Plant Cell, Tissue and Organ Culture56: 79–83, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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The effect of growth regulators on shoot propagation and rooting ofcommon lavender (Lavandula veraDC)

L.B. Andrade, S. Echeverrigaray∗, F. Fracaro, G.F. Pauletti & L. RotaInstitute of Biotechnology, University of Caxias do Sul, R. Francisco G. Vargas, 1130; 95001-970; Caxias do Sul,RS, Brazil (∗requests for offprints)

Received 23 September 1998; accepted in revised form 8 April 1999

Key words:axillary bud breaking, aromatic plant,in vitro propagation,Lavandula angustifolia, nodal explants

Abstract

Nodal segments from micropropagated plants were used to evaluate the effect of growth regulators on theinvitro shoot proliferation and rooting ofLavandula veraDC. The highest multiplication rate was obtained usingMS medium supplemented with 1.0 mg l−1 of TDZ (2.25µM) or BA (2 µM). Hyperhydricity occurred at highconcentrations of these growth regulators. Rooting of the plantlets was obtained in all the media evaluated. How-ever, rooting rates and root growth increased with increased concentrations of NAA and the reduction of the saltstrength of the media. The plantlets were successfully transferred to soil and grown to maturity, exhibiting a normaldevelopment, with high uniformity and no evidences of somaclonal variation.

Abbreviations:AC – activated charcoal; BA – benzyladenine; IBA – indolbutyric; KIN – kinetin; MS – Murashigeand Skoog (1962); NAA – naphthaleneacetic acid; TDZ – thidiazuron; 2ip – 2-isopentenyladenine

Introduction

Common lavender (Lavandula veraDC = Lavandulaofficinalis Chaix = Lavandula angustifoliaMill.) isan important ornamental and essential oil producingplant. Lavender can be propagated by seeds or bystem cuttings. The propagation by seeds is slow andthe plants exhibit too much variation in growth rateand oil composition to be commercially used. Thepropagation by woody stem cuttings is slow, with poorrooting of several clones, and risks of modificationof morphological and chemical characteristics by re-peated vegetative propagation (Moutet, 1980; Panizzaand Tognoni, 1991). Thus, micropropagation throughaxillary budding is proposed to be an alternative forvegetative propagation, allowing for the multiplicationof selected genotypes and chemiotypes.

At the moment, lavenders and lavandins have beenstudied regarding micropropagation as well as second-ary metabolite formation in cell cultures. Quazi (1980)reported the micropropagation through axillary budsof bothL. veraandL. latifolia. However, the multiplic-

ation rate achieved by this investigation was very low.More recently, several reports have been published onthe micropropagation of lavandins (Lavandula vera×Lavandula latifolia), L. latifolia (Sánchez-Gras andCalvo, 1996) andL. stoechas(Nobre, 1996). InL.latifolia adventitious bud formation was induced fromseedling explants indirectly through callus (Calvo andSegura, 1989). Cell cultures and secondary metaboliteformation were studied in different species of lavender(Segura and Calvo, 1991). However, there are not suc-cessful reports, to date, onin vitro vegetative propaga-tion of common lavender. In the present study weevaluate the effect of different plant growth regulatorson shoot proliferation and rooting ofLavandula veraDC and propose a system for the micropropagation ofthis species.

Material and methods

Plants ofLavandula veraDC, selected from a cultivarcommercialized by ISLA S.A., were grown in a green-

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house. Axillary buds measuring 2 to 3 mm in lengthwere excised from the donor plant, from Septemberto February, and used to initiate the cultures. Theexplants were throughly washed with a 70% ethanolsolution for 1 min and subsequently surface-sterilizedwith l g l−1 sodium hypochlorite solution for 20min. Following rinsing with sterile distilled water, theexplants were implanted vertically on the culture me-dium in glass tubes (2 cm diameter). The MS mediumconsists of MS salts (Murashige and Skoog, 1962),0.4 mg l−1 thiamine, 100 g l−1 inositol, 3% of sucroseand 0.6% agar. For the initial multiplication the MSmedium was supplemented with 0.5 mg l−1 (2 µM)of BA. The cultures were maintained at 25±2 ◦C with16-hour day light at an intensity of 10–20µmol m−2

s−1 (Phillips TLD 36W/84). Nodal segments with twoaxillary buds, fromin vitro propagated plants, wereused as starting material for the evaluation of growthregulator effects on the multiplication and rooting ofplants.

