In Vitro Cell. Dev. Biol.—Plant 38:260–267, May– June 2002 DOI: 10.

1079/IVP2001259
q 2002 Society for In Vitro Biology
1054-5476/02 $10.00+0.00

A NOVEL PATHWAY FOR RAPID SHOOT REGENERATION FROM THE PROXIMAL ZONE OF THE
HYPOCOTYL OF MELON (CUCUMIS MELO L.)

S. CURUK1,4†, C. ELMAN1, E. SCHLARMAN2, O. SAGEE2, I. SHOMER3, S. CETINER4‡, D. J. GRAY5, AND V. GABA1*

Departments of 1Virology, 2Citriculture and 3Food Science, ARO Volcani Center, POB 6, Bet Dagan 50250, Israel, 4Laboratory of Plant
Biotechnology, University of Cukorova, Adana, Turkey, 5Mid-Florida Research & Education Center, IFAS, University of Florida, 2725 Binion
Road, Apopka, FL 32703-8504

(Received 9 February 2001; accepted 12 October 2001; editor A. Pretová)

Summary
Hypocotyl explants of melon (Cucumis melo L.) are capable of regenerating multiple shoots on Murashige and Skoog
(1962) medium, augmented with 4.4 mM benzyladenine. Regeneration from the hypocotyl is much more rapid than the more
commonly reported regeneration from cotyledonary explants, producing shoots within 2 wk compared to more than a month
required for cotyledon explants. The rapid regeneration response depends on the presence of a fragment of the cotyledon
remaining attached to the hypocotyl. Controls were performed to ensure that the regeneration was not due to the presence of
the shoot apical bud of the melon seedling after explant production. Scanning electron microscopy revealed that
microsurgery to remove the apical bud left no excess bud material. Regeneration from the proximal part of the hypocotyl was
due to production of a new shoot apical meristem, observed by histology. The apical meristem can be produced before leaf
primordia in regeneration from the hypocotyl, in contrast to the regeneration process from the melon cotyledon.
Key words: organogenesis; bud regeneration; shoot apex; cotyledon; cucurbits.

Introduction
The Cucurbitaceae is an economically important family, including
species such as melon, cucumber, watermelon and squash. Recently,
genetic engineering has enabled improvement of this plant family by
introducing new genes for virus resistance to squash (Cucurbita
pepo ) (Tricoli et al., 1995) and melon (Cucumis melo L.) (Fuchs et al.,
1997). Regeneration of adventitious shoots in vitro is a critical stage
of genetic engineering. Organogenesis is the process by which new
shoots are created in vitro, by cell division and differentiation,
eventually leading to a new shoot apical meristem and subsequent
shoot elongation. Plant transformation processes often depend on the
pathway of regeneration of various tissues, whether the regeneration
route goes through a callus phase, or is ‘direct’ regeneration from the
explanted tissue, or is a type of axillary regeneration (see Draper
et al., 1988; Mukhopadhyay et al., 1992; George, 1993; Gaba et al.,
1996a).
Melon has been a popular cucurbit species for studies of in vitro
regeneration and genetic transformation. Buds and shoots have been
obtained in vitro directly from melon cotyledons (Dirks and van
Buggenum, 1989; Niedz et al., 1989) and leaves (Kathal et al., 1988;
Yadav et al., 1996), and indirectly from callus derived from
cotyledons (Moreno et al., 1985; Orts et al., 1987; Chee, 1991;
*Author to whom correspondence should be addressed: Email vpgaba@
volcani.agri.gov.il
†Current address: University of Mustafa Kemal, Faculty of Agriculture,
Department of Horticulture, 31034 Antakya-HATAY, Turkey.
‡Current address: Sabanci University, Faculty of Engineering and Natural
Sciences, 81474 Tuzla, Istanbul, Turkey.
Molina and Nuez, 1995) and hypocotyls (Kathal et al., 1986; Molina
and Nuez, 1995). Embryogenesis in melon directly from
cotyledonary explants (Oridate et al., 1992; Gray et al., 1993), or
from callus (Oridate and Oosawa, 1986), has also been reported.
Shoot multiplication from apical (Adelberg et al., 1993) or lateral
(Ohki et al., 1991) buds of melon was reported as forms of axillary
multiplication. Regeneration responses are genotype dependent
within this species (Orts et al., 1987; Oridate et al., 1992; Gray et al.,
1993). Genotype can have profound effects on processes dependent
on regeneration, such as micropropagation (George, 1993) and plant
genetic transformation, as is already known in melon (see Gaba et al.,
1996a) and many other species. For this reason it is important to
have information on a variety of different regeneration pathways to
enable manipulation of plant tissues to obtain the desired response
in vitro.
We report here a novel direct shoot regeneration response in
melon (Cucumis melo L.). Regeneration occurs from the proximal
zone of the hypocotyl, in the absence of the shoot apex, with a
fragment of the cotyledon attached. Morphological and anatomical
characteristics of the rapid regeneration response from the hypocotyl
are presented. To our knowledge this is the first report of direct in
vitro regeneration from the hypocotyl of Cucumis species.
Materials and Methods
Plant material and culture. Seeds of the Israeli melon (Cucumis melo L.)
cv. Revigal had the seed coats removed, were surface-sterilized for 20 min in
a solution of 1% sodium hypochlorite (with two drops of Tween 20 per 100 ml
solution), and washed four times with sterile distilled water. Seeds were
germinated in disposable 15 £ 90 mm diameter Petri dishes, on 25 ml MS

