Int. J. Plant Sci. 157(5):554-560. 1996.

? 1996 by The University of Chicago. All rights reserved.

1058-5893/96/5705-0004$02.00

REGENERATION
OFTOMATO ESCULENTUM
(LYCOPERSICON MILL.):SOMATIC AND
EMBRYOGENESIS
SHOOTORGANOGENESIS
FROMHYPOCOTYL
EXPLANTS
INDUCED WITH6-BENZYLADENINE
PHILIP 0. NEWMAN, SANKARAN KRISHNARAJ, AND PRAVEEN K. SAXENA'
Department of Horticultural Science, University of Guelph, Guelph, Ontario NIG 2W1, Canada

In vitro regeneration of plants was achieved in four cultivars of tomato (Lycopersicon esculentum Mill.). The explants
were 10-mm hypocotyl sections, excised from 2-wk-old seedlings grown on Murashige and Skoog (MS) medium
supplemented with or without 6-benzyladenine (BA). The hypocotyl explants were subcultured on MS medium without
growth regulators or on MS media supplemented with 5, 10, 20, or 40 ,uM BA. Regeneration through somatic em-
bryogenesis and shoot organogenesis occurred in explants of all treatments, even on hypocotyls from seedlings grown
on basal medium and subcultured to medium without growth regulators. Explants from seedlings grown on BA-
supplemented medium produced the highest frequency of regenerants when subcultured to media containing BA. In
contrast, BA had an inhibitory effect on explants obtained from seedlings grown on media lacking BA. Light micro-
scopic observations confirmed that regeneration occurred via both somatic embryogenesis and shoot organogenesis
from the subepidermal cortical tissue. Developing somatic embryos were encased in a distinct protoderm, had a sus-
pensor-like structure, and were loosely attached to the hypocotyl tissue. Somatic embryos and shoots developed into
complete plants on a medium lacking growth regulators.

Introduction mato and (ii) to obtain conclusive histological evi-

Tomato (Lycopersicon esculentum Mill.) is an im-
portant food crop, for which successful applications of
in vitro regeneration and genetic transformation have
already been implemented for genetic improvement
(Lindsey 1992). Shoot organogenesis has been
achieved in tomato for explants from many different
tissues: apical meristems, cotyledons, stems, petioles,
leaves, anthers, and inflorescences (Young et al. 1987;
Branca et al. 1990; Compton and Veilleux 1991).
However, there have been very few reports of somatic
embryogenesis in tomato, indicating that classical pro-
tocols for achieving embryogenesis have not been suc-
cessful (Young et al. 1987; Lamproye et al. 1990; Kob-
litz 1991). Lamproye et al. (1990, p. 129) reported the
formation and precocious germination of "bipolar
structures comparable to somatic embryos" on hypo-
cotyl explants cultured on various concentrations of 6-
benzyladenine (BA) in combination with a synthetic
auxin analog, [benzo(b)selenienyl-3]acetic acid. This
IAA analog was synthesized by a complex chemical
reaction that replaced the nitrogen atom in the IAA
indole moiety with a selenium atom (Hofinger et al.
1980). Young et al. (1987) also observed formation of
shoot primordia and somatic embryos on excised zy-
gotic embryos cultured on media containing BA and
yeast extract. However, they relied solely upon scan-
ning electron microscopy to identify regenerating
structures, and stated that the distinction between
shoots and somatic embryos was not clear. In neither
of these reports was the frequency of regeneration
mentioned.
The specific objectives of the present investigation
were (i) to develop an effective protocol for high-fre-
quency regeneration in four different cultivars of to-
'Author for correspondence and reprints. Fax 519-767-0755; E-
mail psaxena@Pevbhort.uoguelph.ca.
Manuscript received August 1995; revised manuscript received April
1996.
dence indicating that both forms of regeneration
(namely, shoot organogenesis and somatic embryogen-
esis) occurred simultaneously on hypocotyl explants.
Material
andmethods
SEEDLINGCULTURE
Seeds of four tomato cultivars-Manitoba, MH 6203 VF,
Orange Queen, and Sweet Million-were obtained from
Stokes Seeds Ltd., St. Catharines, Canada. The seeds were
placed in a tea bag holder to prevent floating and surface-
sterilized for 15 min by placing them in a beaker and agi-
tating with a stir bar in 200 mL of half-strength commercial
bleach (2.5% sodium hypochlorite, v/v), with two drops of
"Tween 20" as a surfactant. The seeds were then rinsed
three times with sterile distilled water and cultured at the
rate of 10 seeds per petri dish (25 mm X 100 mm) contain-
ing 25 mL of appropriate media. Seventy seeds of each cul-
tivar were grown on each treatment.
Seeds were cultured on three different media treatments:
MS (Murashige and Skoog 1962) basal medium (MS 0) and
MS medium supplemented with either 50 pLMor 80 FM
BA). All three treatments were supplemented with B-5 vi-
tamins (Gamborg et al. 1968) and 3% sucrose and were so-
lidified with 0.3% Gelrite (w/v). The pH was adjusted to 5.8
before autoclaving at 121?C for 20 min.
The petri dishes were sealed with Parafilm (American Na-
tional Can, Greenwich, Conn.). The cultures were incubated
in a growth room, maintained at 240 ? 1?C, and a 16-h
photoperiod (20-30 pLmolesm-2 s-') was provided by cool-
white fluorescent lamps (Phillips Canada, Scarborough,
Ont.). Hypocotyl explants were taken from the seedlings 14
d after initial culture.
HYPOCOTYLCULTURE
Hypocotyl sections (ca. 10 mm long) were excised from
the region immediately above the crown of the seedlings.
Because the hypocotyl explants were taken only from the
region nearest the crown, only one explant was taken from
each seedling. For the seedlings grown on MS 0, two cuts
had to be made: one excision at the crown region to remove
the roots and the other 10 mm higher on the hypocotyl. For
seedlings grown on either MS + 50 FiM BA or MS + 80

