Agrobacterium tumefaciens-mediated transformation and regeneration of muskmelon (Cucumis melo L.)‎

Abstract EN

Agrobacterium-based plasmid vectors allow the transfor-mation 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, lind?n) and ?-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.