# Patent

## Title
Manipulation of plant senescence using modified promoters

## Abstract

The present invention relates to methods of manipulating senescence in plants. The invention also relates to vectors useful in such methods, transformed plants with modified senescence characteristics and plant cells, seeds and other parts of such plants.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/605,214, filed Oct. 23, 2009, issued as U.S. Pat. No. 8,399,739, which is a continuation-in-part of U.S. patent application Ser. No. 11/789,526, filed Apr. 24, 2007, now abandoned, and which is also a continuation-in-part of International Patent Application No. PCT/AU2008/000566, filed Apr. 21, 2008. The contents of the foregoing are hereby incorporated herein by reference in their entireties.


STATEMENT REGARDING SEQUENCE LISTING
The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 40774_Sequence_Final—2013-02-26.txt. The text file is 80 KB; was created on Feb. 26, 2013; and is being submitted via EFS-Web with the filing of the specification.
BACKGROUND
The present invention relates to methods of manipulating senescence in plants. The invention also relates to vectors useful in such methods, transformed plants with modified senescence characteristics and plant cells, seeds and other parts of such plants.
Leaf senescence involves metabolic and structural changes in cells prior to cell death. It also involves the recycling of nutrients to actively growing regions.
The regulation of plant and plant organ senescence by cytokinins has important agricultural consequences. Elevated cytokinin levels in leaves tend to retard senescence. A number of promoters have been used to regulate the expression of the ipt gene, whose product (isopentenyltransferase) catalyses a key step in cytokinin synthesis. However, in general, transgenic plants over-expressing the ipt gene have been reported to have retarded root and shoot growth, no root formation, reduced apical dominance, and reduced leaf area.
It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.
In one aspect, the present invention provides a method of manipulating senescence in a plant, said method including introducing into said plant a genetic construct including a modified myb gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
The manipulation of senescence relates to the plant and/or specific plant organs. Senescence of different plant organs, such as leaves, roots, shoots, stems, tubers, flowers, stolons, and fruits may be manipulated. The manipulation of plant and plant organ senescence may have important agricultural consequences, such as increased shelf life of e.g., fruits, flowers, leaves and tubers in horticultural produce and cut flowers, reduced perishability of horticultural crops, increased carbon fixation in senescence-retarded leaves leading to enhanced yields, enhanced biomass production in forage plants, enhanced seed production, etc.
“Manipulating senescence” generally relates to delaying senescence in the transformed plant relative to an untransformed control plant. However, for some applications it may be desirable to promote or otherwise modify senescence in the plant. Senescence may be promoted or otherwise modified for example, by utilizing an antisense gene.
An effective amount of said genetic construct may be introduced into said plant, by any suitable technique, for example by transduction, transfection or transformation. By “an effective amount” is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.
By a “modified myb gene promoter” is meant a promoter normally associated with a myb gene, which promoter is modified to delete or inactivate one or more root specific motifs and/or pollen specific motifs in said promoter.
While applicant does not wish to be restricted by theory, it is postulated that deletion or inactivation of one or more root specific motifs in said myb gene promoter may alleviate or overcome the problem of leaky expression of the gene encoding a cytokinin biosynthetic enzyme in plant meristems, which may affect root development in some species of plants. It is also postulated that deletion or inactivation of one or more pollen specific motifs in said myb gene promoter may alleviate or overcome the problem of leaky expression of the gene encoding a cytokinin biosynthetic enzyme in pollen, which may affect pollen development in some species of plants.
Preferably the modified myb gene promoter is a modified myb32 gene promoter. Preferably the modified myb gene promoter is from Arabidopsis, more preferably Arabidopsis thaliana. 
A suitable promoter which may be modified according to the present invention is described in Li et al., Cloning of three MYB-like genes from Arabidopsis (PGR 99-138) Plant Physiology 121:313 (1999), the entire disclosure of which is incorporated herein by reference.
By a “root specific motif” is meant a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 5 nucleotides, which directs expression of an associated gene in the roots of a plant.
Preferably the root specific motif includes a consensus sequence ATATT or AATAT.
Preferably, between one and ten, more preferably between three and eight, even more preferably between five and seven root specific motifs are deleted or inactivated, preferably deleted, in said myb gene promoter.
The root specific motifs may be deleted by removing individual motifs or by removing a fragment of the promoter containing one or more motifs. For example, all or part of the region between nucleotides 1 and 530, preferably between nucleotides 110 and 530 of the Arabidopsis thaliana myb gene promoter may be deleted.
The deletion may be effected by cutting the nucleic acid, for example with restriction endonucleases, and ligating the cut ends to generate a promoter with a fragment removed.
For example, a modified Arabidopsis thaliana myb gene promoter may be prepared by removing a fragment between the Xcm1 site at positions 162-176 and the SspI site at positions 520-525. This generates a modified myb gene promoter with 6 of the 7 root specific motifs deleted. Alternatively, all 7 of the root specific motifs may be deleted, for example by deleting the region upstream of the SspI site at positions 520-525, or by deleting the region between nucleotides 1 and 120 together with the region between the XcmI site at positions 162-176 and the SspI site at positions 520-525.
A root specific motif may be inactivated by adding, deleting, substituting or derivatizing one or more nucleotides within the motif, so that it no longer has the preferred consensus sequence.
Preferably the modified myb gene promoter includes a nucleotide sequence selected from the group consisting of the sequences shown in FIGS. 2, 3 and 4 hereto (Sequence ID Nos: 2, 3 and 4, respectively) and functionally active fragments and variants thereof.
By a “pollen specific motif” is meant a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 4 or 5 nucleotides, which directs expression of an associated gene in the pollen of a plant.
Preferably the pollen specific motif includes a consensus sequence selected from the group consisting of TTCT and AGAA.
Preferably, between one and thirty, more preferably between three and fifteen, even more preferably between four and ten pollen specific motifs are deleted or inactivated, preferably deleted, in said myb gene promoter.
The pollen specific motifs may be deleted by removing individual motifs or by removing a fragment of the promoter containing one or more motifs. For example, all or part of the region between nucleotides 1 and 540, preferably between nucleotides 390 and 540 of the Arabidopsis thaliana myb gene promoter may be deleted.
The deletion may be effected by cutting the nucleic acid, for example with restriction endonucleases, and ligating the cut ends to generate a promoter with a fragment removed.
For example, a modified Arabidopsis thaliana myb gene promoter may be prepared by removing a fragment between the XcmI site at positions 162-176 and the SspI site at positions 520-525. This generates a modified myb gene promoter with 4 of the 23 pollen specific motifs deleted. Alternatively, 10 of the pollen specific motifs may be deleted, for example by deleting the region upstream of the SspI site at positions 520-525.
A pollen specific motif may be inactivated by adding, deleting, substituting or derivatizing one or more nucleotides within the motif, so that it no longer has the preferred consensus sequence.
Preferably the modified myb gene promoter includes a nucleotide sequence selected from the group consisting of the sequences shown in FIGS. 2, 3 and 4 hereto (Sequence ID Nos: 2, 3 and 4, respectively) and functionally active fragments and variants thereof.
In a further aspect of the present invention there is provided a method of enhancing biomass in a plant, said method include introducing into said plant a genetic construct including a myb gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
The myb gene promoter or a functionally active fragment or variant thereof may be a full length myb gene promoter or a modified myb gene promoter.
The full length myb gene promoter may be a myb32 gene promoter. Preferably the myb gene promoter is from Arabidopsis, more preferably Arabidopsis thaliana. Most preferably the myb gene promoter includes a nucleotide sequence selected from the group consisting of the sequence shown in FIG. 1 hereto (Sequence ID No: 1) and functionally active fragments and variants thereof.
A suitable promoter is described in Li et al., Cloning of three MYB-like genes from Arabidopsis (PGR 99-138) Plant Physiology 121:313 (1999).
The modified myb gene promoter may be a modified myb gene promoter as hereinbefore described.
By “enhancing biomass” is meant enhancing or increasing in a transformed plant relative to an untransformed control plant a growth characteristic selected from the group consisting of total leaf area, cumulative leaf area, leaf growth dynamics (i.e., number of leaves over time), stolon length, percentage of flowering plants and seed yield per flower or per area sown. “Enhancing biomass” also includes reducing or decreasing percentage stolon death in a transformed plant relative to an untransformed control plant.
In particular, applicants have found that while the seed weight (i.e., weight of thousand seeds) of transgenic plants according to the present invention was indistinguishable from non-transgenic control plants, the total seed yield expressed on the basis of per flower or per area sown was significantly higher in the transgenic plants when compared with non-transgenic control plants of equivalent flowering intensity.
By “functionally active” in relation to a myb gene promoter or modified myb gene promoter is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating senescence in a plant by the method of the present invention. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, most preferably at least 300 nucleotides.
By a “gene encoding an enzyme involved in biosynthesis of a cytokinin” is meant a gene encoding an enzyme involved in the synthesis of cytokines such as kinetin, zeatin and benzyl adenine, for example a gene encoding isopentyl transferase (ipt), or an ipt-like gene such as the sho gene (eg. from petunia). Preferably the gene is an isopentenyl transferase (ipt) gene or sho gene. In a preferred embodiment, the gene is from a species selected from the group consisting of Agrobacterium, more preferably Agrobacterium tumefaciens; Lotus, more preferably Lotus japonicus; and Petunia, more preferably Petunia hybrida. 
Most preferably the gene includes a nucleotide sequence selected from the group consisting of the sequences shown in FIGS. 6, 8 and 10 hereto (Sequence ID Nos: 5, 7 and 9) sequences encoding the polypeptides shown in FIGS. 7, 9 and 11 hereto (Sequence ID Nos: 6, 8 and 10), and functionally active fragments and variants thereof.
By “functionally active” in relation to a gene encoding a cytokinin biosynthetic enzyme is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating senescence in a plant by the method of the present invention. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence, to which the fragment or variant corresponds more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes or nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. For example, the functionally active variant may include one or more conservative nucleic acid substitutions of a sequence shown in FIG. 6, 8 or 10, the resulting functionally active variant encoding an amino acid sequence shown in FIG. 7, 9 or 11, respectively. Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 500 nucleotides.
The genetic construct may be introduced into the plant by any suitable technique. Techniques for incorporating the genetic constructs of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation and combinations thereof. The choice of technique will depend largely on the type of plant to be transformed, and may be readily determined by an appropriately skilled person.
Cells incorporating the genetic construct of the present invention may be selected, as described below, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
The methods of the present invention may be applied to a variety of plants, including monocotyledons [such as grasses (e.g., forage, turf and bioenergy grasses including perennial ryegrass, tall fescue, Italian ryegrass, red fescue, reed canary grass, big bluestem, cordgrass, napiergrass, wildrye, wild sugarcane, Miscanthus), corn, oat, wheat and barley)], dicotyledons [such as Arabidopsis, tobacco, soybean, clovers (e.g., white clover, red clover, subterranean clover), alfalfa, canola, vegetable brassicas, lettuce, spinach] and gymnosperms.
In a further aspect of the present invention there is provided a vector capable of manipulating senescence in a plant, said vector including a modified myb gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in the biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
In a still further aspect of the present invention there is provided a vector capable of enhancing biomass in a plant, said vector including a myb gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in the biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
The myb gene promoter or a functionally active fragment or variant thereof may be a full length myb gene promoter or a modified myb gene promoter, as described herein.
In a preferred embodiment of this aspect of the invention, the vector may further include a terminator; said promoter, gene and terminator being operably linked.
By “operably linked” is meant that said promoter is capable of causing expression of said gene in a plant cell and said terminator is capable of terminating expression of said gene in a plant cell. Preferably, said promoter is upstream of said gene and said terminator is downstream of said gene.
The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the plant cell.
The promoter, gene and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.
The vector, in addition to the promoter, the gene and the terminator, may include further elements necessary for expression of the gene, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptII) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.
As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g., GUS assays), thin layer chromatography (TLC), northern and western blot hybridisation analyses.
Those skilled in the art will appreciate that the various components of the vector are operably linked, so as to result in expression of said gene. Techniques for operably linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.
In a further aspect of the present invention there is provided a transgenic plant cell, plant, plant seed or other plant part, with modified senescence characteristics or enhanced biomass. Preferably said plant cell, plant, plant seed or other plant part includes a vector according to the present invention. Preferably the transgenic plant cell, plant, plant seed or other plant part is produced by a method according to the present invention.
The present invention also provides a transgenic plant, plant seed or other plant part derived from a plant cell of the present invention.
The present invention also provides a transgenic plant, plant seed or other plant part derived from a plant of the present invention.
The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.