554
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 NEWMAN ET AL.-EMBRYOGENESIS
p,M BA, only one excision was made. These seedlings failed
to develop roots after germination, so the base of the seed-
ling was left intact and the hypocotyl was excised 1.0 cm
above the base.
Media preparation for hypocotyl culture was similar to
seedling culture, with the exception that concentrations of 0,
5, 10, 20, and 40 ,uM BA were used. Four explants were
cultured per petri dish containing 25 mL of the medium.
Three petri dishes (12 explants) per treatment were used for
each cultivar. The dishes were sealed with Parafilm and in-
cubated under the same conditions as described for seedling
cultures. The numbers of regenerated structures were count-
ed under a dissection microscope 4 wk after the incubation
of the explants.
STATISTICALANALYSIS
The experiment was designed in a split-plot design with
seven replications for seedling cultures (main plot treatment)
and three replications for hypocotyl cultures (subplot treat-
ment). The data were analyzed using the general linear mod-
el PROC-GLM for PC SAS (SAS Institute 1995) and the
means were compared by least significant differences (LSD)
at 1% level of probability.
LIGHT MICROSCOPIC
STUDIES
The explants were harvested at various intervals over the
4-wk incubation period, so that sections could be obtained
of the structures at different stages of development. Explants
were taken both from the control group (seedlings grown on
MS 0 and subcultured on MS 0) and from the BA treatments.
The methods for sample preparation and histology have been
described (Malik and Saxena 1992). Cross sections of 10-
,m thickness were cut, using a Spencer 820 microtome. The
sections were stained with safranin 0 and counterstained
with alcian green and examined under a light microscope.
Results
SEEDLING GROWTH
In all of the cultivars tested, germination was greater
than 90%, regardless of medium composition. Seeds
cultured on either 50 or 80 p,M BA germinated, but
the roots failed to elongate; instead a swollen area re-
sulted at the base of the hypocotyl, i.e., crown region
(fig. 1A, arrow). Seedlings grown on MS 0 had normal
root systems (fig. IA, right). After 2 wk the shoot
length was only 1-2 cm for BA-grown seedlings but
5-6 cm for MS 0-grown seedlings (fig. IA). In addi-
tion, only the cotyledonary leaves were visible on the
BA-grown seedlings, while two or three true leaves
were visible on seedlings grown on MS 0 treatment
after 2 wk. Callusing or regeneration were not ob-
served on the seedlings prior to the excision of hy-
pocotyl explants in any treatment.
REGENERATION FROM HYPOCOTYL EXPLANTS
Regeneration was observed on explants taken from
BA-grown seedlings as well as on MS 0-grown seed-
lings for all of the subculture media. The regenerated
structures became visible 2-3 wk after subculture, and
appeared to be of two distinct types when viewed un-
der a dissection microscope: (1) structures of the first
type were either globular or heart-shaped and had few-
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AND ORGANOGENESIS IN TOMATO 555
er trichomes (fig. iB, arrows) and (2) structures of the
second type were shootlike, with one or two leaves
which were more like true leaves than cotyledonary
leaves in their morphology, and had numerous tri-
chomes (fig. IC, arrow). The structures resembling
stages of somatic embryos developed a proper root and
shoot system, and complete plantlets were obtained
(fig. ID).
Hypocotyl explants taken from MS 0 seedlings had
structures developing only from the two cut ends of
the hypocotyl explant. No callusing was observed on
any explants taken from MS 0 seedlings, regardless of
the subculture medium. Regeneration from hypocotyls
of BA-grown seedlings occurred mostly from the
swollen crown region, and to a lesser extent at the cut
surface, and along the length of the explant. Shootlike
and embryo-like structures were observed at the crown
region and in the middle portion of the explants, but
only the shootlike structures were observed at the cut
surface. A small amount of compact, yellow-green,
nodular callus was also observed at the swollen crown
region when the explants were subcultured on BA.
Higher concentrations of BA in the subculture medium
resulted in a marginal increase in callusing.
EVIDENCE OF SHOOT ORGANOGENESIS
Histological evidence of regeneration via shoot or-
ganogenesis was found both on explants from the con-
trol group (explants from seedlings grown on MS 0
and subcultured on MS 0) and on explants from BA-
grown seedlings. Based on cross sections of the hy-
pocotyl explants, the shoots appeared to originate from
the cortex of the hypocotyl, and were characterized by
a group of meristematic cells (fig. 2A, arrow). These
cells further developed to form a well-defined vascu-
lature (fig. 2B, arrow). Since the explant was taken
from well below the cotyledonary node, it was unlike-
ly that this shoot could have originated from an axil-
lary bud. Consecutive sections of the regenerated
structures clearly demonstrated that these structures
were unipolar and therefore did not fulfill the require-
ment for bipolarity of a somatic embryo.
Further evidence of shoot organogenesis was ob-
served by the presence of a vascular connection from
the unipolar structure to the vascular cylinder of the
hypocotyl (fig. 2C) and the presence of xylem vessels
originating from the stele of the hypocotyl explant (fig.
2C, arrow). Longitudinal section through a shoot pri-
mordium arising from cortical tissue of the hypocotyl
showed the two leaf primordia and the shoot apex, as
well as the vascular connections to the leaf primordia
(fig. 2D). There was, however, no evidence of a root
primordium or of a layer of cells resembling an em-
bryonic protoderm, and thus these structures did not
resemble a somatic embryo.
EVIDENCE OF SOMATIC EMBRYOGENESIS
Histological evidence of somatic embryogenesis
was found on explants from MS 0- and BA-grown
seedlings. Somatic embryos at the early globular stage
556 INTERNATIONAL JOURNAL OF PLANT SCIENCES

