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Micropropagation of Cunila incisa Benth., a potential source of 1,8-cineole.
Agostini, G.; Echeverrigaray, S.
Instituto de Biotecnologia, Universidade de Caxias do Sul, CP 1352, CEP 95001-970, Caxias do Sul,RS, Brasil.
ABSTRACT: Micropropagation of Cunila incisa Benth., a potential source of 1,8-cineole. Cunila incisa
Benth. (family Lamiaceae, subfamily Nepetoideae, tribe Mentheae) is one of the 12 South-American species
of the genus Cunila. The essential oil of this aromatic perennial scrub is characterized by high concentration of
1,8-cineole. Leaves and flowers infusions are used in popular medicine for the treatment of chronic cough and
respiratory diseases. C. incisa can be propagated by seeds and cuttings, but the application of these conventional
propagation methods are limited by poor seed viability, low germination rate and scanty rooting of stem cuttings.
In this context, we report a protocol for the micropropagation and rooting of this species. Axillary buds of adult
selected plants of Cunila incisa Benth. were used to evaluate the effect of growth regulators and culture media
on micropropagation. The highest propagation rate was obtained using Murashigue and Skoog (MS) medium
supplemented with 4.4 µmol . L-1 of benzyladenine. High concentrations of BA resulted in hyperhydricity.
Auxine/cytokinin combinations were not benefic to the micropropagation process. Rooting was achieved on
MS medium without growth regulators. Indolbutyric acid addition to rooting media was detrimental for both
proliferation and rooting. Micropropagated plants transferred to the field showed normal morphology, and were
more vigorous than stem rooting derived plants.
Key words: Cunila incisa, in vitro culture, growth regulators, poejo
INTRODUCTION
The genus Cunila Royen ex L., family Lamia-
ceae, subfamily Nepetoideae, tribe Mentheae (Can-
tino et al., 1992) consists of 22 species with two
centers of distribution: Mexico with 10 species (Gar-
cia-Peña, 1989) and the southern part of South Ame-
rica with 12 species. The South American species
are divided into three sections: Incana, Incisa and
Spicata. C. incisa is one of the two species of the
Incisa section (Coelho de Souza, 1997).
C. incisa grows at the margins of the forest in
the highlands (300-1000m) of southern Brazil. It is a
perennial scrub with 1-2m high with ramified stem
and branches. Their leaves (1-3cm long and 0.8-1.5cm
width) are peciolate, ovoid to rhomboid with charac-
teristic incised margins. The small white to cream
flowers (3mm long) are grouped in cimules fixed to
the branches by short peduncles. Leaves, flowers
and stalk are covered by glandular trichomes (Bor-
dignon, 1997).
Hydrodistillation of the aerial parts of C. inci-
sa yielded 1 to 1.3% (w/w) of an essential oil with a
characteristic high content of 1,8-cineole (50-60%)
(Bordignon et al., 1996). This monoterpene poses
secretolytic, secretomotoric, bronchospasmolytic,
antiphlogistic and antiseptic properties (Duke et al.,
1994) that justify the popular application of this plant
for the treatment of chronic cough and respiratory
diseases (Simões et al., 1994).
The in vitro culture and micropropagation of
aromatic and medicinal plants has proved to be an
important alternative for the rapid multiplication of
selected genotypes or chemotypes (Bajaj et al.,
1988). The micropropagation of Cunila galioides (Fra-
caro and Echeverrigaray, 2001) allowed the establish-
ment of a germplasm collection and the multiplicati-
on of plants with different chemical characteristics
(Echeverrigaray et al., 2003). C. incisa can be pro-
pagated by seeds and cuttings, but the application
of these conventional propagation methods are limi-
ted by poor seed viability, low germination rate and
scanty rooting of stem cuttings. Therefore, the mi-
cropropagation of this species is highly desirable for
its application in the commercial production of es-
sential oil.
In the present paper, we report the effect of
culture media and growth regulators on the micro-
propagation through axillary buds of selected adult
plants of C. incisa.
