Genetic Resources and Crop Evolution 50: 245–252, 2003.  2003 Kluwer Academic Publishers. Printed in the Netherlands. 245 Differential morphogenetic responses, ginsenoside metabolism and RAPD patterns of three Panax species A. Mathur 1 , A.K. Mathur 1, *, R.S. Sangwan 2 , A. Gangwar 1 and G.C. Uniyal 3 1 Division of Genetic Resources & Biotechnology, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India; 2 Division of Plant Physiology & Biochemistry, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India; 3 Division of Instrumentation, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India; * Author for correspondence (e-mail: cimap@ satyam.net.in; e-mail: cimap@ cimap.org; phone: 91 -522 -327212 / 91 -522 342676; fax: 91 -522 -342666) Received 7 August 2001; accepted in revised form 7 October 2001 Key words: Genetic diversity, Ginsenosides, Morphogenesis, Panax, RAPD analysis Abstract Genetic and metabolomic demarcations between two Indian and one American congeners of Genus Panax have been discerned. Genomic DNA was isolated from the root derived callus cultures of these species and amplified by AP-PCR. RAPD analyses of the DNA with six most responding arbitrary oligonucleotide decamers provided a total of 70 reproducible bands for computation of the similarity matrix amongst the Panax species. Only 18 of these were monomorphic giving an estimate of about 74% polymorphism among the test species examined. The similarity coefficient values based on the amplification pattern support an equidistant position of the three test species. The molecular demarcations between the species are also manifested in terms of their characteristic cultural requirements, in vitro growth kinetics, regeneration competence and ginsenoside complement of their calli. The Indian congeners i.e. P. sikkimensis and P. pseudoginseng were distinguishable by higher proportions of ginsenoside Rf and Ro (40% and 20%, respectively) in the crude saponin fractions. Furthermore P. quinquefolium calli mainly accumulated ginsenoside Rb 2 and Rg 1 , whilst P. sikkimensis callus was rich in Rd fraction. P. quinquefolium showed high similarity with P. sikkimensis with respect to plasticity and totipotency for somatic embryogeny whereas P. pseudoginseng callus was highly recalcitrant and lacked regenerative capacity. The chemical and genetic fingerprints alongwith morphogenetic responses of the three species under controlled in vitro environment strongly advance the case of P. sikkimensis as an independent species, rather than a conglomerate of location specific variety or sub-species of P. pseudoginseng. The findings are also of relevance to formulations and evaluation of ginseng-based health foods. Introduction Ginseng is the common name referred to species of the genus Panax (Araliaceae). Traditionally, ginseng has been valued as a potentiating oriental herbal medicine. It has gained tremendous global trade and recognition as health food supplement in the present day world (Choi 1988; Haster 1998; Haughton 1999). Dried root powder of this plant is widely used as a health tonic for anti-stress, anti-fatigue, aphrodisiac and anti-ageing properties (Choi 1988; Nocerino et al. 2000). Frequently the term ginseng is referred to a single species i.e., P. ginseng C.A. Meyer (Oriental or Korean ginseng), that is mainly cultivated in Korea, China and Japan. However, phytochemical analyses of some other species of the genus Panax have revealed them to synthesize the same class of biologically active saponins (ginsenosides) as synthesized by P. ginseng. These congener species include P. quinquefolium L. (American ginseng), P. pseudoginseng Wall. (Sanchi ginseng), P. japonicus C.A. Meyer and P. notoginseng (Burkill) F.H. Chen. These 246 species are also now being increasingly used as substitutes for traditional ginseng prescriptions (Ngan et al. 1999). The Indian species of Panax i.e. P. sikkimensis Ban. and P. pseudoginseng have also been shown to accumulate similar type of ginsenosides in their roots. Our group has also reported earlier the production of such ginsenosides even in callus cultures of these two Indian species (Mathur et al. 1999, 2000). But, the taxonomic demarcation between the two Indian species of the genus Panax is not adequately defined. Often these two species have been botanically referred as synonyms. Particularly, the ambiguity lies with respect to P. sikkimensis which has been stated by many workers as P. pseudoginseng var. angustifolius (Burkill) Li. (Hu 1976; Cannon 1979; Mehta and Haridasan 1992). On