Protoplasma (2000) 211:51-63
PROTOPLASMA
Organogenesis from Solanum melongena L. (eggplant) cotyledon explants is
associated with hormone-modulated enhancement of polyamine biosynthesis
and conjugation
V. Scoccianti 1,,, E. Sgarbi 2, D. Fraternale 1, and S. Biondi 3
1Istituto Botanico, Universit~ di Urbino, Urbino, : Dipartimento di Biologia, Sezione Orto Botanico, Universitg di Modena e Reggio
Emilia, Modena, and 3Dipartimento di Biologia, Universit~ di Bologna, Bologna
Received May 18, 1999
Accepted September 16, 1999
Summary. Eggplant (Solanum melongena L. cv. Violetta lunga 2)
cotyledon explants grown on hormone-free medium (controls) or
on medium containing either naphthaleneacetic acid alone (root
forming) or in combination with zeatin riboside (shoot forming)
showed minor differences in free polyamine titres during culture.
In contrast, conjugated polyamines (particularly those in the
trichloroacetic acid-soluble fraction) accumulated only in hormone-
treated explants, but not in controls. The extent and the temporal
changes in soluble-conjugate levels differed between root-forming
and shoot-forming explants; in the former, accumulation began
earlier (within 1 day of culture) and reached the highest levels. In
both organogenic programmes, maximum conjugate accumulation
occurred just before and during organ emergence. Adventitious
roots and shoots were formed along the cut surfaces. The regions
closest to these ("borders") displayed a significantly higher ratio
of conjugated to free spermidine and/or putrescine than the
nonorganogenic regions ("centres") of the explant. Ornithine decar-
boxylase activity was higher than arginine decarboxylase activity
both in control and hormone-treated explants. However, both activ-
ities increased markedly on day 2 of culture in the presence of hor-
mones. Thereafter ornithine decarboxylase activity remained high in
shoot-forming explants, but not in root-forming ones. Putrescine
oxidising activity was also enhanced by exogenously supplied hor-
mones starting from day 4 of culture. This activity remained high up
to day 12 in the presence of auxin plus cytokinin, whereas it peaked
on day 6 in auxin-treated explants. Spermidine oxidising activity
was the only enzyme activity which was consistently higher in con-
trols than in hormone-treated tissue. Differences between the
two organogenic programmes with respect to temporal changes in
polyamine content, and putrescine biosynthetic and oxidative activ-
ities are discussed in relation to the timing of organ formation. The
latter was monitored both histologically and macroscopically.
Keywords: Solanum melongena; Polyamines; Organogenesis;
Arginine decarboxylase; Ornithine decarboxylase; Amine oxidases.
* Correspondence and reprints: Istituto di Botanica, Universit5 di
Urbino, Via Bramante 28, 1-61029 Urbino, Italy.
9 Springer-Verlag 2000
Printed in Austria
Abbreviations: PA polyamine; Put putrescine; Spd spermidine; Spm
spermine; NAA naphthaleneacetic acid; ZR zeatin riboside; TCA
trichloroacetic acid; ODC ornithine decarboxylase; ADC arginine
decarboxylase.
Introduction
Putrescine (Put), spermidine (Spd), and spermine
(Spm), the m o s t c o m m o n aliphatic p o l y a m i n e s (PAs)
in plants, are implicated in m a n y g r o w t h and develop-
m e n t a l processes and have thus b e e n p r o p o s e d as a
n e w class of plant g r o w t h regulators. This is b e c a u s e
altered levels of PAs and activities of their biosynthetic
e n z y m e s have b e e n associated with changes in g r o w t h
and d e v e l o p m e n t in n u m e r o u s plants ( E v a n s and
M a l m b e r g 1989, Torrigiani 1996). Results concerning
the effects of e x o g e n o u s l y supplied PAs or specific
inhibitors of P A biosynthetic e n z y m e s have led to
similar conclusions. The class of e n z y m e s involved in
P A catabolism (diamine and p o l y a m i n e oxidases) and
the p r o d u c t s of their activity also play an i m p o r t a n t
physiological role in plants (Federico and Angelini
1991). I n the last ten years, s o m e researchers have
emphasised the i m p o r t a n c e of t r i c h l o r o a c e t i c ( T C A ) -
or perchloric acid-soluble c o n j u g a t e d PAs. These are
primarily h y d r o x y c i n n a m i c acid amides (Flores and
M a r t i n - T a n g u y 1991), which are particularly a b u n d a n t
in the r e p r o d u c t i v e organs of t o b a c c o ( C a b a n n e et al.
1981) and in t o b a c c o plants infected with t o b a c c o
mosaic virus (Martin-Tanguy 1985, Torrigiani et al.
1997), suggesting a role for these c o m p o u n d s in repro-
duction, disease resistance, and stress.
52 V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis
M a n y studies have also addressed the question of
P A i n v o l v e m e n t during in vitro morphogenesis, b o t h
organogenesis and e m b r y o g e n e s i s (e.g., Fienberg et al.
1984; Torrigiani et al. 1987, 1989; Tiburcio et al. 1988).
U n d e r s t a n d i n g the role of PAs in such d e v e l o p m e n t a l
processes is h a m p e r e d by the scarcity of m u t a n t s in P A
m e t a b o l i s m and action; on the o t h e r hand, n e w insight
is only recently b e c o m i n g possible with plants trans-
genic for the P A biosynthetic genes ( K u m a r et al.
1997). O t h e r a p p r o a c h e s essentially rely on the use of
P A biosynthesis inhibitors, application of e x o g e n o u s
PAs, and correlative data on t e m p o r a l and spatial
changes in P A levels associated with the different
phases of morphogenesis. I n a few cases, r e g e n e r a t i n g
and n o n r e g e n e r a t i n g explants of the same species
( A r i b a u d et al. 1994, M a r t i n - T a n g u y et al. 1988), or
even tissues of the same explant ( L e s h e m et al. 1991,
P e d r o s o etal. 1997), have b e e n c o m p a r a t i v e l y
analysed. G i v e n that m a n y different experimental
systems have b e e n used (different species, tissues,
physiological stages, h o r m o n a l treatments, and m o r -
p h o g e n i c p r o g r a m m e s ) , no clear picture e m e r g e s f r o m
the m y r i a d data available. A further complication
arises f r o m the fact that n o t all studies have t a k e n con-
jugated PAs into consideration.
In the present study, cellular levels of free and
conjugated PAs, the activity of the two key e n z y m e s
responsible for Put biosynthesis (arginine and
ornithine decarboxylase), and Put and Spd oxidative
activity were analysed in c o t y l e d o n explants of
S o l a n u m m e l o n g e n a L. cultured in the absence
( n o n o r g a n o g e n i c controls) or in the p r e s e n c e of
e x o g e n o u s h o r m o n e s - auxin alone or auxin plus
cytokinin - which induce r o o t or shoot formation,
respectively. The d e v e l o p m e n t a l s e q u e n c e and the
effect of treating explants with e x o g e n o u s Put or Spd
were also investigated.
The m o s t striking difference b e t w e e n control and
h o r m o n e - t r e a t e d explants consisted in the rapid and
consistent a c c u m u l a t i o n of soluble conjugated PAs
occurring in the latter but n o t in the former. This pos-
itive correlation, whose significance is still unclear,
b e t w e e n high soluble-conjugate levels and organised
g r o w t h was further s u p p o r t e d by the fact that the
explant portions directly involved in the o r g a n o g e n i c
process ( b o r d e r regions) generally displayed an
observably higher ratio of conjugated to free PAs than
the n o n o r g a n o g e n i c centres. Differences b e t w e e n the
two o r g a n o g e n i c p r o g r a m m e s with respect to the time
course of P A c o n t e n t as well as Put biosynthetic and
oxidative activity a p p e a r to be related to the different
timing of organ formation.
Material and methods
Plant material
Commercial seeds of eggplant (Solanum melongena L. cv. Violetta
lunga 2) were sterilised in a 5% sodium hypochlorite solution for
30 rain under vacuum, washed five times in sterile water and sown