WElll

Fig. 1 Regeneration of plants from tomato hypocotyl cultures. A, Comparison of shoot length and root development of a seedling grown
on 50 ,uM BA (left) and a seedling of the same age grown on MS 0 (right). Note extensive root development and shoots on MS 0-grown
seedlings, while seedlings grown on 50 puMBA show reduced shoot length and no root (arrow) development (x 0.5). B, Globular-stage
somatic embryos (arrows) developing on a hypocotyl explant as observed under a dissecting microscope (x 25). C, Shoots regenerating on
the decapitated shoot end of a hypocotyl taken from a seedling grown on 50 ,uM BA. Note the numerous trichomes on the shoots (arrows)
(X 4). D, A plantlet that developed from a somatic embryo excised at the cotyledonary stage (x 0.3).

were characterized by a well-defined uniform proto- QUANTITATIVE OBSERVATIONS

derm, consisting of rectangular cells (fig. 3A), and a
suspensor-like structure at its base (fig. 3B). Darkly The average numbers of structures formed per ex-
stained meristematic cells (fig. 3A) and a strand of plant in each of the treatments and cultivars are shown
meristematic cells between the poles were present in in figure 4. Based on the observations during the early
the late globular-stage somatic embryos. Heart-shaped development of the shoots and somatic embryos, over
and late cotyledonary-stage somatic embryos were 80% of the structures formed were somatic embryos
loosely attached to the epidermal cells of the hypocotyl from seedlings grown on media containing BA. As
mother tissue and were frequently detached during described earlier, visual observations of the cultures
sectioning (fig. 3C-E). revealed that the shoots developed only from the cut
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 NEWMAN ET AL.-EMBRYOGENESIS AND ORGANOGENESIS IN TOMATO 557