260
 REGENERATION FROM MELON HYPOCOTYL
(Murashige and Skoog, 1962) medium with 3% sucrose, MS vitamins and
5 g l21 Micro agar (Duchefa) or 8 g l21 TC Agar (JRH Biochemicals, Lenexa,
KS), at 25 ^ 18C; 16-h photoperiod, 30–40 mmol m22 s21 cool white
fluorescent light.
After 4 d explants were prepared. The hypocotyl was excised 0.25–1 mm
from the cotyledon, then the cotyledons were cut perpendicular to the main
axis and the distal parts discarded. The proximal part of the cotyledons was
stood on its hypocotyl stub, and a scalpel blade forced flat between the
proximal cotyledon remnants, separating them. The apical bud of the seedling
was removed using a stereomicroscope (‘de-budding’). This method produces
hypocotyl explants (Figs. 1a and 5c) consisting of 0.25–1 mm of hypocotyl
cut longitudinally with a fragment of the cotyledon, but no shoot apex.
Explants were incubated on 15 £ 90 mm Petri dishes with 25 ml of MS
medium with 3% sucrose, MS vitamins and 8 g l21 TC agar (JRH
Biochemicals or Sigma A1296), with 4.4 mM benzyladenine (BA) (MSBA1
medium), under the incubation conditions described above.
Experiments with cotyledon explants use only explants derived from the
part of the cotyledon proximal to the apex of the seedling. Cotyledon explants
were cut as described in Gaba et al. (1996b) and incubated abaxial side down
on MSBA1 medium as above.
Explants were observed daily by stereomicroscope, and samples taken
every 2– 3 d for morphological observations. Results were consistent, and the
developmental sequence was always similar, but the timing of the responses,
even within an experiment, was variable.
After 3–4 wk in culture the regenerating area was excised from primary
explants and transferred to 150 £ 25 mm tubes containing 10 ml elongation
medium, consisting of MS medium with 3% sucrose, MS vitamins and 8 g l21
agar (Sigma A1296), with 0.44 mM BA and 2.89 mM gibberellic acid (GA3)
for another 3–4 wk. Shoots of $15 mm were excised and rooted in MS
medium with 3% sucrose, MS vitamins and 8 g l21 agar (Sigma A1296),
without growth regulators for about 4 wk.
Histology. Explants were fixed in formalin–acetic acid–alcohol (FAA),
dehydrated in tertiary butyl alcohol, embedded in Paraplast, and stained with
iron haematoxylin (Sass, 1958). Sections (8 mm) were cut by rotary
microtome, and observed using a light microscope (Gaba et al., 1999).
Scanning electron microscope. Explants were fixed in a buffered 4%
glutaraldehyde solution (0.1 M, pH 7, sodium cacodylate trihydrate),
dehydrated in 100% ethanol, critical point dried, and stuck onto metal
plates prior to sputter coating with gold to 20 nm thickness (Balzers Union).
Explants were viewed using a Jeol model JSM-T330A scanning electron
microscope at 50–500 £ magnification.
Statistical analysis. Statistical analysis was accomplished by transform-
ations of the percentage values according to Bartlett (1947). Means were
separated from each other by Tukey’s honestly significant difference (HSD)
test (Compton, 1994).
Results and Discussion
Morphology of regeneration from the hypocotyl. Initial explants
were prepared from 4-d-old germinating seedlings from which the
shoot apex and associated leaves of the original explant were
removed (Fig. 1a). This type of explant now proceeds through a
defined developmental series. The stub of the hypocotyl grows
longer, thicker and deeper with the sides of the hypocotyl stump
enlarging 6-fold within the first 2 – 5 d (Fig. 1b). The area of
regeneration is not visibly differentiated at this stage. A swelling
develops on the upper part of the hypocotyl (seen here with a single
early protuberance) (Fig. 1c). Swelling is visible on 20% of explants
after 3 d, and develops in 45% of explants after 6 d in culture.
Protuberances then develop on the bulge, starting at the distal end of
the bulge (Fig. 1c). The protuberances are visible on 10% of explants
after 3 d, 40% after 6 d, and 80% after 8 d on MSBA1 medium. A
shoot apex is occasionally observed amongst the protuberances
