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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 , subfamily , tribe ) 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 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 in vitro culture and micropropagation of aromatic and medicinal plants has proved to be an The genus Cunila Royen ex L., family Lamia- important alternative for the rapid multiplication of ceae, subfamily Nepetoideae, tribe Mentheae (Can- selected genotypes or chemotypes (Bajaj et al., tino et al., 1992) consists of 22 species with two 1988). The micropropagation of Cunila galioides (Fra- centers of distribution: Mexico with 10 species (Gar- caro and Echeverrigaray, 2001) allowed the establish- cia-Peña, 1989) and the southern part of South Ame- ment of a germplasm collection and the multiplicati- rica with 12 species. The South American species on of plants with different chemical characteristics are divided into three sections: Incana, Incisa and (Echeverrigaray et al., 2003). C. incisa can be pro- Spicata. C. incisa is one of the two species of the pagated by seeds and cuttings, but the application Incisa section (Coelho de Souza, 1997). of these conventional propagation methods are limi- C. incisa grows at the margins of the forest in ted by poor seed viability, low germination rate and the highlands (300-1000m) of southern Brazil. It is a scanty rooting of stem cuttings. Therefore, the mi- perennial scrub with 1-2m high with ramified stem cropropagation of this species is highly desirable for and branches. Their leaves (1-3cm long and 0.8-1.5cm its application in the commercial production of es- width) are peciolate, ovoid to rhomboid with charac- sential oil. teristic incised margins. The small white to cream In the present paper, we report the effect of flowers (3mm long) are grouped in cimules fixed to culture media and growth regulators on the micro- the branches by short peduncles. Leaves, flowers propagation through axillary buds of selected adult and stalk are covered by glandular trichomes (Bor- plants of C. incisa. dignon, 1997). Hydrodistillation of the aerial parts of C. inci- MATERIAL AND METHOD sa yielded 1 to 1.3% (w/w) of an essential oil with a Five field plants of Cunila incisa Benth. collec- characteristic high content of 1,8-cineole (50-60%) ted at Veranópolis, Rio Grande do Sul, Brazil, and (Bordignon et al., 1996). This monoterpene poses selected for their high oil yield and growing characte- secretolytic, secretomotoric, bronchospasmolytic, ristics, were used as the donor plants in the micro- antiphlogistic and antiseptic properties (Duke et al., propagation experiments. Shoot segments with 3 to 1994) that justify the popular application of this 4 nodes were collected during spring (September- for the treatment of chronic cough and respiratory October). The nodal segments were washed with 70% diseases (Simões et al., 1994). 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- Recebido para publicação em agosto/2004 Aceito para publicação em julho/2006 rile distilled water axillary buds measuring 2 to 4 mm

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were excised and implanted vertically on the culture All the experiments were conducted in rando- media. mized design with 40 explants per treatment. The Four basal media were used: MS (Mu- evaluation of the number of shoots per explant, sho- rashige and Skoog, 1962), QL (Quoirin and Lepoivre, ot length, percentage of rooted plantlets, number of 1977), LS (Linsmaier and Skoog, 1965), and B5 roots per explant, and root length, were made after (Gamborg et al., 1968). All the media contained 30 g 30 days in culture. The data were submitted to sta- L-1 sucrose and 0.7% agar. The pH of the media was tistical analysis by ANOVA and the means compa- adjusted to 5.8 prior to autoclaving. Cultures were red by the Tukey´s test. incubated in a culture room at 25±2 °C with 16 h photoperiod under a photon flux of 20 µmol m-2 s-1 RESULT AND DISCUSSION provided by cool white fluorescent lamps (Phillips TLT In a first experiment, axillary buds of adult 40W/75). Cultures were transferred onto fresh me- plants of C. incisa were inoculated on MS supple- dium every 30 days. mented with different concentrations of BA. This ex- To evaluate the effect of culture media axillary periment (Table 1) showed that the presence of BA buds were inoculated on MS, B5, LS and QL supple- on the culture media positively influence shoot proli- mented with 4.4 µmol . L-1 of BA. The effect of ben- feration and explant viability of C. incisa, with a sig- zyladenine concentration (0 to 17.6 µmol . L-1) was nificant increase of the number of shoots per explant evaluated on MS basal medium. MS medium with with the increment of BA concentration from 0 to 4.4 4.4 µM BA supplemented with naphthaleneacetic acid µmol . L-1. On high concentrations of BA (8.8 and (1.0 and 2.7 µmol . L-1), indoleacetic acid (2.8 µmol . 17.6 µmol . L-1) the explants and new shoots showed L-1) or indolebutyric acid (2 µmol . L-1), was used to signs of hyperhydricity indicating an upper limit on evaluate the effect of cytokinin/auxin combination on BA concentration for the multiplication of this speci- the micropropagation. es. High concentrations of cytokinins have been re- Microshoots (2 to 3 cm long) with 4 to 6 no- ported to be one of the most important factors invol- des obtained on MS medium supplemented with 4.4 ved in the induction and/or development of hyperhydri- µmol . L-1 of BA were used to evaluate the effect of city during in vitro culture (Ziv, 1991; Kadota and Nii- indolebutyric acid (0 to 9.8 µmol . L-1) on rooting. mi, 2003). This physiological disorder was previous- Rooted plantlets were washed in tap-water and trans- ly observed in C. galioides (Fracaro and Echeverri- ferred to plastic chambers containing a sterilized garay, 2001). The highest number of shoots per ex- mixture of sand and soil (1:1), covered with a plastic plant and the taller plantlets were obtained on 4.4 cap that was gradually opened during the acclimati- µmol . L-1 of BA, a concentration that is on the range on period of 15 days. Acclimatized plants were trans- recommended for most species of the Lamiaceae ferred to the greenhouse and then to outdoor conditi- family, but is lower than that reported for C. galioides ons. (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.

