Scientiu Horticulturae, 11 (1979) 223-227 223

Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

IN VITRO PROPAGATION OF WATERMELON

LEE R. BARNES
Department of Horticultural Science, Raleigh, North Carolina 27650 (U.S.A.)
(Accepted for publication 23 March 1979)
Paper 6065 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh

ABSTRACT

Barnes, L.R., 1979. In vitro propagation of watermelon. Scientio Hortic., 11: 223-227.

A 3-stage, in vitro propagation system for diploid watermelon (Citrullus lonatus

(Thumb.) Matsum. and Nakai) has been developed which is also applicable to the economic
production of triploid (seedless) watermelon transplants.
Stage I involves the stimulation of axillary-bud development from excised seedling
shoot tips (l-3 mm) by a high cytokinin (K)/low auxin (IAA) ratio (4.6 pmol/l K
0.26 ~mol/l IAA) on a modified Linsmaier and Skoog medium. An average of 4.5 axillary
shoots of sufficient size to subculture (> 15 mm) were obtained from each explant in
5 weeks. These shoots were induced to root during 3 weeks subculture on Stage II
medium containing 11.5 pmol/l IAA. Well-rooted plantlets were successfully transplanted
to a I : 1 peat : sand (v/v) substrate and gradually “hardened-off” to greenhouse conditions
(Stage III) for 3 weeks, after which they could be transferred to field conditions.
Alternatively, axillary shoots from Stage I could be returned to fresh media with
9.3 Mmol/l K and 0.28 Lcmol/l IAA, where an average of 10.3 axillary shoots could be
obtained after 5 weeks.
Cost estimates for producing 10 000 finished transplants per week project an approx-
imate cost of 16 dollar cents per transplant.

INTRODUCTION

The use of in vitro techniques for rapid clonal propagation has greatly
increased in the last 10 years (Murashige, 1974). The development of an
in vitro propagation system for diploid watermelon, which is applicable to
the production of triploid (seedless) watermelon transplants, will be described
in this paper.
Seedless watermelons were first produced by Kihara (1951) during the
late 1930’s. These watermelons exhibit many desirable characteristics be-
sides the novelty of seedlessness, as reported by Kihara (1951), Andrus et al.
(1971), Henderson (1977) and Henderson and Jenkins (1977). Production
difficulties and high seed costs limit the commercial use of triploid water-
melons (Andrus et al., 1971). Grosses to produce triploids yield only 150-
200 seeds per melon (Andrus et al., 1971) with an average cost of 1.6-2.5
dollar cents per seed, compared to 0.064-0.1 dollar cents per seed for open
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pollinated diploids (Henderson, personal communication, 1978). Additional-

ly, the triploid seeds exhibit erratic germination rates and cannot be direct-
seeded. Seeds must be germinated in carefully controlled conditions and later
transplanted to the field, requiring additional expense (Kihara, 1951).
A review of the literature revealed no attempts to use in vitro techniques
to propagate watermelons, although such techniques have been used to study
the mycoflora of watermelon seeds (Sohi and Maholay, 1974) and the effects
of cytokinins on excised watermelon cotyledons (Bhandari and Sen, 1971).

