Alkaloids and neutral sapogenins in hairy roots and normal plant organs. a mg g-1 (Dry wt.).
1. Introduction
Another group of alkaloids has been detected in
2. Materials and methods
2.1. General experimental procedures
Plant materials were purchased from local nurseries (UK); salts for media preparation were obtained from Sigma (UK); PCR reagents were obtained from Perkin Elmer (UK); primers from VH Bio Ltd (UK); Perkin Elmer GeneAmp 480 thermal cycler (UK) was used for DNA amplification; Column chromatography was performed on silica gel (Merck, 70-230 mesh) and Sephadex LH-20 (Sigma, UK); TLC was performed on precoated TLC plates with silica gel 60 F254 (Merck, 0.25 mm, USA); solvents for chromatography were reagent grade; 1H and 13C-NMR spectra (400 and 100 MHz, respectively) were acquired on Bruker dpx 400 spectrometer using pyridine-d5 or D2O as solvents; samples for SEM were gold coated in Polaron E5350; SE micrographs were taken using a Joel JSM T220 scanning electron microscope; standard solasodine (Koch-Light laboratories, UK) and diosgenin (Sigma, UK).
2.2. Establishment of transformed root cultures
Transformed roots were obtained by infecting surface sterilised leaf and stem segments with
2.3. Confirmation of transformation by Polymerase Chain Reaction (PCR)
Genomic DNA was extracted from 100 mg (fresh wt.) of putative hairy roots and normal non-transformed roots (as controls) using commercially available plant DNA extraction kit (Nucleon-Phytopure, UK). These DNAs were used as templates for the reaction. PCR was performed with
2.4. Growth rate analysis
Growth rate analysis, for some root lines which showed good growth characteristics in liquid media, was determined by both dry weight and the dissimilation methods according to Schripsema et al., 1990.
2.5. Scanning electron microscopy
Fresh hairy root material was fixed with 1.5% glutaraldehyde (GA) in 0.05 M sodium cacodylate buffer, pH 7.0, for 45 min. After 1 - 2 min in vacuum (26 mm Hg, 3.46 kPa) the fixative was substituted by 3% GA in 0.1 M cacodylate buffer, pH 7.0 for 2 h. The material was then post-fixed in 1% aqueous solution of osmium tetroxide for 2 h. All treatments were carried out at room temperature. The fixed material was dehydrated in graded ethanol series, dried by the critical point drying method and sputter coated with gold before observation in the electron microscope (Ascenão et al., 1998).
2.6. Extraction and isolation of steroidal compounds
Fresh root material (400 g), grown in B5 liquid media and incubated in dark, was extracted with cold methanol (MeOH) overnight (1 l x 3). The combined methanolic extracts were concentrated under vacuum at 40 C, partitioned between distilled water and petroleum ether, CHCl3 and finally with
2.7. Determination of total steroids
2.7.1. Determination of steroidal bases
A spectrophotometric assay adopted from a method described by Briner, 1969 and modified by Crabbe & Fryer, 1982, was followed. The method depends on formation of a coloured complex of the steroidal bases with methyl orange, after acid hydrolysis of the glycosides in the extract and its spectrophotometric measurement at 425 nm. Determination of the concentration of test samples was achieved by constructing a calibration curve using standard solasodine base.
2.7.2. Determination of neutral sapogenins
A specific spectrophotometric analysis method for the determination of the total steroidal sapogenins, after acid hydrolysis, was followed. This method is based on chromophore formation with a reagent composed of anisaldehyde and sulphuric acid. The produced colour has one absorbance peak at 430 nm. The method is capable of determining different sapogenin types of different structures, irrespective of differences in stereochemistry at rings E and F, rings A/B conformation, presence or absence of unsaturation at C5-C6 or presence of keto or hydroxyl groups at C-3. Other compounds like sterols, triterpenoid sapogenins and/or sugars from glycosides, do not interfere with the determination. The reaction with the chromogen is believed to be restricted to rings E and F (Baccou et al., 1977). Determination of the concentration of test samples was achieved by constructing a calibration curve using standard diosgenin.
