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The Corroboree


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Everything posted by tripsis

  1. tripsis

    Post your track of the day

    Post whatever music track or tracks you like the most today. Give us all something worth listening to. Magyar Posse - Whirlpool of Terror and Tension http://www.youtube.com/watch?v=3o3LzDdA8Pg Androcell - Neurosomatic Circuit
  2. tripsis

    Micropropagation techniques

    I posted this elsewhere a while ago, but forgot to do it here. Figure some of you might find the information in it useful. I'm yet to begin attempting any micropropagation, but will do so one of these days...and it's going to be great! -------------------------------------------------------------------------------------------------------------------- SALVIA SPP.: TISSUE CULTURE, SOMATIC EMBRYOGENESIS, MICROPROPAGATION AND BIOTRANSFORMATION SPIRIDON E.KINTZIOS Department of Plant Physiology, Faculty of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece INTRODUCTION Although Salvia, one of the most commercially important and widely cultivated medicinal plants, is a perennial, it does not last above three or four years without degenerating, so that plantations should be renewed at least every four years (Grieve, 1994). In addition, and in spite of the fact that considerable progress has been made in the field of the in vitro production of various secondary metabolites, such as rosmarinic acid and cryptotanshinone (see I. Hippolyte and Em. Panagiotopoulos, in this volume), the application of biotechnological methods for the propagation of these species is rather limited. This might be due to the fact that most Salvia species can be easily propagated by cut tings and layers, most frequently in the spring and in the autumn, by pulling off or pegging down shoots from three-year-old plants (Grieve, 1994). Some species, like S. sclarea, the clary sage, are propagated by seed, but for most species this method of propagation is limited by the rather low seed germination rate. There is, however, an increasing interest in the development of efficient protocols for the tissue culture and micropropagation of certain Salvia species, in order to establish a relatively fast system for producing disease-free and true-to-type clonal (and therefore uniform) plants from outstanding genotypes. In addition, there is substantial evidence that plant secondary metabolite production can be enchanced by the in vitro induction of morphogenesis, so that in vitro regenerated plants can be a useful resource for pharmacologically active compounds. Tissue culture techniques could also be invaluable for the conservation of endangered or rare Salvia species, such as S. pratensis, a perennial restricted to a few isolated populations in the Netherlands (varying in size from 10 to 1500 flowering plants) (Ouborg and Van Treuren, 1995). The present report focuses on a concise presentation of the various methods developed for the induction of callus, organogenesis and somatic embryogenesis as well as plant regeneration for micropropagation and breeding purposes of some Salvia species. Furthermore, the accumulation of secondary metabolites (in particular rosmarinic and lithospermic acid) in in vitro differentiated tissues is reviewed. GENERAL Optimization of the tissue culture procedure in Salvia has called for the employment of different culture media (especially growth regulators) and culture conditions (such as light and temperature) for each separate species. However, some features of the culture process are essentially the same for most of the cases studied: Explant Source Several kinds of explants have been used for the establishment of tissue cultures from Salvia species, such as seeds (S. miltiorrhiza—Waldemar, 1996), shoot tips (S. miltiorrhiza—Morimoto et al., 1994), shoots with axillary buds (S. canariensis— Luis et al., 1992), young leaves (S. officinalis and S. fruticosa—intzios et al., 1996, 1998), embryos (interspecific species of S. sclarea—Rusina et al., 1997) and seedlings from in vitro-germinated seeds (S. miltiorrhiza—Miyasaka et al., 1989, Gao et al, 1996). Explant Disinfection Seeds and shoot explants have been regularly surface-sterilized in 1–2% solutions of sodium or calcium hypochlorite (15 min-1 hr) usually followed by immersion in 70% ethanol (30s). Luis et al. (1992) additionally used Benlate (at a concentration of 1g/ l) for the surface-sterilization of stem explants from mature S. canariensis plants. Finally, a surface-sterilization for 12 min in 0.1% (w/v) mercuric chloride solution, containing 1–2% Tween-80, is recommended for leaf explants (Kintzios et al., 1996, 1998) derived from field-grown plants. When tissue-culturing certain Salvia species, explant browning can be an serious problem: explants develop necrotic areas which in some cases lead to expiant decline and death. This effect is both genotype- and culture medium-dependent: For instance, in S. officinalis, application of a-napthaleneacetic acid (NAA) and 6- benzyladenine (BA) generally stimulated callus formation, but also promoted explant necrosis (Kintzios et al., 1996). For the same species, the development of necrotic symptoms on explants was highly negatively correlated with callus induc tion. This effect was more profound at low light intensities (50 μmol m-2 s-1). On the contrary, browning of the S. fruticosa explants did not have any observable effect on callus induction: almost all declined explants were able to dedifferentiate as a response to culture. The addition of ascorbic acid (at an optimal concentration of 10 mg/l) to the culture medium greatly reduced the frequency of the necrotic symptoms. Culture Medium The basal medium of Murashige and Skoog (MS medium) (1962) has been most frequently employed in the tissue culture of Salvia. Other types of media (usually of a lower ionic strength), such as Almacigo’s medium (Mederos and Lopez, 1991), White medium (White, 1943) or B5 medium (Gamborg et al., 1968) have been occasionally used. Culture Conditions For culture initiation, explants of various Salvia species have been incubated at 23– 25 °C, over a 16 hr photoperiod and under a photosynthetic photon flux density (PPFD) of 25–250 μmol m-2 s-1 (depending on the particular species). SPECIES-SPECIFIC PROTOCOLS Salvia miltiorhizza Callus induction Callus cultures of S. miltiorrhiza have been successfully induced from in vitro grown seedlings (epicotyles and hypocotyles) (Miyasaka et al., 1989, Waldemar, 1996) and shoot tips (Morimoto et al., 1994) on a solid MS medium supplemented with either 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (Kin) (seedling tissues) or 2.5 μM indolebutyric acid (IBA) and 1.3 μM BA (shoot tips) (usually under a PPFD of ca. 170 μmol m-2 s-1). In the latter case, adventitious shoot induction was always concomitant with callus formation, but replacement of IBA with 2,4-D enhanced the further induction of callus from in vitro regenerated petioles. Shoot and bud induction Shoot structures have been induced either through callus or directly on cultured explants. Regeneration of shoots from callus cultures has been achieved on solid MS medium supplemented with either 0.5 mg/l indoleacetic acid (IAA) and 4.6 μM Kin (seeling-derived callus) (Waldemar, 1996) or 4.6 μM Kin and 1.4 μM gibberellic acid (GA3) (formation of multiple shoot complexes from petiole-derived callus), under a PPFD of ca. 170 μmol m-2 s-1 (Morimoto et al., 1994). Seedling tissues (from sterile germinated S. miltiorrhiza seeds) have been used for the direct induction of adventitious bud clumps on MS medium supplemented with 2.6 μM BA and 0.5 mg/l IAA, under a PPFD of 36 μmol m-2 s-1 (Gao et al., 1996). Root induction Rooting of shoot cultures (several weeks-old) has been usually achieved on a growth regulator-free solid MS medium (Morimoto et al., 1994, Waldemar, 1996) or on solid B5 medium with 1 μM IBA (Gao et al., 1996). Regenerated plants were potted in either soil-leaf mould (1:4) or vermiculite and cultivated for 3–10 weeks (23–25 °C, 16:8 h, 125–200 μmol m-2 s-1) before being carried over to the greenhouse or the field for further hardening and cultivation. Tissue culture has been proven a very efficient propagation method for S. miltiorrhiza. Fifty four fertile plants could be regenerated from a single seed (Waldemar, 1996) while more than 107 clonal plants could be theoretically obtained from a single shoot tip during a total period of 33–37 weeks (including the time necessary for hardening transplanted regenerated plants). Secondary metabolite accumulation Aerial parts of both seed- and stem regenerated S. miltiorrhiza plants demonstrated a higher content of phenolic acids (rosmarinic and lithospermic acid) than parental plants. The inverse was true for the roots, but, as Morimoto et al. (1994) mentioned, this could simply be due to the age difference of the plant material: parent plants were much older (at least three-years old) than regenerants (only a few weeks old). Accumulation of phenolic acids was essentially the same in shoots either regenerated in vitro from callus cultures or clonally propagated from shoots (Morimoto et al., 