Optimising in vitro culture and Agrobacterium tumefaciens-mediated transformation protocols of tobacco BY-2 cells

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2012, Vol. 57, No. 8, pp. 128-137 This paper is available online at OPTIMISING IN VITRO CULTURE AND Agrobacterium tumefaciens-MEDIATED TRANSFORMATION PROTOCOLS OF TOBACCO BY-2 CELLS Nguyen Tuong Van1, Le Quynh Lien1 and Nguyen Thanh Van2 1Institute of Biotechnology, Vietnam Academy of Science and Technology 2Faculty of Biology, Hanoi National University of Education Abstract. In order to set up a system for production of recombinant proteins using suspension cell culture of Nicotiana tabacum cv. Bright Yellow 2 (BY-2), in vitro culture and genetic transformation protocols of such cells were optimised at the laboratory of Plant Cell Biotechnology, Institute of Biotechnology. Our experiments indicated that the development rate of BY-2 cells depended on initial cell concentrations. The best culture condition was the formula F5 with 1:20 dilution at the starting point (2.5 mL of initial cells/50 mL culture medium) which reached the exponential phase after 5 days and had maximum biomass of 1.206 g/mL after 9 days. Agrobacterium tumefaciens-mediated transformation procedure of BY-2 cells was optimised by monitoring transient gusA gene expression. There is a positive correlation between the amount of BY-2 cells and the density of Agrobacterium tumefaciens in coculture medium. The transformation efficiency was the highest at formulars 0.6-1, 0.8-4 and 1.0-5 (bacteria OD-plant cell formular). Keywords: BY-2 cells, Agrobacterium tumefaciens-mediated transformation, in vitro culture, GUS transient expression, coculture 1. Introduction Recombinant proteins are increasingly important components of medicine and applied chemistry. They are needed for a vast range of applications including therapeutics, vaccines, diagnostics and enzymes. Currently, most recombinant proteins originate from genetically engineered bacteria. Other sources are eukaryotes like yeast, human or animal cell lines or even transgenic animals. Compared to these systems, the production cost Received April 9, 2012. Accepted September 7, 2012. Biology Subject Classification: 362 196. Contact Nguyen Thanh Van, e-mail address: 128 Optimising in vitro culture and Agrobacterium tumefaciens-mediated transformation protocols... using plants is low and there is safety from contamination from pathogenic agents such as prions or viruses. In addition, plant cells, like microbes, can be maintained in simple media but, like animal cells, they can synthesize complex human proteins and glycoproteins, which are more similar to their native structural form when compared to the same proteins produced in yeast and filamentous fungi [3]. The application of this technology is dependent on the availability of efficient systems for the transfer of foreign genetic material into host cells. In recent years, much attention has been paid to plant cell culture. The Tobacco BY-2 cell line is the most widely used cell line used to test the production of recombinant proteins because of its relatively homogenous, high growth rate, and easily transformable either by particle bombardment or by co-cultivation with Agrobacterium tumefaciens. BY-2 is a cell line of tobacco that was induced from a pith of Nicotiana tabacum L. cv. Bright Yellow No.2 in 1968. This cell line was made by Dr. Kawashima at the Hatano Tobacco Experimental Station of the Japan Tobacco and Salt Public Cooporation [5]. The cells are relatively large and grow as long chains. BY-2 cells are rapidly growing cells. These cells can multiply up to 100 times within a week under conventional cell culture conditions [8]. There are hundreds of scientific papers published using this line for transformation work. For example, transformation by Agrobactium tumefaciens [2] and gene gun [5]. However, transformation frequencies were said to depend on a range of factors including the physiological state of the cultured cells, Agrobacterium strains and the coculture time period. According to An [1], maximum transformation frequency was obtained with exponentially growing plant cells. Yu and et al. [10] reported that transformed BY-2 cells were easily obtained when the calli were used, but it was difficult to produce transformed BY-2 cells from suspension-cultured cells. The right state of active BY-2 cells for transformation also differs from one to another and is reported to depend on culture conditions. The culture media could be MS [8] or Linsmair and Skoog (LS) [1] or Gamborg (B5) [9]. The presence of auxin for proliferation of BY-2 is needed in all cases, but the type of auxin could have an affect. Klein and et al. [5] obtained the best BY-2 culture in the presence of naphthaleneacetic acid - NAA(1 mg/l), N6-benzyladenine - BA (0.1 mg/l), and dichlorophenoxyacetic acid – 2.4D (0.1 mg/l), while An [1] and Nagata and Kumagai [8] used only 2,4D (0.2 mg/l). BY-2 cell suspension culture grows well in darkness at a temperature of from 25◦C to 28◦C undergoing shaking. An [1] held the culture at 28◦C, shaking it at 150 rpm. Nocarova and Fischer [9] used 26◦C in an incubator with shaking at 115 rpm. Agrobacterium tumefaciens strain is also considered to be an important factor. An [1] reported that Agrobacterium strains A281 containing pTi-Bo542 were better than the strain PC2760 containing pAL4404. Nocarova and Fischer found C58C1 more effective [9]. GUS (beta-glucuronidase) has been frequently used as a reporter gene to assess transformation efficiency using BY-2 cell culture because 129 Nguyen Tuong Van, Le Quynh Lien and Nguyen Thanh Van it can be easily and sensitively assayed using fluorometric methods. It is remarkably stable with a long half-life in living cells and tissue extracts and it enables histochemical analysis that will yield information about tissue-organ-specific localization in transgenic plants. In this research as a part of the project: “Development of fundamental technology for the production of antigens using rapid growing plant cell culture and virus-based expression vectors”, we focused on optimizing culture conditions of BY-2 cells at the Institute of Biotechnology of Hanoi and establishing their genetic transformation protocol using Agrobacterium tumefaciens. A BY-2 tobacco cell suspension culture with 5 different initial concentrations (0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL) was used to optimize the in vitro culture. Agrobacterium-mediated transformation protocol of BY-2 was optimized by using the Agrobacterium tumefaciens C58C1 strain habouring binary vector pPTN289_Gus at 3 different concentrations (OD = 0.6, 0.8, 1.0). Transformed BY-2 cells were selected on the culture medium with kamamycin as the selected agent and the transformation efficiency was measured as percentage of GUS positive cells (transformation frequency) and GUS concentration and activity. 2. Content 2.1. Time and place of study This study was done from November 2010 to July 2011 at the laboratory of Plant Cell Biotechnology, Institute of Biotechnology, Vietnam Academy of Science and Technology. 2.2. Materials and methods 2.2.1. Materials Bacterial strain and plasmid: Agrobacterium tumefaciens (strain C58C1) containing the binary plasmid pPTN289_GUS (Nebraska University, USA) was used for gene transformation. Plant material and growth conditions:Tobacco Bright Yellow 2 (BY-2) cells (kindly provided by Dr. Erwin Witters, Plant Biochemistry Laboratory, Anwept University, Belgium) were cultured in 50 mL of modified MS medium (4.3 g/L MS salt, 1 mg/L thiamine, 100 mg/L myo-inositol, 210 mg/L Miller’s KH2PO4, 0.1 mg/L 2,4-D and 30 g/L sucrose, pH 5.6). Cells were grown in an orbital shaker (130 rpm) at 27◦C in the dark and sub-cultured weekly by transferring 1 mL of the suspension into 50 mL of fresh medium [7]. 130 Optimising in vitro culture and Agrobacterium tumefaciens-mediated transformation protocols... 2.2.2. Methods * BY-2 growth curve: Seven days after the initial culture, 0.5 - 2.5 mL of BY-2 cells were sub-cultured in 50 mL of fresh MS medium forming five dilute formulas F1 - F5 at 1:100, 1:50, 1:33.3, 1: 25 and 1:20 respectively. BY-2 cells of each formula were collected daily by centrifuging 20 mL of suspension at 3000 rpm for 5 minutes. The growth of BY-2 cells was determined based on the weight of these cells. * BY-2 transformation: Experiment formulas: We carried out this study using 15 experiment samples and 5 control samples as described in Table 1. Table 1. Experiment samples Formulas F1 F2 F3 F4 F5 Bacteria 0.6 0.6-1 0.6-2 0.6-3 0.6-4 0.6-5 concentration 0.8 0.8-1 0.8-2 0.8-3 0.8-4 0.8-5 1.0 1.0-1 1.0-2 1.0-3 1.0-4 1.0-5 - Agrobacterium tumefaciens suspension for transformation: A single colony of Agrobacterium tumefaciens containing pPTN289_GUS was inoculated into 10 mL of LB medium containing kanamycine (50 µg/mL) at 28◦C, shaken at 220 rpm overnight. Before plant transformation, 2 mL of a bacterial suspension that was cultured overnight was placed into 10 mL of LB medium containing kanamycine (50 µg/ml) for 2 hours and the concentration was adjusted to OD600 of 0.6, 0.8, and 1.0 in LB. - Cocultivation: Five mL of BY-2 cells cultured for six days were used for each transformation. One hundred µL bacterial suspension and 5 µL of 20 mM acetosyringone were subsequently added to BY-2 cells in 100 mm-petri plates and co-cultured for two days under dark condition at 25◦C. Plates containing only BY-2 cells served as negative controls. -Washing: After two days, the control and transformed BY-2 cells were transferred from each plate into a 15 mL centrifuge tube and the plate was rinsed with an additional 5 - 7 mL of MS medium which was then added to the centrifuge tube. MS medium