EP1603384A1 - Reduced stature plants - Google Patents
Reduced stature plantsInfo
- Publication number
- EP1603384A1 EP1603384A1 EP04721594A EP04721594A EP1603384A1 EP 1603384 A1 EP1603384 A1 EP 1603384A1 EP 04721594 A EP04721594 A EP 04721594A EP 04721594 A EP04721594 A EP 04721594A EP 1603384 A1 EP1603384 A1 EP 1603384A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- scion
- plant according
- rootstock
- gibberellin
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
- A01H6/7418—Malus domestica, i.e. apples
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8291—Hormone-influenced development
- C12N15/8297—Gibberellins; GA3
Definitions
- the present invention relates to the control of plant size through the gibberellin- biosynthetic pathway.
- large trees can produce large quantities of fruit or nuts, for example, such things as harvesting the crop and effectively targeting insecticidal sprays, can be difficult.
- smaller trees are not only easier to harvest from and to spray, but can often be more prolific, as well as produce fruit of higher quality, and are easier to manage than the traditional large trees.
- Growth retardants are generally critical for the control of vigour in fruit trees, but there is a general desire to avoid the use of chemicals, wherever possible. Further, the commonly used retardant, daminozide, was voluntarily withdrawn by the manufacturer in 1989 from use as a fruit tree retardant, owing to concerns over its possible toxicity. The use of paclobutrazol has also been restricted, owing to its extreme persistence in the soil. Other growth retardants, such as Regalis (proliexadione calcium), are currently in trial, but await approval.
- rootstocks which were either tailored to the local conditions, such as providing drought or pest resistance, or which were particularly vigorous.
- the need to use dwarfing rootstocks generally outweighs any possibility of selecting rootstocks for other reasons.
- WO 01/6677.7 discloses the over-expression of giberrellic acid (GA) 20-oxidase to increase growth in trees.
- GA giberrellic acid
- WO 03/006655 discloses that a mutation in the rice C-20 oxidase gene leads to semi-dwarfism.
- EP 1254958 also discloses that mutants of GA 3-oxidase genes lead to semi- dwarfism in rice.
- the present invention provides a dwarf plant, comprising a rootstock and a scion grafted thereon, wherein gibberellin levels in the scion are reduced.
- the plants of the invention are preferably trees, but generally be any woody plant where a scion is graftable, or grafted, onto a rootstock. While the plant created in this manner may consist of two varietals of a single species, or even one varietal wherein the scion exhibits reduced levels of gibberellin, but where the rootstock is not so restricted, it may also comprise a rootstock generally from within the same or a related genus, the only requirement being that the scion be capable of "taking" on the rootstock. Thus, provided that the scion can grow when grafted onto the rootstock, it is immaterial from which species either comes.
- the rootstock does not restore full growth potential to an inhibited scion, given that it is the rootstock that is used to inhibit the growth of the scion, rather than selecting a dwarf tree from which to take a scion.
- the invention permits the utilisation of any rootstock for the cultivation, for example, of commercially viable, perennial fruit or nut crops as scions.
- a rootstock for purposes other than to dwarf the resulting tree.
- This assists in reducing the requirement for insecticides, and allows the rootstock to be selected for suitability to the local terrain, or any other desirable trait, which may be something as simple as vigour, but may alternatively, or in addition, include such properties as drought resistance, pest and disease resistance or toxin resistance, such as salinity resistance, for example, thereby reducing artificial inputs and pollution.
- invigorating rootstocks such as M25, could be used which provide resistance to soil-borne pests and diseases and better anchorage than existing commercial rootstocks.
- Inhibition of the gibberellin biosynthetic pathway can take place at any suitable point, but it is generally preferred to restrict the interference to the latter stages of the biosynthetic pathway, in order to obtain moderate, rather than extreme, reductions in growth.
- the GA pathway is complex, and it is preferred to limit interference to the 20-oxidase, 2-oxidase and 3-oxidase, but it is particularly preferred to inhibit the 20-oxidase.
