Title of Invention

A METHOD FOR MODIFYING THE LENGTH OF A FIBER OF A COTTON PLANT

Abstract Methods and means and provided for modulating fiber length in fiber producing plants such as cotton by altering the fiber elongation phase. The fiber elongation phase may be increased or decreased by interfacing with callose deposition in plasmodesmata at the base of the fiber cells.
Full Text

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Methods and means for altering fiber characteristics in
fiber-producing plants.
Field of the invention
Hie invention relates to the field of agriculture, more specifically towards the use of molecular biology techniques to alter fiber producing plants, particularly cotton plants and/or accelerate breeding of such fiber containing plants. Methods and means are provided to increase fiber length, particularly lint fiber length or to decrease the length of fuzz fibers. Methods are also provided to identify molecular markers associated with fiber length in a population of cotton varieties and related progenitor plants.
Background art
Much of the high quality fiber for the textile industry is provided for by cotton. About 90% of cotton grown worldwide is Gossypium hirsutvm L., whereas Gossypium barbadense accounts for about 8%. Consequently, the modification of cotton fibers characteristics to better suit the requirements of the industry is a major effort in breeding by either classical methods or by genetically altering the genome of cotton plants. Goals to be achieved include increased lint fiber length, strength, dyability decreased fuzz fiber production, fiber maturity ratio, immature fiber content, fiber uniformity and micionaire.
WO0245485 describes methods and means to modulate fiber quality in fiber-producing plants, such as cotton, by modulating sucrose synthase activity and/or expression in such plants.
US6472588 and WO0117333 provides methods for increasing the quality of cotton fiber produced from a cotton plant by transformation with a DNA encoding sucrose phosphate synthase. The fiber qualities include strength, length, fiber maturity ratio, immature fiber content, fiber uniformity and nricronaire.

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WO9508914 discloses a fiber producing plant comprising in its genome a heterologous genetic construct The genetic construct comprises a fiber-specific promoter and a coding sequence encoding a plant peroxidase, such as a cotton peroxidase.
WO9626639 provides methods whereby encoding sequence preferentially directing gene expression in ovary tissue, particularly very early in fruit development, are utilized to express plant growth modifying hormones in cotton ovule tissue. The methods permit the modification of the characteristics of boll set in cotton plants and provide a mechanism for altering fiber quality characteristics such as fiber dimension and strength.
US5981834, US5597718, US5620882, US5521708 and US 5495070 all disclose a method for genetically engineering a fiber-producing plant and the identification of cDNA clones useful for identifying fiber genes in cotton. The cDNA clones are useful in developing corresponding genonric clones from fiber producing plants to enable genetic engineering of cotton and other plants using these genes. Coding sequence from these isolated genes are used in sense or antisense orientation to alter the fiber characteristics of trangenic fiber producing plants.
Published US patent applications US2002049999 and US2003074697 both disclose cotton plants of the genus Gossyphim with unproved cotton fiber characteristics. The cotton plant has an expression cassette containing a gene coding for an enzyme selected from the group consisting of endoxyloglucan transferase, catalase and peroxidase so that the gene is expressed in cotton fiber cells to improve the cotton fiber characteristics.
US5880110 produces cotton fibers with improved fiber characteristics by treatment withbtassinosteroids.
WO 01/402S0 provides methods for improving cotton fiber quality by modulating transcription factor gene expression.

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WO 96/40924 provides novel DNA constructs which may be used as molecular probes or inserted into a plant host to provide for modification of transcription of a DNA sequence of interest during various stages of cotton fiber development The DNA constructs comprise a cotton fiber transcriptional initiation regulatory region associated with a gene, which is expressed in cotton fiber. Also is novel cotton having a cotton fiber, which has a natural color, introduced by Hie expression in cotton fiber cell, using such a construct, of pigment genes.
EP0834566 provides a gene which controls the fiber formation mechanism in cotton plant and which can be used for industrially useful improvement
However,, there is still need for alternative methods and means to attar fiber characteristics of fiber-producing plants such as cotton, which, may be further combined wife, any of other methods to alter fiber characteristics. Such a method is described in the embodiments and paragraphs described hereinafter.
of the invention
In one embodiment of the invention a method for modifying a fiber of a fiber-producing plant, such as cotton, is provided comprising the step of altering a fiber cell elongation phase by modulating deposition of callose at the neck of the plasmodesmata at die base of the fiber cell
In another embodiment of the invention a method for increasing the length of a fiber of a fiber producing plant, such as cotton is provided, comprising the step of introducing a chimeric gene into a cell of the fiber producing plant; wherein the chimeric gene, when expressed in the cell of the fiber-producing plant increases die fiber elongation phase and increases die deposition of callose. The chimeric gene may comprise die following operably linked DNA elements:
- a plant expressible promoter, preferably a plant expressible promoter which
. controls transcription preferentially in die fiber cells such as a fiber-specific
beta tubulin promoter from cotton, a fiber-specific actin promoter from cotton,
a fiber specific promoter from a lipid transfer protein gene from cotton, a

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promoter from an expansin gene from cotton or a promoter from a chitinase gene in cotton;
* a transcribed DNA region, which when transcribed yields a double-stranded RNA molecule capable of reducing the expression of a gene endogenous to the fiber producing plant, the gene being involved in callose removal from the plasmodesmata, such as a 13-p-glucanasc gene which is expressed at the base of the fiber cell, at the end of the fiber elongation phase in the fiber producing plant, and the RNA. molecule comprising a first and second RNA region wherein
- the first RNA region comprises a nucleotide sequence of at least 19
consecutive nucleotides having at least about 94% sequence identify to die
nucleotide sequence of die endogenous gene;
- the second RNA region comprises a nucleotide sequence complementary
to the 19 consecutive nucleotides of the first RNA region;
- fiie first and second RNA region are capable of base-pairing to form a
double stranded RNA molecule between at least the 19 consecutive
nucleotides of the first and second region; and
- a 3' end region comprising transcription termination and polyadenylation
signals functioning in cells of flic plant
The endogenous gene may encode a protein comprising the ammo acid sequence of SEQ ID 4 or it may comprise the nucleotide sequence of SEQ ID No 1 or the first RNA region may comprise a nucleotide sequence of at least 19 consecutive nucleotides having at least about 94% sequence identity to the nucleotide sequence of SEQ ID No 1.
In another embodiment of the invention the chimcric gene may comprise
- a plant-expressible promoter, preferably a plant-expressible promoter which
controls transcription preferentially in the fiber cells, such as a fiber-specific
beta tubulin promoter from cotton, a fiber-specific actin promoter from cotton,
a fiber specific promoter from a lipid transfer protein gene from cotton, a ' promoter from an expansin gene from cotton or a promoter from a chitinase gene in cotton;

