Plant Cell, Tissue and Organ Culture 45: 153-158, 1996. 153 © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Stimulation of the growth and solamargine production by Solanum paludosum multiple shoot cultures using a new culture medium

H. E1 Badaoui l, E Morard 2 & M. Henry l* 1 Laboratoire de Botanique et Mycologie, Facultd des Sciences Pharmaceutiques et Biologiques, 5, Rue Albert Lebrun, B.P. 403, 54001 Nancy cedex, France; 2 Laboratoire d'Ingdnigrie agronomique, Ecole Nationale Supdrieure d'Agronomie, 45, avenue de Muret, 31000 Toulouse, France. (* requests for offprints)

Received 2 May 1995; accepted in revised form4 March 1996

Key words: multiple shoot culture, macronutrient optimized culture medium, solamargine production, Solanum paludosum

Abstract

Organogenic callus cultures of Solanum paludosum were obtained from root, hypocotyle and cotyledon explants of plantlets cultured in sterile conditions. These callus cultures developed multiple shoots which proliferated in Murashige and Skoog basal liquid medium. These multiple shoots produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits.

The optimization of the macronutrient composition of the liquid medium was performed by a method derived from the plant composition. This approach results in the establishment of an appropriate medium (SPOM medium) suitable for the improvement of both growth and solamargine production by multiple shoot cultures ofS. paludosum.

Introduction

Previously, Valverde et al. (1993) reported the pres- ence of about 3% of solamargine, based on the dry weight, in the unripe fruits of a north-eastern brasil- ian shrub, Solanum paludosum Moric. The presence of steroidal alkaloids was established from a work per- formed on unripe fruits of S. paludosum by identifica- tion of solasodine, the aglykone obtained by hydrolysis of the crude extract and not directly from the glycoal- kaloids (Battacharyya, 1984).

Production of steroidal glycoalkaloids by in vitro cultures of S. paludosum have been studied previously (El Badaoui, 1993). The best production of steroidal glycoalkaloids by in vitro proliferating tissues with- out any previous selection was obtained with multiple shoot cultures.

Our purpose was to optimize the production of sola- margine by multiple shoot cultures. The optimization of the production of secondary metabolites by plant cell and organ cultures has been described by many authors. The strategy commonly used for the optimiza- tion of both growth and secondary metabolite produc-

tion is as follow: first the selection of high producing cell lines, second the definition of the best composition of the culture medium, and third the establishment of the adequate environmental conditions (temperature, aeration, light, etc).

Here we chose to study the composition of the cul- ture medium. Generally, two strategies were used to define the balance between the different mineral and organic elements for the optimal growth of the plant cells or tissues. One consists in creating a new com- position of the mineral and organic nutrients for one species by studying the balance effect of each of them on the others. One of the best known example is the work of Murashige and Skoog (1962) who established an optimal nutrient composition of the culture medium for rapid growth with tobacco callus cultures. Another strategie consisted in keeping the formula of a cul- ture medium previously defined for one species and in trying to adapt it to another by changing the con- centrations of some of the different nutrients until the composition supports good growth for the plant species under investigation.

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In this paper, we present a strategy for developing an in vitro culture medium for plant tissue culture. The macronutrient concentrations were optimized by determining the whole plant mineral composition. This applied to Solanum paludosum gave a culture medium optimum for cell growth and enhanced the production of glycoalkaloids.

Materials and Methods

Establishment of the suspension multiple shoot cultures

Seeds of S. paludosum Moric. obtained from wild plants of the region of Joao Pessoa, Paraiba (north- eastern state of Brasil) were surface-sterilized and germinated in the dark on Heller's medium (Heller, 1969) giving the first plantlets a month later. Sterile root, hypocotyle and cotyledon explants from sterile plantlets of 2 to 3 cm high were set out on a solid culture medium of Murashige and Skoog's (MS) mineral nutri- ents (Murashige and Skoog, 1962), Gamborg's vita- mins (Gamborg et al., 1968) containing 30 g 1-1 glu- cose, 8 g 1- l agar, at pH 5.7, an auxin, 3-indolylacetic acid (IAA), 3- indolylbutyric acid (IBA) or naphtale- neacetic acid (NAA), and a cytokinin, zeatin, kinetin, benzylaminopurine (BA) or isopentenyladenine (IPA), in various concentrations. According to the phyto- hormonal balance the different explants regenerated directly shoots, roots or gave proliferating unorganized calluses.