- GA 2-oxidase inactivates GA's, so that a scion in which GA 2-oxidase is over-expressed will exhibit reduced levels of GA, in accordance with the present invention.
- inhibition of the production of GA is meant that at least one form of GA necessary for growth of the scion is produced in a lesser quantity than in the corresponding wild type. This may take the form of complete blocking of synthesis of that GA, or a reduced production, such as between 10 and 50%, for example. This reduction may also be achieved by suitable expression, or over-expression, of a degradative enzyme in the scion.
- the enzyme may be inhibited by any suitable means, hi plants, this is generally restricted to transformation, especially using Agrobacterium tumefaciens, and methods for using this organism are well documented. Transformation of cropping trees, such as fruit or nut trees, is readily achieved by taking fresh samples of healthy, growing leaves, incubating with a suitably transformed preparation of A. tumefaciens, then cultivating the transformed tissue. Incorporating a suitable marker, such as kanamycin resistance, enables positive transformants to be selected.
- the transformation may take the form of incorporating a targeted mutation or a gene encoding an inhibitor for the selected enzyme, for example.
- the antisense DNA is suitable preceded by an appropriate promoter and may be accompanied by a termination sequence.
- the size of the antisense sequence is not important to the present invention, provided that the sequence is capable of binding the target rnRNA, in vivo, in a manner sufficient to trigger the dsRNA digestion response.
- the amount of nucleotides required to achieve a dsRNA response varies according to the technique used, and will be apparent to those skilled in the art.
- a typical length for an interfering RNA (RNAi) may be between about 15 and several hundred nucleotides, but short lengths of about 25 are known to be effective as RNAi's in effecting suppression.
- a recognition motif in a ribozyme may only require about 10 - 15 nucleotides, for example.
- Such techniques are referred to herein as gene silencing, and may also be achieved by transforming the scion tissue with a gene encoding a suitable tr ⁇ fls-acting ribozyme targeted against the rnRNA of the GA 20-oxidase gene, for example.
- the endogenous gene is not being duplicated but, rather, an internal fragment of Ms20oxl is being expressed which appears to efficiently trigger PTGS (c.f. Han Y.H. and Grierson D. (2002) Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato. Plant J., 29, 509-519).
- PTGS c.f. Han Y.H. and Grierson D. (2002) Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato. Plant J., 29, 509-519).
- the present invention also contemplates silencing by co-expression, using a partial or full length sequence.
- Other forms of inhibition may be used, as appropriate, and may include the expression of suitable antibodies, for example, such as single chain antibodies directed against a target GA to sequester, and thereby inactivate, that GA.
- isozymes located throughout even an individual plant there are various isozymes located throughout even an individual plant, and it is preferred to target those isozymes primarily found in shoot tips. Isozymes located in anthers but not in shoot tips, for example, are unlikely to have any significant effect on the overall size of the cultivar. It will be appreciated that individual enzymes or families of enzymes may be targeted by a single RNAi, for example, by suitable selection of a consensus sequence.
- the expression product effecting the gene silencing is associated with a tissue specific promoter, such as an SSU promoter.
- dwarfing is achieved by suppression of GA 20-oxidase gene expression.
- lower endogenous levels of bioactive GA's were produced in the growing areas of the plant.
- GA 20-oxidases catalyse three successive oxidative reactions in the conversion of GA 12 to GA 9 via GA 15 and GA 24 , and of GA 53 to GA 2 o via GA 44 , and GA 19 .
- GA 1 and GA 25 are formed as inactive side-products (reaction scheme, infra).
- GA 9 and GA 20 are then converted by GA 3-oxidases (3 ⁇ -hydroxylation) to bioactive GA.
- MdGA20oxl (corresponding to 20oxl) is expressed in developing embryos, shoot tips, and young leaves, with very low expression in ovary tissue just after pollination. In contrast, MdGA20ox2 is expressed in stamens and unpollinated late balloon stage ovaries. Therefore, it is unlikely that silencing MdGA20ox2 would cause dwarfism, as expression was not detected in shoot tips or young leaves.
