WO1993007272A1 - Transgenic plants - Google Patents

Transgenic plants Download PDF

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Publication number
WO1993007272A1
WO1993007272A1 PCT/AU1992/000528 AU9200528W WO9307272A1 WO 1993007272 A1 WO1993007272 A1 WO 1993007272A1 AU 9200528 W AU9200528 W AU 9200528W WO 9307272 A1 WO9307272 A1 WO 9307272A1
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plant
tuber
dna construct
transgenic
according
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PCT/AU1992/000528
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French (fr)
Inventor
David Stuart Letham
Kim Rochelle Stevenson
Guo-Qing Tao
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Calgene Pacific Pty. Ltd.
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Priority to AUPK873091 priority
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Publication of WO1993007272A1 publication Critical patent/WO1993007272A1/en

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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1085Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291Hormone-influenced development
    • C12N15/8295Cytokinins

Abstract

The present invention relates generally to transgenic plants and is particularly directed to transgenic tuber plants and to DNA constructs useful for producing same. The DNA constructs comprise a first nucleotide sequence corresponding to a promoter capable of functioning in a plant and a second nucleotide sequence under control of said promoter and encoding a molecule capable of enhancing levels of a cytokinin in said plant.

Description

TRANSGENIC PLANTS

The present invention relates generally to transgenic plants and is particularly directed to transgenic tuber plants and to DNA constructs useful for producing same.

Tubers are a swollen part of a stem or root which are usually modified for storage. Many such tuber plants have assumed immense commercial importance in agriculture and horticulture. Important examples include potato, sugar beet, sweet potato, onion, garlic, artichoke and Dahlia,

Other plants, such as grasses and cereals, also use modified stems as important storage organs, particularly for storage of carbohydrate. These storage organs are important because they increase the capacity of the plant to sustain periods of stress through mobilisation of the stored carbohydrates and other compounds.

Stored reserves are also important in contributing to the grain yield of cereals (Borrell et aL, 1989) and the field value of pasture grasses and legumes.

The potato is perhaps the best example of an economically important plant with a modified storage organ, in this case a tuber.

The potato is one of the world's most valuable food crops. The current production level of potatoes alone is estimated to be worth $US90 billion. By volume, potato ranks fourth in the world after rice, wheat and maize with about 300 million tonnes annually produced. Potato has been commercially produced in Europe and the USA for over 200 years, but it is a relatively new crop for many of the developing countries, although now, potato production is increasing in developing countries at a rate nearly twice that of most other food crops. Tuber yield is a key determinant of the profitability of a tuber plant crop and yield is closely linked to tuber number and/or tuber weight on each plant. Tuber initiation is a function of genotype and various environmental conditions, but especially night temperature. Since night temperatures of less than 20°C are required for tuber initiation, tuber production, such as potato, in hot developing countries is restricted to elevated locations or the lowlands for short periods during the "cooler" part of the year.

Cytokinins have been implicated in the control of tuber initiation in plants such as potato. The ability to control the level of a cytokinin in such plants is, therefore, important for controlling tuberization.

The cloning and characterisation of the isopentenyl transferase (ipt) gene from Agrobacterium tnmefaciens has led to the introduction of this ^ene into the DNA of plant cells. The unregulated production of cytokinins in tissues transformed with the ipt gene has typically resulted in complete inhibition of root formation (Smigocki and Owens, 1988). Recently, Medford et aL (1989) and Smigocki (1991) have reported rooted transgenic tobacco plants where the ipt gene was placed under the control of a heat-inducible promoter. In both these cases, although the effect of the inducers on gene expression was dramatic (i.e. remarkably elevated levels of cytokinin after heat treatment), the heat-inducible promoters appeared to be rather non-specific in that the plants exhibited phenotypes associated with excess cytokinin even without thermal induction.