on half-strength MS medium (Murashige and Skoog 1962) supple-
mented with 10 g of sucrose per litre and 0.8% agar. Seeds were
allowed to germinate at 26 + 1 ~ under a 16 h photoperiod.
In vitro culture conditions
Cotyledons were excised from 19-day-old seedlings, sectioned trans-
versely into three parts, and placed on MS medium containing 30 g
of sucrose per litre, 0.8% agar, and different hormonal conditions
as follows: (a) hormone-free, (b) 10-6 M naphthaleneacetic acid
(NAA), (c) 2 • 10 8M NAA plus 7 • 10-7 M zeatin riboside (ZR). In
some experiments, polyamines (Put, Spd) or 13-OH-ethylhydrazine
were added to the media. The cultures were incubated at 26 • 1 ~
under a 16 h photoperiod with cool white fluorescent lamps (Philips
TLD 36W/33; irradiance, 50 gmol/m<s).
Histological analysis
Cotyledon explants at the time of excision (day 0) and after 4, 6, 8,
10, and 12 days in culture on hormone-free, auxin-, or auxin- and
cytokinin-containing media were collected. The border regions of
each explant were excised and fixed in 3 % glutaraldehyde in 50 mM
phosphate buffer, pH 7, postfixed with aqueous 1% OsO~, dehy-
drated in a graded acetone series and embedded in Durcupan ACM
(Fluka Chemic AG, Buchs, Switzerland). Each step was conducted
in a microwave oven according to Gutmann and Feucht (1991).
Cross sections, 1.5 gm thick, parallel to the cut surface of the explant,
were cut with a Reichert Jung Ultracut E microtome, stained with
toluidine blue, and observed and photographed under a Leitz
Orthoplan microscope.
Polyamine analysis
At 0, 1, 2, 4, 6, 8, 10, and 12 days in culture, a 2 mm wide strip was
excised parallel to the two cut ends of each explant. These strips
("borders") and the remaining parts ("centres") of the explants
were weighed separately, immediately frozen in liquid nitrogen, and
kept at -80 ~ until use. Tissue was extracted in 3 volumes of 5%
(w/v) TCA. After centrifugation (15000 g, 15 rain), aliquots of the
supernatant were dansylated and analysed by thin-layer chromato-
graphy on precoated Silicagel 60 plates (Merck) with cyclohexane-
ethylacetate (3 : 2, v/v) as solvent. Spots comigrating with standard
Put, Spd, and Spin were scraped off, eluted with 2 ml of acetone
and their relative fluorescence measured (excitation wavelength,
360 nm; emission wavelength, 505.5 nm) with a Jasco FP-770 spec-
trofluorometer (Torrigiani et al. 1987). Aliquots of the TCA super-
natant and pellet were hydrolysed with 6 N HC1 at 110 ~ overnight
in order to release and then determine, following the same procedure
as described above, TCA-soluble and -insoluble conjugated PAs.
ADC and ODC activity assays
Samples (300-500 rag) were homogenised on ice in 5 volumes of
100 mM Tris-HC1, pH 8.5, containing 50 gM pyridoxalphosphate,
V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis
and centrifuged at 18000 g for 30 min at 4 ~ Ornithine decarboxy-
lase (ODC) and arginine decarboxylase (ADC) activity assays were
performed essentially as described by Bagni et al. (1983). They
consisted in measuring the ~4CO2 evolution from 7.4kBq of
L-[1-14C]ornithine (specific activity, 2.11 GBq/mmol; Amersham,
Milano, Italy) or DL-[U54C]arginine (specific activity, 11 GBq/
mmol; Amersham), respectively after a 2 h incubation at 37 ~ of
aliquots of the supernatant.
Oxidative activity assay
Oxidative activity was assayed by a radiometric method that mea-
sures Aa-[14C]pyrroline and 1,5-[14C]diazabicyclononane formation
from [a4C]putrescine and [~4C]spermidine, respectively. Explants
were homogenised in 3 volumes of 100 mM potassium phosphate
buffer, pH 8, containing 2 mM dithiothreitol, and centrifuged at
18000 g for 30 min. Aliquots of the supernatant and resuspended
pellet were pre-incubated separately at room temperature for
45 rain and then incubated at 37 ~ for 30 min with 7.4kBq
[14C]putrescine (specific activity 4.37 GBq/mmol; Amersham) or
[a4C]spermidine (specific activity, 4.07 GBq/mmol; Amersham),
22.5 gg of catalase, and 10 mM unlabelled putrescine or spermidine.
After stopping the reaction with 2% Na2COB, the a4C-labelled prod-
ucts were extracted in 2 ml of toluene; a 0.5 ml aliquot of the solvent
was then placed in scintillation cocktail (Ultima Gold; Canberra
Packard, Groningen, the Netherlands) before radioactivity was mea-
sured in a scintillation counter (LS 1800; Beckman).
Statistical analysis
Each experiment was performed at least twice. Differences between
means of number of roots per explant or explant fresh weight were
analysed by Student's t-test.
Results
Morphogenesis and developmental sequence
On hormone-free medium, cotyledon explants re-
mained green and healthy for approximately 12-14
days, but never regenerated any organs nor formed
any visible callus. On the contrary, in both hormonal
treatments 90-100% of the explants exhibited an
organogenic response. On medium containing N A A
alone, adventitious roots were formed along the entire
basal cut surface together with some friable callus. The
first roots emerged after 6-8 days in culture with a
mean number of 7.8 + 1.3 roots per explant on day 9.
On medium containing N A A plus ZR, adventitious
buds were formed. Primordia became visible from
around day 10 in culture, initially along the basal cut
surface but later also along the opposite (distal) one.
There was some callus formation all around the
explant.
The general histology of the explants at day 0 is
shown in Fig. 1 a. In cross section cotyledons display
an upper and lower epidermis with stomata and 8
layers of mesophyll (palisade and spongy) consisting
53
of highly vacuolised cells with small intercellular
spaces.
After 4 days in culture on hormone-free medium
(Fig. i b), an increase in intercellular spaces and abun-
dant starch accumulation were observed in both the
palisade and spongy mesophyll. Later on these
explants lost the normal foliar anatomy and appeared
more disorderly. Larger intercellular spaces and starch
accumulation also occurred in hormone-treated
explants; these modifications increased in intensity
during culture, particularly in shoot-forming explants.
In the latter, the first cell divisions were initially
observed within the spongy mesophyll in close prox-
imity to the minor veins (Fig. lc). Later (day 6),
parenchyma cells increased in size, and well-defined
aggregates of dividing cells appeared (Fig. 1 d). These
gave rise asynchronously to differently sized meriste-
moids in the central zone of the mesophyll (Fig. i e).
Shoot-forming explants also formed a conspicuous
amount of callus, leading to the disruption of both epi-
dermal layers (Fig. I f).
In root-forming explants, diffuse meristematic activ-
ity was evident by day 4 (Fig. 2 a); well-defined clusters
of dividing cells occurred in the subepidermal layer
and around the veins, particularly involving the bundle
sheath cells (Fig. 2b). These aggregates either formed
strands, as a result of periclinal divisions, or were glob-
ular (Fig. 2c). By day 6, explants grown on medium
containing N A A alone had well-developed meriste-
moids that soon differentiated into root primordia and
roots (Fig. 2d).
Free- and conjugated-poIyamine levels
At the time of excision (day 0), the border regions of
explants contained a large amount of free Put (Figs.
3-5). This was by far the predominant PA, followed by
Spd and Spm (Fig. 5). Conjugated PAs in the TCA-
soluble fraction were absent.
In control explants grown on hormone-free
medium, free Put titres remained fairly constant
throughout culture (Fig. 3). On the contrary, Spd levels
fell abruptly on day i (down to 42% of the day 0
value), continued to decline up to a minimum on day
6, and remained low thereafter. Spm, which was
present only at time 0 at a concentration of about
14 nmol/g fresh weight, later disappeared; soluble con-
jugates were never detectable.
In the organogenic tissue slices ("borders") of
root-forming explants (Fig. 4A), free Put declined
54 V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis
V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis 55

