscyamine content were made according to the protocol developed by Amdoun et al. .
Results and Discussion
1. Selection of the efficient hairy root line:
Among the five best lines obtained from HR, line S9 proved most effective for biomass and HS content. It was, therefore, selected for further work.
Kinetics of growth and accumulation of hyoscyamine in the RH in the S9:
The Figure 1 shows that the growth cycle is divided into three phases: 1 - lag phase corresponding to the first two days of culture, where a lack of biomass growth is recorded. The HS is detected only in trace forms (0.1 mg / g DM). 2 - Exponential phase corresponding to the period extending between the 5th and the 15th day of culture. The maxima are reached in the 15th day of incubation for biomass (0.26 g DW) and the 20th day for HS (8 mg / g DW). 3 - Phase of decline, it occurs after the 15th day for biomass and the 20th day for HS content. In view of these results the 14th day of culture was selected for the elicitation.
Elicitation by salt (NaCl):
Biomass and HS content were measured 24 hours after elicitation. The salt did not appear to have a significant effect on the biomass, but HS content was highly stimulated. With 1 or 2 g / l NaCl the HS content was three times higher than the control (respectively are: 3.54 and 4.53 mg / g DW). The concentration of 3 g / l caused the opposite effect (Figures 2A,2B).
Combined effect of elicitation and permeabilization:
The results show an unfavourable effect of Tween 20 on the biomass at all concentrations. T20 induced a decrease of around 25% of the dry weight compared to the control. The combination JA/T20 had a marked effect on the HS content. The best results were obtained combining all levels JA with T20 at concentrations of 0.5 and 1.5%. Beyond 1.5% of T20, the HS content decreased significantly. The most favourable combination is that which associate 10 [micro]M of JA and 1.5% of T20 (Figures 3A, 3B). The HRs in the presence of T20 were brown; this phenomenon indicates a beginning of necrosis (Figure4).
Variability in hairy root lines:
There are no major differences in terms of biomass between the studied hairy root lines. The same observation was made by Yu et al. , who did not observe significant differences between 7 hairy root lines of Solanum aviculare in terms of biomass and accumulation of steroids alkaloids. In the roots of whole plants hyoscyamine is the predominant alkaloid . It seems that the hairy roots cultured in vitro have a similar alkaloid biosynthetic profile to that of roots of plants from which they derive [20,22,21]. Our results corroborate those of the previous authors since we detected only hyoscyamine in all the root lines examined.
The kinetics of growth and accumulation of hyoscyamine in the S9 line follows the model described by Oksman-Caldentey et al.  for hairy roots of Hyoscyamus muticus, but with a slight lag time that could be due to the growing conditions, the characteristics of the inoculum, or the intrinsic properties of the plant material. Our results follow the model that growth and accumulation of secondary metabolites are parallel where the highest levels of HS content are achieved in stationary phase of growth . Maldonado-Mendoza et al.  also reported that the alkaloid content in hairy roots of D. stramonium is low during the exponential phase of growth, but reaches its maximum value during the stationary phase.
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Elicitation by salt stress:
According to the results obtained we noticed that moderate salt stress (1-2 g / l) caused a remarkable increase in the HS content in hairy roots. However, higher dose (3 g / l) induced the opposite effect on the HS content. Our findings corroborate those obtained by Brachet et al.  showing an increased HS rate at the uppermost leaves following application of salt stress on plants of D. innoxia. In addition, our results showed no adverse effect of salt on the biomass after 24 hours of application. This suggests that, the increase of HS content was due to a real stimulation of biosynthesis of this alkaloid rather than a simple concentration of the product in a reduced biomass .
Combination of JA and the T20:
In the present study, our results failed to indicate clearly the stimulatory effect of JA on HS accumulation. However, Amdoun et al. (in press), using mathematical modelling to determine the best pair of AJ concentration and exposure time (ET) of hairy roots of D. stramonium, were able to define precisely the dose and the optimal exposure time (AJ = 0.06 mM and TE = 24 hours) for an increase of about 182% in HS content.
Boitel-Conti et al.  reported that hairy root permeabilization depends on both the concentration and application time of the permeabilization agent; and the maximum alkaloids released into the culture medium (100% over control) was obtained after treatment of hairy roots by T20 (at 5%) for 30 hours of incubation.
Our results show that total content of hyoscyamine was the most important with all concentrations of JA combined with the T20 at concentrations of 0.5 and 1.5%. With these combinations, the percentage of HS content released into the medium culture varied between 73.77 and 92.30. However, the combination of JA (10 [micro]M) x T20 (1.5%) was most favorable for the total content of HS (HS in HR + HS release into the culture medium). These results corroborate those of Khelifi et al. .
This study has shown the diversity of behavior of different HR lines derived from genetic transformation of explants within the same species. Before any optimization work, it is necessary to select the most efficient lines for their growth as well as for their ability to produce the desired alkaloid. Indeed, this study showed that among the five lines produced, one of them (S9) was distinguished by the two criteria. It was selected for the study of the growth kinetics and HS content.
The results showed that the exponential growth phase for the biomass is between the 5th and 15th day of culture for the biomass, and between the 10th and 20th day for the biosynthesis of HS. This allowed to define the better time to apply elicitation in order to optimize the HS content. In the case of the S9 HR line, the 14th day of culture was selected for the elicitation and permeabilization.
The elicitation by different concentrations of NaCl showed various reactions of the selected HR line (S9). However, the concentration of 2g/l was shown most efficient for the accumulation of the HS in HRs. In addition, the combination of the JA and T20 also showed various reactions; and the best couple consisted in combining AJ (10 |M) with T20 (1.5%) to induce a good accumulation of HS in the HRs.
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Khelifi L., LRGB/Ecole Nationale Superieure Agronomique, Algiers, Algeria. EmailL: email@example.com Khelifi L., Zarouri B., Amdoun R., Harfi B., Morsli A;, and Khelifi-Slaoui M.
LRGB/Ecole Nationale Superieure Agronomique, Algiers, Algeria.
Khelifi L., Zarouri B., Amdoun R., Harfi B., Morsli A;, and Khelifi-Slaoui M.: Effects of Elicitation and Permeabilization on Hyoscyamine Content in Datura Stramonium Hairy Roots.
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|Title Annotation:||Original Article|
|Author:||Khelifi, L.; Zarouri, B.; Amdoun, R.; Harfi, B.; Morsli, A.; Khelifi-Slaoui, M.|
|Publication:||Advances in Environmental Biology|
|Date:||Jan 1, 2011|
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