Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbiological Systems
Plant Genetic Engineering: Methodology
Conclusion

Introduction/32.html">Genetic Engineering makes it possible to introduce foreign genes into plant Cell cultures, followed by the regeneration of whole, fertile plants from selected transformed Cells. In nature, plant transformation is mediated by soil Bacteria *Agrobacterium tumefaciens*. Upon plant infection, a specific chemical compound begins to synthesize within the host. Responding to this chemical signal, *A. tumefaciens* attaches to The plant cell membrane, after which a portion of the bacterial plasmid (Ti plasmid)—known as T-DNA—is transferred into the plant Cell Nucleus. The T-DNA integrates into the plant genome and undergoes expression. It contains genes encoding Enzymes for phytohormone synthesis, which trigger plant cell enlargement and proliferation. Furthermore, plant cells start producing opines encoded by the T-DNA, which can be utilized exclusively by *A. tumefaciens*. Thus, Evolutionary Processes have shaped a mechanism that turns a plant cell into a "factory" producing a carbon and nitrogen source (opine) exclusively for the metabolic needs of *A. tumefaciens*.

To harness the natural ability of *A. tumefaciens* to penetrate plant cells for delivering cloned genes, modified Ti Plasmids were developed. Phytohormone and opine METABOLISM genes were excised from the T-DNA, and this engineered T-DNA was inserted into a plasmid capable of stable Replication in *E. coli*. The target Gene embedded within the T-DNA was thus delivered into the Nucleus of the recipient plant cell along with it. In a binary system, a shuttle vector carrying the gene cloned into the T-DNA is introduced into an *A. tumefaciens* strain that harbors a modified plasmid with the genes required for T-DNA transfer into the plant cell (vir genes). Additionally, a cointegrative system was designed, involving the Introduction of a shuttle vector into *A. tumefaciens*, where it recombines with a non-oncogenic Ti plasmid carrying vir genes to form a single plasmid containing both functional vir genes and the T-DNA with the cloned gene. The T-DNA region of *A. tumefaciens* has been widely utilized to introduce genes into various plants; unfortunately, this system is not universally applicable to all plant species. Microprojectile bombardment (biolistics) also serves as an efficient method for DNA delivery into diverse plant cells.

To ensure the expression of foreign genes introduced into plant cells, plant promoters were employed. Various promoters functioning exclusively in specific plant Tissues or at defined developmental stages were identified via the expression of a promoterless reporter gene following its integration into the plant chromosomal DNA. Methods were also devised to integrate foreign genes directly into chloroplast or Mitochondrial DNA, allowing the encoded protein to be synthesized right inside these Organelles. Finally, to address public concerns, strategies for removing marker genes from Transgenic Plants were successfully established.

References

An G., Y. Kim. 1993. Techniques for isolating and characterizing plant Transcription promoters, enhancers, and terminators, p. 155—166. In B. R. Glick, J. E. Thompson (ed.), Methods in Plant Molecular Biology and Biotechnology. CRC Press, Boca Raton, Fla.

Carrer H., P. Maliga. 1995. Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene. Bio/Technology 13: 791—794.

Christou P. 1992. Genetic Transformation of crop plants using microprojectile bombardment. Plant J. 2: 275-281.

Dale E. C., D. Ow. 1991. Gene transfer with subsequent removal of the Selection gene from the host genome. Proc. Natl. Acad. Sсi. USa 88: 10558-10562.

Goldsbrough A. P., C. N. Lastrella, J. І. Yoder. 1993. Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Bio/Technology 11: 1286—1292.

Gruber M. Y., W. L. Crosby. 1993. Vectors for Plant transformation, p. 89—119, In B. R. Glick, J. E. Thompson (ed.). Methods in Plant Molecular Biology and Biotechnology’. CRC Press, Boca Raton, Fla.

