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Micropropagation of Chamomilla recutita (L.) Rauschert: A shock treatment model with growth regulators
Scientia Horticulturae 109 (2006) 160–164 www.elsevier.com/locate/scihorti
Micropropagation of Chamomilla recutita (L.) Rauschert: A shock treatment model with growth regulators Alice Sato, Sharon S. de Lima *, Vanessa R. Affonso, Maria Apparecida Esquibel, Celso L.S. Lage Laboratory of Plant Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Cidade Universita´ria, CEP: 21.949-900, Rio de Janeiro, RJ, Brazil Received 28 January 2005; received in revised form 21 November 2005; accepted 7 March 2006
Abstract A protocol for in vitro clonal propagation of a Brazilian variety of chamomile (Chamomilla recutita (L.) Rauschert) is described. Nodal segments were submersed in liquid MS basal medium containing 1.0–4.0 mg l 1 GA3 or thidiazuron (TDZ) or 0.5–4.0 mg l 1 2,4-D for 2 h. After that, they were transferred to solid MS basal media without growth regulators and maintained for 2 months. This process avoided the callus development which was undesirable for shoot multiplication. The best treatment for shoot proliferation and plant rooting contained 1.0–2.0 mg l 1 gibberellin (GA3) or 0.5 mg l 1 2,4-D. Plants treated with 0.5 mg l 1 2,4-D were subcultured for half-strength nitrogen solid MS medium and they were acclimatized two months later. The hardening process in the greenhouse was difficult and time consuming, however, 54% of plants survived and were successfully adapted to field conditions. # 2006 Elsevier B.V. All rights reserved. Keywords: Tissue culture; Growth regulators; Medicinal plant; Chamomile
1. Introduction Herbal medicine practice has had a remarkable increase in the last decade (Calixto, 2000). Some plants are consumed mainly due to their well-known therapeutic actions, despite the existence of scientific support. Additionally, herbal medicines can proceed from non-standardised protocols to handle preparations based on plant material. This being the fact, active ingredients can vary quantitatively and qualitatively, depending on the plant species, its origin, and its harvesting period (Currier et al., 2000). Techniques on tissue culture of medicinal plants have been directed to the production of special metabolic, genetic transformation, germplasm conservation or development of protocols for plant micropropagation (Rout et al., 2000; Lima et al., 2001). Standardised in vitro culture has been considered a promising tool to obtain homogeneous plant material to serve as appropriate source of drugs. Chamomilla recutita (L.) Rauschert (Asteraceae) is a widespread European plant known as chamomile. Medicinal
* Corresponding author. Tel.: +55 21 2562 6643; fax: +55 21 2280 8193. E-mail address: [email protected] (S.S. de Lima). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.03.004
teas prepared from the flowering plant are used in folk medicine to treat gastrointestinal diseases and its active ingredients are indicated as containing carminative, antispasmodic or sedative activities (Correˆa Ju´nior, 1998). The essential oil present in the flower heads of the plant is responsible for its major pharmacological effects (WHO, 1999). Any report about micropropagation of C. recutita, a Brazilian variety is available. Therefore, the present investigation was undertaken to develop an effective method for in vitro multiplication, which could result in high adaptation to field conditions. 2. Material and methods 2.1. Establishment of in vitro cultures Seeds (Isla1 Pak) were used as source of plant material. Seeds’ surface were sterilised in 50% commercial bleach solution for 30 min, rinsed three times for 5 min in distilled water, dipped in 70% ethanol and rinsed three times for 5 min in sterile distilled water. The disinfected seeds were inoculated on glass tubes (10 cm 1.5 cm) containing sterilised water and paper filtering as a support. Seeds were maintained in a growth
A. Sato et al. / Scientia Horticulturae 109 (2006) 160–164
room at 25 2 8C under a 16/8 h light/dark photoperiod (Silvania daylight fluorescent lamps, 23 mmol m 2 s 1). 2.2. Culture treatments with growth regulators Initially, 1.0 cm length nodal segments containing 2–4 buds were excised from in vitro culture seedlings and placed in glass bottles (500 ml) containing 50 ml of MS (Murashige and Skoog, 1962) added to 3% sucrose and solidified with 0.8% agar–agar. In order to obtain at least 30 shoots for each treatment, plantlets were subcultured in the same kind of culture medium until enough shoots were available to establish the experiments. Preliminary tests were conducted to examine growth regulators effects on the development of C. recutita. Thus, nodal segments were transferred to solid MS media supplemented with either thidiazuron (TDZ; 0.1, 0.5 and 1.0 mg l 1), indole-3-acetic acid (IAA; 0.1, 0.5 and 1.0 mg l 1), kinetin (Kin; 0.5 and 1.0 mg l 1) or gibberellin (GA3; 0.5 and 1.0 mg l 1). After the results of the preliminary tests were obtained, a new experiment was done. Nodal segments were excised from plantlets previously cultured in solid MS media, and submitted to a ‘‘shock’’ of growth regulators in liquid MS media supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5, 1.0, 2.0 or 4.0 mg l 1), GA3 (1.0, 2.0 or 4.0 mg l 1) or TDZ (1.0, 2.0 or 4.0 mg l 1) for 2 h, under shaking. After that, nodal segments were transferred to solid MS medium without adding growth regulators. Cultures were maintained in the growth room under the conditions mentioned above. The number of shoots per explant, shoot height (cm), percentage root formation and percentage callus development were recorded after 2 months. Thirty random explants were used per treatment, and each experiment was performed twice. Data were submitted to ANOVA analysis (analysis of variance) and the means were compared by the Tukey’s test at 5% significance. 2.3. Acclimatization After the two-month period of culture in solid MS medium, vigour was evaluated for each plantlet group obtained from the different hormone treatments. According to the growth parameters indicated in the item above, plantlets that were shocked on liquid MS medium supplemented with 0.5 mg l 1 2,4-D for 2 h were chosen as the experimental group to proceed to the acclimatization protocol. To avoid excessive stress of the plants while transferring them to soil, those plantlets were sub-cultured for additional 2 months in halfstrength nitrogen MS medium (1/2 N) containing 1.5% sucrose, and solidified with 0.8% agar, before being acclimatized. Whenever a rooted plantlet appeared, it was removed from the culture medium, the roots were gently washed in tap water, and then transferred to a seed pot containing soil conditioner. They were kept under greenhouse conditions for four weeks before being transferred to outdoor conditions. Plantlets were irrigated every day with the filtered and UV sterilised water.
