Journal of Food, Agriculture and Environment




In vitro reliable regeneration protocol of tomato plants and efficient transformation system for transient gene expression in transgenic tomato varieties resistance to fungi


Author(s):

Mohei El-Din Solliman 1,2*, A. A. Al-Khateeb 2, S. A. Al-Khateeb 2, Heba Allah Mohasseb 1, Ahmed  Elkazzaz 1

Recieved Date: 2016-06-10, Accepted Date: 2016-08-30

Abstract:

Tomato considers as the second important cultivated crop after potato, worldwide. The main objective of this study was to produce transgenic plants of tomato  using a plant antifungal gene (defensin). During this study, we cloned plant defensin (AFP) gene amplified from Phaseolus vulgaris. The sequence of pdf gene was deposited in GENBANK with the accession number (KJ939334.1). The AFP gene was amplified using direct PCR from total DNA extract of P. vulgaris. Reliable regeneration protocol for tomato  has been optimized in vitro. The regeneration occurred directly or indirectly from hypocotyl, cotyledon and leaf segments (explants). Tomato callus showed the embryonic like structures giving regenerated plants which developed to whole plantlets in vitro. The plantlets have been adapted to laboratory conditions successfully grown in pots filled with fertilized peatmoss outdoor in greenhouse. In plant transformation technology use of transient expression of a gene of interest from a foreign source is a valuable technique. There remained an urgent need to develop a transient assay technique to decrease the time to get the expression of any gene of interest in tomato plant tissues or fruits This method, called fruit agroinjection, β-glucuronidase (GUS) protein transient expression in the tomato fruit, with high expression levels in fruits. During this experiment we used Agrobacterium tumefaciens (strain LBA4404) carrying a plant binary plasmid having reporter GUS gene under CaMV 35S promoter (pRI201-AN-GUS) and the AFP under CamV 35S promoter (pRI201-AN-GUS-AFP). For efficient gene expression assay agroinfiltration is a powerful and trustworthy tool and can be utilized with many plant species. We are reporting out designed protocol for transient gene expression analysis and the expected outcomes of our study in tomato plants and fruits. Fruits agroinjection was assayed in our case study: the defensing- β-glucuronidase (AFP-GUS) genes expressions in tomato, we prove the efficient of this reliable technology as a tool for assay transgene expression in tomato plants. Our system provides a rapid, efficient and economical assay of gene function in tomato fruits tissues.

Keywords:

Tomato, agroinfiltration, Agrobacterium, transformation, defensing- (AFP) gene


Journal: Journal of Food, Agriculture and Environment
Year: 2016
Volume: 14
Issue: 3&4
Category: Agriculture
Pages: 29-36


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