1994). Interestingly, accumulation of phenolic acids in regenerated plants transferred after rooting to vermiculite for 5 weeks, then cultured in soil for another 5 weeks, was higher than in plants cultivated continuously in vermiculite only. Callus cultures themselves, on the other hand, demonstrated a rather poor accumulation potential, which was ca. 3–4 times lower than in regenerated plants. Salvia officinalis and S. fruticosa The common sage is the most representative of the Salvia species. It has been cultivated for culinary and medicinal purposes for many centuries in Europe and Middle-East (Grieve, 1994). It is a very variable species, possessing remarkable curative properties. Many kinds of sage have been used as substitutes of tea. Salvia fruticosa is a sage species endemic to the Mediterranean region, commonly used as a substitute of tea. Callus induction and somatic embryogeneis Kintzios et al. (1996, 1998) reported on the induction of somatic embryogenesis from leaf explants of Salvia officinalis and S. fruticosa on a MS medium supplemented with 1.8–18 μZM 2,4-D and Kin or 10.5–21μ M NAA and BA. Only explants from young plants (having 6–8 leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m-2 s-1) favoured callus formation and induction of somatic embryos. S. fruticosa responded to culture much better than S. officinalis. Callus tissue was formed on explants of this species at a 95–100% rate under both low and high light intensities. The callus derived from S. fruticosa also grew at a faster rate than the one from S. officinalis explants (Fig. 1). Higher callus induction rates were obtained for S. officinalis when equimolar auxin and cytokinin concentrations were used. This was also the case for 2,4-D and Kin, but only when applied at the lowest (1.8 μM) or the highest (18μM) concentrations. A novel growth pattern was observed for S. fruticosa callus: it maintained a much higher growth rate throughout the period of investigation with only an intermediary slight growth reduction between the 2nd and the 3rd week after callus formation. For the latter species, higher PPFD values had an observable beneficial effect on callus proliferation. Globular somatic embryos were readily formed on callus tissue of both species after 3 weeks in culture (Fig. 2). Only S. officinalis embrya, induced on 10.5 μM NAA and 10.5 μM BA, and S. fruticosa embrya, induced on 10 μM NAA and 21 μM BA, were able to further develop on the same medium until heart- and torpedo-shaped forms (1–2 mm long) appeared. On the contrary, globular embryos of both species induced on 4.5 μM 2, 4-D and 4.5 μM Kin developed further only after being transfered to a medium with no growth regulators at all. The PPFD value had no effect on the maturation process. In every case, 1–2 mature embryos were counted per callus tissue. Secondary metabolite accumulation Maximum rosmarinic acid accumulation in S. officinalis callus cultured on 4.5 μM 2, 4-D and 4.5 μM Kin coincided with the onset of somatic embryo induction (e.g. at the beginning of the 3rd week after culture initiation), as well as during final embryo development and maturation (6–7 weeks after culture initiation) (Fig. 1). Thus, the process of tissue redifferentiation in vitro was assossiated with an enhanced phenolic acid accumulation, as already aforementioned for S. miltiorrhiza. Salvia sclarea Salvia sclarea, the clary sage, is a biennial plant yielding an oil (clary oil) with a highly aromatic odor, which is under increasing attention for its use in aromatherapy. After the essential oil is removed the crude material is a source of sclareol which is converted to the sclareolide; both are used commercially in the manufacture of ambergris perfumes and as inhibitors of growth of rust fungi (Grieve, 1994, Dweck in this volume). Banthorpe et al. (1990) reported on the establishment of undifferentiated friable, white callus and derived cell suspension lines from stem explants of a sterile S. sclarea plant on a MS medium supplemented with either 4.5 μM 2, 4-D and 0.5 μM Kin or 5.4 μM NAA and 0.5 μM Kin. A callus induction rate of ca. 80% was observed. Consequently, cell suspensions were established on MS liquid medium supplemented with 4.5 μM 2, 4-D and 0.5 μM Kin. The cultures were used in order to study the in vitro accumulation of sclareol. Rusina et al. (1997) used the method of isolated embryo culture for producing interspecific hybrids of S. sclarea with the wild species S. scabiosifolia, S. grandiflora and S. aethiopis. Embryo growth in vitro and the frequency of hybrid plantlets were affected by the stage of embryo development, the composition of the medium and the genotype of the parents. Optimum for hybrid production were modified White medium and the torpedo stage of embryo development. The percentage of hybrids produced was 12.2 to 38.3% depending on the cross. A study of the plants in the field enabled the selection of those with the highest essential oil content. S. canariensis This species, which is endemic of the Canary islands, contains several diterpenes with considerable antibiotic and antioxidant activities. Luis et al. (1992) induced axillary buds on stem segments taken from mature 13-year-old S. canariensis plants on a modified Almacigo’s medium supplemented with 0.01 μM BA and 0.01 μM NAA. Multiplication and elongation of the axillary buds was achieved on modified MS medium containing lower levels of BA (23–25 °C, 16 hr, 25 μmol m-2 s-1). CONCLUSION Existing experience with tissue culture of Salvia sp. is limited o a small number of species and mainly concerns callus induction from various explants in order to facilitate the in vitro production of secondary metabolites. S. miltiorrhiza is probably the only species where different approaches to plant regeneration in vitro have been successfully taken. However, progress in somatic embryogenesis and recent research on the technology of synthetic seeds, along with other advanced aspects of tissue culture (e.g. protoplast culture and fusion, creation of autotetraploid lines) could offer to a significant involvement of biotechnology to the propagation and breeding of the genus Salvia. REFERENCES Banthorpe, D.V, Brown, J.T, Morris, G.S. (1990). Accumulation of the anti-fungal diterpene sclareol by cell cultures of Salvia sclarea and Nicotiana glutinosa. Phy’tochemistry, 29/1, 2145–2148. Gamborg, O.L., Miller, R.A., Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res, 50, 151–158. Gao, S.L., Zhu, D.N., Cai, Z.H., Xu, D.R. (1996). Autotetraploid plants from colchicinetreated bud culture of Salvia miltiorrhiza Bge. Plant Cell. Tiss. Org. Cult. 47, 73–77. Grieve, M. (1994). A Modern Herbal. (Leyel CF, ed.), Tiger Books International, London. Kintzios, S., Nicolaou, A., Skoula, M., Drossopoulos, J., Holevas, C. (1996). Somatic embryogenesis and in vitro rosmarinic acid production from mature leaves of Salvia officinallis and S. fruticosa biotypes collected in Greece. Eucarpia Series: Beitr. Z. Zuchtungsforschung, 282–285. Kintzios, S., Nicolaou, A., Skoula, M. (1998). Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures. Plant Cell Rep. (in press) Luis, J.G., Gonzalez, A.G., Andres, L.S., Mederos, S. (1992). Diterpenes from in vitro-grown Salvia canariensis. Phytochemistry, 31, 3272–3273. Miyasaka, H., Nasu, M. and Yoneda, K. (1989). Salvia miltiorrhiza: In vitro production of cryptotanshinone and ferruginol. In YPS Bajaj (ed.), Biotechnology in Agriculture and Forestry, Vol. 7, Springer-Verlag, Berlin, pp. 417–430. Mederos, S.., Lopez, C.I. (1991). Acta Horticult., 289, 135. Morimoto, S., Goto, Y. and Shoyama, Y. (1994). Production of lithospermic acid B and rosmarinic acid in callus tissue and regenerated plantlets of Salvia miltiorrhiza. J. Natl. Prod., 57, 817–823. Murashige, T., and Skoog, F. (1962). A revised method for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, 472–497. Ouborg, N.J. and Van Treuren, R. (1995). Variation in fitness-related characters among small and large populations of Salvia pratensis, J. Ecology, 83, 369–380. Rusina, L. V, Bugara, A.M. and Bugaenko, L.A. (1997). Using the method of isolated embryo culture for producing interspecific hybrids of sage. Fiziol. I Biokhimiya Kul. Ratenii 29, 121–123. Waldemar, B. (1996). Micrpropagation and phenolic acids production of Salvia miltiorrhiza Buzge. Eucarpia Series: Beitr. Z. Zuchtungsforschung, 327–329. White, P.R. (1943). Nutrient deficiency studies and an improved inorganic nutrient for cultivation of excised tomato roots. Growth, 7, 53–65. -------------------------------------------------------------------------------------------------------------------- A related article. PRODUCTION OF SECONDARY METABOLITES USING LIQUID CULTURE OF SALVIA PLANTS: UP-TO-DATE REPORTS AND SCALE-UP POTENTIAL EMMANOUIL PANAGIOTOPOULOS, MARIA SKAPETI AND CHRISOSTOMOS KAPETANOS Department of Plant Physiology, Faculty of Agricultural Biotechnology, Agricultural University of Athens, Ieva Odos 75, 118 55 Athens, Greece INTRODUCTION Since ancient times, most of the plants in the Lamiaceae family have been mentioned for their pharmaceutical and therapeutical abilities. Usually by mixing with other species or by drinking the extract from boiled