Plant Cell, Tissue and Organ Culture 49: 157–160, 1997. 157c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Research note

In vitro regeneration of Minthostachys andina (Brett) Epling - a Boliviannative species with aromatic and medicinal properties

Jose Antonio Castillo1 & Miguel Jordan2*1Facultad de Ciencias y Tecnologia, Universidad Mayor de San Simon, Cochabamba, Bolivia; 2Departamento deEcologia, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile (*requestsfor offprints)

Received 06 February 1996; accepted in revised form 29 May 1997

Key words: in vitro regeneration, organogenesis, clonal propagation, essential oils, Labiatae

Abstract

Various explants of Minthostachys andina (Brett.) were evaluated for their morphogenic potential under in vitroculture conditions. Axillary buds derived from 2 year-old plants grown in MS-medium supplemented with 4.4 or8.8 �M BA and 0.054�M NAA, initiated shoot growth and new shoot formation. Under subculture in NN medium,shoots were rooted in the presence of NAA (1.6, 2.7 or 5.3 �M) alone or in combination with IBA (9.8 �M),and the regenerated plantlets were later acclimatised in the greenhouse. Also, polynodal segments from seedlingsinitiated multiple shoots and plantlets when initially cultured in presence of NN-liquid salt medium supplementedwith 2.2-17.7 �M BA or 4.5-13.6 M TDZ in combination with different auxin-like growth regulators and after afinal transfer for root initiation. The same types of responses were found in hypocotyl and leaf explants, whichproduced adventitious shoots in the presence of TDZ. The use of antioxidants helped to prevent browning andfavoured organogenesis.

Abbreviations: BA – 6-benzylaminopurine; NAA – �-naphthaleneacetic acid; IAA – indole-3-acetic acid; IBA– indole-3-butyric acid; GA3 – gibberellic acid; CH – casein enzymatic hydrolysate (Sigma); Glu – L-glutamine;MS – Murashige and Skoog-medium (1962); NN – Nitsch and Nitsch-medium (1969); TDZ – 1-phenyl-3-(1,2,3,-thidiazol-5-yl)urea (thidiazuron).

Minthostachys andina (Brett.) Epling, a member of themint family (Fam Labiatae), is an important Andeanhighlands species. It shows wide medicinal properties(Cardenas, 1969) and has been used in traditional Boli-vian medicine and agriculture for food preservation.Known as “muna blanca”, this native species growsat altitudes up to 2500 - 3500 m. Its properties areattributed to the presence of essential oils in the stems,flowers and leaves (0.67-1.72% FW). Pulegone, men-thone and 1,8-cineol have been reported as the mainproducts (Munoz et al., 1990). However due to its natu-ral low rate of regeneration, variability in the essentialoils (Munoz et al., 1993), and the need for industri-al utilisation, the mass propagation of this species isrequired. Several studies on in vitro regeneration of

plants within the family Labiatae have been reported;Mentha (Van Eck and Kitto, 1990), Coleus (Sharma etal., 1991), Pogostemon (Hart et al., 1990), Rosmarinus(Misra and Chaturvedi, 1984), Salvia (Miyasaka et al.,1985), and recently of Minthostachys mollis (Chebelet al., 1995), but no results have been reported for M.andina. This work describes the morphogenic poten-tials found in different explant types of this specieswhen placed under a range of in vitro culture condi-tions aiming to enhance asexual propagation.

Two year-old plants 10-20 cm in height and seedsof M. andina were collected in the high altitude val-leys (2800 to 3500 metres above sea level) close toCochabamba, Bolivia during September 1993 and Jan-uary 1994. This material was grown under greenhouse

JS: PIPS No. 142141 BIO2KAP

*142141* ticu2256.tex; 7/08/1997; 12:20; v.7; p.1

158

Table 1. Effect of different growth regulators on the morphogenic response of buds and polynodal segments of M andina.

