CA2335522A1 - Water stress or salt stress tolerant transgenic cereal plants - Google Patents
Water stress or salt stress tolerant transgenic cereal plants Download PDFInfo
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- CA2335522A1 CA2335522A1 CA002335522A CA2335522A CA2335522A1 CA 2335522 A1 CA2335522 A1 CA 2335522A1 CA 002335522 A CA002335522 A CA 002335522A CA 2335522 A CA2335522 A CA 2335522A CA 2335522 A1 CA2335522 A1 CA 2335522A1
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- 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.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically 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
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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- 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
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- 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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
Abstract
The present invention relates to a transgenic cereal plant and to a cereal plant cell or protoplast transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant. Another aspect of the present invention is a method of conferring water stress or salt stress tolerance to a cereal plant including transformi ng a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis. The present invention also relates to a method of increasing tolerance of a cereal plant to water stress or salt stress conditions, the method including increasing levels of an enzyme for proline biosynthesis in the cereal plant. Yet another aspect of the present inventio n is a transgenic cereal plant transformed with a plasmid that confers water stress or salt stress tolerance to the cereal plant.
Description
WATER STRESS OR SALT STRESS
TOLERANT TRANSGENIC CEREAL PLANTS
This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/090.61. filed June 2~. 1998.
FIELD OF THE INVENTION
The present invention relates to transgenic cereal plants which are transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant and a method of increasin~.~ or conferring=
water stress or salt stress tolerance to a cereal plant.
BACKGROUND OF THE INVENTION
Environmental stresses. such as drought. increased salinity of soil. and extreme temperature. are major factors in limiting plant growth and productivity. The worldw°ide loss in yield of three major cereal crops. rice. maize (corn). and wheat due to water stress (drought) has been estimated to be over ten billion dollars annually. Salt stress and drou~=ht stress are the two most important abiotic stresses. Of the 4.870 million hectares of agricultural land in the world. 930 million ( 19% of total) are salt-affected areas (FAO Quarterly Bulletin of Statistics. Vol. 9'/a (1996)). Moderate levels ofsalt content in the soil (such as ~0 mM) cause a substantial decrease in the yield of crops.
High levels of salt in the soil (higher than 100 or I ~0 mM) are not at all suitable for planting most cereal crops. Approximately 5.2% of the agricultural lands are under drought stress (FAO Quarterly Bulletin of Statistics. Vol. 9 e/4 ( 1996)). and the loss of crop yield is also very significant.
In practical terms. rice is the most important crop because a high percentage of the worlds population depends on it for their staple food.
To~~ether with wheat and corn. these three cereal crops constitute the major source of food and calories to feed the people. With an increase in population and a decrease in arable land. there is a real possibility of a food shorta~~e by the year 2030. Therefore, it is essential to frilly utilize plant biotechnolo~~v to improve plants and produce more food.
Breeding of stress-tolerant crop cultivars represents a promisin~~ strate~~y t~
tackle these problems (Epstein et al.. "Saline Culture of Crops: A Genetic Approach."
WO 991bb785 PCT/US99/14336 Science. 210:399-404 ( 1980)). However. conventional breeding is a slow process for generatin~~ crop varieties with improved tolerance to stress conditions.
Limited ~~ermplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species are additional problems encountered in conventional breeding.
s Recent progress in plant ~Tenetic transformation and availability of potentially useful genes characterized from different sources make it possible to generate stress-tolerant crops using transgenic approaches (Tarczynski et al.. "Stress Protection of Transgenic Tobacco by Production of the Osmolyte Mannitol." Science. 29:508-~ 10 (1993):
Pilon-Smits et al.. "Improved Performance of Trans~~enic Fructan-Accumulating Tobacco Under Drought Stress.' Phvsioi. Plant. 107:1?-130 (1990). Transformation of cereal plants with a~~ronomicallv useful genes that increase tolerance to abiotic stress is one important way to minimize yield loss. For example. it would be highly desirable to produce trans~?enic rice plants that can give reasonable yield when grown in marginal or waste lands that contain relatively high levels of' salt. such as 100-1 ~0 mM.
in the soil.
Characterization and cloning of plant genes that confer stress tolerance remains a challenge. Genetic studies revealed that tolerance to drought and salinity in some crop varieties is principally due to additive gene effects (Akbar et al..
"Breeding For Soil Stress.~~ In Pro~~ress in Rainfed Lowland Rice. International Rice Research Institute.
manila. Philippines, pp. 263-272 (1986): Akbar et al.. "Genetics of Salt Tolerance in Rice.' In Rice Genetics. International Rice Research Institute. Manila.
Philippines. pp.
399-409 ( 1986)). However. the underlying molecular mechanism for the tolerance has never been revealed. Physiological and biochemical responses to high levels of ionic or nonionic solutes and decreased water potential have been studied in a variety of plants.