Primary calluses, formed 3 to 6 weeks later, were transferred onto the same media and subcultured every month under the same conditions. After 5 weeks, small green nodules appeared on the calluses and break from them giving buds. These buds were transferred in 250 ml Erlenmeyer flasks containing 100 ml of liquid MS medium with different combinations of auxins and cytokinins. Some of them gave multiple shoot cul- tures. Suspension cultures were obtained directly from the calluses and put in liquid MS medium with other combinations of phytohormones.

All the cultures were performed at 25°C under flu- orescent light (40 #mol.m-Z.s - l 12-h photoperiod).

Plant chemical analysis

Plant mineral analysis A whole plant of Solanum paudosum was weighed and dried. Macronutrient contents were analysed by classical methods (Morard et al., 1990) after a sulfuric acid mineralisation for total nitrogen and nitric acid for: N and P by automatic absorption spectrophotometry, S by turbidimetry and K, Ca, Mg by atomic absorption spectrophotometry.

Extraction, purification and quantitative determination of solamargine The extraction and the purification of solamargine were described previously (Valverde et al., 1993). The method was derived from Cham & Wilson (1987). An acidic aqueous extract of dried defatted powdered plant material was subjected to repeated (x4) precipi- tation of the glycoalkaloids with NH4OH at pH 9-10 followed by solubilization in acidic aqueous solution. The quantitative determinations of solamargine were accomplished by HPTLC using silica gel 60 F254 plates 10xl0cm (Merck Ref.5628). The plates were devel- oped in a Camag horizontal chamber 10x 10cm with the following solvent system: CHC13/MeOI-I/4% aqueous NHaOH (6:3:0.1). Solamargine spots were revealed by spraying a 10% sulfuric acid solution in methanol. The colour intensity of the spots on the plates was measured with a Vernon densitometer at 420 nm. Solamargine was conclusively detected by comparison of its migra- tion with that of an authentic sample run under identi- cal conditions. The determination of solarmargine was achieved by comparison with a calibration graph of the compound from 2 #g to 24 #g. The intensity of the spots was found to be linear over 10 measurements at different concentrations. For analysis at least 5 g of dried tissue was used and the method was quantitative and reproducible.

Results

Multiple shoot culture

Multiple shoot cultures of S. paludosum were best obtained from plantlets grown in aseptic conditions than from adult plants. As a tropical plant, S.paludosum harvested in its original country was frequently very contaminated both inside and outside the tissues. The calluses obtained from adult plants rapidly produced

phenolic compounds which inhibited the cell growth. The first step in the production of mutiple shoots was the emergence of green calluses. Morphological dif- ferences could be seen between the calluses obtained from the different parts of the plantlets. The callus- es were more chlorophyllaceous from root explants than from cotylodons or hypocotyls. The root calluses showed more numerous and proliferating buds from which the multiple shoots developed. Conversely cal- luses from hypocotyles and cotyledons gave rise to less buds but with more developed primary leaves. Primary buds and shoots were best obtained with zeatin either at 10--SM with IAA (10--6M) or at 10--6M with IBA (10--TM). These buds isolated from the green calluses on solid medium were transferred in 250 ml Erlenmeyer flasks in the same but liquid media (100 ml). Differences in growth and in tissue necrosis due to a phenolic compound production were observed from the different genotypes of the different seeds. One of these cultures gave the best results in liquid medium and was choosen for the assay of solamargine produc- tion.

