1. HORTSCIENCE 26(7):908-910. 1991.
Plant Regeneration from Cotyledons
of Cucumis melo ‘Topmark’
Paula P. Chee
Molecular Biology Research, The Upjohn Company, 301 Henrietta
Street, Kalamazoo, MI 49007
Additional index words. muskmelon, plant tissue culture, organogenesis, cantaloupe
Abstract. A procedure for the regeneration of muskmelon (Cucumis melo L.) CV.
Topmark via shoot organogenesis from cotyledon explants is described. The best in-
duction medium for a morphogenic response was MS salts and vitamins medium with
BA at 1.0 mg·liter-1. Further vegetative bud development was completed by transfer-
ring organogenic tissue to MS medium containing BA at 0.05 mg·liter-1. The shoots
were rooted in MS medium containing NAA at 0.01 mg·liter -1. Morphologically normal
plantlets were obtained. Chemical abbreviations used: 6-benzylaminopurine (BA); in-
doleacetic acid (IAA); naphthaleneacetic acid (NAA).
The literature descriptions of tissue culture for ≈15 min. Seed coats were removed man-
methods applied to muskmelon are few. ually. De-coated seeds were surface steri-
Blackmon and Reynolds (1982) first docu- lized for 10 min with 10% (v/v) solution of
mented the development of adventitious shoots commercial bleach containing a trace amount
from cotyledon and primary leaf explants. of Tween-20 (Sigma, St. Louis) spreader and
Moreno et al. (1985), Kathal et al. (1988), then rinsed three times with sterilized dis-
Dirks and Buggenum (1989), and Niedz et tilled water. Sterilized decoated seeds were
al. (1989) also reported that muskmelon plants germinated on MS medium (Murashige and
could be regenerated from cotyledon and leaf Skoog, 1962) containing 0.1% activated
explants. Oridate and Oosawa (1986) ob- charcoal (optional) and at 26C with a 16-h
served plantlet regeneration from suspension photoperiod under standard cool-white flu-
culture-derived somatic embryos. These pro- orescent lamp (4 klx). Unless otherwise stated,
cedures are not necessarily applicable to other all media were supplemented with 3% su-
cultivars (D. Gonsalves, personal commu- crose and solidified with 0.6% Phytagar
nication) because of the genotype specificity (Gibco). The pH of all media was adjusted
of the methods. This reort describes a tech- to 5.8 before they were autoclave at 121C
nique for the regeneration of whole musk- for 20 min.
melon plants from cotyledon explants C. melo Cotyledons from 9- to 10-day-old seed-
‘Topmark’ through shoot organogenesis. The lings grown in vitro were cut longitudinally
same procedure was also applicable to ‘Per- into halves and placed on induction medium
lita’ muskmelon, another netted cultivar. with the abaxial side down. Induction me-
Seeds of ‘Topmark’ (Asgrow Seed Co., dium was composed of MS medium (Mu-
Kalamazoo, Mich.) were soaked in tap water rashige and Skoog, 1962) supplemented with
either BA (0.0, 0.5, 0.75, 1.0 mg·liter -1) or
Received for publication 5 June 1990. The cost of
IAA at 1.5 mg·liter -1 and kinetin at 6.0
publishing this paper was defrayed in part by the mg·liter -1 (I-K medium; Moreno et al., 1985).
payment of page charges. Under postal regula- Explants were incubated at 26C under dif-
tions, this paper therefore must be hereby marked fuse cool-white fluorescent lamps (4 klx).
advertisement solely to indicate this fact. One hundred explants were evaluated per
908 HORT SCIENCE , VOL . 26(7), JULY 1991

2. Fig. 1. Organogenic tissue derived from coty-
ledon explant. Fig. 3. Shoot formation from organogenic tis-
sues.
Fig. 2. Organogenic tissue with developing veg-
etative buds.
organogenic tissues lost morphogenic poten-
tial and produced friable callus when serially
subculture on I-K medium. Shoot devel-
opment was subsequently promoted by
treatment. After 3 weeks, cultures were transfer of the organogenic tissues to MS with
evaluated for the production of organogenic 0.05 mg BA/liter. Forty percent of the or-
sectors. To resolve statistical differences be- ganogenic tissue then produced many shoot
tween treatments, data from two independent buds (Fig. 2). Shoots developed within 20 Fig.4. Regeneration stages of muskmelon from
experiments were entered into a contingency days of transfer to this latter medium (Fig. cotyledon explant. (Upper left) Initiation of or-
table and x2 values were computed. Agar- 3). Further shoot development was encour- ganogenic tissue. (Upper right) Differentiation
solidified MS medium supplemented with one aged by transfer of organogenic tissue to of organogenic tissue. (Lower left) Plantlet de-
velopment. (Lower right) Regenerated musk-
of four levels of NAA (0.0, 0.001, 0.01, 0.1 Magenta boxes (Magenta Corp., Chicago) melon plant.
mg·liter -1) was evaluated for rooting of containing four sheets of sterile paper towel
shoots. When plantlets developed an exten- saturated with ≈20 ml of MS liquid medium
sive root system, they were transplanted to containing BA at 0.05 mg·liter -1. Fresh me- The differences between the present results
pots containing a planting mix and covered dium was added to boxes every 2 weeks. and the earlier ones are the cultivar used,
with clear polyethylene storage bags for Developing shoots, ≈1 cm in length, were concentration of growth regulators, and the
hardening-off. Subsequently, the regener- dissected from the mass of organogenic tis- ability to propagate organogenic tissues. We
ated plants were potted in soil and grown in sues and transferred to rooting medium. The used cotyledons of ‘Topmark’ and ‘Perlita’
a greenhouse. remainder of the organogenic tissue was re- as explant sources; while the others used cot-
Two types of tissue were produced by the turned to liquid medium in Magenta boxes yledons of ‘Hale’s Best Jumbo’ (Niedz et
cotyledon explants, a compact green organ- for further shoot development. The differ- al., 1989), ‘Accent’, ‘Galia’, 4215, ‘Presto’,
ogenic tissue and a green friable callus. The ences in the frequency of root development and ‘Viva’ (Dirks and van Buggenum, 1989).
