Plant Cell Reports (1999) 19 :88–91 Q Springer-Verlag 1999

Á. Tantos 7 A. Mészáros 7 J. KissimonG. Horváth 7 T. Farkas

The effect of triacontanol on micropropagationof balm, Melissa officinalis L.

Received: 3 December 1997 / Revised: 24 February 1998 / Accepted: 26 February 1999

Communicated by D. Dudits

Á. Tantos, A. Mészáros, J. Kissimon, G. Horváth (Y)Department of Plant Physiology, University of Horticulture andFood Industry, P.O. Box 53, H-1502 Budapest, Hungarye-mail: ghorvath6hoya.kee.huFax: c36-1-209-6388

T. FarkasInstitute of Biochemistry, Biological Research Center,P.O. Box 521, Szeged, Hungary

Abstract Triacontanol, a long-chain primary alcoholwas found to be an effective growth regulator in themicropropagation of balm, Melissa officinalis. In boththe multiplication and the rooting phase, concentra-tions of 2, 5, 10 and 20 mg triacontanol per liter wereapplied. After 4 weeks of culture, the fresh weight ofshoots was measured in the multiplication phase androot formation, photosynthetic activity, chlorophyllcontent and the fresh and dry weights of shoots wereanalyzed in the root induction phase. In the multiplica-tion phase, 5 mg/l triacontanol was found to be theoptimal concentration, while in the rooting phase 2 mg/lwas the most effective. Triacontanol increased thenumber and length of roots, and it enhanced shootgrowth, fresh weight, and the chlorophyll content, but ithad no effect on the dry weight and the photosyntheticactivity of the plants. Results of our work demonstratethat triacontanol can be applied as an effective growthregulator in the tissue culture of balm.

Key words Melissa officinalis 7 Triacontanol 7Growth regulator 7 Tissue culture 7 Micropropagation

Abbreviations IAA Indole-3-acetic acid 7 BAP6-Benzylaminopurine

Introduction

Triacontanol was discovered in 1933 as a component ofthe epicuticular waxes of alfalfa, Medicago sativa(Chibnall et al. 1933), but it was only in 1959 thatCrosby and Vlitos demonstrated its stimulating effecton plant growth. During the past decades numerousexperiments have been carried out with triacontanol inorder to prove its usefulness in enhancing crop yield.Results available in the literature show that triacon-tanol indeed increases vegetative growth, chlorophyllcontent and dry weight in various plants (Srivastavaand Sharma 1990; Ries and Wert 1982).

As a growth regulator, triacontanol can also beconsidered as a potential agent for use in tissue culturebecause it can be autoclaved in solution. In the litera-ture, however, no or very few experiments are availablewith respect to the effect of triacontanol in micropropa-gation and tissue culture, respectively. Yun and Kim(1986) used triacontanol in combinations with variouschemicals to test their effects on callus formation andregeneration in rice, Oryza sativa, but no significantactivity of triacontanol was found. Later, Ma et al.(1990) studied the effect of different growth regulatorson Fuji apple, Malus domestica cv ’Fuji’ in tissueculture and found that triacontanol had a significantstimulating effect on the in vitro growth of this plant.No other investigations involving the in vitro applica-tion of triacontanol are available. The main purpose ofthe work presented here was to explore the applica-bility of triacontanol in the micropropagation of balm,Melissa officinalis. Our results demonstrate that tria-contanol can be effectively applied in both the multipli-cation and rooting phases of balm micropropagation.

Materials and methods

Triacontanol [CH3(CH2)28CH2OH], a long-chain primary alcohol,was obtained from SIGMA. It is soluble in ether and chloroformbut practically insoluble in polar solvents like water. Therefore,

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Fig. 1 The effect of triacontanol on shoot growth in the multipli-cation phase of the micropropagation of Melissa officinalis

2 mg triacontanol was dissolved in 1.5 ml chloroform containing 2drops of Tween 20, and this stock solution was gradually dilutedwith distilled water to the final volume of 400 ml.

Plant material

Sterile cultures of balm, Melissa officinalis L., were used in allexperiments. Tips of actively growing shoots were excised andplaced on various culture media as indicated below.