Mineral plant contents

The macronutrient content of S. paludosum plants grown in wild conditions in Brasil was determined. Ten one-year-old plants were harvested and each of them was dried, ground separately and the min- eral content of each plant was analysed (three times) giving the following results: potassium 178 rag/plant (4.5 meq/plant), calcium 308 rag/plant (15.2 meq/plant), magnesium 41 mg/plant (3.4 meq/plant), nitrogen 289 mg/plant (20.6 meq/plant), phosphorus 55 mg/plant (1.8 meq/plant) and sulfur 152 mg/plant (3.2 meq/plant).

The mineral nutrient concentration of the medium was established on the same ratios as found in the whole plant as used in nutrient formulation for soi- less culture (Morard, 1995). The ionic association of anions and cations (Table 1) was selected according to the solubility of the corresponding salts and the com- mercially available products used for making mineral nutrient solutions: S04 - - and Mg ++ associated with magnesium sulfate, nitrate furnished as calcium and potassium nitrates, phosphate as potassium orthopho's- phate. A theoretical composition of nutritive solution could be established and was expressed in g.1--1 : KNO3 (0.50), Ca(NO3)2 4 H20 (3.54), KH2PO4 (0.27), MgSO4 7H20 (0.74). This medium was called SPOM (Solanum paludosum optimized macronutrients). A

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Table 1. Compositions of the macronutrient solutions of the media used for the multiple shoot culture of Solanum paludo- s u m .

a) SPOM medium

(meq/l) NO 3 H2 PO 4 SO n Cations

K + 5 2 7

Ca ++ 15 15

Mg + + 3 3

Anions 20 2 3 25

b) MS medium

(meq/1) NO 3 H2PO 4 SO n CI - Cations

K + 18.8 1.25 20.05

Ca ++ 3 3

Mg ++ 1.5 1.5

NH 4 + 20.6 20.6

Anions 39.4 1.25 1.5 3 45.15

comparison with MS medium showed: an approxi- matly half total salt concentration, a decrease in the potassium and nitrate concentrations and an increase in the calcium concentration, a lack of ammonium and chloride elements.

Improvement of the culture growth and the solamargine production

The multiple shoot culture used for this study was sub- cultured on Murashige and Skoog's (MS) medium with glucose (30g.l-I), zeatin (10-6M) and IBA (10 -7 M). Three different mineral media were used: MS medium, half strength MS medium (MS 1/2) and SPOM solution. To this macronutrient solution were added the micronu- trient solution of Murashige and Skoog's medium and the vitamin solution of Gamborg's medium. The time courses of growth in batch culture (Erlenmeyer flasks) were given in Figure 1. The growth curves show a lag phase for both MS media which does not exist with SPOM solution. The exponential phase lasts for nine days for MS medium, eleven days for MS 1/2 medium, and fourteen days with the SPOM solution. The maxi- mum biomass production was reached at the same date for all three nutritive solutions but the maximal produc- tion of biomass was much higher for the SPOM solu- tion with more than 2 grammes of biomass dry weight in 100 ml of tissue suspension compared to only 1.3 g of dry weight produced with the MS medium. Thus the SPOM solution was more satisfactory for the biomass production of S. paludosum shoot cultures. The SPOM

156

25 T

~ 1,5.

0,5'

0 : : : : : : : : : : : " : : : "- " : : : : 0 1 2 3 4 5 8 7 8 9 10 t l 12 13 t4 15 16 t7 18 l g 20 2t

Days

Fig. 1. Batch growth of a multiple shoot cultured in modified MS medium, modified MS 1/2 medium and SPOM medium. All the three media contained zeatin 10-6M and IBA t0-TM. Multiple shoot culture was obtained from calluses initiated from root explants (3 flasks harvested for each point; maximal standard deviation of 0.2 g DW!I00ml).

