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Ciência Rural

Print version ISSN 0103-8478

Cienc. Rural vol.24 no.3 Santa Maria Sept./Dec. 1994

https://doi.org/10.1590/S0103-84781994000300013 

THE EFFICIENCY OF SHOOT AND PLANTLET FORMATION OF Cephaelis ipecacuanha AFTER THREE SUBCULTURES IN VITRO

 

EFICIÊNCIA NA REGENERAÇÃO DE BROTOS E FORMAÇÃO DE PLÂNTULAS DE Cephaelis ipecacuanha EM TRÊS SUBCULTIVOS IN VITRO

 

Osmar Alves Lameira1 Marly Pedroso da Costa1 José Eduardo Brasil Pereira Pinto2

 

 

SUMMARY

Multiple adventitious shoot formed from internodal segments of Cephaelis ipecacuanha cultured 25 days on Gamborg basal medium (GAMBORG et al., 1968) supplemented with 6.66mM 6-benzylaminopurine there was a maximum of nine shoots per segment and an average of five shoots per segment formed. The presence of gibberellic acid in the subculture media promoted shoot elongation in all treatments. The shoots attained 3cm in height and rooting of 100% after 35 days of culturing upon Murashige and Skoog's basal medium (MS), added with 4.92mM indole-3-butyric acid, 0.87m gibberellic acid and 0.1% activated charcoal. Further growth was accelerated after the transfer to 1/2 MS without growth regulators. Rooted plantlets transferred to potting soil could be successfully established.

Key words: medicinal plant; micropropagation, Ipecac.

 

RESUMO

Brotos adventícios foram regenerados de segmento internodal de Cephaelis ipecacuanha em três subcultivos. Após 25 dias de cultivo em meio básico de Gamborg (GAMBORG et al., 1968), o meio contendo 6,66mM, 6-benzilaminopurina (BAP) induziu até 9 brotos por segmentos na cultura inicial e uma média de 5 brotos no subcultivo. O ácido giberélico no meio de cultura promoveu um alongamento em todos os tratamentos. Os brotos alcançaram uma altura de 30mm e 100% de enraizamento após 35 dias de cultivo no meio basal de Murashige & Skoog (MURASHIGE & SKOOG, 1962) - MS, suplementado com 4,92mM ácido indolbutírico, 0,87mM ácido giberélico e 0,1% de carvão ativado. Houve um crescimento acelerado após cultivar os brotos em meio 1/2 MS sem regulador de crescimento. As plântulas foram transferidas para vaso e aclimatadas com sucesso.

Palavras-chave: planta medicinal, micropropagação, Cephaelis.

 

 

INTRODUCTION

Cephaelis ipecacuanha (Ipecac) is a perennial herb with medicinal use, grows in humid forests of Brazil. The roots of this plant contain emetine and cephaline and are widely used in folk medicine, mainly as an expectorant, a diaphoretic, and most importantiy, an excellent amoebacid (GUPTA, 1971).

The propagation of ipecac by seeds in rarely used because of the large amount of time required for germination, at least six months. Precious attempts to propagate C. ipecacuanha through root cuttings managed to increase the percentage but failed to improve root growth (GATONNI, 1959; KALYANASUNDARAM, 1968).

Two studies have been reported on the propagation of C. ipecacuanha through tissue culture (IKEDA et al., 1988; YOSHIMATSU & SHIMOMURA, 1991). This paper describes the results of an investigation on shoot and plantlet formation of C. ipecacuanha (Ipecac) after three subcultures in vitro.

 

MATERIALS AND METHODS

The explant sources were internodal segments of ipecac plantiets obtained from preliminary experiments in vitro incubated in Gamborg medium B5 (GAMBORG et al., 1968). The segments, approximately 5mm long were excised and incubated in 25 x 100mm test tubes containing 10ml of Gamborg medium, salts and vitamins (B5) supplemented with 0.22, 2.22, 6.66 or 13.22mM 6-benzylaminopurine (BAP) and 2% (w/v) sucrose. The liquid media were (filter paper bridge) adjusted to pH 5.8 before autoclaving at 121 °C for 15 minutes. All cultures were maintained at 28 ± 1°C under a 16h light photoperiod at 20-25Wm2 for 4 weeks. The shoots obtained were subcultured three times at 3-week intervals under the same conditions and the media were supplemented with 0.87mM gibberellic acid (GA3).

To measure root induction and growth, the shoots that had attained 1cm in height were transferred individually into 100ml Erlenmeyer flasks containing 30ml of Murashige and Skoog's (MURASHIGE & SKOOG, 1962) basal medium (MS), supplemented with combinations of 4.92, 14.76mM indole-3-butyric acid (IBA), 0.87mM GA3, 0.1% or 0.3% activated charcoal. Finally, after 15 days the shoots were transferred to 1/2 MS solid medium, consisted of a half macro salt formulation and 3% (w/v) sucrose and supplemented with 0.1% activated charcoal and without activated charcoal and without growth regulators. The regenerated plantiets were transferred to polyethylene bags containing a mixturer of soil, manure and sawdust (1:1:1) and cultivated under shady conditions. Each experiment was consisted of 20 explants per treatment. Duncan's multiple range test was used to compare treatment means.

 

RESULTS

The effect of BAP on shoot multiplication are shown in Table 1. After 4 weeks of incubation, shoots were observed on all media in use. The most effective basal medium for shoot multiplication was B5 medium containing 6.66mM BAP which induced a mean of 5.4 shoots per segment. The shoots induced on medium containing higher concentration of BAP were smaller in height than the shoots induced in lower concentration (data not shown). The presence of GA3 in the subculture media promoted shoot elongation in all treatments (Table 2). The subcultures were less efficient for shoots multiplication than initial culture for all media, decreased gradually until the second subculture, except for medium containing 0.22mM BAP.

