Plant Cell Reports
Plant Cell Reports (1986) 5 : 17-18
© Springer-Verlag 1986
Tissue culture propagation of Mentha spp. by the use of axillary buds E. L. Rech and M. J. P. Pires National Center for Genetic Resources, CENARGEN/EMBRAPA, P.O. Box 10.2372, 70.770-Brasilia-DF, Brazil Received July 29, 1985 / Revised version received November 13, 1985 - Communicated by I. K. Vasil
ABSTRACT A method is described for rapid multiplication from axillary buds of six Mentha species. Nodal segments from one-year old plants were grown on Murashige and Skoog medium (BMS), supplemented with BAP (i.0; 2.0 rag/l) and KIN (i.0 mg/l) and kept at 28 + - I-°C under fluorescent light for 30 days. After this period, several- shoots (15-20 shoots per explant) with roots were produced which were placed in soil for further growth. I~TRODUCTION The importance of tissue culture propagation in a germplasm bank is twofold. Firstly, it allows long term storage and conservation of plant material which does not normally produce seeds. Secondly, it provides an easy and inexpensive way for international exchange of disease-free material (Murashige, 1977; Withers, 1980; Chomchalow and Sahavacharin, 1981 ). Importance of such techniques in medicinal plant research has been reported particularly for the production of chemical substances by "in vitro" culture (Tabata, 1977). Mentha spp. were originally cultivated in Eastern Asia, mainly in Japan and China (Bersaghi, 1945), and their economic importance is due to the production of mint oil as raw material for pharmaceutical and cosmetic uses as well as for flavouring foods, beverages and tobacco (Sec. Est. Agric. PR., 1978). Few reports about tissue culture of mints have been published. These are related to certain conditions for cell suspension cultures and callus formation (Lin and Staba, 1961; Wang and Stapa, 1963). In the present work, an attempt was made to establish experimental conditions for rapid multiplication of six Mentha species. MATERIALS AND METHODS The following experimental plants were obtained from the Germplasm Bank of Medicinal Plants at the National Center for Genetic Resources/EMBRAPA, Brasilia, Brazil: Mentha arvensis L. (BRA 000060; BRA 000078), Mentha piperita L. (BRA 000086), Mentha pulegium L. (BRA 000094), Mentha spicata L. (BRA 000108) and Mentha virides L. (BRA 000124). Single-node stem segments (ca. 1.0 cm in lenght)
Offprint requests to: E. L. Rech
with intact leaves were removed from one year old plants and surface sterilized with 20% con~mercial bleach (brand Q'boa containing 5% sodium hypochlorire) for 20 min. The material was thoroughly washed twice in sterile distilled water. Explants were prepared by trimming the cut ends and were transferred to a test tube (150 x 25~m) containing 20 ml of standard Murashige & Skoog, 1962 medium (BMS), plus 3% sucrose and one of the following growth regulators: a -Naphthaleneacetic acid (NAA), 2,4-dichlorophenoxy_ acetic acid (2,4-D), Indole-S-acetic acid (IAA), Kinetin (KIN) and 6-Benzylaminopurine (BAP). Each growth regulator was tested at three concentrations: 0.5; 1.0 and 2.0 mg/l. The final culture media were adjusted to pH 5.7 and mixed with 0.6% agar prior to autoclaving at 1.5 Atm for 20 min. The cultures were incubated in a growth chamber at 28 + I-°C with 12 h photoperiod (1500 Lux). Approximately after 30 days, the plantlets were transferred to plastic pots (8 x 8 x 6 cm) containing vermiculite and then covered with plastic bags. Hoagland's solution was applied once a week. These potted plants were grown at 25 + I-°C with 12 h photoperiod (1500 Lux) for two weeks before transfer to soil. RESULTS AND DISCUSSION On the basal medium (BMS) without growth regu_ lators, roots and shoots were produced from axillary buds of the nodal explant (Table i). In the presence of BAP and KIN proliferation of axillary buds was observed (Fig. ib). Shoot proliferation was more (15-20 shoots per explant) on media with i.O and 2.0 mg/l of BAP and 2.0 mg/i of KIN than that observed in the control (Table I). The multiplication rate was increased when nodal segments from these shoots were subcultured. Roots were normal in both original and subcultured nodal segments. The fact that roots were formed almost at the same time as shoot proliferation, reduced greatly the period for'transfer of the plantlets to soil since no subsequent rooting step was required (Fig. id). As shown in the Fig. ic, M. virides, M. pulegium and M. piperita were more prolific in terms of shoot proliferation and rooting. The other species did not produce large number of shoots and
18 roots. All plants appeared to be normal.