Experimental design

In the routine micropropagation process of balm, axillary shootinduction takes place from both shoot tips and nodes in the multi-plication phase. Newly formed shoots can be separated and 2- to3-cm-long shootlets, or in the case of longer shoots tips of thesame size, are allowed to form roots in the root induction phase.In our experiments basal Murashige-Skoog medium (1962)supplemented with 1 mg/l BA and 0.5 mg/l IAA was used in themultiplication phase, and hormone-free basal medium was ap-plied in the root induction phase. Both the multiplication androoting media were supplemented with 0, 2, 5, 10 and 20 mg tria-contanol per liter and sterilized by autoclaving at 121 7C, 1.2 barfor 20 min. Cultures were grown in 200 ml jars containing 30 ml ofmedium.

Five shoot tips were placed into each jar. Four jars repre-sented one treatment, and each treatment was repeated threetimes. Growth conditions were as follows: illumination suppliedby cool-white fluorescent tubes at a light intensity of57 mM7m–27s–1 for 16 h/day with a day/night constant tempera-ture of 23 7C.

For evaluation of the effect of triacontanol, the number andlength of shoots and roots, fresh and dry weights, chlorophyllcontent and fluorescence induction kinetics were measured after4 weeks of culture. Chlorophyll content of the plantlets was esti-mated according to Arnon (1949).

Chlorophyll fluorescence induction kinetics were measuredusing a portable Hansatech Plant Efficiency Analyser (PEA), andas kinetic parameters the Fv/Fm, Fi/Fv and Fd/Fs ratios wereevaluated (Bolhár-Nordenkampf and Öquist 1993).

Student’s T-test was used for statistical evaluation of experi-mental data. Number of data (n), standard deviation (SD) andlevels of significance (P) are presented in the figures.

Results

Effects of triacontanol in the multiplication phase ofbalm micropropagation

As shown in Fig. 1, even the lowest (2 mg/l) concentra-tion of triacontanol resulted in a maximal increment intotal shoot numbers, and higher triacontanol concen-trations up to 20 mg/l did not give any further increasein shoot numbers. In contrast, the increasing triacon-tanol concentration induced a slight but continuousincrease in both the fresh weight and the number ofnodes of plantlets. These data indicate that during themultiplication phase of balm micropropagation triacon-tanol significantly enhances the shoot growth and thenumber of nodes with a reduced length of internodes.

Fig. 2 The effect of triacontanol on the root growth of plants inthe root-generating phase during micropropagation of balm

Triacontanol-induced alteration in the root inductionphase

As shown in Fig. 2, the number of roots significantlyincreased with 2 mg/l triacontanol but any furtherincrease in its concentration resulted in a decrease inroot numbers. Although triacontanol was found to beless effective at higher concentrations, it still inducedmore roots than were found with the untreated control.In contrast, triacontanol at concentrations up to 5 mg/lreduced the length of roots while at concentrations of10 or 20 mg/l again increased root length.

Shoot growth was also evaluated in the rootingphase, and similar alterations were found in both shootlength and number of nodes after triacontanol treat-ment (Fig. 3). Triacontanol positively affected shootgrowth and increased the number of nodes. The longestshoots were formed in the medium containing 10 mg/ltriacontanol, whereas the highest number of nodeswere found at 20 mg/l triacontanol. This latter concen-tration, however, slightly reduced the length of shoots.

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Fig. 3 Triacontanol-induced shoot growth of plantlets during theroot-generating phase

Fig. 4 Alterations in the fresh and dry weights of plantletsinduced by triacontanol during the root-inducing phase

Branching of control plants occurred very rarely onhormone-free media. In contrast, triacontanol inducedthe formation of one or two lateral shoots at all concen-trations, which was characterized by the fresh and dryweight of the plants (Fig. 4). Although the fresh weightof the plantlets was higher at all concentrations of tria-contanol than that of the control, triacontanol had nodramatic effect on the dry weight. Since the color of theplantlets was different at various triacontanol concen-trations, the chlorophyll contents of leaves were alsomeasured. As shown in Fig. 5, the chlorophyll contentof plantlets was increased stepwise up to 10 mg/l of tria-contanol in the medium. The elevated chlorophyllcontent was less pronounced in plants grown on amedium containing 20 mg/l triacontanol. As measuredby fluorescence induction the high chlorophyll contentwas not associated with an increase in the photosyn-thetic functions of plantlets at any of the triacontanol