2.8. Investigation of calystegines content
2.8.1. Sample preparation
Oven dried samples (70 C), 10 g each, of roots grown under light and in dark were extracted with 50% aqueous methanol (100 ml x 3) for 12hr. The hydro-methanolic extract in each case was filtered and concentrated to a syrupy consistency under vacuum at 40C. The extracts were partially purified by ion exchange chromatography on a strong acidic resin, Dowex-50W in the H+ form. Each extract was loaded onto glass columns packed with the resin and unbound material (sugars and phenolic compounds) was washed through with distilled water. The bound fraction (basic compounds and calystegines) was eluted with 2M ammonium hydroxide solution. Ammonia was removed from the samples by evaporation under vacuum at 40C. Each sample was then processed for GC-MS analysis as follows.
2.8.2. Sample derivatisation for GC-MS
Small aliquots of the above-purified bound fraction (1ml each) were freeze-dried. The freeze-dried samples were silylated using 100µl mg-1 of Sigma-Sil A reagent (Sigma, UK), which consisted of a mixture of trimethylchlorosilane [(CH3)3 Si Cl], hexamethyldisilizane [(CH3)3 Si NH Si (CH3)3] and pyridine in the ratio of 1:3:9. Samples were mixed using a vortex mixer and then heated at 55C for 15 minutes. The reaction mixture was cooled down to room temperature for at least one hour and then centrifuged at 2 x 103 rcf for 20 minutes to remove any precipitate formed during the reaction. The supernatants were then transferred to GC vials and analysed by GC-MS as follows: a BPX5 25m capillary column, 0.22mm ID, film thickness 0.25m (SGE Ltd., UK) was used, helium at a pressure of 10psi. Temperature programme started with an isothermal hold at 180 C for 5 minutes, followed by a linear temperature rise to 300 C at 10 C/min. The final temperature was held for 10 minutes and the total length of the program was 27 minutes. Samples were introduced at 1.0 µl per injection. EI-MS of the column effluent was carried out on Perkin-Elmer Q-Mass 910 Benchtop Mass Spectrometer with quadruple mass filter system. The system was set to a constant temperature of 280C. The effluent from the gas chromatograph was transferred to the mass spectrometer via a temperature controlled line set at 250 C.
2.8.3. Large scale fractionation
The calystegine-containing fraction from 500g fresh hairy roots of
2.8.4. GC-MS data for calystegines
Calystegine A3: Rt (4.95 minutes), EI/MS (positive mode) at m/z (rel. intensity) 286 (8), 244 (20), 170 (12), 156 (100), 147 (9).
Calystegine A5: Rt (4.54 minutes), EI/MS (positive mode) at m/z (rel. intensity) 286 (15.4), 244 (17), 169 (15), 156 (100), 147 (17).
Calystegine B1: Rt (7.28 minutes), EI/MS (positive mode) at m/z (rel. intensity) 373 (19.5), 332 (29), 285 (6), 258 (23), 244 (100), 168 (17), 147 (26), 129 (63).
Calystegine B2: Rt (8.70 minutes), EI/MS (positive mode) at m/z (rel. intensity) 284 (11), 259 (14), 229 (8), 217 (100), 204 (8), 156 (20), 147 (21).
Calystegine B1 glucoside: Rt (14.65 minutes), EI/MS (positive mode) at m/z (rel. intensity) 430 (38), 316 (100), 287 (38), 217 (76), 205 (38), 156 (25), 147 (79).
Calystegine A3 (A5) glycoside: Rt (14.74 minutes), EI/MS (positive mode) at m/z (rel. intensity) 286 (100), 244 (6), 217 (12), 204 (12), 169 (8), 156 (65), 147 (19), 129 (8).
2.8.5. 13C data for Calystegine B2
(100 MHz, D2O and drops of acetone-D6 for signal calibration, values in ppm): 90.0 (C1), 74.5 (C2), 74.6 (C3), 77.3 (4), 55.5 (C5), 21.3 (C6), 28.4 (C7).
