title: Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes.
abstract: Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. Effective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5-5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40-85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.
authors.affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479, Poznań, Poland. tpni@igr.poznan.pl.
authors.forename: Tomasz
authors.identifier: http://orcid.org/0000-0002-5754-483X
authors.initials: T
authors.lastname: Pniewski
authors.affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479, Poznań, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Czyż
authors.affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479, Poznań, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wyrwa
authors.affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479, Poznań, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Bociąg
authors.affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479, Poznań, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Krajewski
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 05-216, Warsaw, Poland.
authors.forename: Józef
authors.initials: J
authors.lastname: Kapusta
mesh_terms: D006514:Hepatitis B Surface Antigens / Q000378:metabolism*; D018545:Lactuca / Q000235:genetics* / Q000254:growth & development*; D058634:Molecular Farming / Q000379:methods*; D030821:Plants, Genetically Modified; D046509:Tissue Culture Techniques / Q000379:methods*
chemical_list: D006514:Hepatitis B Surface Antigens
keywords: HBV surface antigens; HBsAg; Lettuce; Micropropagation; Oral vaccine; Plant material standardisation