MATERIAL AND METHOD
Five field plants of Cunila incisa Benth. collec-
ted at Veranópolis, Rio Grande do Sul, Brazil, and
selected for their high oil yield and growing characte-
ristics, were used as the donor plants in the micro-
propagation experiments. Shoot segments with 3 to
4 nodes were collected during spring (September-
October). The nodal segments were washed with 70%
ethanol for 30 sec and surface sterilized with 1 g L-1
sodium hypochlorite solution containing 0.1% Twe-
en 20 (v/v) for 20 min. After repeated rinsing with ste-
rile distilled water axillary buds measuring 2 to 4 mm
Recebido para publicação em agosto/2004
Aceito para publicação em julho/2006
Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.186-189, 2006.
187
were excised and implanted vertically on the culture
media.
Four basal media were used: MS (Mu-
rashige and Skoog, 1962), QL (Quoirin and Lepoivre,
1977), LS (Linsmaier and Skoog, 1965), and B5
(Gamborg et al., 1968). All the media contained 30 g
L-1 sucrose and 0.7% agar. The pH of the media was
adjusted to 5.8 prior to autoclaving. Cultures were
incubated in a culture room at 25±2 °C with 16 h
photoperiod under a photon flux of 20 µmol m-2 s-1
provided by cool white fluorescent lamps (Phillips TLT
40W/75). Cultures were transferred onto fresh me-
dium every 30 days.
To evaluate the effect of culture media axillary
buds were inoculated on MS, B5, LS and QL supple-
mented with 4.4 µmol . L-1 of BA. The effect of ben-
zyladenine concentration (0 to 17.6 µmol . L-1) was
evaluated on MS basal medium. MS medium with
4.4 µM BA supplemented with naphthaleneacetic acid
(1.0 and 2.7 µmol . L-1), indoleacetic acid (2.8 µmol .
L-1) or indolebutyric acid (2 µmol . L-1), was used to
evaluate the effect of cytokinin/auxin combination on
the micropropagation.
Microshoots (2 to 3 cm long) with 4 to 6 no-
des obtained on MS medium supplemented with 4.4
µmol . L-1 of BA were used to evaluate the effect of
indolebutyric acid (0 to 9.8 µmol . L-1) on rooting.
Rooted plantlets were washed in tap-water and trans-
ferred to plastic chambers containing a sterilized
mixture of sand and soil (1:1), covered with a plastic
cap that was gradually opened during the acclimati-
on period of 15 days. Acclimatized plants were trans-
ferred to the greenhouse and then to outdoor conditi-
ons.
All the experiments were conducted in rando-
mized design with 40 explants per treatment. The
evaluation of the number of shoots per explant, sho-
ot length, percentage of rooted plantlets, number of
roots per explant, and root length, were made after
30 days in culture. The data were submitted to sta-
tistical analysis by ANOVA and the means compa-
red by the Tukey´s test.
RESULT AND DISCUSSION
In a first experiment, axillary buds of adult
plants of C. incisa were inoculated on MS supple-
mented with different concentrations of BA. This ex-
periment (Table 1) showed that the presence of BA
on the culture media positively influence shoot proli-
feration and explant viability of C. incisa, with a sig-
nificant increase of the number of shoots per explant
with the increment of BA concentration from 0 to 4.4
µmol . L-1. On high concentrations of BA (8.8 and
17.6 µmol . L-1) the explants and new shoots showed
signs of hyperhydricity indicating an upper limit on
BA concentration for the multiplication of this speci-
es. High concentrations of cytokinins have been re-
ported to be one of the most important factors invol-
ved in the induction and/or development of hyperhydri-
city during in vitro culture (Ziv, 1991; Kadota and Nii-
mi, 2003). This physiological disorder was previous-
ly observed in C. galioides (Fracaro and Echeverri-
garay, 2001). The highest number of shoots per ex-
plant and the taller plantlets were obtained on 4.4
µmol . L-1 of BA, a concentration that is on the range
recommended for most species of the Lamiaceae
family, but is lower than that reported for C. galioides(Fracaro and Echeverrigaray, 2001).
TABLE 1. Effect of benzyladenine concentration on the micropropagation of C. incisa.
* Means followed by the same letter are not significantly different (p<0.05) using Tukey’s test.
Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.186-189, 2006.
