University of Ljubljana
Biotechnical Faculty
Agronomy Department
TRANSFORMATION OF TOBACCO (Nicotiana
tabacum L.) AND BUSH MONKEYFLOWER
(Mimulus aurantiacus Curtis)
Nik Susič, Jana Murovec, Borut Bohanec
Nik Susič, Biotechnical Faculty, Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia (e-mail: [email protected])
Jana Murovec, Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, 1000 Ljubljana, Slovenia (e-mail: [email protected])
Borut Bohanec, Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, 1000 Ljubljana, Slovenia (e-mail: [email protected])
Introduction
• Recombinant DNA technology aimed at
developing improved varieties of many plants.
• DNA can be introduced from other species of
plants, animals and bacteria
• traditionally, genetic diversity was achieved
solely by crossing and selecting genotypes within
species.
• Requirements for genetic engineering:
• development of new scientific methods
• tools for genetic manipulation
• cloning a large number of genes
Tobacco as a model system for tissue
culture and genetic engineering
• Tobacco (Nicotiana tabacum L.) is an
extremely versatile vehicle for all
aspects of cell and tissue culture
research.
• The majority of discoveries in the field
of plant cell culture, tissue culture and
molecular biology have originated from
experiments with tobacco plants:
• in vitro culture medium (Murashige and
Skoog, 1962.),
• first transgenic plants (Zambryski et al.,
1983),
• gene expression and gene stability
experiments,
• expression of transgenes from a variety
of organisms
• commercial products and applications
Mimulus aurantiacus
• Mimulus aurantiacus Curtis (bush
monkeyflower) is a perennial
flowering sub-shrub, growing in
western parts of the United States of
America.
• An emerging model system for
studies of evolutionary and
ecological functional genomics
• an efficient system for stable
transformation is necessary for
comprehensive genetic studies
• testing of candidate genes and/or
promoters that regulate flavonoid
biosynthetic pathways in vivo
resulting in a specific floral
pigmentation (adaptive trait?)
• Introduction of specific genes into the plant
host genome:
• without the use of vector (protoplast fusion,
chemical agents, electroporation, particle
bombardment or biolistics)
• using vectors (viruses, Agrobacterium spp.)
• Agrobacterium tumefaciens most commonly
used
Agrobacterium tumefaciens
• a soil bacterium
• pathogenic to a range of
dicotyledonous plant species
• formation of crown galls or
tumours
• transfers a portion of its DNA
(T-DNA) into the nuclear
genome of the host plant (Tiplasmid)
• Development of plant
transformation system
• Binary Ti vectors:
• disarmed Ti plasmid, containing
vir genes (Ti-helper), while the
T-DNA region with the gene(s)
to be transferred is located on
another smaller plasmid – the
binary plasmid
(adapted from Griffiths et al, 2008; 743-744.)
Materials and methods
• Leaf explants or hypocotyls were inoculated with a
suspension of A. tumefaciens LBA4404 cells.
• Binary Ti vector coding for β-glucuronidase (uidA) marker
gene and kanamycin resistance (nptII) gene (for tobacco) or
hygromycin resistance (hptII) gene (for M. aurantiacus).
• Washing of explants after co-cultivation and transfer to a
selective medium containing antibiotics kanamycin /
hygromycin B and timentin (adventitious shoot
regeneration).
• in vitro plant culture on Murashige and Skoog (MS)
medium with vitamins + 30 g/l sucrose and solidified with 8
g/l agar
• examination of regenerants by histochemical GUS assay.
Results
Transformation of tobacco
• an experiment to determine the
optimal concentration of
kanamycin in the selective media
used for plant transformations
• monitoring GUS expression of
putative transformants at different
antibiotic concentrations
• The results showed increased
adventitious shoot regeneration at
lower antibiotic concentrations.
• A correlation between higher
kanamycin concentration and
higher GUS expression –
progressively higher antibiotic
concentrations leads to more
stringent selection
Kan (mg/l)
50
100
200
300
12
100
80
8
60
6
40
4
GUS positive samples (%)
average no. of shoots / explant
10
20
2
0
0
1
2
3
4
C1
C2
C3
variant
Graphic representation of the relationship between the concentration of kanamycin in the
regeneration medium and the average number of regenerated shoots per explant (black) or
transformation frequency as determined by GUS histochemical assay (grey) in tobacco. Variants 1,
2, 3 and 4 represent: 50, 100, 200 and 300 mg l-1 kanamycin, respectively. C1 is negative (300 mg
l-1 kanamycin) and C2 positive (0 mg l-1 ) control. C3 represent transformants growing without
antibiotic selection.