3. Results and discussion
3.1. Transformation
Secondary metabolites production, in general, needs a certain degree of tissue differentiation, something that is obviously lacking in dedifferentiated
Roots incubated under photoperiod showed greening and those incubated under continuous light regenerated small shoots within four weeks (Fig. 1, C). Roots grown in different liquid media, MS, B5 and SH (Fig.1, B) displayed typical properties of transformed roots, i.e. fast growth, high degree of branching, abundant root hairs and lack of positive geotropism. Insertion of the root inducing plasmid was confirmed by PCR using primers specific to
3.2. Growth characteristics of the cultures
Growth rate analysis of the transformed root cultures was performed using two methods. The first, depended on determination of the dry mass accumulation over a period of four weeks and the second, involved determination of the growth characteristics by the dissimilation method i.e. determination of the loss of carbon dioxide due to carbohydrate consumption. Growth rate determination by the dissimilation method was carried out in three different liquid media (MS, B5 and SH) either under dark or light conditions. None of the cultivation media was inhibitory to the growth of the transformed roots, but differences were observed in the growth rate among these media. Roots showed best growth characteristics in MS media incubated under light conditions and in Gamborg’s B5 media under dark conditions (Fig. 3, A & B). The cultures showed a very short lag phase of less than two days and continued an active exponential phase for 17 days before entering a stationary phase. Generally, roots grown under dark conditions grew more actively, except for roots grown in SH media (Fig. 3, A & B). Growth rate determination by dry mass accumulation method (Fig.4) showed a similar pattern of growth with a 45-fold increase in root mass per flask, with an inoculum size of 200 mg fresh roots. The results demonstrate the importance of selection of the hairy root clones and also the possibility to manipulate the cultures through media selection and incubation conditions. The different clones are derived from different transformation events as a result of the insertion of the T-region of the root inducing plasmid, which is known to be a random process. This variability makes the hairy root system as amenable and flexible to manipulation as the dedifferentiated cell cultures, but having the advantage of still being an organised system with certain degree of maturation that allows expression of the enzymatic systems leading to the different secondary metabolic pathways.
3.3. Scanning Electron Microscopy (SEM)
SEM of the transformed roots (Fig. 5) showed high degree of branching and large number of root hairs. Some of the hairs appeared as long unicellular tubes, while others like short papillae covering the majority of the roots surfaces, especially those growing near and out of the surface of the media. Cut surfaces of the roots, revealed the structure of young dicot root with abundant starch granules and microcrystals of calcium oxalates in the cortical parenchyma (not shown). These cell inclusions which are characteristic features of plants belonging to the family
3.4. Investigation of the produced secondary metabolites
3.4.1. Steroidal compounds
Chromatographic investigation (TLC) of the methanolic extracts of the transformed roots and different parts of the non-transformed plant (roots, aerial parts and fruits) showed almost similar pattern of secondary metabolites with one major alkaloidal spot and several minor ones. Analytical HPLC (data not shown) revealed much more complex profile of the hairy root extracts. No qualitative differences were observed for transformed roots grown in different liquid media or for roots grown in the dark and those grown in light, except for the formation of higher amounts of less polar compounds in the roots grown in dark (higher percentage of chloroform extractives, data not shown). Chromatographic separation of the
carbon at 100.8, correlated to a carbon resonance at 78.6 (C3). The downfield shift observed for C3 (about 8.0 ppm) from that published for free aglycones (70.5 - 71.3 ppm) confirmed glycosylation at this carbon (Toshinori et al., 1993; Ripperger, 1996). Mutually the proton at C3 of the steroid nucleus (multiplet at 3.90) correlated to the sugar anomeric carbon at 100.8. The proton and carbon shifts of the other two sugars (Tables 1 and 2) suggested that they are rhamnoses. Cross peaks in the HMBC spectrum between the anomeric protons of the two rhamnoses ( 6.40 and 5.86, br s) and carbon signals at 78.4 ppm and 79.1 confirmed that those sugars were attached to glucose through (1``2`) and (1``` 4`) linkages. Other important correlations in the sugar region were observed between the anomeric protons of rhamnoses and C5 and C3 of both sugars and also between protons at C6 of the same sugars and the corresponding C4 and 2