FIGURES
FIG. 1 shows the nucleotide sequence of the promoter from myb32 gene (atmyb32) from Arabidopsis thaliana (Sequence ID No: 1), Atmyb32 promoter sequence with MYB type, pollen specific and Root specific motifs highlighted. WAACCA (underline/italics) MYB1AT; GTTAGTT (bold/box) MYB1LEPR; CCWACC (box) MYBPZM; GGATA (italics) MYBST1; AGAAA (underline) POLLEN1LELAT52; ATAT (bold) ROOTMOTIFTAPOX1.
FIG. 2 shows an Atmyb32 promoter sequence variant (Atmyb32xs) with the XcmI-SspI plant sequence deleted. MYB type, pollen specific and Root specific motifs are highlighted. WAACCA (underline/italics) MYB1AT; GTTAGTT (bold/box) MYB1LEPR; CCWACC (box) MYBPZM; AGAAA (underline) POLLEN1LELAT52; ATATT (bold) ROOTMOTIFTAPOX1 (Sequence ID No: 2).
FIG. 3 shows an Atmyb32 promoter variant sequence with all root motifs deleted. MYB type, pollen specific and Root specific motifs are highlighted. WAACCA (underline/italics) MYB1AT; CCWACC (box) MYBPZM; AGAAA (underline) POLLEN1LELAT52 (Sequence ID No: 3).
FIG. 4 shows an Atmyb32 promoter variant sequence with the SspI site upstream sequence deleted. MYB type, pollen specific and Root specific motifs are highlighted. WAACCA (underline/italics) MYB1AT; CCWACC (box) MYBPZM; AGAAA (underline) POLLEN1LELAT52 (Sequence ID No: 4).
FIG. 5 shows Motifs in Atmyb32 and Atmyb32xs promoter sequences.
FIG. 6 shows the nucleotide sequence of the isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens (Sequence ID No: 5).
FIG. 7 shows the deduced amino acid sequence of the isopentyl transferase gene from Agrobacterium tumefaciens (Sequence ID No. 6).
FIG. 8 shows the nucleotide sequence of the isopentyl transferase gene from Lotus japonicus (Sequence ID No. 7).
FIG. 9 shows the deduced amino acid sequence of the isopentyl transferase gene from Lotus japonicus (Sequence ID No. 8).
FIG. 10 shows the Nucleotide sequence of the cytokinin biosynthesis Sho gene from Petunia hybrida (Sequence ID No. 9).
FIG. 11 shows the Deduced amino acid sequence of the cytokinin biosynthesis Sho gene from Petunia hybrida (Sequence ID No. 10).
FIG. 12 shows PCR and Southern DNA analysis of atmyb32::ipt transgenic white clover (Trifolium repens) plants. a) The T-DNA region of patmyb32:ipt showing restriction enzyme sites and location of the probes used for Southern hybridization analysis. b) Ethidium bromide stained 1% agarose gel of the PCR amplified 599 bp nptII and 583 bp ipt products. c) Southern blot hybridization with HindIII digested total genomic DNA isolated from PCR positive white clover plants hybridized with the ipt probe. d) Southern blot hybridization with HindIII digested total genomic DNA isolated from PCR positive white clover plants hybridized with the nptII probe. Lanes 1-2: two independent kanamycin resistant cv. Haifa regenerants, code: Hmi01, Hmi08 respectively; Lanes 3-12: twelve independent kanamycin resistant cv. Irrigation regenerants, codes: Imi06, Imi07, Imi08, Imi09, Imi10, Imi11, Imi12, Imi14, Imi16, Imi18 respectively; Lane C: non-transformed white clover; Lane P: positive control plasmid patmyb32ipt.
FIG. 13 shows RT-PCR analysis of ipt mRNA expression in atmyb32::ipt transgenic white clover (T. repens) plants. Lane 1-11 are samples from 11 independent transgenic lines with corresponding plant codes as in FIG. 4.8; Lane C, Control non-transformed plant; Lane P, plasmid as positive control. Total RNA was isolated from leaf tissues. Total RNA (13 μg) was used for each reverse transcription reaction and 1⁄5 of RT product was amplified by PCR. DNA products on the gel on the right were amplified by 2×30 cycles intensive PCR. No reverse transcriptase was added to the corresponding RT-PCR reaction loaded into alternate lanes.
FIG. 14 shows a senescence bioassay of excised leaves from atmyb32::ipt transgenic white clover (T. repens) plants. At least 30 leaves were collected from each line from similar positions on stolons of plant lines. A. The number of yellowing leaves as a fraction of the total number of excised leaves. B. Typical appearance of leaves kept on water under light for two weeks. Key to plant lines: HC, IC and Hmg, Img, non-transformed and atmyb32::gusA transgenic plants (cv. Haifa and Irrigation) respectively; 01 and 08, atmyb32::ipt transgenic Haifa lines Hmi01 and Hmi08 respectively; 11, 12, 16 and 18 atmyb32::ipt transgenic Irrigation lines Imi11, Imi12, Imi16 and Imi18 respectively.
FIG. 15 shows A) General plant morphology, B) Normal shoot development, and C) Normal root development in atmyb32::ipt transgenic white clover (T. repens) (right) plants compared to control plants (left).
FIG. 16 shows vector details for pBMVkAtMYB32-900::ipt [Gene: Isopentyl transferase (IPT); Vector: pBMVkAtMYB32-900::ipt-nos (backbone pPZPRCS2); Selectable marker: spec; Plant selectable marker cassette: 35S::kan::35ST; Gene promoter: AtMYB32-900; Gene terminator: nos.].
FIG. 17 shows nucleotide sequence of vector pBMVkAtMYB32-900::ipt-nos (Sequence ID No. 11).
FIG. 18 shows vector details for pBMVkAtMYB32xs::ipt-nos [Gene: Isopentyl transferase (IPT); Vector: pBMVkAtMYB32XS::ipt-nos (backbone pPZPRCS2); Selectable marker: spec; Plant selectable marker cassette: 35S::kan::35ST; Gene promoter: AtMYB32-xs; Gene terminator: nos.].
FIG. 19 shows nucleotide sequence of vector pBMVkAtMYB32xs::ipt-nos (Sequence ID No. 12).
FIG. 20 shows vector details for pBMVhAtMYB32-900::ipt-nos [Gene: Isopentyl transferase (IPT); Vector: pBMVhAtMYB32-900::ipt-nos (backbone pPZPRCS2); Selectable marker: spec; Plant selectable marker cassette: 35S::hph::35ST; Gene promoter: AtMYB32-900; Gene terminator: nos.].
FIG. 21 shows nucleotide sequence of vector pBMVhAtMYB32-900::ipt-nos (Sequence ID No. 13).
FIG. 22 shows vector details for pBMVhAtMYB32xs::ipt-nos [Gene: Isopentyl transferase (IPT); Vector: pBMVhAtMYB32XS::ipt-nos (backbone pPZPRCS2); Selectable marker: spec; Plant selectable marker cassette: 35S::hph::35ST; Gene promoter: AtMYB32-xs; Gene terminator: nos.].
FIG. 23 shows nucleotide sequence of vector pBMVhAtMYB32xs::ipt-nos (Sequence ID No. 14).
FIG. 24 shows vector details for pBSubn-AtMYB32-900::ipt-nos [Gene: Isopentyl transferase (IPT); Vector: pBSubn-AtMYB32-900::ipt-nos (backbone pPZPRCS2); Selectable marker: spec; Plant selectable marker cassette: Ubi::bar::nos; Gene promoter: AtMYB32-900; Gene terminator: nos.].
FIG. 25 shows nucleotide sequence of vector pBSAtMYB32900::ipt-nos (Sequence ID No. 15).
