Nicotiana tabacum
Nicotiana tabacum
Common Name : Tobacco
Scientific Name : Nicotiana tabacum
Eukaryotic Multicellular Plant
Biosafety level : BSL1
Benefits and Applications
The commercial tobacco industry is widely known for its manufacture of cigarettes, cigars,
chewing tobacco, etc. Over recent years, Nicotiana tabacum has become a plant model system
for tissue culture and genetic engineering.
Nicotiana tabacum offers unique properties of high biomass yield-high soluble protein levels
compared with many other model and crop species, an attractive feature for a protein
production platform.
Tobacco also offers various ways of expressing proteins of interest, such as transient based
expression via agrobacterium or viral induction.
Tobacco is neither a food nor feed crop, thus reducing the likelihood of transgenic material
contaminating the food or feed chains. These features have made tobacco particularly well
suited for plant-based production of biopharmaceutical products.
Tobacco has also been employed for the culture and fusion of plant protoplasts, providing
invaluable information on ways to explore the potential of somatic hybridization in other crops.
Tobacco BY2 cell line has played an important role in the developments in the field of molecular
farming for the expression and/or production of recombinant proteins, vaccines and antibodies
are gaining significance for industrial use and human healthcare.
Other advantages include:
Faster production time from gene to protein
Fast doubling time of 11-15 hrs
Low capital investment
Low operating costs
Easily scaled operations
Presence of post-translational protein modifications specific to eukaryotes, such as folding,
glycosylation, or assembly of multimeric proteins
Low pathogenic and endotoxic contamination
High homogeneity
Maintenance
The Tobacco BY-2 cell line is a suspension culture derived from a callus induced from a stem of
Nicotiana tabacum L. cultivar Bright Yellow 2. BY-2 cells are maintained in a modified Linsmaier
and Skoog (mLS) medium (pH 5.8) in dark at 27°C in rotatory shaker(130 rpm) and subcultured
at one-week intervals.
Agrobacterium mediated transformation of BY2 cells
Agrobacterium overnight culture is spun down and redissolved in 1-2 ml MS liquid medium (4.4
g/l MS+salts+vitamins, 30 g/l sucrose, 0.2 mg/l 2,4 D, pH5.7 with KOH). 40 ml 3 day old BY2
cell suspension culture is filtered using Whatman filter, and cells are resuspended in the same
volume of MS liquid medium. 4 ml of cell suspension was mixed with 100 Âμl agrobacterium
culture and transferred to the Petri dish with Whatman filter paper on the bottom. Petri dishes
are sealed with parafilm and incubated for 2 to 3 days at 28ºC in the dark without shaking.
After incubation filter papers are washed with 3×10 ml liquid MS +500 Âμg/ml Cefotaxime and
transferred to selective plates containing MS agar + kanamycin or hygromycin ((4.4 g/l
MS+salts+vitamins, 30 g/l sucrose, 1 mg/l 2,4 D, pH5.7 with KOH, 8 g/l agar, 50 Âμg/ml
kanamycin or 20 Âμg/ml hygromycin). Washing liquid of all plates with the same transformation
vector (containing suspension cells) was collected in a 50 ml tube and cells were spinned down
at 500 rpm for 3 min. Pellets are resuspended in 30 ml liquid MS +500 Âμg/ml Cefotaxime,
spinned down again and resuspended in 4 ml/plate. Cells are transferred to selective plates
containing washed Whatman filter paper and spread out evenly over the filter paper. Plates are
incubated for 6 days at 28ºC in the dark.
Growth of transformed cells on Zinc gradient. After 6 days of selection on MS+kanamycin or
hygromycin plates, filter papers were cut into two and transferred to selective plates (MS agar +
antibiotics) containing different concentrations (0, 50, 100, 200, 300 or 500 ÂμM) of zinc (see:
table 1). Plates were incubated for two weeks at 28ºC in the dark.
Analysis of calli Plates are analyzed using fluorescence binoculars. Additionally, cells were
resuspended, put onto a microscope slide and analyzed.