These results demonstrate the metabolic stability of the cultures, where preliminary investigations showed no major differences between hairy roots and the original plant. Other work on
A. | B. | C. |
Sample | Alkaloids | Sapogenins |
B5 media in dark | 4.29a | 2.52a |
B5 media under light | 0.60 | 1.24 |
SH media in dark | 2.90 | 1.19 |
SH media under light | 0.96 | 0.59 |
MS media in dark | 0.57 | 1.89 |
MS media under light | 0.52 | 1.50 |
Normal roots | 4.14 | 1.37 |
Aerial parts | 3.28 | 2.11 |
H | ppm | H | ppm |
1 | 1.01, | 24 | 1.57-1.59 a, |
2 | 1.86 a, 2.11 a | 25 | 1.89, |
3 | 3.90, | 26 | 2.83 a, |
4 | 2.71, | 27 | 0.81, |
5 | - | 1` | 4.94, |
6 | 5.34 | 2` | 4.37 a, |
7 | 1.46 a, | 3` | 4.33-4.39 a, |
8 | 1.52-1.54 a, | 4` | 4.33-4.39 a, |
9 | 0.92, | 5` | 3.66 a |
10 | - | 6` | 4.10, 4.23, |
11 | 1.46-1.47 a, | 1`` | 6.40 |
12 | 1.12, | 2`` | 4.69, |
13 | - | 3`` | 4.55, |
14 | 1.10 a, | 4`` | 4.33, |
15 | 1.56 a, | 5`` | 4.93-4.99 a, |
16 | 4.18 a, | 6`` | 1.78 |
17 | 1.82-1.84 a, | 1``` | 5.86 |
18 | 0.83 | 2``` | 4.84, |
19 | 1.06 | 3``` | 4.64, |
20 | 1.96-2.02 | 4``` | 4.39, |
21 | 1.10, | 5``` | 4.93-4.99 a, |
22 | - | 6``` | 1.64, |
23 | 1.75 a, |
3.4.2. Calystegines
A mixture of calystegines could be identified in hairy root cultures of
which is very close to the retention time of B1-glucoside (14.4 minutes), suggested that it might also be a glycoside. The mass fragmentation pattern (experimental, section 2.8.4) was typical of trihydroxylated nortropane derivatives (calystegine-A group). Hence, this compound is suggested to be calystegine A3 or/A5 glycoside. It would be impossible to distinguish A3 and A5 glycosides without purification and NMR analysis. However this A-glycoside would be a novel compound.
Other major unidentified compounds were also detected in the GC-MS trace of the bound fraction of the hairy root cultures. They are likely to be also novel polyhydroxy alkaloids according to their chromatographic behaviour on ion exchange resins and mass fragmentation patterns and are not accumulated in the whole plant (data not shown). It is worth noting that calystegines A3, A5, B1 and B2 have been identified before in non-transformed plants (Asano et al., 2001). The same research group also identified calystegine N1 that had not been detected in our root cultures. The glycosylated derivatives of calystegines B1 and A-type are reported here for the first time from transformed root cultures of
It is evident, from the data given above (experimental section 2.8.4), that the mass fragmentation pattern of the TMS derivatives of the different calystegines, mainly followed that described for the O-TMS derivatives (Molyneaux et al., 1996). Major fragment ions due to the loss of TMS-OH (-90 mu), were observed (ions at m/z: 286, for calystegines A3 and A5; 373, for calystegine B1 and 284, for calystegine B2).
In the case of A-type calystegines, the base peak was due to the 2-substituted pyrrolinium ion formed via cleavage of the six-member ring (fragment ion at m/z 156). For the B-type
calystegines, the base peak was at m/z 244, which was also due to the formation of the 2-substituted pyrrolinium ion in the case of calystegine B1, while in the case of calystegine B2 this fragment ion was detected at a lower abundance (see experimental, section 2.8.4). Calystegine B2 showed a base peak ion at m/z 217. This is still a common ion fragment for TMS derivatives of polyhydroxylated compounds and sugars (De Jongh et al., 1969). Other fragments, which are characteristic of sugars, were also observed at m/z 204,147 and 129. The structures of these fragments are illustrated in Fig. (8). Surprisingly, the mass fragmentation pattern of calystegine B1-glucoside seems to have mainly proceeded
4. Conclusion
Hairy root cultures could be successfully established for
References
- 1.
Alvarez M. Talou J. Paniego N. Giulietti A. 1994 Solasodine production in transformed organ cultures (roots and shoots) of Cav. Biotechnology Letters,16 393 396 - 2.
Argolo A. Charlwood B. Pletsch M. 2000 The regulation of solasodine production by transformed roots of Solanum aviculare. Planta Medica,66 448 451 - 3.
Asano N. Yokoyama K. Sakurai M. Ikeda K. Kizu H. Kato A. Arisawa M. Hoke D. Drager B. Watson A. A. Nash R. J. 2001 Dihydroxynortropane alkaloids from calystegine-producing plants. ,57 721 726 - 4.
Asano N. Kato A. Miauchi M. Kizu H. Tomimori T. matsui K. Nash R. Molyneux R. 1997 Specific -galactosidase inhibitors, N-metyl calystegines: structure-activity relationships of calystegines from . European Journal of Biochemistry,248 296 303 - 5.