MS basal media and a 1/4 salt strength MS mediumwere used in the present study. The effect of growthregulators on the micropropagation was evaluated onMS basal medium supplemented with different con-centrations of BA, kinetin, 2iP, TDZ, and NAA. Theeffect of auxin concentration (IBA and NAA), as wellas salt composition and activated charcoal on rootingwere evaluated. Rooted plantlets were washed in tap-water and transferred to plastic chambers containinga sterilized mixture of sand and soil (1:1), coveredwith a plastic cap that was gradually opened duringthe acclimation period of 20 days. Acclimatized plantswere transferred to the greenhouse and then to outdoorconditions during spring.

Results and discussion

Growth regulator effect on shoot proliferation andgrowth

Initially, in vitro propagated plantlets were obtained bythe inoculation of axillary buds from actively growingvegetative plants on MS medium supplemented with0.5 mg l−1 of BA. This medium is similar to thatused by Panizza and Tognoni (1991) for the establish-ment of in vitro cultures of lavandins. In order to testwhether different growth regulator compositions in thepropagation medium influences the axillary bud prolif-eration of lavender, stem node explants obtained fromthe in vitro propagated plants were transferred to MS

media supplemented with different cytokinins, aloneor in combination with 0.2 mg l−1 of NAA. After 30days in culture the response was evaluated. The resultspresented in Table 1 and with examples in Figure 1,show that the media supplemented with BA and TDZwere more effective in promoting shoot developmentthan those without growth regulators or supplemen-ted with kinetin or 2iP. TDZ, a substituted phenylurea showing cytokinin-like activity (van Nieuwekerket al., 1986), gave the highest number of shoots perexplant, shoot growth and percentage of shoot pro-ducing explants. However, the price of this growthregulator restrict its use for commercial micropropaga-tion of lavender. Increased levels of BA and TDZ wereaccompanied by an increase in shoot multiplication.These results did not confirm those obtained by Pan-izza and Tognoni (1991) and Nobre (1996) in axillarybud proliferation of nodal cuttings of lavandin andLavandula stoechas, respectively, as they observed noeffect of BA on the multiplication rate.

Hyperhydricity was observed under high concen-trations of TDZ (1.0 mg l−1) and BA (1.0 and 2.0mg l−1). Although several factors are generally in-volved in the induction and/or development of shoothyperhydricity (Ziv, 1991; Debergh et al., 1992), anexcess of cytokinins has been reported to be a ma-jor factor involved on shoot hyperhydricity duringinvitro vegetative propagation of several plant species.Hyperhydricity, a potentially limiting factor in plantpropagation, was previously reported inLavandulastroechas(Nobre, 1996), but was never reported inLavandula vera. The hyperhydricity was not over-come by the addition of 0.2 mg l−1 of NAA into themedia.

In general, the addition of NAA to the multiplic-ation media resulted in a significant reduction on thenumber of shoots per explant. In contrast, NAA didnot affect shoot length. These results contrast withthose previously reported forLavandula vera(Quazi,1980) andLavandula latifolia (Quazi, 1980; Calvoand Segura, 1989), in which a significant increasein the number of shoot was promoted on media sup-plemented with a combination of a cytokinin (BA)and an auxin (IAA or NAA). The different resultswe observed may be done to the response of differ-ent genotypes toin vitro conditions, specifically togrowth regulators. The interaction between genotypeand growth medium has been reported in many plantspecies (Bajaj, 1986).

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Figure 1. Effect of growth regulators on thein vitro multiplication ability of a selected clone of common lavender (Lavandula veraDC). KIN-Kinetin.

Table 1. Axillary budding of common lavender on MS medium supplemented with variousgrowth regulators. Evaluation was made after 30 days in culture.

Cytokinin NAA Shoot length No of shoots Total Explants

(mg l−1) (mg l−1) (cm) per explant weight (g) producing

shoots (%)