 

 

 

 

The effects of combination off IBA, GA3 and activated charcoal on plantiet formation are shown in Table 2. Root induction was observed 10 to 15 days after incubation. The media containing 4.92mM IBA were the most effective for root formation. The combination of 14.76mM IBA, 0.87mM GA3, and 0.3% activated charcoal was the least efficient for root formation. After 5 weeks of culturing the plantlets formed on medium containing 4.92mM IBA, 0.87 GA3 and 0.1% activated charcoal attained 30mm in height, were more easily rooted and produced more roots per shoot. The root growth was accelerated after the transfer to new medium with half macro salt formation without growth regulators. All rooted plants showed over 95% of establishment efficiency when transplanted to soil.

 

DISCUSSION

The higher concentrations of BAP (6.66 and 13.22mM) resulted in greater efficiencies of shoot multiplication than the lower concentrations. It was not observed the inhibition of shoot formation by cytokinin concentration over 2.32mM as reported by YOSHIMATSU & SHIMOMURA (1991). IKEDA et al. (1988) also did not observe this inhibition when 13.94 and 23.23mM BAP or kinetin were used in combination with 0.05mM napthaleneacetic acid (NAA) in nodal segments of ipecac.

It was observed that shoots formed on medium supplemented with lower concentration of BAP in the initial incubation were taller and had larger leaves than the shoots regenerated on higher BAP concentration. These observations were noted under similar conditions by YOSHIMATSU & SHIMOMURA (1991). In this study the level of GA3 used in the subcultures promoted shoot elongation in all media, which made the establishment of subsequent subculture easier. The levei used in this experiment (Tabel 2) is among the concentrations tested for shoot elongation by other researchers (PALEG, 1965; MERTZ, 1966). Occasionally, a low level of GA3 may be useful since it permits some extension growth (KRIOKORIAN, 1982). The gradual decrease in shoot multiplication observed during subcultures demonstrated that the multiplication capacity of certain species decreased rapidly after several subcultures and the morphogenetic potential may eventually be fully lost (HU & WANG. 1983).

The better efficiency for root regeneration and growth is obtained on low concentration of auxin and the elongation is stimulated when the level of auxin and gibberellin are balanced (MERTZ, 1966). The results obtained with the levels of auxin and gibberellin used in this investigation confirm this efficiencies. The higher concentration of IBA inhibited root formation, although the height of plantlet had not been affected. In the experiment of IKEDA et al. (1988) this inhibition was not observed in concentrations over 5.71mM of indole-3-acetic acid (IAA). However, EVANS (1976) reported that the root formation could be inhibited when shoots were cultivated for long periods under auxin or higher concentrations of growth regulators. The results obtained with the higher concentrations of IBA in this investigation confirm this report.

For plantlet formation the presence in vitro of activated charcoal can have inhibitory or stimulatory effects on growth (FRIDBORG et al., 1978). In this study the combination of activated charcoal, IBA and GA3 in adequate levels stimulated this effect and the root number obtained was more than the obtained by IKEDA et al. (1988).

 

ACKNOWLEDGEMENTS

We thank Mr. Brendan Walsh, Mr. Kaleb Kleppner and Ruth Steins for critical review of manuscript. This work was partially supported by grants from the Japan International Cooperation Agency.

 

REFERENCES

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FRIDBORG, G., PEDERSEN, M., LANDSTROM, L. et al. The effect of activated charcoal on tissue cultures: absorption of metabolites inhibiting morphogenesis. Physiology Plantarum, v. 43, p. 104-106, 1978.         [ Links ]

GAMBORG, O.L, MILLER, R.A. and OJIMA, K. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cellular Research, V. 50, p. 151-158,1968.         [ Links ]

GATONNI, L.A. La raicilla a ipecacuanha. Boletim do Ministério da Agricultura, Comércio e Indústria, Panamá, p. 1-32, 1959.         [ Links ]

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IKEDA, K., TESHIMA, D., AOYAMA, T., et al. Clonal propagation of Cephaelis ipecacuanha. Plant Cell Reports, V. 7,p. 288-291, 1988.         [ Links ]

KALYANASUNDARAM, S. Effect of boron and indole-butyric acid on rooting of ipecac root cuttings. Madras Agriculture Journal, V. 56, p. 812-820, 1968.         [ Links ]

KRIOKORIAN, A. Cloning higher plants from aseptically cultured tissues and cells. Biology Raview, v. 57, p. 151-218, 1982.         [ Links ]

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MURASHIGE, T. and SKOOG, F. A revised medium for rapid growth and biossays with tobacco tissue cultures. Physiology Plantarum, M. 15, p. 473-497, 1962.         [ Links ]

PALEG, L.G. Physiological effects of gibberellins. Annual Review Plant Physiology, V. 18, 291-322, 1965.         [ Links ]

YOSHIMATSU, K. and SHIMOMURA, M. Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard. Plant Cell Reports, v. 9, p. 567-570, 1991.         [ Links ]

 

 

1Engenheiro Agrônomo, MsC, EMBRAPA/CPATU, Belém, PA.

2Professor Titular, Departamento de Agricultura, Escola Superior de Agricultura de Lavras, ESAL, Caixa Postal 37, 37200-000 - Lavras-MG.

Recebido para publicação em 01.03.94 Aprovado em 07.06.94

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