Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-11-04
2003-03-11
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S469000, C800S294000
Reexamination Certificate
active
06531649
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to transgenic sugar beet plants capable of tolerating herbicide treatment with glyphosate as active ingredient.
2. Background of the Related Art
Weeds in sugar beet fields are a major problem for the farmer. They compete with the crop thus reducing yield. Today, no single herbicide is able to effectively control all weeds without harming the sugar beet crop itself (Miller et al, J. Sugar Beets Res. 26: 3-4, 1989). In practice, farmers use mixtures of herbicides, which also reduce growth of the crop. Meanwhile the number of weed species having developed resistance to said herbicides continues to increase (Schweizer et al, J. of Sugar Beet Research 28: 1-23, 1991) thereby aggravating the problem of weed control in sugar beet fields.
Roundup® is a broadspectrum, environmentally preferable herbicide inhibiting the growth of both weed and crop species. In the context of the present invention one liter of a herbicidal Roundup® solution comprises 360 g of its active ingredient (a.i.) glyphosate (the common name of N-phosponomethyl-glycine) which is taken up by foliage. So far no glyphosate resistant weed has developed in over 20 years of use (Holt et al., Annu. Rev. Plant Physiol., 1993); additionally no natural tolerance to glyphosate has been found in sugar beet. However, pre-emergence use of Roundup® seems to be more efficient for weed control in sugar beet fields than a combination of herbicides often used in sugar beet agriculture, consisting of phenmediphan, metamitron and ethofumesate (Madsen et al, Weed Res. 35: 105-111, 1995).
Glyphosate inhibits the biosynthesis of aromatic amino acids, through irreversible binding to 5-enolpyruvylshikimate-3-phosphate synthase (epsps). Within the chloroplast this enzyme catalyzes the reaction of shikimate-3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate-3-phosphate and phosphate. Approximately one week after application of the herbicide, visible effects can be seen including wilting, yellowing followed by complete browning, deterioration of plant tissue, and decomposition of the roots.
To impart glyphosate tolerance to crop species, focus has been on the introduction into plants of epsps genes capable of increasing glyphosate tolerance. Besides plants bacteria and fungi naturally express epsps enzyme activity. The cp4/epsps from Agrobacterium sp. CP4 was found to confer tolerance to glyphosate (Barry et al., “Inhibitors of Amino Acid Biosynthesis: Strategies for Imparting Glyphosate Tolerance to Crop Plants”, in: Biosynthesis and Molecular Regulation of Amino Acids in Plants, Singh et al (eds), American Society of Plant Physiologists, pages 139-145, 1992). Introduction of the cp4/epsps gene into soybean and oilseed rape yielded tolerance to foliar application of the herbicide under field conditions (Delannay et al., Crop Sci. 35: 1461-1467, 1995; Padgette et al., Crop Sci. 35: 1451-1461, 1995).
Glyphosate oxidase reductase (gox) isolated from Achromobacter sp. strain LBAA (Barry et al., supra) degrades glyphosate into aminomethyl phosphonic acid, a compound non-toxic for the plant. A combination of the cp4/epsps and glyphosate oxidase (gox) genes has been successfully used to obtain transgenic wheat (Zhou et al., Plant Cell Rep. 15: 159-163, 1995) tolerant to glyphosate.
SUMMARY OF THE INVENTION
The object of the present invention is to provide sugar beet plants which tolerate glyphosate in doses sufficiently high to effect optimal herbicidal activity. Such plants can be further improved by backcrossing with elite sugar beet lines to optimize agronomic properties such as yield, pathogen resistance, etc.
Sugar beets may be transformed using
Agrobacterium tumefaciens
mediated transformation (Fry et al, Third international congress of plant mol. biol., Tuscon, Ariz., USA; D'Halluin et al, Bio/Technology 10: 309-314, 1992; Konwar, J. Plant Biochem & Biotech 3: 37-41,1994). Agrobacterium-mediated transformation often results in more than one copy of the T-DNA being integrated into the plant's genome. The gene to be integrated is preferably introduced into the T-DNA such that it becomes located close to the T-DNA right border which, contrary to the left border, will almost always be transferred to the plant.
Plants according to the present invention tolerate treatment with more than about 3×6 liters of the herbicide Roundup® per hectar (about 18 liters per hectar). The total standard dose to obtain good weed control varies between 4 and 6 liters per hectare, depending on weed pressure. At these concentrations herbicide treatment exerts no detectable effect on plant vigour and leaf chlorosis. The tolerance exhibited by the plants according to the invention is conferred by a transgenically expressed cp4/epsps enzyme activity. A preferred embodiment of the present invention has been deposited with the National Collections of Industrial and Marine Bacteria Limited (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland UK, on Oct. 24, 1997, under the Accession No. 40905. This deposit will be maintained in the NCIMB depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request or for the effective life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant has satisfied all the requirements of 37 C.F.R §§1.801-1.809, including providing an indication of the viability of the sample. Applicant imposes no restriction on the availability of the deposited material from the NCIMB; however, Applicant has no authority to waive any restriction imposed by law on the transfer of biological material or its transportation in commerce.
The present invention thus relates to a sugar beet plant including the descendants thereof expressing cp4/epsps enzyme activity. In particular the invention relates to a sugar beet plant including the descendants thereof tolerating the treatment with about 4 to about 18 liters Roundup® per hectar.
Plants according to the present invention can be obtained by routine Agrobacterium mediated transformation using a transformation vector comprising between right and left T-DNA border sequences a piece of DNA as described in SEQ ID NO: 5 encoding i.a. cp4/epsps.
It was surprisingly found within the scope of the present invention, that a transformation event (RRMax) lacking left and right T-DNA border sequences within the transgenic genome and resulting in deletion of a considerable part of the transformation vector DNA while retaining the cp4/epsps encoding DNA provides superior glyphosate tolerance. In particular a piece of DNA as characterized by SEQ ID NO: 1 is found integrated into a highly repetitive region of the genome simlutaneously replacing part of said repetitive genomic sequence. The genomic DNA directly adjacent to that part of the transgene sequence which in the transformation vector used is linked to the T-DNA right border sequence, has the sequence given in SEQ ID NO: 2. The genomic DNA directly adjacent to the other end of the integrated transgenic DNA has the sequence given in SEQ ID NO: 3. The complete DNA sequence of the newly formed genomic DNA arrangement is given in SEQ ID NO: 4.
Accordingly, the present invention relates to a sugar beet plant including the descendents thereof wherein DNA characterized by the nucleotide sequence of SEQ ID NO: 1 forms part of the plant's genome and said nucleotide sequence preferably replaces highly repetitive DNA sequences within the plant's genome.
Preferred herein is a sugar beet plant including the descendents thereof wherein those parts of the genome directly linked to said nulceotide sequence are characterized by the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
The herbicide tolerance engineered into the transgenic seeds and plants mentioned above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in descendant plants. Generally said maintenance and propagation ma
Mannerloef Marie
Steen Per
Tenning Paul Peter
Mehta Ashwin
Nelson Amy J.
Syngenta Participations (AG)
Vrana Bruce
LandOfFree
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