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%
- a DNA region encoding a p-l»3 ghican synthase protein, such as a DNA
region comprising the nucleotide sequence of SEQ ID No 2; and
- a 3'end region comprising transcription termination and polyadenylation
signals functioning in cells of the plant
In yet another embodiment of the invention, a method for decreasing the length of a fiber of a fiber producing plant; comprising flic step of introducing a chimeric gene into a cell of file fiber producing plant, wherein the chimeric gene, when expressed in fiie cell of flie fiber-producing plant decreases the deposition of callosc and decreases fiie fiber elongation phase.
It is also an object of fee invention to provide a method for identifying allelic variations of the genes encoding proteins involved in fiber elongation in a population of different genotypes, cultivars or varieties of a particular plant species, preferably a fiber-producing plant species, which are correlated either alone or in combination with the length of fibers produced, comprising file steps of
(a) Providing a population of different varieties or genotypes of a particular plant
species or interbreeding plant species comprising different allelic forms of the
nucleotide sequences encoding callose synthase or (5-1,3 glucanase,
particularly of SEQ ID No 1 or SEQ ID 2;
(b) Determining parameters related to fiber length for each individual of the
population;
(c) Determining the presence of a particular allelic form of the nucleotide
sequences encoding callose synthase or £-1,3 glucanase, particularly of SEQ
IDNolorSEQID2;
(d) Correlating the occurrence of particular fiber length with the presence of a
particular allelic form of the mentioned nucleotide sequence or a particular
combination of such allelic forms.
In yet another embodiment, the invention provides the chimeric genes as herein described, as well as cells of a fiber producing plant comprising such chimeric genes.

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It is also an object of the invention to provide fiber- producing plants, such as cotton, and seeds or progeny comprising a cbimeric gene according to the invention, particularly fiber producing plants which have increased fiber length or increased drought resistance;
Hie invention also provides fibers produced according to the methods of the invention.
Brief description of the fignres
Figure 1 is a graphic representation of the evolution of the fiber length in time for three tetraploid cotton cuWvars with short, normal and long fibers (Gh-r, Gh-c and Gb, respectively). The X-axis represents days after anthesis (DAA), whereas the Y-axis represents the fiber length in cm. The triangles represent the fiber length for Gb, the closed circles represent fiber length for Gh-c and the open circles represent Gh-r. The closure of the plasmodesmata is indicated by the horizontal bars (open bar for Gh-c; closed bar for Gb; plasmodesmata do not close in Gh-r).
Figure 2. Localization of callose in plasmodesmata at the fiber base in cuitivar Gh-c, at 10 DAA (panels B, E and F) when plasmodesmata woe closed, at 5 DAA (panel A and D) before plasmodesmata are closed, or at 20 DAA (panel C) when pi&5ijjGu£Suiata arc rc-cpsnsd Panels A to C: aniline blue fluorescent labeling. Panels D to E: immuno-gold labeling with monoclonal antibody against callose (light-microscope). Panel F; immuno-gold labeling with monoclonal antibody against callose (electron-microscope). Arrows indicate the fiber base, where callose deposition may occur, f: fiber, sc: seed coat
Figure 3: Northern analysis of mRNA, prepared from developing fibers 6,12 and 20 DAA and 6-d seed, in Gh-r, Gh-c and Gb cotton cultivars. The used probes are either a cDNA clone of 3-1,3 glucanase comprising the sequence of SEQ ID No 1 (upper panel) or a cDNA clone of fM»3 glucanase comprising the sequence with Accession number AI728205).

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IWafled description nf different emhftiBments
Each cotton Hnt fiber is a single cell that elongates to 2.5 to 3.0 cm form the seed coat epidermis within -16 days after anthesis(DAA). Roan et aL 2001 (The Plant Cell 13: pp 47-60) found that this elongation process was controlled by gating of the plasmodesmata and coordinated expression of sucrose and K+ transporters and expansin. Single celled cotton fibers interconnect with the underlying seed coat only at their base regions, where a high number of plasmodesmata axe present Plasmodesmata are the intercellular cytoplasmic connections that act as gates controlling molecular trafficking from cell to cell. During the rapid elongation ph
(-10 to ~16 DAA), the symplastic connection was disrupted, allowing the rapid buildup of a turgor in the fiber cell, which is higher than in the underlying seed coat cells, by active solute import into the fiber celL Coordinated with the cell wall loosening (into alia by expansins), tins higher turgor pushes out the fiber cell to its length. Ruan et aL also examined the possibility that callose deposition at die neck region of the plasmodesmata was implicated in the closing thereof; but found no correlation using a monoclonal antibody against callose, between the deposit of callose and the closing an4 reopening of the plasmodesmata.
The current invention is based on the observations by the inventors that on the one hand, the length of the period of the closing of plasmodesmata in different cotton cultivars is correlated with the variation in fiber length, and on the other hand, that callose deposition is involved in the closure of the plasmodesmata. Additionally, it was observed that the timing and the level of expression of a fiber-specific P-1,3-ghicanase gene among three cotton cultivars differing in fiber length correlated with the degradation of callose in the plasmodesmata.
Thus, in one embodiment of the invention, a method is provided for altering the length of a fiber of a fiber-producing plant, such as a cotton plant, comprising die step of altering the fiber cell elongation phase by modulating the deposition of callose at the neck of the plasmodesmata at the base of the fiber celL

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Conveniently, the deposition of callose may be altered by introduction of a chimeric gene capable of modulating the deposition of callose at the neck of the plasmodesmata at the base of the fiber celL Ibis maybe achieved eg. by increasing the expression level and/or the activity of the encoded product of a gene involved in callose removal, such as a p-1,3 ghicanase. Deposition of callose may also be altered by increasing the expression level and/or the activity of Hie encoded product of a gene involved in callose synthesis and accumulation) such as a P-13 gtucan synthase (callose synthase).
In one embodiment, the drimeric gene may encode a silencing RNA molecule or an inhibitory RNA molecule, capable of reducing the expression of a gene involved in callose removal from the plasmodesmata at the base of the fiber cell to increase fee fiber length. Such reduction of the expression of a gene involved in callose removal should occur preferably through post-transcriptional silencing. However, it will be dear that even when an inhibitory SNA molecule decreases the expression of a gene involved in callose removal through post-transcriptional silencing, such an SNA molecule may also exeat other functions within a cell, such as guiding DNA methylation of the endogenous gene involved in callose removal, again ultimately leading to decreased expression of that gene. Also, expression of endogenous genes involved in callose removal may be reduced by transcriptional silencing, e.g. by using RNAi or dsRNA targeted against the promoter region of the endogenous gene involved in callose removal.
Several methods are available in the art to produce a silencing RNA molecule, ie. an RNA molecule which when expressed reduces the expression of a particular gene or group of genes, including the so-called "sense" or "antisense" RNA technologies.
Thus in one embodiment, the inhibitory RNA molecule encoding drimeric gene is based on the so-called antisense technology. In other words, the coding region of the chimeric gene comprises a nucleotide sequence of at least 20 consecutive nucleotides of the complement of the nucleotide sequence of the endogenous gene involved in callose removal of the plant Such a chimeric gene may be constructed by operably linking a DNA fragment comprising at least 20 nucleotides from a gene involved in