188
The effect of four culture formulations on the
micropropagation of C. incisa is shown in Table
2. The explants cultivated on MS, B5 and LS
media produced more shoots than those
cultivated on QL medium. This difference may
be associated to the high NO3/NH4 relation
(85:15) of the last medium, developed for the
multiplication of woody plants (Quoirin and
Lepoivre, 1977). Nitrate/ammonium relation is
essential for the nitrogen supply, and pH control
during in vitro culture, drastically affecting
culture performance (George, 1996).
TABLE 2. Influence of culture medium composition on the micropropagation of C. incisa.
* Means followed by the same letter are not significantly different (p<0.05) using Tukey’s test.
Although non-significant differences were
detected among LS, MS, and B5 cultures with
respect to the number of shoots per explant and
shoot length, the low explant viability (65%), and
high hyperhydricity (23%) observed on LS, and
the hyperhydricity (18%) and leaves yellowing
exhibited by B5 grown plantlets, limits their use.
Considering all the parameters and the aspect of
the plantlets, the best results were obtained on MS
medium.
The effect of combinations of auxine and
cytokinin was evaluated, and the results obtained are
present in Table 3. The best results were obtained on
MS medium supplemented with 4.4 µmol . L-1 BA,
and with the combination of 4.4 µmol . L-1 BA and 2.0
µmol . L-1 IBA. Higher concentrations of IBA (4.0 and
6.0 µmol . L-1), IAA (5.6 µmol . L-1), and NAA (5.4
µmol . L-1) in combination with 4.4 µmol . L-1 BA
induced callus and a stunt development of the
plantlets (data not shown).
TABLE 3. Cytokinin/auxine combination on the micropropagation of C. incisa.
* Means followed by the same letter are not significantly different (p<0.05) using Tukey’s test.
Plantlets with 2 to 3 cm, obtained on MS
medium with 4.4 µmol . L-1 BA, were transferred to
rooting media with different concentrations of IBA.
As can be observe in Table 4, the four traits evaluated
were negatively affected by IBA, with an inverse
relation between IBA concentration and both
proliferation and rooting parameters. Considering
these results the best system for in vitro rooting of
C. incisa plantlets was MS medium without growth
regulators. These data differs from those obtained
with C. galioides, in which 0.5 to 2.4 µmol . L-1 IBA
positively stimulated rooting (Fracaro and
Echeverrigaray, 2001). The uses of ½ and ¼ MS salt
concentration did not affect root induction but
negatively affect both plantlet and root growth (data
not shown).
Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.186-189, 2006.
189
TABLE 4. The effect of IBA concentration on the in vitro rooting of micropropagated plantlets of C. incisa.
IBA 
concentration 
(µmol . L-1) 
N° of shoots per 
explant* 
Shoot length* 
(cm) 
N° of roots per 
explant* 
Root length* 
(cm) 
Explants 
producing 
callus (%) 
0.0 3.08 ± 0.76 a 3.43 ± 0.55 a 4.58 ± 1.27 a 2.25 ± 0.57 a 0 
0.5 2.65 ± 0.60 ab 2.20 ± 0.42 ab 2.30 ± 0.68 ab 2.16 ± 0.69 a 0 
2.4 2.50 ± 0.61 ab 1.89 ± 0.42 b 1.35 ± 0.56 b 1.65 ± 0.26 ab 0 
4.9 1.47 ± 0.21 b 1.44 ± 0.14 b 1.47 ± 0.57 b 1.31 ± 0.49 ab 35 
9.8 1.06 ± 0.06 b 0.99 ± 0.24 b 0.86 ± 0.29 b 0.97 ± 0.32 b 100 
 * Means followed by the same letter are not significantly different (p<0.05) using Tukey’s test.
A total of 155 rooted plantlets with 3.30 ± 0.30 cm
height were transferred to plastic boxes for
acclimatizing. Ninety five percent (95%) of these
plantlets survive the acclimatization period and were
transferred to outdoor conditions with 100% success.
These plants were cultivated during one year
exhibiting normal morphological and phenological
characteristics. Compared with stem cutting derived
plants, the micropropagated plants were more
vigorous, with larger leaves, and a higher number of
branches per plant.
ACKNOWLEDGEMENT
This work was supported by a grant form the
University of Caxias do Sul and CAPES, Brazil.
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