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3. Results 3.1. In vitro establishment and growth regulators on culture The disinfecting method was 100% successful in eliminating any seed contamination. After 13 days, 24.5% of the seeds germinated, as considered by emission of the first radical. The preliminary test with growth regulators TDZ, IAA, Kin or GA3 added to solid MS medium showed no satisfactory results. The nodal segments cultured in those media frequently developed callus, and the few shoots, whenever formed, soon initialized a senescence process, which was evidenced after about 5 weeks of culture. Except for the shock treatments with 2.0 mg l 1 TDZ or with 4.0 mg l 1 GA3, all other concentrations of growth regulators enhanced shoot formation. The greatest shoot number (3.31 per explant) and the highest shoots (4.61 cm in length) were obtained from nodal segments submitted to the shock treatment with 1.0 mg l 1 2,4-D. The increase in 2,4-D concentrations up to 4.0 mg l 1, as well as its lowest concentration (0.5 mg l 1), reduced both the shoot formation and elongation. Nodal segments treated with 1.0 mg l 1 GA3 or 4.0 mg l 1 2,4-D developed three shoots per explant. Yet, the media supplemented with either 1.0–2.0 mg l 1 GA3 or 0.5– 4.0 mg l 1 2,4-D did not cause plants to attain statistically different results. Besides, shock treatment with 2.0 mg l 1 TDZ produced both the lowest number of shoots (1.93 per explant), and elongation (3.42 cm). Treatment with either 1.0 mg l 1 TDZ or with 2.0 mg l 1 GA3 resulted in plant 4.25 cm tall in average, and no statistical difference was found in the number and length of shoots. C. recutita shoots succeeded to root with all treatments, although 2.0 mg l 1 GA3 resulted in the highest rooting percentage (100%), and no callus was observed after treatment with this growth regulator. The increase in TDZ concentration from 1.0 to 4.0 mg l 1 promoted the increase of both the rooting and callus percentage. Conversely, increasing the 2,4-D concentration caused the plantlets to reduce rooting. The percentage of callus formation was the highest with 2.0 mg l 1 2,4-D treatment, as well as 1.0 mg l 1 TDZ (data not shown). The ideal interval of time for subcultures was 2 months for all treatments with growth regulators. The best media tested contained 1.0–2.0 mg l 1 GA3 or 0.5 mg l 1 2,4-D in terms of what was observed during the proliferation and rooting phases (Fig. 1). Ex vitro rooting phase appeared not to be necessary. The complete morphologic results are summarized in Table 1. 3.2. Acclimatization Plantlets developed in solid MS media without previous shock did not survive acclimatization. Treatment of plantlets with liquid MS media plus 0.5 mg l 1 2,4-D, nevertheless, caused plantlets to overcome such stressing period. A preadaptation to autotrophic conditions can be stimulated on in vitro plants (Grattapaglia and Machado, 1998), and an attempt
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A. Sato et al. / Scientia Horticulturae 109 (2006) 160–164
Fig. 1. Morphologic aspect of plants submitted to shock with growth regulators in liquid media for 2 h. After having been shocked or not (control), the plants were transferred to solid MS media, where they were maintained for 2 months: (a) MS medium free of growth regulators, (b) MS medium added of 1.0 mg l 1 GA3, (c) MS medium added of 2.0 mg l 1 GA3, or (d) MS medium added of 0.5 mg l 1 2,4-D. Bar = 1.0 cm.