Based on accumulated experimental observations and theoretical consideration.
one 2~ su~~gested mechanism that may underlie the adaptation or tolerance of plants to osmotic stresses is the accumulation of~compatible. low molecular weight osmolytes such as sugar alcohols. special amino acids. and glycine hetaine (Greenway et al..
"Mechanisms of Salt Tolerance in Nonhalophytes." A1111t1. Rev. Plant Pl3vsiol.. 31: 149-190 ( 1980): Yancey et al.. "Livin~~ With Water Stress: Evolution of Osmolyte System." Science. 217:
1214-12~~
( 1982)). In particular, proline level is know to increase in a number of plants and bacteria under drought or salt stress. Recently. a trans~~enic study has demonstrated that accumulation of the su''ar alcohol mannitol in trans~~enie tobacco conferred protection against salt stress fTarczvnski et al.. "Stress Protection of Trans~wnic Tobacco by WO 99/66785 PCT/US99/~4336 - J
Production of the Osmolye Mannitol." Science. ?~9:~0$-~ 10 ( 1993)). Two recent studies using a trans~~enic approach have demonstrated that mmabolic en~~ineerin~~ of the ~~lvcine betaine biosynthesis pathway is not only possible but also may eventuallw lead to production of stress-tolerant plants (Holmstrom et al.. "Production of the E.schcmichicr coli Betaine-Aldehyde Dehydrogenase, An Enzyme Required for the Synthesis of the Osmoprotectant Glycine Betaine, in Transgenic Plants." Plant .1.. 6:749-7~$ ( 1994):
Rathinasabapathi et al., "Metabolic Engineerin~~ of Glvcine Betaine Synthesis:
Pfant Betaine Aldehyde Dehydro;~enases Lacking Typical Transit Peptides are Tar~~eted to Tohacco Chloroplasts Where they Confer Betaine Aldehvde Resistance." Planta.
193:1 ss-162 ( 1994)).
In addition to metabolic changes and accumulation of low- molecular wei~Yht compounds, a large set of genes is transcriptionallv activated which leads to accumulation of new proteins in vegetative tissue of plains under osmotic stress conditions. including the late embryogenesis abundant (L1:A? family.
dehvdrines. and 1 ~ COR47 (Skriver et al., "Gene Expression in Response to Ahscisic Acid and Osmotic Stress." Plant Cell. 2:503-512 ( 1990), Chandler et al.. "Gene L:xpression Re~~ulated by Abscisic Acid and its Relation to Stress Tolerancc.~~ AI71111. Rev: Plant Phvsiol. Plant Mol.
Biol.. :~~:1 13-141 ( 1994)). The expression levels of a Illllllhel' of ~~enes hale been reported to be correlated with desiccation. salt. or cold tolerance of ditterent plant varieties of the same species. It is generally assll117ed that sh'tSS-111dlICed prOle111S n 7l~~ht play a role in tolerance. but the functions of many stress-responsive ~.~enes are unknown.
Elucidatin~~ the fiu~ction of these stress-responsive ~~enes and enzymes involved in the biosynthesis of stress-induced osmolytes will not only advance the understanding of plant adaptation and tolerance to environmental stresses. but also may 2~ provide important information for designing new strate~_ies for crop improvement (Chandler et al.. "Gene Expression Regulated by Abscisic Acid and its Relation to Stress Tolerance." Annu. Rev. Plant Phvsiol. Plant Mol. Biol., =t~:1 13-141 ( 1994)).
Several genes that encode key enzymes involved in the biosynthesis of specific osmolvtes (such as mannitol, proline. or glycine betain e) have been introduced into tobacco cells. The regenerated transgenic tobacco plants showed partial tolerance to drought and to salt stress (Tarezynski et al.. "Stress Protection of~Trans~~elzic Tobacco by Production of Osmotic Mannitol," Science. ?s9:~0$-~ 1 U ( 1993 ): Kishor et al..
..~'.~1't\pl'eSSloll Of ~1~-tyrroline-3-carboxvlate Svnthetaw Increases Praline Production WO 99/66785 PCT/US99/1d336 _:
and Confers Osmotolerance in Transgenic Plants." Plant Physiol.. 108:1387-1394 ( 199 1:
Lilies et al.. "Enhanced NaC1 stress tolerance in trans~~enic tobacco expressing bacterial choline dehydrogenase."' Biotech., 14:177-180 ( 1966)). I-Iowever. only transgenic tobacco was used for these studies. and similar work on producing stress-tolerant cereal S crop plants has not been carried out. It is not clear whether these genes in transgenic cereal plants will enhance salt or drought tolerance since the physiology of dicot plants.
such as tobacco. is very different from monocots. such as cereal plants. Thus.
only experimentation using cereal crop plants can provide the answer.
The present invention is directed to overcoming the above-noted deficiencies in the prior art.
SUMMARY OF THE INVENTION
The present invention relates to a trans~~enic cereal plant transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or 1 ~ salt stress tolerance to the plant.