There is a need, therefore, to utilise the benefits of the ipt or like gene in elevating cytokinin levels while not suppressing root formation. There is also a need to manipulate tuber plants, such as potato plants, to generate a transgenic plant with elevated cytokinin levels yet retaining at least normal root formation capability. In accordance with the present invention, there is provided a fusion between the chalcone synthase (chs promoter from Antirrhinum majus and the ipt coding sequence. This construct can be used to produce transgenic tuber plants with useful agronomic characteristics. In particular, the construct can be used to produce transgenic potato plants having improved tuber production. This type of construct is an example of a wider range of constructs which can be usefully employed in generating transgenic plants with improved properties.

Accordingly, one aspect of the present invention provides a DNA construct comprising a first nucleotide sequence corresponding to a promoter capable of functioning in a plant and a second nucleotide sequence under control of said promoter and encoding a molecule capable of enhancing levels of a cytokinin in said plant.

Preferably the plant is a tuber plant such as but not limited to potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia, Most preferably the plant is potato.

The present invention is described and exemplified using the chs. promoter from A. majus (Sommer and Saedler, 1986) and the ipt coding sequence (Barker et aL, 1983; Heidekamp et a , 1983). This is done with the understanding, however, that the present invention extends to other functionally equivalent promoters or to other coding sequences which, like the ipt gene, result in elevated cytokinin levels. Sources of other genes that could act to increase levels of active cytokinins include Agrobacterium rhizogenes and Pseudomon s. Examples of alternative sources of promoters include genes such as those described in Keller et aL (1989) and Yang and Russell (1990) which are expressed in stem tissues. Reference herein, therefore, to the ______ promoter and/or ipt gene or coding sequence is taken to include reference to other functionally similar promoters and /or genes.

Accordingly, in a preferred aspect of the present invention there is provided a DNA construct comprising a first nucleotide sequence corresponding to the chϋ promoter or a functional equivalent thereof and a second nucleotide sequence under the control of said promoter encoding ipt or a functional equivalent thereof, wherein said DNA construct is capable of expressing said ipt gene or functional equivalent thereof in a tuber plant.

The first nucleotide sequence corresponding to the chs. promoter may encode the entire naturally occurring promoter sequence or may contain single or multiple nucleotide substitutions, deletions and/ or additions to the naturally occurring sequence provided such changes in nucleotide sequence result in a functional promoter. One skilled in the art will readily recognise that the promoter can be subject to a variety of mutational events and the effect of such events on the expression of sequences under the control of the promoter screened. Accordingly, reference herein to the chs. promoter, includes reference to the naturally occurring promoter and to any functional mutants or derivatives thereof. Examples of such mutants or derivatives include those resulting in constitutive expression, super-inducible expression and altering the control and/or inducibility of the promoter.

The second nucleotide sequence of the present invention is located "downstream" of the chs. promoter and, hence, is under control thereof. Accordingly, the chs. promoter directs transcription and ultimate expression of the second nucleotide sequence. In accordance with the present invention, the second nucleotide sequence corresponds to the naturally occurring ipt gene of A. tumefaciens or to any single or multiple nucleotide substitutions, additions and/or deletions, provided that any resulting mutant or derivative of the naturally occurring sequence encodes a polypeptide which is functionally similar to the ipt gene product and results in elevated cytokinin levels in a host plant. All altered but functional ipt genes are encompassed by the present invention. The present invention also extends to the recombinant product of the ipt gene or its mutants or derivatives when expressed by the chs. or functionally equivalent promoter.

The first and second nucleotide sequences of the present invention are referred to herein as a "DNA construct". The DNA construct of the present invention may exist alone or in combination with a larger DNA construct such as a vector molecule. Such a vector molecule may be replicable in prokaryotic and/or eukaryotic cells and may contain other coding promoter and/or regulatory sequences. Preferably, the vector is capable of facilitating entry of itself and/or a DNA construct into the genome of a plant cell.

In a most preferred embodiment, a promoter fragment from the A. majus chs gene is fused to the A. tumefaciens ipt gene to give plasmid pCGP275. The fusion is conveniently accomplished using in vitro mutagenesis to introduce a restriction site between the transcriptional and translational start sites of the chs. gene. The introduction of the restriction site can be accomplished by a single or multiple base change but preferably only a single base is changed. In a most preferred embodiment, an Xbal site is introduced by a single base change (C→A) at position +35 of the chs. gene.

