In VitroCell.Dev.Biol.

--Plant34:249-251,July~September1998
9 1998Societyfor In VitroBiology
1054-5476/98 $05.00+0.00

MICROPROPAGATION OF MINTHOSTACHYS MOLLIS (H.B.K.) GRIESEB. AND ESSENTIAL

OIL COMPOSITION OF CLONALLY PROPAGATED PLANTS

ANGELICA DELV. CHEBEL, ADOLFINAR. KOROCH, HECTOR R. JULIANI, JR., HECTOR R. JULIANI,1
AnD VICTORIO S. TRIPPI

Laboratorio de Biotecnologfa Vegetal, Facultad de CienciasAgropecuarias, Universidad Nacional de Crrdoba, P.O. Box 509, (5000)
Crrdoba, RepablicaArgentina (A. d. V. C., A. R. K., H. R. J., E S. T.); Cdtedra de Farma~ognosia, Facultad de Ciencias Qufmicas,
Universidad Nacional de Crrdoba, Suc. 16 C.C. 61 (5016) Crrdoba, Rept~blicaArgentina (H. R. J.).

(Received 18 July 1997; accepted 12 March 1998; editor M. K. Redenbaugh)

SUMMARY

Cultures of Minthostachys mollis were established from nodal explants obtained under aseptic conditions. Explants were
cultured on Murashige and Skoog (MS) half-strength medium containing 6-benzyladenine (BA), and/or naphthaleneacetic
acid (NAA). Optimum numbers of healthy shoots were induced on media containing 0.05 ~tM NAA plus 2.2 ~M BA; higher
concentrations caused more hyperhydricity and less extension. Rooting was achieved on half-strength MS medium with
0.05 ~tM NAA. Plantlets were acclimatized and successfully transferred to soil. Essential oil composition of the regenerated
plants were determined by gas chromatography/mass spectrometry and little differences were found in the essential oil
composition with the plant grown in the wild.
Key words: aromatic plant; in vitro culture; multiple shoots; terpene.

INTRODUCTION to micropropagate M. molli~ and to study the essential oil composition

Medicinal and aromatic plants constitute an important group
among the economic plants (Arora and Engels, 1993). Due to the
increasing population, especially in developing countries, many nat-
ural growing sites and ecosystems are being destroyed and a number
of plant species are threatened. Efforts are necessary to domesticate
a large number of medicinal plants collected in the wild (Franz,
1993).
In Argentina, many native aromatic plants are collected from the
wild. As a result, the natural populations of medicinal plants has
seriously declined and in some cases endangered. Among these me-
dicinal plants is Minthostachys mollis, a member of the Lamiaceae
family, which occurs in the hilly regions of central Argentina; it is
popularly known as "peperina." This aromatic shrub is used in pop-
ular medicine as a digestive, antispasmodic, and antidiarrheic (So-
raru and Bandoni, 1978). The leaves are also used for preparing
liquors and in mixtures for teas and nonalcoholic beverages. The
volatile oil of M. mollis from Tucuman (Arg.) contains carvone (65%)
as the main component, pulegone (17%), menthone (9%), and lim-
onene (4%) (Retamar, 1986); whereas, the essential oil of M. mollis
from Crrdoba and San Luis provinces was characterized by the pres-
ence of menthone (53%) and pulegone (33%) (Fester et al., 1961).
The latter has been cited as an hepatotoxic monoterpene (de Vincenzi
et al., 1995).
In vitro culture has gained considerable importance during recent
years in view of its possible application for the production of aromatic
plants (Mulder-Krieger et al., 1988). The aim of the present work is
1To whom correspondence should be addressed.
of the clonally propagated plants.
MATERIALSAND METHODS
Seeds of M. mollis were used for this study. Seed samples were collected
in Los Condores (Crrdoba, Republic of Argentina). The seeds were surface
disinfected in a 1% wt/vol sodium hypochlorite solution for 20 rain. After
three rinses in sterile distilled water, the seeds were placed on half-strength
MS medium (Murashige and Skoog, 1962), without growth regulators, to ob-
tain aseptically grown seedlings. Shoot tip and nodal segments from 5-wk-
old sterile plantlets were used to initiate micropropagation. Multiplication
was performed on half-strength MS medium. The shoot tips and nodal seg-
ments were transferred to shoot multiplication medium with growth regulators
(Table 1).
In all the experiments reported here, the pH of the media was adjusted to
5.7-5.8 and 0.7% Difco Bacto-agar and 1.5% sucrose were added before
autoclaving for 20 min at 121~ C. Cultures were incubated under continuous
cool-white fluorescent light (50 p.mol'm-Z's i photon flux) at 24 + 3~ C. The
experimental, design was fully randomized with 3 replicates per treatment and
12 explants per replicate. Regression analysis was employed to analyze trend
and relationship between variables. The number of axillary shoots arising
from each explant were counted after 4 wk, and treatment means were cal-
culated.
For rooting, all shoots up to 1.5 cm long were transferred to half-strength
MS medium supplemented with 0.05 or 0.5 I.tM naphthaleneacetic acid
(NAA), 0.05 or 0.5 }aMindole-3-butyric acid (IBA), or 0.06 or 0.6 ~tMindole-
3-acetic acid (IAA). Rooting percentage as well as the number of roots per
shoot were evaluated 4 wk later.
For acclimatization, rooted plantlets were washed in warm water to remove
agar from the roots and transferred to pots containing a sterile mixture of
soil:vermiculite (50:50 vol/vol) and placed in the culture room. Glass covers
were used to ensure high humidity around the plants at the initial stage of
growth and were gradually opened day-by-day during the acclimatization
period. The percentage of plants established ex vitro was evaluated.
For comparison purposes, wild plants were compared with those accli-
matized plants that were cultivated in an experimental field (Facultad de