The present invention also relates to a cereal plant cell or protoplast transformed with a nucleic acid encoding an enzyme For proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant regenerated from said cereal plant cell or protoplast.
Another aspect of the present inyen lion is a method of conferrin~~. water stress or salt stress tolerance to a cereal plant including.: transforming, a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis.
The present invention also relates to a method of increasin'.! tolerance of a cereal plant to water stress or salt stress conditions. the method includin~~
increasing 2~ levels of an enzyme for proline biosynthesis in the cereal plant.
The present invention allows the production of cereal plants with increased tolerance to water stress (drought) and salt stress. Thus, an enzy°tne for proline biosynthesis can be used as a molecular tool for ~~enetic crop improvement by conterrin~~
stress tolerance.
WO 99/66785 PCT/US99/t4336 DETAILED DESCR11'TION OF THE INVENTION
The present invention relates to a trans~~enic cereal plant transformed with a nucleic acid encodiny~ an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant.
Suitable nucleic acids encoding an enzyme for proline biosynthesis include the P.SC ~S ~~ene of mothbean and a feedback-inhibition insensitive mutant.
P~C,S-129A. of the PLC'S gene. The sequence of the PLC'S gene can be found in Kishor et al..
''Overexpression of 0~-pyrroline-~-carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Trans~,enic Plants." Plant Phvsiol., 108:1387-1394 (1990.
which is hereby incorporated by reference. and the sequence of the PSC'S-129A
mutant gene can be found in Zhan~~ et al.. "Removal of Feedback Inhibition of P.SC',S
in Plants."
J. Bioi. Chem.. 270:20491-20496 (1990. which is hereby incorporated by reference.
Cereal which can be transformed in accordance with the subject invention are members of the family Gr~crinir7ecre (also known as Pocrc~eoc~). and include rice (~~enus Or;yvu). wheat. maize (corn). barley. oat. sorghum. and millet. Preferably.
the cereal is rice. w=heat. or corn. and most preferably the cereal is rice. Many species of cereals can be transformed. and within each species the numerous subspecies and varieties can be transformed. For example. within the rice species is subspecies lndica rice (W
y=a .smiou ssp. Indica). which includes the varieties IR36. IR64. IR72. Pokkali. Nona Bokra.
KDML10~. Suponburi 60. Suponburi 90. Basmati 38~. and Pusa Basmati 1. Another rice subspecies is Japonica. which includes Nipponbere. Kenfeng and Tainun~~ 67.
Examples of suitable maize varieties include A188. B73. VA?2. L6. L9. K1. 509. X922, 482. HNP.
and 1GES. Examples of suitable wheat varieties include Payon. Anza. Chris.
Coker 983.
FLA301. FLA302. Fremont and Hunter.
2~ Having identified the cereal plant of interest. plant cells suitable for transformation include immature embryos. calli. suspension cells. and protoplasts. It is particularly preferred to use suspension cells and immature embryos.
These cereal plant cells are transformed with a nucleic acid. which could be RNA or DNA and which is preferably cDNA. encodin~~ an enzyme for proline biosynthesis. The nucleic acid can be biolo~~icallv isolated or synthetic. In the followin~~
Examples, a key enzyme for proline biosynthesis. ~~-pyrroline-~-carboxylate synihase (PSCS). is encoded by the I'~C'S'~ene of mothbean. Howeewr. other genes encoding an etlzyme for proiine biosynthesis. includin~~ a feedback-Illhlbltl(lil lnSeI1S1t1Ve n7uta17t ot: the P~C:S ~~ene. P.SC'.S-129A. can also be utilized.
Transformation of plant cells can be accomplished by using a plasmid.
The plasmid is used to introduce the nucleic acid encoding an enzyme for proline ~ biosynthesis into the plant cell. Accordingly. a plasmid preferably includes DNA
encodin~~ an enzyme for proline biosynthesis inserted into a unique restriction endonuclease cleavage site. Heterologous DNA, as used herein. refers to DNA
not normally present in the particular host cell transformed by the plasmid. DNA
is inserted into the vector using standard cloning procedures readily known in the art.
This generally involves the use of restriction enzymes and DNA li~~ases. as described by Sambrook et al.. Molecular Clonin~~: A Laboratory Manual. 2d edition. Cold Sprin~~ Harbor Laboratory Press. Cold Sprin~~ Harbor. New York ( I 989). which is hereby incorporated by reference. The resulting plasmid which includes nucleic acid encoding an enzyme for proline biosynthesis can then be used to transform a host cell. such as an A~nohucter°ium and/or a plant cell. (See generally. Plant Molecular Biolo~~v Manual. 2nd Edition. Gelvin et al., Eds.. Kluwer Academic Press. Dordrecht. Netherlands ( 1994). which is hereby incorporated by reference).
For plant transformation. the plasmid preferably also includes a selectable marker for plant transformation. Commonly used plant selectable markers include the hy';romvcin phosphotransferase (hp!) gene. the phosphinothricin acetyl transferase ~~ene (hur). the ~-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS). neomycin ~'-O-phosphotransferase gene (np/ II). or acetolactate svnthase ~~ene (ALS).
