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Micropropagation techniques

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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!

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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.

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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.

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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.

Huang, M.T., Ho, C.T., Wang, Z.Y., Ferraro, T., Lou, Y.R., Stauber, K., Ma, W., Georgiadis,

C., Laskin, J.D., Conney, A.H. (1994). Inhibition of skin tumorigenesis by rosemary and its

constituents carnosol and ursolic acid. Cancer Research, 54, 701–708.

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(1998). Comparative study of seventeen Salvia plants: Aldose reductase inhibitory activity

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Li, L.N., Tan, R., Chen W.M. (1984). Salvianolic acid A, a new depside from roots of Salvia

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Kluwer Academic, Dordrecht, pp. 700–705.

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Protocols for Establishment of an In Vitro Collection

of Medicinal Plants in the Genus Scutellaria

Ian B. Cole , Faisal T. Farooq , and Susan J. 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).

(B) 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.

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, C. , Hodgson , K. , Murch , S.J. , and

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Sohn , D.H. (2002). Scutellaria baicalensis

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on chemical mediator production in peritoneal

excudate cells and immunoglobin E of

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--------------------------------------------------------------------------------------------------------------------

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:

salvia3_20040601142656.jpg

salvia_phase4_totally_hardened_off_20040508010247.jpg

salvia4_2_20040413005206.jpg

salvia_20040302232251.jpg

salvia1_20040217134513.jpg

Lophophora fricii:

Callus culture

fricii_callus3_20040229232312.jpg

Callus culture with shoot formation

fricii_callus_buttons_20040217234447.jpg

Lophophora phase 1:

peyote_phase1_20040217235201.jpg

Lophophora phase 3:

loph_phase3_20040217134733.jpg

Lophophora jourdaniana phase 2:

fricii_phase2_20040217142801.jpg

Trichocereus pachanoi phase 1 explants:

phase1_2_20040214010018.jpg

phase1_20040214005959.jpg

T. pachanoi phase 3:

pedro_phase3_20040217134807.jpg

T. pachanoi phase 3 explants:

phase2_20040214010559.jpg

phase2-2_20040214010539.jpg

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?

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I havent seen the article from teonanacatl, but i lifted this from mycotopia a while back.

For Lophophora Williamsii medium:

Medium

---------

Base:

Standard Murashig and Skoog basal salt

Vitamines:

Niacin 0.5 mg/l

Thiamine Hcl 0.5 mg/l

Pyrodoxine Hcl 0.5 mg/l

I-Inostitol 100 mg/l

Dextrose:

30 g/l

Agar:

10 g/l

Combine 100ml of above with:

1 ppm (1ug/ml) 2,4-D (2,4-Dichlorophenoxyacetic acid)

5 ppm (5ug/ml) BA (6-Benzylaminopurine aka. benzyl adenine)

To start your Lophophora tissue before you subculture onto the above medium, use the same mix, but change the following:

include: 2,4-D (same concentration, 1ppm)

add: 10-15% liquid endosperm

exlude: 5 ppm 6-Benzylaminopurine

Lighting should be a 16/8 day/night cycle as well...

The cultures also like blue light, not so much the red.

From this initiation medium, to the subcluture medium you could experiment and find something better maybe?

Or i have a PDF of MICROPROPAGATION OF CACTUS (Opuntia ficus-indica) that i can send you if you like.

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Seems like pretty decent information. What do they mean by "liquid endosperm" I wonder?

Now to find sources for hormones in Australia...

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Seems like pretty decent information. What do they mean by "liquid endosperm" I wonder?

Liquid endosperm usually = coconut water. That's *water*, not milk. Don't buy it from the supermarket! You can prepare it yourself, it requires cooking and filtering.

http://plant-tc.cfans.umn.edu/listserv/1998/log9803/msg00205.html

http://www.sigmaaldrich.com/life-science/molecular-biology/plant-biotechnology/tissue-culture-protocols/media-preparation.html#coconut

But really, buy it in, save yourself time and hassle. Coconut water is what's called a biological indeterminate, inasmuch as it's chemical composition can vary widely between batches

I got a copy of the paper from Teon, but it didn't work for me and I didn't keep a copy. Even different batches of seed can give different results, the media didn't agree with my stock

The media was used to try to initiate callus and regenerate plants from 2yo stock in-vitro plants and looked like this:

Lophophora callus induction

Murashige and Skoog Basal Media ( with vitamins ) + 30g/L sucrose + 1mg/L 2-4D MS + 10% coconut water + 7g/L gelcarin pH5.8 under coolwhite lights 16/8 at 24C

Lophophora callus prolif

Murashige and Skoog Basal Media ( with vitamins ) + 30g/L sucrose + 1mg/L 2-4D + 5mg/L BAP + 10% coconut water + 7g/L gelcarin pH5.8 under coolwhite lights 16/8 at 24C

I got callus but no regen, which is pretty much the case once you've pushed your plants too far into bat country when initiating the callus

Am just including this so you know it's been tried unsuccessfully in my case. It's almost identical to the one from Mycotopia but there is a slight difference with the vitamins- which sometimes can make a difference, lophs are weird about vitamins IME. Worth a shot.