plant leafs, they have been used by folk medicine as well as modern medicine until today. As a result, the research concerning Salvia plants is focused on the medicinal activity of individual substances that can be found in extracts from aerial parts or from roots of the plant. The majority of the active—worthy of investigation substances are products of metabolic pathways that apparently are not involved directly in growth or development, known as secondary metabolites. Those secondary plant products appear to function mainly as defences against predators and pathogens. As we can see, there is a great interest in using antioxidant, antimicrobial, antiviral and even anticancer substances that are products of “natural factories”, the plant cells. Natural products often have less side effects, when applied in normal dosages and they are more acceptable in a wide range of consumers. The main disadvantage of natural substances is the small quantity that can be extracted from very large quantities of plant parts. Also, some species are difficult to cultivate in a wide range of environmental conditions and if they grow they give a poor product yield. Lining up against those disadvantages, tissue culture and especially liquid culture seem to give a potential to future production of plant secondary metabolites. SECONDARY METABOLITES OF SALVIA PLANTS Categories and Structure of Secondary Metabolites There are three major groups of secondary products according to their biosynthetic way: terpenoids, phenolics and nitrogen containing compounds. Terpenoids Terpenoids are lipids synthesised from acetyl-CoA via the mevalonic acid pathway. They consist of five-carbon units that have the structural frame of the isopentane. The taxonomy is based on the number of the C5 containing units of the terpenoid. So the two C5-unit containing terpenoids are called monoterpenes (10 carbon atoms), the three C5-unit containing terpenoids are called sesquiterpenes (15 carbon atoms), the four C5-unit containing terpenoids are called diterpenes (20 carbon atoms) and so on (triterpens-30 carbon atoms, more than 40 carbon atoms-polyterpenes). Some terpenoids occur in primary metabolism pathways as in-between products of primary production, such as some plant growth regulators (absisic and gibberellic acid) and cell membrane components (steroids from triterpenes). Most of plant terpenes are used as defence molecules having toxic activity that prevent herbivorous insects and higher animals from eating plant tissues. Phenolics Phenolic compounds are aromatic substances mainly formed via the sikimic acid or the malonic acid pathway in various ways. This group has the characteristic of a hydroxyl joined to an aromatic ring. It is a rather heterogeneous group because some phenolics are water soluble, others can be solved only in organic solvents and some are insoluble polymers. They play various roles in the plant’s physiology, such as defence against pathogens and herbivorous, mechanical enforcement and attraction to pollination insects. Nitrogen containing compounds Most of those secondary products are synthesized by common amino acids. In this group we can find many plant defense substances, such as alkaloids and cyanogenic glycosides. Plants from Salvia species do not contain any worthy substance from this category of secondary metabolite. Main Secondary Metabolites in Salvia Plants The following compounds of Salvia plants that belong to the phenolics group have been studied: Rosmarinic acid (RA) Chemically known as a-O-caffeoyl-3, 4-dihydroxyphenillactic acid, rosmarinic acid belongs to the phenolics group and it is characterised as a phenylpropanoid (Fig. 1). It was identified for the first time in rosemary extracts (Scarpati et al., 1958) and it is known for its antioxidant activity. Rosmarinic acid is biosynthesised through the condensation of caffeic acid and 3, 4-dihydroxyphenyllactic acid. Two precursor amino acids—phenylalanine and tyrosine—are involved in this biosynthetic procedure (Peterson and Alfermann, 1988). Quantities of phenolics indicating rosmarinic acid have been extracted from various Salvia plant cells such as S. officinalis and S. fruticosa, with different concentrations of the phenylalanine precursor in the medium (Panagiotopoulos et al. manuscript in preparation). The approximate quantitative determination of RA has been done by measuring the absorption at 333 nm, the characteristic wavelength of phenolic rings of the callus ethanolic extract. Another report (Hippolyte, 1990, see also Chapter 16 in this volume) gave an alteration of production by 50% with different hormonal balances in the medium for S. officinalis suspension culture. Lithospermic acid B (LSA) Studies about lithospermic acid B (Fig. 2) as a Salvia plant extract have been made mainly in roots and rhizomes of S. miltiorrbiza Bunge. One of them (Fung et al., 1993) achieved isolation to > 95% purity by HPLC from the aqueous extract of the roots of the plant. This study involved a demonstration of the myocardial salvage effect of LSA. Another report gave a comparative reference of seventeen different Salvia species and varieties that gave a wide range of amounts of LSA, from 0.3 μtg/ mg of extract at S. deserta to 258.3 μg/mg at S. paramiltiorhiza f. purpureo ruba, measured by LC-MS analysis (Kasimu et al., 1998). Salvianolic acids Salvianolic acids A (Sai A), B and K are some of the different chemical structures in Salvia plants (Figs 3, 4, 5), all belonging to the phenolics group. Observed for the first time in roots of S. miltiorrhiza B. (Li et al., 1984), Sai A was investigated for its protective action against peroxidative damage to biomembranes (Liu et al., 1992). Some interesting work has been made, involving anti-oxygen radicals activities in rats (Lin and Liu, 1991), effects of Sai A on oxygen radicals released by rat neutropilis and on neutrophilic action (Lin et al., 1996) and generally antioxidant activities of salvianolic acid. Quantitative determination gave considerable amounts (29.28 μg/mg) of salvianolic acid K in S. deserta, compared to other species (Kasimu et al., 1998). Concerning the terpenoids group, there have been reported many of them in Salvia plants. Particularly worth-mentioning is a report on the isolation of twenty one abietatriene diterpenes grouped in five classes and found in only two Salvia species : S. canariensis and S. mellifera (Moujir et al., 1993). Those compounds were evaluated for structure-antimicrobial activity. Most of known Salvia terpenoids have been evaluated for anti-fungal, antiviral and generally antimicrobial activity. We are going to refer to the most common terpenoids which are: Tanshinones An extensive report on tanshinones, (Hu and Alfermann, 1993) in hairy root cultures of S. miltiorrhiza B., gave an quantification of tanshinone I (Fig. 6), tanshinone IIA (Fig. 7), tanshinone IIB (Fig. 8), tanshinone V (Fig. 9), tanshinone VI (Fig. 10), cryptotanshinone (Fig. 11) and dihydrotanshinone I (Fig. 12), by means of a quantitative HPLC. The cultures were established by sterile grown plants whose roots were transformed by infection of five strains (only four gave roots) of Agrobacterium rhizogenes. Production of cryptotanshinone was achieved by a series of suspension cultures with callus from seedlings of S. miltiorrhiza B., comparing an original MS medium without Fe-EDTA and a simplified medium that finally gave better production of cryptotanshinone (110±4.86 mg/l) (Miyasaka et al., 1989). Ferruginol In the previous reference (Miyasaka et al., 1989), the production of ferruginol (Fig. 13) was also achieved but with a better production in the original medium (69.3±.77 mg/l). Hu and Alfermann (1993), also recorded the production of ferruginol from transformed root cultures of Salvia miltiorrhiza B., by leaf explants without hormones in the medium, reaching a maximum of approx. 