(Fig. 1d). After 10 d in culture about 30% of explants have leaves,
rising to 67% with leaves after 13 d (Fig. 1e), mixed with buds. Such
buds may develop into early multiple shoots (Fig. 1f ). Axillary buds
become visible at the base of the larger buds (Fig. 1f ). After 30 d
261
small multiple shoots and their leaves cover the area between the
hypocotyl and the cotyledon (Fig. 1g). Callus becomes visible at both
ends of the explant (Fig. 1g). Transfer of areas with small multiple
shoots to elongation medium produces a group of multiple shoots
after another 4 wk (Fig. 1h). Shoots excised from the group of
multiple shoots and placed on rooting medium grow and produce
roots after 4 wk (Fig. 1i ), and are transferred to the greenhouse for
hardening.
Kinetics of regeneration from hypocotyl compared to cotyledon
explants. Regeneration from melon hypocotyl explants is more
rapid than from proximal cotyledon explants (Gaba et al., 1999). The
appearances of both protuberances and leaves are more rapid on the
hypocotyl explants. After 15 d, 96% of hypocotyl explants
regenerated a leaf or shoot, and 46% regenerated a shoot; whereas
at this time no regeneration of leaves or shoots occurred from
cotyledon explants (Fig. 2). After 21 d most hypocotyl explants
produced multiple shoots or at least a single shoot (87% together);
but no visible shoot regeneration was evident from proximal sections
of the cotyledon, and only 12.5% regenerated visible leaves from
protuberances at this stage (Fig. 2).
The rapidity of the shoot regeneration response of hypocotyl
explants is surprising. The speed of response may be due to the
ability of young undifferentiated cells in the proximal zone of the
hypocotyl exposed by explant preparation to redirect their
developmental program, and thereby produce new shoot apical
meristems. In a closely related melon cultivar, buds are only
observed by histology developing on cotyledon explants in culture
after 15 d (Gaba et al., 1999). It takes 6– 8 wk to regenerate shoots
visible to the naked eye from cotyledons of cv. Revigal and related
cultivars (this work; Gaba et al., 1999), and then only following
subculture. Shoot regeneration requires 4 – 6 wk from watermelon
cotyledons (Compton and Gray, 1993), 3 – 4 wk (Yahav et al., 1996)
or 7 wk (Kathal et al., 1988) from melon leaf explants. However, the
most proximal part of the cotyledon gave rapid regeneration in
cucumber (Cucumis sativus L.), with multiple shoots regenerated
after 15 d in culture, although microscopic in size (Gambley and
Dodd, 1990). Rapid differentiation of shoot meristems has also been
noted on the most proximal end of Brassica cotyledons (Sharma and
Bhojwani, 1990).
Development of explants that were not de-budded. The apex and
joint between the hypocotyl and the cotyledon are clearly visible
under low magnification on explants that have not been de-budded
(Fig. 3a). The shoot apex (apical bud with surrounding leaves) is
visible by SEM (Fig. 5a). The partner explant prepared from the
other cotyledon of the same seed does not have a shoot apex, but has
an indentation from the apex (visible in the SEM, Fig. 5b). Within 3 d
a leaf is visible on 30% of the explants that have not been de-budded
(Fig. 3b), when no leaves are visible on de-budded explants. Two
days later the apical bud with a leaf on either side is visible (Fig. 3c).
The hypocotyl stub has enlarged massively, and the space in the
middle has not yet been filled by the development of the swelling
seen later in the de-budded samples (compare to Fig. 1c). After 8 d
individual shoots are visible in 42% of explants that have not been
de-budded, and not at all on de-budded explants. Later, early
multiple shoots are observed (12 d; Fig. 3d). Development of
explants that were not de-budded was therefore more rapid than from
de-budded explants, showing the effect of a pre-existing shoot apex.
Often a single shoot with a large leaf is found, surrounded by
subsidiary buds (16 d; Fig. 3e), or occasionally a short compact shoot
262 CURUK ET AL.