Asano N. Nash R. J. Molyneaux R. Fleet G. 2000 Sugar-mimic glycosidase inhibitors: Natural occurrence, biological activity and prospects for therapeutic application. ,11 1645 1680 - 6.
Asano N. Oseki K. Tomioka A. Kizu H. Matsui K. 1994 N-containing sugars from and their glycosidase inhibitory activities. Carbohydrate Research,259 243 255 - 7.
Ascenão L. Figueiredo A. C. Barroso J. Pedro L. Schripsema J. Deans S. Scheffer J. 1998 : Morphology of the glandular trichomes, essential oil composition and its biological activity. International Journal of Plant Science,159 31 38 - 8.
Atta-ur-rahman N. choudhary M. 1993 Steroidal Alkaloids, In: , Alkaloids and Sulphur Compounds, Waterman, P. (ed.), Vol. VIII, chapter 13,473 510 Academic Press, London. - 9.
Baccou J. Lambert F. Sauvaire Y. 1977 A spectrophotometric method for determination of total sapogenins. ,102 458 465 - 10.
Briner J. 1969 Determination of total steroid bases in species. Journal of Pharmaceutical Science,58 258 259 - 11.
Chilton M. Tepfer D. Petit A. David C. Tempè J. 1982 A. inserts T-DNA in the genome of the host plant root cells. Nature,295 432 434 - 12.
Crabbe P. Fryer C. 1982 Evaluation of chemical analysis for the determination of solasodine in . Journal of Pharmaceutical Sciences,71 1356 1362 - 13.
De Jongh D. Radford T. Hribar J. Hanessian S. Bieber M. Dawson G. Sweeley C. 1969 Analysis of trimethylsilyl derivatives of carbohydrates by gas chromatography and mass spectrometry. ,91 1728 1740 - 14.
Delmotte F. Delay D. Cizeau J. Guerin B. Leple J. 1991 vir-inducing activities of glycosylated acetosyringone, acetovanillone, syringaldehyde and syringic acid derivatives. Phytochemistry,30 3549 3552 - 15.
Drewes F. Van Staden J. 1995 Initiation of and solasodine production in hairy root cultures of Scop. Plant Growth Regulation,17 27 31 - 16.
Ehmke A. Eilert E. 1993 (bittersweet): Accumulation of steroidal alkaloids in the plant and different in vitro systems, In: Biotechnology in Agriculture and Forestry 21, Medicinal and Aromatic Plants IV, Bajaj, Y. (ed.),339 352 Springer-Verlag, Berlin. - 17.
Gamborg O. miller R. Ojima K. 1968 Nutrient requirements of suspension cultures of soybean root cells. ,50 151 158 - 18.
Georgiev M. Pavlov A. Bley T. 2007 Hairy root type plant systems as sources of bioactive substances. Applied Microbiology and Biotechnology,74 1175 1185 - 19.
Ghyesen G. Herman L. Breyne P. Van Montagu M. Depicker A. 1989 as a tool for the genetic transformation of plants, In: Genetic transformation and expression,151 174 Intercept Ltd., England. - 20.
Tropane derivatives from Calystegia sepium. Phytochemistry,Goldman A. Milat M. Ducort L. Lallemand J. Maille M. Lepingle A. Charpin I. Tepfer D. 29 2125 2127 - 21.
Hamill J. Lidgett A. 1997 Hairy root cultures: Opportunities and key protocols for studies in metabolic engineering. In: , Doran, P. (ed.),1 29 Harwood Academic Publishers, Australia. - 22.
Hamill J. Rounsley S. Spencer A. Todd G. Rhodes M. J. C. 1991 The use of the polymerase chain reaction in plant transformation studies. ,10 221 224 - 23.
Hegnauer R. 1989 Solanaceae, In: , Vol. VI,403 452 Verlag, Switzerland. - 24.
Hooykaas P. Klapwijk M. Nuti M. Schilperoort P. Rorsch A. 1977 Transfer of the Ti plasmid to avirulent Agrobacteria and Rhizobium ex planta". Journal of General Microbiology,98 477 484 - 25.
Ikenaga T. Oyama T. Muranaka T. 1995 Growth and steroidal saponin production in hairy root cultures of . Plant Cell Reports,14 413 417 - 26.
