ENHANCED CLONAL PROPAGATION AND ALKALOID BIOSYNTHESIS IN CULTURES OF RAUWOLFIA. Key Words: Rauwolfia serpentina, alkaloids, callus, embryoid, in vitro

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1 Pak. J. Pl. Sci. 13 (1): 5-56, 2007 ENHANCED CLONAL PROPAGATION AND ALKALOID BIOSYNTHESIS IN CULTURES OF RAUWOLFIA Ihsan Ilahi 1, Fazal Rahim 2 and Mussarat Jabeen 2 Department of Botany, Kohat University of Science & Technology, Kohat, Pakistan Department of Botany, University of Peshawar, Pakistan Abstract Stem nodal explants of Rauwolfia serpentina were inoculated on MS medium supplemented with 0.5 or 1.0 mg/l of BAP and 0.5 or 1.0 mg/l of 2,-D for axillary shoot regeneration. 3-5 axillary shoots developed on the dormant bud. Culture of the nodal segments on MS containing 2.0 mg/l of BAP +1.0 mg/l of 2,-D or 2.0 mg/l of BAP +1.0 mg/l of IAA, resulted into copious callus induction along with meristemoid structures. Shoots regenerated from these meristemoids on sub culturing to MS supplemented with 1.0 mg/l BAP +0.5 mg/l IAA +5.0 mg/l AS or 1.0 mg/l BAP +0.5 mg/l NAA +5.0 mg/l AS Shoots regenerating frequency increased on MS containing 1.0 mg/l BAP +5, 10 or 15 mg/l AS Embryoid like structures were formed on the callus when sub cultured on MS containing 1.0 mg/l each of BAP and Kn. Rooting of the young shoots was obtained with 1.0, 2.0 or.0 mg/l of IBA. Further, the calli were analyzed for indole alkaloid production. All the major indole alkaloids were detected in appreciable quantities. Key Words: Rauwolfia serpentina, alkaloids, callus, embryoid, in vitro Introduction Rauwolfia serpentina Benth. has attracted worldwide attention and used for centuries in the Ayurvedic and Unani systems of medicines in South Asia. The drug is collected from wild plants. The cultivation of Rauwolfia plant is very difficult for various reasons, one being formation of a large proportion of non-viable seeds (Mitra, 1976). In Pakistan the plants are raised in nurseries, which are devoid of the specific environmental requirement, thus they do not contain the important alkaloid reserpin (Ilahi & Akram, 1987). The recent and rapid advances in the development of plant tissue culture techniques have allowed large scale cultivation of plant tissues and exploration of their biosynthetic capabilities. For example, a number of aromatic, pharmaceutical, industrial, food etc. products have been obtained as a result of cell and tissue culture in a number of species (Pareek, 2006). Presently shikonin production is being undertaken by Metsui Petrochemicals in Japan utilizing Lithospermum erythrorhizon cell cultures (Staniforth et al., 200). In vitro propagation of Rauwolfia serpentina was reported by various workers (Ilahi & Akram, 1985 A & B; Ilahi & Akram, 1986; Ilahi, 1995; Ilahi et al. 1995). The crude drug named ajmaline, which contains extracts of Rauwolfia plant material, has been marketed with good results in the homeopathic system of medicine. Since then efforts are made to naturalize this plant in our local environment, so that we may fulfill Rauwolfia drugs demand from the local market. Thus the present studies were initiated with two objectives viz. one for the development of protocol for rapid and large scale multiplication of the plant material, so that it could substitute the cultivation by seed which is

2 6 Clonal propagation of Rauwolfia Ihsan Ilahi et al. not a sure method due to production of non-viable seeds in the nature. The second aim was to raise cultures for production and isolation of important alkaloids. Secondary metabolites production viz. alkaloids, isoprenoids, plant phenolics synthesis and isolation have been reported in cultures of various plants (Butcher, 1977; Staba, 1980). Materials and Methods Stem segments of Rauwolfia serpentina were obtained from the Medicinal Plant Garden of the Department of Botany, University of Peshawar. The explants were thoroughly washed with tap water and then immersed in 1% solution of mercuric chloride for three minutes. They were then rinsed thoroughly with sterile distilled water before inoculation. Murashige & Skoog (MS) medium (1962) containing 5% sucrose was used as a basal culture medium for stem explants. The MS medium was suitably supplemented with various levels of auxins and cytokinins (detailed in the subsequent text). The medium was solidified with agar at 0.9%. The medium was autoclaved at 15 lb/sq inch pressure for 15 minutes at C. The cultures were maintained at C under 16hour photoperiod provided by fluorescent light. Procedure for chemical analysis followed was that of WAGNER