Although the second nucleotide sequence of the present invention is directly controlled, i.e. expression directed, from the first nucleotide sequence, this expression may further be controlled by another regulatory sequence such as encoding a regulatory gene. Alternatively, the other nucleotide sequence may encode or be a cis. controlling element. This is particularly useful if what is desired is to control developmentally the expression of the second nucleotide sequence or to induce expression during certain environmental conditions, climatic periods or certain months of the year. The stimulus to induce the overall expression may be environmental, developmental or may require the addition of external growth or other stimulatory factors.

Another aspect of the present invention is directed to a transgenic tuber plant carrying the DNA construct as hereinabove contemplated. More particularly, this aspect of the present invention provides a transgenic tuber plant carrying a DNA construct comprising a first nucleotide sequence corresponding to a promoter capable of functioning in said plant and a second nucleotide sequence under the control of said promoter and encoding a molecule capable of enhancing levels of a cytokinin in said plant.

In a preferred aspect of the present invention there is provided a transgenic tuber plant carrying a DNA construct comprising a first nucleotide sequence corresponding to the chs, promoter or a functional equivalent thereof and a second nucleotide sequence under the control of said promoter encoding ipt or a functional equivalent thereof, wherein said DNA construct is capable of expressing said ipt gene or functional equivalent thereof in said plant. Preferably, the tuber plant is potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia, Most preferably, the tuber plant is potato.

The DNA construct may be introduced into the tuber plant ϊia any number of convenient routes including mobilisation by Agrobacterium, transformation, microprojectile bombardment, micro-injection and electroporation of individual or groups of cells followed by plantlet regeneration.

Another aspect of the present invention contemplates a method for producing a transgenic tuber plant comprising preparing transgenic cells from a tuber plant carrying a DNA construct comprising a first nucleotide sequence corresponding to the chs. promoter or functional equivalent thereof and a second nucleotide sequence under the control of said promoter encoding ipt or a functional equivalent thereof and then regenerating a tuber plant from said transgenic cells.

In accordance with the present invention, the transgenic tuber plants exhibit any one or more of the following properties: increased level of endogenous cytokinin(s); increased tuber number and/or weight; increased stem diameter; increased plant height; increased leaf size; delayed leaf senescence; increased photosynthetic capacity of leaves thereby increasing the ability of the plant to support an increased tuber load and hence increase tuber yield. In addition, expression of the second nucleotide sequence of the DNA construct results in elevated cytokinin levels in stem tissue, such as developing stem tissue. Since stem tubers develop on the stem, the elevated cytokinin levels are directly implicated as causing at least an increase in number and/or weight of tubers per plant. However, expression need not be confined to the stem tissue and may also occur, for example, in leaves. Indeed, in some circumstances, it may be advantageous to have expression in leaves and stem to facilitate delayed leaf senescence as well as increased tuber initiation. The delayed leaf senescence may allow the plant to further support the increased tuber load and/or number.

Additionally, the insertion of the DNA construct of the present invention can lead to different transgenic effects which may result in different phenotypes. Although not intending to limit the present invention to any one theory as to mode of action, the different transgenic events may explain the varied properties (see above) observed in the transgenic plants. An additional property in some plants is the miniaturisation of tubers, such as observed in Figure 8. This may or may not be associated with an increase in number of tubers. Such miniature tubers may be important as specialised food items in the culinary arts.

The present invention is particularly described with reference to stem tubers. Examples of plants in this category include potato, onion, garlic and artichoke. However, the use of the term "stem tuber" should be construed in its broadest sense and include plants with modified stems such as bulbs, corms, rhizomes and cereals. Transgenic cereals having stems with increased thickness would be of value, for example, for wind resistance. In the floriculture industry, tulip, lily, freesia, gladiolus and Dahlia all have modified stems which are encompassed by the term "stem tubers" as used herein.