FIG . 1. Regeneration from de-budded hypocotyl explants. All samples have the adaxial surface upwards, with the distal part of the
explant towards the top of the page. a, Initial explant from the cotyledon of a 4-d-old germinated melon cv. Revigal seedling. The shoot apex
and associated leaves have been removed. The white arrow ‘H’ marks the part of the hypocotyl remaining after explant preparation. The
cotyledon remnant is labeled ‘C’. The black arrow indicates the semicircular joint between the hypocotyl and the cotyledon ðbar ¼ 1 mmÞ. b,
Explant after 5 d in culture. Asterisks label the sides of the hypocotyl stump, and the now empty space between the sides is marked with an
arrow. Regeneration will occur slightly above the point indicated by the arrow ðbar ¼ 0:5 mmÞ. c, Explant after 10 d in culture. Swelling on
the hypocotyl is marked with a three-headed arrow. A regenerating protuberance is marked with an arrow. Regeneration occurs in the area
marked between the two arrows ðbar ¼ 2 mmÞ: d, A shoot apex (marked ‘S’) on an explant cut from a 7–10-d-old seedling. Explants from
this age of seedling regenerate callus (visible in the foreground) rapidly ðbar ¼ 0:15 mmÞ: e, Explant after 14 d in culture with buds (arrows)
and leaves ðbar ¼ 2:5 mmÞ: f, Close-up of an explant after 12 d in culture with several buds close together (buds marked by arrows). Further
small axillary buds formed at the base of the large buds are visible ðbar ¼ 1 mmÞ: g, Explant after 32 d in culture. Small multiple shoots and
leaves cover the area between the hypocotyl (H) and the cotyledon (C) ðbar ¼ 4 mmÞ: h, Multiple shoots regenerating from an explant 4 wk
after transfer to elongation medium ðbar ¼ 6 mmÞ: Shoots are marked with white arrows. The original explant, now greatly swollen, is
marked with a black arrow. i, A regenerated plant of cv. Revigal from a hypocotyl explant, rooting in vitro ðbar ¼ 10 mmÞ:

with a swollen base (Fig. 3f ) with buds visible around the base. In
later stages of development the appearances of de-budded explants
and those that have not been de-budded are similar.
Dissections of the basal fragment of the cotyledon from the
hypocotyl. The hypocotyl part can be surgically separated from the
most proximal part of the cotyledon. The cotyledon fragment
regenerates (Fig. 4a), resembling the regeneration of protuberances
found after a similar period (16 d) with proximal cotyledon explants
(Gaba et al., 1999). After 16 d in culture the separated hypocotyl
fragment has enlarged as if it were still attached to the rest of the
 REGENERATION FROM MELON HYPOCOTYL 263

FIG . 2. Kinetics of regeneration responses of a variety of explants of
melon. Regeneration of a shoot, or leaf or shoot, was recorded after 15 d and
21 d on MSBA1 medium of hypocotyl explant (de-budded), cotyledon,
hypocotyl fragment excised from the hypocotyl explant, cotyledon fragment
excised from the hypocotyl explant. Regeneration of the hypocotyl explant
was significantly different from that of the other explants for each response,
for each time reported, by Tukey’s HSD test ðP , 0:05Þ. There was no
significant difference between the regeneration of the explants except for the
hypocotyl, by the HSD test ðP , 0:05Þ. Minimum significant difference
values according to the HSD test at P , 0:05 were 13.56 (for regeneration of
a shoot after 15 d), 12.53 (leaf or shoot after 15 d), 13.79 (shoot after 21 d),
36.66 (leaf or shoot after 21 d).
explant (Fig. 4b); the sides have also risen up, leaving a space in the
center. Swelling develops rapidly on the excised hypocotyl section,
and 50% of the explants showed swelling after 6 d. Swelling did not
develop as much as with the intact explants, and a knob emerged at
the point where there is normally regeneration of buds and shoots
(Fig. 4b). After 15 d in culture no leaves regenerated from either
cotyledon or hypocotyl fragments (Fig. 2); but 29% of the cotyledon
fragments regenerated leaves after 21 d (Fig. 2), and the excised
hypocotyl produced protuberances (probably leaf primordia; Gaba
et al., 1999) in 60% of explants, along with a low level of leaf
production (Fig. 2), after which development did not continue.
The requirement for attachment of a fragment of the cotyledon to
the hypocotyl for regeneration from the hypocotyl could be due to two
factors. Firstly, the damage due to the separation of the organs may
prevent regeneration. Alternatively, factors (possibly growth
substances) may be transmitted from the cotyledons to the hypocotyl
enabling regeneration. For example, cotyledon-produced factors are
involved in the photo-control of elongation of the cucumber
hypocotyl (Black and Shuttleworth, 1974). Regeneration from Vigna
cotyledonary nodes can to some extent depend on attachment of part
of the cotyledon (Sen and Guha-Mukherjee, 1998).
Histology of regeneration from the hypocotyl. Histological
examination of regeneration from the hypocotyl was undertaken.
Sections were made from melon hypocotyl samples in which no ‘bud’