et al. (198). One gm of the freeze dried material of callus, shoot or root was grinded with 10% NH 3 solution for about one hour. Then mixed with methanol and left over night. It was filtered and the residue was again extracted 2 times with methanol. The methanol phase was evaporated under vacuum and the residue applied to thin layer plates coated with Merck silica gel 60 F 256 of 0.5 mm thickness. The plates were developed in AL-1 solvent system viz. Toluene: ethylacetate: diethylamine (70:20: 0) and observed under UV light (25 and 360 nm). The bands were compared with the references. Some of the plates were sprayed with Dragendroff reagent for development of specific colours. The bands were also eluted for spectrophotometric studies. Results A) Propagation of plants from axillary buds. The propagation of plants from axillary buds has proved to be the most generally applicable and reliable method of in vitro propagation in Rauwolfia serpentina, as the regeneration of the plant is very difficult from seeds and other sources. The seeds are mostly non-viable due to abortive embryos (Mitra, 1976; Akram & Ilahi, 1986). To achieve in vitro propagation, nodal segments were inoculated from actively growing shoots of field grown plants and cultured on MS (Murashige & Skoog) medium containing 0.5 mg/l each of BAP(Benzyl Amino Purine) and 2,-D (2,-Dichlorophenoxy Acetic Acid). After four weeks of culture period, the dormant axillary buds sprouted and developed into 3- vigorously growing shoots. These shoots attained a length approximately -6 cm and developed nodes after about 3 weeks. The development of 3- shoots from a single node indicated that the original bud might have been induced to divide and produce more than one shoot under the influence of these growth hormones. When the concentration of both 2,-D and BAP was raised to a level of 1.0 mg/l each, this time only a single shoot developed from the bud after four weeks. The suppression of axillary bud sprouting could be due to higher concentration of 2, -D.

3 Clonal propagation of Rauwolfia Ihsan Ilahi et al. 7 To enhance the sprouting of axillary buds, BAP and NAA (Naphthalene Acetic Acid) were supplemented at a level of 0.5 mg/l each. This time too, 3- axillary shoots sprouted from the bud after four weeks. These axillary shoots were as strong as those developed under the influence of BAP and 2,-D. In addition some whitish green callus got induced at the cut ends of the explants (Table 1). For further proliferation of the above axillary shoots, MS medium was supplemented with a higher concentration of BAP i.e. 1.0 mg/l, while NAA was supplied at a level of 0.5 mg/l. After about four weeks, a cluster of multiple shoots got regenerated on the explant (Table 1: Fig. 1). At this combination of BAP 2 and NAA, the number of shoots formed was greater than those of BAP and 2,-D. Hence this combination proved to be ideal for multiple shoot production from nodal explants. The plantlets could be induced to root as described in section E. B) Callogenesis It is evident from the literature that callus tissues are usually initiated from organ explant under the influence of growth regulators. As detailed in Materials and Methods section, the explants were inoculated on MS medium containing 5% sucrose. After a culture period of -6 weeks no activity like callus or shoot formation was noticed in the explants. As no callus could be induced on the plain basal medium, therefore, in the present study nodal segments were subjected to various concentrations of growth regulators for callus induction and its further growth. Aseptic stem nodal explants of Rauwolfia serpentina were inoculated onto MS medium supplemented with various combinations of phytohormones for callus induction (Table 1). Three to four vigorously growing shoots formed on the explant. However, there was no callus formation. Hence the concentration of phytohormones was raised to 1.0 mg/l each of BAP and 2,-D for callus induction. After a culture period of six weeks, a copious cream coloured shiny callus got induced on the explant. The rate of callus formation was slow in the first two weeks. However, the callus exhibited good growth and covered the entire explant within four weeks. Shoot primordia also got developed on the callus, which revealed its organogenic nature (Table 1). To sustain the continuous growth of this callus, further subcultures were made onto MS containing 2.0 mg/l of BAP and 1.0 mg/l of 2,-D. After six weeks of culture, an excellent loose textured callus got developed on the pre-existing callus. The major portion