MATERIALS AND METHODS

Seeds of diploid watermelon ‘Charleston Gray’ were soaked overnight in

sterile distilled water and surface disinfected in 10% hydrogen peroxide with
0.1% Tween 20, a surfactant, for 15 min, rinsed 3 times with sterilized water
and allowed to germinate in sterilized petri plates in the dark at 30°C. When
the cotyledons had split open the seed coat in 7-10 days, the seedlings were
disinfected with 10% Chlorox (with 0.1% Tween 20) for 10 min followed by
3 water rinses. All additional preparations involved aseptic techniques under
a forced-air microfilter hood. Explants were prepared by making a transverse
cut through the hypocotyl, just basal to the cotyledonary node, thus remov-
ing the radicle. One cotyledon was removed by cutting or gently tearing,
resulting in an easily handled explant consisting of the seedling shoot tip
(epicotyl d-3 mm) attached to the remaining cotyledon. The explant was
placed on 20 ml of Linsmaier and Skoog (1965) basal medium (L&S) in
125-ml glass bottles. The basal medium contained the following addenda:
100 mg/l myo-inositol; 100 mg/l sodium monophosphate; 30 gm/l sucrose;
growth regulators 6-furfurylaminopurine (K=kinetin) and indoleacetic acid
(IAA) as specified for each of the propagation stages. The pH was adjusted
to 5.5 with 1 M sodium hydroxide before the addition of 8 gm/l Bacto agar.
Media were autoclaved for 3 min to dissolve the agar, poured into bottles
and then autoclaved for 20 min at 1.05 kg/cm’ pressure and 125°C.
Three distinct propagation stages were necessary for a high propagation
rate and successful transfer to soil. Optimum growth regulator concentra-
tions and cultural conditions were determined as follows:

Stage I Stages Ii...n Stage II Stage III

Initial Additional Rooting and Establishment
multiplication multiplication preparation of transplant
for transfer

Explant Seedling Axillary Axillary shoots Plantlets

shoot tips shoots and (>15 mm) (>25 mm)
(l-3 mm) shoot tips
(>lO mm)
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Duration 5 weeks 5 weeks 3 weeks 3 weeks

Media Nutrient Nutrient agar Nutrient 1:l

agar (L&S) (L&S) solution with peat : sand
vermiculite (v/v)
substrate

Growth 4.4 pmol/l K; 9.3 pmol/l K; 11.5 pmol/l No exogenous

regula- 0.28 fi mol/l 0.28 pmol/l IAA growth regu-
tors IAA IAA lators

Cultural Sterile Sterile Sterile Non-sterile

conditions culture, culture, culture, culture,
1000-2000 1000-2000 10 000 lux, 10 000 lux,
lux, lux, 28 + 5°C 27 * 3°C day
28 f 5°C 28? 5°C 21 f 3°C night

RESULTS AND DISCUSSION

Preliminary experiments involved explant selection, media choice and de-

termining optimum growth regulator levels. Callus could be readily obtained
from all tissues tested, but attempts to stimulate neoformation of adventitious
shoots were unsuccessful, although numerous plant parts were tested over a
wide range of different growth regulators. However, excised axillary buds
from greenhouse-grown vines developed into multiple shoots when cultured
with 4.4 pmol/l K, producing shoots of sufficient size (>15 mm) to sub-
culture after 2 or 3 months in culture. Later it was found that seedling shoot
tips (l-3 mm) with attached cotyledon would produce an average of 4.5 +
1.4 axillary shoots of sufficient size (>15 mm) to subculture after 5 weeks.
Additional Stages Ii...n subcultures, using excised axillary shoots and
shoot tips, resulted in an average of 2.6 axillary shoots per explant after
35 days. To increase the number of shoots obtained, levels of K and sucrose
were increased (Table I). The maximum number of axillary shoots in 35 days
was obtained by increasing the K level to 9.29 ymol/l and maintaining the
sucrose level at 3%. In general, higher levels of K resulted in a greater number
of short shoots, whereby higher levels of sucrose resulted in a reduced number
of long shoots. In other experiments, levels of K greater than 25 pmol/l
reduced the number and length of elongating axillary shoots, many of which
were deformed.
Shoots greater than 20 mm in length were induced to root during Stage II
in 2-3 weeks by subculturing to liquid L&S medium with 11.5 pmol/l IAA,
but without K or agar. The use of liquid media with a vermiculite substrate
for support and aeration of the shoot during Stage II resulted in a significantly
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TABLE I

Number and average length of axillary shoots on explants from Stage I,, subcultures
grown for 35 days with increased levels of K and sucrose

Sucrose No. of axillary Average length

~molil) (S) shoots of shoots (mm)

