AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION AND REGENERATION OF MUSKMELON (CUCUMIS MELO L.)

Abstract

Agrobacterium-based plasmid vectors allow the transformation of a wide range of plant species by capitalizing on anatural bacterial system to introduce DNA into the nucleargenome of plants. A regeneration protocol for muskmelon(Cucumis melo cv. Hales Best Jumbo) was established usingsegments of cotyledon as explants, and combined this protocolwith A. tumefaciens-mediated transformation to producetransgenic muskmelon plants. Cotyledon explants excised from 4-day-old seedlings of muskmelon and cut into four segments afterremoved 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 (glufosinatammonium, lindan) and β-glucuronidase gene (gusA) as reporterfor 20 min. Then, transferred to regeneration medium, MSmedium supplemented with different combinations of plantgrowth regulators (pH 5.0), and incubated in a growth room at25°C with a photoperiod of 16h light/8h dark for 3 days. Anovernight bacterial culture and low pH medium (5.0) used duringco-cultivation, to improve the transformation efficiency. For shootinduction, explants were transferred onto MS medium containeddifferent 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 theAgrobacterium was the key of successful regeneration andtransformation after co-cultivation. After co-cultivation on MSmedium with a low pH, explants were transferred to selectivemedium, with higher pH 5.8, containing 500 mg/l claforan andglufosinat ammonium in concentrations ranged from 0 to10 mg/l(0, 1, 2, 3, 4, 5, 8 and 10 mg/l) to know the optimum selectiveconcentration 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 fromco-cultivated cotyledons with Agrobacterium died under theconditions of high concentration more than 3 mg/l glufosinatammonium. The medium was changed every two weeks till shootswere induced. All shoots rooted on MS medium supplementedwith 0.3 mg/l indole-3-butyric acid (IBA). The expression of theintroduced gene construct was confirmed by GUS staining ofcallus and shoot segments. Finally, transgenic muskmelon plantswere produced efficiently by inoculating pieces of cotyledons asexplants with A. tumefaciens strain LBA4404 harboring pRGG bar plasmid. The methods have been used to develop a moreefficient transformation and regeneration procedures by usefulgenes, such as herbicide-resistance, that can be introduced intomuskmelon by indirect insertion of such genes using the geneticengineering approach.