of the callus was creamy white, while some portion of the callus showed signs of browning, which may be due to longer culture period (Table 1). No shoot formation occurred in this combination of the growth regulators used. A combination of BAP and IAA (Indole Acetic Acid) were also tried for callus induction. A slight greenish white callus was obtained from the stem cuttings on MS medium supplemented with 0.5 mg/l each of BAP and IAA (Table 1). This callus was isolated from the explants and subcultured onto fresh MS medium in which BAP and IAA were supplied at a level of 1.0 mg/l each. A slight increase in the callus mass was observed. However, when BAP and IAA were supplied at a concentration of 2.0 mg/l and 1.0 mg/l respectively, enhancement in the callus growth was noticed. After a culture period of four weeks, the callus exhibited green colour and compact texture. Nonetheless, meristemoid-like structures were also developed on the callus which indicated its organogenic nature (Table 1: Fig.2). The callus so obtained was further subcultured to shoot differentiating media for regeneration (shoot regeneration section).

4 8 Clonal propagation of Rauwolfia Ihsan Ilahi et al. C) Shoot regeneration on callus A variety of hormonal combinations were tried to induce plantlet regeneration in the callus obtained on MS medium containing BAP + IAA and BAP +2,-D. When the basal medium was supplemented with 1.0 mg/l BAP in combination with 0.5 mg/l IAA and 5.0 mg/l AS, copious callus proliferation occurred within two passages (each passage of four weeks). At the third subculture, adventitious buds got initiated from the callus (Table 2). The frequency of shoot regeneration was enhanced when BM was supplemented with 1.0 mg/l BAP and 5.0 mg/l of AS (Adenine Sulphate). After about four weeks of culture, 0-50 adventitious shoots were obtained from the callus. Nonetheless, the organogenic callus also exhibited good growth compared with the callus developed on MS supplemented with BAP and IAA or 2,-D but no AS. With a view to promote further shoot proliferation, the basal medium was incorporated with a higher concentration of AS i.e. 10 mg/l, while the concentration of BAP was maintained at the same level i.e. 1.0 mg/l.this combination of BAP and AS did not exhibit a good effect on callus growth. However, the frequency of adventitious shoot formation got increased and an average of 50 shoots per callus mass were obtained (Table 2; Fig. 3). A tremendous increase in shoot regeneration was noticed when the concentration of AS was further increased to a level of 15 mg/l in addition with 1.0 mg/l of BAP. Approximately shoots per culture were obtained (Table 2). Further sub culturing of this organogenic callus on the same medium retained its active growth and shoot regenerating capability for an indefinite period. When the calli induced under the influence of BAP and 2,-D were transferred onto MS containing 1.0 mg/l BAP in combination with 0.5 mg/l NAA and 5.0 mg/l of AS, about 0 adventitious shoots were observed on the callus after four weeks (Table 2). With an increase in NAA level to 1.0 mg/l, there was a decrease in the shoot forming capacity of the callus and an average shoots per callus were noticed after four weeks. However, good callus growth was obtained (Table 2). Prolonged culture, nonetheless, resulted in browning of the callus. It could be concluded that a higher concentration of cytokinins (BAP and AS) and a lower concentration of auxins (IAA and NAA) help in shoot regeneration in organogenic callus. It is evident from the experiment that BAP at 1.0 mg/l in combination with AS at 10.0 or 15.0 mg/l gave the highest frequency of shoot regeneration. C) Regeneration through embryoids The callus previously formed on BAP and IAA was sub cultured on MS medium containing 1.0 mg/l BAP and 1.0 mg/l of Kn. A reasonable amount of callus was induced after four weeks. Furthermore, various kinds of structures were also discernable on this callus. Examination of these structures revealed their embryoid nature. Some of these structures were rod shaped, thus probably resembling the torpedo stage. The others were of a heart shaped and resembling another developmental stage of the embryos (Table 2). The heart and rod shaped embryoid like structures were isolated along with a small piece of callus and inoculated to a freshly prepared medium containing 0.5 mg/l each of IAA and Kn (6-furfurylamino purine). After a culture period of six weeks, roots and shoots got initiated on the embryoids, which then got transformed into complete plantlets (Table 2; Fig.