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callose removal, isolated or identified as described elsewhere in tins application, in inverse orientation to a plant expressible promoter and 3* end formation region involved in transcription termination and polyadenylation. It wfll be clear that there is no need to know the exact nucleotide sequence or the complete xnicleotide sequence of such a DNA fragment fiom the isolated gene involved in callose removaL
In another embodiment, the inhibitory RNA molecule encoding chimeric gene is based on fee so-called co-suppression technology. In other words, the coding region of the chimeric gene comprises a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence of the endogenous gene involved in callose removal of the plant Such a chimeric gene may be constructed by operably linking a DNA fragment comprising at least 20 nucleotides fiom a gene involved in callose removal, isolated or identified as described elsewhere in this application, in direct orientation to a plant expressible promoter and 3* end formation region involved in transcription termination and polyadenylation. Again, it will be clear that there is no need to know the exact nucleotide sequence or the complete nucleotide sequence of such a DNA fragment fiom the isolated gene involved in callose removaL
The efficiency of the above mentioned chimeric genes in reducing the expression of the endogenous gene involved in callose removal may be further enhanced by the inclusion of DNA element which result in the expression of aberrant, unpolyadenylated inhibitory SNA molecules or results in the retention of the inhibitory RNA molecules in the nucleus of the cells. One such DNA element suitable for that purpose is a DNA region encoding a self-splicing ribozyme, as described in WO 00/01133 (incorporated by reference). Another such DNA element suitable for that purpose is a DNA region encoding an RNA nuclear localization or retention signal, as described in PCT/AU03/00292 published as WO03/076619 (incorporated by reference).
A convenient and very efficient way of downregulating the expression of a gene of interest uses so-called double-stranded RNA (dsRNA) or interfering RNA (RNAi), as described e.g. in WO99/53050 (incorporated by reference). In this technology, an RNA molecule is introduced into a plant cell, whereby the RNA molecule is capable of forming a double stranded RNA region over at least about 19 to about 21

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nucleotides, and whereby one of the strands of this double stranded KNA region is about identical in nucleotide sequence to the target gene ("sense region"), whereas the other strand is about identical in nucleotide sequence to the complement of the target gene or of the sense region fantisense region**). It is expected that for silencing of the target gene expression, the nucleotide sequence, of the 19 consecutive nucleotide sequences may have one mismatch, or the sense and antisense region may differ in one nucleotide. To achieve the construction of such SNA molecules or the encoding cMmeric genes, use can be made of the vector as described in WO 02/059294.
Thus, in one embodiment of the invention, a method for increasing the length of a fiber of a fiber producing plant, such as cotton, is provided comprising the step of introducing a chimeric gene into a cell of the fiber producing plant, wherein the chimeric gene comprises the following opcrably linked DNA elements:
(e) a plant expressible promoter, preferably a plant expressible promoter which
controls transcription preferentially in the fiber cells;
(f) a transcribed DNA region, which when transcribed yields a double-stranded
KNA molecule capable of reducing the expression of a gene endogenous to the
fiber producing plant; the gene being involved in caUose removal from the
plasmodesmata, and the RNA molecule comprising a first and second RNA
region wherein
i) the first RNA region comprises a nucleotide sequence of at least 19
consecutive nucleotides having at least about 94% sequence identity to the
nucleotide sequence of the endogenous gene; ii) the second RNA region comprises a nucleotide sequence complementary
to the at least 19 consecutive nucleotides of the first RNA region; iif) the first and second RNA region are capable of base-pairing to form a
double stranded RNA molecule between at least the 19 consecutive
nucleotides of fee first and second region; and
(g) a 3' end region comprising transcription termination and polyadenylaticm
signals functioning in cells of the plant
As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups

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thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, le.» be embedded in a larger nucleic add or protein. A chimeric gene comprising a DKA region, which is functionally or structurally defined, may comprise additional DKA regions etc.
The length of the first or second RNA region (sense or antisense region) may vary fiom about 19 nucleotides (nt) up to a length equaling the length (in nucleotides) of the endogenous gene involved in callose removal. Hie total length of Hie sense or antisense nucleotide sequence may Urns be at least at least 25 nt, or at least about 50 nt, or at least about 100 nt, or at least about ISO nt, or at least about 200 nt, or at least about 500 nt It is expected that there is no upper limit to file total length of the sense or the antisense nucleotide sequence. However for practical reasons (such as e.g. stability of the chimeric genes) it is expected that the length of the sense or antisense nucleotide sequence should not exceed 5000 nt, particularly should not exceed 2500 nt and could be limited to about 1000 nt
It will be appreciated that the longer the total length of the sense or antisense region, the less stringent the requirements for sequence identity between these regions and the corresponding sequence in the endogenous gene involved in callose removal or its complement Preferably, the nucleic add of interest should have a sequence identity of at least about 75% with die corresponding target sequence, particularly at least about SO %» more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially be identical to the corresponding part of the target sequence or its 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 die 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

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the two optimally aligned sequences winch have identical residues (xlOO) divided by the number of positions compared A gap, Le. 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 SO and a gap extension penalty of 3.
It will be clear that whenever nucleotide sequences of KNA molecules axe defined by reference to nucleotide sequence of corresponding DNA molecules, the thynrine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNAorDNA molecules will be clear from the context of the application,
dsRNA encoding chimeric genes according to the invention may comprise an intron, such as a heteroiogous mtron, located e.g. in the spacer sequence between the sense and antisense SNA regions in accordance with the disclosure of WO 99/53050 (incorporated herein by reference).
As used herein, an "endogenous gene involved in callose removal** is a gene whose expression product regulates or catalyzes the breakdown of callose deposited at a particular location in plants* "Callose" is a long-chain carbohydrate polymer, consisting of (5-1,3-glucan, that seals certain regions, e.g., damaged sieve elements, growing pollen tubes, or plasmodesmata.
As used herein, an "endogenous gene" is a gene that naturally occurs in the species of the fiber-producing plant that has been chosen for modulation of fiber characteristics, or a gene that occurs naturally in a species of another fiber-producing plant but may be introduced into the species of the fiber-producing plant that has been chosen for modulation of fiber characteristics, by conventional breeding techniques.