Information on these selectable markers can be found in Bowers. "Markers for Plant Gene Transfer" in Trans:.~enic Plants. Kung et al.. Eds.. Vol. I . pp. 89-123. Academic Press, NY ( 1993).
2~ which is hereby incorporated by reference.
The plasmid preferably also includes suitable promoters for expression of the nucleic acid encoding an enzyme for proline biosynthesis and for expression of the marker ~~ene. The cauliflower mosaic virus 3~S promoter is commonly used for plant transformation. as well as the rice actin 1 ~~ene promoter. In plasmid p,1S102 used in the followin~~ Examples. the nucleic acid encodin~~ an enzyme for proline biosynthesis is under the control of the constitutive rice actin 1 ;gene promoter and the marker ~_ene (hur) is under control of the cauliflower mosaic virus 3sS promotr-r. Other promoters useful for plant transformation with an enzyme for proline hiosvnthesis include those from the genes encodin~~ ubiquitin and proteinase inhibitor II (PINII ). as well as stress-induced promoters (such as the HVA1 gene promoter of barley. an abscisic acid (ABA)-induciblc promoter. such as ABRCI from barley linked to a rice Act-100 miniri~ai promoter. and a HVA22 intron).
The plasmid designated p.IS 112 has been deposited pursuant to. and in satisfaction of. the requirements of the Budapest Treaty on the International Reco~~nition of the Deposit of Microorganisms for the Purposes of Patent Procedure. with the American Type Culture Collection (ATCC). i 0801 University Boulevard, Manassas. VA
201 10-2209. under ATC C Accession No. on .tune 17. 1999.
For plant transformation. the plasmid also preferably includes a nucleic acid molecule encodin~~ a 3' terminator such as that from the 3~ 11011-COdln~.! re~~ion of genes encoding a proteinase inhibitor. actin. or nopaline svnthase (nos).
Other suitable plasmids for use in the subject invention can be constructed.
For example. ~~enes encoding an enzyme for proline biosynthesis other than the PSC:f 1 S gene of mothbean could be ligated into plasmid ,1S 109 after use of restriction enzymes to remove the P~C :f gene. Other promoters could replace the actin 1 ~~elle promoter present in plasmid .15102. Alternatively. other plasmids in general containing genes encodin~~ an enzyme for proline bioswthesis under the control of a suitable promoter. with suitable selectable markers. can be readily constructed usin~~ techniques well known in the art.
Havin~~ identified the plasmid. one technique of transforming cereal plant cells with a ~~ene which encodes for an enzyme for proline biosynthesis is by contactin«
the plant cell with an inoculum of an A~t,>rvhucnc~rium bacteria transformed with the plasmict comprisin~~ the ~~ene that encodes far the enzyme for proline biosynthesis.
Generally. this procedure involves inoculatin~~ the plant cells with a suspension of the 2~ transformed bacteria and incubating the cells for ~8 to 72 hours on re~~eneration medium without antibiotics at 25-28°C.
Bacteria from the genus A~>mohucncrirun can be utilized to transform plant cells. Suitable species include A~mohcrrterium ~tmrc~Jecicn.s and A~oohcrclenim~i rlri=o~c~nc.s. A~rohur~crirm7lumcyac~icns (e.~~., strains LBA4404 or EHAIOS) is particularly useful due to its well-known ability to transform plants.
In inoculating the cells of cereal plants with .-t ~>ruhocvc~rirrm accordin~~
to the subject invention. the bacteria must be transtormcd with a vector which includes a ~~ene encoding for an enzyme for praline biosynthesis.
WO 99/66785 PCT/US99/~4336 _g-Plasmids. suitable for incorporation in ;a,slrnhcrwerirrnr. which include a gene encoding for an enzyme for proline biosynthesis. contain an origin of replication for replication in the bacterium E5L'l1L'1'IC'ylr(I c'Ull. all Orl~.jlll Of replication for replication in the bacterium fl~,~rohuc~enirrm lumefacic~ns. T-DNA ri~~ht border sequences for transfer of genes to plants. and marker genes for selection of transformed plant cells.
Particularly preferred is the vector pBII?1 which contains a low-copy RK2 origin of replication. the neomycin phosphotransferase (nptll) marker gene with a nopaline synthase (NOS) promoter and a NOS 3' polyadenylation signal. T-DNA plasmid vector pBI121 is available from Clonetech Laboratories. Ine.. 4030 Fabian Wav. Palo Alto.
California 94303. A gene encoding for an enzyme for proline biosynthesis is inserted into the vector to replace the beta-;~lucuronidase (GUS) gene.
Typically. A~~robucleritrnT spp. are transformed with a plasmid by direct uptake of plasmid DNA after chemical and heat treatment. as described by Holsters et al.