5 Clonal propagation of Rauwolfia Ihsan Ilahi et al. 9 ). The resultant plantlets were transplanted to plastic vials containing vermiculite and allowed to continue development in the green house. D) Rooting of young shoots In vitro rooting of microshoots excised from proliferating cultures depends on the species used, on media composition and of the concentration of the auxin used. In our experimental studies the rooting of Rauwolfia serpentina microshoots were carried out on a series of full strength MS medium supplemented with 0.5, 1.0 or 2.0 mg/l of IBA(Indole Butyric Acid). After one week, these shoots were consequently transferred to plain MS medium. Roots got initiated on these shoots at all concentrations after a culture period of three weeks. Fifty percent of the plantlets were rooted when MS was supplied with 2.0 mg/l of IBA. At a concentration of 1.0 and 0.5 mg/l, rooting percentage declined to 20 and 10% respectively (Table 2). To see the effect of higher concentration of auxin (IBA), MS medium was supplied with.0 mg/l of IBA. After four days, these shoots were transferred to plain MS medium. Resultantly root formation was observed after three weeks of culture. However, only 30% of the shoots initiated roots on this treatment (Tab.2). It could be concluded from the results that at low as well as higher concentrations of IBA the percentage of root induction was low, while at a median concentration the rooting percentage was highest i.e. about 50%. F) Chemical analysis An extensive range of secondary metabolites that includes alkaloids, isoprenoids, phenolics etc. have been isolated from cell and tissue cultures. Similarly, during the present studies with the help of chromatography and photospectrometry indole alkaloids were detected in Rauwolfia cultures. The material used was in vitro raised shoots, roots and callus. As evident from Table 3, all major alkaloids were detected. The regenerated shoots exhibited three main alkaloids viz. yohimbine (0.0183%), ajmaline (0.0763%), and ajmalicine (0.079%). Reserpine was also present in a small quantity ( %). Traces of renoxydine were also detected. Callus growing on MS +Kn +2,-D indicated the presence of four major alkaloids in higher concentration than the regenerated shoots (Table 3). In addition two other indole alkaloids viz. renoxydine and reserpigine were also detected in the callus masses. Similarly roots regenerated on MS + Kn +2,-D +TPN exhibited highest quantity of Yohimbine (0.170%) and serpentine (0.1012%) followed by reserpine (0.099%), ajmaline (0.0678%) and ajmalicine (0.095%). Two other alkaloids viz. reserpiline and renoxydine were also detected in small quantities. During the present studies highest percentage of reserpine, ajmaline and ajmalicine was detected in callus tissues. Serpentine was absent in regenerated shoots and callus and isolated only from regenerated roots. This could be probably due to synthesis of indole alkaloid precursor in the root tissue.

