Title of Invention

METHOD TO PRODUCE A DROUGHT TOLERANT COTTON PLANT

Abstract The invention relates to the use of cotton para2 gene or cDNA sequences to obtain stress tolerant cotton plants. Various cotton para2 sequences are also provided.
Full Text FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
COMPLETE SPECIFICATION
(See Section 10)
TITLE
STRESS TOLERANT COTTON PLANTS
APPLICANT
Bayer BioScience N.V.
Techno iogiepark 38 B-9052 Gent Belgium
Nationality: Belgium
The following specification particularly describes the nature of this invention and the manner in which
it is to be performed

Stress tolerant cotton plants.
The following invention relates to modified cotton plants which have a higher degree than counterpart unmodified cotton plants of resistance to adverse growing conditions, particularly abiotic stress conditions such as, but not limited to, low or high temperatures, drought, high -light intensities, chemical pollution, flooding, high salinity, high light intensities, high UV irradiation. Such stress tolerant cotton plants may be obtained by lowering the expression of the endogenous cotton parpl gene(s), particularly under stress conditions, by modifying the activity of endogenous cotton parpl gene(s), by exchanging the endogenous cotton parpl gene for another allele of the parpl gene which provides better stress tolerance, or by any combination thereof.
Description of related art
Poly(ADP-ribose) polymerase (PARP), also known as poly(ADP-ribose) transferase (ADPRT) (EC 2.4.2.30), is a nuclear enzyme found in most eukaryotes, including vertebrates, arthropods, mollusks, slime moulds, dinoflagellates, fungi and other low eukaryotes with the exception of yeast The enzymatic activity has also been demonstrated in a number of plants (Payne et al.9 1976; Willmitzer and Wagner, 1982; Chen et al.,1994; OTarxell, 1995).
PARP catalyzes the transfer of an ADP-ribose moiety derived from NAD+, mainly to the carboxyl group of a glutamic acid residue in the target protein, and subsequent ADP-ribose polymerization. The major target protein is PARP. itself, but also histones, high mobility group chromosomal proteins, a topoisomerase, endonucleases and DNA polymerases have been shown to be subject to this modification.
The PARP protein from animals is a nuclear protein of 113-120 kDa, abundant in most cell types, which consist of three major functional domains: an ammo-terminal DNA-binding domain containing two Zn-finger domains, a carboxy-terminal catalytic domain, and an internal domain which is auto-modified (de Murcia and Menissier de Murcia, 1994; Kameshita et al, 1984; Lindahl et aL9 1995). The enzymatic activity in vitro is greatly increased upon binding to single-strand breaks in DNA. The in vivo activity is induced by conditions that
CONFIRMATION COPY



nuclear localization signal, the automodification domain, and the NAD+- binding domain are conserved in the maize enzyme.
Babiychuk et al (1998) described that two poly (ADP-ribose) polymerase homologies were found in plants, the classical Zn-finger-containing polymerase and the structurally ^iion-~ classical PARP proteins, which lack the characteristic N-terminal Zn-finger domain.
Current nomenclature refers to the classical Zn-finger-containing polymerases as PARP1 proteins (and corresponding parpl genes) whereas the structurally non-classical PARP proteins are currently referred to as PARP2 (and corresponding parp2 genes).
The following database entries identifying experimentally demonstrated and putative poly ADP-ribose polymerase protein sequences, parts thereof or homologous sequences, could be identified: BAD53855 (Oryza sativa); BAD52929 (Oryza sativa); XP_477671 (Oryza sativa); BAC84104 (Oryza sativa); AAT25850 (Zea mays); AAT25849 (Zea mays); NP_197639 (Arabidopsis thaliana); NP_850165 (Arabidopsis thaliana); ML188107 (Arabidopsis ihaliarui); NP_850586 (Arabidopsis thaliana); BAB09119 (Arabidopsis thaliana); AAD20677 (Arabidopsis thaliana); Q11207 (Arabidopsis thaliana); C84719 (Arabidopsis thaliana); T51353 (Arabidopsis thaliana); T01311 (Arabidopsis thaliana); AAN12901 (Arabidopsis tlialiana); AAM13882(rabidopsis thaliana); CAB8Q732 (Arabidopsis thaliana); CAA10482 (Arabidopsis thaliana); AAC79704 (Zea mays): AAC19283 (Arabidopsis thaliana); CAA10888 (Zea mays);.CAA10889 (Zea mays); CAA88288 (Arabidopsis thaliana).
Amor et al (1998) described the involvement of PARP in the, oxidative stress response in plants. The authors showed that in cultured, soybean pells, PARP is involved in responses to mild and severe abiotic stresses, by mediating DNA repair and programmed cell death processes, respectively.
W099/37789 describes compositions and methods for influencing the metabolic state of plant cells. The compositions comprise poly ADP-ribose polymerase genes and portions thereof, particularly the maize poly ADP-ribose polymerase gene as well as antisense nucleotide