249
250 CHEBEL ET AL.

TABLE 1 TABLE2

EFFECT OF GROWTH REGULATOR CONCENTRATION ON SHOOT EFFECT OF AUXINS ON ROOTING IN VITRO REGENERATED
FORMATION AND ELONGATION IN MINTHOSTACHYS MOLLIS SHOOTS OF MINTHOSTACHYS MOLLIS AFTER 4 WK INCUBATION
AFTER 4 WK INCUBATION
Growth Regulator (~tM)* Percentage Rooting Mean Root Number b + Standard Error (SE)
NAA ~ (I~M) BAd 0aM) Mean Shoots Per Explant b Mean Shoot Length (cm)~
Control 75 4.2 -+ 0.4
0 0 2.8 --- 0.3 ~ 4.7 - 0.6
2.2 2.8 + 0.3 5 + 0.6 NAA
4.4 3.7 + 0.3 3.8 + 0.3 0.05 83.3 ~ 5.3 + 0.5
13.3 3 + 0.4 1.8 + 0.4 0.5 91.6 ~ 5.4 + 0.6
22 4.3 + 0.3 2.9 -+ 0.3 IAA
30.8 2.1 -+ 0.27 0.4 + 0.08 0.06 75 4.6 + 0.4
0.05 0 2.1 - 0.2 2.3 + 0.4 0.6 66 4.1 + 0.4
2.2 5.3 + 0.3 2.8 + 0.2 IBA
4.4 3.3 + 0.2 3.1 + 0.5 0.05 66 4.4 + 0.4
13.3 3.5 + 0.5 2.3 + 0.3
0.5 50 4.4 + 0.3
22 3.8 + 0.2 1.4 + 0.2
30.8 2.8 + 0.2 1.8 + 0.3
~ at 5% (Test for comparing proportions).
0.27 0 2.5 + 0.2 1.1 + 0.3 bThe response for root number was described using a lineal model. Re-
4.4 3.1 + 0.2 2.1 + 0.3 gression: Y NAA = 11.274 + 1.116x, P = 0.0006; R 2 = 0.3851; Y IAA
13.3 3.2 -4-_0.3 1.6 + 0.4 = 18.062 + 1.970x, P = 0.0253; R 2 =0.1992; Y IBA = 7.986 + 0.632x,
22 3.4 + 0.3 2.7 + 0.4 P = 0.1538; R 2 = 0.1538.
30.8 2.6 -+ 0.2 1.1 + 0.1 cNAA = naphthaleneacetic acid; IAA = indole-3-acetic acid; IBA =
indole-3-butyric acid.
0.1 0 1.8 + 0.4 2.3 -+ 0.6
4.4 2.8 + 0.2 1.8 + 0.3
13.3 3.2 + 0.2 2.1 -+ 0.4
22 3.1 + 0.4 3.1 + 0.6 RESULTS AND DISCUSSION
30.8 3.5 -+ 0.3 1.9 + 0.3
There was a significant increase in shoot n u m b e r when the level
0.53 0 2.4 + 0.4 2.2 -+ 0.4
2.2 2.4 + 0.2 2.9 + 0.6 of 6-benzyladenine (BA) increased (Table 1). However, greater
4.4 2.8 -+ 0.3 1.8 -+ 0.4 amounts of hyperhydricity were found in cultures containing higher
22 2.9 +-+-0.5 1.8 + 0.4 concentrations of BA (22 ~tM BA). The combination of 0.05 laM NAA
1.07 0 2.4 + 0.3 2.9 -+ 0.6 + 2.2 I.tM BA induced a high n u m b e r of shoots per explant, em-
2.2 2.1 + 0.1 1.8 -+ 0.3 phasizing the superiority of this m e d i u m . Shoot length was generally
4.4 1.7 + 0.3 2.1 - 0.7 inverse to n u m b e r of shoots. M a x i m u m shoot length was initiated
22 2 + 0.25 0.5 - 0.02
with 2.2 ~tM BA or m e d i u m without growth regulators.
After 15 d of culture in rooting media, shoots cultured in m e d i a
aMeans + standard error.
bThe response for shoot number and shoot length was described using a with auxins developed roots. Best root development and the highest
quadratic model (Y shoot = 1.789 + 0.092 • NAA + 0.944 • BA - 0.015 rooting percentage (91.6%) was achieved on half-strength MS me-
• NAA • BA - 0.037 • NAA2 - 0.114• BA2; P < 0.001; n 2 = 0.1487. d i u m s u p p l e m e n t e d with NAA (Table 2). W h e r e a s transferring to an
cy length = 5.447 - 1.553 • NAA - 0.154 • BA + 0.109 • NAA • BA
auxin-free m e d i u m reduced the n u m b e r of rooted shoots to 75%.
+ 0.149XNAA 2 - 0.064• P < 0.0001; R 2 = 0.1746); as indicated