"Transfection and Transformation of ~~~rvhae~e~rimn mme~f~rcic.~ns.'" Mol.
Gen. Genet..
1 ~ 163:181-187 ( 1978), which is hereby incorporated by reference: by direct uptake of plasmid DNA after electroporation. as described by Shen et al.. "Efficient Transformation ofA~nrnhucJc~ritrnt spp. by High Voltage Electroporation."' Nucleic Acids Research.
17:838 ( 1989). which is hereby incorporated by reference: by triparental conju4~ational transfer of plasmids from Esc~herichiu coli to .4~ruhcrctc~ritu~~ mediated by a Tra+ help strain as described by Ditta et al.. "Broad I-lost Range DNA Clonin<~ System for Gram-negative Bacteria: Construction of a Gene Bank of Rhcohilmr molilo~i."' Pror.
Natl. Acad.
Sci. USA. 77:7347-73~ 1 ( 1981 ). which is hereby incorporated by reference;
or by direct eonjugational transfer from E.scherichiu cvli to A~I'Ohuclc'!'11u71 as described by Simon et al.. "A Broad Host Range Mobilization System for i» rims Genetic En;~ineering:
2~ Transposon Muta~~enesis in Gram-negative Bacteria." Biotechpolo~y. 1:784-791 ( 198?).
which is hereby incorporated by reference.
Another method for introduction of a plasmid containing nucleic acid encodin~~ an enzyme for proline biosynthesis into a plant cell is by transformation of the plant cell nucleus. such as by particle bombardment. As used throughout this application.
particle bombardment (also known as biolistic transformation) of the host cell can be accomplished in one of several ways. The first involves propelling inert or biolo~~ically active particles at cells. This technique is disclosed in U.S. Patent Nos.
4.9~1~.Oi0.
6.036.006. and 5.100.79?. all to Salltot'd et al.. which are hereby incorporated by reference. Generally. this procedure involves propellin~~ inert or biologicaily active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the plasmid can be introduced into the cell by coating the particles with the plasmid containing the heterologous DNA. Alternatively, the target cell can be surrounded by the plasmid so that the plasmid is carried into the cell by the wake of the particle.
Biologically active particles (e.g.. dried bacterial cells containing the plasmid and heterologous DNA) can also be propelled into plant cells.
A further method for introduction of the plasmid into a plant cell is by transformation of plant cell protoplasts (stable or transient). Plant protoplasts are enclosed only by a plasma membrane and will therefore take up macromolecules like heterolo~~ous DNA. These engineered protoplasts can be capable of regenerating whole plants. Suitable methods for introducing heterolo~,ous DNA into plant cell protoplasts include electroporation and polyethylene glycol (PEG) transformation. As used throughout this application. electroporation is a transformation method in which.
generally. a high concentration of plasmid DNA (containing heterologous DNA) is added to a suspension of host cell protoplasts and the mixture shocked with an electrical field of 200 to 600 V/cm. Following electroporation. transformed cells are identified by growth on appropriate medium containing a selective agent.
As used throughout this application. transformation encompasses stable transformation in which the plasmid is integrated into the plant chromosomes.
In the Examples which follow. rice has been transformed usin~~ biolistic transformation. Other methods of transformation have also been used to successfully transform rice plants. including the protoplast method (for a review, see Cao et al..
2~ "Regeneration of Herbicide Resistant Transgenic Rice Plants Following Microprojectile Mediated Transformation of Suspension Culture Cells." Plant Cell Rep.. 11:586-( 1992). which is hereby incorporated by reference). and the ,~lgrv6crcterium method (Hiei et al.. "Efficient Transformation of Rice (Ori_cr .suliru L.) Mediated by Agrobcrclerirrnr and Sequence Analysis of the Boundaries of the T-DNA.' The Plant .iournaf.
6:271-?82 ( 1994). which is hereby incorporated by reference). Biolistic transformation has also been used to successfully transform maize (for a review. see Mackey et al..
"Transgenic Maize," /r~ Transsenic Plants. Kung et al.. Eds.. vol. ?. pp. ? 1-33 ( 1993 ).
which is hereby incorporated by reference) and wheat (see U.S. Patent No. ~.~0~.76~ to Vasil et al.. which is herehv° incorporated by reference).
Once a cereal plant cell or protoplast is transformed in accordance with the present invention. it is re;~enerated to form a transgenic cereal plant.
Generally.
regeneration is accomplished by culturing transformed cells or protoplasts on medium containing the appropriate gro~-th regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of ~l~rrobcrc~enium or other comaminants and to select for the development of transformed cells or protoplasts. Following shoot initiation. shoots are allowed to develop in tissue culture and are screened for marker ;gene activity.
In suitable transformation methods. the cereal plant cell to be transformed can be in ri~ro or in vioo. i.e. the cereal plant cell can be located in a cereal plant.
The invention also provides seed produced by the transgenic cereal plant.
The invention is also directed to seed. which upon germination. produces the transgenic cereal plant.