Kittipongapatana N. Hock R. Porter J. 1998 Production of solasodine by hairy root cultures of Forst. Plant Cell, Tissue and Organ Culture,52 133 143 - 27.
Mathé J. R. Mathé I. Botz L. Koch L. 1986 Possibilities of alkaloids production in European temperate zone. Acta Horticultura,188 193 201 - 28.
Mathé J. R. Mathé I. 1979 Variation in Alkaloids in L., In: The Biology and Taxonomy of the Solanaceae, Hawkes, J., Lester, R. & Skelding, A. (eds),211 222 Academic press, London. - 29.
Metcalf C. Chalk L. 1957 Solanaceae, In: . Vol. II.,965 978 Clarendon Press, UK. - 30.
Molyneaux R. Nash R. Asano N. 1996 The chemistry and biological activity of calystegines and related nortropane alkaloids, In: : Chemical and Biological Perspectives, Pelletier, S. (ed.), volume II, chapter 4,303 343 Elsvier Science Ltd., UK. - 31.
Murashige T. Skoog F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. ,15 473 497 - 32.
Nash R. Rotschild M. Porter A. Watson A. Waigh R. Waterman P. 1993 Calystegines in and Datura species and the death’s head hawk-moth (Acherontia atropus). Phytochemistry,34 1281 1283 - 33.
Porter J. 1991 Host Range and implication of plant infection by . Critical Reviews in Plant Sciences,387 421 - 34.
Rhodes M. Robins R. Hamill J. Parr A. Walton N. 1987 Secondary product formation using -transformed “hairy root” cultures. IAPTC Newsletters,53 2 15 - 35.
Ripperger H. 1996 22 26 epiminocholestane alkaloids with unusual (20R) configurations from species. Phytochemistry, 41, 1629-1631. - 36.
Rönsch H. Schreiber K. 1966 Alakloide-LXXII. Uber 1- und -solamarin, zwei neue tomatidenol glycoside aus Solanum dulcamara L. Phytochemistry,5 1227 1233 - 37.
Sato Y. Miller H. Mosettig E. 1951 Degradation of solasodine. ,73 5009 5011 - 38.
Schenk R. Hildebrandt A. 1972 Medium and techniques for induction of monocotyledonous and dicotyledonous plant cell cultures. , 50,199 204 - 39.
Schimming T. Tofern B. Mann P. Richter A. Jennett-Siems K. Drager B. Asano N. Gupta M. Correra M. Eich E. 1998 Distribution and taxonomix significance of calystegines in the Convolvulaceae. ,49 1989 1995 - 40.
Schripsema J. Meijer A. Van Iren F. Hoopen H. Verpoorte R. 1990 Dissimilation curves as a simple method for the characterisation of growth of plant cell suspension cultures. ,22 55 64 - 41.
Stachel S. Messens E. Van Montagu M. Zambryski P. 1985 Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in tumefaciens. Nature,318 624 629 - 42.
Subroto M. Doran P. 1994 Production of steroidal alkaloids by hairy roots of and the effect of gibberelic acid. Plant Cell, Tissue and Organ Culture,38 93 102 - 43.
Tepfer D. Goldman A. Pamboukdjian N. Maille M. Lepingle A. Chevalier D. Denarie J. Rosenberg C. 1988 A plasmid of 41 encodes catabolism of two compounds from root exudates of Calystegia sepium. Journal of Bacteriology,170 1153 1161 - 44.
Toshinori N. Teruhiko Y. Junko O. Sadao S. 1993 Steroidal alkaloids from tomato stock. ,34 1153 1157 - 45.
Usubillaga A. Aziz I. Tettamanizi M. Waibel R. Achenbach H. 1997 Steroidal alkaloids from . Phytochemistry,44 537 543 - 46.
Watson A. Fleet G. Asano N. Molyneaux R. Nash R. 2001 Polyhydroxylated alkaloids, natural occurrence and therapeutic applications. ,56 265 295 - 47.
Willuhn G. 1966 Undersuchungenzur chemi-schen differenzierung bei S. L. I. Genetische fixierung der unter-schiedlichen steroidalkaloid fÜhrung. Planta Medica,14 408 420 - 48.
Yu S. Kwok K. Doran P. 1996 Effect of sucrose, exogenous product concentration and other culture conditions on growth and steroidal alkaloid production by hairy roots. Enzyme and Microbial Technology,18 238 243