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Agrobacterium-mediated Transformation of Nicotiana tabacum cv. Xanthi Leaf Explants

AR Ana Lilia Rosales-Campos
AG Abel Gutiérrez-Ortega

Published: Jan 20, 2019 DOI: 10.21769/BioProtoc.3150 Views: 11435

Edited by: Adam Idoine Reviewed by: Joëlle Schlapfer

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Abstract

Plant transformation has become an important technology for the large-scale production of a number of goods, ranging from vaccines and pharmaceutical products to biofuel. A plant that is frequently used for this purpose is Nicotiana tabacum due to several advantages: it can be easily regenerated, its genome is completely sequenced and genetic transformation is straightforward. We describe here the most common method for tobacco genetic transformation, which involves Agrobacterium tumefaciens. Many plasmid vectors are compatible with this bacterium, allowing the transformation of a wide range of plant species, as well as the insertion of more than two genes of interest in the plant genome. Furthermore, the capability to transfer a single copy DNA fragment to its host reduces the probability to switch off the gene or genes of interest. Here, we present a simple and reliable methodology to transform tobacco leaf explants using Agrobacterium tumefaciens to insert two genes of interest.

Keywords: Agrobacterium tumefaciens search Genetic transformation search In vitro culture search

Background

Plants have been exploited as expression bioreactors for vaccine antigens, antibodies for immunotherapy, nutritional supplements, enzymes, and even peptides of interest with industrial applications (Sharma and Sharma, 2009). They require relatively low financial investment to conduct initial studies, constituting a large advantage for biotechnology entrepreneurs (Yao et al., 2015). After the first plant transformation reports (Fraley et al., 1983; Herrera-Estrella et al., 1983), protocols have not changed a lot using Agrobacterium as a method for this purpose; nevertheless, other methods have been developed to enhance transformation efficiency, such as biolistics. Currently, tobacco is among the most important plant platforms for protein expression; its cells, as other eukaryotic cells, can perform post-translational modifications to the proteins of interest and tobacco plants can be easily regenerated by organogenesis or embryogenesis; additionally, its genome is completely sequenced. Current protocols allow for the insertion of multiple transgenes, requiring only one selectable marker (Wang, 2015). Our protocol is based on Curtis et al. (1995) and involves co-cultivation Agrobacterium-Nicotiana tabacum, like another protocol with algae (Pratheesh et al., 2014) or fungi. We were able to transform Nicotiana tabacum with a binary plasmid that contained two expression cassettes for the simultaneous expression of two viral antigens and confirmed that both genes were expressed by RT-PCR analysis in transformed plants.

Materials and Reagents

  1. 15 ml centrifuge tubes
  2. Plastic pots (Home depot, catalog number: 504092)
  3. Scalpel
  4. Parafilm
  5. Aluminum foil
  6. Disposable plastic Petri plates
  7. Electroporation cuvettes 0.2 cm (Bio-Rad, catalog number: 1652086)
  8. Plastic vessels for plant tissue culture, 1 L (Sigma-Aldrich, catalog number: B8648)
  9. Vessel for plant tissue culture, 100 ml (Sigma-Aldrich, catalog number: V8630)
  10. Tobacco seeds cv. Xanthi
  11. Agrobacterium tumefaciens strain LBA4404
  12. pCAS35S plasmid vector 
  13. Binary plasmid vector pCAMBIA2300gp5-m 
  14. Murashige and Skoog basal medium w/vitamins (PhytoTechnology Laboratories, catalog number: M519)
  15. Distilled water 
  16. Cefotaxime (Sigma-Aldrich, catalog number: C7039)
  17. Kanamycin (Thermo Fisher, catalog number: 11815024)
  18. Acetosyringone (3’5’Dimethoxy-4’hydroxyacetophenone) (Sigma-Aldrich, catalog number: D134406)
  19. YM medium broth (Amresco, catalog number: J904)
  20. Agar (Difco, catalog number: 281230)
  21. 1 N NaOH
  22. 1 N HCl
  23. 96% ethanol
  24. Methanol
  25. Sodium hypochlorite (household bleach 6%)
  26. Phytagel (Sigma-Aldrich, catalog number: P8169)
  27. Sucrose (Sigma-Aldrich, catalog number: S5391)
  28. Glycerol
  29. Benzyl aminopurine (BAP) (Sigma-Aldrich, catalog number: B3408)
  30. Benzyl aminopurine (BAP) stock (1 mg/ml) (see Recipes)
  31. Acetosyringone 100 mM (see Recipes)
  32. MS basal medium (see Recipes)
  33. MS co-cultivation medium (see Recipes)
  34. MS selection medium (see Recipes)
  35. MS rooting medium (see Recipes)

Equipment

  1. Micropipettes
  2. 250 ml flasks
  3. Glass containers
  4. Curved and straight tweezers
  5. pH meter (Bante 210)
  6. Orbital mixer
  7. Analytical balance (A&D, catalog number: 11100-80)
  8. Autoclave
  9. Vortex
  10. Gene pulser II electroporator (Bio-Rad) 
  11. Laminar flow hood
  12. Growth chamber 24 °C with 12/12 h photoperiod
  13. Greenhouse

Procedure

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant transformation > Agrobacterium

Molecular Biology > DNA > Transformation

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