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A target gene involved in caUose removal from piasmodesmata at the base of fiber cells in fiber-producing plants such as cotton, is an endo-13-p-ghicanase gene that is naturally expressed at the base of the fiber cell, at the end of fiber elongation phase*. An example of such a l,3-{3-glucanase gene from cotton, is a gene encoding a protein comprising the amino acid sequence of SEQ ID No 4 or comprising the nncleotide sequence of SEQ ID I (or Genbank Accession number D88416). Shimuzu et aL (Plant Cell Physiology 38 (3), pp 375-378, L997) have described that the level of mRNA for endo-13-P-gbcanase was very low in elongating fiber cells, but increased gradually at the onset of secondary wall synthesis, accompanying the massive deposition of cellulose, but characterized this endo-l,3~P-gtocanase activity as required for the deposition of cellulose. The current invention has correlated the endo-l^-P-ghicanasc activity with caHose removal in different cotton variety with different lengths of fiber elongation phases.
Variants of the endo-l,3-P-ghicanase gene involved in removal of caUose from plasmodesmata at the base of elongating fiber cells, such as the endogenous genes coding for endo-l»3-p~glucanase from fiber producing plants different from cotton, maybe found by stringent hybridization using the nucleotide sequence of SEQ ID No 1, or a pact thereof comprising at least about 25 or SO consecutive nucleotides of SEQ ID No 1 or the complementary nucleotide sequences thereof as a probe.
"Stringent hybridization conditions1" as used herein means that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfete, and 20 jig/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 x SSC at approximately 65 °C, preferably for about 10 minutes. Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), particularly chapter 11.

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Such variant sequences may also be obtained by DNA amplification using oligonncteotidea specific for eado-13-P-glucanase gene as primers, such as but not limited to oKgonucleotides comprising or consisting of about 20 to about 50 consecutive nucleotides of the nucleotide sequence of SEQ ID 1 or its complement Variant sequences incfrT^ modificatkms of a sequence by addition, deletion or substitution of nucleotides.
As used herein, the term "promoter" denotes my DNA which is recognized and bound (directly or indirectly) by a DNA-dependent RNA-polymerase during initiation of transcription. A promoter includes the transcription initiation site, and binding sites for transcription initiation factors and RNA polymeiase, and can comprise various other sites (e.g», enhancers), at which gene expression regulatory proteins may bind.
As used herein the tenn ^lant-expressibte which
is capable of controlling (initiating) transcription in a plant celL This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e«, certain promoters of viral or bacterial origin such as the CaMV35S, die subterranean clover virus promoter No 4 or No 7, or T-DNA gene promoters and the like.
A plant-expressible promoter that controls initiation and maintenance of transcription preferentially in fiber cells is a promoter that drives transcription of the operably linked DNA region to a higher level in fiber cells and the underlying epidermis cells than in other cells or tissues of the plant Such promoters include the promoter from cotton from a fiber-specific {J- tubulin gene (as described in WO0210377), the promoter from cotton from a fiber-specific actin gene(as described in WO0210413), the promoter from a fiber specific lipid transfer protein gene from cotton (as described in US5792933), a promoter from an expansin gene from cotton (WO9830698) or a promoter from a chitinase gene in cotton (US2003106097) or the promoters of the fiber specific genes described in US6259003 or US6166294.

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As mentioned above, deposition of callose, to increase me closing of the plasmodesmata at the base of the fiber cell and consequently increase the fiber elongation phase, may also be alteted by increasing the expression level and/or the activity of Hie encoded product of a gene involved in callose synthesis and accumulation, such as a p-1,3 ghican synfhase (callose synthase).
Thus in another embodiment, a method is provided to increase fiber length comprising introduction of a chimeric gene into cells of a fiber-producing plant of a chimeric
gene comprising
. a plant-expressible promoter, preferably a plant-expressible promoter which controls transcription preferentially in the fiber cells;
- a DNA region encoding a $-1,3 ghican synthase protein; and
- a 3'end region comprising transcription termination and polyadenylation signals
functioning in cells of the plant
A suitable DNA region encoding a (3-1,3 ghican synthase protein is a DNA region comprising the nucleotide sequence of SEQ ID No 2 (Geribanlt Accession number AI730469) or encoding a protein comprising the amino add sequence of SEQ ID No 3.
Alternative DNA regions encoding a $-1,3 ghican synthase protein may be found in nucleotide sequence databases such as die entries with the following identification numbers AF085717 (Gossypium hirsutam) AY324384 (Oryza sativa (japonica cultivar-group); NMJ79940, NM_121303, NM_116593, NMJ79622, NMJ23045, NMJ16736, NMJ15772, NMJ12317, NM_111596, NM.100528, NMJ00436 (Arabidopsis thaliana); AY17766S (Hordeum vulgare subsp. vulgare); BQ702515 (Pinus taeda); BQ696956, BG625796 BG625791, BG317521, BF516675 (Pinus taeda); CA935202 (Gfycine max) CA900204, CA900203, CA900202 (Phaseolus coccineus); BI978498 (Rosa chinensis); BU964672, BU927399 (Gfycine max); > AL750S22 (Pinus pinaster); BQ081239, BQ080234 (Gfycine max); AJ43O780 (Vitis vinifera)\ BM270236, BF066990, BG651282, BG509952, BG363511, BG359433, BG157340, BM086291 (Gfycine max); AF237733 (Arabidopsis thaliana); BE644560 (Suaeda maritima subsp. salsa); BE040372 (Oryza sativa).

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Variants of these sequences may be obtamed by substitution* deletion or addition of particular mtcleotides, and such variants may also be suitable for the currently described methods and means, particularly if they retain endo-p-13-glucan synthase activity.
It will be clear that the methods and means described herein to increase the length of fibers in fiber-producing plants may be combined with each other to further increase the length of fibers.
The methods of the current application may of course also be combined with other methods to altar fiber characteristics as known in the art
In one embodiment of the invention, the methods of the current application are combined with those described in WO02/45485 whereby fiber quality in fiber producing plants, such as cotton* is modified by modulating sucrose synthase activity and/or expression in such plants. In a particular embodiment, cotton plants are provided comprising a chimeric gene as herein described, which when expressed in the cells of the fiber producing plants, such as the fiber base cells, increases die fiber eleongation phase and increases die deposition of callose and further comprising a chimeric gene as described in WO02/45485 (herein incorporated by reference) which when expressed results h* iiK-t^as^d «xprK&sinn or activity of sucrose synthase. It is expected that the combined expression of the genes will result in a synergistic effect on the increase of the fiber length and/or strength.
The chimeric genes may be introduced by subsequent transformation into cells of one plant, or may be combined into cells of one plant by crossing between plants comprising one chimeric gene each.
I Furthermore, the invention also provides cotton plants, combining (a) natural occurring allele(s) of the fiber preferential /3-1,3-glucanase gene(s) associated with longer closing of the plasmodesmata gates and with long fiber length and (a) natural