Also encompassed by the present invention are transgenic cereal plants transformed with fragments of the nucleic acids encoding an enzyme for proline biosynthesis of the present invention. Suitable fragments capable of conferring water stress or salt stress tolerance to cereal plants can be constructed by using appropriate restriction sites. A fragment refers to a continuous portion of the encodin~,~
molecule for an enzyme for proline biosynthesis that is less than the entire molecule.
Non-essential nucleotides could be placed at the ~- and/or 3~ ends of the fragments (or the full length molecules encodin~~ an enzyme for proline biosynthesis) without affecting the functional properties of the fra~~ment or molecule (i.e.
in increasing water stress or salt stress tolerance). For example, the nucleotides encoding an enzyme for proline biosynthesis may be conjugated to a signal (or leader) sequence at the N-terminal end (for example) of the enzyme for proline biosynthesis which co-translationally or post-translationally directs transfer of the enzyme for proline biosynthesis. The nucleotide sequence may also be altered so that the encoded enzvrne is ~0 conju;~ated to a linker or other sequence for ease of synthesis.
purification. or identification ofthe enzyme.
The present invention also relates to a cereal plant cell or protoplast transformed with a nucleic acid encodin~~ an enzyme for proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant ryenerated from said cereal plant cell or protoplast. Unce transformation has occurred. the cereal plant cell or protoplast can be regenerated to form a transgenic cereal plant.
Preferably. the nucleic acid encodin~~ an ellzvllle for proline biosynthesis ~ is controlled by a strong promoter to effect maximum expression of an enzyme for proline biosynthesis. or by a stress-induced promoter to eftect induction of the promoter in response to stress conditions. In one embodiment. the trans~~enic cereal plant cell or protoplast or plant is transformed with the nucleic acid encodin~z the promoter. such as the rice actin 1 gene promoter. by providing a plasmid w '111011 II1CILIdeS DNA
encoding an enzyme for proline biosynthesis and the promoter.
The transgenic cereal plant cell or protoplast or plant cato also be transformed with a nucleic acid encoding a selectable marker. such as the hcn-~.~ene. to allow for detection of transformants. and with a nucleic acid encoding the caulitlower mosaic virus 3iS promoter to control e~cpression of the hur '~enc. Other selectable markers include ~~enes encoding EPSPS. nptIl. or ALS. Other promoters include those from ~_enes encoding actin 1. ubiquitin, and PINII. These additional nucleic acid sequences can also be provided by the plasmid encoding the enzyme for praline biosynthesis and its promoter. Where appropriate. the various nucleic acids could also be provided by transformation with multiple plasmids.
While the nucleotide sequence refs-:rred to herein encodes an enzyme for proline biosynthesis. nucleotide identity to previously sequenced enzymes for proline biosynthesis is not required. As should be readily apparent to those skilled in the art.
various nucleotide substitutions are possible which are silent mutations (i.e.
the amino acid encoded by the particular codon does not chan~~e). It is also possible to substitute a nucleotide which alters the amino acid encoded by a particular codon. where the amino acid substituted is a conservative substitution (i.e. amino acid "homology' is conserved).
It is also possible to have minor nucleotide and/or amino acid additions.
deletions. andlor substitutions in the enzyme for proline biosynthesis nucleotide and/or amino acid sequences which have minimal influence on the properties. secondary structure.
and hvdrophilic/hvdrophobic nature of the encoded enzymes for proline biosynthesis. These variants are encompassed by the nucleic acid encodin~~ an enzyme for proline bi~,svnthesis accordin~~ to the subject invention.
WO 99/66785 PCT/US99/~4336 _ 1~ -The present invention is also directed to a transgenic cereal plant regenerated from the transgenic cereal plant cells or protoplasts: as well as to seed produced by the trans~,~enic cereal plants.
Another aspect of the present invention is a method of conferring water S stress or salt stress tolerance to a cereal plant includin~~ transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis.
In a preferred embodiment, the method further includes regenerating the transformed cereal plant cell or protoplast to form a transgenic cereal plant.
The present invention also includes seed produced by the trans'~enic cereal plant.
The present invention also relates to a method of increasing tolerance of a cereal plant to water stress or salt stress conditions. the method including increasin~~
levels of an enzyme for proline biosynthesis in the cereal plant.
In a preferred embodiment, the plasmid is desi~~nated p.IS102. p.IS107. or pJS112 (See Examples 1 and 2).