FIG. 26 shows generation of transgenic canola containing the pBMVhATMYB3-900::ipt-nos and pBMVhATMYB32xs::ipt-nos. A. Canola seeds are germinated in vitro; B. Hypocotyl sections are excised from 7-day-old seedlings and inoculated with an Agrobacterium suspension; C&D. Regeneration from inoculated hypocotyl sections under hygromycin selection; E-J. Transgenic T0 canola plants carrying the pBMVhATMYB3-900::ipt-nos and pBMVhATMYB32xs::ipt-nos vectors.
FIG. 27 shows a process for biolistic transformation of wheat.
FIG. 28 shows biolistic transformation of wheat (Triticum aestivum L. MPB Bobwhite 26). Donor plant production (A & B); zygotic embryo isolation (C&D); Regeneration under glufosinate selection (E-G); Root formation under selection (H); T0 plants growing under containment glasshouse conditions for recovery of transgenic offspring (I).
FIG. 29 shows contained field trial of transgenic white clover plants expressing chimeric Atmyb32::ipt genes.
FIG. 30 shows comparative assessment of growth rates and growth dynamics of transgenic white clover plants expressing chimeric Atmyb32::ipt genes with non-transgenic control white clover plants. A) Growth Rates B) Growth Dynamics C) Growth Characteristics (after 45 days).
 Transgenic white clover  Non-transgenic control white clover
(light) (dark)
FIG. 31 shows flowering intensity (i.e., number of ripe flower per m2) of transgenic white clover plants expressing chimeric Atmyb32::ipt genes (i.e., LXR 12, LXR 18 and LXR 11) and non-transgenic control white clover plants (i.e., WT) under contained field conditions.
FIG. 32 shows seed weight (i.e., weight of thousand seeds, in grams) of transgenic white clover plants expressing chimeric Atmyb32::ipt genes (i.e., LXR 12, LXR 18 and LXR 11) and non-transgenic control white clover plants (i.e., WT) under contained field conditions.
FIG. 33 shows seed yield per flower (in milligrams) of transgenic white clover plants expressing chimeric Atmyb32::ipt genes (i.e., LXR 12, LXR 18 and LXR 11) and non-transgenic control white clover plants (i.e., WT) under contained field conditions.
FIG. 34 shows seed yield per area (in kg/ha) of transgenic white clover plants expressing chimeric Atmyb32::ipt genes (i.e., LXR 12, LXR 18 and LXR 11) and non-transgenic control white clover plants (i.e., WT) under contained field conditions.
FIG. 35 shows generation of transgenic alfalfa plants containing the chimeric pBMVkATMYB3-900::ipt-nos and pBMVkATMYB32xs:: ipt-nos genes A. Petiole explants from alfalfa clones C2-3, C2-4 and 19-17 are used for inoculation with an Agrobacterium suspension and lead to the production of transformed embryogenic calli following selection in presence of kanamycin; B-D. Regeneration of transgenic alfalfa plantlets carrying chimeric genes from pBMVkATMYB3-900::ipt-nos and pBMVkATMYB32xs::ipt-nos vectors, from somatic embryos grown in vitro.
FIG. 36 shows PCR analysis of transgenic canola plants (T1 LXR canola plants Line 4). Genomic DNA was isolated from different transgenic canola plants of T1 LXR04 lines and subjected to PCR using primers specific for A. the selectable marker (hph) or B. the candidate gene of interest (IPT).
FIG. 37 shows expression analysis of the IPT gene in T1 transgenic canola plants (T1 LXR canola relative IPT leaf expression).
FIG. 38 shows transgenic canola displaying delay of detached cotyledon senescence as compared to wild-type control cotyledons 7 days following detachment.
FIG. 39 shows the senescence score of T1 transgenic canola cotyledons at 7 days following detachment.
0 (dark solid column)=No Visible senescence; 1 (checkered column)=Pale Green; 2 (wavy-lined column)=Some Yellowing;  3 (light solid column)=Mostly Yellow; 4 (diagonally-lined column)=Completely Yellow.
FIG. 40 shows transgenic canola displaying delay of detached juvenile leaf senescence as compared to wild-type control cotyledons 14 days following detachment.
FIG. 41 shows senescence score of T1 transgenic canola juvenile first leaves at 14 days following detachment.
0 (dark solid column)=No Visible Senescence; 1 (checkered column)=Pale Green;  2 (wavy-lined column)=Some Yellowing 3 (light solid column)=Mostly Yellow; 4 (diagonally-lined column)=Completely Yellow; 5 (horizontally/vertically-lined column)=Rotting/Necrosis.
FIG. 42 shows Southern hybridisation analysis of transgenic wheat lines. Lanes include: MW—molecular weight; WT—wild-type; Transgenic wheat lines LXR3, LXR4, LXR13, LXR16 and positive plasmid controls containing 10 and 20 pg.
FIG. 43 shows expression analysis of independent T1 transgenic wheat lines (IPT quantitative expression in wheat). Quantitative transcript values were determined in femtomoles (fmol) per microgram of RNA using a standard curve derived from plasmid DNA containing the target sequence. Samples represent high, medium and low expression classes for candidate gene IPT.
FIG. 44 shows phenotypic variation of glasshouse grown T1 transgenic wheat plants. A. T1 LXR 13 wheat plants displaying normal phenotype as compared to null control wheat plants. B. T1 LXR 04 wheat plants displaying stunted phenotype and increased flag leaf number as compared to null control wheat plants.
FIG. 45 shows transgenic wheat displaying delay of leaf senescence as compared to null control leaves 7 days following detachment.
FIG. 46 shows phenotypes of the T2 transgenic canola plants expressing the chimeric atmyb32::ipt transgene (i.e., LXR lines), compared to wild-type non-transgenic control canola plants (i.e., WT), 150 DAP (left) and 225 DAP (right).
FIG. 47 shows flowering intensity in transgenic canola plants expressing the chimeric atmyb32::ipt transgene (i.e., LXR lines), compared to wild-type non-transgenic control canola plants (i.e., WT).
FIG. 48 shows southern hybridisation analyses of T2 transgenic canola lines (i.e., LXR 7.1 lines) expressing the chimeric atmyb32::ipt transgene. WT: wild-type, negative, non-transgenic control.
FIG. 49 Relative expression level of atmyb32::ipt transgene in T2 transgenic canola lines (i.e., LXR 6.6-, 7.1- and 8.9 T2 lines). WT: wild-type, negative, non-transgenic control.
FIG. 50 Flowering intensity and seed yield in T2 transgenic canola lines expressing the chimeric atmyb32::ipt transgene (i.e., lines 6.6-, 7.1- and 8.9) compared to wild-type, negative, non-transgenic control (i.e., WT).
FIG. 51 Seed weight of T2 transgenic canola lines expressing the chimeric atmyb32::ipt transgene (i.e., lines 6.6-, 7.1- and 8.9) compared to wild-type, negative, non-transgenic control (i.e., WT).