Table 1 Effect of growth regulators ‘‘shocking’’ treatment on in vitro development of Chamomilla recutita Plant growth regulator (mg l 1)
Number of shoots per explants a
Shoot lengtha (cm)
MSb
2.35 0.20 bcd
3.88 0.13 bcd
98.9
–
GA3 1.0 2.0 4.0
3.03 0.32 a 2.71 0.30 abc 2.23 0.17 cd
3.71 0.17 cd 4.25 0.18 ab 3.55 0.09 d
93.9 100 84.3
– – –
TDZ 1.0 2.0 4.0
2.70 0.32 abcd 1.93 0.19 d 2.68 0.26 abcd
4.25 0.33 ab 3.42 0.12 d 3.95 0.19 bcd
74.1 90.3 95.4
51.8 58.1 59.1
2,4-D 0.5 1.0 2.0 4.0
2.82 0.26 3.31 0.40 3.11 0.15 3.04 0.22
3.87 0.13 4.61 0.26 4.04 0.11 3.52 0.14
94.7 88.5 83.6 75.0
15.8 15.4 97.1 45.8
ab a a a
bcd a bc d
Rooting (%)
Callus development (%)
Plants were treated in liquid media added of growth regulators for 2 h and then transferred to solid MS, where they were cultured for 2 months. a Values are mean standard error. Values in same column followed by the same letter (a, b, c, d) do not differ statistically at p 0.05. b MS—control medium, without adding growth regulators.
to increase survival rates of plants was made by decreasing the amounts of nitrogen and sucrose on MS media before acclimatization. Plants from this pre-adaptation treatment were actually less susceptible to dehydration and 54% of the plants succeeded in survival. After the first 3 months on the field, it was advisable not to expose plants to direct sunlight, to avoid dehydration. Until the achievement of tissue ‘‘hardening’’, the leaves should not be wet or they could die, and nothing except a moist soil was maintained throughout the initial period. After 1 month on natural field conditions, 90% of them flowered at the same time in August, reaching 33.0 cm mean length and the flower heads attained an average of 8.0 0.5 mm in diameter. Acclimatized plants were morphologically normal. 4. Discussion At first the plants were maintained, throughout the culture, in solid medium supplemented with growth regulators, and the
initial results did not appeared interesting to proceed with micropropagation. Calluses were often formed from the cultured explants, which was undesirable for a successful micropropagation. Other previous studies with in vitro culture of Chamomilla spp. were concerned with callus culture (Reichling and Becker, 1976; Reichling et al., 1984; Magiatis et al., 2001), somatic embryogenesis and in vitro flowering (Kintzios and Michaelakis, 1999), viability of cells after cryopreservation (Cellarova et al., 1992), and mRNA expression in culture shoot primordia (Ashida et al., 2002). In any case, the appearance of callus in such cultures was considered a problem. Plant multiplication was the main target of the present work, and it was necessary to find ideal conditions for explants to develop without forming callus. Therefore, the plants were submitted to a ‘‘shock’’ with growth regulators for 2 h, as a means to cause a stressful situation. The growth regulators were used as an agent capable of evoking physiological responses necessary for morphogenesis, and to consequently improve the yield of the culture.
A. Sato et al. / Scientia Horticulturae 109 (2006) 160–164
The callus formation could be decreased when the nodal segments were submitted to a shock with growth regulators. If different times of exposure to the growth regulators were used, they could possibly elicit proportionally correspondent plant answers, with more or less intensity. And it is to mention that different varieties of the same species can present different patterns of development when they are submitted to the same treatment (Grattapaglia and Machado, 1998). Pretreatment with high or relatively high concentrations of growth regulators can improve organogenesis or somatic embryogenesis in other species as has been previously described. Pretreatment in cucumber (Cucumis sativus L.) leaves with 14 mM 2,4-D for 3 weeks increased the embryogenic-callus production. (Kuijpers et al., 1996). Pretreatment of chilli pepper (Capsicum annuum) leaves with high cytokinin concentration (129 mM 6-benzylaminopurine for 24 h) and later transferring onto solid ‘standard induction medium’ can improve the embryo proliferation (Kintzios et al., 2000). Growth regulator pretreatment improves somatic embryogenesis in the leaves of squash (Curcubita pepo L.) and melon (Cucumis melo L.). Leaves were pretreated with 113.1, 226.2 or 452.4 mM 2,4-D, 46.5, 93 or 186 mM Kin or a combination of both at above concentrations for 6, 24 or 48 h. Exposure of leaf explants to at least 452.4 mM 2,4-D for 6 h (squash) or 226.2 mM 2,4-D for 48 h (melon), 46.5 mM Kin for 48 h (squash) or 186 mM Kin for 6 h (melon), and subculture onto ‘species-specific standard induction medium’ significantly promoted somatic embryo induction (Kintzios et al., 2002). Passamonti et al. (1998) established a micropropagation protocol for the European varieties of C. recutita, using the traditional protocol of in vitro culture, with the growth regulators added to the solid culture media. Additionally, they have reported the best medium for proliferation phase one that contained 0.5 mg l 1 kinetin, although the plants were not able to rooting in vitro. Considering the multiplication rates, the present work showed results quite similar to those observed by Passamonti