culture assay, using a cold germination assay, by determination of the concentration of any one of reactive oxygen species, NAD or ATP or by any other stress tolerance assay. The nucleotide sequence of the parpl gene or parpl cDNA may comprises the nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 18, SEQ ID No. 19 or SEQ ID No. 20* or a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 13, SEQ ID No.:21 or SEQ ED No.:22 or a variant thereof.
It is another object of the invention to provide a method to produce a stress tolerant cotton plant comprising the steps of providing one or more double stranded RNA molecules to cells of the cotton plants, wherein the double stranded RNA molecules comprise two RNA strands, one RNA strand consisting essentially of an RNA nucleotide sequence of 20 to 21 consecutive nucleotides selected from the nucleotide sequence of a parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species; and identifying a cotton plant comprising die double stranded RNA molecule or molecules which is more resistant to abiotic stress conditions than a same cotton plant which does not comprise the double stranded RNA molecule or molecules. The double, stranded RNA may be,provided to the cells by integrating a chimeric gene into the genome of a cell, the chimeric gene comprising a DNA region comprising at least 20 consecutive nucleotides selected from the nucleotide, sequence of a .parp2 gene -or porp2 cDNA frqm.a cottop species or from a species related to a cotton progenitor species in antisense or sense orientation; operably linked to a plant expressible promoter and a DNA region comprising a transcription teiminatipn and polyadenylatfon signal functional in plants. The nucleotide sequence of the parpl gene or parpl cDNA may comprises the nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.; 11, SEQ ED No.: 12, SEQ ID No.: 18, SEQ ID No.: 19 or SEQ ID No.:20 or a nucleotide sequence encoding a protein, comprising the amino acid sequence of SEQ JD No.: 13, SEQ ID No.: 21 or SEQ, ID No. :22 or a variant thereof.
It is yet another object of the.inyention to provide a method to identify;cotton para2 DNA, fragments comprising thq steps of providing genomic DNA or cDNA obtainable from a cotton



identifying in each plant line of the population a. parpl allele according to the method of claim 21; analyzing the stress resistance of each plant line of the population and identifying those cotton plant lines ; and correlating the increased stress resistance in a plant line to the presence of a specific parpl allele. The cotton parpl allele may be introduced into a cotton plant line of choice to obtain stress tolerant plants.
Also provided is a method to identify a stress resistant cotton plant comprising the following steps: initiate a fiber tissue culture from the cotton plant; subject the fiber tissue culture to a stress condition, such as increased temperature, preferably in the rajjge of 45 to 50°C, for a selected period of time, preferably in the range of 2 to 4 hours; and compare fiber initation or elongation in the culture to fiber initiation or elongation in a, culture initated from a control plant and subject to the stresses conditions.
It is yet another object of the invention to provide an isolated DNA fragment encoding a protein comprising the amino acid sequence of SEQ ID No,: 13, SEQ ID,No.: 20, SEQ ID No,:21 or SEQ ID No,: 15, or comprising the nucleotide sequence selected from the group of any one of the nucleotide sequences of SEQ ID New 5, SEQ ID NO: 6, SEQ ID No,: 7, SEQ ID No.: 8, SEQ ID NO.: 9, SEQ ID No.: 10, SEQ ID No,: 11 SEQ IDNO.: 12, SEQ JP No.: 18, SEQ ID No.: 19 or SEQ ID No. :20.
The invention further provides a chimeric gene comprising the following operably linked DNA fragments: a plant expressible promoter; a transcribable DNA region comprising a first DNA region comprising at least 20 consecutive nucleotides selected from the nucleotide sequence of a parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species in sense orientation and a second DNA region comprising at least 20 consecutive nucleotides selected from the nucleotide sequence? of a parp2 gene or parp2 cDNA from a cotton species or from a species related to a cotton progenitor species in sense orientation, whereby an ,RNA molecule produced by transcription of the transcribed DNA region is capable of forming a double stranded RNA region by base-pairing between an RNA region corresponding to the first DNA region and an RNA region corresponding to the second RNA region; and a DNA region comprising a transcriprion termination and polyadenylation