Were the people who posted the media recipe at Mycotopia the same people who posted the pics? The pics are very lovely, but sometimes they're using a different media to the people who may have just posted the media and never have tried it. Worth checking

Note: ALWAYS always always ( and I can't stress this enough ) check to see which version of Murashige and Skoog is used in a paper. The terms basal salts and basal media can refer to two different formulations. They can even be wrongly attributed to each other. One has vitamins in it, one doesn't. When looking to make up or purchase media always definitely check with the publication authors if you're uncertain. Get them to supply catalogue numbers so you can check against a supplier's catalogue. I've seen experienced scientists make this mistake, though not for a while mind you :)

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Ebay or the kitchen TC web site link i sent you.

I did send you the link didn't i???

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Interesting that they use coconut endosperm to stimulate growth in other plants! Thanks for the links and info DL, very helpful. :)

I got callus but no regen, which is pretty much the case once you've pushed your plants too far into bat country when initiating the callus

What do you mean by bat country?

Not too much to lose from experimenting. Obviously micropropagation of Lophs can be done, so I guess it's just a question of working out the best method and refining it.

When looking to make up or purchase media always definitely check with the publication authors if you're uncertain. Get them to supply catalogue numbers so you can check against a supplier's catalogue

How would you go about doing that?

Yes, you did send me that link shortly. I'll have a look at it again .:)

Also, cheers for the link dworx. Looks like a decent option for someone just starting out.

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Interesting that they use coconut endosperm to stimulate growth in other plants! Thanks for the links and info DL, very helpful. :)

My pleasure :)

What do you mean by bat country?

Getting callus is easy. Often easier than getting cells to do something organised,like organise into roots and shoots suitable for more work. For a lot of species you just throw a little 2-4D at it. Or mess with the media hormone levels some other way and you'll have callus

Getting the callus quality and type you want is way harder. If you add too many hormones ( bat country ), or the wrong kind, your callus could be too mushy, or contain the wrong kind of cells ( you might be after scutellum callus, for example, but get hypocotyl ). It could also die. Or just refuse to do anything but replicate more callus.

Callus is easy for a plant to make, it requires less energy and direction than becoming something organised

Even if you get good looking callus from your species, changing the hormones/ media/ culture parameters to something which can regenerate can be a challenge. Often you'd start by dropping the main media and sugar levels and putting in some auxin if you are working on a species without a protocol. But not always :)

This makes it sound way more complicated than getting the actual experiment done. You can have a lot of theories but you can only work so many experiments at once, just do it and make good notes

Really, just always save enough material back so you can run another experiment if you need to and you'll get the hang of it. If something has gone too far, realistically it's better to start on the material you didn't put in the current experiment than it is to try to rescue something that's too far gone and is as confused by now as you are

Not too much to lose from experimenting. Obviously micropropagation of Lophs can be done, so I guess it's just a question of working out the best method and refining it.

You got it. I'm gunna help you out here because you're such a nice bloke. The best paper I've found is in Spanish. And the way you select and cut your explants makes all the difference. Young bloke from the forums here worked it out, he did better than me on it at my place ( and he deserved it too )

Keep good notes- record everything you do when you start. Even little stuff you might not think is relevant. Eventually you'll work out your own shorthand and figure how many of all those details you'll need later to make sense of your own notes 5 years on.

If you're wanting a squillion lophs, it might just be quicker to seed raise them. If you want to learn about tissue culture, lophs are a good way to start. Take it from seed, as fresh as possible. Find a container to sterilise them in so they don't just float off when you pour the bleach off ( I use a 30ml polycarb tube with stainless mesh on either end, teabags are a pain ). Usually 2 min in 1.5% final concentration of scentless household bleach + a drop of scentless detergent will do small seeds nicely

Rinse 3 x with sterile water, put them on sterile #1 filter paper to dry in the hood ( keeps bacterial contam down ). Plate 'em up and watch 'em grow!

And let us know how you do. Good luck!

Also, cheers for the link dworx. Looks like a decent option for someone just starting out.

It does hey, wonder how they manage the flow hood thing? Do they assume you have one? Or do they throw in a perspex tunnel?

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No worries I was looking at this a couple of years ago, there are people doing it in Australia with natives @ home, so I guess its not all that hard, just a time thing for me, but I knew they sold kits on ebay and their are others selling them from their own websites....

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No worries I was looking at this a couple of years ago, there are people doing it in Australia with natives @ home, so I guess its not all that hard,

It's not hard, It's disciplined but the discipline becomes habit after a bit.

Australian natives can be tricky buggers, I'm glad people have persisted and had success with them at home, or even just lucked out ( which happens a lot ) and done well

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Thanks for the articles guys. I'm looking at getting into this to get some interesting plants passed around more in the community. The main problem I am having is what sort of containers are suitable. I contacted Cospack (found thru Darklight's advice and Google) but have received no response.