et al. (1998). The ideal interval to subculture chamomile Brazilian variety was 2 months. Plants had their leaves turned to yellow if were maintained for more than the indicated period in the same aged medium and, sometimes, the shoots died. They could not improve under such a situation. The pre-adaptation to autotrophic conditions was crucial to achieve successful acclimatization. Conditions leading to callus formation either can render an overwhelming plant acclimatization, or may even impair it. A small callusing percentage cannot be a hindrance for plant acclimatization. Some authors connect flower head diameter and soil fertility, as seen in Bezzi et al. (1991), who examined nine different chamomile cultivars and observed flower mean diameters as large as about 31.10 mm in good fertile soil. In the present work, any fertilisers were used on soil; moreover, the largest flower head diameters were obtained as soon the flowers showed up. It is worth noting here, the flowering synchronism attained in acclimatised plants circumventing daily harvest. In the most continuously flowering cultivars, for instance, the Mandirituba Brazilian variety, harvesting is demanded every 8 days, raising the production costs (Correˆa Ju´nior, 1998). Plants
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reached 33.0 cm mean length when setting up flowering, whereas for Mandirituba variety, the flowering period began with plants as high as about 24.0 cm. Overall, the present results for plant development have been considered as reasonable ones, in comparison to cultures of chamomile found abroad, for instance, 63.0 and 87.6 cm for Bona and Egito, respectively (Correˆa Ju´nior, 1998). Altogether, the present collected data suggests that successful in vitro culture and rapid propagation of the Brazilian variety of the C. recutita are possible once the explants are exposed to the controlled conditions with the growth regulators. Acknowledgements The authors thank CAPES, FAPERJ and FAPERJ-RECOPE for financial support, and they also thank Professor Claudia Lage from Carlos Chagas Filho Biophysics Institute—Federal University of Rio de Janeiro, for English revision. References Ashida, Y., Nishimoto, M., Matsushima, A., Watanabe, J., Hirata, T., 2002. Molecular cloning and mRNA expression of geraniol-inducible genes in cultured shoot primordia of Matricaria chamomilla. Biosci., Biotechnol. Biochem. 66 (11), 2511–2514. Bezzi, A., Aiella, N., Ghidini, G., 1991. La coltivazione della camomilla nell’Italia settentrionale; vari tipi di camomilla comune coltivati in Trentino-Alto adige, vol 19. L ‘Informatore Agrario, Verona, pp. 27–32. Calixto, J.B., 2000. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz. J. Med. Biol. Res. 33, 179–189. Cellarova, E., Cernicka, T., Vranova, E., Brutovska, R., Lapar, M., 1992. Viability of Chamomilla recutita (L.) Rauschert cells after cryopreservation. Cryo Lett. 13 (1), 37–42. Correˆa Ju´nior, C., 1998. Influeˆncia das adubac¸o˜es orgaˆnica e quı´mica na produc¸a˜o de Chamomilla recutita (L.) Rauschert e do seu o´leo essencial. In: Ming, L.C., Scheffer, M.C., Correˆa Ju´nior, C., Barros, I.B.I., Mattos, J.K.A. (Eds.), Plantas medicinais, aroma´ticas e condimentares – Avanc¸os na pesquisa agronoˆmica, vol.1. Universidade Estadual Paulista, Botucatu, SP, pp. 129–163. Currier, S.J., Johnston, P.D., Gorelick, K.J., 2000. Herbal medicines. Sci. Med. 40–43. Grattapaglia, D., Machado, M.A., 1998. Micropropagac¸a˜o. In: Torres, A.C., Caldas, L.S., Buso, J.A. (Eds.), Cultura de tecidos e transformac¸a˜o gene´tica de plantas, vol.1. Embrapa-SPI/Embrapa-CNPH, Brası´lia, pp. 183–260. Kintzios, S., Michaelakis, A., 1999. Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomille (Chamomilla recutita L.). Plant Cell Rep. 18 (7–8), 684–690. Kintzios, S., Drossopoulos, J.B., Shortsianitis, E., Peppes, D., 2000. Induction of somatic embryogenesis from young, fully expanded leaves of chilli pepper (Capsicum annuum L.): effect of leaf position, ilumination and explant pretreatment with high cytokinin concentrations. Sci. Horti. 85, 137–144. Kintzios, S., Sereti, E., Bluchos, P., Drossopoulos, J.B., Kitsaki, C.K., LiopaTsakalidis, A., 2002. Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Curcubita pepo L.) and melon (Cucumis melo L.). Plant Cell Rep. 21, 1–8. Kuijpers, A.M., Bouman, H., Klerk, G.J., 1996. Increase of embryogenic-callus formation in cucumber by initial culture on high concentration of 2,4dichlorophenoxyacetic acid. Plant Cell Tiss. Org. Cult. 46, 81–83. Lima, S.S., Esquibel, M.A., Henriques, A.B., Silva, F.O., Silva, P.H.B., Lage, C.L.S., 2001. Monoclonal culture of erva-de-bicho (Polygonum acre HBK
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var. aquatile) for commercial scale production of a phyto-therapeutic medicine. Braz. J. Med. Plant. 4 (1), 51–55. Magiatis, P., Michaelakis, A., Skaltsounis, A.L., Haroutounian, S.A., 2001. Volatile secondary metabolite pattern of callus cultures of Chamomilla recutita. Nat. Prod. Lett. 15 (2), 125–130. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Passamonti, F., Piccioni, E., Standardi, A., Veronesi, F., 1998. Micropropagation of Chamomilla recutita (L.) Rauschert. Acta Horti. 457, 303–310.