Brief description of the Figures
Figure 1 is a schematic representation of pTMTOl, a T-DNA vector comprising a chimeric gene which upon transcription yields a double stranded RNA molecule capable of reducing the expression of cotton PARA2 genes. The following abbreviations are used: LB: left T-DNA border; 3'nos: transcription termination and polyadenylation signal from the nopaline synthase gene of A. tumefaciens T-DNA; 2mepsps: double mutant 5-enol-pyruvylshikimate-3-phosphate synthase protein from corn; TPotpC: transit peptide; PcsvmvX, Y, Z: first, second and third part of Cassava vein mosaic virus promoter; P35S2: Cauliflower mosaic virus 35 S promoter; parp2Gh: part of the cotton parp2 nucleotide sequence; Pdk-intron: Intron 2 from the pdk-intron. of Flaveria trinervia; OCS-terminator: transcription teiminatiqn and polyadenylation signal from the octopine synthase gene of A tumefaciens T-DNA; RB: right T-DNA border; NPTI-fragment: portion of the nptl antibiotic resistance gene; ORI ColEl: origin of replication of ColEl plasmid; ORI pVSl: origin of replication of pVSl replicpn.
Figure 2: Graphical representation of the cold germination assay. For each transgenic event (indicated by the number on the X-axis and by the pattern) the percentage of seedlings germinating at 16°C are indicated for the homozygous (H) and azygous (h) segregated populations. :.,.,. .;.;,,,,
Figure 3: Graphical representation of the conductivity of the mediiun after incubation in the presence of different concentrations of paraquat for control cotton plants (♦) or for transgenic cotton lines comprising zparpl silencing construct (■).
Figure 4 is an alignment of the various amino acid sequences obtained for parpl from cotton. GY1; amino acid sequence (3EQ ID NO: 21) encoded by genomic DNA variant one (SEQ ID NO; 19); cDNA: amino acid sequence (SEQ ID NO: 12) encoded by cDNA; GV2: amino acid sequence (SEQ ID NO: 22) encoded by genomic DNA variant two (SEQ ID NO: 20).



SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 19 or SEQ ID No.: 20. Other examples of such paip2 cotton sequences include the nucleotide sequences encoding a cotton PARP2 gene comprising e.g. the amino acid sequence of SEQ ID No.: 13 or of SEQ ID No.: 21 or of SEQ ID No.:22.
However, it will be immediately clear to the person skilled in the art that the exemplified nucleotide sequences or parts thereof can be used to identify further parpl genes or parpl cDNAs in other cotton plants, in cotton varieties other than Cooker312 or in cotton-progenitor related plants, and that such nucleotide sequences or parts thereof may also be used e.g. to increase the stress tolerance in cotton plants. The exemplified nucleotide sequences could be used to select:
i) a DNA fragment comprising a nucleotide sequence encoding the amino acid
sequence of SEQ ID No.: 13 for use as a probe;
ii) a DNA fragment comprising the nucleotide sequence of any one of SEQ ID No,: 5,
SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10,
SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No,: 19 or SEQ ID No.: 20 for use as a
probe :\ ■ ■ - . ■. ,■: ,.,■,
iii) a DNA fragment or oligonucleotide comprising a nucleotide sequence consisting of between 20 to 1382 consecutive nucleotides selected from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.: 13 for use as a probe; iv) a DNA fragment or oligonucleotide comprising a nucleotide sequence consisting of between 20 to 2000 consecxitive nucleotides selected from a nucleotide sequence encoding the amino acid sequence of SEQ ID Nos: 21 or 22 foruseasaprobe v) a DNA fragment or oligonucleotide comprising a nucleotide sequence consisting of between 20 to 2000 consecutive nucleotides selected from a nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 19 or SEQ ID No,: 20 for use as a probe; vi) an oligonucleotide sequence haying a nucleotide sequence comprising between 20 to 200 consecutive nucleotides selected from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.: 13 for use as a primer in a PCR reaction;







complement. However, it is preferred that the nucleic acid of interest always includes a sequence of about 19 consecutive nucleotides, particularly about 25 nt, more particularly about 50 nt, especially about 100 nt, quite especially about 150 nt with 100% sequence identity to the corresponding part of the target nucleic acid. Preferably, for calculating the sequence identity and designing the corresponding sense or antisense sequence, the number of gaps should be * minimized, particularly for the shorter sense sequences.
For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xl 00) divided by the number, of positions,compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues, The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970). The computer-assisted sequence alignment above, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madision, Wisconsin, USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference, to; nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application.
It has been demonstrated that the minimum requirement for silencing a particular target gene is the presence in the silencing chimeric gene nucleotide sequence of a nucleotide sequence of about 20-21 consecutive nucleotides long corresponding to the target gene sequence, in which at least 19 of the 20-21 consecutive nucleotides are identical to flie corresponding target gene sequence, "19 out of 20 consecutive nucleotides" as used herein refers to a nucleotide sequence of .20 consecutive nucleotides selected from the target gene having one mismatch nucleotide.