Cheers!

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Thank you again DL, it is very helpful to have someone to talk to who actually has experience with plant tissue culture. :)

It makes sense that getting a callous culture would be the easy part, seeing as it's basically just a mass of relatively undifferentiated cells dividing without a specific purpose. thus it also makes sense that regenerating that callous would be more challenging.

Taking notes is definitely something I need to work on, so it's good that you've stressed the importance of it. Something I'll be sure to practice when I begin. :)

A couple of questions on equipment/technique. You mentioned a flowhood. I've got plenty of experience with fungal tissue cultures, but the media preparation is far simpler than with plants and much less precise, as is the entire process. Also, a flowhood is not necessary, although obviously preferred. I don't own a flowhood and can't see myself having one for a fair amount of time. Will a glovebox do with plant tissue culture? Also, regarding taking/selecting explants (I can't read Spanish), I've never understood how people manage to take a sterile explant from something as thin as a leaf. Is it more of a reliance on chemicals, such as fungicides and antibiotics, that allow non-sterile explants to be used to induce a callous culture? E.g. some of the explants from the cacti in those MushMush photos I posted above have intact cuticle and all, which I would have expected to be covered with contaminants.

While I love Lophs, having a squillion of them is not my aim with micropropagation, although I do like the idea of propagating them in different (and novel) ways. I'd like to have the ability to propagate rare species/varieties/clones of various plants so that they are more widely available to the community. Micropropagation of Ephedra species would be interesting. :)

IceCube, try Plasdene Glass Pak. Much the same stock, different company.

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It makes sense that getting a callous culture would be the easy part, seeing as it's basically just a mass of relatively undifferentiated cells dividing without a specific purpose. thus it also makes sense that regenerating that callous would be more challenging.

Spot on

Taking notes is definitely something I need to work on, so it's good that you've stressed the importance of it. Something I'll be sure to practice when I begin. :)

You should see some of mine, especially the earlier ones. Some contain stuff like " oh shit, I was pretty crazy when I did that experiment, can't remember whether the hormone stock was in date or not. May have used IBA instead of IAA. It worked, but repeat at a later date to confirm" or " bloody ferment flies hanging round in the ethanol rinse in the flow hood. Could be contam probs, monitor"

Putting stuff into context can give you a trigger which might help you remember later. They're your notes at this stage, they're not the intellectual property of a corporation or academic institution. Go crazy. It's a part of the creative process :)

A couple of questions on equipment/technique. You mentioned a flowhood. I've got plenty of experience with fungal tissue cultures,(snip

Will a glovebox do with plant tissue culture?

Hell yes. You can even work under a perspex tunnel. I'm still trying to find a pattern for one as mine was er.. borrowed ten years ago. Cost about $50 these days. I want one too, so if I can find one I'll put the pattern up. Great for low volume work up to 100 tubes a day

Also, regarding taking/selecting explants (I can't read Spanish),

You don't need to. Scientific terms and abbreviations are mostly interchangeable, as are Latin names. A lot of the rest can be sorted using Babelfish, at least to the point where you can write up a protocol to try

I've never understood how people manage to take a sterile explant from something as thin as a leaf. Is it more of a reliance on chemicals, such as fungicides and antibiotics, that allow non-sterile explants to be used to induce a callous culture? E.g. some of the explants from the cacti in those MushMush photos I posted above have intact cuticle and all, which I would have expected to be covered with contaminants.

One of the main theories of tissue culture is that every cell is totipotent- ie each cell can, under the right condition, replicate and differentiate into any other cell or organ of that species. In practice this isn't the case yet or we'd be using zip drive livers instead of caning the one we were born with. But with the right protocol you only need a few cells of the right type and in the right media to get the ball rolling. So yeah, in some cases even a whole leaf is a big bite to start with

Surface sterilisation is a multi-headed beast. Careful pretreatment of the plant will help lower the nasties you will do battle with. Starting from seed is in a lot of cases much easier ( and in the beginning it's good to plan for the easy stuff cos there are lots and lots of variables you need to think about as you learn). And you can sterilise a larger portion of an explant and then cut away aseptically so you only have a few cells of the type you want ( go the apical meristem ). Media can also play a part in decreasing contam risks ( reducing to say 50% sugar levels until established ). Repeated subcultures in the early phase to out-run contamination. Sonication. Benomyl presoak. Etc. You probably won't encounter these in your work with Lophs at this point. Surface sterilisation can be tricky if the obvious things don't work out but it's usually pretty basic.

There are a lot of things you can do to reduce contam in the planning phases. Some of these will kill your stuff. It's trial and error if you don't have a protocol, and believe me I've lost a lot of biomass. A successful surface sterilisation for a new species without a protocol which has a 70% mortality rate is considered an outstanding success if the rest of the explants proliferate. Like I said, just make sure you don't use up all your explants in the one experiment- keep some back

Micropropagation of Ephedra species would be interesting. :)

I've done it, but had a process problem at the last point which I couldn't resolve before seed stock became more available. Quicker to get seed stocks out there, given the deflasking hassles you could have too.But give it a go

IceCube, try Plasdene Glass Pak. Much the same stock, different company.

