Plant Cell Reports (1998) 17: 661–663
C. Magioli · A. P. M. Rocha · D. E. de Oliveira
E. Mansur
Efficient shoot organogenesis of eggplant (Solanum melongena L.)
induced by thidiazuron
Received: 1 March 1997 / Revision received: 29 October 1997 / Accepted: 15 November 1997
Abstract Morphogenesis from several explant types ex-
cised from in-vitro-grown plantlets of a Brazilian eggplant
(Solanum melongena L.) variety (F-100) was evaluated in
response to thidiazuron (TDZ). Leaves and cotyledons
were found to be the most responsive explants. Optimal
shoot bud induction rates (75–100 buds/explant) were
achieved in the presence of 0.2 µM TDZ. Organogenic calli
were transferred to growth regulator free MS medium be-
fore shoot excision. Rooting was induced on half-strength
MS medium supplemented with 0.6 µM IAA.
Key words Eggplant · Solanum melongena ·
Organogenesis · Thidiazuron
Abbreviations BAP 6-Benzylaminopurine · IAA Indol-
eacetic acid · MS medium Murashige and Skoog medium ·
TDZ thidiazuron
Introduction
In vitro regeneration of different explant types from sev-
eral eggplant cultivars has been reported both via embryo-
genesis (Matsuoka and Hinata 1979; Gleddie et al. 1983;
Rao and Singh 1991; Saito and Nishimura 1994; Sharma
and Rajam 1995) and organogenesis (Kamat and Rao 1978;
Matsuoka and Hinata 1979; Fassuliotis et al. 1981; Alli-
chio et al. 1982; Gleddie et al. 1983; Mukherjee et al. 1991;
Sharma and Rajam 1995). The response to culture condi-
Communicated by S. Gleddie
C. Magioli · D. E. de Oliveira
Universidade Federal do Rio de Janeiro,
Instituto de Biologia, LGMV/DG,
Rio de Janeiro, Brazil
A. P. M. Rocha · E. Mansur (½)
Universidade do Estado do Rio de Janeiro,
Instituto de Biologia,
LABMIT/DBCG-Rua São Francisco Xavier,
524-PHLC, sala 505-Rio de Janeiro, Brazil
E-mail: mansur@uerj.br
© Springer-Verlag 1998
tions is reported to differ markedly depending on cultivar,
explant type, and culture medium (Fassuliotis et al. 1981;
Allichio et al. 1982; Gleddie et al. 1983; Sharma and Rajam
1995).
Increased numbers of adventitious shoots have been re-
ported by several investigators using only 6-benzylami-
nopurine (BAP) (Gleddie et al. 1983; Sharma and Rajam
1995), zeatin (Gleddie et al. 1983), or kinetin (Gleddie et
al. 1983; Mukherjee et al. 1991). Although most of the pub-
lished protocols resulted in relatively low regeneration fre-
quencies of a maximum of 7 shoots/explant (Gleddie et al.
1983; Mukherjee et al. 1991), Sharma and Rajam (1995)
observed the production of 20 shoots/explant for one of the
cultivars tested.
Thidiazuron (TDZ) is a substituted phenylurea that is
commercially used as a defoliant for cotton, and which also
produces high cytokinin-like activity in in-vitro-cultivated
cells (Wang et al. 1986; Fiola et al. 1990; Saxena et al.
1992). The mechanism of TDZ action is partly related to
the inhibition of cytokinin degradation by cytokinin oxi-
dase, resulting in increased levels of endogenous cytoki-
nin (Hare and Van Staden 1994). TDZ has proven to be
very effective in inducing in vitro shoot regeneration of
several species such as kiwi fruit (Suezawa et al. 1988),
apple (Fasolo et al. 1989), grape (Matsuta and Hirabaya-
shi 1989), pear (Leblay et al. 1991), pea (Bohmer et al.
1995), peanut (Kanyand et al. 1994) and several woody
species (Huetteman and Preece 1993). The present work
was designed to determine the effect of TDZ on in vitro
morphogenesis of eggplant. The effect of TDZ concentra-
tions on bud induction, shoot elongation, and the subse-
quent development of roots was examined.