nucleotides, which serve as guide sequence in the degeneration of die corresponding mRNA (reviewed by Baulcombe, 2004). Thus, in another embodiment, the invention is drawn to a method for producing a stress tolerant cotton plant comprising the steps of:
a) providing one or more double stranded RNA molecules to cells of the cotton plants, wherein the double stranded RNA molecules comprise two RNA strands, one RNA strand consisting essentially of an RNA nucleotide sequence of 20 to 21 consecutive nucleotides selected from the nucleotide sequence of a parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species; and
b) identifying a cotton plant comprising these double stranded RNA molecule or molecules which is more resistant to abiotic stress conditions than a same cotton plant which does not comprise the double stranded RNA molecule or molecules.
The mentioned 20-21 nt long dsRNA sequences are also generated in the course of conventional antisense RNA mediated silencing or sense RNA mediated silencing. Thus, in another embodiment of the invention, a method is provided for producing stress tolerant cotton plants, comprising the step of providing to cells of the cotton plant a chimeric gene comprising, operably linked, the following DMA fragments
a) a plant expressible prompter;
b) a DNA region comprising at least 20 consequtive nucleotides selected from the nucleotide sequence of a parpl gene or para 2 cDNA from a cotton species or from a species related to a cotton progenitor species in antisense or in sense orientation;
c) a DNA region comprising a transcription termination and polyadenylatipn signal furictipn in plants.
The rnenuopeqannsense or,sense nucleotide regions may thus be about from about 21 nt to about 5000 nt long,, such as 21,nt, 40 nt, 50 nt, lOOnt, 200 nt, 300nt, 500nt, 1000 nt, or even . about 2000 nt or larger.in length. Moreover, it is not required for the purpose of the invention that the nucleotide sequence of the used inhibitory parpl gene molecule or the encoding region of the chimeric gene is completely idenitical or complementary to the endpgenous cotton parp2 gene the expression of which, is targeted to be reduced in the cotton plant cell The longer the sequence, theless stringpnl; the requirement for the overall sequence identity is. Thus, the sense







SEQ ID No 4: nucleotide sequence of the oligonucleotide Plx suitable to amplify a part of
cotton parp2 gene or cDNA. SEQ ID No 5: partial cDNA sequence of cotton parp2 gene (containing PARP signature)
variant 1. SEQ ID No 6: partial cDNA sequence of cotton paip2 gene (containing PARP signature)*
variant 2. SEQ ID No 7: partial cDNA sequence of cotton parp2 gene (containing PARP signature)
variant 3. SEQ ID No 8: partial cDNA sequence of cottonparp2 gene (part 2) Yariant 1. SEQ ID No 9: partial cDNA sequence of cotton parp2 gene (part 2) variant 2. SEQ ID No 10; partial cDNA sequence of cotton parp2 gene (part 2) variant 3. SEQ ID No 11: partial cDNA sequence of cotton parp2 gene (part 2) variant 4. SEQ ID No 12: partial nucleotide sequence of cotton parp2 cDNA (fused). SEQ ID No 13: partial amino acid sequence of cotton PARP2 protein. SEQ ID No 14: nucleotide sequence of T-DNA region of vector pTMTOl. SEQ ID No 15: variants of the partial amino acid sequence of cotton PARP2 protein. SEQ ID No 16: oligonucleotide primer 1 used for the preparation of a cotton parpl specific
probe. SJEQ YD No 17: oligonucleotide primer 2 used for the preparation of a cotton parpl specific
probe SEQ ID No 18: nucleotide sequence of a cotton parpl specific probe. SEQ ID No 19: nucleotide sequence of genomic DNA comprising a cotton parpl gene variant
SEQ ID No 20: nucleotide sequence of genomic DNA comprising a cotton para2 gene variant
2... ,. . . . ._.. .,
SEQ ID No 21: amino acid sequence of the protein structure which can be encoded by SEQ ID
NO19
SEQ ID No 22: amino acid sequence of the protein structure which can be encoded by SEQ ID
No 20. SEQ ID No.23: cDNA copy of the mRNA of cotton parpl gene yariant 1 SEQ ID No 24: cDNA copy of the mRNA of cotton parpl gene variant 2