gradually during culture, reaching one third of the
day 0 value on day 12. Spd decreased rapidly during
the first 4 days, reaching a minimum on day 6 (Fig.
4B). Later, it increased up to 2 or 3 times this level
but still remained well below the initial value. After
day 0, spermine was no longer detectable, The most
dramatic change occurring in the PA pattern of rhizo-
genic explants was the appearance of TCA-soluble
conjugated Put and Spd already after 1 day in culture
(Fig. 4A, B). This was followed by a large and rapid
increase in these conjugates; maximum accumulation
of conjugated Put occurred between days 4 and 8,
while conjugated Spd peaked on day 4 but then grad-
ually declined, so that by day 12 it was no longer
detectable. TCA-insolnble conjugated Put, which also
appeared on day 1, was always less than the free or
soluble conjugated form; its levels were fairly stable
with a slight increase between days 6 and 10 (Fig. 4A).
On shoot-inducing medium, the organogenic
borders exhibited similar changes in the pattern of free
amines; thus, Put levels underwent a gradual decline
(Fig. 5A), while Spd decreased abruptly during the
first 4 days when it reached a minimum value which
persisted up to day 12 (Fig. 5B). Unlike rhizogenic
explants, in shoot-forming explants free Spm was
present throughout culture without significant varia-
tions, while soluble conjugated Spin exhibited a large
peak on day 2 (Fig. 5C). Like the rhizogenic ones,
shoot-forming explants also accumulated conspicuous
amounts of TCA-soluble conjugated Put which, how-
ever, appeared only from day 4 with a first peak on day
6 followed by a second, larger increase on day 12.
These explants did not at any time accumulate as
much of this conjugate as did root-forming ones. Con-
jugated Spd also appeared later (day 6), practically
disappeared again on day 8, and only occurred in rel-
evant amounts on day 12 (Fig. 5 B). Later on (day 17),
it declined to values comparable to those of day 6
(Table 1). Also in these exptants, the only amine
present in the TCA-insoluble fraction was Put. It was
detectable from day 1, increased up to day 6, and after
day 10 tended to disappear again (Fig. 5 A).
A comparative analysis of free-PA levels in
organogenic borders versus nonorganogenic centres of
shoot-forming explants indicated that on days 4 and
12 borders had somewhat higher levels of Put (ca.
twofold) than centres, and that Spd and Spin were
not significantly different (Table 1). Later in culture