I use lots of the Cospak H1135 babyfood jars ( dunno if that's the catalogue number, it's printed on the bottom of the jar ) with Magenta B cap lids. The lids are exxy but last for years. Also 30ml polycarb tubes with screw cap lids from Technoplas. But if you look around there might be better ghetto tek containers, just make sure they can be sterilised at 15psi- the vendor should be able to supply that information

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How would you go about doing that?

Contacting the authors? If there is a published protocol the authors contact details will be correct at the time of publication. Or you could google their names and see where they are now

Before you hit Send and fire the email off, confirm exactly what you're confused about by referencing the paper and quoting it in the text of your email. Don't do it from memory, or you risk falling over silly.

ie. in your paper " Micropropagation of Dragibus curiosa via axillary proliferation " published Journal of Alchemic Quackery 1986 you stated on page 786 that that you used "Murashige and Skoog media", but I can't find a reference to which MS media was used in either the body of the text or the bibliography. Is it possible to clarify this- was it basal salts or basal medium? Do you have a record of the supplier's catalogue number for this product?

Throw something in about how useful you found the paper, or some other nice, sincere thing, it helps. Then and only then can you press Send

Lots of scientists get thrilled if you write them sensible emails asking relevant questions about their work, even if you're a beginner. Some even write back. I got a lot of such help in my early days

If you get a reply, use it to cross reference the original paper, go to the supplier's web site and check everything against their tech specifications for that product, they're usually on there somewhere or available by email on request

Edited by Darklight

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It's encouraging to hear that the such scientific techniques can still be recorded in any way that works. Better to take notes that make sense to you and flow easily than to take notes that you think sound/look good and are next to useless.

Hell yes. You can even work under a perspex tunnel. I'm still trying to find a pattern for one as mine was er.. borrowed ten years ago. Cost about $50 these days. I want one too, so if I can find one I'll put the pattern up. Great for low volume work up to 100 tubes a day

What do you mean by a perspex tunnel? I tried searching for it on Google but didn't get much out of it.

You don't need to. Scientific terms and abbreviations are mostly interchangeable, as are Latin names. A lot of the rest can be sorted using Babelfish, at least to the point where you can write up a protocol to try

Do you have the article/author name? I think based on what you said, I'd be able to get at least the general idea of what is being described in it.

One of the main theories of tissue culture is that every cell is totipotent- ie each cell can, under the right condition, replicate and differentiate into any other cell or organ of that species. In practice this isn't the case yet or we'd be using zip drive livers instead of caning the one we were born with. But with the right protocol you only need a few cells of the right type and in the right media to get the ball rolling. So yeah, in some cases even a whole leaf is a big bite to start with

All plant cells are supposedly totipotent, except perhaps seed coats and the such. But as you say, in practice the reality is different. So it then becomes more about taking an explants from an area with undifferentiated or slightly differentiated cells (such as meristematic tissue), in the hopes that these cells with survive pretreatment and go on to become a callous culture?

Surface sterilisation is a multi-headed beast. Careful pretreatment of the plant will help lower the nasties you will do battle with. Starting from seed is in a lot of cases much easier ( and in the beginning it's good to plan for the easy stuff cos there are lots and lots of variables you need to think about as you learn). And you can sterilise a larger portion of an explant and then cut away aseptically so you only have a few cells of the type you want ( go the apical meristem ). Media can also play a part in decreasing contam risks ( reducing to say 50% sugar levels until established ). Repeated subcultures in the early phase to out-run contamination. Sonication. Benomyl presoak. Etc. You probably won't encounter these in your work with Lophs at this point. Surface sterilisation can be tricky if the obvious things don't work out but it's usually pretty basic.

I suppose this is what I hadn't got my head around yet. I have rarely tried to surface sterilise fungal fruitbodies when cloning them, so don't really think about it when I think about taking an explant. Repeated subculturing is something I would have expected. Sonication...hadn't even come across the term before! You'd either have to have access to a lab or be a dedicated enthusiast to make use of it. Benomyl...where would you get it? It's banned in most countries. It would be damned useful to have, but a nasty and very dangerous fungicide to have around the house. I suppose the point is that there are various pretreatments that can be done depending on where the explant is being taken from and what works/doesn't work for it, thus the explant does not need to be from interior tissue to be successful.

I've done it, but had a process problem at the last point which I couldn't resolve before seed stock became more available. Quicker to get seed stocks out there, given the deflasking hassles you could have too.But give it a go

Very interesting. Do you find xerophytes are harder to work with than mesophytes, or does it not make a difference? I'm wouldn't consider seed to be easily available for most species still, so propagating what is here seems like a good idea. But a clone army isn't good either, as the option to breed plants should always be there, at least I think so.

Finally, do you use a micropipette for measuring small and precise quantities of the chemicals needed?