formed in it (Table i). Intense callus production without shoot and root formation was promoted at all concentrations of 2,4-D and at 1.0 and 2.0 mg/l of NAA (Fig. la) in all species. On the other hand, NAA at all concentrations and IAA at 1.0 and 2.0 mg/l induced both callus and axillary shoots. Sugisawa and Ohnishi (1976) showed that essential oil can be produced from cell suspension cu!~J~es of Perilla sp. leaves. The technique proposed here for callus formation in Mentha spp. can be of practical use in a similar manner. Also, this technique can be used for exchange and conservation of disease-free mint germplasm. Table 1 - Effect o£ different concentrations of NAA, 2,4-0,
IAA,
KIN and BAP on shoot, root and callus production
from
nodes of Memtha spp. Concmm-
Growth ~egulators
trations
(mg/l)
NAA
2,4-0
IAA
KIN
BAP
a
0
AS+R
AS+R
AS+R
AS+R
AS+R
0.5
C+AS
C
AS+R
AS+R
SP+R
1.0
C
C
AS+C+R
SP+R
SP+R
2.0
C
C
AS+C+R
SP+R
SP+R
a For each concentration of growth regulators, 20 explants were used
AS = a x i l l a r y shoot development; SP = shoot proliferatlon; R = root formation;
C = callus.
ACKNOWLEDGEMENTS We would like to thank Dr. H.M. Rocha and Dr. L. Creppy for discussion and comments and Mr. Nestor Lima for photography. REFERENCES
d Fig.l.a - Emergence of callus from nodal segments of M. arvensls, cultured 4 weeks on BMS + 2,4-0 (2 mg/l), b-Shoot proliferation From the explant on BMS +BAP (2 mg/l).
c-Development of six mint species (from the l e f t to the night): M. vlrldes (BRA 000124); M. arvensls (BRA 000078); M. spleata (8RA 000108), M. arvensls (BRA 000060); M. pulegium (BRA 000094) and M. plperlta
(BRA 000086). d-45 d~y-old plant of M. arvensis.
Under the experimental conditions, 40 days were required for the production of a new plantlet. Production of callus was observed in the presence of all the auxins tested, nevertheless, no shoots were
Bersaghi C (1945) Cultura da Menta. Secretaria de Agricultura, Industria e ComSrcio do Estado de Sac Paulo, S2p. Chomchalow N, Sahavacharin 0 (1981) In: Rao AN (ed) The role of tissue culture in the development of Medicinal Plants and Spices, Proc. Symp on tissue culture of Economicaly Important Plants, Singapore. pp. 162-166. Lin NL, Staba EJ (1961) Lloydia 24:189. Misawa M (1977) In: Barz W, Reinhard E, Zenk ~{£ (eds) Plant Tissue Culture and its Bio-technological Application, Springer-Verlag, Berlin Heidelberg, New York, pp. 17-26. Murashige T (1977) Hort Sci 12: 127-130. Murashige T, Skoog F (1962) Physiol Plant 15: 473-498. Secretaria do Estado da Agricultura do Paran~. Departamento de Economia Rural (1978) Mentha (Mentha arvensis L. ) Curitiba, S9p. Sugisawa H, Ohnishi Y (1976) Agr Biol Chem 40: 281232. Tabata M (1977) In: Barz W, Reinhard E, Zenk MH (eds) Plant Tissue Culture and its Bio-technological Application, Springer-Verlag. Berlin Heidelberg. New York, pp. 8-16. Wang CJ, Staba EJ (1963) J Pharm Sol 52: 1058-1062. Whithers LA (1980) Tissue Culture Storage for genetic conservation IBPGR tech Rpt, FAO, Rome.