Table L Free and TCA-soiuble conjugated PA content in organogenic borders and

non0rganogenic centres of shoot-forming explants of S. melongena at different times in
cultures

Time ~ i f i d Concentration (nmol/g fresh weight) of PAs"

region
free soluble conjugated

Put Spd Spm PUt Spd Spm

Day 4
Border 333 _+60 55 + 9 19 _+3 228 _+43 traces 15 _+3
Centres 182 _+8 73 _+3 16 _+1 46 _+2 traces 34 + 6
Day 12
Borders 223 _+40 75 _+10 14 +_3 409 _+80 traces 6 _+1
Centres 106 -+ 12 80 + 13 8+2 134 _+4 traces 24 _+8
Day 17
Borders 62_+9 30+4 10_+2 368+_73 15-+2 48-+9
Centres 67_+3 16_+1 9-+1 141_+8 11_+2 22_+3

Values are means with standard deviation

Fig. l a - f . Histology of X raelongena cotyledon exptants during in vitro culture; transverse sections stained with toluidine blue. a Cotyle-
don from a 20-day-old seedling at the time of excision (day 0). x220. b Explant after 4 days on hormone-free medium showing starch accu-
mulation and large intercellular spaces, x220. e Explant after 4 days in culture on medium supplemented with NAA and Z R showing cell
divisions near the minor veins (arrow). x160. d Shoot-forming explant showing dusters of dividing cells (arrows). x160. e Meristemoids at
day 8 (x160), and f callus proliferation disrupting the two epidermal layers (x160)
56 V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis

Fig. 2a-d. Histology of root-forming S. melongena cotyledon explants during in vitro culture; transverse sections stained with toluidine
blue. a and h Explant at day 4 showing areas of meristematic activity (a, x160) and strands of dividing cells in a perivascular position (short
arrows) and in subepidermal layers (long arrow). Starch accumulation is evident in adaxial layers (b, x200), c Globular meristemoids in
subepidermal layers (x160). d Root primordia in 6-day-old explant (x160)
V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis 57

1000 800

800
600
t/.
600 u.

o 400 r 400
E
i-
0
E
200 e..
2O0
0 i I T i 9 i

0 2 4 6 8 10 12 0 I m I I I I I

Time (days) 200

Fig. 3. Temporal changes in free Put (m) and Spd (0) levels in
B
border regions of S. melongena cotyledon explants during culture on 150
hormone-free medium I..k
"T,
100
"6
E
1000 " 50
A
800
50
" 600 C
40
0 U.
E 400 "7,
30
{-

"6 20
200 E
e-
10
"P I I I I I
0
200 0 2 4 6 8 10 12
Time (days)

150 Fig. 5. Temporal changes in free (e) and soluble (m) and insoluble
(&) conjugated Put (A), Spd (B), and Spm (C) levels in border
n regions of S. melongena cotyledon explants during culture on shoot-
"7, inducing medium
m
100
O
E
(day 17), both regions had similar Put and Spm titres,
50 while borders had more Spd than centres. Even more
striking differences between borders and centres were
observed with respect to TCA-soluble conjugated-Put
0 I I I I I "P"

levels (Table 1). In 4-day-old shoot-forming explants,

0 2 4 6 8 10 12
the former had 5-fold higher levels of this conjugate
Time (days) than the latter. Smaller yet consistent differences
Fig. 4. Temporal changes in free (e) and soluble (m) and insoluble were also observed in 12- and 17-day-old explants.
(A) conjugated Put (A) and Spd (B) levels in border regions of Consequently, the ratio of conjugated to free Put dif-
S. melongena cotyledon explants during culture on root-inducing fered markedly between borders and centres, and
medium
increased with time in culture: 0.7, 1.8, and 5.9 in
borders, 0.25, 1.3, and 2.1 in centres on days 4, 12, and
17, respectively.
58 V. Scocciantiet al.: Organogenesisfrom eggplantexplants and polyaminebiosynthesis

Table 2. Ratio of conjugatedto free PA in organogenicborders and 2000

nonorganogenic centres of S. melongena cotyledon explants at dif- "T.
e- A
ferent times on root-inducingmedium ~ 1500

Day Put Spd 1000

O1
Borders Centres Borders Centres
500
E
1
2
4
0.3
0.3
2.0
0.2
0.2
0.0
0.8
1.8
7.4
0.9
0.5
0.5
e~

i i
Zl
6 1.8 1.1 5.3 0.5 2000-
8 1.7 0.5 1.9 0.2
e- B I
r 1500

U.
1000
01
As shown in Table 2, the ratio of soluble conjugated 500
to free Put was always higher in borders than in e~

centres also in root-forming explants, especially from i I

d a y 4 onwards. Furthermore, conjugated Spd in

borders prevailed (about 2-fold) over the free form
starting from day 2, but never in centres where this
conjugate was scarce.
A D C and O D C activities
O D C activity was much higher (7- to 10-fold) than
A D C activity at the time of excision and throughout
culture in all three treatments. In control explants,
neither activity changed significantly during culture
and the rather low values of day 0 were maintained
throughout (Fig. 6). In contrast, both activities in-
creased after 2 days in hormone-treated explants. In
shoot-forming explants (Fig. 6), ODC activity contin-
ued to rise up to day 6 and remained high thereafter,
while root-forming explants exhibited a significantly
lower activity on days 6 and 12 compared to that of
day 2 (Fig. 6). A D C activity followed a similar pattern
to that of O D C in both organogenic programmes.
Put and S p m oxidation
Put and Spd oxidative activities were measured in
both the supernatant and the pellet with the former
prevailing over the latter in most cases; particulate
activity was especially abundant on days 4 and 6 in
hormone-treated explants (Fig. 7). Total (supernatant
plus pellet) diamine (Put) oxidase activity displayed
only minor variations during time in culture in control
explants (Fig. 7). On the contrary, this activity in-
creased significantly on day 4 in caulogenic and on
day 6 in rhizogenic explants (Fig. 7). In the former, it
20O0
C
1500
[,K
1000
01
o 50O
E
o.
0 2 6 12
T i m e (days)
Fig. 6. Time course of ADC (grey column) and ODC (white
column) activities in control (A), shoot-forming (B), and root-
forming (C) cotyledonexplants of S. melongena during culture
remained high up to day 12, whereas in the latter it
tended to decline.
The pattern of Spd oxidation was markedly differ-
ent: it was very low on day 0 and then increased dra-
matically only in control explants where it peaked on
day 2 and remained well above day 0 values up to day
6 (Fig. 8). In hormone-treated explants, it increased
significantly only on days 2 and 4 in root-forming and
shoot-forming explants, respectively (Fig. 8).
Treatment with exogenous Spd, Put or diamine
oxidase inhibitor
Exogenous Spd supplied at physiological concentra-
tion (equivalent to the endogenous level in day 0
explants) exerted a deleterious effect on rhizogenesis
in terms of both the percentage of organogenic
V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis 59