6 50 Clonal propagation of Rauwolfia Ihsan Ilahi et al. Fig: 1. Formation of multiple shoots on nodal explants of Rauwolfia serpentina when cultured on MS medium supplemented with BAP (1.0) and NAA (0.5 mg/i). The cultures were raised in cooled incubators at 25 ± 0 C with 16/8 hrs light/dark cycle provided by fluorescent tubes. Fig: 2. Organogenic callus and embryoids formation in R. serpentina cultured on MS fortified with BAP and 1AA at 2.0 and 1.0 mg/l respectively.

7 Clonal propagation of Rauwolfia Ihsan Ilahi et al. 51 Fig: 3 Increase in adventitious shoot formation under the influence of BAP and AS. Other cultural conditions were the same in Fig: 1 Fig: Induction of roots on regenerated shoots (See text for details)

8 52 Clonal propagation of Rauwolfia Ihsan Ilahi et al. Discussion The in vitro multiplication of Rauwolfia serpentina shoots through the sprouting of axillary buds is the most commercially viable means of micro-propagation. It is also useful for increasing number of shoots, which originally differentiated in vitro. The auxin 2,-D and cytokinin BAP were supplemented to basal MS medium. Best growth of axillary shoots was obtained on MS containing 0.5 mg/l each of BAP and 2,-D. Three to four vigorously growing shoots were developed on the explant, which was due to the multiplication of the original bud. However, at higher concentration of 2,-D (1.0 mg/l) along with 1.0 mg/l BAP, bud multiplication frequency got reduced and only a single shoot was developed. A cluster of multiple shoots was also developed on the nodal explants under the influence of 1.0 mg/l BAP and 0.5 mg/l NAA. The number of shoots formed on this combination of phyto-hormones was greater than that of BAP and 2, -D. Similarly, Mathur et al. (1993) induced multiple shoots on nodal segments from in vitro shoots of Rauwolfia serpentina, cultured on MS medium containing 1.0 mg/l BAP and 0.1 mg/l NAA. Induction of callus on stem explant took place on MS medium supplemented with 1.0 or 2.0 mg/l BAP +1.0 mg/l of 2,-D or 2.0 mg/l BAP +1.0 mg/l IAA. Excellent organogenic callus was achieved under the influence of 2.0 mg/l BAP +1.0 mg/l 2,-D. Meristemoid-like structures appeared under the treatment of 2.0 mg/l BAP and 1.0 mg/l IAA. Similar to our results, Ilahi & Akram (1993) obtained good callus growth on Rauwolfia serpentina explants using a combination of NAA (1.0 mg/l), Kn (0.5 mg/l) in addition to 10% CM. Regeneration of adventitious shoots was obtained on MS medium supplemented through different sets of hormonal combinations as detailed in Table 2. High shoot regeneration was observed when MS was supplemented with 1.0 mg/l of BAP and 5.0 or 10.0 or 15.0 mg/l of AS. Low concentration of NAA (0.5 mg/l) supplied along with BAP and AS had no stimulatory or inhibitory effect on shoot regeneration, but at high concentration (1.0 mg/l) shoot regeneration frequency was reduced and callogenesis was promoted. Thus it reveals that for shoot regeneration a low concentration of auxin and high concentration of cytokinin is necessary. Similarly, regeneration of shoots from black locust callus tissue has been obtained on MS containing BAP alone (5-25 mg/l) or in combination with NAA ( mg/l) (Wang et al., 1985; Han & Keathly 1989; Han et al., 1990) Embryoid-like structures were also obtained on the callus cultured under the influence of 1.0 mg/l of BAP and Kn. Complete plantlets were obtained when these embryoids were cultured onto MS containing 0.5 mg/l each of IAA and Kn. Similarly, somatic embryos can be obtained in a number of species, notable among them being cocoa (Pence et al., 1979), coffee (Monaco et al,. 1977), Sweet gum (Sommer & Brown, 1980) etc. Rooting ability of micro shoots obtained in the present studies was satisfactory. Auxin like IBA was supplemented through MS medium. Highest rooting frequency was obtained at 2.0 mg/l concentration. IBA supplemented at.0 mg/l also gave satisfactory rooting. As compared to our results, IBA at a concentration of 2.5 mg/l has been used for rooting, on shoots of many woody plants and it had been reported to be suitable for the rooting of Pistacio cutting (Al Barazi & Schwabe, 1982).

