Adenine Sulphate and L-Glutamine Enhance Multiple Shoot Induction from
Cotyledon Explants of Melon (Cucumis melo L. cv. Swarna)
M. Muruganantham1, A. Ganapathi1, N. Selvaraj2, R. Prem Anand1, A.Vasudevan1, and
G.Vengadesan1
1
Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620
024, Tamil Nadu, and 2Department of Botany, Periyar E.V.R college, Thiruchirappalli - 620 023.
Tamil Nadu, India. Email: ganabiotech@rediffmail.com
Introduction. Cucumis melo L.is a popular
commercial as well as household vegetable in India.
Among various uses, it is mainly known for fresh
salad and pickling. However, this economically
important crop is susceptible to a number of
devastating insects and diseases (7) including papaya
ringspot virus and cucumber green mottle mosaic
virus, which seriously limit productivity. Hence, this
crop deserves improvement in terms of disease
resistance, abiotic stress and higher yield. Melon is
known to be recalcitrant to regeneration (1,2). The
development of tissue culture protocols is one of the
solutions to address these problems. The present
study was conducted to produce multiple shoots in a
local cultivar of melon by using important additives,
adenine sulphate (AdS) and glutamine, in the culture
medium.
acid (GA3) (0.5mg/l). The cultures were maintained
at 25 ± 2º C under white fluorescent light (Philips
India Pvt. Ltd.) with a photon flux of (30µ mol m-2
s-2) at a 16h photoperiod.
Materials and Methods. The Indian melon cultivar
Swarna (Indo-American Hybrid Seeds (India) Pvt.
Ltd., Bangalore, India) was used in the present study.
Seeds were surface sterilized by the usual
sterilization procedure (3). The seed coats were
aseptically removed and the two cotyledons were
separated from the embryonic axis. Mature
cotyledons were used as explants. The cotyledonary
pieces with intact proximal ends (0.5 cm) were
inoculated in Murashige and Skoog (MS) medium (4)
containing 0.8% bacto agar, 3% sucrose and
combinations of the growth regulators BA, Kinetin
(Kn), and the additives AdS and L-glutamine. Every
treatment was tested at five different concentrations
(mg/l): BA – 0.5, 1.0, 1.5, 2.0, 2.5. Kn - 0.5, 1.0, 1.5,
2.0, 2.5. AdS – 5, 10, 15, 20, 25. L-Glutamine - 5, 10,
15, 20, 25.
Effect of BA with additives. Multiple adventitious
shoot buds (Fig. 1) were initiated from the proximal
end of the explants after 2-3 weeks of inoculation in
MS medium containing BA in combination with AdS
and glutamine. A maximum number of 30.6
shoots/explant was produced with BA (2.0mg/l), AdS
(15mg/l) and glutamine (15mg/l) in two subcultures
(Table 1). The present study revealed that a
combination of additives (AdS and glutamine) with
optimal concentration of BA (2.0mg/l) promoted the
highest shoot bud induction as well as adventitious
shoot production (Fig 2). The regenerated shoots
were elongated in MS medium with (0.5mg/l) GA3
(Fig 3). This protocol is rapid, less time consuming
and highly reproducible, and could be applied in
genetic manipulation of this cultivar by
transformation methodologies.
Results and Discussion.
Effect of cytokinins.
Multiple adventitious shoot buds were induced from
the proximal end of the cotyledons in MS medium
containing BA (2.0mg/l) or Kn (1.5mg/l) after 10-12
days of inoculation. A maximum number of shoots
(12.2/explants) was obtained after 3-4 weeks of
culture supplemented with only BA (2.0 mg/l) (Table
1). Lower numbers of shoots were produced in
medium containing Kn (1.5mg/l). Similar results
were observed by others in melon (3,5) and in
cucumber (6). The shoots were subcultured in
medium with the same composition.
To facilitate shoot elongation, the regenerated shoots
were grown in MS medium fortified with gibberellic
Cucurbit Genetics Cooperative Report 25: 22-24(2002)
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Cucurbit Genetics Cooperative Report 25: 22-24(2002)
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Table 1. Effect of plant growth regulators and additives on multiple shoot induction from mature
cotyledon explants of melon (C. melo cv. Swarna) cultured on MS medium.
Treatment
(mg/l)
BA
Kn
BA + AdS
BA + L-Glutamine
BA + AdS + L-Glutamine
Kn + AdS
Kn + L-Glutamine
Kn + AdS + L-Glutamine
2.0
1.5
2.0 + 15
2.0 + 15
2.0 + 15 + 15
1.5 + 15
1.5 + 15
1.5 + 15 + 15
Number of
shoots/explants
Shoot length
(cm)
12.2 b
2.0 h
9.2 c
8.0 d
30.6 a
6.3 e
5.0 f
4.0 g
6.2 b
n.d.
5.0 c
4.3 d
7.9 a
4.0 de
2.3 f
2.0 g
Means with the same letter in a column are not significantly different according to Duncan’s
Multiple Range Test at the 5% level.
n.d.- not determined.
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