AGROINJECTION TECHNIQUE FORTRANSIENTGENE EXPRESSION AS RAPID AND HIGHLY EFFICIENT METHOD FOR POTATO AGROBACTERIUM-BASED TRANSFORMATION BY CRY1CA GENE

摘要

Potato is one of the major food crops and considered the fourth most important food crops worldwide.The insect and fungal infections are the significant constraints for potato production in the Arab countries,causing yield losses reaching more than 20%.We have used and developed a rapid and low-cost transient transformation assay by using in-planta agroinfiltratoin in potato seeds slide,which transformation efficiency is higher than those of the conventional transient assays are.Polymerase chain reaction (PCR) was used to amplify a single copy of Bacillus thuringiensis Crystal Protein CrylCa (crylCa) gene from extracted DNA to screen for the presence or absence of this gene.Potato slices were agroinjected with plasmid pRI201-AN-GUS DNA for transient assay of P-glucuronidase gene (GUS).The transformation was performed using the disarmed A.tumefaciens strain LBA4404 harboring a binary vector pRI201-AN-GUS- CrylCa.Genetically modified potato plants were selected on kana-mycin-containing Murashige and Skoog medium (MS-media) and subcultured later to MS-media supplemented with 1.0 mg 1-1 6-Benzylaminopurine (BA) and 1.5 mg 1-1 -Naphthaleneacetic acid (NAA).A.tumefaciens strain LBA4404 carried out the plant binary vector pRI201-AN-GUS,and pRI201-AN-GUS-CrylCa were used for transient expression assay.The Synthetic CrylCa (crylCa) gene,under the control of a CaMV 35S and the caste,also contained P-glucuronidase (GUS) gene.Agroinjection is an efficient transient uidA expression assay for reporter gene and analysis of candidate genes in plants.The successful transformation of CrylCa gene potato plants will contribute to increasing yield and food security in the future.Finally we describe here a protocol for the genetic transformation of potato based on agroinjection using A.tumefaciens carrying a plasmid (pRI201-AN-GUS-CrylCa) expressing a reporter uidA gene in addition to describing transient expression evidence based on histological and molecular assays.Successful transformation of transgenic potato plants will contribute to increasing the yield and food security in Saudi Arabia in the near future.