- the present invention also provides a process for obtaining a dwarf plant, wherein the plant is as defined and comprises a rootstock and a scion grafted thereto, the term "dwarf indicating that the plant is of reduced stature by comparison with non-dwarfed plants of the same cultivar from which the scion is derived, comprising transforming the cultivar to exhibit reduced levels of gibberellin.
- the process will generally comprise transforming cultivar tissue, typically meristem, or stem, cells and culturing the tissue to obtain a plant, or plants.
- the invention extends to such transformed tissue, whether it be the cells, callus or a plant capable of providing a scion.
- Methods for reducing the levels of GA are as exemplified above and elsewhere herein, and preferably comprise inhibiting or blocking expression of GA 20-oxidase, such as by PTGS, especially by RNAi.
- the invention further provides plants produced by this process, as well as crops obtained from plants of the invention, and stands of such plants.
- the following reaction sequence shows the late stages of the GA biosynthetic pathway involved in the production of bioactive GA 1)4j3;7 .
- Both the non 13-hydroxylation pathway and the 13-hydroxylation pathway can occur in any particular species, but one or the other may predominate.
- GA 1 and GA 25 occur as minor by-products and have no known biological activity.
- the GA 20-oxidase, 3-oxidase and 2-oxidase genes exist as multiple gene families in the various species analysed so far. These dioxygenase enzymes utilise 2-oxoglutarate as a co-substrate, require Fe 2+ , and are stimulated by ascorbate.
- Figure 1 shows the pDH51 vector as used to fuse the 20oxl fragment with the cauliflower mosaic virus 35S promoter
- Figure 2 shows the pSCV1.2 binary vector.
- the 35S-20o l -expression cassette was blunt ligated into the Smal site;
- Figure 3 shows pSCV1.2-20oxl construct T-DNA showing the 20oxl anti-sense (-») and sense ( ⁇ — ) expression cassette inserted into the Smal site by blunt ligation;
- FIG. 4 shows dwarfism in transgenic plants where silencing of endogenous GA 20-oxidase gene expression had occurred
- Figure 5 show that the dwarf scions remained dwarfed after a growth season, even on invigorating M25 rootstock.
- Figure 6 compares the relative effects on mean overall height and mean internode length between rootstocks, expressed as a percentage of the tallest non-transgenic control means.
- Agrobacterium tumefaciens strain EHAIOI (containing the appropriate pSCV construct) was streaked from a frozen stock onto LB agar plates containing ZSmg.i "1 neomycin, 50mg.r ! chloramphenicol, 25mg.l _1 gentamycin, and incubated at 28°C.
- the bacterial solution was aliquoted in 10ml volumes into sterile 15ml Falcon tubes (Sarstedt) and incubated at 20°C with gentle shaking for 5 hours. During this time, leaf disks approximately 7mm in diameter were cut with a sterile number 2 size cork borer from young healthy expanding leaves and placed abaxial side down onto BNZ 5-1-1 regeneration medium (5mg.l -1 BAP [Sigma- Aldrich], lmg.i "1 NAvA [Sigma- Aldrich], lmg.l “1 TDZ [Sigma-Aldrich], 4.4g.l ⁇ 1 MS salts [Sigma-Aldrich], 40g.l "1 sorbitol [Sigma- Aldrich], pH5.2, and 2.5g.l "1 Phytagel [Sigma-Aldrich]), contained in Petri dishes for holding until required.
- the linearised vector was blunted as before.
- the linearised vector was de-phosphorylated with calf intestinal alkaline phosphatase (CLAP, Promega) for 30 minutes at 37°C according to the manufacturer's instructions. After de-phosphorylation of the free ends to prevent self re-ligation, the vector was phenol: chloroform extracted and re-suspended in water. To determine an approximate concentration, a small aliquot was run out on an agarose (Seakem) gel.