Alright, that's surely enough questions for now. I'll do some reading. :)

:wub:

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Contacting the authors? If there is a published protocol the authors contact details will be correct at the time of publication. Or you could google their names and see where they are now

Before you hit Send and fire the email off, confirm exactly what you're confused about by referencing the paper and quoting it in the text of your email. Don't do it from memory, or you risk falling over silly.

ie. in your paper " Micropropagation of Dragibus curiosa via axillary proliferation " published Journal of Alchemic Quackery 1986 you stated on page 786 that that you used "Murashige and Skoog media", but I can't find a reference to which MS media was used in either the body of the text or the bibliography. Is it possible to clarify this- was it basal salts or basal medium? Do you have a record of the supplier's catalogue number for this product?

Throw something in about how useful you found the paper, or some other nice, sincere thing, it helps. Then and only then can you press Send

Lots of scientists get thrilled if you write them sensible emails asking relevant questions about their work, even if you're a beginner. Some even write back. I got a lot of such help in my early days

If you get a reply, use it to cross reference the original paper, go to the supplier's web site and check everything against their tech specifications for that product, they're usually on there somewhere or available by email on request

Great, thanks for that. The only times I tried to find out the contact details of some authors was a few months back in India, regarding a couple of papers I had read, but there was no contact info and I couldn't find any by searching the web for them either. Probably a good thing really. I expect it must be done differently here. :)

Yes, I expect if I were a scientist publishing articles and I received emails asking for details and leaving positive remarks, I would appreciate it too.

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What do you mean by a perspex tunnel? I tried searching for it on Google but didn't get much out of it.

Me either. They rock too. Wish I could get the dimensions on my old one.

They're clear, shaped like half a hexagon, with the bottom cut out. You sterilise them, everything that goes in them, and have some 70% ETOH soaked paper towells on the bottom. Sterilise your arms and hand before you put them in from the sides. They're cheap and incredibly portable and they work

Do you have the article/author name? I think based on what you said, I'd be able to get at least the general idea of what is being described in it.

Sorry, it was a theoretical article for a theoretical question

All plant cells are supposedly totipotent, except perhaps seed coats and the such. But as you say, in practice the reality is different. So it then becomes more about taking an explants from an area with undifferentiated or slightly differentiated cells (such as meristematic tissue), in the hopes that these cells with survive pretreatment and go on to become a callous culture?

Yep. But don't get so hung up on callus culture as a means of mass replication. It is usually easier for most species to use axillary proliferation. Callus is generally good for fast replication but has the disadvantage of causing mutations and instability ( ie refusal to regenerate ) in the long term.

http://en.wikipedia.org/wiki/Somaclonal_variation

I suppose this is what I hadn't got my head around yet.

Don't worry about it except on a case by case basis. Try the standard stuff first, what's in the protocol, what usually works. Then add new steps incrementally. This will be easy because you will have kept enough plant material back with each experiment to work with in a new experiment ;)

Sonication...hadn't even come across the term before! You'd either have to have access to a lab or be a dedicated enthusiast to make use of it.

Buy a sonicated jewellery cleaner. Cost about $100 and do effectively the same thing for our purpose

http://www.sonicsonline.com/jewelrycleaners.html

Lots of lab gear has ghetto tek equivalents

Benomyl...where would you get it?

I think Sigma has it still under it's chemical name. You used to be able to get it in only in 5kg lots, and it lasts forever. Practically every lab has or knows of a stash somewhere

I suppose the point is that there are various pretreatments that can be done depending on where the explant is being taken from and what works/doesn't work for it, thus the explant does not need to be from interior tissue to be successful.

Exactly. But work it backwards too- you want to set up an explant that is as clean as possible so you can muck around with different types of tissue just in case the thing you're trying doesn't work

Very interesting. Do you find xerophytes are harder to work with than mesophytes, or does it not make a difference?

Mmm, I've never given it any thought. But no, the explant source is more important than anything. Dirty parent plants are a pain in the long term because ultimately you get less material from them to work with and you lose a lot in the process. Sometimes you run out of plant before you run out of ideas

But a clone army isn't good either, as the option to breed plants should always be there, at least I think so.

Spot on. Try to emphasise re-establishing diversity in a population when working on endangered species. In the old Mitragyna days we emphasised that the clones were single source and some back breeding was necessary to re-establish a solid genetic base. A few people took that up in the US and it's a worked

Finally, do you use a micropipette for measuring small and precise quantities of the chemicals needed?