EXAMPLES
Example 1
Atmyb32 Promoter Sequence and Promoter Sequence Variants
The Atmybb32 promoter sequence and variants thereof are shown in FIGS. 1-4.
Example 2
Cytokinin Biosynthesis Genes
Examples of cytokinin biosynthesis genes suitable for use in the present invention are shown in FIGS. 6, 8 and 10. Suitable genes also include those encoding the polypeptides shown in FIGS. 7, 9 and 11.
Example 3
Production of Transgenic White Clover Plants
Transgenic white clover plants (Trifolium repens cv. Haifa and Irrigation) were produced by Agrobacterium-mediated transformation using a binary vector carrying the chimeric atmyb32::ipt gene (FIG. 12a). The transgenic plants were screened by PCR using ipt and nptII primers (FIG. 12b). HindIII digested genomic DNA samples subjected to Southern DNA hybridization analysis showed that the DNA fragments greater than 4.4 kb were detected in all lanes by both ipt and nptII probes, demonstrating the presence and integration of full-length T-DNA into the white clover genome (FIG. 12). Transgenic lines Hmi01, Imi06, Imi11, and Imi18 (Lane 1, 3, 5, 8 and 12 respectively) appeared to have a single copy of full-length T-DNA integrated in the genome. Other transgenic lines had multiple copies of the atmyb32::ipt transgene.
Example 4
IPT Gene Expression in Transgenic White Clover Plants
The expression of the atmyb32::ipt transgene in transgenic white clover (T. repens) plants was assessed by RT-PCR. The ipt mRNA was detected in leaf tissues of all atmyb32::ipt transgenic white clover plants examined, with varying levels of PCR products detected (FIG. 13).
Example 5
Delayed Detached Leaf Senescence in Transgenic White Clover Plants
Experiments were performed to assess detached leaf senescence of atmyb32::ipt transgenic plants. Rapid yellowing was observed in detached leaves from non-transformed and atmyb32::gusA transgenic white clover plants of both cultivars within one week. Transgenic lines Hmi01, Hmi08, Imi16 and Imi18 showed delayed senescence while Imi11 and Imi12 showed no sign of yellowing by the end of 7 days. After two weeks, the leaves of all atmyb32::ipt transgenic plants were much greener than those of non-transformed and atmyb32::gusA control transgenic plants (FIG. 14). The degree of senescence in excised leaves was in the order HC, Hmg>Hmi01>Hmi08 for cv. Haifa, and IC and Img>Imi16>Imi18>Imi11 and Imi12 for cv. Irrigation. HC is Haifa untransformed control, Hmg is Haifa atmyb32::gusA control, IC is Irrigation untransformed control, 1 mg is Irrigation atmyb32::gusA control. Hmi01, Hmi08, Imi16, Imi18, Imi11 and Imi12 are independent atmyb32::ipt transgenic white clover plants from the cultivar Haifa (H) and Irrigation (I), respectively.
Example 6
Plant Morphology and Root Development in Transgenic White Clover Plants
Normal plant morphology as well as normal shoot and normal root development was observed in atmyb32:ipt transgenic white clover plants (FIG. 6), thus indicating that the regulated expression of the ipt gene under control of the atmyb32 promoter did not negatively affect neither rooting nor apical dominance of the transgenic white clover plants (Table 1).