encoded by the variant sequences contained the so-called PARA signature (TGYMFGKG) which is conserved in all PARP proteins.
On the basis of the amplified sequences, a new (non-degenerated) primer was designed which would allow amplification of the upstream part of the parp2 cDNA:
Plx: 5'-CAAGAGGAAACAGlTCACAGTGAAGC-3, (SEQEDNo.:4).
Using cDNA and PCR conditions as described above, with the exception that only 35 cycles were performed, and oligonucleotides Plx and Pic as primers, a DNA fragment of about 600 bp was amplified, which overlapped with the previously amplified part of the parpl cDNA, and constituted the part of the cDNA of parpl upstream of the previously amplified fragment. Again, variant sequences were identified (SEQ ID Nos 8-11).
SEQ ID No. 11 represents the nucleotide sequence of the fused para gene parts. SEQ ID No.: 13 includes the amino acid sequence of the encoded protein PARP2 by the nucleotide sequence of SEQ ID No.: 12.
Southern hybridizations were performed with genomic DNA of A-genome diploid cotton plants, and AD tetraploid plants. Two bands of which only one was present in the A-genome diploids, could be observed using several restriction enzyme digestions.
Example 2: Construction of a T-DNA vector containing a PARP2 silencing gene
An amplified. DNA fragment comprising the PARP signature as described in Example 1 was used to construct a chimeric gene which upon transcription yields an RNA molecule comprising a sense and antisense DNA sequence from the amplified DNA fragment, and which could basepair to form a double stranded RNA molecule. Such a chimeric gene can be used to reduce; the expression ofpqrpl in cotton. To this end the following DNA fragments were operably linked using standard recombinant DNA techniques:
• a fragment, including the promoter region of,the Cauliflower.Mosaic Virus 35S transcript (Odell et al 1985)(SEQ ID No.: 14 from nucleotide 2686 to nucleotide 3191)



The T-DNA vector was introduced into Agrobacterium tumefaciens comprising a helper Ti-plasmid. Cotton plants were transformed using the obtained A. tumefaciens strain, according to the protocol as described in US patent 6,483,013.
Example 3: Analysis of transgenic cotton plants harboring a PARA2 silencing gene.
Different transgenic cotton lines, comprising the chimeric gene as described in Example 1 were obtained. Transgenic plant lines were analyzed on molecular level using Southern blot analysis. Similarly,the plant lines are analyzed for parp2 RNA expression using Northern blot and for presence of PARP2 protein using e.g. ELISA or Western blotting. An indication of PARP activity can be obtained using e.g. the TUNEL assay which visualizes single stranded DNA breaks.
Transgenic plant lines of TO generation were backcrossed with Coker 312 plants, to reduce potential somaclonal variation in the resulting transformed plant lines.
The segregating populations of selfed transgenic cotton lines were analysed for the presence of the transgene in homozygous, or heterozygous form, or the absense of the transgene using real-tianePCR
The different plant lines are subjected to various forms of stresses. Either homologous populations of transgenic plants are compared to untransfonned reference plants, or segregating populations are used, followed by detennination of the homozygous, heterozygous and azygous plant lines using standard techniques.
A first assay is the "cold germination assay" whereby seeds are germinated on sandy soil at a temperature of 5°C. Similar test may also be used as described by Schulze et al.t 1996, Schulze et al 1996, Duesterhaus et al, 1999 or Duesterhaus et al, 2000