300 250
"T, c
e,"
A 'E 2 0 0 A
200 15o
I.I.
1.1., 9r, 100
"7
100 50
O
E
c
E
" 0 I I
n
0
9,re-, 2 5 0
300
"E 2 0 0 B
"7, B T
~

E -T..,.
,.i- 15o
1"
200 I,,I..
9r, 100
O'J

I.I.
i!~i!~i~i!i!il 50
% 100 E
= 0
ili!i i ii/!!i .......
E 9";f..,, 2 5 0

0 i i | i = a "~= 2 0 0
C

'.r, 300 15o

e.. I,.I.
C 100
i.o
200 '~ 50
E
!.1.. " 0
.7
100 0 1 2 4 6
o
E Time (days)
c
0 i
R ! i !
i!iii i i Fig. 8. Time course of soluble (grey column) and particulate (white
column) Spd oxidative activity in control (A), shoot-forming (B),
0 1 2 4 6 8 12
and root-forming (C) cotyledon explants of S. rnelongena during
Time (days) culture

Fig. 7. Time course of soluble (grey column) and particulate (white

column) Put oxidative activity in control (A), shoot-forming (B), Table 3. Organogenic response and fresh weight of S. rnelongena
and root-forming (C) cotyledon explants of S. melongena during cotyledon explants after 9 days in culture on root-inducing medium
culture (control) supplemented with 0.4 mM spermidine starting from day
0or5

