Full Length Research Paper
of an efficient Agrobacterium tumefaciens-mediated
leaf disc transformation of spine gourd (Momordica dioica
Roxb. ex Willd)
and Ill-Min Chung*
Department of Applied Life Science, Konkuk University, Seoul
143-701, South Korea.
*Corresponding author. E-mail:
Tel: +82-2-450-3730. Fax: +82-2-446-7856.
6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid;
GA3, gibberellic acid; GUS,
β-glucuronidase; IBA, indole 3-butyric acid; LB,
Luria-Bertani; npt II, neomycin phosphotransferase
polymerase chain reaction; RT-PCR, reverse
transcription-PCR; MSB5, Murashige and
Skoog basal salt mixture + B5 vitamins.
Accepted 15 November, 2011
Spine gourd (Momordica dioica Roxb. ex Willd) is a
medicinally and economically important plant and also used
as vegetable. In this study, we established an
Agrobacterium tumefaciens-mediated transformation
procedure for M. dioica. Leaf explants were incubated
with A. tumefaciens strain
LBA4404 containing a binary vector
carrying the reporter gene β-glucuronidase intron (GUS-INT)
and the marker gene neomycin phosphotransferase (NPTII). Following
co-cultivation, leaf explants were
cultured on Murashige and Skoog
(MS) + Gamborg et al., (B5) medium
supplemented with 6.6 μM 2,4-dichlorophenoxyacetic acid
(2,4-D) combined with 3.3 μM 6-benzylaminopurine (BAP)
containing 100 mg L–1 kanamycin and 300 mg L-1
carbenicillin. Kanamycin-resistant calluses were induced
from the leaf explants after three weeks. Shoot regeneration
was achieved after transferring the calluses onto fresh
selection medium 8.8 μM BAP and 2.2 μM
2,4-D. Transgenic shoots were excised from callus and
elongated in MS medium fortified with 3.0 μM gibberellic
acid (GA3), 100 mg L-1 kanamycin
and 300 mg L-1
carbenicillin. Finally, the shoots were rooted on
MS basal medium supplemented with 3.0 μM indole 3-butyric
acid (IBA) and 100 mg L-1 kanamycin. High
transformation frequency was achieved by using three-day-old
precultured leaf explants. Furthermore, the presence of
acetosyringone (200 μM), infection of explants for 30 min
and three days of cultivation proved to be critical factors
for greatly improving the transformation efficiency.
Incorporation and expression of the transgenes confirmed by
polymerase chain reaction (PCR), Southern blot analysis,
reverse transcription (RT)-PCR and GUS histochemical assay.
Using this protocol, transgenic M. dioica plants can
be obtained in approximately three months with a high
transformation frequency of 9%.
Agrobacterium tumefaciens, acetosyringone, growth
regulators, GUS, genetic transformation, Momordica dioica.