EXAMPLES
Example 1 - Construction of the p.ISI07 Plasmid for Plant Transformation Plcrvmicl C'ons~rtrclim p.lS 107 was constructed by isolating a 2.4 kb .Sell fra~~ment containin~~
mothbean (1~~l~rilCl LlC'UI?III'IJIIU L.) P.iC:ScDNA from the plasmid pUbiP~CS
(Hu et al.. "<A
Bifunctional Enzyme (0~-pyrroline-~-carboxylate synthetase) Catalyzes the First Two Steps in Pro Biosynthesis in Plants." Proc. Natl. Acad. Sci. I1SA. 89:934-938 (1992).
which is hereby incorporated by reference). This DNA fragment was blunted with Klenow DNA polymerise and subcloned into the .Smcrl site of~ the expression vector pJS104 (Su et al.. "Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants.'" Plant Phvsiol.. 1 17:913-922 ( I 998). 'which is hereby incorporated by reference) to create pJS107. In p.1S107. the P~C'.S-codiiy~
sequence was downstream of a stress-inducible promoter complex (desi~~aoated as AIPC-ABA
inducible promoter complex). AIPC includes a 49 by ABA-responsive element from the barley Hru?2 gene (Shen et al.. "Functional Dissection Of An Abscisic Acid (ABA)-Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containin~~ A G-Boy And A Novel C'r.s-ACtlll~~ Element.' Plant Cell. 7:?9i-3U7 ( 199;). which is hereby incorporated by reference). a 180 by minimum rice actin ;.gene promoter (Su et al..
"Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants." Plant Physiol.. 117:913-9?2 ( 1998). which is hereby incorporated by reference).
and a Hrcr?? intron (Shen et al.. "Functional Dissection Of An Abscisic Acid (ABA)-Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containing A G-Boa And A Novel C'i.s-Acting Element." Plant Cell. 7:295-307 ( 199;).
which is hereby incorporated by reference). p,lS 107 also contains the bur cassette.
which was used for selection of transgenic calli and plants in the presence of the herbicide.
Bialaphos.
I0 Ti-crrrsformmion vjRice C.'e!ls ~-irh cr rLlolhheun P.iC;S cD;VA (Zhu et al.. "Overexpression Of A 'P~C',S' Gene And Analysis Of Tolerance To Water And Satt Stress In Transgenic Rice." Plant Science 139:1-48 (1998). which is hereby incorporated by reference) The procedure and media used for the establishment of suspension cells 1 S was according to a previously described method (C ao et ai., "Assessment Of Rice Genetic Transformation Techniques. In Rice Biotechnolo~_v. Toenniessen et al.. Eds..
CAB
International. Oxon. UK, pp. 175-198 ( 1991 ): Cao et al.. "Regeneration Of Herbicide Resistant Transgenic Rice Plants Following Microprojectile-Mediated Transformation Of Suspension Culture Cells."' Plant Cell Rep.. 1 1:586-191 ( 1992). which are hereby 20 incorporated by reference). Dehusked rice seeds (l)rwcr su river L. var.
Nipponbare) were used for callus induction. Following growth in suspension cultures. pJS107 was introduced into suspension culture cells by the biolistic method. The cells were cultured and selected in KPR medium (Can et al., "Assessment Of Rice Genetic Transformation Techniques. In Rice Biotechnolo~~v. Toenniessen et al.. Eds.. CAB
International. Oxon.
?5 UK. pp. 175-198 (1991 ). which is hereby-incorporated by reference) containing 8 mg per liter Bialaphos. The resistant calli were transferred to MS regeneration medium to regenerate into plants. Plants regenerated from the same resistant callus were regarded as clones of the same line. Re~~enerated plants were transferred into soil and grown in the greenhouse (32°C day/2?°C night. with supplemental photoperiod of 10 hours).
30 Plasmid p.lS 107 (ABRC 1 /Act-100 promoter/Hva22 intron/P.SCS
cDNA/Pin2 3'//~SS promoter/hur/Nos 3") was introduced into rice suspension cells usin~~
the biolistic-mediated transformation method.
Rcy~cnc~rcuion cmcJAnulu~~i.s of Truns~~cnir Plums A number of transgenic plants ((O you smivu L.) were generated. and four lines with relatively low transgene copy number were analyzed. The results are shown in Table I , below.
Table 1. Analysis of Transgenic Rice Plants Transformed With a Moth bean P~CS
cDNA.
Southern PSCS Proline pg/gShoot Fresh Plant Blot Activitya _ Fresh LeafWeight (g)h Copy Number Control 0 - 27 0.80 N22 5 ++ 45 1.22 N~2 3 48 0.82 N60 2 +++ 71 1. ~ 1 N70 2 ++++ (g 1.90 a P~CS activity was assayed based on the conversion of [~''C] glutamate to [~'C]
proline: TLC separation.
b Eight-week-old plants (4-10 per line) were stressed with no water for 6 days.
then water 1 day. Four cycles (28 days).
Thus, this data indicated that transgenic rice plants produced an increased level of the PSCS enzyme activity as well as proline content (measured by using a colorimetric method) in leaves.