Halfter U-, P. C. Morris, L. Willmitzer. 1992. Gene targeting in Arabidopsis thaliana. Mol. Gen. Genet. 231: 186-193.

lshida Y., H. Saito, S. Ohta, Y. Hiei, T. Kimari, T. Kumashiro. 1996. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat. Biotechnol. 14: 745-750.

Jefferson R. A., T. A. Kavanagh, M. W. Bevan. 1987. GUS fusions: ß-glucuronidase as a sensitive and versatile Gene Fusion marker in higher plants. EMBO J. 6: 3901-3907.

Klein T. M., E. D. Wolf, R. Wu, J. C. Sanford. 1987. High-velocity microprojectiles for delivering Nucleic Acids into living cells. Nature (London) 327: 70-73.

Krüger-Lebus S., I. Potrykus. 1987. A simple and efficient method for direct gene transfer to Petunia hybridia without electroporation. Plant Mol. Biol. Rep. 5: 289—294.

Miki В. L., P. F. Fobert, P. J. Charest, V. N. Iyer. 1993. Procedures for introducing foreign DNA into plants, p. 67—88. In B. R. Glick, J. E. Thompson (ed.), Methods in Plant Molecular Biology and Biotechnology. CRC Press, Boca Raton, Fla.

Mitsuhara I., M. Ugaki, H. Hirochika, M. Ohshima, T. Murakami, Y. Gotoh, Y. Katayose, S. Nakamura, R. Honkura, S. Nishimiya, K. Ueno, A. Mochizuki, H. Tanimoto, H. Tsugawa, Y. Otsuki, Y. Ohashi. 1996. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol. 37: 49—59.

Ow D. W., К. V. Wood, M. DeLuca, J. R. de Wet, D. R. Helinski, S. H. Howell. 1986. Transient and stable expression of the firefly luciferase gene in PLANT CELLS AND transgenic plants. Science 234: 856-859.

Paszkowski J., M. Baur, A. Bogucki, I. Potrykus. 1988. Gene targeting in plants. EMBO J. 7: 4021-4026.

Pausl К. P. 1995. Plant biotechnology for crop improvement. Biotechnol. Adv. 13: 673—693.

Potrykus I. 1990. Gene transfer to cereals: an assessment. Bio/Technology 8: 535—542.

Potrykus I. 1991. Gene transfer to plants: assessment of published approaches and results. Annu. Rev. Plant Physiol. 42: 205—225.

Southgate E. M., M. R. Davey, J. B. Power, R. Marchant. 1995. Factors affecting the GENETIC ENGINEERING OF plants by microprojectile bombardment. Biotechnol. Adv. 13: 631—651.

Vain P., J. de Buyser, V. Bui Trang, R. Haicour, Y. Henry. 1995. Foreign delivery into monocotyledonous species. Biotechnol. Adv. 13:653—671.

Walden R., J. Shell. 1990. Techniques in plant molecular biology—progress and problems. Ear. J. Biochem. 192: 563—576.

Walden R., R. Wingender. 1995. Gene-transfer and plant-regeneration techniques. Trends Biotechnol. 13: 324-331.

Yoder J. I., A. P. Goldsbrough. 1994. Transformation system for generating marker-free transgenic plants. Bio/Technology 12: 263—267.

Zambryski P. 1988. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu. Rev. Genet. 22: 1—30.

Zambryski P., J. Tempe, J. Schell. 1989. Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56: 193-201.

Review Questions

1. Why is the Ti plasmid from Agrobacterium tumefaciens suitable as a vector for transferring foreign genes into plant chromosomal DNA?

2. What is the difference between binary and cointegrate vector systems?

3. What are reporter genes, and how are they used in plant cell transformation?

4. What is the microprojectile bombardment method used for plant transformation?

5. Describe in detail how you would isolate a ROOT tissue-specific plant promoter.

6. How can a foreign gene be integrated into chloroplast DNA?

7. How can a transgenic plant lacking a marker gene be obtained?

8. How can The activity of a plant promoter be enhanced?