Fig.2 Histological evidence of shoot organogenesis in hypocotyl cultures of tomato. A, B, Cross section of a hypocotyl explant showing
the vasculature of the hypocotyl as well as the meristematic zone and the vascular bundle of the regenerating shoot that originated by
organogenesis from the cortex of the hypocotyl (affows) (x 40). C, A developing shoot primordium, with vascular attachments to the vascular
bundle of the hypocotyl explant (x 40). Note the xylem tracheids (arrow) in the developing shoot. D, A shoot primordium developing on a
hypocotyl explant (x 40). Note the development of shoot apex and vascular strands extending into the leaf primordia.

end of these explants, whereas somatic embryos orig-
inated from the swollen crown region and along the
length of the hypocotyl explant. Also, the cut ends
produced only two or three shoots (fig. 1C), whereas
more than 10 somatic embryos developed in a ring
around the swollen crown region of the hypocotyl ex-
plants grown on BA (fig. iB).
The average numbers of regenerating structures
formed in each treatment varied from five to 20 per
explant, except for the cultivar Sweet Million, in
which the number of structures varied from one to five
per explant (fig. 4). The highest numbers of regener-
ants were observed on explants taken from seedlings
grown either on 50 or 80 ,uM BA and subcultured on
either 20 or 40 p,M BA. However, the structures
formed in these treatments were smaller and developed
more slowly compared to those formed on subculture
media containing lower concentrations of BA. Among
the seedling treatments, there were no significant dif-
ferences between 50 and 80 ,uM BA treatments in any
cultivar. In all cultivars, explants taken from seedlings
grown on MS 0 had significantly fewer regenerated
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structures than explants grown on either 50 or 80 ,uM
BA.
Discussion
The protocol developed for high-frequency regen-
eration of tomato cultivars was straightforward, as the
hypocotyl explants were easily obtained and the in-
duction media consisted of simple hormone-free me-
dium, or the one supplemented with BA, a readily
available synthetic cytokinin. The three most impor-
tant results of this study were (1) the simultaneous
formation of shoots and somatic embryos, (2) the for-
mation of shoots and somatic embryos in the control
group, which were not treated with exogenous growth
regulators at any stage of the experiment, and (3) the
development of a reliable and simple procedure for
obtaining somatic embryos in tomato and the appli-
cability of the culture conditions to each of the four
cultivars tested.
The frequency of regeneration observed in the pres-
ent study was as high as 20 structures per explant, with
ca. 80% of them being somatic embryos. Also, the
558 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig.3 Histological evidence of somatic embryogenesis in hypocotyl explants of tomato. A, A late globular-stage somatic embryo, with
two dark-staining meristematic poles and a well-defined protoderm separating it from the surrounding tissue (X 100). B, A globular stage,
separated from the surrounding tissue by a layer of rectangular cells (protoderm) and with a short suspensor-like structure at one end (arrows)
(x 100). C, A cross section through nodular structures formed on hypocotyl explants that were taken from seedlings grown on either 50 or
80 ,uM BA and subcultured on 20 p.M BA (x 30). D, E, A cotyledonary stage (X 40) and heart-shaped (x 40) somatic embryos that have
separated from the hypocotyl explant during sectioning, respectively.