Materials and methods
Seeds of eggplant (F-100) were obtained from Agroceres Ltda. and
stored at 4°C. The seeds were washed in distilled water containing
0.02% Tween 80 and surface-sterilized for 25 min in a 5% sodium
hypochlorite solution. After three rinses with sterile distilled water,
the seeds were inoculated on half-strength (Murashige and Skoog
662
1962) (MS) basal medium supplemented with 1.5% (wt/vol) sucrose
for germination. Media pH was adjusted to 5.8 with 1 N NaOH be-
fore adding agar (Sigma) at a concentration of 7 g/l. Growth regula-
tors were added to the medium before autoclaving at 120°C for
15 min. Plant material was maintained in a growth chamber at
28±2°C under a 16-h photoperiod regime provided by cool-white
fluorescent lamps (General Electric) with a photon fluency of
36 µmol · m–2 · s–1
Hypocotyl, epicotyl, and node segments (5 mm long) as well as
leaf and cotyledon segments (50 mm2) were excised from 25- to 30-
day-old seedlings. Explants were cultured for 30 days on MS medi-
um plus 3% sucrose and 0.7% agar (SIGMA) and supplemented with
0.05, 0.1, 0.2 or 0.4 µM TDZ. After 30 days culture, calli formed in
the presence of 0.2 µM TDZ were fragmented and transferred either
to hormone-free MS basal medium or to MS medium supplemented
with 0.01 µM or 0.2 µM TDZ, and cultured for 15 days. Excised
shoots (1.5–2.0 cm long) were transferred to half-strength MS basal
medium plus 3% (wt/vol) sucrose and 0.6 µM indoleacetic acid (IAA)
for root initiation. The rooted plantlets were transferred to a green-
house after 2 weeks in a phytotron chamber.
The efficiency of regeneration was measured as follows: percent-
age of explants that developed buds, number of buds and number of
shoots per explant. As the high number and proximity of buds im-
paired accurate counting, a class system corresponding to the ap-
proximate number of buds per explant was adopted as follows: class
1, 1–25; class 2, 25–50; class 3, 50–75; and class 4, 75–100 buds/ex-
plant. Twenty explants were analyzed in each experiment and each
treatment was repeated at least twice. Confidence intervals were giv-
en for α=0.05 using the t-test.

Results and discussion

Leaf and cotyledon were significantly more responsive

than the other explants (P<0.05), considering both the fre-
quencies of organogenic calli induction and the number
of buds regenerated per explant (Fig. 1). The mean num-
ber of buds/explant induced by 0.2 µM TDZ, on both ex-
plant types, was higher than we obtained previously for the
same variety in response to BAP (data not shown).
Reports on in vitro morphogenesis of eggplant usually
describe hypocotyl (Kamat and Rao 1978; Matsuoka and
Hinata 1979) and leaf explants (Gleddie et al. 1983; Muk-
herjee et al. 1991) as the most responsive types. However,
previous comparative studies on the responsiveness of dif-
ferent explant types were undertaken only by Allichio et
al. (1982) and Sharma and Rajam (1995). The first authors
obtained the best responses for leaf and cotyledon explants
in six eggplant cultivars and the latter authors obtained
their best results using hypocotyl explants, except for cul-
tivar Pusa Kranti, in which cotyledonary explants were the
most responsive.
The development of buds into shoots from cotyledon or
leaf explants was studied by transferring calli induced in
the presence of 0.2 µM TDZ to the following media: MS
without growth regulators or MS supplemented with 0.01
or 0.2 µM TDZ. The highest rate of shoot development was
observed on cotyledon-derived calli maintained on MS me-
dium supplemented with 0.2 µM TDZ (Table 1).
Although the number of induced shoots from both cot-
yledon and leaf explants was significantly higher upon con-
tinued exposure to 0.2 µM TDZ (P<0.05), we found that
they were short and failed to develop roots in response to
Fig. 1 TDZ-induced organogenesis from different explants of egg-
plant after 30 days of culture initiation, showing the percentage of
explants that developed buds (A) and the number of buds per explant
(B), corresponding to four classes: class 1, 1–25 buds; class 2,
25–50 buds; class 3, 50–75 buds and class 4, 75–100 buds per ex-
plant
Table 1 Influence of the shoot-inducing medium on the develop-
ment of adventitious shoots formed from leaf and cotyledon explants.
Buds induced on MS medium supplemented with 0.2 µM TDZ for
30 days were then subcultured on either MS basal medium or MS
medium supplemented with 0.01 or 0.2 µM TDZ
TDZ concentration Mean number of shoots/explant (±SE)
(µM) in the shoot
induction medium Leaf Cotyledon
0 11.6 (±1.3) 17.6 (±3.2)
0.01 15.6 (±3.6) 16 (±2.4)
0.2 18.3 (±1.1) 23.3 (±5.4)
several root inducing media. TDZ is reported to increase
the levels of endogenous cytokinins by, at least partly, in-
hibiting the action of cytokinin oxidase (Hare and Van Sta-
den 1994). It is therefore possible that the inability to form
roots observed in these shoots was due to endogenous cy-
tokinin levels. This idea is supported by the observation
that shoots excised after subculture of calli on MS supple-
mented with 0.01 µM TDZ rooted at a frequency of 6% on
half-strength MS supplemented with 0.6 µM IAA and 3%

sucrose. Moreover, highly efficient root induction (70%)
was achieved when calli were maintained on hormone-free
medium before shoot excision for a period of 2 weeks af-
ter bud induction by TDZ. This treatment was essential for
the recovery of whole plants, probably by decreasing the
endogenous levels of cytokinins in the shoots. Plants which
were produced in this manner were successfully transferred
to soil and were phenotypically normal.
