Enhancement of stem elongation ...
139
ENHANCEMENT OF STEM ELONGATION AND
1
FLOWER BUD REGENERATION BY SALICYLIC ACID
Walter Handro 2 , Celina M. Mello 4 , M.A. Manzano 4 and Eny I.S. Floh 3
Plant Cell Biology Laboratory, Dept of Botany, Inst. of Biosciences,
University of São Paulo, C.P. 11461, São Paulo, SP, 05422-970.
ABSTRACT- Application of salicylic acid to plants
induces a large variety of responses, among them
flowering and tuberization. We investigated the effects
of SA on these processes using leaf discs of
Streptocarpus nobilis (a short-day plant) and stem
segments of Ullucus tuberosus, which form tubers
under short days. Salicylic acid does not replace the
effect of short days to induce flowering and
tuberization in such species. Under short days,
however, the number of floral buds in leaf discs of
Streptocarpus was significantly increased with
addition of salicylic acid (0.5-2.0 µM), and floral
scapes were significantly longer than controls. When
stem segments of Ullucus were cultured on medium
with salicylic acid (0.5 - 1.0 µM), the axillary buds
developed shoots significantly more elongated than
controls.
Additional index terms: morphogenesis, salicylic
acid, Streptocarpus, Ullucus.
ESTIMULO DO ALONGAMENTO
CAULINAR E REGENERAÇÃO DE
GEMAS FLORAIS PELO ÁCIDO
SALICÍLICO.
RESUMO - A aplicação do ácido salicílico em plantas
induz uma grande variedade de respostas, entre elas
a floração e a tuberização. Foram investigados os
efeitos do ácido salicilico nesses processos
usando-se discos foliares de Streptocarpus nobilis
(uma planta de dias curtos) e segmentos caulinares
de Ullucus tuberosus, que formam tubérculos em dias
curtos. Verificou-se que o ácido salicilico não substitui
o efeito dos dias curtos para indução de floração e
tuberização nessas espécies. Em dias curtos,
entretanto, o número de gemas florais regeneradas
em discos foliares de Streptocarpus aumentou
significativamente com a adição de ácido salicílico
1
Recebido em 30/07/1996 e aceito em 28/04/1997.
2
Biologist, Full Professor, Dr.
3
Biologist, Dr.
4 diameter) were prepared from leaves previously
Graduate Students
R. Bras. Fisiol. Veg., 9(2):139-142, 1997.
COMMUNICATION
(0,5-2,0µM), e os escapos florais foram
significativamente mais longos que os controles.
Quando segmentos caulinares de Ullucus foram
cultivados em meio com ácido salicílico (0,5-1,0 µM)
as gemas axilares produziram ramos
significativamente mais longos que os controles.
Termos adicionais para indexação: ácido salicílico,
morfogênese, Streptocarpus, Ullucus.
INTRODUCTION
Recent reviews have demonstrated that salicylic
acid has a relevant role in the control of several
physiological and biochemical processes in plants,
and it is considered a phytohormone by some authors
(Raskin 1992, 1995; Gross & Parthier, 1994). Among
the morphogenetic processes affected by salicylic
acid are flowering and tuberization; e.g., salicylic acid
enhances flowering in Lemna (Khurama & Cleland,
1992), hastens flower initiation in Phaseolus (Lagoa &
Pereira, 1991) and induces tuberization in potato
(Koda et al., 1991).
In our laboratory we have used Streptocarpus
nobilis to study in vitro flowering (Handro, 1977; 1983)
and Ullucus tuberosus, a plant that produces aerial
tubers (Asahira & Nitsch, 1968), to study in vitro
tuberization. Preliminary experiments showed that
salicylic acid does not replace photoperiodic
conditions to induce flowering and tuberization, but
affects the stem growth of Ullucus, and the
neoformation of buds in Streptocarpus. This work was
developed to characterize the effects of salicylic acid
on growth and regenerative process in these
experimental systems.
MATERIAL AND METHODS
Culture of leaf discs of Streptocarpus nobilis
Streptocarpus plants were obtained from seeds and
kept vegetative from germination under long-day
conditions (16:8 h, light:dark). Leaf discs (10 mm
140 Handro et al.
FIGURE 1- Effect of salicylic acid on bud regeneration
in leaf discs of Streptocarpus nobilis, after 6 weeks
under short days. A - % of cultures with buds; B -
number of buds per culture. Number of floral buds in
salicylic acid treatments differs significantly from
controls (confidence interval at 95%). o floral buds n
vegetative buds.
sterilized with 5% sodium hypochlorite (5 min) and
cultured on a medium containing Knop’s
macroelements (Gautheret, 1959), microelements
and vitamins (Nitsch & Nitsch, 1965), benzyladenine
(0.35 g.L-1 ), IAA (0.1 mg.L-1 ), adenine (1.5 mg.L-1)
and sucrose (20 g.L-1). The pH was adjusted to 5.5 and
then the medium was gelled with 0.8 g.L-1 agar.
Salicylic acid was added via filtration through Millipore
membranes after autoclaving. The cultures were kept
either under short days (8:16 h, light:dark), or long
days (16:8 h, light:dark), photon flux 45 µMm-2s-1, and
temperature 26 1oC. After 5 weeks, the number of
vegetative and floral buds were counted. Each
treatment consisted of 20 tubes with one explant in
each, and was repeated once more, with similar
results.
Culture of stem segments of Ullucus
A sterile plant population used as explant source
was originated by aseptic culture of stem segments
developed in Ullucus tubers. This population has been
maintained by successive cultures of stem segments.
Explants were stem segments 10 mm in length, with a
node, cultured on MS basal medium (Murashige &
Skoog, 1962), without growth regulators, with sucrose
R. Bras. Fisiol. Veg., 9(2):139-142, 1997.
(20 g.L-1), agar (0.8 g.L-1), pH 5.6. Salicylic acid was
added to the autoclaved medium, after filtration
through Millipore membrane. Cultures were
maintained under conditions of 16:8 h, light:dark, at 26
 1oC. Each treatment consisted of 5 flasks (50 x 100
mm) with three explants in each. After 5-6 weeks in
culture, results were recorded as the length of the
longest new branch, and total fresh matter.
RESULTS AND DISCUSSION
Under short day conditions, leaf discs of
Streptocarpus nobilis give rise to flower and vegetative
buds, with flower buds predominating. Salicylic acid
enhanced both the number of cultures with floral buds
(Fig. 1, A), and the number of flower buds per culture
(Fig. 1, B) in all concentrations. Regarding the
vegetative buds, effects of salicylic acid were not
evident. In salicylic acid treated cultures, the length of
floral scapes were markedly higher than controls (Fig.
2). Under long days less than 50% of cultures
regenerated buds and all these were vegetative. Only
1.0 µM salicylic acid treatment was stimulatory for
regeneration; no effects on stem elongation were
observed (data not shown).
In the stem segments of Ullucus tuberosus cultured
in vitro the nodal bud develops, giving rise to a new
shoot with branches. The stem segment (explant) also
roots profusely. Figure 3 shows the aspect of control
and salicylic acid treated plants Fig. 4 and the results
corresponding to the stem length and fresh matter of
the aerial part. These results show clearly the
stimulatory effect of salicylic acid.
Our results, obtained in two very different materials,
show a common aspect, the stimulatory effect of
salicylic acid on stem elongation. There are few
reports on salicylic acid effects on growth: it affects
growth in Vicia faba (Manthe et al., 1992), gravitropic
curvature in maize (Medvedev & Markova, 1991) and
FIGURE 2- Leaf discs of Streptocarpus nobilis after 7
weeks under short days. Left, controls; right, 0.5 µM
salicylic acid treated plants.
 Enhancement of stem elongation ... 141

