Agrobacterium- Mediated Transformation of Tobacco Plants using Walnut Polyphenol Oxidase Gene

Abstract

Polyphenol oxidase (PPO) is a well-recognized copper-containing enzyme which can catalyze oxidation of a great variety of phenolic compounds. PPO is found in diverse microorganisms, plants, and animals. To examine the anti-pathogenic role of PPO in walnut (Juglans regia L.), Agrobacterium tumefaciens strain LBA4404 was used to transform tobacco (Nicotiana tobacum L.) explants. Recombinant binary vector pBI121, containing the walnut JrPPO gene and the nptII gene as a selectable marker, was incorporated into A. tumefaciens. MS medium supplemented with 50 mg/l of kanamycin and 200 mg/l of cefotaxime was used as a selection medium. Plantlets were regenerated from putatively transgenic calli and polymerase chain reaction and Southern blot analysis were performed to confirm the integration of JrPPO into the genome. To evaluate the function of PPO in pathogen resistance, transgenic tobacco plants were exposed to Pseudomonas syringae pv. tabaci. According to symptom progress and quantitative analyses, although there was no significant difference in transgenic tobacco, but mean comparison showed that disease severity of transgenic tobacco was less than wild plants. Finally, it may be concluded that PPO has a notable function in the resistance process in walnut, but tobacco transgenic plants might not be a suitable test plant to examine the resistance role of PPO in walnut.