Example 4: Field trials with transgenic cotton lines
Different homozygous transgenic cotton lines, as well as corresponding null lines, identified as described in Example 3, were used in field trials comparing plots which received full-time irrigation were compared with plots which were only irrigated in the beginning of the grSwtlf season, thereby subjecting the cotton plants to significant heat stress. A hail storm destroyed part of the field, making interpretation of the results difficult. Nevertheless, it appeared that a few transgenic lines looked healthier and had more vegetative growth, i.e. appeared more vigorous.
Example 5: Analysis of transgenic cotton lines using a cold germination assay.
Transgenic cotton lines were selfed and the segregating progeny population was analyzed as
described in Example 3 for progeny plants which were either homozygous or which were
azygous. 50 seeds from either homozygous plants or from azygous plants for each event were
sown in sand. The trays were incubated at a constant temperature of 16°C for 21 days, when
germinated seedlings were counted. Germination of cotton seeds is sensitive to temperatures
lower than 18°C. At the same time 50 seeds from the same seedlots as mentioned above were
grown.qn sand but incubated at 26°C during the day and 21°C at night for 12 days. The
number of emerging seedlings was counted and used to correct the data for the cold
germination test for any effect of seed lot quality,
Figure 4 represents, data for 11 different events comparing the.homozygous lines, with azygous lines. Particularly the homozygous transgenic lines indicated as line 7, 9 and 11 performed very well, as almost no loss of germination during the stress could be observed.
Example 6: Analysis of transgenic cotton lines for tolerance to of paraquat treatment
Leaves from the three transgenic cotton lines identified in Example 5 as performing well in the cold germination assy were subjected to a paraquat tolerance assay, in comparison with leaves



The main difference between the polypeptides encoded by the genomic clones and the (incomplete) polypeptide encoded by the cDNA clone is the presence of an additional 26 amino acids stretch in the polypeptides encoded by the genomic clones (both variants) (SEQ ED No.: 21 from AA 444 to AA 469).
GV1 polypeptide moreover has an N-terminal extension (SEQ ID No 21 from AA 1 to AA 65) while it lacks a stretch of 48 amino acids present in GV2 (SEQ ID No 22 from AA 174 to AA 221). A similar stretch of amino acids (except the 4 AA; VLQK) are also absent from the polypeptide encoded by the cDNA clone. In addition the GV1 polypeptide has an insertion of about 11 amino acids in its C-terminal part (SEQ ID No 21 from AA 644 to AA 664)
Preferred target regions to include into the silencing constructs according to the invention may therefore be the nucleotide sequences encoding a polypeptide having the amino acid sequence of SEQ ID No.: 13 from 7 to 26; SEQ ID No.: 13 from 31 to 238; SEQ ID No.: 13 from 239 to 412; SEQ ID No.: 13 from 413 to 423; SEQ No.: ID 13 from 425 to 460.











3) The method according to claim 1, wherein said nucleotide sequence of said parpl gene or parpl cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 13 or SEQ ID No.: 21 orSEQ ID No.: 22.
5 4) The method according to claim 1, wherein said nucleotide sequence of said parpl gefie or' parpl cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 15.
5) The method according to any one of claims 1 to 3, wherein said first and said second DNA
) region comprise at least 50 consecutive nucleotides.
6) The method according to any one of claims 1 to 3, wherein said first and said second DNA
region comprise at least 200 consecutive nucleotides.
! 7) The method according to any one of claims 1 to 5, wherein said transgenic cotton plant which is more resistant to abiotic stress conditions than an untransformed cotton plant is identified using a fiber tissue culture assay.
8) The method according to any one of claims 1 to 5, wherein said transgenic cotton plant
1 which is more resistant to abiotic stress conditions than an untransformed cotton plant is
identified using a cold germination assay.
9) The method according to any one of claims 1 to 5, wherein said transgenic cotton plant which is more resistant to abiotic stress conditions than an untransformed cotton plant is identified by determination of the concentration of any one of reactive oxygen species, NAD or ATP.
10) A method to produce a stress tolerant cotton plant comprising the steps of:
a), providing one or,more, double stranded RNA molecules to cells of said cotton plants, wherein said double stranded RNA molecules comprise two RNA strands, one RNA strand consisting essentially, of an RNA nucleotide sequence of 19 out of 20 to 21



b) a transcribable DNA region comprising
i) a first DNA region comprising at least 19 out of 20 consecutive nucleotides selected from the nucleotide sequence of a parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species in sense orientation; ii) a second DNA region comprising at least 19 out of 20 consecutive nucleotides* selected from the nucleotide sequence of a parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species in sense orientation,
whereby an RNA molecule produced by transcription of said transcribed DNA region is capable of forming a double stranded RNA region by base-pairing between an RNA region corresponding to said first DNA region and an RNA region corresponding to said second RNA region; and c) a DNA region comprising a transcription termination and polyadenylation signal functional in plants.
14) The method according to any one of claims 10 to 13, wherein said nucleotide sequence of
said parpl genp or para2 cDNA comprises the nucleotide sequence of any one of SEQ ID
No.: 5, SEQ ID No.; 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ IP No.: 10,
SEQ ID No.; 11,SEQ ID No.: 12, SEQ ID No.: 18, SEQ ID No.: 19 or SEQIDNo.: 20.
15) The method according to any one of claims 10 to 13, wherein said nucleotide sequenpe of said parp2 gene ox para2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 13 or SEQ H) No.: 21 or SEQ ID No.: 22.
16) The method according to any one of claims 10 to 13, wherein said nucleotide sequence, of said parpl gene or para2 DNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 15.
17) A method to produce a stress tolerant cotton plant comprising the steps of