In accordance with the present invention, the transgenic potato plants produce an increased yield (i.e. weight and/ or number) of tubers. Furthermore, when potato tuberization occurs under non-inducing conditions, such transgenic potatoes will be very useful for cultivation in warmer climates such as developing countries.

The present invention also extends to tubers, such as potatoes, produced from the transgenic plants herein described.

The present invention is further described by reference to the following non- limiting Figures and Example.

In the Figures:

Figures 1(a) and (b) are a schematic representation of the introduction of an Xhal site at position +35 (indicated with *) of the chs. sequence to form plasmid pCGP263.

Figures 2(a) and (b) are a schematic representation of the reconstruction of the chs. promoter from plasmid pCPG263 to form plasmid pCGP267.

Figure 3 is a schematic representation showing the introduction of an Xbal site upstream of the ipt translation initiatipn codon.

Figures 4(a) and (b) are a schematic representation showing the formation of the chsript fusion pCGP274.

Figure 5 is a schematic representation showing the Ti-binary construct pCGP275.

Figure 6 is a graphical representation showing the altered phenotypes of ten chsript transgenic potato plants.

Figure 7 is a photographic representation showing increased tuber production by potato plants 2-8, K-l and K-2 transformed with pCGP275, compared to a non-transgenic control plant (W) at the same age.

Figure 8 is a photographic representation showing the production of a large number of small tubers (over 100) by a potato plant transformed with pCGP275 (K-4) compared to a non-transgenic control plant (W) of the same age.

Figure 9 shows photographic representations of transgenic potato plants transformed with pCGP275 compared to a non-transgenic control plant (W) of the same age: a: shows the "shooty" spreading phenotype of K-4; b: shows prolonged photosynthetic capacity (delayed leaf senescence) of K-2; c: shows increased plant height and leaf size of K-l and K-2.

Figure 10 is a photographic representation showing an autoradiograph of a Southern blot of potato DNA probed with a fragment of the ipt gene. Each lane contained lOμg DNA isolated from potato leaves, digested with EcoRl. C = untransformed control; 2-1 and K-4 = transgenic potato plants transformed with the pCGP275 construct. Filters were hybridized and washed under conditions of high stringency.

EXAMPLE 1. MATERIALS AND METHODS

Bacterial strains

The Agrobacterium tumefaciens strains used were K61 and LBA4404 (Hoekema et aL, 1983).

The plasmid pCGP275 (Figure 5) was introduced into Agrobacterium by adding 5μg of DNA to lOOμL of competent Agrobacterium cells. The competent cells were prepared by inoculating 50mL of MG/L (Garflnkel and Nester, 1980) and growing for 16 hours with shaking at 28 °C. The cells were then pelleted and resuspended in 0.5mL of 85% (v/v) 100 mM CaCl2/15% (v/v) glycerol. The DNA-Agrobacterium mixture was frozen by incubation in liquid N2 for 2 minutes and then allowed to thaw for 5 minutes at 37°C. The DNA/bacterial mix was then placed on ice for a further 10 minutes. The cells were then mixed with lmL of MG/L media and incubated with shaking for 16 hours at 28°C. Cells of A. tumefaciens carrying the plasmids were selected on MG/L agar plates containing 100 ug/mL gentamycin.

Potato transformation

Transformation of potato (cv. Desiree) leaf segments followed standard protocols and transformed plants were identified by their ability to grow on kanamycin-containing media.

Construction of pCGP275

A promoter fragment from the Antirrhinum majus chalcone synthase (chs) gene (Sommer and Saedler, 1986) was fused to the A. tumefaciens ipt gene (Barker et aL, 1983; Heidekamp et aL, 1983) to give pCGP275.

Construction of the fusions was facilitated using in vitro mutagenesis to introduce an Xh l site between the transcriptional and translational start sites of the chs. gene. This was accomplished with a single base change (from C to A) at position +35 of the A. majus chs gene (Sommer and Saedler, 1986). Prior to mutagenesis, a small fragment of the chs.5' sequence was subcloned to avoid undesired mutations occurring at other sites in the chs. sequence.