I do now, but you don't need to to start. Back then I used a shotgun shell reloading scale- beam type ( extremely accurate to about 1/15 gram, cost about $50 ) and diluted all my chems so they could be measured with a 2 or 5ml syringe. instead of having to measure 5mg of something, I could dilute it to 5mg/ml in whatever solvent was appropriate and dispense it that way

Only downside was that I frequently had to make up new stocks of stuff that went off in storage. But hey, when you buy Thiamine.HCl in 100g lots it takes a long time to go through it at 10mg/ml every month :)

Alright, that's surely enough questions for now. I'll do some reading. :)

You ask really cool questions, I really appreciate your interest :) but don't overthink it or you'll wear your energy for it on the theory rather than learning the practical side. Have a go at it, I'll talk you through as you proceed if you like, we can do it here and other people will learn from it too

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They're clear, shaped like half a hexagon, with the bottom cut out. You sterilise them, everything that goes in them, and have some 70% ETOH soaked paper towells on the bottom. Sterilise your arms and hand before you put them in from the sides. They're cheap and incredibly portable and they work

So similar to a sturdy glovebox, but one that can be completely sterilised? You'd need a pretty sizeable autoclave to fit something large enough to accomodate hands and equipment inside. I like the idea of being able to have a portable sterile workspace.

Sorry, it was a theoretical article for a theoretical question

:lol:

Gotcha. Point taken anyway. :)

Yep. But don't get so hung up on callus culture as a means of mass replication. It is usually easier for most species to use axillary proliferation. Callus is generally good for fast replication but has the disadvantage of causing mutations and instability ( ie refusal to regenerate ) in the long term.

Had to look axillary proliferation up. Basically it's taking an apical/axillary growth point with the meristematic tissue present and placing it onto the media to induce axillary budding? Such as depicted here:

peyote_phase1_20040217235201.jpg

So doing this skips the need to create a callous culture, but allows for the quick propagation of many buds/plantlets by utilising the plants own budding mechanisms? With less succulent plants, once a bud has grown, do you then cut it and strike it like you would an ordinary cutting?

It's interesting that mutations can occur during callous culture growth. This could lead to interesting new variants, but I suppose more often than not, this isn't the aim.

Buy a sonicated jewellery cleaner. Cost about $100 and do effectively the same thing for our purpose

Should have thought about that. Have heard of them used in mycological applications before.

I think Sigma has it still under it's chemical name. You used to be able to get it in only in 5kg lots, and it lasts forever. Practically every lab has or knows of a stash somewhere

You're right, they do sell it! It's strange after searching for it on several occasions that I never found it there. Considering the risks associated with it, do you recommend using it or are there now safer alternatives? Because it is a largely mould-specific fungicide, at least when the dosage rate is low, it can be very useful in mycology, but surely targetting specific types of fungi is necessary when surface sterilising explants?

Exactly. But work it backwards too- you want to set up an explant that is as clean as possible so you can muck around with different types of tissue just in case the thing you're trying doesn't work

Makes sense. So interior tissues would be preferable as there should be less contaminants present, but the option to surface sterilise exterior tissues shouldn't be dismissed, as there may be other cell types present which could work better?

I do now, but you don't need to to start. Back then I used a shotgun shell reloading scale- beam type ( extremely accurate to about 1/15 gram, cost about $50 ) and diluted all my chems so they could be measured with a 2 or 5ml syringe. instead of having to measure 5mg of something, I could dilute it to 5mg/ml in whatever solvent was appropriate and dispense it that way

That's a good idea. I'd like to get a micropipette, but they're not cheap. If dissolving in solvents such as ethanol or acetone, can/will this affect the culture, or does it evaporate off?

You ask really cool questions, I really appreciate your interest :) but don't overthink it or you'll wear your energy for it on the theory rather than learning the practical side. Have a go at it, I'll talk you through as you proceed if you like, we can do it here and other people will learn from it too

I really appreciate your help. :) For now, all I can do is study the theory as I'll be going to NZ for a month in a couple of weeks, so won't be able to start until after that. But once I do, I'll be sure to document my progress here, and will no doubt have more questions as I actually begin the practical side.

One other question, when using seeds as your starting material, is the idea to germinate them in-vitro, then cut the seedling up to make further cultures from?

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So similar to a sturdy glovebox, but one that can be completely sterilised? You'd need a pretty sizeable autoclave to fit something large enough to accomodate hands and equipment inside.

Perspex doesn't autoclave well I don't think. You surface sterilise it with 70% ethanol ( 70% metho is good for most things )

Basically it's taking an apical/axillary growth point with the meristematic tissue present and placing it onto the media to induce axillary budding? Such as depicted here:

Yep :)I think in cactus it's also called aureole activation, not sure if there is some kind of technical difference between those two terms

So doing this skips the need to create a callous culture, but allows for the quick propagation of many buds/plantlets by utilising the plants own budding mechanisms? With less succulent plants, once a bud has grown, do you then cut it and strike it like you would an ordinary cutting?

Yes. Or proliferate it further and then strike them in rooting media

It's interesting that mutations can occur during callous culture growth. This could lead to interesting new variants, but I suppose more often than not, this isn't the aim.

Not if you're trying to sell uniform plants for say, cropping or ornamental purposes. Tho producing mutations via callus is a common in-vitro breeding tactic

You're right, they do sell it! It's strange after searching for it on several occasions that I never found it there. Considering the risks associated with it, do you recommend using it or are there now safer alternatives? Because it is a largely mould-specific fungicide, at least when the dosage rate is low, it can be very useful in mycology, but surely targetting specific types of fungi is necessary when surface sterilising explants?