3.4 mg/g of dry weight. The most productive strain of the Agrobacterium rhizogenes was used for inducing hairy roots from the leaf explants. Sclareol Another diterpene, sclareol (lab-14-en-8, 13(S)diol) (Fig. 14) is used commercially in the manufacture of ambergris perfumes and is also a potent inhibitor of growth of rust fungi (Baily et al., 1975). Accumulation of sclareol by cell cultures of S. sclarea has been achieved at rates (μg/g per day) varying from 0.2 to 6% of those found in the parents plants. The maximum accumulation was shown to take place near the entry to the exponential growth phase (Banthorpe et al., 1990). Carnosic (carnosolic) acid and carnosol It has been reported that carnosol (Fig. 15) and carnosic acid (Fig. 16), among many other antioxidants, can give about 90% of the antioxidant activity of rosemary plants (Rosmarinus officinalis L.) (Aruoma et al., 1992). Studies showed that carnosic acid gave the strongest inhibitory effect, among other substances extracted from R. officinalis L. (including carnosol), on HIV-1 protease in cell-free assays (Paris et al., 1993). Skin tumorigenesis was inhibited by carnosol, again from rosemary plants (Huang et al., 1994). In Salvia species, those two diterpenes where isolated from seven-day-old in vitro grown plantlets of S. canariensis, though neither carnosol nor carnosic acid could be obtained from 25-day-old plantlets (Luis et al., 1992). Underlying that many other terpenoids (as well as phenolics) have been also isolated from Salvia plants and studied for various biological activities, we point out the interest in producing those medicinally important substances on industrial scale. SECONDARY METABOLITE PRODUCTION USING LIQUID CULTURE Liquid Culture and the Mechanisms of Plant Secondary Metabolism Suspension cultures have been frequently used for the production of secondary metabolites from various plants. The usual procedure is to transfer a callus from a solid medium to a liquid one (i.e. in conical flasks), establishing a suspension culture. Many works have been made to determine the parameters for the optimisation of secondary metabolite production, varying medium composition, culture conditions and using different plant species and varieties. In each plant two conditions have to be met before a considerable production of secondary metabolites can be occur: the various enzymes directly involved in secondary metabolism have to be inducted and also sufficient supply of precursors from primary metabolism is necessary before product accumulation can be observed. Primary metabolism not only supplies secondary metabolism, but many of the same precursors are important to the synthesis of cell constituents. For that reason there is often a competition for these precursors between the growth process and secondary metabolism. As a consequence, synthesis of secondary metabolites occurs especially when growth is slow or absent, often after the completion of a differentiation process (van der Plas et al., 1995). It is often observed that secondary production is induced when plant cells are under stress, a reaction that refers to the physiological role of secondary metabolites. Liquid culture has the advantage of easier control of culture conditions so that with continuous suspension cultures we can obtain specific growth stage treatments to enhance the production of the secondary metabolite of interest. Enzymatic mechanisms were examined in rosmarinic acid (RA) formation in Anchusa officinalis cell suspension cultures (Mizukami and Ellis, 1991). As expected, the RA concentration increased progressively during the late linear growth phase, reaching a maximum in the stationary phase. The same metabolite (RA) in Salvia officinalis and S. fruticosa gave similar results concerning the phase that the maximum production was achieved (Panagiotopoulos et al. manuscript in preparation). In the same work the use of phenylalanine precursor, as well as higher sucrose quantities in the liquid medium gave a slight support to the production of RA. Suspension cultures were also used by Hu and Alfermann (1993) for the production of diterpenoids (tanshinones and ferruginol) from Agrobacterium rhizogenes—trans formed leaf segments of Salvia miltiorrhiza. In this study a considerable percentage of the secondary metabolites was found in the liquid medium. In another report of Miyasaka et al. (1989) involving in vitro production of cryptotanshinone and ferruginol, secondary metabolites passed from the plant cells to the medium in a remarkable percentage, especially when immobilized cells were cultured (see below). Cell Immobilization in Liquid Culture There has been much interest in the use of immobilized cultured plat cells for biotransformation and production of secondary metabolites. According to Miyasaka et al. (1986), the production of the diterpenes cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza was successful. The two diterpenes were produced continuously by immobilized cells using a two-stage culture method, with normal medium for growth and then medium without Fe-EDTA to suppress cell growth an induce the production of the two metabolites. For immobilization, cells were entrapped on calcium alginate beads (mean diameter of 4mm) and after 25 days their productivity was calculated. In comparison with the free cell culture, production of cryptotanshinone and ferruginol by immobilized cells was about 39 and 61% respectively, of the yield obtained from the free cells. Nevertheless, about 74% of cryptotanshinone was released into the medium, whereas only 25% was released by free cells. A similar “release effect” (the mechanism of this effect is unknown) was also reported for alginate entrapped cultured cells of Catharanthus roseus (Brodelius et al., 1979). After the re-use of the immobilized cells, they retained their viability but the total production of the diterpenes was lower, proba bly due to accumulated lipophilic metabolites. This considerable attribute of some particular secondary substances can give a different perspective to the production of individual metabolites with continuous cell suspensions, by simplifying the isolation process of the desirable secondary product, without the need of reestablishment of the culture after extracting the product. CONCLUSION—PERSPECTIVES FOR SCALE-UP PRODUCTION The commercial production of natural products from cell or tissue culture has long been a goal for plant biotechnology. Though a great deal of effort has been applied, only a handful of process has come to commercial fruition, principally in Japan, and even in those instances production units operate at the limits of economic viability. In spite of apparent lack of progress at the commercial level it is generally agreed that the potential of the synthesis of high value natural products from plant cell cultures is tremendous (Stafford and Fowler, 1991). Bioreactors are in-wrought with the large scale production of secondary metabolites, so it is obvious that study and optimisation of liquid culture can lead to the desirable result. Stirred tank and airlift bioreactors are the most common in plant cell cultivation for secondary metabolites production. An example of bioreactor configuration developed by Wilson et al. (1990) is seen at Figure 17. At this point we can mention a 200 1 bioreactor configured as a module spiral stirrer that produces rosmarinic acid from cultivated cells of Coleus blumei. Large scale processes based upon plant cell cultures have been reported even up to 75m3 of a stirred tank bioreactor, growing cells of Rauwolfia serpentina (Westphal, 1990). Two-stage process formats, including cell immobilization, seem to be the most possible and effective way to produce extracellular secondary metabolites from Salvia plants. Already evaluated bioreactor configurations can be tested in Salvia plants, giving to individual secondary substances the potential for an industrial scale production. REFERENCES Aruoma, O.I., Halliwell, B., Aeschbach, R., Loligers, J. (1992). Antioxidant and prooxidant properties of active rosemary constituents: carnosol and carnosic acid. Xenobiotic, 22, 257–268. Bailey, J.A., Carter, G.A., Burden, R.S., Wain R.L. (1975). Nature, 255, 328. Banthorpe, D.V., Brown, J.T., Morris, G.S. (1990). Accumulation of the anti-fungal diterpene sclareol by cell cultures of Salvia sclarea and Nicotiana glutinosa. Phytochemistry, 29/7, pp.2145–2148. Brodelius, P., Deus, B., Mosbach, K., Zenk, M.H. (1979). Immobilized plant cells for the production and transformation of natural products. Febs Letters, 103/1, 93–97 Fowler, M.W., Stafford, A.M. (1992). Plant Cell Culture, Process Systems and Product Synthesis. In M.W. Fowler, G.S. Warren, and M. Moo-Young, (eds.) Plant Biotechnology, Pergamon Press, Oxford, pp. 79–98. Fung, L.F., Zeng, L.H., Wu, J., Wong, H.N.C., Lee, C.M., Hon, P.M., Chang, H.M., Wu, T.W. (1993). Demonstration of the myocardial salvage effect of lithospermic acid B from the aqueous extract of Salvia miltiorrhiza. Life Sciences, 52, pp. PL 239–244. Hippolyte, I. (1990). Utilisation chez la Sauge (Salvia officinalis L.) de techniques de culture de tissus pour la multiplication vegetative par l’ isolement d’ apex et la production d’ acide rosmarinique par des suspension cellulaires. PhD thesis. U.S.T.L. Montpeller II. Hu, B.Z., Alfermann, A.W. (1993). Diterpenoid production in hairy root cultures of Salvia miltiorrhiza. Phytochemistry, 32/3, pp. 699–703. 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Effects of salvianolic acid A on oxygen radicals released by rat neutrophilis and on neutrophil function. Biochemical Pharmacology., 51, pp. 1237– 1241. Liu, G.T., Zhang, T.M., Wang, B.E., Wang, Y.W. (1992). Protective action of seven natural phenolic compounds against peroxidative damage to biomembranes. Biochem Pharmacol, 43, 147–152. Luis, J.G., Gonzalez, A.G., Andres, L.S., Mederos, S. (1992). Diterpenes fron in vitro-grown Salvia canariensis. Phytochemistry, 31/9, pp. 3272–3273. Miyasaka, H., Nasu, M., Yamamoto, T., Endo, Y., Yondea, K. (1986). Production of cryptotanshinone and feruginol by immobilized cultured cells of Salvia miltiorrhiza. Phytochemistry, 25/7, 1621–1624 Miyasaka, H., Nasu, M., Yoneda, K. (1989). Salvia miltiorrhiza : In vitro production of cryptotanshinone and feruginol. In Y.P.S. Bajaj (ed.), Biotechnology in Agriculture and Forestry, Vol. 7, Medical and Aromatic Plants II, Springer-Verlag, pp. 417–430. Mizukami, H., Ellis, B.E. (1991). Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension culrtures. Plant cell reports, 10, 321–324. Moujir, L., Gutierrez-Navarro, A.M., Andres, L.S., Luis, J.G. (1993). Structure-antimicrobial activity relationships of abietane diterpenes from Salvia species. Phytochemistry, 34/6, pp. 1493–1495. Paris, A., Strukelj, B., Renko, M., Turk, V. (1993). Inhibitory effect of carnosolic acid on HIV- 1 protease in cell-free assays. Journal of Natural Products, 56/8, pp. 1426–1430. Peterson, M., Alfermann, A.W. (1988). Z Naturforsch, 43C, 501–510. Scarpati, M.J., Oriente, G. (1958). Isolamento e costituzione dell’ acido rosmarinico. Ricerca Sci, 28/11, 2329–2333. Stafford, A., Fowler, M.W. (1991). Plant cell culture and product opportunities. Agro-Industry Hi-Tech, 2, 19–23. Van der Plas, L.H.W., Eijkelboom, C., Hagendoorn, M.J.M. (1995). Relation between primary and secondary metabolism in plant cell suspensions. 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Murch Summary The study of medicinal plants has many unique challenges and special considerations. These plants are studied for their specific chemistry, or pharmacologic activity. Plants are highly sensitive to their environment and respond through changes in their chemistry. To date, one of the biggest problems in the study of medicinal plants has been the acquisition of consistent, positively identified material for chemical analysis. Successful protocols for the collection, identification, and establishment of medicinal plants species in tissue culture is invaluable for future studies. This protocol outlines methods to establish Scutellaria baicalenisis , and Scutellaria lateriflora from commercial seed sources, and collection and establishment of Scutellaria racemosa from wild populations. Key words: Medicinal plants, Scutellaria, Medicinal biotechnology, In vitro propagation, Germplasm collection 1. Introduction The genus Scutellaria is a diverse and widespread genus within the family Lamiaceae , the mint family, with more than 350 species worldwide, covering geographic regions from Alaska to South America, from Siberia to Japan, and throughout Europe, the Middle East, and Asia ( 1 ) . Species within the genus Scutellaria have been widely used by many cultures to treat a variety of different ailments. Scutellaria baicalensis (Huang-qin) remains one of the most commonly prescribed herbs in both traditional Chinese medicine and in Japanese Kampo medicine ( 2, 3 ) . More than 275 scientific reports have appeared since 2000, outlining the medicinal effectiveness of the extracts of Huang-qin in the prevention and treatment of many disorders including prostate cancer ( 4 ) , hepatic cancer ( 5 ) , HIV ( 6 ) , and neurodegeneration ( 7 ) . Animal studies of S. baicalensis supplementation to the diet indicated protection against aflatoxin –B1-induced liver mutagenesis ( 8 ) , inhibition of liver fibrosis ( 9 ) , inhibition of hemin-nitrite-H 2 O 2 induced liver damage ( 10 ) , and reduction of symptoms of Type 1 allergic reactions ( 11 ) . Scutellaria lateriflora , also known as “mad dog” or “skullcap,” is a native North American species that was used by the Cherokee, Applachian, and Iriquois as a treatment for anxiety, psychosis, neurologic disease, and feminine difficulties ( 12, 13 ) . Currently, skullcap is sold in North America as a tea; tonic or capsules made from dried aerial parts, and is used to treat epilepsy, St Vitus’s dance, insomnia, anxiety, neuralgia, and withdrawal from tranquilizers or barbiturates ( 14, 15 ) . Flavonoids found in S. lateriflora also inhibited [3H]-LSD binding to 5-HT7 (sero-tonin) receptors ( 15 ) and reduced anxiety levels in rats ( 16 ) . Scutellaria racemosa , is a native of Central and South America that has recently become an invasive species in the southern United States ( 17, 18 ) . Ethnobotanic evidence suggests that the Cauca peoples of Columbia and Ecuador use specifically selected ecotypes of S. racemosa in a ceremonial or narcotic preparation. Bianchi et al. ( 19 ) demonstrated that extracts of S. racemosa had neuroprotective activity in stressed animal models but very little research has been done with this species, in part because of a lack of available plant material for study. One of the major requirements for research is to identify specific medicinally active phytochemicals from Scutellaria species is the development of optimized protocols for growth, production, harvest, handling, and for preventing the loss of wild germplasm (20, 21). Controlled environment production systems have the potential to provide a continuous supply of consistent plant material, free from pathogens or abiotic contamination [(22), (see Note 1)]. Previous reports have provided detailed methods for in vitro establishment and production of S. baicalensis(3, 23, 24). However, the potential of many species including S. lateriflora, S. racemosa and their ecotypes remains unexploited. In many cases, species of Scutellaria are identified only by wild collection and herbaria vouchers. For example, TROPICOS, the database of the Missouri Botanical Garden, lists approximately 800 field collections of Scutellaria identified by dried shoot and floral tissues, with GPS coordinates locating populations and corresponding maps that locate the site of collection. Recently, these resources were used to identify ten new species of Scutellaria in Mesoamerica, an indication of the rich genetic resources remaining undiscovered in the genus (25). 2. Materials 2.1. Plant Material In this chapter we describe standardized, efficient protocols for in situ field collections, species identification, in vitro establishment, regeneration, and controlled environment production of axenic cultures of three Scutellaria species namely S. baicalensis, S. lateriflora and S. racemosa. S. baicalensis and S. lateriflora seeds were obtained from Richter’s Herb’s, Goodwood, ON ( see Note 2 ); S. racemosa material was collected from wild populations in Florida. The location of populations of desired plant species was facilitated by herbarium databases such as Missouri Botanical Garden’s W3TROPICOS database ( http:// http://www.mobot.mobot.org/W3T/Search/vast.html'>http://www.mobot.mobot.org/W3T/Search/vast.html ), Florida State University ( http://www.herbarium.bio.fsu.edu/search-specimens.'>http://www.herbarium.bio.fsu.edu/search-specimens. php ), Fairchild Tropical Garden ( http://www.virtualherbarium.'>http://www.virtualherbarium. org/vh/db/index.htm ), The New York Botanical Garden (http:// sciweb.nybg.org/science2/virtualherbarium.asp) and from the University of South Florida’s Atlas of Florida Vascular Plants database ( http://www.plantatlas.usf.edu/'>http://www.plantatlas.usf.edu/ ) ( 26 ) . In the case of S. racemosa , the databases have records of about 27 populations, which have been collected in North America between 1974 and 2002. We chose to sample populations in Florida for several reasons including: access to populations on public lands, ease of collection, persistence of the population since first description and geographic distribution. S. racemosa was collected from three sites in Florida in January of 2005 ( Fig. 2b ) . 2.2. Field Collection Supplies Conditions in the field are highly dynamic and change frequently. Check the weather of the area in which you will be working and dress appropriately. No matter what the weather predictions are; bring a raincoat. Additionally it is important to understand risks from wild animals, spiders, insects, or snakes. Immunizations are a good idea if you are working in an area with the risk of malaria or hepatitis. Once in the field you will need to have all of the necessary supplies for collecting material and getting it safely back to the laboratory. An assortment of Ziploc bags, plastic 25- or 50-mL tubes, and paper towels or newspaper should be in your pack as well as a weather-proof field notebook, camera, and several pencils. To physically harvest plant material, it is important to have a large knife or machete, pruners or secateurs, and a small pocket-knife for delicate cuts ( see Note 3 ). 2.3. Building a Plant Press Plant presses are typically constructed of two pieces of slatted wood 12″ × 18″ ( ~ 30 × 45 cm) filled with pieces of corrugated cardboard and blotter paper or newspaper to provide air ventilation and absorb moisture. The plant press is secured with webbed straps [ ( 27 ) ; Fig. 2d ]. 2.4. Surface Sterilization Solutions Solutions used in surface sterilization of seeds and tissues include commercially available bleach ( ~ 5.25% sodium hypochlorite) are diluted with water, and made into 10 and 20% solutions. Other chemicals include: 90% ethanol, the detergent Tween-20 (Phytotechnology Laboratories Inc.; Lexana, KS); and Plant Preservation Mixture (PPM) which was purchased from Plant Cell Technology Inc., (Washington DC). 2.5. Tissue Culture Media and Culture Vessels All media are prepared in 1 L media bottles with screw-on caps. The ingredients of media are 30 g/L sucrose, 4.33 g/L MS salts, and 1 mL/L Gamborg’s B5 vitamin solution (Sigma; Canada). The pH of the media is adjusted to 5.75 using 1 N NaOH, and 1 N HCl solutions. The plant growth regulators indoleacetic acid and indolebutyric acid are added to media prior to autoclaving. To solidify media, 7 g/L agar is added (Laboratory Grade Agar; Fisher Scientific, Mississauga, ON). All media are autoclaved at 121°C and 20 lb pressure for 25–30 min. Seeds are germinated in sterile clear Petri plates (Fisher- Scientific,;Canada). Plant material is grown in Phytotech P700 culture boxes (Phytotechnology Laboratories Lexana, KS). 