- Plasmid DNA was purified using a Plasmid Midiprep kit (Qiagen). Prepared constructs were transferred to the Agrobacteri ⁇ im tumefaciens strain ⁇ HA101 by electroporation in an ⁇ . coli Pulser Transformation Apparatus (Biorad) using 100 to 300ng of plasmid DNA according to the manufacturer's instructions. Competent Agrobacterium cells were prepared using the method described by McCormac et al. (1998).
- the levels of native bio-active GA were reduced in Greensleeves apple (dessert variety) by co-suppression and anti-sense strategies with a 314bp long DNA fragment (SEQ ID NO. 1, supra) of an endogenous GA 20-oxidase gene sequence (referred to as '20oxl '). This caused dwarfism in some of the resulting transgenic plants where silencing of endogenous GA 20-oxidase gene expression had occurred ( Figure 4).
- the GA 20- oxidase gene used was MdGA20oxl (corresponding to 20oxl) and is expressed in developing embryos, shoot tips, and young leaves, with very low expression in ovary tissue just after pollination.
- transgenic dwarf scions of two lines were grafted onto MM 106 and M25 stock. After one growth season, it was apparent that the dwarf scions did indeed remain dwarfed, particularly on invigorating M25 rootstock ( Figures 5 and 6).
- Figure 6 compares the relative effects on mean overall height and mean internode length between rootstocks, expressed as a percentage of the tallest non-transgenic control means.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Botany (AREA)
- Physiology (AREA)
- Medicinal Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Endocrinology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0306154.6A GB0306154D0 (en) | 2003-03-18 | 2003-03-18 | Reduced stature trees |
GB0306154 | 2003-03-18 | ||
PCT/GB2004/001210 WO2004082369A1 (en) | 2003-03-18 | 2004-03-18 | Reduced stature plants |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1603384A1 true EP1603384A1 (en) | 2005-12-14 |
Family
ID=9954979
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP04721594A Withdrawn EP1603384A1 (en) | 2003-03-18 | 2004-03-18 | Reduced stature plants |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1603384A1 (en) |
CA (1) | CA2519397A1 (en) |
GB (1) | GB0306154D0 (en) |
WO (1) | WO2004082369A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2727564C (en) * | 2008-06-13 | 2017-09-12 | Performance Plants, Inc. | Methods and means of increasing the water use efficiency of plants |
CN104160907B (en) * | 2014-08-06 | 2016-03-09 | 福建省农业科学院果树研究所 | A kind of method improving shaddock class graft seedling growth efficiency |
CN108728420B (en) * | 2017-04-24 | 2021-10-29 | 中国科学院分子植物科学卓越创新中心 | Gene for regulating crop dwarfing and yield and application thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
USPP4926P (en) * | 1981-02-09 | 1982-11-09 | Mccormick's Fruit Tree Co. Inc. | Apple tree |
USPP5508P (en) * | 1983-04-21 | 1985-07-09 | James L. Glazier | Apple tree |
GB9311147D0 (en) * | 1993-05-28 | 1993-07-14 | Long Ashton Research Station | Regulation of plant growth |
WO2000009722A2 (en) * | 1998-08-10 | 2000-02-24 | Monsanto Company | Methods for controlling gibberellin levels |
-
2003
- 2003-03-18 GB GBGB0306154.6A patent/GB0306154D0/en not_active Ceased
-
2004
- 2004-03-18 EP EP04721594A patent/EP1603384A1/en not_active Withdrawn
- 2004-03-18 WO PCT/GB2004/001210 patent/WO2004082369A1/en not_active Application Discontinuation
- 2004-03-18 CA CA002519397A patent/CA2519397A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO2004082369A1 * |
Also Published As
Publication number | Publication date |
---|---|
GB0306154D0 (en) | 2003-04-23 |
CA2519397A1 (en) | 2004-09-30 |
WO2004082369A1 (en) | 2004-09-30 |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: JAMES, DAVID, JOHN Inventor name: BULLEY, SEAN Inventor name: HEDDEN, PETER Inventor name: PHILLIPS, ANDREW, LEONARD |
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