Plant Cell Reports (1986) 5 : 17-18
© Springer-Verlag 1986
Tissue culture propagation of Mentha spp. by the use of axillary buds E. L. Rech and M. J. P. Pires National Center for Genetic Resources, CENARGEN/EMBRAPA, P.O. Box 10.2372, 70.770-Brasilia-DF, Brazil Received July 29, 1985 / Revised version received November 13, 1985 - Communicated by I. K. Vasil
ABSTRACT A method is described for rapid multiplication from axillary buds of six Mentha species. Nodal segments from one-year old plants were grown on Murashige and Skoog medium (BMS), supplemented with BAP (i.0; 2.0 rag/l) and KIN (i.0 mg/l) and kept at 28 + - I-°C under fluorescent light for 30 days. After this period, several- shoots (15-20 shoots per explant) with roots were produced which were placed in soil for further growth. I~TRODUCTION The importance of tissue culture propagation in a germplasm bank is twofold. Firstly, it allows long term storage and conservation of plant material which does not normally produce seeds. Secondly, it provides an easy and inexpensive way for international exchange of disease-free material (Murashige, 1977; Withers, 1980; Chomchalow and Sahavacharin, 1981 ). Importance of such techniques in medicinal plant research has been reported particularly for the production of chemical substances by "in vitro" culture (Tabata, 1977). Mentha spp. were originally cultivated in Eastern Asia, mainly in Japan and China (Bersaghi, 1945), and their economic importance is due to the production of mint oil as raw material for pharmaceutical and cosmetic uses as well as for flavouring foods, beverages and tobacco (Sec. Est. Agric. PR., 1978). Few reports about tissue culture of mints have been published. These are related to certain conditions for cell suspension cultures and callus formation (Lin and Staba, 1961; Wang and Stapa, 1963). In the present work, an attempt was made to establish experimental conditions for rapid multiplication of six Mentha species. MATERIALS AND METHODS The following experimental plants were obtained from the Germplasm Bank of Medicinal Plants at the National Center for Genetic Resources/EMBRAPA, Brasilia, Brazil: Mentha arvensis L. (BRA 000060; BRA 000078), Mentha piperita L. (BRA 000086), Mentha pulegium L. (BRA 000094), Mentha spicata L. (BRA 000108) and Mentha virides L. (BRA 000124). Single-node stem segments (ca. 1.0 cm in lenght)
Offprint requests to: E. L. Rech
with intact leaves were removed from one year old plants and surface sterilized with 20% con~mercial bleach (brand Q'boa containing 5% sodium hypochlorire) for 20 min. The material was thoroughly washed twice in sterile distilled water. Explants were prepared by trimming the cut ends and were transferred to a test tube (150 x 25~m) containing 20 ml of standard Murashige & Skoog, 1962 medium (BMS), plus 3% sucrose and one of the following growth regulators: a -Naphthaleneacetic acid (NAA), 2,4-dichlorophenoxy_ acetic acid (2,4-D), Indole-S-acetic acid (IAA), Kinetin (KIN) and 6-Benzylaminopurine (BAP). Each growth regulator was tested at three concentrations: 0.5; 1.0 and 2.0 mg/l. The final culture media were adjusted to pH 5.7 and mixed with 0.6% agar prior to autoclaving at 1.5 Atm for 20 min. The cultures were incubated in a growth chamber at 28 + I-°C with 12 h photoperiod (1500 Lux). Approximately after 30 days, the plantlets were transferred to plastic pots (8 x 8 x 6 cm) containing vermiculite and then covered with plastic bags. Hoagland's solution was applied once a week. These potted plants were grown at 25 + I-°C with 12 h photoperiod (1500 Lux) for two weeks before transfer to soil. RESULTS AND DISCUSSION On the basal medium (BMS) without growth regu_ lators, roots and shoots were produced from axillary buds of the nodal explant (Table i). In the presence of BAP and KIN proliferation of axillary buds was observed (Fig. ib). Shoot proliferation was more (15-20 shoots per explant) on media with i.O and 2.0 mg/l of BAP and 2.0 mg/i of KIN than that observed in the control (Table I). The multiplication rate was increased when nodal segments from these shoots were subcultured. Roots were normal in both original and subcultured nodal segments. The fact that roots were formed almost at the same time as shoot proliferation, reduced greatly the period for'transfer of the plantlets to soil since no subsequent rooting step was required (Fig. id). As shown in the Fig. ic, M. virides, M. pulegium and M. piperita were more prolific in terms of shoot proliferation and rooting. The other species did not produce large number of shoots and
18 roots. All plants appeared to be normal.