3. Methods 3.1. Seed Germination 1. Seeds are surface sterilized by immersion in 95% ethanol for 30 s, and then in 20% bleach containing 2 drops per 100 mL Tween-20 for 18 min. Seeds are then rinsed at least three times with sterile distilled water ( see Note 4 ). 2. Seeds are germinated in Petri dishes containing 25 mL 0.8% agar containing 4 mL PPM. 3. Seed germination occurs after incubation in a dark growth chamber at 24°C for 14 d. 3.2. Identification and Collection of Scutellaria Racemosa (Pers.) from Wild Populations for In Vitro Culture 3.2.1. Preparations for Field Collections 1. All field collections should be performed with appropriate permissions and in compliance with the Convention on Biological Diversity (CBD) and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In the case of S. racemosa , the plant is an obligate weed in Florida, it is not an endangered species and therefore not covered by the CITES treaty and there are no restrictions on collection from public lands. However, collection of any plant material from private lands should always be avoided unless permission to collect is requested and granted. 2. It is necessary to obtain appropriate permits for transport of any field collections of plant material from the site of collection to the research laboratory. Plant import permits are issued by the Plant Protection agency of each country. For details of this process and application forms, please see APHIS/USDA ( http://www.aphis.usda.gov/plant_health/permits/index.'>http://www.aphis.usda.gov/plant_health/permits/index. shtml ) or the Canadian Food Inspection Agency, Plant Protection Branch ( http://www.inspection.gc.ca/english/for/'>http://www.inspection.gc.ca/english/for/ pdf/c5256e.pdf ). For wild collections of S. racemosa in Florida, USA and subsequent transport to the research laboratory in Canada, permits were required to allow the collection and transportation of seeds and plant meristems ( see Note 3 ). 3. There are a number of effective resources that are helpful in identifying unique characteristics of the plant. Such resources include herbaria specimens and published monographs that are available as published books or journals including Systematic Botany Monographs published by the American Society of Plant Taxonomists ( http://herbarium.lsa.umich.edu/ SBMweb/index.html ), and the Kew Bulletin ( http://www. kew.org/publications/kewbulletin.html ). An example of a herbarium voucher for S. racemosa is seen in Fig. 1 . Scutellaria is highly heterophyllus genus however; S. racemosa is easily identifiable in the field by the consistently hastate leaves and flower corollas less than 7 mm long [ ( 1 ) ; Fig. 2a ]. 3.2.2. Collection of Scutellaria in the Field 1. The field notebook is essential for accurate records of plant collection, preparation of herbarium vouchers and creating a record of plant locations. It is important to use a notebook with cloth, waterproof, all-weather writing paper and to write field notes in pencil that will not run when wet. The following information is crucial to the plant collection process: (a) Collector(s) full name(s). ( Date of collection. © Detailed location: country, state, county or province, roads, road junctions, mile markers, distance to cities or towns, elevation, and GPS coordinates. (d) Habitat: type of plant community and other plants growing in the area. (e) Plant habit: the form of the plant (herb, vine, tree shrub) and its height (f) Frequency: is the plant rare, occasional, frequent, or common? (g) Plant description: record any characteristics which may be lost upon drying such as aroma, flower colour, fruit colour, or leaf orientation. (h) Unique collector number: assign each collection an individual number. 2. Field notes from the collection of S. racemosa are shown in Fig. 2c . In brief, field collection of S. racemosa consisted of collection of seeds and meristems. Initially, all plant material was photographed to provide a visual record. Seeds were collected from fully mature, dehisced flower heads. About 100–200 seeds per flower stalk should be collected into plastic 50-mL tubes. Seeds should be collected from at least three different plants at random and details of the collection should be recorded in the field notebook. For meristem collections, healthy stems, about 15- to –35-cm long are excised from the growing plants using secateurs or a knife. It is important to select healthy material from as many different plants in the population as possible however, this may be difficult if populations are in decline or dormant. 3. Following harvest, plant material should be transported quickly. For S. racemosa , the most effective means of transport was immersion of the cut surface in a container of clean, tepid water. Store the container with cuttings out of the direct sun in a cool place and transport to tissue culture facility within 24 h. 3.2.3. Making Herbarium Vouchers Herbarium vouchers create a permanent record of your collections and should be stored at institutes in many different locations ( see Note 5 ). In order to comply with the requirements of different herbaria, all specimens must be prepared according to well-established international guidelines. It is important to file herbarium vouchers of field collections at four or five different herbaria for comparison and to avoid loss if one herbarium suffers an unavoidable catastrophe. Specimens must be properly dried and pressed for mounting as described below ( see Note 5 ). 1. For herbaceous species, stems about 31-cm long are appropriate for herbarium vouchers. Stems should be complete with attached leaves and roots, as well as flowers and/or fruits if possible. For larger specimens, aquatic plants or cacti etc., different processing of the tissues may be required. 2. Stems are arranged onto a 27 × 38-cm piece of newspaper in a lifelike manner (i.e., shoots toward the top of the sheet and roots toward the bottom which allows observation of all morphological structures, especially reproductive, including both sides of leaves and several stages of development of shoots, or flowers). Fold newspaper over the stems ( Fig. 2d ) ( see Note 6 ). 3. A label should be prepared for all herbarium specimens. The label is usually 4.5 -wide with the collector number, collector’s name, collection date, and location of collection corresponding to records in the field notebook. 4. The newspaper with the plant material protected is carefully folded and placed in press between sheets of blotter paper and cardboard. Straps are tightened on plant press and the press is dried using one of several different techniques. For most applications with small herbaceous plants, dry under light using three 60–120 W incandescent light bulbs for 24–36 h and transport to herbarium for mounting ( see Note 7 ). 5. Replicate herbarium voucher specimens should be donated to herbaria in at least four different locations in order to ensure that the samples will be preserved in perpetuity ( see Note 8 ). 3.3. Creation of an In Vitro Germplasm Collection of Scutellaria Racemosa from Field Collections 1. Stems of S. racemosa collected in the field are transported to the tissue culture laboratory within 24 h of collection ( see Note 5 ). 2. Stems are cut into 4- to 5-cm pieces and surface sterilized with a 20% solution of bleach and two drops of Tween-20 for 30 min, washed with sterile distilled water 3 times and cultured onto basal culture media (MSO) for shipping to final destination ( see Note 6 ). 3. Subculture of the field collected plant material onto fresh MSO is required within 3 d of shipping. Meristem sections (0.5–1.0 cm) are excised from the field collected shoots and subcultured onto fresh MSO medium in Phytotech P700 Culture Boxes. All cultures are incubated in a growth chamber with a 16-h photoperiod under cool-white light, 22–45 m mol/m 2 /s at 25°C. Shoot apices were subcultured every 3 wk ( Fig. 1f ) . It is interesting to note that several cultures of Scutellaria produced profusions of shoots after 4–6 wk of culture under these conditions ( Fig. 1f ) ( see Note 6 ). 3.3.1. Maintenance of the In Vitro Collection of Scutellaria 1. Once a collection of germplasm of Scutellaria species has been established, the plants provide tissue for optimization and phytochemical quantification experiments. All cultures must be transferred to MSO medium devoid of any growth regulators prior to bioassays, optimization studies or chemical analysis ( see Note 9 and Note 10 ). 2. Plants kept on MSO medium with a 16-h photoperiod will need to be subcultured every 6 wks to maintain the collection. 3. More rapid growth of Scutellaria plantlets in culture is achieved by supplementation of the MSO medium with 1.0 m M kinetin. 4. Rooting of the in vitro plantlets is facilitated by supplementation of the medium with 2.5 mM indoleacetic acid (IAA) or 0.5 mM indolebutyric acid (IBA). 