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The effect of four culture formulations on the (85:15) of the last medium, developed for the micropropagation of C. incisa is shown in Table multiplication of woody plants (Quoirin and 2. The explants cultivated on MS, B5 and LS Lepoivre, 1977). Nitrate/ammonium relation is media produced more shoots than those essential for the nitrogen supply, and pH control cultivated on QL medium. This difference may during in vitro culture, drastically affecting be associated to the high NO3/NH4 relation 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 The effect of combinations of auxine and detected among LS, MS, and B5 cultures with cytokinin was evaluated, and the results obtained are respect to the number of shoots per explant and present in Table 3. The best results were obtained on shoot length, the low explant viability (65%), and MS medium supplemented with 4.4 µmol . L-1 BA, high hyperhydricity (23%) observed on LS, and and with the combination of 4.4 µmol . L-1 BA and 2.0 the hyperhydricity (18%) and leaves yellowing µmol . L-1 IBA. Higher concentrations of IBA (4.0 and exhibited by B5 grown plantlets, limits their use. 6.0 µmol . L-1), IAA (5.6 µmol . L-1), and NAA (5.4 Considering all the parameters and the aspect of µmol . L-1) in combination with 4.4 µmol . L-1 BA the plantlets, the best results were obtained on MS induced callus and a stunt development of the medium. 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 C. incisa plantlets was MS medium without growth medium with 4.4 µmol . L-1 BA, were transferred to regulators. These data differs from those obtained rooting media with different concentrations of IBA. with C. galioides, in which 0.5 to 2.4 µmol . L-1 IBA As can be observe in Table 4, the four traits evaluated positively stimulated rooting (Fracaro and were negatively affected by IBA, with an inverse Echeverrigaray, 2001). The uses of ½ and ¼ MS salt relation between IBA concentration and both concentration did not affect root induction but proliferation and rooting parameters. Considering negatively affect both plantlet and root growth (data these results the best system for in vitro rooting of not shown).

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TABLE 4. The effect of IBA concentration on the in vitro rooting of micropropagated plantlets of C. incisa. IBA N° of shoots per Shoot length* N° of roots per Root length* Explants concentration explant* (cm) explant* (cm) producing (µmol . L-1) 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 Duke, J.A. Biologically-Active Compounds in Important height were transferred to plastic boxes for Spices. In: Spices, Herbs and Edible Fungi. acclimatizing. Ninety five percent (95%) of these Amsterdam, Elsevier. 1994, p. 225-250. plantlets survive the acclimatization period and were Echeverrigaray, S.; Fracaro, F.; Santos, A.C.A.; Paroul, N.; Wasum, R.; Atti-Serafini, L. Essential oil composition transferred to outdoor conditions with 100% success. of south Brazilian populations of Cunila galioides and These plants were cultivated during one year its relation with the geographical distribution. exhibiting normal morphological and phenological Biochem. Syst. Ecol. v.31: 467-475, 2003. characteristics. Compared with stem cutting derived Fracaro, F.; Echeverrigaray S. Micropropagation of Cunila plants, the micropropagated plants were more galioides, a popular medicinal plant of south Brazil. vigorous, with larger leaves, and a higher number of Plant Cell Tiss. Org. Cult. v.64: 1-4, 2002 branches per plant. Gamborg, O. L.; Miller, R. A.; Ojima, H. Nutrient requirements of suspension cultures of soybean root ACKNOWLEDGEMENT cell. Exp. Cell Res. v.50: 151-158, 1968. Kadota, M.; Niimi, Y. Effect of cytokinin types and their concentration on shoot proliferation and This work was supported by a grant form the hyperhydricity in in vitro pear cultivar shoots. Plant University of Caxias do Sul and CAPES, Brazil. Cell Tiss. Org. Cult. v.72: 261-265, 2003 Linsmaier, E. M.; Skoog, F. Organic growth factor REFERENCE requeriments of tobacco tissue culture. Physiol. Plant. v.18: 100-127, 1965. Murashige, T.; Shoog, F. S. A revised medium for rapid Bajaj, Y. P. S.; Furmonowa, M.; Olszowska, O. growth and bioassay with tobacco tissue culture. Biotechnology of the micropropagation of medicinal Physiol. Plant. v.15: 473-497, 1962. and aromatic plants. In: Bajaj, Y.P.S. (ed) Quoirin, M.; Lepoivre, P. Etude de milieus adaptes aux Biotechnology in Agriculture and Forestry, Vol. 4, cultures in vitro de Prunus. Acta Hortic. v.78: 437-442, Medicinal and Aromatic Plants I. Berlin, Springer- 1977. Verlag, 1998, p. 60-103. Simões, C. M. O.; Mentz, L. A.; Schenkel, E. P.; Irgang, B. Bordignon, S. A. L.; Schenkel, E. P.; Spitzer, V. The essential E.; Stehmann, J.R. Plantas da medicina popular no oil of Cunila incisa (Lamiaceae)- a rich source of 1,8- Rio Grande do Sul. Ed. Universidade/UFRGS. 1994. cineole. Química Nova v.19: 105-107, 1996. Ziv, M. Vitrification: morphological and physiological Cantino, D.; Harley, R. M.; Wagstaff, S. J. Genera of disorders of in vitro plants. In: Debergh, P. C.; Labiatae: status and classification. In: Harley, R. M.; Zimmerman, R. H. (eds). Micropropagation– Reynolds, T. (eds) Advances in Labiatae Science. Technology and application. Dordrecht, Kluwer Kew, Royal Botanical Garden,. 1992, p. 511-522. Academic Publish. 1991, p. 45-69.

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