Explant & culture Growth regulators (�M) Multipl. Shoot % % Root % Calluses

mediaa BA TDZ NAA IBA IAA GA3 Shoots/ length Elong. formation Calluses sizeb

explant (mm) shoots

Buds 4.44 — — — — — 2.5 9.5 100 0.0 0.0 -

(solid MS- medium) 4.44 — 0.05 — — — 2.5 11.5 100 0.0 13.3 ++

4.44 — 0.05 — — 0.14 1.8 7.3 95 0.0 23.0 ++

8.87 — 0.05 — — — 2.0 4.0 80 0.0 16.6 ++

4.44c — 0.05 — — 0.14 2.6 7.8 88 0.0 0.0 ++

8.87c — 0.05 — — — 2.3 8.7 90 0.0 0.0 -

Polynodal Segments

(liquid 1/2 streng MS-

medium) 2.22 — 0.05 — — 1.26 4.5 n.e. 100 0.0 00

“stationary culture” 3.56 — 1.7 3.44 — 0.03 2.4 n.e. 88 5.0 30 ++

4.44 — 1.81 4.9 — 0.03 1.1 n.e. 72 5.5 22 +

17.74 — 1.07 — — 1.87 3.8 n.e. 100 0.0 15 ++

— 4.54 — — 0.85 — 3.7 n.e. 95 0.0 30 ++

— 9.08 — — 0.85 — 3.8 n.e. 81 4.7 33 +

4.44 13.6 — — 0.85 — 2.2 n.e. 95 0.0 70 ++

a: Treatments with 15-20 replicates and 21-28 induction days. All media with 0.1% w/v PVP-360.b: No callus formation; + Moderate callus; ++ Large callus; n.e. not evaluated.c: Besides PVP, these treatments were additionally supplemented with 100 mg l�1 of phloroglucinol and 50 mg l�1 of adenine hemisulfate.

conditions (25 �C, 50% RH) supplemented to a 18h-light photoperiod at 92 �mol m�2 s�1 provided by“daylight” 400 W sodium-lamps (P. L. Light Systems,Canada). For all explants, surface disinfection of a pre-liminary incubation treatment with a mixture of Bayle-ton 25% WP 2,5 g l�1, Captan 80% WP 1,0 g l�1 andTriton X-100 0.5 ml l�1 stirring for 15 m, followedby soaking the explants in commercial Na-hypoclorite(final conc. 3%) for 5 min, then washed twice in anautoclaved antioxidant solution (citric acid and ascor-bic acid 250 mg l�1 each) for 10 min and finally rinsedin sterile distilled water. For in vitro culture, all growthmedia tested (as indicated below) were supplement-ed with Glu (100 mg l�1) and CH (200 mg l�1), pHadjusted to 5.5 and autoclaved at 121 �C for 15 min.The explants were cultured either in Pyrex tubes (25x 135 mm), 100 ml Erlenmeyer flasks (stationary cul-ture) closed with aluminum foil, or in Magenta contain-ers maintained in a growth chamber at 23�1 �C anda 12h-light photoperiod provided by white fluorescenttubes (Philips TL-40/45) producing 57 �mol m�2 s�1

at plant level. Various types of explants including buds,polynodal and hypocotyl segments and leaf sections,were tested to evaluate their morphogenic potential.

Culture conditions that promoted axillary budgrowth, multiple-shoot formation and roots on buds

and polynodal sections are summarised in Table 1.Buds, (6-10 mm in length, obtained from plants grownin the field), cultured in modified MS-medium (0.7%w/v agar, Sigma Type A), containing BA (4.4 or 8.8�M) only or in combinations with NAA and/or GA3,initiated multiple shoots. After subculture, the root-ing of elongated shoots was promoted in presence ofIBA (9.8 �M) and/or NAA (1.6, 2,7, or 5.3 �M) onNN-medium (supplemented with 15 mg l�1 Ca pan-thotenate, 1.5% sucrose, 0.1% w/v PVP-360, 0.6%w/v agar) ranging 66-90% with 6.1-8.7 roots/explant.Root length was 5.2-9.8 mm after 35 days (Figure 1a). The regenerated plantlets showed good adaptationafter transfer to greenhouse conditions on a sterilisedpotting substrate composed of peat and perlite 1:1 v/v,weekly fertilized with a 1/10 conc. Knop solution.