the other hand, Banerjee (1968), Bennet and Sharma (1983) have classified P. sikkimensis as a separate Panax species on the basis of its rhizome structure, leaf and fruit morphology. Interestingly, we have also observed that the two Indian congeners of Panax behave quite differently with respect to their in vitro growth requirements, biogenesis and accumulation pattern of ginsenoside factions in their callus cultures (Mathur et al. 1999, 2000, 2001). The present study was, therefore, undertaken to further discern the taxonomic identity and genetic demarcation between P. pseudoginseng and P. sikkimensis in terms of their relative AP-PCR amplification patterns, ginsenosides metabolism and morphogenetic behaviour of their calli. Panax quinquefolium was also included in this study to estimate the genetic distance and heterogeneity between Indian species of Panax and their American counterpart at the callus level of organisation. Materials and methods Establishment of callus cultures Callus cultures from root explants of P. quinquefolium, P. pseudoginseng and P. sikkimensis were raised as per our earlier protocol (Mathur et al. 1999). biomass (fresh weight) was calculated as percent increment over the initial inoculum and was represented as Growth Index (G.I.). For extracting crude ginsenosides, the freshly harvested callus tissue was extracted overnight with MeOH and the procedure was repeated 4 times. The extracts were pooled and concentrated to dryness at 60 8C. The residue was dissolved in 10 ml of H 2 O and extracted with diethyl ether at room temperature. The aqueous fraction was collected and further extracted with water saturated n-BuOH. Finally, the n-BuOH fraction was collected, concentrated on a flash evaporator and weighed to determine the crude ginsenoside content. The crude ginsenosides, thus obtained were compositionally analysed using TLC and HPLC as reported earlier (Mathur et al. 1994, 1999). DNA isolation from root callus cultures Genomic DNA from callus of all the three species was isolated by a modified CTAB procedure (Sangwan et al. 1998). Briefly 3.0 g callus was ground in liquid nitrogen, homogenised with 23CTAB buffer and extracted for 30 min at 65 C in a waterbath. The homogenate was added with one volume of CHCl 3 : Isoamyl alcohol (24:1) and aqueous phase was collected after centrifugation. The preparation was added with 1 / 10th volume of 10% CTAB buffer and reextracted with equal volume of CHCl 3 : Isoamyl alcohol (24:1) as above. The aqueous phase was collected and mixed with 1.5 volumes of CTAB precipitation buffer at room temperature and allowed to stand for 45 min. DNA precipitates collected by centrifugation were washed thrice with absolute ethanol. The DNA preparation was dried under vacuum, redissolved in sterile water, made RNA-free using RNAase A (10 mg / ml) treatment at 37 C for 1 h and deproteinised using CHCl 3 : Isoamyl alcohol. The DNA was electrophoressed on 0.8% agarose gel and checked for quality of the preparation (intactness and purity) by visualizing on a UV-transilluminator after staining with ethidium bromide. The genomic DNAs of the species, thus obtained were employed as template for polymerase chain reaction (PCR) amplification profiling. Growth and ginsenoside measurements Amplification conditions Fresh weight of the callus at various stages (5–50 days of culture) of growth was measured by weighing the freshly harvested tissue after carefully removing the adhered agar from the callus. The increase in The template genomic DNA was subjected to randomly primed-PCR (RAPD) analysis using ten-mer random primers essentially as reported previously Table 1. Differences in cultural behaviour and nutritional / hormonal requirements of callus cultures of three Panax species. Species Morphology of explant Source (roots) Medium for callus induction and maintenance Medium for SE induction Medium for SE maturnation Medium for plantlet conversion P. quinquefolium Fleshy thick, carrot like tap roots MS 1 2,4-D(0.5) MS 1 Kn(5.0) 1 NAA(0.25 1 charcoal (1%) 1 ]MS 2 P. pseudoginseng Tuberous, fasicled, fusiform, dark brown roots Globose, knotted with ring-like persistent scales MS* 1 2,4-D(1.0)** 1 Kn (0.25) (callus friable pale white) MS 1 2,4-D(1.0) 1 Kn(0.25) 1 CH (3 g/l) (Callus compact, light brown) MS 1 2,4-D (1.0) 1 Kn (0.25) (callus fragile, shiny with purple pigmentation) Not obtained; highly recalcitrant Not obtained; highly recalcitrant Not obtained; highly recalcitrant MS 1 2,4-D(0.5) MS 1 2,4-D(0.5) 1 ]MS 1 BAP (0.5) 1 GA 3 2 P. sikkimensis 1 BAP (0.5) 1 GA 3 (0.5) (0.5) 6 IBA (2.0) * Murashige and Skoog (1962) based medium modified afterMathur et al. (1994); ** All values in Parentheses are in mg / l 247 248 Table 2. Comparative growth kinetics and qualitative and quantitative metabolism of callus cultures of three Panax species. Species Culture age (wk) Growth Index (G.I.)