4. Notes 1. In vitro-grown plant material has many advantages, especially for importing and exporting plants across borders. Axenic, sterile shoots are free from many of the strict regulations surrounding the shipping of cuttings, bare roots, seeds, or other plant parts. 2. In the commercial marketplace, adulteration of Scutellaria lateriflora seeds with Teucrium , (germander) has been reported and selection of a reputable seed source is crucial to the success of both experiments and commercialization efforts. Further, the germination of some seeds under greenhouse conditions followed by comparison of the plants with herbarium vouchers is required for positive identification of the species. 3. This is a very basic set of supplies for the field. It is necessary to make many decisions on what to bring, based on where the work will take place. 4. Scutellaria seeds responded very well to this basic surface sterilization protocol. However, some species require more extensive methods to remove pathogens from the surface of the seeds. 5. Another important use of herbarium vouchers is the ability to study and observe the plants of interest to assist in identifying plants in the field. This section is a brief description of herbarium voucher mounting and preparation. Such work is considered to be an art. For further information please refer to ( 27 ) . 6. Ideally, plant material would be cultured immediately upon harvest. However, because of the nature of field collection, this is not always possible. Often material must be transported prior to culture. It is important to keep plant material damp either in a vessel of water, or wrapped in wet paper towels or newspaper. It is also important to keep plant tissues safe from extreme environmental conditions such as direct sunlight or snow. These extreme conditions can also affect the material while it is stored in a backpack, or clothing. 7. The value of a herbarium voucher is determined by the care in which the plant material is dried, pressed, and mounted. If the stem tissue is dried in a manner which shows many morphologic characteristics, the voucher will be invaluable for later studies. For detailed information on this process please see reference ( 27 ) . 8. There are many different drying methods and drying-oven designs. Some of which are very simple, such as the field method suggested above, whereas other oven designs can be very complicated. It is important to use the best design for the project. If many vouchers will need to be prepared, a larger oven may be necessary. 9. In these studies, Scutellaria was subcultured by excising stem segments, with 1–2 nodes, about 5-mm in length. These nodal segments were then aseptically placed on fresh media in a laminar flow hood. 10. For phytochemical studies , it is common to need large quantities of plant material for controls and all of the treatments in a given experiment. We found that subculturing Scutellaria into 750-mL culture vessels allowed the plantlets to grow larger and supplied much more material for extractions and chemical analysis. It is very important to culture material onto media free from plant growth regulators (auxins and cytokinins) for several weeks prior to phytochemical analysis. Some of these compounds can affect the chemical profile of samples, and could skew the data. References 1 . Paton , A. (1990) . A global investigation of Scutellaria (Labiatae) . 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Planta Medica . 68 , 128 – 132 . 6 . Li , B.Q. , Fu , T. , Yan , Y.D. , Baylor , N.W. , Ruscetti , F.W. , and Kung , H. (1993) . Inhibition of HIV infection by baicalin-a flavonoid compound purified from Chinese herbal medicine . Cell Mol Biol Res . 339 , 119 – 124 . 7 . Li , F.Q. , Wang , T. , Pei , Z. , Liu , B. , and Hong , J.S. (2004). Inhibition of microglial activation by the herbal flavonoid baicalein attenuates inflammation-mediated degeneration of dopaminergic neurons . J Neur Trans . 112 , 331 – 347 . 8 . de Boer , J.G. , Quiney , B. , Walter , P.B. , Thomas , C. , Hodgson , K. , Murch , S.J. , and Saxena , P.K. (2005.) Protection against aflatoxin- B1-induced liver mutagenesis by Scutellaria baicalensis Mut . Res . 578 , 1 – 22 . 9 . Nan , J.X. , Park , E.J. , Kim , Y.C. , Ko , G. , and Sohn , D.H. (2002). Scutellaria baicalensis inhibits liver fibrosis induced by bile duct litigation on carbon tetrachloride in rats . J Pharm Pharmaco . 54 , 555 – 563 . 10 . Zhao , Y. , Li , H. , Gao , Z. , Gong , Y. , and Xu , H. (2000). Effects of flavonoids extracted from Scutellaria baicalensis Georgi . On hemin-nitrite-H2O2 induced liver injury. Eu J Pharm . 536 , 192 – 199 . 11 . Lim , B.O. (2003). Effects of wogonin, wogonoside and 3,5,7,2 ¢ ,6 ¢ -pentahydroxyflavone on chemical mediator production in peritoneal excudate cells and immunoglobin E of rat mesenteric lymph node lymphocytes . J Ethnopharm . 84 , 23 – 29 . 12 . Moerman , D.E . (1998). Native American Ethnobotany . Timber Press , Portland, OR . 13 . Johnson , T . (1999). CRC Ethnobotany Reference . CRC , Boca Raton, FL . 14 . Foster , S . and Tyler , V.E . (1999). Tyler’s honest herbal . New York , Haworth Herbal Press, NJ . 15 . Gafner , S.C. , Bergeron , L.L. , Batcha , J. , Reich , J. , Arnason , J.T. , Burdette , J.E. , Pezzuto , J.M. , and Angerhofer , C.K. (2003). Inhibition of [3H]-LSD binding to 5-HT7 receptors by flavonoids from Scutellaria lateriflora . J Nat Prod . 66 , 535 – 537 . 16 . Awad , R. , Arnason , J.T. , Trudeau , V. , Bergeron , C. , Budzinski , J.W. , Foster , B.C. , and Merali , Z. (2003). Phytochemical and Biological analysis of Skullcap (S cutellaria laterifolia L.): a medicinal plant with anxiolytic properties . Phytomed . 10 , 640 – 649 . 17 . Kral , R. (1973). Some notes on the flora of the southern states, particularly Alabama and Tennessee . Rhodora . 75 , 366 – 410 . 18 . Krings , A. and Neal , J.C. (2001) . South American Skullcap ( Scutellaria racemosa : Lamiaceae) in the Southeastern United States . SIDA . 19 , 1171 – 1179 . 19 . Bianchi , A . (2006). Attivita antidepressiva di due specie di Scutellariae Colombiane . VI Convegno AMIAR . Torino, Italy . 20 . Murch , S.J ., KrishnaRaj , S ., and Saxena P.K . (2000). Phytopharmaceuticals: Problems, Limitations and Solutions . Sci Rev Alt Med . 4 , 33 – 38 . 21 . Saxena , P.K ., Cole , I.B ., and Murch , S.J . (2007) . Approaches to quality plant based medicine: Significance of chemical profiling . In: Applications of plant metabolic engineering . ( Verpoote , R ., ed.), Springer , NY , pp. 303 – 323 . 22 . Murch , S.J. , KrishnaRaj , S. , and Saxena , P.K. (2000) Phytopharmaceuticals:Mass-Production, Standardization and Conservation . Sci Rev Alt Med . 4 , 39 – 43 . 23 . Zobayed , S.M.A. , Murch , S.J. , Rupasinghe , H.P.V. , de Boer , J.G. , Glickman , B.W. , and Saxena , P.K. (2004). Optimized system for biomass production, chemical characterization and evaluation of chemo-preventive properties of Scutellaria baicalensis Georgi . Plant Sci . 167 , 439 – 446 . 24 . Cole , I.B. , Saxena , P.K. , and Murch , S.J. (2007). Medicinal biotechnology in the genus Scutellaria . In Vitro Cell. Dev. Biol. Plant . 43 , 318 – 327 . 25 . Pool , A. (2006). New species of Scutellaria (Lamiaceae) from Mesoamerica . Novon . 16 , 388 – 403 . 26. Wunderlin, R. P., and B. F. Hansen. (2004). Atlas of Florida Vascular Plants (http:// http://www.plantatlas.usf.edu/'>http://www.plantatlas.usf.edu/). (Landry, S. M. and Campbell, K. N., ed.), Florida Center for Community Design and Research. Institute for Systematic Botany, University of South Florida, Tampa, FL. 27 . Bridson D . and Forman , L . (2004). The Herbarium Handbook . The Royal Botanic Gardens, Kew Press , Richmond, BC . -------------------------------------------------------------------------------------------------------------------- What I find really interesting about the second article, the one about the production of secondary metabolites in Salvia spp. using liquid cultures, is that if one had the desire and right equipment, one could theoretically produce, lets say, terpenoids or diterpenoids, without even growing the plant which would usually produce them. Personally, I would prefer to grow the plant, but I find it intriguing nevertheless. Some pics of micropropation with an ethnobotanical twist. Salvia clones from micropropagation: Lophophora fricii: Callus culture Callus culture with shoot formation Lophophora phase 1: Lophophora phase 3: Lophophora jourdaniana phase 2: Trichocereus pachanoi phase 1 explants: T. pachanoi phase 3: T. pachanoi phase 3 explants: While searching for information on micropropagation here, I found a post by teonanacatl saying that he had an article on the micropropagation of Lophophora species specifically that he was going to pass on to darklight. Does anyone have a copy of that article?
  3. I bought this from SAB a while back as a KK Trichocereus uyupampensis. I've looked at a few images online and it looks a decent match for the species, but again, it looks like it has T. cuzcoensis traits in it too. Are the two species closely related? This is mine: From Trout: From M S Smith Opinions?
  4. tripsis