No previous report has appeared on the use of TDZ for
in vitro regeneration of eggplant. The most effective TDZ
concentration (0.2 µM) for eggplant was lower than the lev-
els adopted for other species (2–200 µM) (Huetteman and
Preece 1993). Higher concentrations caused reduction
in bud formation and necrosis. Similar inhibitions in bud
number by high concentrations were also found in Pseu-
ditsuga menziesii (Goldfarb et al. 1991) and pear (Leblay
et al. 1991).
Acknowledgements The authors are indebted to Drs. W. R. Krul
and M. A. Costa Lima for helpful suggestions and critical reading of
the manuscript. C. Magioli is a MSc. fellow supported by CAPES
and FAPERJ and A. P. M. Rocha is an undergraduate student sup-
ported by CNPq/PIBIC.
References
Allichio R, Del Grosso E, Boschieri E (1982) Tissue cultures and
plant regeneration from different explants in six cultivars of Sol-
anum melongena. Experientia 38:449–450
Bohmer P, Meyer B, Jacobsen H-J (1995) Thidiazuron-induced high
frequency of shoot induction and plant regeneration in protoplast
derived pea callus. Plant Cell Rep 15:26–29
Fasolo F, Zimmerman RH, Fordham I (1989) Adventitious shoot for-
mation on excised leaves of in vitro grown shoots of apple cul-
tivars. Plant Cell Tissue Organ Cult 16:75–87
Fassuliotis G, Nelson BV, Bhatt DP (1981) Organogenesis in tissue
culture of Solanum melongena cv. Florida Market. Plant Sci Lett
22:119–125
Fiola JA, Hassan MA, Swartz HJ, Bors RH, McNicols R (1990) Ef-
fect of thidiazuron, light fluence rate and kanamycin on in vitro
shoot organogenesis from excised Rubus cotyledons and leaves.
Plant Cell Tissue Organ Cult 20:223–28
663
Gleddie S, Keller W, Setterfield G (1983) Somatic embryogenesis
and plant regeneration from leaf explants and cell suspensions of
Solanum melongena (eggplant). Can J Bot 61:656–666
Goldfarb B, Howe GT, Bailey LM, Strauss SH, Zaerr JB (1991) A
liquid cytokinin pulse induces adventitious shoot formation from
Douglas-fir cotyledons. Plant Cell Rep 10:156–160
Hare PD, Van Staden J (1994) Inhibitory effect of thidiazuron on the
activity of cytokinin oxidase isolated from soybean callus. Plant
Cell Physiol 35:1121–1125
Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for
woody plant tissue culture. Plant Cell Tissue Organ Cult 33:
105–119
Kamat MG, Rao PS (1978) Vegetative multiplication of eggplants
(Solanum melongena) using tissue culture techniques. Plant Sci
Lett 13:57–65
Kanyand M, Dessai AP, Prakash CS (1994) Thidiazuron promotes
high frequency regeneration of peanut (Arachis hypogaea) plants
in vitro. Plant Cell Rep 14:1–5
Leblay C, Chevreau E, Raboin LM (1991) Adventitious shoot regen-
eration from in vitro leaves of several pear cultivars (Pyrus com-
munis L.). Plant Cell Tissue Organ Cult 25:99–105
Matsuta N, Hirabayashi T (1989) Embryogenic cell lines from so-
matic embryos of grape (Vitis vinifera L.). Plant Cell Rep 7:
684–687
Matsuoka H, Hinata K (1979) NAA-induced organogenesis and em-
bryogenesis in hypocotyl callus of Solanum melongena. J Exp
Bot 30:363–370
Mukherjee SK, Rathinasabapathi B, Gupta N (1991) Low sugar and
osmotic requirements for shoot regeneration from leaf pieces of
Solanum melongena L. Plant Cell Tissue Organ Cult 25:13–16
Murashige T, Skoog F (1962) A revised method for rapid growth and
bioassays with tissue cultures. Physiol Plant 15:473–497
Rao PVL, Singh B (1991) Plantlet regeneration from encapsulated
somatic embryos of hybrid Solanum melongena L. Plant Cell Rep
10:7–11
Saito T, Nishimura S (1994) Improved culture conditions for somat-
ic embryogenesis using an aseptic ventilative filter in eggplant
(Solanum melongena L.). Plant Sci 102:205–211
Saxena PK, Malik CA, Gill R (1992) Plantlet formation from isolat-
ed protoplasts to Solanum melongena. Planta 187:421–424
Sharma P, Rajam MV (1995) Genotype, explant and position effects
on organogenesis and embryogenesis in eggplant (Solanum me-
longena). J Exp Bot 46:135–141
Suezawa K, Matsuta N, Omura M, Yamaki S (1988) Plantlet forma-
tion from cell suspensions of kiwifruit (Actinidia chinensis
Planch, var. chinensis). Sci Hort 37:123–128
Wang SY, Steffens GL, Faust M (1986) Breaking bud dormancy in
apple with a plant bioregulator, thidiazuron. Phytochemistry
25:311–317