Polynodal segments with 4 nodes derived from 12days-old seedlings, cultured in 100 ml flasks con-taining 2.0 ml liquid modified 1/2 half-strength MS-medium (stationary culture), also showed, multipleshoot formation and simultaneously roots in the pres-ence of various growth regulators (Table 1). The newshoots when transferred to NN-medium with the samegrowth regulators as indicated before, initiated rootsin approx. 50% of the explants after 35 days, alsoallowing plantlet recovery in greenhouse conditions.

ticu2256.tex; 7/08/1997; 12:20; v.7; p.2

159

Figure 1. (a) Rooted plantlets derived from elongated buds. (b) Leaf explants with multiple shoot formation in presence of TDZ and IBA(bar=1.0 mm). (c) Cross-section showing multiple shoots initiated from the palisade leaf parenchyma (bar=1.0 mm). (d) Bud elongation fromshoots initiated from leaf and hypocotyl segments. (e) Hypocotyl callus with shoot initiation.

Multiple shoot formation also occurred in the lam-ina of leaf explants derived from 12-day old and 3-month old plants grown in the greenhouse (Figure 1b,c). Responses were observed in explants growing onmodified MS or NN-media (0.7% w/v agar). From arange of concentrations tested (2.2, 4.5, 7.7 and 11.3

�M) only the highest level of TDZ combined with 2.4�M IBA proved morphogenic, leading to adventiveshoots. This effect was enhanced when one of the fol-lowing antioxidants: glutathione, 15 and 25 mg l�1; L-cysteine, 15 mg l�1; PVP 50 mg l�1 or aminooxyaceticacid, 10 mg l�1; was included. Under these conditions,

ticu2256.tex; 7/08/1997; 12:20; v.7; p.3

160

multiple shoot formation occured in 8.8-22.7% of theexplants. After subculture on 1/2 strength MS-liquidmedium in presence of 1.1 �M BA, 0.27 �M NAAand 0.02 �M GA3, these shoots evidenced fast growth,branching and root initiation (Figure 1d). Finally, mul-tiple shoots were also observed in hypocotyl sectionsgrowing on NN-medium after 5 weeks’ culture (Fig-ure 1e); their highest inductive responses was (23.6%),besides rooting (24.1%), in presence of 4.5�M TDZ incombination with 0.8 �M IAA. In conclusion, despitethe fact that this species experimented a strong brown-ing effect in vitro, (partially overcome with antiox-idants), different types of explant examined in thiswork performed organogenesis easily favouring asex-ual multiplication.

Acknowledgements

The authors thank the A.W. Mellon Foundation forfunding and are also grateful for the valuable help givenby Mme. A. Goreux.

References

Cardenas M (1969) Manual de Plantas Economicas de Bolivia. 2a

ed. Ed. Los Amigos del Libro. Cochabamba, Bolivia (291p)Chebel A, Koroch A, Juliani H & Trippi V (1995) Micropropagacion

de Minthostachis mollis (H.B.K) Griseb. a traves del cultivo detejidos. Proc. REDBIO’95. Puerto Iguazu, Argentina

Hart JW, Woodcock GJ & Wilson L (1970) Culture and sesquiterpeneanalyses of cells and regenerated plantlets of Pogostemon cablinBenth. (Patchouli). Ann. Bot. 34: 789–798

Misra P & Chaturvedi HC (1984) Micropropagation of Rosmarinusofficinalis L. Plant Cell Tiss. and Org. Cult. 3: 163–168

Miyasaka H, Nasu M, Yamamoto T & Yoneda K (1985) Productionof ferruginol by cell suspension cultures of Salvia miltiorrhiza.Phytochem. 24: 1931–1933

Munoz S, Collin GJ, Gagnon M & Soriano J (1990) The essential oilsof Hedeoma mandoniana Wedd and of Minthostachys andina(Brett) Epling. J. Ess. Oil Res. 2: 61–66

Munoz S, Soriano J, Collin GJ, Jean FI & Deslauriers H (1993) Vari-ability in the composition of the essential oils of Minthostachysandina in central Bolivia. Phytochem. 33: 123–127

Murashige T & Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497

Nitsch JP & Nitsch C (1969) Haploid plants from pollen grains.Science 163: 85–87

Sharma N, Chandel KPS & Srivastava VK (1991) In vitro propa-gation of Coleus forskohlii Briq., a threatened medicinal plant.Plant Cell Rep. 10: 67–70

Van Eck JM & Kitto SL (1990) Callus initiation and regeneration inMentha. HortSci. 25: 804–806

ticu2256.tex; 7/08/1997; 12:20; v.7; p.4