. Tantos 7 A. Mszros 7 J.

Kissimon
G. Horvth 7 T. Farkas

The effect of triacontanol on micropropagation

of balm, Melissa officinalis L.

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

Abstract Triacontanol, a long-chain primary alcohol

was found to be an effective growth regulator in the
Introduction
micropropagation of balm, Melissa officinalis. In both
Triacontanol was discovered in 1933 as a component of
the multiplication and the rooting phase, concentra-
the epicuticular waxes of alfalfa, Medicago sativa
tions of 2, 5, 10 and 20 mg triacontanol per liter were
(Chibnall et al. 1933), but it was only in 1959 that
applied. After 4 weeks of culture, the fresh weight of
Crosby and Vlitos demonstrated its stimulating effect
shoots was measured in the multiplication phase and
on plant growth. During the past decades numerous
root formation, photosynthetic activity, chlorophyll
experiments have been carried out with triacontanol in
content and the fresh and dry weights of shoots were
order to prove its usefulness in enhancing crop yield.
analyzed in the root induction phase. In the multiplica-
Results available in the literature show that triacon-
tion phase, 5 mg/l triacontanol was found to be the
tanol indeed increases vegetative growth, chlorophyll
optimal concentration, while in the rooting phase 2 mg/l
content and dry weight in various plants (Srivastava
was the most effective. Triacontanol increased the
and Sharma 1990; Ries and Wert 1982).
number and length of roots, and it enhanced shoot
As a growth regulator, triacontanol can also be
growth, fresh weight, and the chlorophyll content, but it
considered as a potential agent for use in tissue culture
had no effect on the dry weight and the photosynthetic
because it can be autoclaved in solution. In the litera-
activity of the plants. Results of our work demonstrate
ture, however, no or very few experiments are available
that triacontanol can be applied as an effective growth
with respect to the effect of triacontanol in micropropa-
regulator in the tissue culture of balm.
gation and tissue culture, respectively. Yun and Kim
(1986) used triacontanol in combinations with various
Key words Melissa officinalis 7 Triacontanol 7
chemicals to test their effects on callus formation and
Growth regulator 7 Tissue culture 7 Micropropagation
regeneration in rice, Oryza sativa, but no significant
activity of triacontanol was found. Later, Ma et al.
Abbreviations IAA Indole-3-acetic acid 7 BAP
(1990) studied the effect of different growth regulators
6-Benzylaminopurine
on Fuji apple, Malus domestica cv Fuji in tissue
culture and found that triacontanol had a significant
stimulating 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 of
the work presented here was to explore the applica-
bility of triacontanol in the micropropagation of balm,
Melissa officinalis. Our results demonstrate that tria-
Communicated by D. Dudits contanol can be effectively applied in both the multipli-
. Tantos, A. Mszros, J. Kissimon, G. Horvth (Y) cation and rooting phases of balm micropropagation.
Department of Plant Physiology, University of Horticulture and
Food Industry, P.O. Box 53, H-1502 Budapest, Hungary
e-mail: ghorvath6hoya.kee.hu Materials and methods
Fax: c36-1-209-6388
T. Farkas Triacontanol [CH3(CH2)28CH2OH], a long-chain primary alcohol,
Institute of Biochemistry, Biological Research Center, was obtained from SIGMA. It is soluble in ether and chloroform
P.O. Box 521, Szeged, Hungary but practically insoluble in polar solvents like water. Therefore,
2 mg triacontanol was dissolved in 1.5 ml chloroform containing 2
drops of Tween 20, and this stock solution was gradually diluted
with distilled water to the final volume of 400 ml.

Plant material

Sterile cultures of balm, Melissa officinalis L., were used in all

experiments. Tips of actively growing shoots were excised and
placed on various culture media as indicated below.

Experimental design

In the routine micropropagation process of balm, axillary shoot

induction takes place from both shoot tips and nodes in the multi-
plication phase. Newly formed shoots can be separated and 2- to
3-cm-long shootlets, or in the case of longer shoots tips of the
same 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 the
multiplication phase, and hormone-free basal medium was ap-
plied in the root induction phase. Both the multiplication and
rooting media were supplemented with 0, 2, 5, 10 and 20 mg tria-
contanol per liter and sterilized by autoclaving at 121 7C, 1.2 bar
for 20 min. Cultures were grown in 200 ml jars containing 30 ml of
medium.
Five shoot tips were placed into each jar. Four jars repre-
sented one treatment, and each treatment was repeated three
times. Growth conditions were as follows: illumination supplied
by cool-white fluorescent tubes at a light intensity of
57 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 and
length of shoots and roots, fresh and dry weights, chlorophyll
content and fluorescence induction kinetics were measured after
4 weeks of culture. Chlorophyll content of the plantlets was esti-
mated according to Arnon (1949).
Chlorophyll fluorescence induction kinetics were measured
using a portable Hansatech Plant Efficiency Analyser (PEA), and
as kinetic parameters the Fv/Fm, Fi/Fv and Fd/Fs ratios were
evaluated (Bolhr-Nordenkampf and quist 1993).
Students T-test was used for statistical evaluation of experi-
mental data. Number of data (n), standard deviation (SD) and
levels of significance (P) are presented in the figures.
Fig. 1 The effect of triacontanol on shoot growth in the multipli-
cation phase of the micropropagation of Melissa officinalis
Triacontanol-induced alteration in the root induction
phase
As shown in Fig. 2, the number of roots significantly
increased with 2 mg/l triacontanol but any further
increase in its concentration resulted in a decrease in
root numbers. Although triacontanol was found to be
less effective at higher concentrations, it still induced
more roots than were found with the untreated control.
In contrast, triacontanol at concentrations up to 5 mg/l
reduced the length of roots while at concentrations of
10 or 20 mg/l again increased root length.
Shoot growth was also evaluated in the rooting
phase, and similar alterations were found in both shoot
length and number of nodes after triacontanol treat-
ment (Fig. 3). Triacontanol positively affected shoot
growth and increased the number of nodes. The longest
shoots were formed in the medium containing 10 mg/l
triacontanol, whereas the highest number of nodes
were found at 20 mg/l triacontanol. This latter concen-
tration, however, slightly reduced the length of shoots.

Results

Effects of triacontanol in the multiplication phase of

balm micropropagation

As shown in Fig. 1, even the lowest (2 mg/l) concentra-

tion of triacontanol resulted in a maximal increment in
total shoot numbers, and higher triacontanol concen-
trations up to 20 mg/l did not give any further increase
in shoot numbers. In contrast, the increasing triacon-
tanol concentration induced a slight but continuous
increase in both the fresh weight and the number of
nodes of plantlets. These data indicate that during the
multiplication phase of balm micropropagation triacon-
tanol significantly enhances the shoot growth and the
number of nodes with a reduced length of internodes.
Fig. 2 The effect of triacontanol on the root growth of plants in
the root-generating phase during micropropagation of balm