by a significant p-value. Moreover, root systems developed from N A A treatments were larger
4NAA = naphthaleneacetic acid; BA = 6-benzyladenine. and more vigorous than those from the other treatments.
The establishment of in vitro grown plants in soil was easily
achieved. After initial acclimatization, 9 0 % of the plantlets devel-
oped in mature plants when transferred to the greenhouse. Plants
Ciencias Agropecuarias). Four samples of both plants were steam distilled.
derived from in vitro culture did not show noticeable differences in
Fresh leaves and stems were hydrodistilled (60 min) in a Clevenger appa-
their appearance.
ratus. The fresh oils were analyzed using a Q-mass 910 Perkin Elmer mass
spectrometer coupled to a gas chromatograph equipped with an SE-30 (meth- The analysis of the clonally propagated plants showed that the
ylsilicone) capillary column (30 m • 0.3 mm, 0.25 ~tm film thickness). The essential oil composition was similar to that of plants grown in the
flow rate of helium was 1 ml/min. The oven temperature program was 60 ~ C wild, both plants contained high levels of m e n t h o n e and pulegone
(1 min), then 4 ~ C/min to 240 ~ C (30 min). Individual identifications were (Table 3). However, the relative concentration of pulegone was higher
made by matching their 70 eV mass spectra with those found in literature and menthone was lower in the cultivated plants; as well, ot pinene
and in mass spectral libraries. Whenever possible, coelution gas chromatog- and piperitenone were also lower. The variation in the oil composition
raphy (GC) with reference compounds was used for confirmation of the results. could be attributed to differences in soil conditions and altitude. It
GC was carried out on a Konik KNK 3000 HRGC gas Chromatograph
was observed in M. mollis from Venezuela that the altitude modified
equipped with an SE 30 capillary column idem GC/MS. The flow rate of N2
the essential oil composition, and the authors concluded that the oil
was set at 2 ml/min. Injector and detector temperatures were maintained at
240 ~ C. The column temperature was programmed from 80 ~ C (after 2 min) composition could be d e p e n d e n t on climatic conditions (Rojas and
to 220 ~ C at 5 ~ C/rain and the final temperature was held for 15 min. The Usubillaga, 1995). Our results resemble those described by Fester
quantification of chromatograms was performed by an SP4290 Spectra Phys- et al. (1961), who observed the presence of m e n t h o n e and pulegone
ics integrator. in high a m o u n t s (53% and 33%, respectively).
 MICROPROPAGATION OF MINTHOSTACHYS MOLLIS 251

TABLE 3 ACKNOWLEDGMENTS
ESSENTIAL OIL COMPOSITION (%) OF FIELD-GROWN AND This study was financially supported by CONICOR, ACADEMIA NA-
MICROPROPAGATED PLANTS OF MINTHOSTACHYS MOLLIS CIONAL DE AGRONOMIA Y VETERINARIA AND SECyT (UNC).

Terpenes Wild Plants CultivatedPlants

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