TABLE 1





Transformant
Cultivar
Construct
ipt copy No
Phenotype



















Hmi01
Haifa
Atmyb32::ipt
1
Normal


Hmi08
Haifa
Atmyb32::ipt
>3
Normal


Imi06
Irrigation
Atmyb32::ipt
1
Normal


Imi07
Irrigation
Atmyb32::ipt
3
Normal


Imi09
Irrigation
Atmyb32::ipt
>3
Normal


Imi10
Irrigation
Atmyb32::ipt
>3
Normal


Imi11
Irrigation
Atmyb32::ipt
1
Normal


Imi12
Irrigation
Atmyb32::ipt
2
Normal


Imi16
Irrigation
Atmyb32::ipt
2
Normal


Imi18
Irrigation
Atmyb32::ipt
1
Normal









Normal plant morphology and normal rooting was observed in ten independent atmyb32::ipt transgenic white clover lines analysed. Estimated ipt gene copy numbers in the ten independent atmyb32::ipt transgenic white clover lines are shown.
Example 7
Generation of Vectors for Plant Transformation
Four binary vectors have been generated for Agrobacterium-mediated transformation of plants (FIGS. 16-19). Each vector has a pPZP200 vector backbone (Hajdukiewicz et al., 1994) and contains either chimeric Atmyb32-900::ipt-nos or Atmyb32-xs::ipt-nos with or without a chimeric 35S::nptII-35st or 35S::hph-35st selectable marker cassettes.
One transformation vector has been constructed for biolistic transformation (FIGS. 20 and 21). The transformation vector contains chimeric Atmyb32-900::ipt-35st with a chimeric Ubi::bar-nos selectable marker cassette.
The Atmyb32 promoter, promoter variant Atmyb32xs, the isopentyl transferase gene and terminators 35st and nos were amplified by PCR using GatewayTM (Invitrogen) adapted primers and cloned into a pDONR221 entry vectors. These were subsequently cloned using recombination into destination vectors containing the conventionally cloned selectable marker cassettes. All vectors were fully sequenced following strict quality assurance protocols.
Example 8
Agrobacterium-Mediated Transformation of Canola
Brassica napus 
Binary vectors pBMVhATMYB3-900::ipt-nos (FIG. 20) and pBMVhATMYB32xs::ipt-nos (FIG. 22) containing chimeric ipt genes under control of Atmyb32 promoter (FIG. 1) and Atmyb32xs variant promoter sequence with deleted root-specific motifs (FIG. 2) were used for Agrobacterium-mediated transformation of Brassica napus hypocotyl segments (FIG. 26).
Brassica napus seeds are surface sterilised in 70% ethanol for 2 minutes, washed 3 times in sterile water then further surface sterilised in a solution containing 1% (w/v) Calcium hypochlorite and 0.1% (v/v) Tween 20 for 30 minutes. The seeds are washed at least 3 times in sterile water and planted in 120 ml culture vessels containing a solidified germination medium containing 1× Murashige and Skoog (Murashige and Skoog Physiol. Plant, 15: 473-497, 1962) macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 2% (w/v) sucrose at a pH of 5.8 with the addition of 4 g/L Gelrite. The vessels are incubated at 25° C. under 16 h light/8 h dark conditions for 7 days to encourage germination.
After 7 days, seedlings of Brassica napus (whole seedlings) are transferred to a liquid medium consisting of 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 3% (w/v) sucrose at a pH of 5.8. Seedlings are grouped together and the roots and cotyledons removed prior to cutting the hypocotyls into 7-10 mm sections and plating on 9×1.5 cm petri dishes containing a preconditioning medium consisting of 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 3% (w/v) sucrose at a pH of 5.8 solidified with 6.4 g/l Bacto-Agar.
Hypocotyl sections are cultured for 24 hours prior to inoculation with an Agrobacterium suspension OD600=0.2 for 30 minutes consisting of 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 100 μM Acetosyringone, 3% (w/v) sucrose at a pH of 5.8.
Following inoculation, hypocotyl sections are blotted on sterile paper towels and transferred to 9×1.5 cm petri dishes containing 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 100 μM Acetosyringone, 1 mg/L 2,4-D, 3% (w/v) sucrose at a pH of 5.8 solidified with 8 g/l Bacto-Agar. Explants are incubated at 25° C. under 16 h light/8 h dark conditions for 72 hours for co-cultivation.
Following co-cultivation, 20-30 hypocotyl explants are transferred to 9×1.5 cm petri dishes containing a solidified selection medium consisting of 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 1 mg/L 2,4-D, 3% (w/v) sucrose at a pH of 5.8 solidified with 8 g/l Bacto-Agar, supplemented with 250 mg/l timentin and 10 mg/l hygromycin to select for hygromycin-resistant shoots. Plates are incubated at 25° C. under 16 h light/8 h dark conditions.
After 7 days hypocotyl explants are transferred to 9×2.0 cm petri dishes containing a solidified regeneration media consisting of 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 1 mg/L 2,4-D, 3% (w/v) sucrose at a pH of 5.8 solidified with 8 g/l Bacto-Agar, supplemented with 4 mg/l BAP, 2 mg/l Zeatin, 5 mg/l Silver Nitrate, 250 mg/l timentin and 10 mg/l hygromycin. Plates are incubated under direct light at 25° C. under fluorescent light conditions (16 hr light/8 hr dark photoperiod; 55 μmol m−2 sec−1) for 4 weeks to encourage shoot development.
Regeneration is monitored weekly and hypocotyl explants transferred to fresh 9×2.0 cm petri dishes containing solidified regeneration media, RM supplemented with 4 mg/l benzyladenine, 2 mg/l zeatin, 5 mg/l silver nitrate, 250 mg/l timentin and 10 mg/l hygromycin for 6-8 weeks to encourage shoot development.
Hygromycin-resistant (Hygr) shoots are transferred to 120 ml vessels containing solidified root induction medium, RIM1, consisting of 1× Murashige and Skoog macronutrients, 1× micronutrients and B5 organic vitamins, supplemented with 500 mg/L MES, 1 mg/L 2,4-D, 1% (w/v) sucrose at a pH of 5.8 solidified with 8 g/l Bacto-Agar supplemented with 250 mg/l timentin. Shoots are incubated under direct fluorescent light at 25° C. (16 hr light/8 hr dark photoperiod; 55 μmol m−2 sec−1) to encourage shoot elongation and root development over 4-5 weeks. All Hygr shoots with developed shoot and root systems are transferred to soil and grown under glasshouse conditions.
Example 9
Biolistic Transformation of Wheat
Triticum aestivum L.
Transformation vectors containing chimeric ipt genes under control of Atmyb32 promoter (FIG. 1) and Atmyb32xs variant promoter sequence with deleted root-specific motifs (FIG. 2) were used for biolistic transformation of wheat (Triticum aestivum L. MPB Bobwhite 26). A representative vector is shown in FIG. 24. A schematic of the procedure for biolistic transformation of wheat is outlined in FIG. 27. The transformation procedure includes the following steps:
Step 1 (Donor Plant Production):
Triticum aestivum (Bobwhite 26) seed is used for the production of donor plant material. Wheat plants are grown in a nursery mix consisting of composted pine bark, perlite and vermiculite, with five plants per pot to a maximum pot size of 20 cm. Plants are kept under glasshouse conditions at approximately 22-24° C. for 12-16 weeks (FIG. 28A). Once the first spike emerges from the flag leaf, plants are tagged and embryos collected from the tallest heads 12-15 days post anthesis.