The various subcloning steps that preceded the mutagenesis are shown in Figures 1(a) and (b). A 5.7kb fragment containing 5' sequence from the ____ majus. chs. gene was removed from plasmid AM3 (Wienand et aL, 1982) by Ecα RI digestion and inserted into the ECΩ. RI site of pBluescript KS M13- (Stratagene). The resulting plasmid, designated pCGP253, was then digested with Seal and Ecα RI and the 2.1 kb fragment which included the chs. initiation codon and 240bp of 5' untranslated leader sequence was ligated with a Scal/Ecα RI digest of pBR322. The resulting pBR322 derivative was designated pCGP260. The plasmid pCGP260 was digested with Estl and Sad and a fragment which included 235bp pBR322 sequence, the chs. initiatioi- codon and 240bp of untranslated leader sequence was ligated to a _____I/Sa__I digest of pBluescript KS M13- digested with the same enzymes to give pCGP262 (Figure lb).

A Xhal site was introduced into pCGP262 at position +35 in the chs. sequence (Sommer and Saedler, 1986) using the Biorad MUTAGENE kit and the synthetic oligonucleotide 5'-CAATCATCTAGAACAACCACTTC-3'. The modified plasmid was designated pCGP263 and the mutagenesis was confirmed by sequence analysis (Figure lb).

The chs. promoter was re-constructed as shown in Figures 2(a) and (b). The plasmid pCGP253 (shown in Figure la) was digested with Eco RI and the 5.7kb chs. fragment inserted into the Ecα RI site of pCGP263 to yield pCGP264. Plasmid pCGP264 contained the large chs. promoter fragment in the same orientation as the mutated fragment (see Figure 2a). Plasmid pCGP264 was then partially restricted with Seal and re-ligated. A plasmid which had lost the 2.1kb Seal fragment was designated pCGP265 (Figure 2b). Plasmid pCGP265 was subsequently digested with Δ__aII and the overhanging 5' end filled in with the Klenow fragment of DNA Polymerase I prior to digestion with Xhal. The 1.2kb promoter fragment was isolated and inserted into pUC19 digested with Smal and Xhal. The resulting plasmid was designated pCGP267.

chs-ipt Fusion Plasmid pCGN1278 contained the ipt gene cloned into the Smal site of pBluescript M13- as a Rsal fragment which included T-DNA from nucleotide 8487 to nucleotide 9836 (Barker el aL, 1983). The ipt gene was removed from pCGN1278 by digestion with Ecα RI and Xhal, the overhanging 5' ends were filled in the Klenow fragment of DNA Polymerase I and the blunt-end fragment was inserted into the Smal site of pBluescript KS Ml 3-. The resulting plasmid was designated pCGP259.

Figure 3 shows the strategy used to introduce a Xhal site 8bp upstream of the ip translation initiation codon. Plasmid pCGP259 was digested with BstXI and EspMI, ligated to the synthetic oligonucleotide 5'- AATTAGATGCAGGTCCATAAGTTTTTTCTAGACGCG-3' which included a Xha.1 site (underlined) and 5' and 3' ends complementary to the respective overhanging ends which remained after digestion of pCGP259. Following ligation, the single stranded gap was filled in using the Klenow fragment of DNA polymerase I. The modified ipt gene-containing plasmid was designated pCGP261 (Figure 3).

To construct the chsript fusion, the chs. promoter fragment was isolated from pCGP267 as a Sacl/Xhal fragment and inserted upstream of the ipt gene in a Sacl/Xhai digest of pCGP261 (Figure 4a). The SacI site in the resulting plasmid, pCGP273, was converted to a Hindlll site by restriction with SacI, digestion of the overhanging 3' end with the Klenow fragment of DNA polymerase I and ligation of a Hindlll linker to give pCGP274 (Figure 4b).

Ti-binary constructs

The Hindlll fragment from pCGP274 containing the chs. promoter fused to the ipt gene was inserted into the Hindlll site of the binary vector pCGN1558 (McBride and Summerfelt, 1990) to give pCGP275 (Figure 5).