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occurring allele(s) of (a) sucrose synthase gene(s) resulting in high expression of sucrose synthase in fiber cells.
It is also expected that the methods described here to increase the fiber elongation phase and fiber length, particularly the reduction of expression of the endogenous gene involved in callose removal, will also lead to increased drought resistance, particularly of the fibers.
For some fiber-producing plants, it may sometimes be beneficial to decrease fiber length, particularly eliminate fiber production. Tins can be achieved according to the invention by shortening the fiber elongation phase through decreased callose deposition or increased callose removal. To tins end, chimeric genes may be introduced which when expressed decrease callose deposition or increase callose removal. Such chimeric genes may comprise the following operably linked DNA
- a plant-expressible promoter, preferably a plant-expressible promoter which
controls transcription preferentially in fiber cells;
• a DNA region encoding a gene involved in callose removal, such as a 0-1,3 glucanase protein; and
- a 3'end region comprising transcription termination and polyadenylation signals
functioning in cells of that plant;
or
- a plant expressible promoter which controls transcription preferentially in fiber
cells;
- a transcribed DNA region, which when transcribed yields a double-stranded SNA
molecule capable of reducing the expression of a gene endogenous to the fiber
producing plant, the gene being involved in callose deposition, such as a callose
synthase, in the plasmodesmata at the base of a fiber cell, and wherein the RNA
molecule comprising a first and second RNA region wherein
- fiie first SNA region comprises a nucleotide sequence of at least 19 consecutive nucleotides having at least about 94% sequence identity to the nucleotide sequence of the mentioned endogenous gene;

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. the second RNA region comprises a nucleotidc sequence complementary to
tbe 19 consecutive nucleotides of the first RNA region; - the first and second RNA region are capable of base-pairing to form a double
stranded RNA molecule between at least fte 19 consecutive nucleotides of the
first and second region; and
- a 3* end region comprising transcription termination and polyadenylation signals functioning in cells of die fiber producing plant
The invention also encompasses the chimeric genes herein described, as well as plants, seeds, tissues comprising these chimeric genes, and fibers produced from such plants.
Methods to transform plants are well known in the art Methods to transform cotton plants axe also well known in the art Agrobacteriwn-mediated transformation of cotton has been described e.g. in US patent 5,004,863 or in US patent 6,483,013 and cotton transformation by particle bombardment is reported e.g. in WO 92/15675.
The chimeric genes may be introduced by transformation in cotton plants from which embryogenic callus can be derived, such as Coker 312, Coker310, Coker 5Acala SJ-5, GSC25U0, FiberMax 819 , Siokra 1-3, T25, GSA7S, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C1, Acala B1644, Acala B1654-26, Acala B1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAM1, Acala Cl, Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, Acala B2724, Acala B4894, Acala B5002, non Acala "picker" Siokra, "stripper" variety FC2017, Color 315, STONEVILLB 506, STONEVULE 825, DP50, DP61, DP90, DP77, DES119, McN235, HBX87, HBX191, HBX107, FC 3027, CHEMBRED Al, CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED Bl, CHEMBRED B2, CHEMBRED B3, CHEMBRED Cl, CHEMBRED C2, CHEMBRED C3, CHEMBRED C4, PAYMASTER 145, HS26, HS46, S1CALA, PIMA S6 and ORO BLANCO PIMA and plants with genotypes derived thereof.
"Cotton** as used herein includes Gossypium hirsutum% Gossypium barbadense% Gossypium arboreum and Gossypium herbaceum.

WO 2005/017157 PCT/AU2004/001076
Nevertheless, the methods and means of die current invention may also be employed for other plant species such as hemp, jute, flax and woody plants, inchiding bat not limited to Phots spp.rPopulus spp^ Picea spp* Eucalyptus spp. etc.
Hie obtained transformed plant can be used in a conventional breeding scheme to produce more transfoimed plants with the same characteristics or to introduce the chimeric gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from tfce transformed plants contain the chimeric genes of the invention as a stable genomic insert and axe also encompassed by die invention.
In wither embodiment, a method for identifying allelic variations of the involved in fiber length and/or drought resistance in a population of different genotypes or varieties of a particular plant species, preferably a fiber-producing plant species, which axe correlated either alone or in combination with the quantity and/or quality of fiber production is provided These method includes the following steps:
a) providing a population of different varieties or genotypes of a particular plant
species or interbreeding plant species comprising different allelic forms of the
nucleotide sequences encoding caUose synthase or p-l»3-glucanase, such as
nucleotide sequences comprising SEQ ID No 1 or 2. The different allelic forms
may be identified using the methods described elsewhere in this application.
Preferably, a segregating population is provided, wherein different combinations
of the allelic variations of the proteins involved in callose deposition and/or fiber
elongation or drought resistance axe present Methods to produce segregating
populations are well known in the art of plant breeding;
b) determining parameters related to fiber length or callose deposition at the neck of
the plasmodesmata at fee base of the fiber cell during fiber elongation or drought
resistance for each individual of the population;
c) determining the presence of a particular allelic form of the nucleotide sequences
encoding {3-1,3-gfcicanase or {3-1,3-glucan synthase such as nucleotide sequences
comprising SEQ ID No 1 or 2, for each individual of fee population; and

WO 2005/017157 PCT/AU2004/001076
d) correlating the occurrence of particular fiber length or callose deposition or drought resistance with the presence of a particular allelic form of the mentioned nucleotide sequence or a particular combination of such allelic forms.
The resulting information may be used to accelerate breeding program varieties with particular fiber or drought resistance characteristics, by detennining the presence or absence of allelic forms, using conventional molecular biology techniques.
*
Allelic forms of the 0-1,3-ghicanase gene associated with increased fiber length may also be identified, isolated and introduced into plants, such as cotton plants, whereby the expression of the endogenous {3-1,3-gtacanase genes has been reduced or eliminated. Such reduction of expression of the endogenous p-13-glucanasc genes can be conveniently achieved by posttranscriptional or transcriptional silencing as herein described, or may be achieved by inactivation, such as by deletion, of the endogenous {3-13-gtocanase genes. Introduction of the allelic forms may be achieved by breeding techniques, or by transformation with the isolated genes.
Biochemical assays for p-13-gtucanase or p-M-glucan synthase, particularly when performed on fiber cells or the underlying seed coat and particularly when performed immediately prior, during and immediately subsequent to fiber cell elongation, may also be used to identify in a population of cotton plant lines, or a population of cotton relatives which sis capable of inteteeftding with cotton nlant lines, or nlant populations resulting fiom wide crosses between cotton and such cotton relatives or in populations of resynthesized cotton lines, those lines with interesting characteristics, particularly those lines which have a relatively low p-l,3-glucanase activity and/or a relatively high j3-13-glucan synthase, particularly at the base of fiber cells immediately prior, during and immediately subsequent to the fiber elongation phase.
The following non-limiting Examples describe chimeric genes for the alteration of fiber characteristics in cotton and uses thereof. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et aL (1989) Molecular Cloning: A Laboratory

WO 2005/017157 PCT/AU2004/001076
Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et aL (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R-D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and BlackweU Scientific Publications, UK.
Throughout the description and Examples, reference is made to the following sequences represented in the sequence listing:
SEQ ID No 1: nucleotide sequence of endo-13-beta ghicanase
SBQ ID No 2: nucleotide sequence of the partial cDNA encoding endo-13-(3-glucan
synthase.
SEQ ID No 3: amino acid sequence encoded by the partial cDNA of SEQ ID No 2. SEQ ED No 4: amino acid sequence encoded by SEQ ID No 1.