I'd use it, dispense with gloves and a dust mask in a draught free area at least, and pour the waste out while wearing shoes ;) There are things that work better on some fungus or in some species, but some of them require that you know and understand the contaminant you're dealing with, which is a world of pain you mightn't need to go into when you start. It's also pretty low toxicity to plants, which is more than I can say for some other stuff.

You're talking using it at 0.1%. In a pretreatment for seeds you know or suspect to be fungus laden you might be looking at 200-300mg in 200-300ml water- and that's for a lot of large seed. Some people use it in media successfully, for example in bamboo, but I've never tried it. It's autoclavable, but I just don't fancy inhaling any vapour ( if there is any ) if I open the autoclave before it is fully cool

Benomyl is systemic, so it gives you a few days grace while it kills off more fungus, during which time you can subculture the seed/ explant again to try to escape any contaminants

I'd use it in conjunction with other things- careful pretreatment and media planning for example

Makes sense. So interior tissues would be preferable as there should be less contaminants present, but the option to surface sterilise exterior tissues shouldn't be dismissed,

Interior tissues are more likely to be sensitive to surface sterilisation processes such as bleach. Check out the apical meristem sterilisation processes for kava, for example. I don't have the ref with me, but it's a good guide to getting clean tissue out of notoriously dirty plants

Oh, here's the ref:

Micropropagation of ‘Awa(Kava, Piper methysticum),J. Kunisaki, A. Araki, and Y. Sagawa, Biotechnology Jan. 2003

as there may be other cell types present which could work better?

You got it

That's a good idea. I'd like to get a micropipette, but they're not cheap. If dissolving in solvents such as ethanol or acetone, can/will this affect the culture, or does it evaporate off?

Mmm, stock solution prep, an important point. Lots of stuff will dissolve well in water, use that as a preference. Other stuff, like plant hormones, needs to be dissolved in a small amount of KOH or ETOH and bought up to volume with water ( check your supplier's tech manual ). Some things can handle ethanol or DMSO. If you're making up 15ml of say a 1mg/ml solution of GA3 you'd dissolve it in say 2ml ethanol max, when it's dissolved you top it up to 15ml with water. At 1-2 mg of GA3 per litre of media you won't be getting much ethanol in there overall

The more you dilute a compound, the easier it is to get close to accurate dispensing with a syringe. There is less margin for error in dispensing say 10mg Thiamine.HCl from a 2mg/ml solution than there is in dispensing it at a 20mg/ml solution

Most stock solutions are stable for up to 3 months, check if you need to fridge or freeze it. Some retain a portion of their activity for longer but 3 months is a good rule of thumb

I really appreciate your help. :) For now, all I can do is study the theory as I'll be going to NZ for a month in a couple of weeks, so won't be able to start until after that. But once I do, I'll be sure to document my progress here, and will no doubt have more questions as I actually begin the practical side.

I look forward to seeing the results. Happy to help. I get a few enquires from people every year and most don't end up taking it further or getting back. It's an incredibly inspiring process to see other people get successful in this and I hope it works for you

One other question, when using seeds as your starting material, is the idea to germinate them in-vitro, then cut the seedling up to make further cultures from?

That's the one :)

Really, now I reckon you have enough information to at least be at the planning and purchasing phase for your Loph work- don't overthink it and waste energy, just start and ask questions on the fly. I'm thinking I'm giving you too much theoretical information here that's not going to be relevant to you just getting started. It's not often at this stage that you will be in such a hurry that something can't wait even a day max.

Everyone experiments in this field- all the time- at every level of professionalism. You only hear about the successes, and then only rarely, usually for reasons of IP. If academic publications contained all the shit anyone had ever tried but which didn't work as well as the stuff which did, it would make bench work more transparent, we wouldn't be re-inventing the proverbial every ten minutes and my life would be way easier.

Mind you scientific publications would go on for much longer and be probably much less clear. But they'd be more fun and inspire more people IMO

When you get back, draw me up a protocol if you like, refer me to a paper and then we can break it down on a case/ step specific basis so you can proceed as you feel comfortable.

Have fun in NZ

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Ah yes, should have thought about that, perspex would not withstand autoclaving. I had the idea that you meant you sterilised the entire unit in an autoclave, which you obviously did not. I just use 70% metho in my glovebox. Is there an advantage to ethanol? Being a restricted chemical I expect it would be far more difficult to source.

Which brings me to a question, how easy/difficult is it to get the necessary additives and chemicals? Are they restricted and if so, how do you get around that as an amateur? Would benomyl be restricted?

I'd use it, dispense with gloves and a dust mask in a draught free area at least, and pour the waste out while wearing shoes ;) There are things that work better on some fungus or in some species, but some of them require that you know and understand the contaminant you're dealing with, which is a world of pain you mightn't need to go into when you start. It's also pretty low toxicity to plants, which is more than I can say for some other stuff.