2. RESULTS

Generation of transgenic potato plants

Transgenic potato shoots carrying the chsript gene fusion were selected on kanamycin-containing media after co-cultivation of leaf discs with either K61/pCGP75 or LBA4404/pCGP275. Rooted shoots from ten separate transformation events were transferred to soil and grown to semi-maturity. Figure 6 summarises some of the phenotypic alterations that resulted from the introduction of the gene fusion. In all ten plants there is a significant increase in tuber yield compared to non-transgenic control plants. For the most part the yield increase is associated with an increase in the number of tubers initiated on the plants (Figures 6 and 7). One of the transgenic plants, K-4 (Figures 8 and 9a) was of particular interest in that it produced over one hundred small tubers. This plant had a more "shooty" spreading phenotype suggestive of decreased apical dominance, but it was able to root normally.

In plants other than K-4, the introduction of the chsript fusion had the effect of increasing plant height and leaf size (Figures 6 and 9c). Most of the transgenic potato plants also had thicker stems than the non-transgenic control suggestive of preferential ipt expression in the stem tissues. The potato plants transformed with chsript fusion also showed delayed leaf senescence (Figure 9b).

These data, therefore, indicate that potato plants transformed with the chsript construct will be able to produce an increased and possibly high yield of tubers and may be able to be cultivated under non-inducing conditions in many of the warmer developing countries.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. REFERENCES

Barker, R.F., Idler, K.B., Thompson, D.V. and Kemp, J.D. Plant Molecular BiΩlΩg3_ 2; 335-350, 1983.

Borrell, A.K., Incoll, L.D., Simpson, R . and Dalling, MJ. Annals, nf Botany 62; 527-539, 1989.

Garfinkel, D J. and Nester, E.W. j___]__act_ ____£_.: 732-743, 1980.

Heidekamp, F., Dirkse, W.G., HiUe, J. and van Ormondt, H. Nucl. Acids Res 11: 6211-6223, 1983.

Hoekema, A., Hirsch, P.R., Hooykaas, P .J. and Schilperoort, RA Mature (London).302; 179-181, 1983.

Keller, B., Schmid, J. and Lamb, C.J. E_______Ω__L 8(5); 1309-1314, 1989.

McBride, K.E. and Summerfelt, K.R. Plant Molecular Biology l_fc 269-276, 1990.

Medford, J.I., Horgan, R., El Sawi, Z. and Klee, H J. The Plant Cell _ 403- 413, 1989.

Smigocki, A.C, Plant Molecular Biology 1 : 105-115, 1991.

Smigocki, A.C. and Owens, L.D. Proc. Natl. Acad. Sci. USA 85: 5131-5135, 1988.

Sommer, H. and Saedler, H. Mol. Gen. Genet. 202: 429-434, 1986. Wienand, U., Sommer, H., Schwarz, Z., Shepherd, N., Saedler, H., Kruezaler, F., Ragg, H., Fautz, E., Hahlbrock, K., Harrison, B., Peterson, P.A. Mol. Gen. GeneL ______; 195-201, 1982.

Yang, N.-S. and Russell, D. Proc. Natl. Acad. Sci. USA 82: 4144-4148, 1990.