* Crude Ginsenoside (% fresh weight) Ginsenoside Fractions (% of crude) Rb Rg Ro Rb:Rg P. pseudoginseng 25 35 45 25 35 45 25 35 45 44.0** 83.4 161.4 151.8 221.3 272.2 175.9 216.2 324.1 0.360 0.641 1.100 0.372 0.684 0.961 0.800 0.976 1.210 16.6 19.0 23.3 46.8 56.6 43.1 38.2 44.9 53.8 34.8 46.3 45.0 8.8 12.7 15.5 38.2 39.4 43.4 11.41 20.80 19.62 0.92 1.00 1.00 2.99 5.14 4.69 0.47 0.41 0.51 5.31 4.45 2.78 1.00 1.13 1.23 P. sikkimensis P. quinquefolium * Inoculum size 5 25% (10 g / 40 ml medium); ** Each value represents mean of 4 replicates (Sangwan et al. 1999). The reaction mixture (25 mL) consisted of Taq polymerase buffer (2.5 mL), MgCl 2 (1.0 mM), dNTPs (400 mM each), Taq DNA polymerase (0.25 units), primers (10 pmoles), template DNA (50 ng) and water to make the final volume. After gentle mixing the polymerase chain reaction assay mixture, amplification was carried out in a thermal cycler (Perkin Elmer, Model 2400) under following conditions: 1 cycle of 5 min. at 94 C, 1.5 min. at 35 C and 15 min. at 10 C; 40 cycles of 1.5 min. at 94 C, 1.5 min. at 35 C and 1 min. at 72 C. Finally extension was completed at 72 C for 5 min. The amplification products were separated electrophoretically on a 1.4% agarose in 1 3 TAE buffer as described earlier (Sangwan et al., 2000). The amplification profiles were visualized and image compared in NightHawk system equipped with a CCD-camera and online PC (pdi, USA) with Diversity Database Programme. The profiles were comparatively analysed using the diversity database software (pdi USA). Results Although in vitro morphogenetic response and media requirements of American ginseng (P. quinquefolium) were quite similar to those of P. sikkimensis, the other Indian species – P. pseudoginseng behaved entirely differently as summarised in Table 1. Besides, the calli of all the three species, exhibited substantial morphological differences in culture. The calli of P. quinquefolium were pale white and fragile in appearance, whilst P. sikkimensis callus was purple, granular and slimmy in appearance. The callus of P. pseudoginseng was very hard, compact and light brown in colour. The surface cells of P. sikkimensis callus also tended to turn violet-red and possessed increased intensity of pigmentation with culture age. In fact, in a separate study (data not presented) a high anthocyanin yielding cell line rich in ‘peonidin’ type of anthocyanin could be isolated from P. sikkimensis calli (Mathur et al, unpublished results). The smear preparations of root tips of seed-grown plants as well as actively growing calli of the three experimental Panax species, were examined cytologically for chromosome count at metaphase. The study indicated that the two Indian species had 2n 5 24 in comparison to 2n 5 4x 5 48 in the cultivated P. quinquefolium in roots of seed-grown plants as well as in calli derived from root explants of these three species. The callus lines of three Panax species also showed varying degrees of similarity and dissimilarity with respect to their growth kinetics (Table 2) and regeneration potential in vitro. The calli of P. quinquefolium were fastest growing, registering a growth index of 175.9 and 324.1 after 25 and 45 days of subculture, respectively. The corresponding G.I. values for P. sikkimensis callus were 151.8 and 272.2 during these periods of growth P. pseudoginseng cultures were very slow growing, exhibiting a growth index (G.I.) of only 161.4 after 45 days of growth cycle. P. quinquefolium and P. sikkimensis calli also showed a close similarity with respect to their organogenetic potential and plantlet regeneration pattern. Calli of both the species exhibited a high tendency to turn embryogenic on a 2,4-D containing medium, but had different hormonal requirements for subsequent maturation and hardening of the resultant somatic embryos (SE). While SE development from globular to heart-shape stage in P. quinquefolium was favoured by 5.0 mg / l Kn and 0.25 mg / l NAA in 1% (w / v) charcoal containing medium, the same was 249 Figure 1. Arbitrarily primed-polymerase chain reaction (AP-PCR) amplification profiles of genomic DNAs from three Panax species. The lanes in the gels (A-E), designated as 1, 2 & 3 respectively