0.1% does seem like a small quantity, but chemicals can still be toxic in tiny amounts (haven't read the MSDS on benomyl yet). How to you dispose of it in a way that doesn't pose a threat to the environment?

The more you dilute a compound, the easier it is to get close to accurate dispensing with a syringe. There is less margin for error in dispensing say 10mg Thiamine.HCl from a 2mg/ml solution than there is in dispensing it at a 20mg/ml solution

That's a good point to remember, that the more a compound is diluted, the smaller the margin of error becomes when measuring the solution out. thanks for the tip. :)

Most stock solutions are stable for up to 3 months, check if you need to fridge or freeze it. Some retain a portion of their activity for longer but 3 months is a good rule of thumb

Great, I was wondering how long a a stock solution would remain stable for. 3 months isn't too short a period of time.

I look forward to seeing the results. Happy to help. I get a few enquires from people every year and most don't end up taking it further or getting back. It's an incredibly inspiring process to see other people get successful in this and I hope it works for you

I hope I have some results to post in the not-too-distant future. I've been wanted to start trying out plant tissue culture for quite a while now, but for various reason haven't got around to it. This thread has definitely reignited that desire. Hopefully I'll have enough money when I'm back from NZ to purchase the necessities for it and get get the ball rolling before I'm back at uni in 2nd semester.

Everyone experiments in this field- all the time- at every level of professionalism. You only hear about the successes, and then only rarely, usually for reasons of IP. If academic publications contained all the shit anyone had ever tried but which didn't work as well as the stuff which did, it would make bench work more transparent, we wouldn't be re-inventing the proverbial every ten minutes and my life would be way easier.

Mind you scientific publications would go on for much longer and be probably much less clear. But they'd be more fun and inspire more people IMO

Ha, scientific publications with all the details of every fail would be messier, but would also be pretty interesting. In a way, it is a shame that scientists don't often publish methods that fail, as we would all learn a lot from it. Science should be, after all, a discovery of knowledge and that should include methods that don't work.

When you get back, draw me up a protocol if you like, refer me to a paper and then we can break it down on a case/ step specific basis so you can proceed as you feel comfortable.

Would love to. :)

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I just use 70% metho in my glovebox. Is there an advantage to ethanol? Being a restricted chemical I expect it would be far more difficult to source.

Metho is fine for most things. Some ppl prefer to make up those plant hormones stocks which are dissolved in ethanol in at least lab-grade ethanol.

Lab grade and above ethanol is a pain to acquire, really at the early stages don't bother, metho is fine

Which brings me to a question, how easy/difficult is it to get the necessary additives and chemicals?

Easy as. You'll need to set up an account with a supplier, but sales of most of those compounds aren't restricted for that use

Would benomyl be restricted?

You'd need an account with Sigma or one of it's re-supplier's. You might need to submit an End User Declaration with the more paranoid of these just to prove you aren't going to eat it or spray it on food

Just a side note for the general public- everyone please behave professionally with suppliers. Do your research, check catalogue numbers and such before you contact them. Places tend to tighten up ordering processes if they get a lot of idiots on the phone and it makes everyone's life harder, including people who might want to set up accounts in future

It doesn't hurt to tell them you've never tried this before but have studied the theory at TAFE or something. No-one minds a beginner and most people enjoy hearing enthusiasm in people's voices. But random stoner ramblings don't cut it when setting up an account

0.1% does seem like a small quantity, but chemicals can still be toxic in tiny amounts (haven't read the MSDS on benomyl yet). How to you dispose of it in a way that doesn't pose a threat to the environment?

I pour it on something I hate. It's not terribly environmentally friendly, but given that I use about 300ml of an 0.1% solution every six months, in the scheme of things it's pretty small

Science should be, after all, a discovery of knowledge and that should include methods that don't work.

Hell yes

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Thanks again Darklight, I plan on putting your advice and help to good use. :) I think I'll start with seeds and shoot proliferation and see how I go from there.

How much do estimate initial costs would be for starting up, for things such as hormones, media, fungicide, etc. and minimal glassware? I seem to be tearing through my money at a gut-wrenching rate and with NZ on the horizon don't see myself with much cash to throw around when I get back.

Hmmm, that's interesting, both those links I posted are restricted articles. I must have been logged into my uni at the time...which is shit, as my password has now been reset as I've been deferred for so long. Does anyone have access to those articles? I forgot to save them.

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tripsis, here are the articles, along with an interesting one I found a few months ago whilst researching tissue culture. We seem to be getting into this at the same time, hopefully we can share info at some stage! My primary interests are micropropagating cacti, acacias and native Erythloxium.

Micropropagation of 21 species of Mexican cacti by axillary proliferation.pdf

In vitro Propagation of Eight Species or Subspecies of Turbinicarpus (Cactaceae).pdf

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation.pdf

Edited by IceCube
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Many thanks for that IceCube. :)

Indeed we do. I just need to get enough money to buy what's needed to start now.

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