WO 2005/017157 PCT/AU2004/001076
Examples
Example 1: Corrdatioii of the dnration of the dosvre of the plasmodesmata with the length of fibers, in different cotton cultivars*
The gating status of fiber plasmodesmata (PD) was examined among three tetraploid cotton cultivate Gh-r,Gh-c and Gb, with short, normal and long fiber, respectively, by using confocal imaging of a membrane-impermeable fluorescent molecule CF. As summarized in Fig 1, the genotype Gh^r, with the shortest fiber, does not close its fiber PD. In contrast, the long fiber genotype, Gb> closes PD earlier and longer than the intermediate line (Gh-c). A tetraploid lintless mutant {fls\ which produces fuzz-like fiber of less than 0.5 cm, does not close its fiber PD.
It was also found that, among the dipkrid progenitor of cotton cultivars, an A genome line Ga closes fiber PD for - 10 d and produces about 1.5 cm long fiber, while a D genome fine, Gt, does not close PD and virtually no fiber elongation occurs.
These data demonstrate that the genotypic differences in duration of PD closure positively correlate with fiber length.
Example 2: Analysis of caUose deposition at the base of the fiber cells in different cotton cultivars.
Hie molecular basis of PD gating is virtually unknown. However, caUose deposition at the neck region of PD has been shown to close PD in several plant systems. Callose deposition was analyzed using a callose-specific stain, aniline blue. The timing and duration of callose deposition in fiber base, judged from fluorescent signals of aniline blue, matched with that of PD closure in Gh-c and Gb. Representative images from the genotype Gh-c axe presented in Fig 2 (A-C). Callose was undetectable in the fiber base at 5 and 20 DAA when PD were open but became readily detectable at 10 DAA when PD closed (Fig 2). Similar results were obtained by using antibody against callose. The inununo-gold labelled callose signals were detected at the fiber base at 10 DAA (Fig 2E), but not at 5 (Fig 2D) or 20 DAA. At the EM level, callose was localized to the PD at the fiber base (Fig 2F).

WO 2005/017157 PCT/AU2004/001076
These results show that callose deposition correlates with the closure of fiber PD.
Example 3: Analysis of the expression of a fiber-specific β-l,3-gliicanase gene (Ghdacl) In cotton.
To analyze the rote of (3-1,3 glucanase in the molecular mechanism of PD closure/ reopening, a partial 0-1,3 glucanase cDNA (GhGlucI) from cotton fiber mRNA was cloned (using the sequence information with Geribaak accession number D88416). Fig 3 shows that the mRNA of this gene is expressed only at 20 DAA (matching with the timing of callose disappearance- see Fig 2 C) and not at 10-12 DAA in Gh-c9 when callose was present at the fiber base (see Figs 2 B and E). Consistently, the GhGlucI was not expressed at - 6 DAA ( Fig 3) when callose has not been produced (Fig 2 A). Significantly, at 20 DAA, the mRNA levels of the gene axe strongest, weaker and undetectable in Gh-rt Gh-c and Gbt representing short, intermediate and long fiber cultivars, respectively (Fig 3). The expression of this gene is fiber specific as its mRNA is undetectable in 6-d young seed (Fig 3), embryo, shoot or root
Taken together, the data show that the expression of GhGlucI is fiber- and developmental-specific. The timing and the level of the GhGlucI expression among the three cultivars differing in fiber length suggest that GhGlucI is responsible for the degradation of callose in the PD, which either prevents PD closure (e.g. in Gh-r) or allows PD to re-open (e.g. in Gh-c)9 hence shortens the elongation period
Example 4: Increasing fiber length by silencing GhGlucI expression in cotton fiber.
A chimeric gene is constructed containing the following DNA elements:
• a CaMV35S promoter
• a sense RNA encoding region corresponding to the nucleotide sequence of SEQ
IDNol
• a antisense RNA encoding region corresponding to the complement of the
nucleotide sequence of SEQ ED No 1.
• A3' nos terminator region

WO 2005/017157 PCT/AU2004/001076
This chimeric gene is introduced into a T-DNA vector together with a selectable bar gene. The T-DNA vector is introduced into Agrobacterium tumefaciens and used to produce transgenic cotton plants.
Transgenic cotton plants comprising the chimeric gene are analyzed as in Examples 1 and 2. The fiber elongation phase is prolonged when compared to the untransfonned control cotton plants, and the fibers are longer than in the untransformed control cotton plants.
The transgenic cotton plants comprising the above described chimeric gene axe crossed with plants comprising the coding sequence of a potato sucrose synthase cDNA (Genbank Accession number Ml 8745) opcrably linked to a subterranean clover stunt virus promoter (S7; WO9606932) and a 3' transcription termination and polyadenylation signal functional in plants, as described in WO 02/45485 (Example 3). Plants comprising both types of chimeric genes are selected and analysed as in Examples 1 and 2.
The fiber elongation phase is prolonged when compared to the untransfoxmed control cotton plants, sucrose synthase expression is higher than in untransformed control cotton plants and the fibers are longer than in the untransfbrmed control cotton plants.
Example 5: Increasing fiber elongation by expression of a chimeric β-1,3 ghiean synthase in cotton fiber.
A chimeric gene is constructed containing the following DNA elements:
• a cotton expansin promoter;
• a B-1,3 ghiean synthase encoding region comprising the nucleotide sequence of
SEQIDNo2;
• a 3' nos terminator region.
This chimeric gene is introduced into a T-DNA vector together with a selectable bar gene. The T-DNA vector is introduced into Agrobacterium tumefaciens and used to produce transgenic cotton plants.

WO 2005/017157 PCT/AU2004/001076
Transgenic cotton plants comprising the chimeric gene axe analyzed as in Examples 1 and 2. The fiber elongation phase is prolonged when compared to the untxansfbrmed control cotton plants, and the fibers axe longer than in the untransformed control cotton plants.