pertain to P. sikkimensis, P. quinquefolium and P. pseudoginseng. Also the lanes labelled as 4,5,6 in gel E correspond to these three species in the same order as above. The primer (10-mer) sequences generating the gel profiles are: gel A, 59 CTGATGCATC 39; gel B, 59 GTCCTACTCG 39; gel C, 59 AGGGGTCTTG3’; gel D, 59 GAAACGGGTG39; gel E, (lanes 1-3), 59 CGCTGTTACC39; gel E (lanes 4–6), 59 GACCGACACG39. Lane M in the gels provides molecular size scale (10 fragments ladder of size range from 1.00 Kb to 100 bp). facilitated by 0.5 mg / l 2,4-D alone in the MS medium in case of P. sikkimensis. Greening of cotyledons and development of shoots from SE in both the species, however occurred on a germination medium comprising half strength MS medium supplemented with 0.5 mg / l each of BAP and GA 3 . Rhizogenesis in embryoderived shoots was favoured by 2 mg / l IBA in liquid shake cultures in case of P. sikkimensis, where as in P. quinquefolium, the induced roots had a tendency to recallus again with numerous secondary embryo formation. On the contrary, P. pseudoginseng calli were highly recalcitrant and failed to respond towards any organogenetic pattern so far. When the three Panax species were compared for their triterpene glycosidal saponins (ginsenosides), it was observed that maximum crude ginsenoside content (0.8% f.wt.) after 25 days of growth was in P. quinquefolium calli followed by P. sikkimensis (0.68% f.wt.) and P. pseudoginseng (0.36% f.wt.). The crude ginsenoside content after 45 days of growth were, however comparable (Table 2). HPLC analysis of the crude fraction revealed that the three species significantly differed with respect to the compositional metabolism of their saponin pool. Broadly, the ginsenosides present in the Panax roots can be classified in two groups (Awang 2000). The ginsenosides of the RB group (i.e. Rb 1 , Rb 2 , R c , and Table 3. Similarity indices of three Panax species based on random prime polymerase chain reaction. Species P. sikkimensis P. quinquefolium P. pseudoginseng Similarity indices (S.I.) P. sikkimensis P. quinquefolium P. pseudoginseng 1.00 0.39 0.38 0.56 1.00 0.39 0.54 0.56 1.00 Similarity values based on Dice Coefficient and Jaccard’s coefficient are presented above and below the diagonal, respectively. Figure 2. UPGAMA based phylogenetic relationships between three Panax species deduced from Jaccard’s similarity matrix of molecular data of DNA polymorphism. 250 R d ) are CNS depressants, anti-stress, aptrodisiac and hypotensive in action. The Rg group of ginsenosides (Rg 1 , Rg 2 , Re and Rf) on the other hand are associated with CNS stimulatory, antifatique and hypertensive actoions of guiseup root (Dou et al. 1998; Awang 2000; Nocerino et al. 2000). In the present study, P. quinquefolium calli maintained almost constant equal proportion of Rb and Rg groups of ginsenoside (Rb:Rg 5 1.0–1.23) throughout the growth period with Rb 2 and Rg 1 as major constituents of the two groups. P. sikkimensis calli, on the contrary, were characterized by preponderance of Rb group ginsenosides (3–5 times more) compared to Rg group. Also, the bulk of Rb ginsenosides was accounted for by Rb 2 and Rd fractions and Rg ginsenosides were dominated by Rf fraction. The composition of crude ginsenoside pool in P. pseudoginseng was marked by 2.0–2.5 fold more accumulation of Rg than Rb group fractions. Callus of this Indian species of Panax was also characterized by very high accumulation of Rf (around 40% of the total crude ginsenosides) and Ro (20% of the crude) fractions of ginsenoside. Another major difference in the biogenetic pattern of different constituents of ginsenosides in Indian and American species was that Indian congeners had relatively very high amount (9–14% of crude) of Rd fraction which was present in very low level in callus of American ginseng (. 2% of the crude), Ginsenoside Rd is mainly responsible for stimulating the secretion of adrenocorticotropic hormone that accounts for the aphrodisiac action of Panax roots. The diversity of the three Panax species at the level of DNA polymorphism was also assessed through arbitrarily primed polymerase chain reaction. The amplification products from six most responding tenmer primers were employed in the molecular differentiation. A representative set of profiles of PCR products is shown in Figure 1. A total number of 70 reproducible band types were scored and analysed for computation of similarity matrix and genetic distance / relatedness (Table 3). Only 18 out of the 70 amplicons were monomorphic, giving