increases the flower size in Campanula (Serek, 1992).

FIGURE 3- Plants of Ullucus tuberosus developed
from stem segments after 8 weeks in culture. A,
control; B, 0.5 µM salicylic acid; C, 1.0 µM salicylic
acid.
On the other hand, acetyl-salicylic acid (AAS) retards
the loss of regenerative capacity in Pennisetum and
increases callus dry matter (Pius et al., 1993).
Stimulatory effects of salicylic acid on bud induction
are also known: salicylic acid induces and increases
the number of buds in moss protonema (Chopra &
Mehta, 1991) and increases the formation of buds
induced by cytokinin in Cucumis (Shetti et al., 1992).
The most typical biological response of salicylic
acid is a systemic signal for induction of disease
resistance (Raskins, 1995). However, the
physiological mechanisms of salicylic acid action in
growth and morphogenetic processes remain
unknown. A decrease of ethylene production after
salicylic acid application, resulting in stimulation of
growth and regenerative capacity, was described by
Serek (1992) and Pius et al., (1993). Kumar & Nanda
(1981a, 1981b) have shown that the higher number of
floral buds induced by salicylic acid was accompanied
by increase in protein synthesis and appearance of
new isozym bands. Other works to be considered
concern the effects of salicylic acid leading to an
inhibition of catalase and accumulation of H2O2 (Chen
et al., 1993, Kauss & Jeblick, 1995). Furthermore,
peroxidase activity could be altered or inhibited by
excess of H2O2 (Nakajima et al. 1991, Acosta et al.
1991). On the other hand, peroxidases are involved in
different aspects related to growth, such as auxin
catabolism, ethylene biosynthesis and cell wall
lignification (Gaspar et al., 1991. Thus, the study of
variations in ethylene, peroxidase activity and H 2O2 in
salicylic acid treated materials could lead to
understanding the mechanism of salycilic acid
induced growth responses.

ACKNOWLEDGEMENTS
To CNPq (Brasil) for grants, and to Dr. G.E. Delgado
(UNPRG, Peru) for the kind supply of Ullucus.

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