iii) a DNA fragment or oligonucleotide comprising a nucleotide sequence consisting of between 20 to 1382 consecutive nucleotides selected from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.: 13 for use as a probe;
iv) a DNA fragment or oligonucleotide comprising a nucleotide sequence consisting of between 20 to 2000 consecutive nucleotides selected from a nucleotide sequence -encoding the amino acid sequence of SEQ ID Nos: 21 or 22 for use as a probe
v) a DNA fragment or oligonucleotide comprising a nucleotide sequence consisting of between 20 to 2000 consecutive nucleotides selected from a nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 19 or SEQ ID No.: 20 for use as a probe;
vi) an oligonucleotide sequence having a nucleotide sequence comprising between 20 to 200 consecutive nucleotides selected from a nucleotide sequence encoding the amino acid sequence of SEQ ID No.: 13 for use as a primer in a PCR reaction;
vii)an oligonucleotide sequence having a nucleotide sequence comprising between 20
to 200 consecutive nucleotides selected from a nucleotide sequence encoding the
amino acid sequence of SEQ ID Nos: 21 or 22 for use as a primer in a PCR
reaction;
viii) an oligonucleotide sequence having a nucleotide sequence comprising between 20 to 200 consecutive nucleotides selected from the nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No,: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 19 or SEQ ID No.: 20 foruse as a primer in a PCR reaction or ,
ix) an oligonucleotide haying the nucleotide sequence, of any one of SEQ ID No.: 1, SEQ ID No.: 2, SEQID No.; 3 , SEQ ID No.; 4, SEQ ID No.: 16 or SEQ ID No.: 17 for use as a primer in a PCR reaction.
x) a fragment which can be amplified from cotton genomic or cDNA using as primers an oligonucleotide as described in vi, vii, viii or ix, such as a fragment comprising the nucleotide sequence of SEQ ID No, 18, for use as a probe.



26) The method according to claim 25, wherein said stress condition comprises subjecting said
fiber tissue culture to increased temperature for a selected period of time.
27) The method according to claim 26, wherein said increased temperature is selected from the range of 45 to 50°C, and said period of time is selected from the range of 2 to 4 hour.
28) An isolated DNA fragment encoding a protein comprising the amino acid sequence of SEQ ID No.: 13, SEQ ID No.: 15, SEQ ID No.:21 or SEQ ID No.:22.
29) An isolated DNA fragment encoding a protein comprising the amino acid sequence of SEQ
ID No.: 15.
30) An isolated DNA fragment comprising the nucleotide sequence selected from the group of any one of the nucleotide sequences of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ IDNo.: 18,SEQ ID No.: 19 or SEQ ID No.: 20.
31) An isolated DNA fragment obtainable by the method of claim 2L .
32) A chimeric gene comprising the following operably linked DNA fragments:

a) a plant expressible promoter;
b) a transcribable DNA region comprising
i) a first DNA region comprising at least 20 consecutive nucleotides selected from the nucleotide sequence of a. parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species in sense orientation;
ii) a second DNA region comprising at least 20 consecutive nucleotides selected from the nucleotide sequence of a parpl gene or parpl cDNA from a cotton species or from a species related to a cotton progenitor species in sense orientation, whereby an RNA molecule produced by transcription of said transcribed DNA region is capable of forming a double stranded RNA region by base-pairing



The chimeric gene according to any one of claims 32 to 34, wherein said nucleotide sequence of said parpl gene or para2 cDNA comprises a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 15.
38) A cotton plant cell comprising the chimeric DNA of any one of claims 32 to 37.
39) A cotton plant consisting essentially of the cotton plant cells of claim 38.
40) A cotton plant obtained by the method of claim 24.
41) A seed of a cotton plant according to claim 39 or claim 40.
42) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 13, SEQ ID No.: 21 or SEQ ID No.: 22 or a part thereof comprising at least 19 out of 20 consecutive nucleotides to increase the stress tolerance of a cotton plant
43) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 15 or a part thereof comprising at least 19 out of 20 consecutive nucleotides to increase the stress tolerance of a cotton plant.
44) Use of a nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID NO,: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 18, SEQ ID No.: 19 or SEQ ID No.: 20 or a part thereof comprising at least 19 out of 20 consecutive nucleotides to increase the stress tolerance of a cotton plant.
45) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ,ID No.: 13 or a,part thereof comprising at least 19 out of 20 consecutive nucleotides to identify a parpl gene or para2 cDNA in a a cotton species, such as Gossypium hirsutum, Gossypium barbaffense, Gossypium arboreum, or Gossypium herbaceum or from a cotton
species related to; a cotton progenitor species such as Gossypium raimondii, Gossypium trilobum and Gossypium gossypioides.

46) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of
SEQ ID No.: 15 or a part thereof comprising at least 19 out of 20 consecutive nucleotides to
to identify a parpl gene or parpl cDNA in a a cotton species, such as Gossypium
hirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium herbaceum or from
a cotton species related to a cotton progenitor species such as Gossypium raimohdii,
Gossypium trilobum and Gossypium gossypioides.
47) Use of a nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7,
SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ
ID No.: 18, SEQ ID No.: 19 or SEQ ID No.:20 or a part thereof comprising at least 19 out
of 20 consecutive nucleotides to identify a parpl gene ox parpl cDNA in a cotton species,
such as Gossypium kirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium
herbaceum or from a cotton species related to a cotton progenitor species such as
Gossypium raimondii, Gossypium trilobum and Gossypium gossypioides.
48) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 13, SEQ ID No. 21 or SEQ ID No.: 22 or a part thereof comprising at least 19 out of 20 consecutive nucleotides to identify a stress tolerant parpl allele in a cotton species, such as Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium herbaceum or from a cotton species related to a cotton progenitor species such as Gossypium raimondii, Gossypium trilobum and Gossypium gossypioides .
49) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No.: 15 or a part thereof comprising at least 19 out of 20 consecutive nucleotides to identify a stress tolerant parpl allele in a cotton species, such as Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium herbaceum or from a cotton
species related to a cotton progenitor species such as Gossypium raimondii, Gossypium

trilobum sad Gossypium gossypioides.
50) Use of a nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7,
SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No.: 12, SEQ

ID No.: 18, SEQ ID No.: 19 or SEQ ID No.: 20 or a part thereof comprising at least 20 consecutive nucleotides to identify a stress tolerant parpl allele in a cotton species, such as Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, or Gossypium herbaceum or from a cotton species related to a cotton progenitor species such as Gossypium raimondii, Gossypium trilobum and Gossypium gossypioides.
51) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of
SEQ ID No.: 13, SEQ ID No.:21 or SEQ ID No.:22 or a part there of comprising at least 19
out of 20 consecutive nucleotides to introduce a stress tolerant parpl allele in a cotton
species.
52) Use of a nucleotide sequence encoding a protein comprising the amino acid sequence of
SEQ ID No.: 15 or a part thereof comprising at least 19 out of 20 consecutive nucleotides
to introduce a stress tolerant parpl allele in a cotton species.
53) Use of a nucleotide sequence of any one of SEQ ID No.: 5, SEQ ID No,: 6, SEQ ID No.: 7,
SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10, SEQ ID No.: 11, SEQ ID No,: 12, SEQ
ID-No-.: 18, SEQ ID No.:19 or SEQ ID No.:20 or a part thereof comprising at least 19 out of
20 consecutive nucleotides to introduce a stress tolerant parpl allele in a cotton species.


Documents:


Patent Number 243312
Indian Patent Application Number 2301/CHENP/2007
PG Journal Number 41/2010
Publication Date 08-Oct-2010
Grant Date 06-Oct-2010
Date of Filing 29-May-2007
Name of Patentee BAYER BIOSCIENCE N.V.
Applicant Address TECHNOLOGIEPARK 38, B-9052 GENT, BELGIUM
Inventors:
# Inventor's Name Inventor's Address
1 REYNARTS, ARLETTE BUISSTRAAT 5, B-9031 DRONGEN, BELGIUM
2 JACOBS, JOHN WILGENSTRAAT 4B, 9820 MERELBEKE, BELGIUM
3 VAN THOURNOUT, MICHEL GROENE POORDREEF 32, 8200 SINT-MICHIELS, BELGIUM
PCT International Classification Number C12N 15/82
PCT International Application Number PCT/EP05/11657
PCT International Filing date 2005-10-27
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 04077984.5 2004-10-29 EUROPEAN UNION
2 60/628,597 2004-11-17 EUROPEAN UNION