Treatment % organogenic No. roots/ Explant fresh

explants and the number of roots per explant (Table exptants explant weight (mg) a
3). This negative influence increased when the PA was
Root-inducing 87.2 5.7 51.6 + 21 (100)
supplied at a critical point of organ formation (day 5). (controls)
Conversely, in the presence of NAA plus ZR, exoge- + Spd day 0 70.9 5.1 40.9 _+12 (79)
nous Spd did not significantly affect the percentage of + Spd day 5 52.7 3.9 34.4 + 10"* (67)
organogenic explants (data not shown), but it acceler- aValues are means with standard error; ** P _<0.01 for significant
ated primordia development into shoots (Fig. 9) and difference from controls. In parentheses, percent of controls
caused a significant fresh-weight increment: 123 + 21
and 181 + 44 mg per explant for controls and explants
Discussion
treated with Spd from day 5 onwards, respectively.
An exogenous supply of Put also inhibited rhizo- Results presented here indicate that free amines in
genesis as did treatment with the diamine oxidase eggplant cotyledon explants essentially underwent a
(DAO) inhibitor [~-OH-ethylhydrazine (Table 4). constant and gradual decrease with time in culture
60 V. Scocciantiet al.: Organogenesis from eggplant explants and polyamine biosynthesis
A B C D
Fig. 9. Cotyledon explants after 18 days in culture on shoot-forming
medium without (A) or with 0.4 mM Spd supplied from day 0 (B),
day 2 (C), or day 5 (D)
irrespective of the hormonal treatment and of the
cotyledon portion (borders or centres). Lack of mean-
ingful differences were also reported between free-PA
levels in organogenic (basal end) and nonorganogenic
(middle) tissues of excised melon cotyledons (Leshem
et al. 1991), and between shoot-forming and non-
shoot-forming Pinus radiata cotyledons (Kumar and
Thorpe 1989). Other types of explants (e.g., tobacco
thin layers) respond, upon introduction in culture, with
an increase in PA titres (Tiburcio et al. 1989;Torrigiani
et al. 1987, 1989); this is determined by hormonal
treatment and presumably raises them to the levels
required for active cell division and organised growth.
The decrease in free PAs observed by us during culture
is probably justified by the fact that, different from
other types of explants, eggplant cotyledons, as
reported for other seedling portions (Fobert and Webb
1988, Kumar and Thorpe 1989, Sharma and Rajam
1995), have very high levels of free PAs at the time of
excision, possibly because they are a storage form of
nitrogen. These levels are probably sufficient to sustain
any form of growth and development, including organ
induction, and are presumably even in excess.
Since, as also supported by present data on A D C
and O D C activities, treatment with exogenous hor-
mones and growth activation in general appears to
represent a strong stimulus towards PA biosynthesis or
at least prevents their depletion (Fobert and Webb
1988), a further increase in the free-amine pool may
have to be counterbalanced by conjugation and oxi-
dation as a means of reducing the active (free) pool of
PAs. This raises the question as to whether or not
soluble conjugated PAs should be considered merely
as secondary metabolites or if, as postulated by some
investigators (Martin-Tanguy 1985), they have a func-
tion in growth regulation. On medium supplemented
with HCAs, proliferative growth of tobacco leaf discs
was enhanced, whereas bud formation was inhibited
(Martin et al. 1985). In Helianthus tuberosus tuber
explants and tobacco thin cell layers, Bagni et al.
Table4. Organogenic response and fresh weight on day 9 of
cotyledon explants of S. rnelongena cultured on root-inducing
medium (control) supplemented with 1 mM Put or with [3-OH-
ethylhydrazine
Treatment % organogenic No. roots/ Explant fresh
explants explant" weight(rag)"
Root-inducing 100 9.9 _+1.2 68.3 +_21 (100)b
(control)
+ Put 64 2.5 + 0.6** 36.7 -+19"* (57.5)
+ DAO
inhibitor
0.5 mM 52 1.9 + 0.5** 34.0 + 16"* (53.3)
2 mM 29 0.8 +_0.6** 26.0 _+11"* (40.7)
a Values are means with standard error; ** P _<0.01 for significant
difference from controls
bIn parentheses, percent of controls
(1994) observed that addition of coumaroylPut had no
significant effect on growth of the former, and strongly
inhibited both bud and callus formation in the latter.
In fact, in tobacco explants, overaccumulation of
soluble conjugated PAs resulting from treatment with
methylglyoxal bis(guanylhydrazone) is strongly dele-
terious to meristemoid and bud primordia formation
(Scaramagli et al. 1999); Wyss-Benz et al. (1990) also
concluded that HCAs do not trigger growth and dif-
ferentiation. Therefore, as long as the function of
HCAs remains unclear, their accumulation in cultured
tissues may be interpreted as a side effect of hormone-
stimulated free-PA production.
On the other hand, results of the present work indi-
cate clearly that PA levels in the soluble conjugated
fraction exhibit marked changes in relation to hor-
monal treatment, the time course of organogenesis,
and organogenic potential of different explant zones.
In tobacco leaf explants, irrespective of the hormonal
treatment, free and conjugated PAs peaked on days
7-10 (Martin-Tanguy et al. 1988). Nonetheless, soluble
conjugated Put titres decreased in this order: 2,4-
dichlorophenoxyacetic acid (2,4-D) alone > 2,4-D plus
benzyladenine (BA) > BA > hormone free. Similar
results were obtained by us even though the auxin and
cytokinin used were different. In leaf explants of
Crysanthernurn rnorifolium, conjugates also appeared
on all hormone-supplemented media after 2 days in
culture and levels were strongly influenced by the hor-
monal treatment (Aribaud et al. 1994). In particular,
both Put and Spd conjugates accumulated to much
higher levels in the presence of indoleacetic acid (root-
forming medium) compared with BA (shoot-forming
v. Scocciantiet al.: Organogenesis from eggplant explants and polyamine biosynthesis
medium). These data are in agreement with present
results. In fact, although conjugated Put increased
sharply starting from day 2 in culture both on NAA-
and NAA-plus-ZR-containing medium, the maximum
levels reached were higher in the former than in the
latter (700 and 320 nmol/g fresh weight, respectively),
suggesting that auxin may play an important role in
causing a rapid induction of this accumulation (also
in relation to the concentration used). The auxin-
plus-cytokinin treatment resulted in accumulation of
conjugated Put (almost 600 nmol/g fresh weight) com-
parable to that induced by auxin alone only later in
culture (day 12). These temporal changes can be cor-
related with the timing of the organogenetic process
which is faster for roots than shoots. In both cases, the
sharp rise in conjugated Put and Spd occurs before
the first roots or buds become visible. Furthermore,
the late rise in Put conjugates in explants treated with
N A A plus Z R could be attributed to the presence of
buds whose putative auxin-synthesising capacity may
have enhanced these levels.
A positive correlation was reported between the
spatial distribution of free and conjugated forms of
Put, Spd, and Spm and the embryogenic capacity of