4.65 3.0 2.6 7.07

9.29 3.0 10.4 5.61
23.23 3.0 10.4 6.40
4.65 4.0 3.0 9.06
9.29 4.0 4.6 6.43
23.23 4.0 5.3 7.10

superior root system (*P<O.O5), better branched with extensive root-hairs,

than when grown on 0.4-1.2s agar. Additionally, there was less damage to
the roots when removed from the vermiculite than from agar and better ex-
plant survival when transferred to the peat:sand mixture. Increasing the light
intensity to 10 000 lux during this stage resulted in considerable growth
during culture, producing a plantlet which could be successfully transferred
to Stage III.
Stage III, establishment of hardened transplants, involved the transfer of
vigorous, well-rooted plantlets to a 1 : 1 peat : sand (v/v) mixture in 5-cm dia-
meter peat pots and gradually “hardening-off” the plantlets to greenhouse
conditions. Attempts to maintain high humidity by using intermittent mist
resulted in poor explant survival. Plantlets could be successfully established
in the greenhouse with partial shading by covering with a clear plastic cup
to maintain high humidity. Over a period of a week, the cups were partially
lifted and tilted to allow air circulation, and later removed for 5- to 6-hour
daily periods. The cups were then completely removed from those plantlets
which exhibited vigorous growth and a lack of wilting. These transplants
required an additional 2 or 3 weeks of growth before they could be success-
fully transplanted to field conditions.
Field experiments comparing seedling with in vitro produced transplants
were conducted and no undesirable effects due to in vitro techniques were
observed during the growing-season or at harvest.
Projected costs were estimated at 16 dollar cents per finished transplant
for a volume of 10 000 transplants per week, based on calculations by
Anderson et al. (1977) for broccoli, but modified to the production rate for
watermelons, and including laboratory and equipment expense, labor and
rental of greenhouse space.
A sufficient number of experiments has been conducted using the less
available triploid seedling shoot tips to demonstrate that the 3-stage system
developed for diploids is easily adaptable for rapid propagation of triploid
watermelon transplants.
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Hindsight suggests that the slower-growing axillary buds from greenhouse-

grown vines should be reconsidered as an initial explant. These buds produce
clumps of multiple shoots which remain small, but can be more easily
handled by division than by working with individual shoots. Subcultures of
these clumps resulted in more uniform explants than those obtained from
seedling shoot tips.
Finally, this propagation system has potential application to other cucur-
bits as a means to rapidly increase particularly desirable clones.

REFERENCES

Anderson, W.C., Meagher, G.W. and Nelson, A.G., 1977. Cost of propagating broccoli
plants through tissue culture. HortScience, 12: 543-544.
Andrus, CF., Seshandri, V.S. and Grimball, P.C., 1971. Production of seedless water-
melons. U.S.D.A. Tech. Bull., 1425.
Bhandari, M.C. and Sen, D.N., 1971. Effect of cytokinin on seedling growth, excised
cotyledons, chlorophylls and protein contents of Citrullus Zanatus (Thumb.). Mansf.
Biochem. Physiol. Pflanzen (Jena), 162: 481-488.
Henderson, W.R., 1977. Effect of cultivar, polyploidy and “reciprocal” hybridization on
characters important in breeding triploid seedless watermelon hybrids. J. Am. Sot.
Hortic. Sci., 102: 293-297.
Henderson, W.R. and Jenkins, SF., 1977. Resistance to anthracnose in diploid and poly-
ploid watermelons. J. Am. Sot. Hortic. Sci., 102: 693-695.
Kihara, H., 1951. Triploid watermelons. Proc. Am. Sot. Hortic. Sci., 58: 217-231.
Linsmaier, E.M. and Skoog, F., 1965. Organic growth factor requirements of tobacco
tissue cultures. Physiol. Plant., 18: 100-127.
Murashige, T., 1974. Plant propagation through tissue cultures. Annu. Rev. Plant Physiol.,
25: 135-166.
Sohi, H.S. and Maholay, M.N., 1974. Studies on mycoflora of watermelon seeds. Indian
J. Mycol. Plant Pathol., 4: 25-28.