Reichling, J., Becker, H., 1976. Tissue-culture of Matricaria chamomilla L. 1. Isolation and maintainance of tissue-culture—preliminary phytochemical investigations. Planta Med. 30 (3), 258–268. Reichling, J., Bisson, W., Becker, H., 1984. Comparative-study on the production and accumulation of essential oil in the whole plant and in the callusculture of Matricaria chamomilla. Planta Med. 50 (4), 334–337. Rout, G.R., Samantaray, S., Das, P., 2000. In vitro manipulation and propagation of medicinal plants. Biotech. Adv. 18, 91–120. WHO–World Health Organization 1999. Flos Chamomillae. In: WHO Monographs on Selected Medicinal Plants, vol. 1. Geneva, pp. 87–94.
Micropropagation of Chamomilla recutita (L.) Rauschert: A shock treatment model with growth regulators Alice Sato, Sharon S. de Lima *, Vanessa R. Affonso, Maria Apparecida Esquibel, Celso L.S. Lage Laboratory of Plant Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Cidade Universita´ria, CEP: 21.949-900, Rio de Janeiro, RJ, Brazil Received 28 January 2005; received in revised form 21 November 2005; accepted 7 March 2006
Abstract A protocol for in vitro clonal propagation of a Brazilian variety of chamomile (Chamomilla recutita (L.) Rauschert) is described. Nodal segments were submersed in liquid MS basal medium containing 1.0–4.0 mg l 1 GA3 or thidiazuron (TDZ) or 0.5–4.0 mg l 1 2,4-D for 2 h. After that, they were transferred to solid MS basal media without growth regulators and maintained for 2 months. This process avoided the callus development which was undesirable for shoot multiplication. The best treatment for shoot proliferation and plant rooting contained 1.0–2.0 mg l 1 gibberellin (GA3) or 0.5 mg l 1 2,4-D. Plants treated with 0.5 mg l 1 2,4-D were subcultured for half-strength nitrogen solid MS medium and they were acclimatized two months later. The hardening process in the greenhouse was difficult and time consuming, however, 54% of plants survived and were successfully adapted to field conditions. # 2006 Elsevier B.V. All rights reserved. Keywords: Tissue culture; Growth regulators; Medicinal plant; Chamomile
1. Introduction Herbal medicine practice has had a remarkable increase in the last decade (Calixto, 2000). Some plants are consumed mainly due to their well-known therapeutic actions, despite the existence of scientific support. Additionally, herbal medicines can proceed from non-standardised protocols to handle preparations based on plant material. This being the fact, active ingredients can vary quantitatively and qualitatively, depending on the plant species, its origin, and its harvesting period (Currier et al., 2000). Techniques on tissue culture of medicinal plants have been directed to the production of special metabolic, genetic transformation, germplasm conservation or development of protocols for plant micropropagation (Rout et al., 2000; Lima et al., 2001). Standardised in vitro culture has been considered a promising tool to obtain homogeneous plant material to serve as appropriate source of drugs. Chamomilla recutita (L.) Rauschert (Asteraceae) is a widespread European plant known as chamomile. Medicinal
* Corresponding author. Tel.: +55 21 2562 6643; fax: +55 21 2280 8193. E-mail address: [email protected] (S.S. de Lima). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.03.004
teas prepared from the flowering plant are used in folk medicine to treat gastrointestinal diseases and its active ingredients are indicated as containing carminative, antispasmodic or sedative activities (Correˆa Ju´nior, 1998). The essential oil present in the flower heads of the plant is responsible for its major pharmacological effects (WHO, 1999). Any report about micropropagation of C. recutita, a Brazilian variety is available. Therefore, the present investigation was undertaken to develop an effective method for in vitro multiplication, which could result in high adaptation to field conditions. 2. Material and methods 2.1. Establishment of in vitro cultures Seeds (Isla1 Pak) were used as source of plant material. Seeds’ surface were sterilised in 50% commercial bleach solution for 30 min, rinsed three times for 5 min in distilled water, dipped in 70% ethanol and rinsed three times for 5 min in sterile distilled water. The disinfected seeds were inoculated on glass tubes (10 cm 1.5 cm) containing sterilised water and paper filtering as a support. Seeds were maintained in a growth
A. Sato et al. / Scientia Horticulturae 109 (2006) 160–164
room at 25 2 8C under a 16/8 h light/dark photoperiod (Silvania daylight fluorescent lamps, 23 mmol m 2 s 1). 