an estimate of about 74% polymorphism amongst the species. The phylogenetic relationship based on Jaccard coefficients and UPGAMA method of clustering (Figure 2) revealed that the three species under reference are almost equidistant from each other. Discussion In India, ginseng is a rare and endangered Himalayan species distributed from Himachal Pradesh to Arunachal Pradesh and Sikkim (1000–2000 m altitude). The taxonomic and herbarium descriptions of Indian ginsengs have been very inadequate (Mehta and Haridasan 1992) and quite often, they are treated as a conglomerate of four Panax species namely P. pseudoginseng, P. sikkimensis, P. burkillianus and P. bipinnatifidus, referred collectively as P. pseudoginseng. Amongst these, the taxonomic status of P. sikkimensis has been most unclear and deserves more closer attention for its taxonomic authentication (Bennet and Sharma 1983). Traditionally, the identification of American and Oriental ginseng species is based on morphological, histological and organoleptic characteristics of their roots and / or rhizome (Shaw and But 1995). Recently the techniques of chemical and DNA fingerprinting have also been employed for this purpose (Wang et al. 1999; Cheung et al. 1994; Dou et al. 1998; Boehm et al. 1999). However, several practical limitations in applying these techniques to field grown tissues have been highlighted. These include differences arisng out of climatic changes, soil chemistry and age of the source plants (Thompson 1991; Dou et al. 1998; Boehm et al. 1999). The present study, therefore, was conducted to ascertain the chemotypic, morphogenetic and genetic demarcation between P. sikkimensis and P. pseudoginseng. The two Indian species of Panax were also compared with their American counterpart for these attributes. The tissues used for making these comparisons were in vitro grown root callus cultures, maintained in controlled identical growth conditions to avoid influence of environmental and / or ontogenic fluctuations. The results of this study clearly indicate that the three Panax species can be easily demarcated from each other by the nutritional requirements for their callus growth and regeneration patterns. P. sikkimensis showed more similarity with P. quinquefolium in terms of faster callus growth and high plantlet regeneration efficiency. P. pseudoginseng, not only required additional nutrient supplement in the form of caseine hydrolysate (3 g / l) for callus growth but, lacks regeneration potential also. In addition, both the Indian species are diploid with somatic chromosome count of 2n 5 24 in comparison to 2n 5 4x 5 48 in American Panax. which was earlier reported to be allotetraploid (Boehm et al. 1999). Our findings on chromosome counts in roots of seed-grown plants and calli derived from such root explants in P. quinquefolium and P. pseudoginseng, are in conformity with earlier observation of Walsh and McClelland (1990), Boehm et 251 al. (1999). However, this is the first report of chromosome count in P. sikkimensis. The chemical fingerprints of the ginsenosides produced and accumulated in the calli of the three experimental species of Panax clearly established the biogenetic dissimilarity between them. In brief, P. sikkimensis is characterised by high Rb:Rg ratio, high Rd fraction (12–15%) and a marked tendency to accumulate anthocyanin pigment. P. pseudoginseng can be demarcated by overproduction of Rf (38–40%) and Ro fractions (18–20%) in the saponin pool, whereas P. quinquefolium calli synthesised Rb 2 and Rg 1 fractions in very high but equal ratio (each about 40% of the crude each). Most importantly, the three species maintained their characteristic ginsenoside fingerprints in their samples that were grown in vitro for more than three years and were constantly analyzed through a 50 days culture cycle after every six months of culture age. This is relevant because earlier reports on identity and amount of various ginsenosides present in field grown leaves and roots of American and Oriental ginsengs and their products have been highly conflicting and variable (Lui and Staba 1980; Tanaka et al. 1986; Proctor and Bailey 1987; Ma et al. 1995; Awang 2000). The arbitrarily primed polymerase chain reaction (AP-PCR) is a powerful technique that has now frequently been used for genetic mapping, genomic fingerprinting and discerning phylogenetic relationships in a wide range of plant species (Walsh and McClelland 1990; Goodwin and Annis 1991; Pang et al. 1992). 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