FIG . 3. Regeneration from hypocotyl explants that have not been de-budded. a, b, d, are presented adaxial surface upwards, with the
distal part of the explant towards the top of the page. a, An explant prepared from a 4-d-old seedling. The apex of the seedling is labeled.
The joint between the hypocotyl and the cotyledon is marked with two arrows. The cotyledon fragment (C) and the hypocotyl stump (H) are
labeled ðbar ¼ 1 mmÞ: b, Explant after 3 d in culture. The leaf covering the apical bud is marked with an arrow. Asterisks mark the sides of
the hypocotyl stump. The cotyledon fragment (C) and the hypocotyl stump (H) are labeled. The ‘H’ is on the hollow part of the stump
ðbar ¼ 1 mmÞ: c, Explant after 5 d in culture, viewed from the cut hypocotyl side. The apical bud with a leaf on either side is marked with an
arrow. Asterisks mark the sides of the hypocotyl stump (H). The ‘H’ is on the hollow part of the stump where swelling will develop
ðbar ¼ 1 mmÞ: d, Explant after 12 d in culture. Arrows indicate shoots. The cotyledon fragment (C) and the hypocotyl stump (H) are labeled
ðbar ¼ 2 mmÞ: e, Explant after 16 d in culture, viewed laterally. A short stem is shown, accompanied by leaves. A subsidiary bud is marked
with an arrow. The cotyledon fragment (C) and the hypocotyl stump (H) are labeled ðbar ¼ 3 mmÞ: f, A short compact shoot with massively
swollen base, marked by a white arrow, regenerated after 30 d in culture, viewed laterally. A bud at the base of the swollen shoot is marked
with a black arrow ðbar ¼ 3 mmÞ:
264 CURUK ET AL.

FIG . 4. Regeneration from dissected explants. a, A surgically removed cotyledon fragment after 16 d in culture. The explant is shown
adaxial surface up, with the most proximal side towards the bottom of the page. Regenerating leaves and protuberances are on the most
proximal edge (arrow) ðbar ¼ 1 mmÞ: b, A hypocotyl section surgically removed from a de-budded hypocotyl explant (as in a ), after 16 d in
culture. The sides have grown (marked with asterisks), leaving a space in the center (black arrow). A regenerated knob has emerged at the
proximal end of the hypocotyl (white arrow) ðbar ¼ 1 mmÞ: c, Transverse section of a de-budded hypocotyl explant after 11 d in culture in
which no development was visible externally by stereomicroscope. A small, meristematic bulge (m) on the adaxial side (at the top) is
opposite the vascular bundle (vb) of the midrib. Parenchymatic cells (p) are between the meristem and the vascular bundle ðbar ¼ 100 mmÞ:
d, The meristem from 3c viewed in close up. The meristem (m) and a parenchyma cell (p) are indicated ðbar ¼ 10 mmÞ: e, Transverse section
of hypocotyl explant after 11 d in culture on which there was a visible knob, showing a young bud structure on a promontory composed
mainly of parenchyma (p), developing in the same position on the explant as in c and d. The shoot apical meristem (m) is visible, amongst
several leaf primordia (arrows) ðbar ¼ 50 mmÞ: f, Close up of e, showing the densely staining cells of the shoot apical meristem (sam) and the
nearest leaf primordium (lp) ðbar ¼ 10 mmÞ:
 REGENERATION FROM MELON HYPOCOTYL 265

FIG . 5. SEM observations of the preparation of explants. All scanning electron microscope photographs are located with the hypocotyl
stub to the top of the page, adaxial surface up. The hypocotyl fragment is labeled ‘H’, and the cotyledon is marked ‘C’. a, An explant (similar
to that of 3a ) that has not been de-budded, immediately after preparation. The shoot apex (apical bud with surrounding leaves) is labeled ‘A’
ðbar ¼ 500 mmÞ: b, An explant from the cotyledon that did not have the apical bud after cutting. A small amount of material from the shoot
apex remains (A), as well as a negative impression of the shoot apex (NI) ðbar ¼ 500 mmÞ: c, An explant following removal of the apical bud
and associated material. A small scar remains on the de-budded sample (arrow) ðbar ¼ 500 mmÞ: d, Higher magnification of the removal
scar from c. The arrow indicates the scar ðbar ¼ 100 mmÞ: e, An explant following removal of the apical bud and associated material. A
wrinkle of material from the shoot apex remains (arrow) ðbar ¼ 100 mmÞ:

development was visible externally by binocular microscope, after
several days on MSBA1 medium. The structure observed in Fig. 4c is
a cross-section of a hypocotyl explant with swelling, very different in
appearance from regenerating cotyledon sections (Gaba et al., 1999).
There is a single small, meristematic bulge on the adaxial side (at the
top of the picture) (Fig. 4c), opposite the vasculature. Hairs are
visible on the epidermis near the meristem. There is parenchyma
between the meristem and the vascular bundle, the parenchyma
constituting most of the swelling observed in Fig. 1c. When viewed
in close up, the deeply stained meristem (Fig. 4d) has the
appearance of an early apical meristem. A bud is observed in
another sample with an externally obvious knob (Fig. 4e), developing
in the same position on the explant as in Fig. 4c, d. The bud is on a
promontory composed mainly of parenchyma, of which only the axial
part is shown (Fig. 4e). Several leaf primordia are visible near the
shoot apical meristem, similar to the mass production of
protuberances on melon cotyledons in culture (Gaba et al., 1999).
The shoot apical meristem and the adjacent leaf primordium stained
densely with meristematic activity (Fig. 4f ).
The development of a new shoot apical meristem was observed by
histology, and therefore does not occur from a pre-existing apical
meristem. The apical meristem may be visible on the melon
hypocotyl before leaf primordia obscure it (Figs. 1d and 4c), in
contrast to the regeneration process from cotyledons (Gaba et al.,
1999) where mass production of protuberances (leaf primordia)
occurs prior to the appearance of a shoot apical meristem.
Examination of the cutting procedure by scanning electron
microscope (SEM). Prior to removal, the shoot apex is conspicuous
on one explant of each pair prepared (Figs. 3a and 5a). The explant
from the cotyledon which did not have the apex after cutting always
266 CURUK ET AL.
has a small amount of apical material remaining (that should be
removed prior to the experiment), and a negative impression of the
shoot apex (Fig. 5b). Following removal of the apex and associated
material, a small scar remains on the de-budded sample (Fig. 5c). At
a higher power the removal scar can be seen to be small, indicating
an effective operation (Fig. 5d), or in the worst case there is a small
wrinkle of material remaining (Fig. 5e). Regeneration from the
hypocotyl occurs even when all existing apical material has been
removed. The complete removal of the shoot apex was demonstrated
here by light microscopy and SEM.
Regeneration from the melon hypocotyl is reminiscent of
regeneration from cotyledonary nodes of legumes in that it is
rapid, produces multiple shoots, gives a high percentage of explant
regeneration, and regeneration may depend on attachment of part of
the cotyledon. However, in the case of the hypocotyl of melon, there
is no pre-existing shoot bud as there is with the cotyledonary node
procedure. It is known that areas adjacent to shoot apices may
regenerate new shoots efficiently, e.g. cucumber (Gambley and
Dodd, 1991), pea (Bean et al., 1997), and cotton (Hemphill et al.,
1998).
In this paper we have demonstrated a new mode of direct in vitro
regeneration in melon from the hypocotyl rather than direct
regeneration from the cotyledon, as it is a more rapid process. This is
the first report of direct organogenesis from the hypocotyl in melon
and to our knowledge in the Cucurbitaceae, although shoot
regeneration from hypocotyl-derived callus has been reported
(Kathal et al., 1986; Molina and Nuez, 1995).
Acknowledgments
Contribution from the Agricultural Research Organization, The Volcani
Center, Bet Dagan, Israel, no. 503/01. This work was supported by research
grant no. US2079-91 from BARD, The United States–Israel Binational
Agricultural Research and Development Fund to V.G. and D.J.G., and by a
grant to V.G. from the Chief Scientist of the Ministry of Agriculture (Israel),
and to S.C. from the Scientific and Technical Research Council of Turkey
(TUBITAK). The authors wish to thank Dr. Irvin Fischer for assistance with
the scanning electron microscopy, and Drs. B. Steinitz and
G. Ananthakrishnan for critical comments on the manuscript. This paper is
dedicated to Prof. Mike Black, of King’s College, University of London, UK, a
great teacher and friend, on his recent retirement, by V. Gaba.
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