Step 2 (Day 1)
Spikes at the desired stage of development are harvested (FIG. 28B). Caryopsis are removed from the spikes and surface sterilised for 20 minutes in a 0.8% (v/v) NaOCl solution and rinsed at least four times in sterile distilled water.
Embryos up to 10 mm in length are aseptically excised from each caryopsis (removing the axis) using a dissecting microscope and cultured axial side down on an osmotic medium (E3maltose) consisting of 2× Murashige and Skoog (1962) macronutrients, 1× micronutrients and organic vitamins, 40 mg/L thiamine, 150 mg/L L-asparagine, supplemented with 15% (w/v) maltose, 0.8% (w/v) Sigma-agar and 2.5 mg/L 2,4-D (FIGS. 28C&D). Embryos are cultured on 60 mm×15 mm clear polypropylene petrie dishes with 15 mL of media. Culture plates are incubated at 24° C. in the dark for 4 hours prior to bombardment. Embryos are bombarded using a BioRad PDS1000 gene gun at 900 psi and at 6 cm with 1 μg of vector plasmid DNA precipitated onto 0.6 μm gold particles. Following bombardment, embryos are incubated overnight in the dark on the osmotic media.
Step 3 (Day 2):
Embryos are transferred to a callus induction medium (E3calli) consisting of 2× Murashige and Skoog (1962) macronutrients and 1× micronutrients and organic vitamins, 40 mg/L thiamine, 150 mg/L L-asparagine, supplemented with 6% (w/v) sucrose, 0.8% (w/v) Sigma-agar and 2.5 mg/L 2,4-D. Embryos are cultured for two weeks at 24° C. in the dark.
Step 4 (Day 16):
After 2 weeks of culture on E3calli, embryos have produced embryogenic callus and are subcultured onto a selection medium (E3Select) consisting of 2× Murashige and Skoog (1962) macronutrients and 1× micronutrients and organic vitamins, 40 mg/L thiamine, 150 mg/L L-asparagine, supplemented with 2% (w/v) sucrose, 0.8% (w/v) Sigma-agar, 5 mg/L of D,L phosphinothricin (PPT) and no plant growth regulators (FIG. 28E-G). Cultures are incubated for further 14 days on E3Select at 24° C. in the light and a 12-hour photoperiod.
Step 5 (Day 30):
After 14 days culture on E3Select, embryogenic callus is sub-cultured onto fresh E3Select for a further 14 days (FIG. 28E-G).
Step 6 (Day 44):
After about 4 weeks on E3Select, developing plantlets are excised from the embryonic callus mass and grown for a further three weeks in 65 mm×80 mm or 65 mm×150 mm polycarbonate tissue culture vessels containing root induction medium (RM). Root induction medium consists of 1× Murashige and Skoog (1962) macronutrients, micronutrients and organic vitamins, 40 mg/L thiamine, 150 mg/L L-asparagine, supplemented with 2% (w/v) sucrose, 0.8% (w/v) Sigma-agar, and 5 mg/L of PPT (FIG. 28H). Remaining embryogenic callus is sub-cultured onto E3Select for another 14 days.
Step 7 (Day 65+):
Regenerated plantlets surviving greater than 3 weeks on RM with healthy root formation are potted into a nursery mix consisting of peat and sand (1:1) and kept at 22-24° C. with elevated humidity under a nursery humidity chamber system (FIG. 28). After two weeks, plants are removed from the humidity chamber and hand watered and liquid fed AquasolTM weekly until maturity. The T0 plants are sampled for genomic DNA and molecular analysis. T1 seed is collected and planted for high-throughput Q-PCR analysis (FIG. 28J).
Example 10
Agronomic Performance of Transgenic White Clover Plants
The agronomic performance of atmyb32::ipt transgenic white clover (Trifolium repens) plants, relative to that of non-transgenic control white clover plants, was evaluated under environmentally controlled growth chamber conditions and in contained field trials (FIG. 29).
Transgenic white clover plants expressing chimeric Atmyb32::ipt genes assessed under controlled growth chamber conditions revealed a significantly enhanced biomass accumulation and reductions in manifestations of senescence, when compared with non-transgenic control white clover plants (FIG. 30). The transgenic white clover plants expressing chimeric Atmyb32::ipt genes showed enhanced total leaf area, increased cumulative leaf area, higher leaf growth dynamics (i.e., number of leaves over time), higher stolon length and increased % flowering plants as well as reduced stolon senescence and death compared with non-transgenic control white clover plants (FIG. 30A-C).
The seed yield performance of 3 independent atmyb32::ipt expressing transgenic white clover plants (i.e., LXR 12, LXR 18 and LXR 11) was also comparatively assessed with non-transgenic control plants (i.e., wild type, WT) under contained field conditions. Two independent atmyb32::ipt expressing transgenic white clover plants (i.e., LXR 12 and LXR 18) with indistinguishable flowering intensity (i.e., number of ripe flowers per m2) to the non-transgenic control plant (i.e., WT) were selected for field evaluation (FIG. 31).
While the seed weight (i.e., weight of thousand seeds) of transgenic white clover plants expressing chimeric Atmyb32::ipt genes (i.e., LXR 12, LXR 18 and LXR 11) was indistinguishable from non-transgenic control white clover plants (i.e., WT) (FIG. 32), the total seed yield expressed on the basis of per flower (FIG. 33), and per area sown (FIG. 34) was doubled in transgenic white clover plants expressing chimeric Atmyb32::ipt genes (i.e., LXR 12 and LXR 18) when compared with non-transgenic control white clover plants of equivalent flowering intensity (i.e., WT).
Example 11
Agrobacterium-Mediated Transformation of Alfalfa
Medicago sativa 
The binary vector pBMVkATMYB32xs::ipt-nos (FIG. 18) containing chimeric ipt genes under control of Atmyb32xs variant promoter sequence with deleted root-specific motifs (FIG. 2) was used for Agrobacterium-mediated transformation of Medicago sativa petiole explants from highly-regenerable alfalfa (M. sativa) clones C2-3, C2-4 and 19-17 (FIG. 35).
Following co-cultivation with Agrobacterium tumefaciens strain LBA 4404 harbouring the binary vector pBMVkATMYB32xs::ipt-nos, the alfalfa explants were washed with medium containing cefotaxime and used for induction of embryogenic callus under selective medium containing 25 mg/l kanamycin. Transgenic embryogenic alfalfa calli were recovered and allowed to regenerate transgenic alfalfa shoots, which were transferred on rooting medium leading to the recovery of transgenic alfalfa plants expressing chimeric ipt genes under control of Atmyb32xs variant promoter (FIG. 35).
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
Example 12
Production of Transgenic Canola Plants
Transgenic canola plants (Brassica napus) were produced by Agrobacterium-mediated transformation using binary vectors (FIGS. 20 and 22) carrying the chimeric atmyb32::ipt gene. The genetic modification has been characterised for the presence of the candidate gene (IP) or the selectable marker (hph) using PCR at the T1 generation (FIG. 36).
