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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image EcoTub: An ecologically safe selection system for transgenic crops based on modified plant-tubulin genes

Background and objectives

There is substantial public concern about a potential spread of antibiotic resistance genes in the soil and intestinal bacteria as a consequence of the use of genetically modified organisms, and in particular of the agricultural use of transgenic crops. A selection system for plant transformation that is exclusively based on genetic information already present in the host plant and that does not require antibiotic resistance genes would avoid any such risk. This project, which has just started, aims to develop such ecologically safe systems for cereals. The project is based upon truncated tubulins lacking the binding site for carbamates. Plants carrying the genes encoding such truncated proteins will be resistant to carbamate herbicides thereby allowing selection. The production of the truncated tubulins will be driven by the tubulin's own promoters within DNA fragments devoid of foreign sequences. The suitability of this selection system will be analysed using rice as a model cereal crop and the rice gene for phytochrome as a test gene.

Rice panicle. image Rice panicle.
Photo: PBIU, University of Leeds.
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Figure 1 shows the response of microtubules (MT) and actin microfilaments (MF) to a depletion of the plant growth regulator auxin. image  


Figure 1 shows the response of microtubules (MT) and actin microfilaments (MF) to a depletion of the plant growth regulator auxin. One can see that the microtubules reorient into a longitudinal direction, with the consequence that the cellulose fibres in the cell wall are laid down in the same direction, such that the length growth of the plant is blocked (the plants become shortened). This is the physiological response, where the tubulin gene TUBA1 is important.

 

In Figure 2, the rice seedlings shown are from the wild type (WT) that have been treated with carbamate herbicides (+EPC) image  


In Figure 2, the rice seedlings shown are from the wild type (WT) that have been treated with carbamate herbicides (+EPC) - note the short-stunted and malformed seedlings in comparison to the untreated (-EPC) control, where the seedlings are straight and long. The carbamates destroy microtubules and thus block cell elongation. If the wild type tubulin is replaced by a mutated form, where only one base is exchanged (leading to a precocious stop codon and a C-terminally truncated product), the carbamate can bind no longer and the respective seedling is straight and long as if it had not experienced the drug (ER31).

 

Such seedlings change their growth patterns, for instance, as shown in Figure 3 image  


Such seedlings change their growth patterns, for instance, as shown in Figure 3: they produce longer organs in the embryo depending on whether they have one (ER31) or two (ER31*) copies of the mutated gene.

 

Figure 4 shows the response of microtubules to the plant growth regulator auxin in the wild type (WT) and in plants with the mutated tubulin gene (ER31). image

Figure 4 shows the response of microtubules to the plant growth regulator auxin in the wild type (WT) and in plants with the mutated tubulin gene (ER31). Microtubules made up of the mutated tubulin are able to produce proper microtubules, but these microtubules no longer respond to the growth regulator, and they maintain a more or less longitudinal orientation.



Approach and methodology

A selection marker cassette consisting of the tubulin promoter, coding sequence for alpha-tubulin conferring resistance to carbamate herbicides, and full-length beta-tubulin will be constructed. Selection and transformation protocols based on carbamates will then be optimised. The selection marker cassette will then be used for cotransformation with rice phytochrome as model and the expression and functionality of the transgene will be assayed.

Throughout this work, the approach will be based on “clean DNA” technology, which involves avoiding the use of extraneous backbone and vector sequences.


Main findings and outcome

This project which is just starting is expected to generate the following results. The sequences of various different rice alpha- and beta-tubulins will be determined and their promoters isolated. Variants of rice alpha-tubulin with a truncated or modified C-terminus will be constructed. Vectors will then be made from these elements.

Protocols for homologous transfection into rice embryos will be developed involving tests for conferred carbamate resistance. The clean-DNA system will then be validated using the rice phytochrome gene. Phytochrome expression and functionality in transformed cells will be investigated and carbamate resistance and phytochrome overexpression during development and propagation of transgenic plants analysed. This will also include tests for potential negative effects of tubulin expression on plant development.


Conclusions

This project will contribute to a clean DNA technology to minimise the risks associated with genetically modified organisms. In addition to the advantages of reduced risk, this should also improve public acceptance of genetic modification technology. In particular, rice plants will be produced that are resistant to a herbicide (carbamate) but that do not contain any DNA sequences of foreign origin.

 

Major publications

Nick P., Yatou O., Furuya M., Lambert A.M., "Auxin-dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC".
Plant J., 6, 1994, pp. 651-663.

Qin X., Gianì S., Breviario D., "Molecular cloning of three rice alpha-tubulin isotypes: differential expression in tissues and during flower development".
Biochem. Biophys. Acta 1354, 1997, pp. 19-23.

Kohli A., Leech M., Vain P., Laurie D.A., Christou P., "Transgene organization in rice engineered through direct DANN transfer supports a two-phase integration mechanism mediated by the establishment of integration hot-spots".
Proc. Natl. Acad. Sci. USA, 95, 1998, pp. 7203-7208.

 

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imageResearch project
 

Contract number
QLK3-2000-00060

Period
October 2000 - September 2003

Coordinator
P. Nick
Institut für Biologie II
Freiburg (DE)

 
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Partners


D. Breviario
Centro Nazionale di Ricercha
Milano (IT)

P. Christou
John Innes Centre
Norwich (UK)

 
 
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