formed in it (Table i). Intense callus production without shoot and root formation was promoted at all concentrations of 2,4-D and at 1.0 and 2.0 mg/l of NAA (Fig. la) in all species. On the other hand, NAA at all concentrations and IAA at 1.0 and 2.0 mg/l induced both callus and axillary shoots. Sugisawa and Ohnishi (1976) showed that essential oil can be produced from cell suspension cu!~J~es of Perilla sp. leaves. The technique proposed here for callus formation in Mentha spp. can be of practical use in a similar manner. Also, this technique can be used for exchange and conservation of disease-free mint germplasm. Table 1 - Effect o£ different concentrations of NAA, 2,4-0,
IAA,
KIN and BAP on shoot, root and callus production
from
nodes of Memtha spp. Concmm-
Growth ~egulators
trations
(mg/l)
NAA
2,4-0
IAA
KIN
BAP
a
0
AS+R
AS+R
AS+R
AS+R
AS+R
0.5
C+AS
C
AS+R
AS+R
SP+R
1.0
C
C
AS+C+R
SP+R
SP+R
2.0
C
C
AS+C+R
SP+R
SP+R
a For each concentration of growth regulators, 20 explants were used
AS = a x i l l a r y shoot development; SP = shoot proliferatlon; R = root formation;
C = callus.
ACKNOWLEDGEMENTS We would like to thank Dr. H.M. Rocha and Dr. L. Creppy for discussion and comments and Mr. Nestor Lima for photography. REFERENCES
d Fig.l.a - Emergence of callus from nodal segments of M. arvensls, cultured 4 weeks on BMS + 2,4-0 (2 mg/l), b-Shoot proliferation From the explant on BMS +BAP (2 mg/l).
c-Development of six mint species (from the l e f t to the night): M. vlrldes (BRA 000124); M. arvensls (BRA 000078); M. spleata (8RA 000108), M. arvensls (BRA 000060); M. pulegium (BRA 000094) and M. plperlta
(BRA 000086). d-45 d~y-old plant of M. arvensis.
Under the experimental conditions, 40 days were required for the production of a new plantlet. Production of callus was observed in the presence of all the auxins tested, nevertheless, no shoots were
Bersaghi C (1945) Cultura da Menta. Secretaria de Agricultura, Industria e ComSrcio do Estado de Sac Paulo, S2p. Chomchalow N, Sahavacharin 0 (1981) In: Rao AN (ed) The role of tissue culture in the development of Medicinal Plants and Spices, Proc. Symp on tissue culture of Economicaly Important Plants, Singapore. pp. 162-166. Lin NL, Staba EJ (1961) Lloydia 24:189. Misawa M (1977) In: Barz W, Reinhard E, Zenk ~{£ (eds) Plant Tissue Culture and its Bio-technological Application, Springer-Verlag, Berlin Heidelberg, New York, pp. 17-26. Murashige T (1977) Hort Sci 12: 127-130. Murashige T, Skoog F (1962) Physiol Plant 15: 473-498. Secretaria do Estado da Agricultura do Paran~. Departamento de Economia Rural (1978) Mentha (Mentha arvensis L. ) Curitiba, S9p. Sugisawa H, Ohnishi Y (1976) Agr Biol Chem 40: 281232. Tabata M (1977) In: Barz W, Reinhard E, Zenk MH (eds) Plant Tissue Culture and its Bio-technological Application, Springer-Verlag. Berlin Heidelberg. New York, pp. 8-16. Wang CJ, Staba EJ (1963) J Pharm Sol 52: 1058-1062. Whithers LA (1980) Tissue Culture Storage for genetic conservation IBPGR tech Rpt, FAO, Rome.