FIG. 36 illustrates PCR analysis of transgenic canola plants. Genomic DNA was isolated from different transgenic canola plants of T1 LXR04 lines and subjected to PCR using primers specific for the selectable marker (hph gene) or the candidate gene of interest (IPT). In FIG. 36A hph specific primers were used to amplify a product from genomic DNA and were visualised on an agarose gel. FIG. 36B demonstrates the use of IPT specific primers to amplify genomic DNA using a fluorescent PCR method. The primers are specific for the target sequence which results in detectable fluorescence that is inversely proportional to the amount of accumulated PCR product.
Example 13
IPT Gene Expression in Transgenic Canola Plants
The expression of the atmyb32::ipt transgene in transgenic canola was assessed using a fluorescent RT-PCR method specific for the target sequence (FIG. 37). The IPT mRNA was detected in tissues and relative expression levels were compared among lines and with null controls. Null controls are progeny lines that have undergone the transformation process but do not contain target sequences after crossing.
Example 14
Delayed Detached Leaf Senescence in Transgenic Canola Plants
Experiments were performed to assess detached leaf senescence of atmyb32::ipt transgenic plants. FIGS. 38 to 41 indicate detached senescence assay data associated with expression of the candidate gene in canola. Assays for detached cotyledons and leaves were conducted to induce aging and asses the senescence phenotype of transformed canola as compared to wild-type controls. At day 7 and 14 of the detached senescence assays the progress of senescence was qualitatively scored for each tissue sample as either 0—no possible signs of senescence; 1—first visible signs of senescence with a paling of the green colour: 2—further progression of senescence with yellowing in colour becoming noticeable; 3—the tissue was mostly yellow in colour, however a pale green colour remained evident; 4—progression to completely yellow in colour; 5—yellow in colour with some bleaching and patches of necrosis (FIGS. 39 and 41).
Example 15
Production of Transgenic Wheat Plants
Genetic transformation of wheat was based on biolistic transformation of zygotic embryos from Triticum aestivum L Bobwhite 26 wheat line as outlined in FIGS. 27 and 28.
The chimeric atmyb32::ipt gene was inserted into the wheat genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome (FIG. 24).
The transformation vector has been fully sequenced (FIG. 25). The genetic modification has been characterised for the presence of the candidate gene by Southern analysis at the T1 generation (FIG. 42).
FIG. 42 illustrates Southern hybridisation analysis of transgenic wheat plants. Genomic DNA was isolated from different T1 lines of transgenic wheat plants and digested with a restriction enzyme to determine candidate gene copy number. The control is non-transformed wild-type Triticum aestivum ‘Bobwhite 26’. Digests were electrophoresed, transferred to nylon membrane and probed with a full-length DIG labelled IPT gene, as a probe. A range of copy numbers was observed.
Example 16
IPT Gene Expression in Transgenic Wheat Plants
RNA was extracted from young leaf tissue of glasshouse grown transgenic T1 wheat plants containing the IPT gene driven by the AtMYB32 promoter and first strand cDNA prepared.
Quantitative expression of the transgene was determined using a probe based qRT-PCR method for the target sequence. Representative examples of high, medium and low expressing lines for each of the constructs are presented in FIG. 43. A primer/probe set designed to the endogenous sucrose synthase gene was also used as a control. All amplification plots of the control gene began with in one cycle of each other indicating differences in the level of detection of the GMOs is due to variation in expression.
Both the PCR primers and probe are specific for the target sequence which results in detectable fluorescence that is proportional to the amount of accumulated PCR product. Serially diluted plasmid DNA containing the target sequence being detected was employed to create a standard curve for quantification.
Example 17
Plant Morphology in Transgenic Wheat Plants
Differences in growth characteristics were observed in the glasshouse within and among transgenic wheat lines. The phenotypes predominantly observed among T1 wheat plants included stunted plant height, tillering intensity, leaf number, as well as vegetative biomass (FIG. 44).
Example 18
Delayed Detached Leaf Senescence in Transgenic Wheat Plants
A detached leaf assay was used to asses induced aging and the senescence phenotype of transformed wheat leaves as compared to null controls (FIG. 45). Null controls are progeny lines that have undergone the transformation process but do not contain target sequences after crossing.
Example 19
Analysis of Transgenic Canola Plants
Transgenic T2 canola plants expressing the chimeric atmyb32::ipt transgene showed a higher number of stems, inflorescences, flowers and mature siliques than control plants. FIGS. 46 and 47 show the numbers of stems, inflorescences, flowers and siliques 150 and 225 days after planting for atmyb32::ipt transgenic canola (i.e., LXR lines) compared to the wild type non-transgenic control (WT).
Molecular analysis of T2 transgenic canola lines (i.e., LXR 7.1 lines) expressing the chimeric atmyb32::ipt transgene showed a single copy of the transgene in all analysed lines (FIG. 48).
Analysis of the expression level of the chimeric atmyb32::ipt transgene in T2 transgenic canola lines revealing a range of expression levels in the 6.6-, 7.1- and 8.9-derived lines. LXR 6.6-derived lines showed highest level of expression of the atmyb32::ipt transgene. No expression observed in the wild-type, negative control line (FIG. 49).
T2 transgenic canola lines expressing the chimeric atmyb32::ipt transgene showed up to a 5 fold increase in flower number and up to a 6 fold increase in seed yield when compared to wild-type, negative, non-transgenic controls (FIG. 50).
Analysis of the seed weight (seed weight/100 seeds) showed up to a 30% increase in seed weight in the transgenic canola plants expressing the chimeric atmyb32::ipt transgene relative to the wild-type, negative, non-transgenic control (FIG. 51).
It will also be understood that the term “comprises” (or its grammatical variants) as used in this specification is equivalent to the term “includes” and should not be taken as excluding the presence of other elements or features.
Documents cited in this specification are for reference purposes only and their inclusion is not an acknowledgement that they form part of the common general knowledge in the relevant art.
Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