9 Clonal propagation of Rauwolfia Ihsan Ilahi et al. 53 In vitro cultures are capable of synthesizing alkaloids and contain a spectrum of these substances which is identical to that found in the whole plant (Bohm, 1980). As evident from the Table No.3 almost all the major Rauwolfia alkaloids were detected in the tissues analyzed. Moreover, reserpine, ajmaline and ajmalicine were detected in higher percentage compared to the mother plant. Similarly, three other alkaloids viz. serpagine, renoxydine and reserpiline were also detected in the cultures. However, percentage of these alkaloids varied when callus, regenerated shoots or roots were analyzed. As a result of present investigation alkaloid concentration has been increased as compared to other reports (Ilahi & Akram, 1993). Production and increase in secondary metabolites has been reported for many plant species (Bhojwani et al., 1983). During the present studies increase in some metabolites and production of certain substances has been reported. This aspect will be reported in another paper. Table 1. Axillary shoots formation and callus induction on stem nodal explant of Rauwolfia serpentina under the influence of different concentrations of phytohormones. Nature of Explant Nodal Segments Phyto-hormones mg/l BAP 2,-D Culture period (weeks) Remarks 3- vigorously growing shoots developed A single shoot sprouted on the node. Copious callus also got induced. NAA BAP shoots sprouted from the bud. Callus = = = BAP 2,-D BAP IAA A cluster of multiple shoots developed on the explant. Excellent, loose textured callus induction. Slight greenish-white callus induction. A slight increase in the callus mass. Green colour callus with meristemoids induction.

10 5 Clonal propagation of Rauwolfia Ihsan Ilahi et al. Table 2. Shoot regeneration, embryoid formation and rooting of the plantlets in Rauwolfia serpentina under the influence of different combination of phytohormones supplied through MS medium. Nature of Explant Callus Phyto-hormones Mg/l BAP IAA AS Culture period (weeks) 8 Remarks Copious callus induction along with shoot regeneration. Callus Embryoids BAP NAA AS BAP Kn IAA Kn shoots regenerated on the organogenic callus. About 50 shoots got developed per callus culture shoots got regenerated per culture. 0 shoots got developed on the organogenic callus shoots got induced per culture. Embryoids like structures formed on the callus. Shoots IBA Complete plantlets formation from these embryoids. 10% of the plantlets were rooted. 20 % of the plantlets were rooted. 50% shoots developed roots. 30% of the shoots produced roots.