Fig. 5 The effect of various triacontanol concentrations on thechlorophyll content of leaves in the root-inducing phase of balmmicropropagation

Table 1 The effect of triacontanol on the fluorescence inductioncharacteristics of micropropagated plants

Treatment Fv/Fm(xBSD)

dFi/Fv(xBSD)

Fd/Fs(xBSD)

Control 0.84B0.002 0.475B0.006 1.41B0.1202 mg/l triacontanol 0.82B0.004 0.521B0.014 1.33B0.2455 mg/l triacontanol 0.82B0.003 0.491B0.003 1.20B0.220

10 mg/l triacontanol 0.83B0.010 0.476B0.004 1.54B0.15520 mg/l triacontanol 0.84B0.001 0.476B0.009 1.56B0.115

concentrations (Table 1). Amongst the various parame-ters of fluorescence induction, the ratio of the variable(Fv) and maximal fluorescence together with the ratioof the intermediary (dFi) and variable fluorescence(Fv) are usually used to characterize the electron trans-port capacity, whereas changes in the ratio of the fluo-rescence decrease (Fd) and steady state (Fs) indicatealterations in the carboxylation capacity of leaves(Bolhár-Nordenkampf and Öquist 1993; Lichtenthalerand Rinderle 1988). Data presented in Table 1,however, showed no significant differences at any tria-contanol concentration, which indicated that triacon-tanol had no direct effect on either electron transportor the carboxylation capacity of micropropagatedbalm.

Discussion

The present work indicates that triacontanol is an effec-tive growth regulator in the micropropagation of balm.During the multiplication phase it increased both thenumber of shoots and nodes as well as the fresh weightof plants. The increase in the fresh weight of the plantswas 50%–106% in the range of 2–20 mg/l triacontanol.These values are much higher than those found in field

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experiments (Srivastava and Sharma 1990, 1991). Inour experiments triacontanol, however, had no notice-able effect on the dry weight of plants, which is incontradiction with the results obtained by Srivastavaand Sharma (1990, 1991). Another interesting result isthe increased chlorophyll content of the plantlets. Infield experiments triacontanol increased the chloro-phyll content by 20–30% (Debata and Murty 1981); inour experiments treatment with 10 mg/l concentrationinduced a 176% increase in the chlorophyll content ofplantlets. The most possible explanation is that micro-propagated plants can take up the triacontanol contin-uously from the culture media and not only occasion-ally, one or two times after spraying the leaves in thefield. The fact that the chlorophyll content wasincreased but not the photosynthetic activity indicatesthat the higher green mass production observed in fieldexperiments (Mahadevappa et al. 1989; Thakur et al.1991) is related to the elevated photosynthetic activitycaused by the high chlorophyll content rather than anincrease in the electron transport or carboxylationactivity. In the case of balm, however, no significantincrease was obtained in dry matter, which clearlyshows that the high chlorophyll content itself does notenhance photosynthetic activity. We assume that in ourcase, the pigment-protein complexes containing theadditionally synthetized chlorophyll molecules werenot able to insert functionally into the photosyntheticunits.

At low concentrations, triacontanol promoted bothrooting and shoot growth of plants, but at the highestconcentration it had a slight inhibitory effect. Corre-sponding data published in the literature also indicatethat triacontanol has an inhibitory effect above certainconcentrations (Srivastava and Sharma 1991; Somenand Seetalakshmi 1991). The plants on the hormone-free medium containing 5 mg/l triacontanol had themost and longest shoots but they had low fresh weight.The inhibitory effect of triacontanol on shoot growthcan also be seen at concentrations of 20 mg/l. Anotherinteresting effect of triacontanol is that it stimulatesbranching not only in combination with otherhormones but also in a hormone-free environment.During the multiplication phase there were four to sixshoots in one bunch and some callus formation couldalways be observed. In a hormone-free medium,however, values were lower, which was due to the factthat only two or three shoots were initiated in onebunch. Our results clearly indicate that triacontanol is a

useful growth regulator in both the multiplication androot-inducing phases of balm micropropagation.

Acknowledgements This work was partially supported by theHungarian National Foundation for Scientific Research (T-017697), the Ministry of Culture and Education (MKM-351) andthe Hungarian Academy of Sciences.

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