Regeneration and infection mediated by Agrobacterium tumefaciens usingexplants of tobacco (Nicotiana tabacum L.) cv. SR1
Abstract (Summary)The modern molecular biotechnology allows the production of several recombinantproteins using different expression systems. Plant-based vaccines may provide anattractive, safe and inexpensive alternative to conventional vaccine production.Antigens, like HBsAg from common infectious organisms like hepatitis-B virus canbe produced in DNA-transformed plants. An expression system that uses plants asbioreactors needs an efficient in vitro regeneration and transformation process.The main aim of the present work was to develop an efficient regeneration andtransformation protocol in order to establish an expression system for theproduction of the HBsAg antigen. Factors influencing regeneration, selection andtransformation frequencies using the Agrobacterium-mediated protocol developedfor Nicotiana tabacum L. cv. SR1 leaf and internodal stem segments wereoptimized, with particular emphasis on important transformation parameters. Forregeneration the ideal kinetine concentration was 1.0 mg.L -1 and 2.0 mg.L -1 forinternodal stem segments and leaf segments respectively. The canamicinatolerance test was highly efficient and the regeneration suppression was achievedat 50 mg.L -1 and at 100 mg.L-1 for leaf segments and internodal stem segmentsrespectively. Although the good performance of leaf segments in regenerationtests, in Agrobacterium mediated transformation experiments, internodal stemsegments were significantly more efficient. Even so, the ideal parameters fortobacco cv.SR1 transformation include explants pre-conditioning for 4 days inregeneration medium, explants immersion for 10 min with Agrobacterium in MSmedium and subsequent co-cultivation of 24 h at semi-solid medium. The explantsshould be washed with the antibiotic cefotaxime (100 mg.L -1 ) and tetracycline (400mg.L -1 ) since this was the less harmful antibiotic combination. The regenerationafter transformation should be done in the absence of kanamycin that negativelyaffects shoots regeneration. The use of the optimized protocol resulted insignificantly higher transformation frequencies. Many of the explants, callus andshoot primordia were chimeric for ? -glucoronidase activity and ranged from non-stainingto solidly staining explants. But solidly transformed plants with integratedT-DNA were not confirmed, such plants should be analyzed by other moleculartechniques to verify the presence of the hbsag gene and the viability of the strainC58C1 of Agrobacterium for effective plant transformation. The demonstratedsuccess of the regeneration and transformation process provides an importantadvance in the strategy to achieve a stable transformation using plants asbioreactor for recombinant expression of the HBsAg antigen.
Advisor:Pedro Canisio Binsfeld
Source Type:Master's Thesis
Keywords: Nicotiana tabacum
Date of Publication:06/27/2005