Transformation of M. aurantiacus
• tested protocol successfully introduced T-DNA
• expression of selective and marker genes
• 6% of explants formed shoots on the selection
medium
• generation of callus tissue in up to 16% of putative
transformed explants (no adventitious shoot
regeneration)
• future optimization of this protocol
Conclusion
• A possibility of developing a robust transformation
system for M. aurantiacus using A. tumefaciens.
• Developing methods for differentiation of callus
tissue observed in the experiment with M.
aurantiacus with the aim of improving transformation
efficiency.
• Detection of GUS expression should be coupled with
PCR, Southern blot and analysis of progeny.
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
An G., Watson B. D., Chiang C. C. (1986). Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System. Plant
Physiology. 81: 301-305.
Barampuram S., Zhang Z. J. (2011). Recent Advances in Plant Transformation. Methods in Molecular Biology, 701: 1-35.
Barton K. A., Binns A. N., Matzke A. J., Chilton M. D. (1983). Regeneration of intact tobacco plants containing full length copies of genetically
engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell, 32 (4): 1033-43.
Ganapathi T. R., Suprasanna P., Rao P. S., Bapat V. A. (2004). Tobacco (Nicotiana tabacum L.) – A model system for tissue culture interventions and
genetic engineering. Indian Journal of Biotechnology, 3: 171-184.
Griffiths A. J. F., Wessler S., Lewontin R., Carrol S. (2008). Genetic engineering. Intoduction to Genetic Analysis, 9th ed., 742-745. New York, USA: W.
H. Freeman and Company.
Jefferson R. A., Kavanagh T. A., Bevan M. (1987). GUS fusion: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants.
EMBO Journal. 6: 3901-3907.
Jube S., Borthakur D. (2007). Expression of bacterial genes in transgenic tobacco: methods, applications and future prospects. Electronic Journal of
Biotechnology, 10 (3): 452-467. Available at: http://www.ejbiotechnology.info/content/vol10/issue3/full/4/
Klein T. M., Wolf E. D., Wu R., and Sanford, J. C. (1987). High velocity microprojectiles for delivering nucleic acids into living cells. Nature, 327: 70-73.
Kump B., Javornik B. (1996). Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench) populations by RAPD
markers. Plant Science, 114: 149-158.
Lee L.-Y., Gelvin S. B. (2008). T-DNA Binary Vectors and Systems. Plant Physiology, 146: 325-332.
Mullineaux P., Hellens R., Klee H. (2000). A guide to Agrobacterium binary Ti vectors. Trends in Plant Science, 5 (10): 446-451.
Murashige T., Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15: 473-497.
Streisfeld M. A., Rausher M. D. (2009). Altered transregulatory control of gene expression in multiple anthocyanin genes contributes to adaptive
flower color evolution in Mimulus aurantiacus. Molecular Biology and Evolution, 26: 433-444.
Wenck A., Pugieux C., Turner M., Dunn M., Stacy C., Tiozzo A., Dunder E., van Grinsven E., Khan R., Sigareva M., Wang W. C., Reed J., Drayton P.,
Oliver D., Trafford H., Legris G., Rushton H., Tayab S., Launis K., Chang Y.-F., Chen D.-F., Melchers L. (2003). Reef-coral proteins as visual, nondestructive reporters for plant transformation. Plant Cell Reports. 22: 244-251.
Zambryski P., Joos H., Genetello C., Leemans J., Montagu M. V., Schell J. (1983). Ti plasmid vector for the introduction of DNA into plant cells without
alteration of their normal regeneration capacity. EMBO Journal, 2 (12): 2143-50.
Žel J. (1996). Specifične metode genske tehnologije pri rastlinah – vnos genov. Published in Biotehnologija, Raspor P. (ed.), 299-308. Ljubljana,
Slovenia: BIA d.o.o.