best induction medium was MS medium due to NAA concentration were significant. We were able to induce organogenic callus
supplemented with 1.0 mg BA/liter; 80% of The highest rooting percentage was obtained tissues by culture of the explants on MS me-
explants cultured on this medium produced in a medium containing 0.01 mg NAA/liter dium with 1.0 mg BA/liter and shoot devel-
organogenic tissue (Table 1). The difference (Table 2). Although medium containing no opment could be promoted by transfer of the
in response between all media formulations NAA was ineffective for root formation, a organogenic tissues to MS medium with 0.05
was highly significant. The difference be- difference between NAA at 0, 0.001, and mg BA/liter. In contrast, Niedz et al. (1989)
tween 1 and 0.75 mg·liter-1 was also highly 0.1 mg·liter-1 could not be resolved (x2 = reported that 75% of the explants were in-
significant (x2 = 19.1**). The least organ- 2 . 6 3N S). The difference between 0.01 duced to form organogenic callus with the
ogenic tissue was generated from explants mg·1iter -1 and all other treatments pooled use of IAA at 0.875 mg·liter-1 and abscisic
cultured on a medium with 0.5 mg BA/liter was highly significant (x2 = 14.96**). The acid at 0.26 mg·liter-1 in combination with
other than from the control. Only one-fourth medium containing NAA at 0.1 mg·liter-1 BA at 1.0 mg as inductive growth regulators.
as many explants with I-K medium produced stimulated callus growth at the shoot bases. Shoot formation (30%) was obtained by
organogenic tissue as those with 1 mg BA/ Rooted plants flowered and produced fruits transfer of shoot buds to MS medium with
liter. with seed. BA at 0.675 mg·liter -1. In addition, Dirks
Organogenic tissues could be serially Organogenesis from cotyledon of Cucu- and van Buggenum (1989) obtained shoot
propagated by subculture on MS with BA at mis melo has been reported earlier (Dirks and formation directly from every cotyledon ex-
0.75 or 1.0 mg·liter -1 (Fig. 1). Generally, van Buggenum, 1989; Niedz et al., 1989). plant cultured on MS medium that contained
HORT SCIENCE , VOL . 26(7), JULY 1991 909

3. BA at 1.0 mg·1iter-1. In the work reported in gene transfer using either Agrobacteria or Kathal, R., S.P. Bhatnagar, and S.S. Bhojwani.
here, the competence of organogenic tissue microprojectile bombardment. 1988. Regeneration of plants from leaf explants
was maintained by serial propagation on MS of Cucumis melo cv. Pusa Sharbati. Plant Cell
Literature Cited Rpt. 7:449-451.
medium with 1.0 mg BA/liter.
Moreno et al. (1985) maintained organo- Blackmon, W.J. and B.D. Reynolds. 1982. In vi- Moreno, V., M. Garcia-Sogo, I. Granell, B. Gar-
tro shoot regeneration of Hibiscus acetosella, cia-Sogo, and L.A. Roig. 1985. Plant regen-
genic tissues of ‘Amarillo Oro’ muskmelon eration from calli of melon (Cucumis melo L.,
musk-melon and winged bean. HortScience
for 1 year using I-K medium. However, when cv. “Amarillo Oro”). Plant Cell Tissue Organ
17:588-589.
organogenic calli of ‘Topmark’ were serially Chee, P.P. 1990a. Transformation of Cucumis sa- Culture 5:139-146.
subculture onto I-K medium, morphogenic tivus via Agrobacterium tumefaciens, p. 85–90. Murashige, T. and F. Skoog. 1962. A revised me-
competency was lost and only friable tissues In: H.J.J. Nijkamp, L.H.W. Van Der Plas, and dium for rapid growth and bioassays with to-
were produced. Such difference in morpho- J. Van Aartrijk (eds.). Progress in plant cellular bacco tissue cultures. Physiol. Plant 15:473-
genic responses is likely due to genetic dif- and molecular biology. Kluwere Academic, The 497.
ferences between the cultivars. The procedure Netherlands. Niedz, R.P., S.S. Smith, K.B. Dunbar, C.T. Ste-
reported here (Fig. 4) may be applicable to Chee, P.P. 1990b. Transformation of Cucumis sa- phens, and H.H. Murakish. 1989. Factors in-
other muskmelon cultivars, as ‘Perlita’ and tivus tissue by Agrobacterium tumefaciens and fluencing shoot regeneration from cotyledonary
the regeneration of transformed plants. Plant Cell explants of Cucumis melo. Plant Cell Tissue
‘Topmark’ responded similarly. Cotyledons and Organ Culture 18:313–319.
Rpt. 9:245-248.
have proven to be an excellent source of Dirks, R. and M. van Buggenum. 1989. In vitro Oridate, T. and K. Oosawa. 1986. Somatic em-
transformation experiments in Cucumis sa- plant regeneration from leaf and cotyledon ex- bryogenesis and plant regeneration from sus-
tivus (Chee, 1990a, 1990b). The simple re- plants of Cucumis melo L. Plant cell Rpt. 7:626- pension callus culture in melon (Cucumis melo
generation method described may be useful 627. L.) Jpn. J. Breed. 36:424-428.
HORT SCIENCE , VOL . 26(7), JULY 1991