was poured into the tube with the cells to make a final volume of 12 mL. The tubes were mixed by gentle inversion, and then the cells were allowed to settle in the centrifuge tube. The supernatant was removed carefully by aspiration. This wash step was repeated two times with MS medium and one more time with MS medium containing cefotaxim (400 µg/mL). Finally, BY-2 cells were diluted in 12 mL of MS medium containing cefotaxim and gently mixed by inversion. - Plating cells on selective and non-selective medium: One mL of control and transformed BY-2 cells were plated onto selection medium [MS containing cefotaxim (400 mg/L) and kanamycine (50 mg/L)]. In addition, 1mL of control cells were cultured 131 Nguyen Tuong Van, Le Quynh Lien and Nguyen Thanh Van in non-selective medium (antibiotic-free MS medium). All cells were grown under dark conditions at 25◦C and collected 6, 12 and 20 days after co-culture for further analysis. * Determination of transformation efficiency -Histochemical assays:BY-2 cells were immersed in an X-Gluc solution containing 10 mM EDTA, 0.1 M Na2HPO4, 0.1 M Na2HPO4 (pH = 7), 0.5 mM K ferricyanide, 0.5 mM K ferrocyanide and 1.0 mg X-gluc per mL (Stomp 1992) at 37◦C for 24 hours in the dark. Chlorophyll was removed from staining samples using 70% ethanol. Transformation efficiency was determined by counting stained cells under a dissecting microscope. - GUS assays (Fluorometric assay): + Protein isolation: One hundred mg of BY-2 cells were collected 6, 12, 20 days after co-culture and homogenized in liquid nitrogen using a manducatory machine at 21 rps for 2 min. Then to each sample was added 100 µL of extraction buffer (50 mM NaPO4 7.0 pH, 1 mM EDTA, 0.1% Triton X-100, 10 mM beta-Mercaptoethanol and 25 µg/mL PMSF). Protein extracts were collected via centrifuging at 13.000 rpm, 4◦C for 10 min and kept at -80◦C until use. + Determination of protein concentration: The total protein content in supernatants was determined according to the method of Bradford [1] with the Bradford protein assay solution (Bradford Laboratories, Hercules, Califs). The standard curve is obtained by measuring the absorbance of 0.01 - 10 mg bovine serum albumin (BSA). + GUS activity: GUS activity was determined [10] with modifications. 15 minutes before assay, 80 µL of assay buffer (extraction buffer containing 2 mM MUG) was prepared in eppendorf tubes and pre-warmed to 37◦C. Then 20 µL of protein extract was added to each tube and incubated for 1 h at 37◦C. The reaction was stopped with 900 µL of 0.2 M Na2CO3 and absorption was measured at 365 nm. 2.3. Results and discussion 2.3.1. Establishment of BY-2 growth curve The course of growth over time was determined over a period of ten days. The fresh biomass of culture was measured every day, except for day one (Figure 1). The growth curve of the cultured cells was affected by the initial dilution of plant cell suspensions. All BY-2 cell formulas achieved maximum cell weight on the eighth day. Specifically, the amount of BY-2 cells in formulas 1, 2, 3, 4, 5 increased slowly during the first 4 days. The log phase appeared from the 4th to the 8th day, followed by the equilibrium phase. The growth curve ended with a slight decrease on the last day. However, there was the direct relationship between the initial concentration of cells and their growth ability. Cells in formula F1 had the least amount of growth while those in the formula F5 grew at the highest level of growth. 132 Optimising in vitro culture and Agrobacterium tumefaciens-mediated transformation protocols... Figure 1. The growth curve of BY-2 cells The second important point is that the different formulas had no significant difference in the period in which the log phase and the equilibrium phase were reached. With the best cell growth formula, F5 with 2.5 mL of initial concentration (1:20 in liquid MS medium), there was a maximum cell weight of 1.206 g/mL. From above results, we recommend the following protocol for BY-2 cell culture (Figure 2). Figure 2. Standard protocol for BY-2 cell in vitro culture 2.3.2. Determination of the BY-2 transformation efficiency After A. tumefaciens transformation, BY-2 cells were screened in selection media containing 50 µg/mL kanamycine and 400 µg/mL cefotaxim. Control BY-2 cells in MS/cefotaxim/kanamycine plates could not grow because they did not harbor the pPTN289_GUS vector with kanamycine resistance ability (negative control) while these cells grew well in MS medium, especially in MS/cefotaxim medium (positive control). Only successfully transformed BY-2 cells could grow in medium containing kanamycine. 