2.2. Culture treatments with growth regulators Initially, 1.0 cm length nodal segments containing 2–4 buds were excised from in vitro culture seedlings and placed in glass bottles (500 ml) containing 50 ml of MS (Murashige and Skoog, 1962) added to 3% sucrose and solidified with 0.8% agar–agar. In order to obtain at least 30 shoots for each treatment, plantlets were subcultured in the same kind of culture medium until enough shoots were available to establish the experiments. Preliminary tests were conducted to examine growth regulators effects on the development of C. recutita. Thus, nodal segments were transferred to solid MS media supplemented with either thidiazuron (TDZ; 0.1, 0.5 and 1.0 mg l 1), indole-3-acetic acid (IAA; 0.1, 0.5 and 1.0 mg l 1), kinetin (Kin; 0.5 and 1.0 mg l 1) or gibberellin (GA3; 0.5 and 1.0 mg l 1). After the results of the preliminary tests were obtained, a new experiment was done. Nodal segments were excised from plantlets previously cultured in solid MS media, and submitted to a ‘‘shock’’ of growth regulators in liquid MS media supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5, 1.0, 2.0 or 4.0 mg l 1), GA3 (1.0, 2.0 or 4.0 mg l 1) or TDZ (1.0, 2.0 or 4.0 mg l 1) for 2 h, under shaking. After that, nodal segments were transferred to solid MS medium without adding growth regulators. Cultures were maintained in the growth room under the conditions mentioned above. The number of shoots per explant, shoot height (cm), percentage root formation and percentage callus development were recorded after 2 months. Thirty random explants were used per treatment, and each experiment was performed twice. Data were submitted to ANOVA analysis (analysis of variance) and the means were compared by the Tukey’s test at 5% significance. 2.3. Acclimatization After the two-month period of culture in solid MS medium, vigour was evaluated for each plantlet group obtained from the different hormone treatments. According to the growth parameters indicated in the item above, plantlets that were shocked on liquid MS medium supplemented with 0.5 mg l 1 2,4-D for 2 h were chosen as the experimental group to proceed to the acclimatization protocol. To avoid excessive stress of the plants while transferring them to soil, those plantlets were sub-cultured for additional 2 months in halfstrength nitrogen MS medium (1/2 N) containing 1.5% sucrose, and solidified with 0.8% agar, before being acclimatized. Whenever a rooted plantlet appeared, it was removed from the culture medium, the roots were gently washed in tap water, and then transferred to a seed pot containing soil conditioner. They were kept under greenhouse conditions for four weeks before being transferred to outdoor conditions. Plantlets were irrigated every day with the filtered and UV sterilised water.
161
3. Results 3.1. In vitro establishment and growth regulators on culture The disinfecting method was 100% successful in eliminating any seed contamination. After 13 days, 24.5% of the seeds germinated, as considered by emission of the first radical. The preliminary test with growth regulators TDZ, IAA, Kin or GA3 added to solid MS medium showed no satisfactory results. The nodal segments cultured in those media frequently developed callus, and the few shoots, whenever formed, soon initialized a senescence process, which was evidenced after about 5 weeks of culture. Except for the shock treatments with 2.0 mg l 1 TDZ or with 4.0 mg l 1 GA3, all other concentrations of growth regulators enhanced shoot formation. The greatest shoot number (3.31 per explant) and the highest shoots (4.61 cm in length) were obtained from nodal segments submitted to the shock treatment with 1.0 mg l 1 2,4-D. The increase in 2,4-D concentrations up to 4.0 mg l 1, as well as its lowest concentration (0.5 mg l 1), reduced both the shoot formation and elongation. Nodal segments treated with 1.0 mg l 1 GA3 or 4.0 mg l 1 2,4-D developed three shoots per explant. Yet, the media supplemented with either 1.0–2.0 mg l 1 GA3 or 0.5– 4.0 mg l 1 2,4-D did not cause plants to attain statistically different results. Besides, shock treatment with 2.0 mg l 1 TDZ produced both the lowest number of shoots (1.93 per explant), and elongation (3.42 cm). Treatment with either 1.0 mg l 1 TDZ or with 2.0 mg l 1 GA3 resulted in plant 4.25 cm tall in average, and no statistical difference was found in the number and length of shoots. C. recutita shoots succeeded to root with all treatments, although 2.0 mg l 1 GA3 resulted in the highest rooting percentage (100%), and no callus was observed after treatment with this growth regulator. The increase in TDZ concentration from 1.0 to 4.0 mg l 1 promoted the increase of both the rooting and callus percentage. Conversely, increasing the 2,4-D concentration caused the plantlets to reduce rooting. The percentage of callus formation was the highest with 2.0 mg l 1 2,4-D treatment, as well as 1.0 mg l 1 TDZ (data not shown). The ideal interval of time for subcultures was 2 months for all treatments with growth regulators. The best media tested contained 1.0–2.0 mg l 1 GA3 or 0.5 mg l 1 2,4-D in terms of what was observed during the proliferation and rooting phases (Fig. 1). Ex vitro rooting phase appeared not to be necessary. The complete morphologic results are summarized in Table 1. 3.2. Acclimatization Plantlets developed in solid MS media without previous shock did not survive acclimatization. Treatment of plantlets with liquid MS media plus 0.5 mg l 1 2,4-D, nevertheless, caused plantlets to overcome such stressing period. A preadaptation to autotrophic conditions can be stimulated on in vitro plants (Grattapaglia and Machado, 1998), and an attempt
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Fig. 1. Morphologic aspect of plants submitted to shock with growth regulators in liquid media for 2 h. After having been shocked or not (control), the plants were transferred to solid MS media, where they were maintained for 2 months: (a) MS medium free of growth regulators, (b) MS medium added of 1.0 mg l 1 GA3, (c) MS medium added of 2.0 mg l 1 GA3, or (d) MS medium added of 0.5 mg l 1 2,4-D. Bar = 1.0 cm.