Claims

CLAIMS:
1. A DNA construct comprising a first nucleotide sequence corresponding to a promoter capable of functioning in a plant and a second nucleotide sequence under control of said promoter and encoding a molecule capable of enhancing levels of a cytokinin in said plant.
2. The DNA construct according to claim 1 wherein the plant is a tuber plant.
3. The DNA construct according to claim 2 wherein the tuber plant is potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia.
4. The DNA construct according to claim 3 wherein the tuber plant is potato.
5. The DNA construct according to claim 1 wherein the first nucleotide sequence corresponds to the chs. promoter or to a functional equivalent thereof.
6. The DNA construct according to claim 1 wherein the second nucleotide sequence encodes ipt or a functional equivalent thereof.
7. A DNA construct comprising a first nucleotide sequence corresponding to the chs. promoter or a functional equivalent thereof and a second nucleotide sequence under the control of said promoter encoding ipt or a functional equivalent thereof, wherein said DNA construct is capable of expressing said ipt gene or functional equivalent thereof in a tuber plant.
8. The DNA construct according to claim 7 wherein said tuber plant is potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia.
9. The DNA construct according to claim 8 wherein the tuber plant is potato.
10. The DNA construct according to claim 1 or 5 wherein said construct is part of a larger DNA construct capable of being maintained in a prokaryotic and/or a eukaryotic cell.
11. The DNA construct according to claim 10 wherein the maintenance is either by integration into the cell genome or by autonomous replication.
12. The DNA construct according to claim 11 having the designation pCGP275, as hereinbefore described.
13. The DNA construct according to claim 11 wherein said promoter and/or said ipt gene or functional equivalents thereof is/ are under the further control of a regulatory sequence.
14. The DNA construct according to claim 13 wherein the further control permits developmental regulation.
15. A transgenic tuber plant carrying a DNA construct comprising a first nucleotide sequence corresponding to a promoter capable of functioning in said plant and a second nucleotide sequence under the control of said promoter and encoding a molecule capable of enhancing levels of a cytokinin in said plant.
16. The transgenic tuber plant according to claim 15 wherein said plant is a potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia.
17. The transgenic tuber plant according to claim 16 wherein said plant is potato.
18. The transgenic tuber plant according to claim 15 wherein the first nucleotide sequence corresponds to the chs. promoter or to a functional equivalent thereof.
19. The transgenic tuber plant according to claim 15 wherein the second nucleotide sequence encodes ipt or a functional equivalent thereof.
20. A transgenic tuber plant carrying a DNA construct comprising a first nucleotide sequence corresponding to the chs. promoter or a functional equivalent thereof and a second nucleotide sequence under the control of said promoter encoding ipt or a functional equivalent thereof, wherein said DNA construct is capable of expressing said ipt gene or functional equivalent thereof in said plant.
21. The transgenic tuber plant according to claim 20 wherein said plant is potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia.
22. The transgenic tuber plant according to claim 21 wherein said plant is potato.
23. The transgenic tuber plant according to claim 15 or 20 wherein said promoter and/or said ipt gene or functional equivalents thereof is/are under the further control of a regulatory sequence.
24. The transgenic tuber plant according to claim 23 wherein the further control permits developmental regulation.
25. The transgenic tuber plant according to claim 15 or 20 exhibiting one or more of the following properties: increased level of endogenous cytokinin(s); increased tuber number and/or weight; increased stem diameter; increased plant height; increased leaf size; delayed leaf senescence; increased photosynthetic capacity of leaves thereby increasing the ability of the plant to support an increased tuber load and increase tuber yield.
26. A transgenic tuber plant carrying a DNA construct corresponding in whole or in part to pCGP275, as hereinbefore described.
27. The transgenic tuber plant according to claim 26 wherein said plant is potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia.
28. The transgenic tuber plant according to claim 27 wherein the transgenic tuber plant is potato.
29. A method for producing a transgenic tuber plant comprising preparing transgenic cells from a tuber plant carrying a DNA construct comprising a first nucleotide sequence corresponding to the chs. promoter or functional equivalent thereof and a second nucleotide sequence under the control of said promoter encoding ipt or a functional equivalent thereof and then regenerating a tuber plant from said transgenic cells.
30. The method according to claim 29 wherein the tuber plant is potato, sugar beet, sweet potato, onion, garlic, artichoke or Dahlia.
31. The method according to claim 30 wherein the tuber plant is potato.
32. The method according to claim 29 wherein expression of said ipt or functional equivalent thereof is /are under the further control of a regulatory sequence.
33. The method according to claim 32 wherein the further control permits developmental regulation.
34. The method according to claim 29 wherein the transgenic cells are prepared by mobilisation with Agrobacterium, transformations, microprojectile bombardment, micro-injection or electroporation.
35. The method according to claim 29 wherein the DNA construct is pCGP275.
36. The method according to claim 29 wherein the transgenic tuber plant is capable of tuberisation under non-inducing conditions.
37. The method according to claim 29 wherein said plant exhibits one or more of the following properties: increased level of endogenous cytokinin(s); increased tuber number and/or weight; increased stem diameter; increased plant height; increased leaf size; delayed leaf senescence; increased photosynthetic capacity of leaves thereby increasing the ability of the plant to support an increased tuber load and increase tuber yield.
PCT/AU1992/000528 1991-10-03 1992-10-02 Transgenic plants WO1993007272A1 (en)