133 Nguyen Tuong Van, Le Quynh Lien and Nguyen Thanh Van Transformed calli resisting antibiotic selection were obtained (Figure 3). Figure 3. Transformation of tobacco-cultured cells with a kanamycin resistance gene The 6-d-old tobacco cells were cocultivated with A. tumefaciens containing pPTN289_GUS and the transformed calli were selected on the agar medium containing 50 µg/mL kanamycine. Upper lane: control cells; lower lane: transformed cells. Because of the presence of the GUS marker gene in the transformation vectors, the blue color of the transformed cells was visible under the dissecting microscope (Figure 4). Under kanamycine selective pressure, only successful transformation cells containing the kanamycine resistance gene survived and developed into callus. Figure 4. Transformed BY-2 cells with GUS activity In order to obtain an exact determination of transformation efficiency, extracellular proteins were prepared to measure GUS activity (Figures 5, 6, 7). There was no distinction among the samples on the first days after transformation. However, the enzyme activity of 0.6-1, 0.6-2 and 0.6-3 samples increased rapidly and in the 0.6-1 sample was seen the highest value, 0.061 pmol of MU.min−1 .mg−1 approximately from the 12th day. In contrast, enzyme activity level of the 0.8-4 sample emerged earlier, on day 6 (Figure 6). 0.8-2 and 0.8-3 samples had a significant increase after 12 days of transformation but decreased later; the 0.8-4 sample was still the best 134 Optimising in vitro culture and Agrobacterium tumefaciens-mediated transformation protocols... compared to other 0.8-OD samples. As expected, low enzyme activity of 0.6-4, 0.6-5 and 0.8-1 was observed, nearly zero until after the 20th day. No transformed BY-2 was detected using the X-Gluc staining method. In the case of 1.0-OD samples (Figure 7), 1.0-3, 1.0-4, 1.0-5 were clearly distinguished from the others by their enzyme activity values. Among them, enzyme activity of the 1.0-5 sample reached a maximum of 0.07 pmol of MU.min−1.mg−1 on the 20th day. Expectedly, the use of both histochemical assay and fluorometric assay led to the same results which confirms the accuracy of this study. One more finding was that transformation efficiency could only be determined after a transformation of more than 12 days. Figure 5. Enzyme activity of 0.6-OD samples Figure 6. Enzyme activity of 0.8-OD samples 135 Nguyen Tuong Van, Le Quynh Lien and Nguyen Thanh Van Figure 7. Enzyme activity of 1.0-OD samples 3. Conclusion In this study, we have optimized the protocol of in vitro tobacco BY-2 cell culture with 2.5 mL of initial BY-2 cell suspension determined over a period of days. Moreover, 0.6-1, 0.8-4 and 1.0-5 samples were found to have the highest transformation efficiency and we also determined that the optimum protocol for BY-2 cell transformation was with the use of A.tumefaciens. Additionally, there was the correlation between BY-2 cell growth and A.tumefaciens density. The known BY-2 cell amount transformed with a suitable density of A.tumefaciens would give a high transformation efficiency: BY-2 cell formula 1 and 0.6 of bacterium OD; formula 4 and 0.8 of bacteria OD; formula 5 and 1.0 of bacteria OD. The protocols optimized in this study are very meaningful for the production of recombinant proteins on a large scale in Vietnam. We suggest that: - pCB301_Kan construct (App1) containing scFv24 gene and kanamycin resistance gene be used as the selectable marker. - BY-2 cells be transformed via A.tumefaciens with pCB301_Kan containing scFv24 gene. In this case, the expression level of scFv24 antibody fragment from transformed cells can be assayed by both Western blot and ELISA. REFERENCES [1] An G., 1985. High efficiency transformation of cultured tobacco cells. Plant Physiol, vol 79, pp. 568-570. 136 Optimising in vitro culture and Agrobacterium tumefaciens-mediated transformation protocols... [2] Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, Vol. 72, pp. 248-254. [3] Hellwig S., Drossard J., Twyman RM., Fischer R., 2004. Plant cell cultures for the production of recombinant proteins. Nat. Biotechnol., Vol. 22(11), pp. 1415-1422. [4] Kim T., Chowdhury M. K. U., and Hazel Y. W., 1997. A quantitative and histological comparison of GUS expression with different promoter constructs used in microprojectile bombardment of peanut leaf tissue. In vitro Cell. Dev. Biol.-Plant, Vol. 35, pp. 51-56. [5] Klein TM., Harper EC., Svab Z., Sanford JC., Fromm ME., Maliga P., 1988. Stable genetic-transformation of intact Nicotiana cells by the particle bombardment process. J. Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 8502-8505. [6] Kost B., Spielhofer P. and Chua N.H., 1998. A GFP–mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J., Vol. 16, pp. 393-401. [7] Mill DR., Lee JM., 1996. A simple, accurate method for determining wet and dry weight concentrations of plant cell suspension cultures using microcentrifuge tubes. Plant Cell Rep., Vol. 15, pp. 634-636. [8] Nagata T., Kumagai F., 1999. Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line. Methods Cell Sci., Vol. 21(2-3), pp. 123-127. [9] Nocarova E. and Fischer L., 2009. Cl