## Claims

1. A method of manipulating senescence in a plant or for enhancing plant biomass, said method comprising introducing into said plant a genetic construct comprising a modified myb gene promoter, said modified myb promoter comprising a nucleotide sequence selected from the group consisting of:
(a) Sequence ID No. 2;
(b) Sequence ID No. 3;
(c) Sequence ID No. 4; and
(d) a functionally active variant of one of the sequences recited in (a), (b) or (c), said functionally active variant having at least approximately 95% identity to Sequence ID No. 2, Sequence ID No. 3 or Sequence ID No. 4;
said modified myb gene promoter being operatively linked to a cytokinin biosynthesis gene.

2. The method according to claim 1, wherein said myb gene promoter is from Arabidopsis.

3. The method according to claim 1, wherein said cytokinin biosynthesis gene is selected from the group consisting of an isopentenyl transferase (ipt) gene and a sho gene.

4. The method according to claim 3, wherein said cytokinin biosynthesis gene is from a genus selected from the group consisting of Agrobacterium, Lotus and Petunia.

5. The method according to claim 4, wherein said cytokinin biosynthesis gene comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos: 5, 7 and 9, sequences encoding the polypeptides having a sequence of Sequence ID Nos: 6, 8 and 10, and a functionally active variant of the Sequence ID Nos: 5, 7 or 9, said functionally active variant having at least approximately 95% identity to Sequence ID No: 5, Sequence ID No. 7 or Sequence ID No. 9.

6. The method according to claim 1, wherein said genetic construct is introduced into said plant by Agrobacterium-mediated or biolistic transformation of plant cells.

7. The method according to claim 6, wherein plant cells incorporating the genetic construct are selected and then cultured to regenerate transformed plants.

8. A method of manipulating senescence in a plant or for enhancing plant biomass, said method comprising introducing into said plant a genetic construct comprising a modified myb gene promoter, said modified myb promoter comprising a nucleotide sequence selected from the group consisting of:
(a) Sequence ID No. 2;
(b) Sequence ID No. 3; and
(c) Sequence ID No. 4;
said modified myb gene promoter being operatively linked to a cytokinin biosynthesis gene.

9. A vector comprising a modified myb gene promoter, said modified myb gene promoter comprising a nucleotide sequence selected from the group consisting of:
(a) Sequence ID No. 2;
(b) Sequence ID No. 3;
(c) Sequence ID No. 4; and
(d) a functionally active variant of a sequence recited in (a), (b) or (c), said functionally active variant having at least approximately 95% identity to Sequence ID No. 2, Sequence ID No. 3 or Sequence ID No. 4;
said modified myb gene promoter being operatively linked to a heterologous cytokinin biosynthesis gene.

10. The vector according to claim 9, further comprising a terminator; said promoter, gene and terminator being operatively linked.

11. The vector according to claim 9, wherein said myb gene promoter is from Arabidopsis.

12. The vector according to claim 9, wherein said cytokinin biosynthesis gene is selected from the group consisting of an isopentenyltransferase (ipt) gene and a sho gene.

13. The vector according to claim 12, wherein said cytokinin biosynthesis gene is from a genus selected from the group consisting of Agrobacterium, Lotus and Petunia.

14. The vector according to claim 9, wherein said cytokinin biosynthesis gene comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos: 5, 7 and 9, sequences encoding the polypeptides having a sequence of Sequence ID Nos: 6, 8 and 10, and a functionally active variant of Sequence ID Nos: 5, 7 or 9, said functionally active variant having at least approximately 95% identity to Sequence ID No: 5, Sequence ID No. 7 or Sequence ID No. 9.

15. A vector comprising a modified myb gene promoter, said modified myb promoter comprising a nucleotide sequence selected from the group consisting of:
(a) Sequence ID No. 2;
(b) Sequence ID No. 3; and
(c) Sequence ID No. 4;
said modified myb gene promoter being operatively linked to a heterologous cytokinin biosynthesis gene.

16. A transgenic plant cell, plant, plant seed or other plant part, with modified senescence characteristics, said plant cell, plant, plant seed or other plant part comprising a genetic construct comprising a modified myb gene promoter, said modified myb gene promoter comprising a nucleotide sequence selected from the group consisting of:
(a) Sequence ID No. 2;
(b) Sequence ID No. 3;
(c) Sequence ID No. 4; and
(d) a functionally active variant of a sequence recited in (a), (b) or (c), said functionally active variant having at least approximately 95% identity to Sequence ID No. 2, Sequence ID No. 3 or Sequence ID No. 4;
said modified myb gene promoter being operatively linked to a cytokinin biosynthesis gene.

17. A transgenic plant cell, plant, plant seed or other plant part derived from the transgenic plant cell according to claim 16.