    RIP andyamine

    Been a long time since I've been on this forum, only just seeing this now. Literally a day too late for the camp it seems. Really sad news to hear. He was far too young to pass, particularly given the circumstances. Much love to all who were close with him.
  5. Wondering if anyone has experience using Pereskia species as grafting stock? Would they confer comparable growth rates to scions as Pereskiopsis species do?
  6. tripsis

    EGA Ticket for sale $350

    Wish I could get the time off for this. So disappointing...
  7. tripsis

    Chlorociboria aeruginascens

    Some shots of some Chlorociboria aeruginascens I found this morning. Unfortunately, I didn't have my camera with me, only my phone.
  8. Looking into small commercial scale propagation of Australian natives for a conservation project, and trying to decide on what kind of greenhouse / shadehouse to build, or whether one is even required. Anyone have any useful input or experience?
  9. I'm going down the path of hardening them off from the outset. Now for the hard part - working out materials, systems, and labour, and providing costings estimates. Short deadline to do all this. If funding is approved, I'll let you know how it goes.
  10. Good tips, thanks for the info. That's what I figure with the tunes too, ultimately bot allowing seedlings to get rootbound is the best approach. Do you make your own media? Or what the native mix from somewhere like ANL be adequate? Given this first stage will be a trial, I'm really looking for optimal success rates to help guarantee that it will be scaled up.
  11. Also, any input on whether those tubes with internal ribs really do promote beneficial root development?
  12. That's right, Skellum, it'll be a diverse range of species from grasses and groundcovers through to trees. I was hoping you'd reply, @waterboy 2.0. That's exactly what I was thinking, grow them hard to reduce mortality later. Shade is definitely a consideration for understory species. What do you think of a tunnel with wire mesh, and shade at one end, to protect from animals? Pallets or pallets on crates as benches?
  13. Interested in hearing how you're using your still air box / how you've made it. I expect that if technique is your problem, using a laminar flow hood will not help you much.
  14. tripsis

    Ethnobotany book collection for sale

    Willing to split at all? Only after one book in there really...
  15. tripsis

    Unable to send PMs

    Many thanks, Torsten.
  16. tripsis

    Unable to send PMs

    Logged on for the first time in a while and tried to send a PM to another member. Unfortunately, the dialogue box for the message body appears to be broken. The dialogue boxes for the recipient name and subject are fine, and can be written in, but the message box isn't present. Can this be fixed, please?
  17. tripsis

    Unable to send PMs

    Attempting to create a new one here, so that's not a solution for me unfortunately.
  18. tripsis

    Unable to send PMs

    I tried that, but the field is still not there, just a message that it needs content in it.
  19. tripsis

    Pleurotus nebrodensis

    Hey Will, if this is the culture you received from me many years ago, I've always questioned whether it really is Pleurotus nebrodensis. If memory serves me correctly, I received the culture from Paul (speedy); I'm not sure how he came to the conclusion it P. nebrodensis. Interestingly, looking into it again now, I see Aloha Medicinals sells a culture of it (and uses one of my photos without permission - seems to be a few sites doing that with my photos), originating from China. This article discusses some of the relevant taxonomy if you're interested. Anyway, good to see you're enjoying the fruits of your labour!
  20. tripsis

    Post your track of the day

    Can't believe this thread is still going. I almost feel bad for starting it. It's become an immense, unwieldy beast, squandering bandwidth on a site that has historically, and continues to, struggle to meet server costs.
  21. Couldn't agree more. Entheogenesis is an incredible collaboration, which brings together people from all walks of life, and with a very diverse set of backgrounds, interests, and approaches to the questions and subjects discussed at these events. Hard to believe how many years have passed since the last outdoor event. I'll be there 100%.
  22. Photos of Ephedra species from a recent trip to Central Asia. Ephedra equisetina or E. intermedia in Kyrgyzstan. Not sure which species. Both occur in Kyrgyzstan and both have red fruit. I only got to see the fruit out of a moving vehicle. E. fedtschenkoae, Kyrgyzstan. E. gerardiana, Tajikistan. Much smaller than the ones I found in India. Found very close to the border of Afghanistan, literally just across one of the border rivers. E. regeliana, Tajikistan. Found in the same area as some of the above E. gerardiana.
  23. tripsis

    Ephedra in Kyrgyzstan & Tajikistan

    The latter, but I knew they would be around and was looking for them the whole time. There were many other beautiful and interesting plants in the region too, but I decided only to post photos of the Ephedra species. And for the record, so that others don't ask, I did not collect any seed.
  24. Been a long time aince I last felt the compulsion to post here, but this article is just so incredible that I had to share it here. Hope some of you find it as much of an incredible read as I did. Bread Wheat Genome Contains “Shocking” Plot Twist By Jennifer Frazer | July 18, 2014 "Wheat P1210892" by Copyright © 2007 David Monniaux - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons. Yesterday scientitsts announced in a quartet of papers in the journal Science that the draft genome of bread wheat -- Triticum aestivum -- had been decoded and mapped. Together with barley, wheat is the crop on which civilization rose in the Fertile Crescent and Egypt some 10,000 years ago. With theses grasses and the help of wild yeast, humans created bread and beer and have rarely looked back (Prohibition and the current gluten-free fad being notable exceptions). I covered the story over at National Geographic. The content of the genome was not a surprise, Robert Bowden, supervisory research plant pathologist at the U.S. Department of Agriculture's Hard Winter Wheat Genetics Research Unit in Manhattan, Kansas, told me. What was unexpected, he said, was what the genome told scientists about the evolution of wheat, as detailed in a second paper released concurrently with the genome by Marcussen et al. In the genome, "we found pretty much what were expecting," Bowden said. "The second paper was the one was the one that was kind of shocking, because we thought we understood a lot about the evolution of wheat.” Indeed, scientists did understand a lot about the evolution of wheat. But they didn't know everything, hampered by a lack of wheat fossils and by the intractably large and repetitive wheat genome, which had resisted sequencing. You can read more about that story over at Nat Geo. For example, scientists had known for some time that wheat is a triple "polyploid", a hybrid of three parent species of wheat who through two accidents of biology had merged two genomes into one to produce emmer or durum wheat (used primarily for pasta today, though probably for different purposes by the ancients), and then two into three to produce bread wheat with a genome three times as big as that of its ancestral genomes. You can read more about this process in a blog post I wrote about polyploidy in plants here. But without a map of the genome, answering questions about how the three parent species of wheat were related to each other (they were presumably close relatives) was difficult to impossible. Then along came the draft wheat genome, and suddenly lots of things were possible. Thomas Marcussen, Odd-Arne Olsen, and Simen Sandve of the Norwegian University of Life Sciences and their colleagues in Norway, Germany, and the UK initially set out to date the two known polyploidy events and find out how the three wheat parents were related to one another – a topic that had been controversial for some time due to the fossil and genome void. They expected a bifurcating tree in which two of the parent species -- they were not sure which two -- were more closely related to each other than the third. Instead, they discovered a more complex situation. Instead of two hybridizations in wheat's past, there now appeared to be three. “We really couldn't make a model that look liked a normal bifurcting tree based on our data,” Sandve said. “We had to try to make it into a network to make an evolutionary model that would fit the data.” Wheat parents Triticum urartu and Aegilops speltoides were equally closely related to Parent #3 Aegilops tauschii and more closely related to A. tauschii than to each other. That could only make sense if the ancestors of T. urartu and A. speltoides had hybridized to produce the ancestor of A. tauschii via a process called monoploid hybridization. This type of hybridization can only take place between two very closely related species. It happens when a normal egg cell from one species meets a normal sperm cell from another and the species are not so distantly related that the sperm can't fertilize the egg. A familiar example is the production of a mule from the mating of a donkey to a horse -- two different species. In that case, the mule is usually infertile, but in the case of wheat, A. tauschii was evidently good to go. In short, the wheat family tree is beginning to look distressingly similar to the Hapsburgs'. "It's complicated": The convoluted family tree of wheat. AA = T. urartu, BB = Ae. speltoides, and DD = Ae. tauschii. AABB = emmer/durum wheat (T. turgidum), and AABBDD= modern bread wheat, T. aestivum. The first speciation event is homoploid hybrid, the second two are polyploid (as seen by chromosome copy increase). Images show extant wheat closely related to respective species. The circles indicate dates of hybridizations in millions of years ago. The lines connecting A and B to D are what's new here. Fig. 3 from Marcussen et al 2014. Click image for source. The discovery fits with other data in plants like sunflowers that seem to show that this type of direct hybridization that seems to have produced A. tauschii – called homoploid hybridization – may be more common in plants than previously thought, Sandve said. Bowden said he was taken aback by these results. “Instead of there being two speciation by hybridization events in the evolution of wheat, there's three, which is shocking. I don't think anybody was expecting that,” he said. “If it's true, and I think it is true, it's a really really unexpected result and shows the power of this method of analysis and leveraging all the data that [the draft genome] produced.” He said they would have to rethink how they approached mining the A. tauschii genome for useful traits for new wheat varieties. Although it no doubt still contains valuable traits, it is no where near as old or independent of the T. urartu and A. speltoides subgenomes as they had thought, the said. In addition, from an evolutionary perspective, he said, it's intriguing that the ancestral T. urartu and A. speltoides genomes hybridized twice in two different ways – once to make homoploid hybrid A. tauschii, and once in polyploid fashion to produce emmer/durum wheat (T. turgidum). “It's very interesting that these two different kinds of event happened with the same two species to start with,” he said. “and it also says 'Wow, this is not very rare. It happened three times just in the evolution of wheat!'” Source.
  25. Since a forum upgrade maybe 6 months or a year ago, the "View New Content" tab hasn't worked for me, instead yielding only "Sorry, no new content found.". If I'm not logged in, it works, but once logged in, there is never any new content. What's going on?