Table 1 Effect of growth regulators ‘‘shocking’’ treatment on in vitro development of Chamomilla recutita Plant growth regulator (mg l 1)
Number of shoots per explants a
Shoot lengtha (cm)
MSb
2.35 0.20 bcd
3.88 0.13 bcd
98.9
–
GA3 1.0 2.0 4.0
3.03 0.32 a 2.71 0.30 abc 2.23 0.17 cd
3.71 0.17 cd 4.25 0.18 ab 3.55 0.09 d
93.9 100 84.3
– – –
TDZ 1.0 2.0 4.0
2.70 0.32 abcd 1.93 0.19 d 2.68 0.26 abcd
4.25 0.33 ab 3.42 0.12 d 3.95 0.19 bcd
74.1 90.3 95.4
51.8 58.1 59.1
2,4-D 0.5 1.0 2.0 4.0
2.82 0.26 3.31 0.40 3.11 0.15 3.04 0.22
3.87 0.13 4.61 0.26 4.04 0.11 3.52 0.14
94.7 88.5 83.6 75.0
15.8 15.4 97.1 45.8
ab a a a
bcd a bc d
Rooting (%)
Callus development (%)
Plants were treated in liquid media added of growth regulators for 2 h and then transferred to solid MS, where they were cultured for 2 months. a Values are mean standard error. Values in same column followed by the same letter (a, b, c, d) do not differ statistically at p 0.05. b MS—control medium, without adding growth regulators.
to increase survival rates of plants was made by decreasing the amounts of nitrogen and sucrose on MS media before acclimatization. Plants from this pre-adaptation treatment were actually less susceptible to dehydration and 54% of the plants succeeded in survival. After the first 3 months on the field, it was advisable not to expose plants to direct sunlight, to avoid dehydration. Until the achievement of tissue ‘‘hardening’’, the leaves should not be wet or they could die, and nothing except a moist soil was maintained throughout the initial period. After 1 month on natural field conditions, 90% of them flowered at the same time in August, reaching 33.0 cm mean length and the flower heads attained an average of 8.0 0.5 mm in diameter. Acclimatized plants were morphologically normal. 4. Discussion At first the plants were maintained, throughout the culture, in solid medium supplemented with growth regulators, and the
initial results did not appeared interesting to proceed with micropropagation. Calluses were often formed from the cultured explants, which was undesirable for a successful micropropagation. Other previous studies with in vitro culture of Chamomilla spp. were concerned with callus culture (Reichling and Becker, 1976; Reichling et al., 1984; Magiatis et al., 2001), somatic embryogenesis and in vitro flowering (Kintzios and Michaelakis, 1999), viability of cells after cryopreservation (Cellarova et al., 1992), and mRNA expression in culture shoot primordia (Ashida et al., 2002). In any case, the appearance of callus in such cultures was considered a problem. Plant multiplication was the main target of the present work, and it was necessary to find ideal conditions for explants to develop without forming callus. Therefore, the plants were submitted to a ‘‘shock’’ with growth regulators for 2 h, as a means to cause a stressful situation. The growth regulators were used as an agent capable of evoking physiological responses necessary for morphogenesis, and to consequently improve the yield of the culture.
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The callus formation could be decreased when the nodal segments were submitted to a shock with growth regulators. If different times of exposure to the growth regulators were used, they could possibly elicit proportionally correspondent plant answers, with more or less intensity. And it is to mention that different varieties of the same species can present different patterns of development when they are submitted to the same treatment (Grattapaglia and Machado, 1998). Pretreatment with high or relatively high concentrations of growth regulators can improve organogenesis or somatic embryogenesis in other species as has been previously described. Pretreatment in cucumber (Cucumis sativus L.) leaves with 14 mM 2,4-D for 3 weeks increased the embryogenic-callus production. (Kuijpers et al., 1996). Pretreatment of chilli pepper (Capsicum annuum) leaves with high cytokinin concentration (129 mM 6-benzylaminopurine for 24 h) and later transferring onto solid ‘standard induction medium’ can improve the embryo proliferation (Kintzios et al., 2000). Growth regulator pretreatment improves somatic embryogenesis in the leaves of squash (Curcubita pepo L.) and melon (Cucumis melo L.). Leaves were pretreated with 113.1, 226.2 or 452.4 mM 2,4-D, 46.5, 93 or 186 mM Kin or a combination of both at above concentrations for 6, 24 or 48 h. Exposure of leaf explants to at least 452.4 mM 2,4-D for 6 h (squash) or 226.2 mM 2,4-D for 48 h (melon), 46.5 mM Kin for 48 h (squash) or 186 mM Kin for 6 h (melon), and subculture onto ‘species-specific standard induction medium’ significantly promoted somatic embryo induction (Kintzios et al., 2002). Passamonti et al. (1998) established a micropropagation protocol for the European varieties of C. recutita, using the traditional protocol of in vitro culture, with the growth regulators added to the solid culture media. Additionally, they have reported