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US6329570B1 (en) 1989-07-19 2001-12-11 Calgene, Llc Cotton modification using ovary-tissue transcriptional factors
WO1995007993A1 (en) * 1993-09-13 1995-03-23 Zeneca Limited Regulation of senescence
EP0758841A1 (en) * 1995-02-28 1997-02-26 Calgene, Inc. Cotton modification using ovary-tissue transcriptional factors
EP0758841A4 (en) * 1995-02-28 1999-02-03 Calgene Inc Cotton modification using ovary-tissue transcriptional factors
EP0804066A1 (en) * 1995-03-29 1997-11-05 Wisconsin Alumni Research Foundation Transgenic plants with altered senescence characteristics
EP0804066A4 (en) * 1995-03-29 1999-01-27 Wisconsin Alumni Res Found Transgenic plants with altered senescence characteristics
US6359197B1 (en) 1995-03-29 2002-03-19 Wisconsin Alumni Research Foundation Transgenic plants with altered senescence characteristics
US6452068B1 (en) 1998-01-28 2002-09-17 The Rockefeller University Chemical inducible promoters used to obtain transgenic plants with a silent marker
US7525012B2 (en) 1998-01-28 2009-04-28 The Rockefeller University Chemical inducible promoters used to obtain transgenic plants with a silent marker
US7230157B1 (en) 1998-01-28 2007-06-12 The Rockefeller University Chemical inducible promoter used to obtain transgenic plants with a silent marker and organisms and cells and methods of using same for screening for mutations
US6114603A (en) * 1998-03-27 2000-09-05 John Innes Center Genetic engineering of sugarbeet plants
WO2000063401A1 (en) * 1999-04-16 2000-10-26 Pioneer Hi-Bred International, Inc. Regulated expression of genes in plant seeds
US6992237B1 (en) 1999-04-16 2006-01-31 Pioneer Hi-Bred International Inc. Regulated expression of genes in plant seeds
US7531723B2 (en) 1999-04-16 2009-05-12 Pioneer Hi-Bred International, Inc. Modulation of cytokinin activity in plants
WO2001034821A2 (en) * 1999-11-12 2001-05-17 The Rockefeller University Chemical inducible promoter used to obtain transgenic plants with a silent marker and organisms and cells and methods of using same for screening for mutations
WO2001034820A2 (en) * 1999-11-12 2001-05-17 The Rockefeller University Chemical inducible promoters used to obtain transgenic plants with a silent marker
WO2001034821A3 (en) * 1999-11-12 2002-05-30 Chua Nam Hai Chemical inducible promoter used to obtain transgenic plants with a silent marker and organisms and cells and methods of using same for screening for mutations
WO2001034820A3 (en) * 1999-11-12 2002-01-17 Univ Rockefeller Chemical inducible promoters used to obtain transgenic plants with a silent marker
WO2002042440A2 (en) * 2000-11-25 2002-05-30 University Of Leeds Regulation of plant growth by modifying the expression of a putative trna-isope ntenyl transferase
WO2002042440A3 (en) * 2000-11-25 2002-10-17 Peter Meyer Regulation of plant growth by modifying the expression of a putative trna-isope ntenyl transferase
EP1520028A2 (en) * 2002-06-19 2005-04-06 BASF Plant Science GmbH Compositions and methods for improving plant performance
EP1520028A4 (en) * 2002-06-19 2006-08-16 Basf Plant Science Gmbh Compositions and methods for improving plant performance

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