11 Clonal propagation of Rauwolfia Ihsan Ilahi et al. 55 Table 3. Percentage of indole alkaloids in root, callus and shoot cultures of Rauwolfia serpentina Material Analyzed Callus Phytohormones mg/l Kn 2,- D Reserpine % Ajmaline % Ajmalicine % Serpentine % - Yohimbine % Others * Roots Shoots Kn 2,-D TPN x References BAP AS or 10 Alkaloids in trace amount: * renoxydine, + reserpline, x serpagine Akram, M. and I. Ilahi. 1985a. Biosynthesis of some important alkaloids by stem cultures of Rauwolfia serpentina Benth. In: Plant Tissue Culture (ed. Ilahi I.), Univ. Peshawar. p: -5. Akram, M. and I. Ilahi. 1985b. Bud differentiation in root callus of regenerated plantlets of Rauwolfia serpentina.in: Plant Tissue Culture (eds. Qureshi. A. and john. I.), PARC, Islamabad. pp: Akram, M. and I. Ilahi Plantlets formation in root callus of Rauwolfia serpentina. Pak. J. Bot., 18: Al-Barazi, Z. and W.W. Schwabe Rooting softwood cuttings of adult Pistacia vera. J. Hortic. Sci. 58: Blojwani, S.S., V. Dhawan and E.C. Cocking Plant Tissue culture. Elseviar, Amsterdam. Bohm, H The formation of secondary metabolites in plant tissue and cell cultures. International Review of Cytology, Supplement 11B: Butcher, D.N Secondary products in tissue cultures. In: Plant Cell Tissue and Organ Culture. Reinert. J. and Bajaj. Y.P.S. (eds) pp: Springer-Verlag, Heidelberg. Han, K.H. and D.E. Keathley Regeneration of whole plants from seedling-derived callus of black locust tree (Robinia pseudoacacia L.). Nitrogen Fixing Tree Res. Rep., 7: Han, K.H., J. M. Davis and D.E. Keathley Differential responses persist in shoot explants which have been regenerated from callus of two mature black locust (Robinia pseudoacacia L.) Tree Physiol., 6: Ilahi, I Rapid micro-propagation of Rauwolfia. Apam News letter. Volume 1:

12 56 Clonal propagation of Rauwolfia Ihsan Ilahi et al. Ilahi, I., F. Rahim and M. Jabeen Induction of somatic embryos and plant regeneration in Rauwolfia serpentina Benth. Pak. J. Plant. Sci., 1 (2): Ilahi, I. and M. Akram Leaf callus culture of Rauwolfia serpentina. Pak. J. Bot., 19: Ilahi, I. and M. Akram Enhanced propagation, root production and alkaloid biosynthesis by cultures of Rauwolfia serpentina. In: Adapted Propagation Techniques for Commercial Crops of the Tropics. Nguyen Thi Quynh and Nguyen Van Hyen (eds). Agri. Publishing House, Hochi Minh City, Vietnam. Mathur, A., P.S. Ahuja and A.K. Mathur Micro-propagation of Panax quiquefoluim, Rauwolfia serpentina and some other medicinal and aromatic plants of India. In: Adapted Propagation Techniques for Commercial Crops of the Tropics. Nguyen Thi Quynh and Nguyen Van Hyen (eds). Agri. Publishing House, Hochi Minh City, Vietnam. Mitra, G.C Studies on the formation of viable and non-viable seeds in Rauwolfia serpentina Benth. Ind. J. Exp. Biol., 1:5-56. Monaco, L.C., M.R. Sondahl, A. Carvalho, O.J. Crocomo and W.R. Sharp Application of tissue culture in the improvement of coffee. In: Reinert, J. & Bajaj, Y.P.S. (eds) Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture. Springer- Verlag, Heidelberg. p Murashige, T and F. Skoog A Revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol., 15: Pareek, L. K Trends in Plant Tissue Culture and Biotechnology. Agribios (India), Jodhpur. Pence, V.C., P.M. Hasegawa and J. Janick Asexual embryogenesis in Threobroma cacae L. J. Am. Soc. Hortic. Sci., 10: Sommer, H.E and C.L. Brown Embryogenesis in tissue culture of sweet gum. For. Sci., 26: Staba, E.J Secondary metabolites and biotransformation. In Staba E.J (eds) Plant Tissue culture as a source of Bio-chemicals. CRC Press, Boca Raton. Staniforth, V., S.Y. Wang, L.F. Shyur and N.S. Yang Shikonins, phyto compounds from Lithospermum erythrorhizon, inhibit the transcriptional activities of human tumor necrosis factor a promoter in vivo. J. Biol. Chem., 279, Wang, Q.Z., J.B. Zhao and B.H. Zhao Fast propagation of superior clones of Robinia pseudoacacia through tissue culture. For. Sci. Technol., (Linye Keji Tongxun) 8: 8-9. Wangwagner, H., S. Bladt and E.M. Zginski Plant drugs analysis.a thin layer Chromatography Atlas. Springer-Verlag, Heidelbug. (Received June 15, 2007; Revised July 25, 2007)