the best medium for proliferation phase one that contained 0.5 mg l 1 kinetin, although the plants were not able to rooting in vitro. Considering the multiplication rates, the present work showed results quite similar to those observed by Passamonti et al. (1998). The ideal interval to subculture chamomile Brazilian variety was 2 months. Plants had their leaves turned to yellow if were maintained for more than the indicated period in the same aged medium and, sometimes, the shoots died. They could not improve under such a situation. The pre-adaptation to autotrophic conditions was crucial to achieve successful acclimatization. Conditions leading to callus formation either can render an overwhelming plant acclimatization, or may even impair it. A small callusing percentage cannot be a hindrance for plant acclimatization. Some authors connect flower head diameter and soil fertility, as seen in Bezzi et al. (1991), who examined nine different chamomile cultivars and observed flower mean diameters as large as about 31.10 mm in good fertile soil. In the present work, any fertilisers were used on soil; moreover, the largest flower head diameters were obtained as soon the flowers showed up. It is worth noting here, the flowering synchronism attained in acclimatised plants circumventing daily harvest. In the most continuously flowering cultivars, for instance, the Mandirituba Brazilian variety, harvesting is demanded every 8 days, raising the production costs (Correˆa Ju´nior, 1998). Plants
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reached 33.0 cm mean length when setting up flowering, whereas for Mandirituba variety, the flowering period began with plants as high as about 24.0 cm. Overall, the present results for plant development have been considered as reasonable ones, in comparison to cultures of chamomile found abroad, for instance, 63.0 and 87.6 cm for Bona and Egito, respectively (Correˆa Ju´nior, 1998). Altogether, the present collected data suggests that successful in vitro culture and rapid propagation of the Brazilian variety of the C. recutita are possible once the explants are exposed to the controlled conditions with the growth regulators. Acknowledgements The authors thank CAPES, FAPERJ and FAPERJ-RECOPE for financial support, and they also thank Professor Claudia Lage from Carlos Chagas Filho Biophysics Institute—Federal University of Rio de Janeiro, for English revision. References Ashida, Y., Nishimoto, M., Matsushima, A., Watanabe, J., Hirata, T., 2002. Molecular cloning and mRNA expression of geraniol-inducible genes in cultured shoot primordia of Matricaria chamomilla. Biosci., Biotechnol. Biochem. 66 (11), 2511–2514. Bezzi, A., Aiella, N., Ghidini, G., 1991. La coltivazione della camomilla nell’Italia settentrionale; vari tipi di camomilla comune coltivati in Trentino-Alto adige, vol 19. L ‘Informatore Agrario, Verona, pp. 27–32. Calixto, J.B., 2000. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz. J. Med. Biol. Res. 33, 179–189. Cellarova, E., Cernicka, T., Vranova, E., Brutovska, R., Lapar, M., 1992. Viability of Chamomilla recutita (L.) Rauschert cells after cryopreservation. Cryo Lett. 13 (1), 37–42. Correˆa Ju´nior, C., 1998. Influeˆncia das adubac¸o˜es orgaˆnica e quı´mica na produc¸a˜o de Chamomilla recutita (L.) Rauschert e do seu o´leo essencial. In: Ming, L.C., Scheffer, M.C., Correˆa Ju´nior, C., Barros, I.B.I., Mattos, J.K.A. (Eds.), Plantas medicinais, aroma´ticas e condimentares – Avanc¸os na pesquisa agronoˆmica, vol.1. Universidade Estadual Paulista, Botucatu, SP, pp. 129–163. Currier, S.J., Johnston, P.D., Gorelick, K.J., 2000. Herbal medicines. Sci. Med. 40–43. Grattapaglia, D., Machado, M.A., 1998. Micropropagac¸a˜o. In: Torres, A.C., Caldas, L.S., Buso, J.A. (Eds.), Cultura de tecidos e transformac¸a˜o gene´tica de plantas, vol.1. Embrapa-SPI/Embrapa-CNPH, Brası´lia, pp. 183–260. Kintzios, S., Michaelakis, A., 1999. Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomille (Chamomilla recutita L.). Plant Cell Rep. 18 (7–8), 684–690. Kintzios, S., Drossopoulos, J.B., Shortsianitis, E., Peppes, D., 2000. Induction of somatic embryogenesis from young, fully expanded leaves of chilli pepper (Capsicum annuum L.): effect of leaf position, ilumination and explant pretreatment with high cytokinin concentrations. Sci. Horti. 85, 137–144. Kintzios, S., Sereti, E., Bluchos, P., Drossopoulos, J.B., Kitsaki, C.K., LiopaTsakalidis, A., 2002. Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Curcubita pepo L.) and melon (Cucumis melo L.). Plant Cell Rep. 21, 1–8. Kuijpers, A.M., Bouman, H., Klerk, G.J., 1996. Increase of embryogenic-callus formation in cucumber by initial culture on high concentration of 2,4dichlorophenoxyacetic acid. Plant Cell Tiss. Org. Cult. 46, 81–83. Lima, S.S., Esquibel, M.A., Henriques, A.B., Silva, F.O., Silva, P.H.B., Lage, C.L.S., 2001. Monoclonal culture of erva-de-bicho (Polygonum acre HBK
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