1,6 o

~" 1,4. ,

1,2

0,8

._~ 0,6 -

~ 0,2

0 1 2 3 4 5 8 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Days

Fig. 2. Solamargine content (mg/100ml of culture medium) of the multiple shoot culture cultivated on the three media.

medium gave an increase of the pH throughout the batch growth, compared to the preliminary decrease seen with the two MS media (data not shown). These results observed for the growth of the multiple shoot culture are rather the same for the solamargine produc- tion (Fig. 2). The maximum of solamargine production was obtained at the 19th day of batch culture with 4.5 mg.l - l for MS medium, 7.8 mg.1- for MS1/2 and 14.5 mg.1- l for SPOM solution corresponding to sola- margine contents 0.038, 0.055 and 0.072% dry weight respectively. No solamargine was excreted into the medium even at the end of the batch culture (data not shown). But as we can see in Figure 2, a decrease in the solamargine content was observed in the culture during the stationary phase of growth leading to the conclusion that solamargine could be transformed or perhaps metabolized.

Discussion

The comparison between the three media revealed the following points. The SPOM solution differs from

the MS solution, first by the phosphate concentration which is enhanced in SPOM by about two fold. Phos- phates are known to be a limiting factor of growth for many species. Improvements of the cell suspen- sion growth were described with the enhancement of the phosphate concentration for Catharanthus roseus (MacCarthy et al., 1980; Merillon et al., 1983) and Anchusa officinalis (De Eknamkul & Ellis, 1985). The increase of the phosphate concentration in the medium is able to reduce the lag phase rending more easy the carbohydrate uptake by the cells (Carew & Krueger, 1977). Nitrogen also is an important factor in the improvement of the tissue growth of batch cultures. Qualitatively NH4 + was found to be an inhibitor of the shikonin metabolism in Lithospermum erythrorhizon cell cultures. NH4 + was described as a growth inhibitor for tissue cultures of Cichorium intibus (Vasseur & Dubois, 1985). NH4 + could be toxic by a rapid acidifi- cation of the medium (Martin et al., 1977) as we could observe for the multiple shoot cultures of S. paludo- sum with MS medium. NO3- is generally described as the best nitrogen source for plant tissue cultures of many species (Matsumoto et al., 1971). SPOM medi-

um without NH4 + ions also confirms these results for

S. paludosum. Some other macronutrients were opti-

mized by the technique presented here. They partic-

ipate in the equilibrium of the nutrient solution. For example, Ca ++ with an increase of 2.5 fold in its concentration replaces the potassium ion. Vasseur &

Dubois (1985) with Cichorium intibus cells showed

that an increase of the calcium concentration in the

culture medium stimulated the growth with an opti-

mal concentration of 18 meq . l - ' as we found for S. paludosum (15 meq. l - i ) in contrast MS calcium con-

centration is only 6 meq.1-I

In this paper we show that the solamargine produc- tion in multiple shoot cultures ofS. paludosum followed

the same pattern as the biomass growth in batch cul-

ture. The same results were described by Drapeau et al. (1986) in Dioscorea deltoidea cell suspension cultures

for the production of diosgenin, a steroidal sapogenin

with a chemical structure is very closely related to sola-

sodine, the genin present in solamargine, and by Nigra

et al. (1987) in callus culture of Solanum eleagnifolium for the solasodine production. This time course is use-

ful for the production of plant secondary metabolites

since the harvest of the maximum of biomass produc-

tion coincides with the maximum of the secondary metabolite production. Such a pattern was reported for many compounds as for example anthraquinone glu-

coside by Cinchona succirubra cell cultures (Khouri

et al., 1986) or pentacyclic triterpenoidal saponins of

Saponaria officinalis cell suspension cultures (Hen-

ry & Guignard, 1982) or of Gypsophila paniculata

multiple shoot cultures (Pauthe-Dayde et al., 1989). Generally, it was interpreted as a dependance of the

metabolism of these compounds directly on the pri- mary metabolism. The following step of such a study

would be the investigation of the regulatory bottle-

necks of the biosynthesis with respect of the different ion concentrations to know what kind of correlation

could exist between the both primary and secondary

metabolisms at the cellular level.

Acknowledgements

Thanks are due to Conselho National de Desen- volvimiento Cientifico e Technologico (CNPq-Brasil) and Comit6 Fran~ais d 'Evaluation de la Coop6ration Universitaire avec le Br6sil (COFECUB-France) for financial support. The authors thank Pr. R. Verpoorte (Gorlaeus Laboratories-Leiden) for his advice,

157

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