Document Type : Research articles.
Author
Department of Genetics, Faculty of Agriculture, Fayoum University, Egypt.
Abstract
Agrobacterium-based plasmid vectors allow the transformation of a wide range of plant species by capitalizing on a
natural bacterial system to introduce DNA into the nuclear
genome of plants. A regeneration protocol for muskmelon
(Cucumis melo cv. Hales Best Jumbo) was established using
segments of cotyledon as explants, and combined this protocol
with A. tumefaciens-mediated transformation to produce
transgenic muskmelon plants. Cotyledon explants excised from 4-
day-old seedlings of muskmelon and cut into four segments after
removed the edges of the cotyledons, co-cultivated with A.
tumefaciens strain LBA4404 harboring binary pRGG bar plasmid.
The plasmid contained phosphinothricin acetyl transferase gene
(bar) as selectable marker for herbicide resistance (glufosinat
ammonium, lindán) and β-glucuronidase gene (gusA) as reporter
for 20 min. Then, transferred to regeneration medium, MS
medium supplemented with different combinations of plant
growth regulators (pH 5.0), and incubated in a growth room at
25°C with a photoperiod of 16h light/8h dark for 3 days. An
overnight bacterial culture and low pH medium (5.0) used during
co-cultivation, to improve the transformation efficiency. For shoot
induction, explants were transferred onto MS medium contained
different combinations of plant growth regulators with pH 5.8,
and 500 mg/l claforan (cefotaximum) was added to the medium,
to prevent the growth of Agrobacterium. The elimination of the
Agrobacterium was the key of successful regeneration and
transformation after co-cultivation. After co-cultivation on MS
medium with a low pH, explants were transferred to selective
medium, with higher pH 5.8, containing 500 mg/l claforan and
glufosinat ammonium in concentrations ranged from 0 to10 mg/l
(0, 1, 2, 3, 4, 5, 8 and 10 mg/l) to know the optimum selective
concentration during shoot formations. The results indicated that,
MS medium supplemented with 1.05 mg/l indole-3-acetic acid
(IAA)+0.6 mg/l 6-benzyladenine (BA)+0.24 mg/l abscisic acid
(ABA) ranked the best in shoot formations. Shoots formed from
co-cultivated cotyledons with Agrobacterium died under the
conditions of high concentration more than 3 mg/l glufosinat
ammonium. The medium was changed every two weeks till shoots
were induced. All shoots rooted on MS medium supplemented
with 0.3 mg/l indole-3-butyric acid (IBA). The expression of the
introduced gene construct was confirmed by GUS staining of
callus and shoot segments. Finally, transgenic muskmelon plants
were produced efficiently by inoculating pieces of cotyledons as
explants with A. tumefaciens strain LBA4404 harboring pRGG bar plasmid. The methods have been used to develop a more
efficient transformation and regeneration procedures by useful
genes, such as herbicide-resistance, that can be introduced into
muskmelon by indirect insertion of such genes using the genetic
engineering approach.
Keywords
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