0.0 0.0 0.63±0.12ef 1.42±0.22de 0.32 78.6

BA 0.5 0.0 1.70±0.21abc 3.35±0.59cd 1.91 92.8

BA 1.0 0.0 1.06±0.27cde 4.42±1.59c 3.15 97.1

BA 2.0 0.0 1.42±0.28c 4.21±1.15c 3.25 71.4

KIN 1.0 0.0 1.07±0.19cde 1.35±0.22de 0.63 78.6

2iP 1.0 0.0 0.54±0.13ef 1.07±0.26de 0.49 71.4

TDZ 0.5 0.0 2.18±0.17a 7.2l±0.89b 5.12 100

TDZ 1.0 0.0 2.07±0.13abc 10.78±0.83a 8.53 100

0.0 0.2 0.35±0.07f 1.07±0.22de 0.19 71.4

BA 0.5 0.2 1.54±0.19bc 3.00±0.81cd 1.68 85.7

BA 1.0 0.2 1.64±0.21abc 4.21±1.03c 3.40 85.7

BA 2.0 0.2 0.75±0.20def 1.78±0.47de 0.66 64.3

KIN 1.0 0.2 1.35±0.31cd 1.14±0.27de 0.99 64.3

2iP 1.0 0.2 0.14±0.09f 0.28±0.19e 0.12 14.3

TDZ 0.5 0.2 2.14±0.10ab 6.85±1.10b 7.06 100

TDZ 1.0 0.2 2.08±0.25ab 5.42±0.83bc 3.92 100

Values with different letters are significantly different (p=0.05) with Duncan’s test.

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Table 2. Effect of MS salt concentration, auxins and activated charcoal on the plant growth androoting rates of micropropagated plantlets ofLavanda veraafter 35 days in culture.

Medium Auxin Explants Shoot length Root length No of roots

(mg l−1) producing (cm) (cm) per explant

roots (%)

MS 0.0 27.3 1.93±0.23c 0.30±0.18c 0.40±0.19d

MS IBA 0.1 67.2 2.00±0.14c 1.01±0.26a 0.80±0.17cd

MS IBA 0.5 61.6 1.98±0.20c 0.71±0.20abc 1.13±0.30cd

MS IBA 1.0 53.5 2.08±0.19c 0.29±0.07bc 1.13±0.42cd

MS NAA 0.2 85.2 2.32±0.38bc 0.73±0.26abc 1.53±0.50bcd

MS NAA 0.5 86.7 4.31±0.49a 0.88±0.20ab 1.73±0.41bcd

MS NAA 1.0 73.4 2.99±0.40b 0.89±0.21ab 2.87±0.69ab

1/4 MS 0.0 62.1 1.82±0.16c 1.24±0.27a 2.00±0.45bc

1/4 MS IBA 0.5 71.8 1.67±0.12c 0.98±0.19a 2.67±0.54ab

1/4 MS NAA 0.2 92.7 1.56±0.14c 0.86±0.21ab 3.60±0.74a

1/4 MS+AC 0.0 35.1 1.73±0.19c 0.32±0.20bc 0.80±0.36cd

1/4 MS+AC IBA 0.5 42.5 1.85±0.13c 0.16±0.07c 0.58±0.22cd

1/4 MS+AC NAA 0.2 40.2 1.45±0.14c 0.31±0.12bc 0.61±0.31cd

Values with different letters are significantly different (p=0.05) with Duncan’s test.

Effect of growth regulators on the rooting of the plants

The plantlets micropropagated on MS with 0.5 mg l−1

BA (1.0 cm) were transferred to MS media, completeor with 1/4 the original salt concentration, supple-mented with different concentrations of IBA or NAA.Rooting occurred readily in all the media (Table 2),fifteen to twenty days after transfer. However, as re-ported for lavandins andLavandula stoechas(Panizzaand Tognoni, 1991; Nobre, 1996), the number of rootsper explant, as well as the percentage of rooting, in-creased with the increasing of the NAA concentration.The frequency of rooting, the number of roots and rootgrowth were higher on 1/4 strength MS medium thanon full strength medium. Similar results have been re-ported for several other plant species (Németh, 1986).In contrast, the shoot length was higher on MS com-plete medium. The addition of activated charcoal tothe media had a negative effect on the percentage ofrooting and root growth, probably due to the partialabsorption of auxin and other nutrients (Weatherheadet al., 1979).

All plantlets acclimatized well first to the green-house and then to outdoor conditions. The establish-ment of micropropagated plants occurred at a highrate (94%). All the 397 plants transferred to the fieldwere grown until maturity, showed high homogeneitywithout evidence of somaclonal variation.

Using MS supplemented with 1.0 mg l−1 of BA asthe multiplication medium and 1/4 MS supplemented

with 0.2 mg l−1 of NAA for rooting, we were ableto obtain more than 500 plantlets from one axillarybud in five months. These system had been used withsame success in other cultivars of common lavender,but was not appropriate for the micropropagation ofL. latifolia L. andL. dentatavar. candicansL. (datanot shown). These results shows that micropropaga-tion through axillary budding is a reliable method forthe rapid multiplication of common lavender, allowingfor the selection and propagation of elite clones.

Acknowledgements

This work was supported by a grant from the Secret-ary of Science and Technology of Rio Grande do SulState, CNPq and CAPES, Brazil.

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