WO 2005/017157 PCT/AU2004/001076
We claim:
1. A method for modiiying a fiber of a fiber-producmg planter
altering a fiber cell elongation phase by modulating deposition of callose at the neck of the plasmodesmata at the base of said fiber celL
2. A method for increasing the length of a fiber of a fiber producing plant,
comprising die step of introducing a chimeric gene into a cell of said fiber
producing plant, wherein said chimeric gene, when expressed in said cell of said
fiber-producing plant increases said deposition of callose and increases said fiber
elongation phase.
3. The method according to claim 2, wherein said chimeric gene comprises the
following operably linked DNA elements:

(a) a plant expressible promoter, preferably a plant expressible promoter which
controls transcription preferentially in said fiber cells;
(b) a transcribed DNA region, which when transcribed yields a double-stranded
RNA molecule capable of reducing the expression of a gene endogenous to
said fiber producing plant, said gene being involved in callose removal from
said plasmodesmata, and said RNA molecule comprising a first and second
RNA region wherein
0 said first RNA region comprises a nucleotide sequence of at least 19
consecutive nucleotides having at least about 94% sequence identity to the
nucleotide sequence of said endogenous gene; ii) said second RNA region comprises a nucleotide sequence complementary
to said 19 consecutive nucleotides of said first RNA region; iii) said first and second RNA region are capable of base-pairing to fonn a
double stranded RNA molecule between at least said 19 consecutive
nucleotides of said first and second region; and
(c) a 3* end region comprising transcription termination and polyadenylation
signals functioning in cells of said plant

WO 2005/017157 PCT/AU2004/001076
4. The metbod according to claim 3, wfaerem said promoter is a fiber-specific beta
tubulin promoter from cotton, a fiber-specific actin promoter from cotton, a fiber
specific promoter from a lipid transfer protein gene from cotton, a promoter from
an expansin gene from cotton or a promoter from a chitinase gene in cotton.
5. Tlie method accordmg to claim 3 or 4, wherein said endogenous gene is a 1,3-0-
gtacanase gene which is expressed at tbe base of said fiber cell, at the end of said
fiber elongation phase in said fiber producing plant
6. Hie method according to claim 5, wfaerem said endogenous gene encodes a
protein comprising the ammo acid sequence of SEQ ID No 4 or wherein said gene
comprises the nucleotide sequence of SEQ ID No 1.
7. Hie method according to anyone of claims 3 to 6, wherein said first RNA region
comprises a nucleotide sequence of at least 19 consecutive nucleotides having at
least about 94% sequence identity to a nucleotide sequence encoding a protein
comprising the amino acid sequence of SEQ ID No 4 or to the nucleotide
sequence of SEQ ID No 1.
8. The method according to claim 2, wherein said chimeric gene comprises
(a) a plant-expressible promoter, preferably a plant-expressible promoter which
controls transcription preferentially in said fiber cells;
(b) a DNA region encoding a 0-1,3 gtucan synthase protein; and
(c) a 3'end region comprising transcription termination and polyadenylation
signals functioning in cells of said plant

9. Hie method according to claim 8, wherein said DNA region encoding said (3-1,3
glucan synthase protein, comprises the nucleotide sequence of SEQ ID No 2.
10. The method according to claim 8 or 9, wherein said promoter is a fiber-specific
beta tubulin promoter from cotton, a fiber-specific actin promoter from cotton, a
fiber specific promoter from a lipid transfer protein gene from cotton, a promoter
from an expansin gene from cotton or a promoter from a chitinase gene in cotton.

WO 2005/017157 PCT/AU2004/001076
11. The metixxl according to any on of claims 2 to 10, wherein said fiber pioducing
plant is cotton.
12. Hie method accoidmg to claim 11, wherein said fiber is a lint fiber.
13. Hie method according to claim 11, wherein said fiber is a fiizz fiber.
14. The method according to claim 3, further comprising introducing a second
chimeric gene, wherein said second chimeric gene comprises the following
operably linked DNA fragments:

(a) a plant-expressible promoter, preferably a plant-expressible promoter which
controls transcription preferentially in said fiber cells;
(b) a DNA regkm encoding a P-13glucansynthase protein; aiid
(c) a 3'end region comprising transcription termination and polyadenylation
signals functioning in cells of said plant
15. The method according to any one of claims 1 to 14, further comprising
(a) growing plants obtained according to said method; and
(b) isolating fibers from said fiber-producing plants

16. A method for decreasing the length of a fiber of a fiber pioducing plant,
comprising the step of introducing a chimeric gene into a cell of said fiber
producing plant, wherein said chimeric gene, when expressed in said cell of said
fiber-producing plant decreases said fiber elongation phase and decreases said
deposition of caUose.
17. Hie method according to claim 16, wherein said chimeric gene said chimeric gene
comprises the following operably linked DNA fragments:

(a) a plant-expressible promoter, preferably a plant-expressible promoter which
controls transcription preferentially in said fiber cells;
(b) a DNA region encoding a 0-1,3 glucanase protein; and

WO 2005/017157 PCT/AU2004/001076
(c) a 3'end region comprising transcription termination and polyadenylation signals functioning in cells of said plant
18. The method according to claim 17, wherein said DNA region encoding said p-1,3
gbcanase protein comprises die nucleotide sequence of SEQ ID No 1.
19. The method according to claim 17 or 18, wherem said promoter is a fiber-specific
beta tubufin promoter from cotton, a fiber-specific actin promoter from cotton, a
fiber specific promoter from a Kpid transfer protein gene from cotton, a promoter
from an expansin gene from cotton or a promoter from a chitinase gene in cotton,
20. The method according to claim 16, wherein said chimeric gene comprises flic
folk)wing operably linked DNA elements:

(a) a plant expressible promoter which controls transcription preferentially in said
fiber cells;
(b) a transcribed DNA region, which when transcribed yields a double-stranded
KNA molecule capable of reducing the expression of a gene endogenous to
said fiber producing plant, said gene being involved in callose deposition in
said plasmodesmata, and said RNA molecule comprising a first and second
RNA region wherein
i) said first KNA region comprises a nucleotide sequence of at least 19
consecutive nucleotides having at least about 94% sequence identity to the
nucleotide sequence of said endogenous gene; ii) said second RNA region comprises a nucleotide sequence complementary
to said 19 consecutive nucleotides of said first RNA region; in) said first and second RNA region are capable of base-pairing to form a
double stranded RNA molecule between at least said 19 consecutive
nucleotides of said first and second region; and
(c) a 3' end region comprising transcription termination and polyadenylation
signals functioning in cells of said plant
21. The method according to claim 20, wherein said promoter is a fiber-specific beta
tubulin promoter from cotton, a fiber-specific actin promoter from cotton, a fiber

WO 2005/017157 PCT/AU2004/001076
specific promoter from a lipid transfer protein gene from cotton, a promoter from an expansin gene from cotton or a promoter from a chitinase gene in cotton.
22. The method according to claim 20 or 21, wherein said endogenous gene is a 1,3-
p-ghican synthase gone which is expressed in said fiber of said fiber producing
plant
23. The method according to claim 22, wherein said endogenous gene encodes a
protein comprising the amino acid sequence of SEQ ID No 3 or wherein said
endogenous gene comprises the nucleotide sequence of SEQ ID No 2.
24. The method according to anyone of claims 21 to 23, wherein said first SNA
region comprises a nucleotide sequence of at least 19 consecutive nocleotides
having at least about 94% sequence identity to a nncleotide sequence encoding a
protein comprising the amino acid sequence of SEQ ID No 3 or to the nucleotide
sequence of SEQ ID No 2.
25. A method for identifying allelic variations of the genes encoding proteins involved
in fiber elongation in a population of different genotypes, cultivars or varieties of
a particular plant species, preferably a fiber-producing plant species, which are
correlated either alone or in combination with the length of fibers produced,
comprising the steps of