somatic embryos tended to be loosely attached to the
mother tissue, and could be easily excised for further
development into plantlets. Another noteworthy aspect
of the present study was the role of preconditioning of
source seedlings in the presence of BA. In the absence
of preconditioning, for hypocotyls taken from seed-
lings grown on MS 0, the best subculture medium was
MS 0, and increasing concentrations of BA in the sub-
culture media resulted in a nonsignificant decrease in
the number of regenerants formed. For explants taken
from seedlings grown on either 50 or 80 ,uM BA, the
opposite was true: poorest regeneration was on MS 0,
and increasing concentrations induced more structures,
with the optimum BA concentrations being either 20
or 40 ,uM. The explants from the seedlings grown on
50 or 80 ,uM BA not only produced higher frequencies
of regeneration but the mode of regeneration also tend-
ed to shift toward somatic embryogenesis. These
trends were common to all cultivars. Thus, BA had a
stimulatory effect both on the induction of embry-
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ogenic cells as well as on the conversion of these cells
into somatic embryos, since the continued presence of
BA in the seed germination and explant culture me-
dium was necessary for optimum regeneration. Inhi-
bition of regeneration by BA in explant cultures de-
rived from seedlings grown in MS 0 is likely to be a
result of supraoptimal levels of BA in the subculture
medium.
The precise reason for the observed effect of pre-
conditioning with BA is not clear, but a modified me-
tabolism or biosynthesis of endogenous cytokinins in
response to BA may be responsible, at least in part.
It is evident from many studies that cytokinins are
synthesized in root tips, and they differ quantitatively
and qualitatively in various parts of the plant (Letham
1986). Inhibition of root development in the BA-pre-
conditioned seedlings is indicative of an altered cy-
tokinin metabolism and transport. Much-limited hy-
pocotyl elongation in preconditioned seedlings as
well as the development of somatic embryos and
 NEWMAN ET AL. -EMBRYOGENESIS AND ORGANOGENESIS IN TOMATO 559

25 25
Manitoba MH6203 VF
a

20 2

I ab~~~~~~~~~~~~~~~~~~~~o

b
~~~io bcc~~~~~
b
cd
Z cd d m

0 0
MSO 0 10 20 40 MS0 0 10 20 40
BAPconcontraon(W) BAPconcntrabon(W)

14 Sweet Million 14 OrangeQueen

.~12 i0 bc 20 40 .~12 0 0 20 4
lo 1 bc

110 M1S mb
ab

bc
o6 a ~~~~~~~~~~~ b
cbc
bc
ab b bc bc

b
bcN b bcN2

bbC bc
bc- bcb 0 10 20 40
20 40MSO
BAPoconcentralon
(uM) BAPoconcentralon
(uM)

Seedling grownon

Ms 0 ~ 5OpM BA 8OpMBA
Fig.4 The average number of regenerants per cultured explant for four of the tomato cultivars tested. Seedlings were grown for 2 wk, on
either MS 0, MS + 50 FM BA, or MS + 80 FM BA. Hypocotyl explants were taken from the 2-wk-old seedlings and subcultured on either
MS 0 or MS + 5, 10, 20, or 40 FLMBA. Regenerants were counted under a dissection microscope 4 wk after subculture. Bars with the same
letter are not significantly different (P = .01).

shoots at different sites on the cultured hypocotyl ex-
plants point to the possibility of auxin involvement
causing localized differences in auxin/cytokinin ra-
tios. It can be speculated that a high cytokinin/auxin
ratio resulting from discontinuation of auxin transport
at the time of explanting the hypocotyl tissue, coupled
with exposure to high BA during subculture, may
have stimulated a limited degree of organogenesis in
a largely embryogenic population of cells. We have
previously shown that seedlings of many species de-
veloped somatic embryos when grown continuously
on media enriched with cytokinins or phenylureas
with strong cytokinin activity, and the somatic em-
bryo development was accompanied by significant
changes in endogenous auxin and other growth reg-
ulators (Murthy et al. 1995; R. Gill et al., unpublished
data).
Since the induction of both shoot organogenesis
and somatic embryogenesis occurs simultaneously,
albeit at different sites, this experimental system
could be useful for studies on factors that favor so-
matic embryogenesis over organogenesis, and vice
versa. Furthermore, the protocol could be useful for
investigating the role of endogenous hormones in re-
generation because of the independence of morpho-
genesis from exogenous growth regulators. For ex-

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560 INTERNATIONAL JOURNAL
ample, the inclusion of the inhibitors of auxin,
cytokinin or ethylene action, biosynthesis or transport
in the explant or seedling media may facilitate the
determination of the effects of these hormones on dif-
ferentiation. In addition, its application to the study
of the developmental biology of morphogenesis and
the simplicity and efficacy of the regeneration pro-
cedure are expected to facilitate gene transfer exper-
iments in tomato.
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Acknowledgments
We express our appreciation to Dr. J. Gerrath and
Mr. B. N. S. Murthy for help in histology and exam-
ining slides and to Ms. Tannis Slimmon for technical
assistance with histology and photography. This re-
search was funded in part by a research grant from the
Natural Sciences and Engineering Research Council of
Canada (NSERC) to Praveen K. Saxena and an
NSERC summer scholarship to Philip 0. Newman.
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