TRANSFORMATION OF TOBACCO (Nicotiana tabacum L.) AND BUSH MONKEYFLOWER

(Mimulus aurantiacus Curtis)

Nik Susič, Jana Murovec, Borut Bohanec

University of Ljubljana Biotechnical Faculty

Agronomy Department

Nik Susič, Biotechnical Faculty, Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia (e-mail: nik.susic@gmail.com) Jana Murovec, Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, 1000 Ljubljana, Slovenia (e-mail: jana.murovec@bf.uni-lj.si) Borut Bohanec, Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, 1000 Ljubljana, Slovenia (e-mail: borut.bohanec@bf.uni-lj.si)

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 (Ti- plasmid)

• 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

0

20

40

60

80

100

0

2

4

6

8

10

12

1 2 3 4 C1 C2 C3

GU

S po

sitiv

e sa

mpl

es (%

)

aver

age

no. o

f sho

ots /

exp

lant

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, non-destructive 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.