(a) providing a population of different varieties or genotypes of a particular plant
species or interbreeding plant species comprising different allelic forms of the
nucleotide sequences encoding caflose synthase or β-1, 3 ghicanase,
particularly of SEQ ID No 1 or SEQ ID 2;
(b) determining parameters related to fiber length for each individual of die
population;
(c) determining die presence of a particular allelic form of the nucleotide
sequences encoding callose synthase or p-13 glucanase, particularly of SEQ
ID No lor SEQ ID 2;

WO 2005/017157 PCT/AU2004/001076
(d) correlating the occurrence of particular fiber length with the presence of a particular allelic form of the mentioned nucleotide sequence or a particular combination of such allelic forms.
26. A chimeric gene as described in any one of claims 3 to 24.
27. A cell of a fiber-producing plant comprising a chimeric gene according to claim
26.
28. A fiber producing plant comprising a chimeric gene according to claim 26.
29. A fiber producing plant according to claim 28, wherein fibers of said plant are
increased in length compared to untransfonned control plants.
30. A fiber producing plant according to claim 28, which has increased drought
resistance.
31. A fiber producing plant according to any one of claims 28 to 30, wherein said
plant is cotton.
32. Seed of the fiber producing plant according to any one of claims 26 to 31, said
seed comprising a chimeric gene according to claim 26.
33. Fibers produced according to the methods of any one of claims 1 to 24.
34. A method for increasing drought resistance in a fiber producing plant, said method
comprising
(a) introducing a chimeric gene into cells of said fiber producing plant wherein said chimeric gene comprises the following operably linked DNA elements: i) a plant expressible promoter which controls transcription preferentially in
said fiber cells;
ii) a transcribed DNA region, which when transcribed yields a double-stranded UNA molecule capable of reducing the expression of a gene endogenous to said fiber producing plant, said gene being involved in

WO 2005/017157 PCT/AU2004/001076
callose removal in said plasmodesmata, and said SNA molecule comprising a first and second SNA region wherein
(1) said first SNA region comprises a nucleotide sequence of at least 19
consecutive nucleotides having at least about 94% sequence identity to
the nucleotide sequence of said endogenous gene;
(2) said second SNA region comprises a nucleotide sequence
complementary to said 19 consecutive nucleotides of said first SNA
region;
(3) said first and second SNA region are capable of base-pairing to form a
double stranded SNA molecule between at least said 19 consecutive
nucleotides of said first and second region; and
in) a 3' end region comprising transcription termination and polyadenyiation signals functioning in cells of said plant
35. A method for increasing the length of a fiber in cotton, comprising introducing a the step of introducing a chimeric gene into a cell of said fiber producing plant, wherein said chimeric gene, when expressed in said cell of said fiber-producing plant increases said fiber elongation phase and increases said deposition of callose, said chimeric gene comprising
(a) a plant expressible promoter;
(b) a transcribed DNA region, which when transcribed yields a double-stranded
SNA molecule capable of reducing the expression of a gene endogenous to
said fiber producing plant, said gene being involved in callose removal from
said plasmodesmata, and said SNA molecule comprising a first and second
SNA region wherein
i) said first SNA region comprises a nucleotide sequence of at least 19 consecutive nucleotides having at least about 94% sequence identity to the nucleotide sequence of SEQ ID No 1;
ii) said second SNA region comprises a nucleotide sequence complementary

to said 19 consecutive nucleotides of said first SNA region; in) said first and second SNA region are capable of base-pairing to form a double stranded SNA molecule between at least said 19 consecutive nucleotides of said first and second region; and

WO 2005/017157 CT7AU2004/001076
(c) a 3' end region comprising transcription termination and polyadenylatbn
signals functioning in cells of said plant; or
said chimeric gene comprising the following operably linked DNA elements: i) a plant-expressible promoter, preferably a plant-expressible promoter
which controls transcription preferentially in said fiber cells; ii) a DNA region encoding a (5-13 ghicanase protein comprising the anrino acid sequence of SEQ ID No 3 or the nucleotide sequence of SEQ ID No 2; and
iii) a 3'end region comprising transcription termination and polyadenylation signals fiinctioning in cells of said plant

Documents:

0884_-chenp-2006-abstract .pdf

0884_-chenp-2006-claims.pdf

0884_-chenp-2006-correspondence-others.pdf

0884_-chenp-2006-description-complete.pdf

0884_-chenp-2006-drawings.pdf

0884_-chenp-2006-form 1.pdf

0884_-chenp-2006-form 3.pdf

0884_-chenp-2006-form 5.pdf

0884_-chenp-2006-pct.pdf

884-chenp-2006 amended pages of specification 02-12-2010.pdf

884-chenp-2006 amended claims 02-12-2010.pdf

884-chenp-2006 other patent document 02-12-2010.pdf

884-CHENP-2006 CORRESPONDENCE OTHERS 15-07-2011.pdf

884-chenp-2006 form-3 02-12-2010.pdf

884-chenp-2006 form-3 09-08-2011.pdf

884-chenp-2006 amended claims 02-08-2011.pdf

884-chenp-2006 correspondence others 09-08-2011.pdf

884-chenp-2006 form-3 02-08-2011.pdf

884-chenp-2006 power of attorney 02-12-2010.pdf

884-chenp-2006 correspondence others 09-12-2010.pdf

884-chenp-2006 correspondence others 02-08-2011.pdf

884-CHENP-2006 CORRESPONDENCE OTHERS 28-10-2010.pdf

884-CHENP-2006 CORRESPONDENCE PO.pdf

884-chenp-2006 examination report reply recieved 02-12-2010.pdf

884-CHENP-2006 FORM-18.pdf

884-chenp-2006 form-3 09-12-2010.pdf


Patent Number 250132
Indian Patent Application Number 884/CHENP/2006
PG Journal Number 50/2011
Publication Date 16-Dec-2011
Grant Date 09-Dec-2011
Date of Filing 13-Mar-2006
Name of Patentee COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION (CSIRO)
Applicant Address Limestone Avenue, Canberra, Australian Capital Territory 2601
Inventors:
# Inventor's Name Inventor's Address
1 RUAN, Yong, Ling 9 Rosella Street, Nicholls, Australian Capital Territory 2913
2 FURBANK, Robert, T. 35 Gillespte Street, Weetangara, Australian Capital Territory 2614
PCT International Classification Number C12N 15/29
PCT International Application Number PCT/AU2004/001076
PCT International Filing date 2004-08-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/495,123 2003-08-15 U.S.A.