different regions of the leaves (Yadav and Rajam
1997) or of the hypocotyls of S. melongena seedlings
(Sharma and Rajam 1995). In fact, the difference
between apical and basal leaf discs was much more
marked with respect to conjugates. In embryogenic
(margins) and nonembryogenic (central) regions of
Camellia japonica leaf explants, free and conjugated
PAs increased significantly after the induction of
embryogenesis. Later in culture, free PAs decreased
in both regions, while soluble conjugated ones (and
insoluble conjugated Put) increased significantly in
embryogenic but not in nonembryogenic regions
(Pedroso et al. 1997).
Despite the presence of conjugates only i n
hormone-treated eggplant cotyledon explants, the
consistently higher ratios of conjugated to free Put and
Spd (the latter only in root-forming explants) which
characterise the organogenic tissues compared with
the nonresponsive parts of the explant indicate that
overall hormonal treatment alone does not account
for conjugate accumulation. The spatial relationship
suggests that local events occurring at the cut ends are
also involved in the induction of this accumulation.
The extent to which localised changes in hormone
levels (uptake and metabolism) regulate conjugate
accumulation requires further studies. Furthermore,
61
since in the absence of exogenously supplied hor-
mones there was no accumulation of conjugates, this
also shows that the stress imposed by excision and in
vitro culture per se is not sufficient to evoke this
response.
Yadav and Rajam (1998) reported that basal leaf
disc portions of S. melongena pretreated with Put
showed improved embryogenesis that was comparable
to that of apical portions. We attempted to further
enhance the organogenic response by supplying
exogenous Put either from the start of culture (day 0)
or from the time when cellular levels of free Put had
declined to half of day 0 values (day 5). Such treat-
ments, however, inhibited the rhizogenic response, sug-
gesting that they lead to supraoptimal levels of free
and/or conjugated forms of the diamine itself or of the
PAs derived from it. With the aim of maintaining high
levels of Put, eggplant explants grown on rooting
medium were also treated with [3-OH-ethylhydrazine,
an inhibitor of DAO activity. In Crysanthemum leaf
discs induced to form buds (Aribaud et al. 1994) or
roots (Martin-Tanguy et al. 1997), addition of 2 mM [3-
OH-ethylhydrazine markedly enhanced Put content
and blocked organogenesis totally. The latter authors
suggested that DAO-mediated Put catabolism is
involved in root induction in this material. In our expe-
rience, treatment with this inhibitor was highly delete-
rious towards rhizogenesis and explant growth; effects
were only slightly reverted by addition of 1 mM Put
(data not shown), and thus difficult to interpret as a
specific response to altered Put metabolism.
Some authors have provided correlative evidence
for the positive involvement of Spd in the initiation of
adventitious buds (Bagni et al. 1993, Tanimoto et al.
1994, Aribaud et al. 1994). Also in the present work,
free-Spd levels in caulogenic explants were always
higher than in rhizogenic ones. This positive correla-
tion is supported by results when explants were
treated with exogenous Spd. In fact, root formation
was strongly inhibited by the presence of this
polyamine, whereas overall growth and shoot devel-
opment in explants treated with N A A plus Z R were
favoured by exogenous Spd. Higher precursor (Spd)
availability could at least partly account for the pres-
ence of free and soluble conjugated Spm in caulogenic
explants.
As previously reported for some in vitro cultured
plant tissues (Aribaud et al. 1994, Altabella et al. 1995)
but not others (Tiburcio et al. 1988, Feirer et al. 1984),
ODC markedly prevailed over A D C activity, the
62 V. Scoccianti et al.: Organogenesis from eggplant explants and polyamine biosynthesis
former considered the enzyme associated with cell
division (Cohen etal. 1982). Both activities were
equally enhanced by the presence of hormones and
started at the same time (day 2). Put oxidising activity
(DAO) in eggplant cotyledon explants coincided with
m a x i m u m A D C and O D C activities in shoot-forming
explants resulting in high and stable free-Put content
between days 4 and 12. Thus, oxidation and biosyn-
thesis together seem to regulate cellular diamine
levels. In rooting explants, a peak in O D C activity cor-
related with the start of conjugated-Put accumulation
as evidence that it was required in order to supply the
precursor (free Put); on the other hand, D A O activity
increased transiently concomitantly with root emer-
gence. C o m p a r e d with controls, h o r m o n e - t r e a t e d
explants frequently had a larger proportion of partic-
ulate amine oxidase activity. Immunohistochemical
studies have provided evidence for the occurrence of
D A O in the cell walls of some plants (Federico et al.
1985) where it may be involved in the polymerisation
of lignin and suberin (Federico and Angelini 1991).
The almost total depletion of the Spd pool in control
explants correlated with the high Spd oxidation activ-
ity of these explants. A similar correlation is m o r e dif-
ficult to establish in explants treated with N A A or
N A A plus Z R where this activity peaked at slightly
different times in culture but whose m a x i m u m values
were quite similar. In fact, the pattern which emerges
with respect to all the enzyme activities assayed here
is that, except for Spd oxidising activity, these are
enhanced by exogenous h o r m o n e s and remain high
for a longer period of time in shoot-forming than in
root-forming explants, m a y b e in relation to the differ-
ent duration of the two organogenic processes.
In conclusion eggplant cotyledon explants are char-
acterised, at the time of excision, by very high contents
of free Put and Spd. Both amines tend to decrease
during culture both in the presence or absence of hor-
mones. Treatment with h o r m o n e s stimulates biosyn-
thesis of Put and, to some extent, its oxidation. Its main
fate, however, appears to be that of becoming conju-
gated to small molecules, probably hydroxycinnamic
acids, thus leading to a rapid and large accumulation
of this diamine in the TCA-soluble conjugate fraction.
Spd, whose biosynthesis is likely to be stimulated as
well, follows a similar fate. A relatively high auxin
concentration (root-forming explants) evokes this
response m o r e rapidly than a low concentration com-
bined with cytokinin (shoot-forming explants). Finally,
although organogenic border regions of the explants
exhibit a sensibly higher ratio of conjugated to free of
Put and Spd (the latter only in root-forming explants),
further studies are required before a possible role
can be assigned to hydroxycinnamic acid amides in
organogenic tissues.
Acknowledgments
The authors are grateful to R Torrigiani for fruitful discussions.This
work was partially supported by funds from MURST (ex-60%) to
V.S. and from the University of Bologna, special project "Molecular
Signals in Differentiation" to S.B.
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