Example 2 - Transformation of Rice Calli with a Moth bean PSCS cDNA and Comparison of an Inducible vs. Constitutive Proline Synthesis in Transgenic Rice Plusmicl Construction Three plasmids were constructed. The components of these plasmids are:
pJS102 (with a constitutive promoter): Rice actin 1 promoter/P~C:ScDNA/Pin 2 3'//355 promoter/hur/Nos 3";
pJSI 12 (with a stress-inducible promoter): ABRC4/Actl-100 promoter/Hmr??
intron/P~C:ScDNA/Pin2 3'//35S promoter/hur/Nos 3': and p,lS 1 10 (with a constitutive promoter and all components as in pJS I 1 ~.
except that a uicl,-1 reporter gene is used in place of the PAC S cDNA in pJS I 12).
_ l; _ Ti-crrzcfirrnnrtiura u~'Ricc~ C'crlli vrirh a ~Llathhcorr~ I',5C :S cD,~~:a The preparation of rice calli. transformation procedure. and regeneration of~
plants were similar to those described in Example 1.
:9ncrlt:si.s ol~Trcrn.s.Qer~ic Plcrnls:
Gro~rth Llr9Gl SIr'C'.S'S' Ti-eutment.s of Plank in Soil Refined and sterilized field soil was used to grow the rice plants in the greenhouse. R~ seeds were germinated in 1/2 MS medium for 7 days. and the 7-day-old seedlings were transplanted into soil in small pots (8x8 inches) with holes in the bottom (4 to 6 plants per pot). The pots were kept in flat-bottomed trays containing water. The seedlin~~s were grown for an additional 2 weeks. and within the third week.
they were tested for Basta resistance. Two Basta-resistant plants with the same plant height per pot were selected for stress treatments. Stress treatments were carried out as follows.
In the first round of stress treatment, water was withheld from the trays for 1 ~ 7 days. and. then, the stressed plants were resupplied with water for 2 days. One or three additional rounds of stress treatments were imposed on the plants. For salt stress. 3-week-old plants were transferred to trays containing.: 300 mM NaC l solution for 20 days. The NaCI solution was changed every 3 days to maintain a constant concentration of NaCI in the soil. The pots containin;~ stressed plants were transferred back to trays containing tap water to allow the stressed plants to recover and grow without stress for 10 days. flfter the I 0 days of recovery. a second round of salt stress was imposed by using the same concentration of NaCI solution for 10 days. Liquid fertilizer (Peters Excel.
N:P:K =
I ~:~: I ~. Scotts Professional Co.) mixed with tap water or NaCI solution was applied to the plants weekly.
Gnomnh Per f mn7crnec~ o~~Trcrnske~nie Plar~ts~ Urtclen ~'~Cllc'r' .S/r'L'.S',S C.'vnditions Since there was no significant difference in ~~rowrth performance between NT plants and uicl.9 plants in seedlings tested. the uicL-1 plants (L3) were chosen as more suitable control plants for the following experiment because Lhey also contained hew and the same promoter cassette as the p.5rs-transgenic plants.
Before initial water stress. all the 3-week-old plants includin~~ the L3 control plants. were tested for Basta resistance. Healthy. Basta-resistant plants with similar plant hei!~ht were selected for analvzin~_ ~~rownh performance. Under non-stress conditions in soil. no significant differences were observed between logs-containing transgenic plants and SIPC'-uiclA control plants in their grow rth performance during the entire period of the experiment. Upon withholding water from the trays. the absolute water content in the soil decreased from 35% to 1?% after 7 days water stress.
Following 2 cycles of the water stress, the leaves of SIPC'-uicl.-1 control plants started to turn yellow.
and the Actl ~.irs plants showed low-growth rate. whereas the SIPC'-p~rs plants with a stress-inducible promoter showed healthy growth. After 4 cycles of water stress. more severe symptoms, such as leaf chlorosis (in both control and Ac~l p.5cs plants) or death of leaf tips (in control plants only), were found. The SIPC'-p.5cs plants still showed a high rate of growth and less-severe leaf chlorosis. Data in Table 2 (top half) show the avera~Te fresh shoot weight and fresh root weight of the plants after 4 cycles of 7 days water stress.
The results indicated that under water stress. the SIPC' p.5cs plants (L~ and L7), which contained a stress-inducible promoter to drive the p~e.s expression, grew much faster as compared to Acrl-p~c~.s plants (L I ). which contained a constitutive promoter for driving theh.ics expression. The difference between using a stress-inducible promoter and a constitutive promoter was highly significant (P<0.01: t = 5.88 to 7.64).
Gruwlh Perprnurnce orTrcrnsgeraic Rice Plcrr~~.s L'r~clc~r S'crll .~rrc~.ss C'ondirion.s To create high soil salinity, 300 mM NaCI solution was added to the trays in which the pots were placed. At an early sta~~e ( 10 d after the initial stress). the control plants (L3) started to wih and the leaves began to turn yellow. whereas the lots transgenic plants still showed healthy growth. After 20 days of NaCI stress. the Acrl-PSC'S
plants (L I ) also started to wilt. Following l0 days of watering to allow recovery and an additional 10 days of 300 mM NaCI stress. more severe damage occurred in both control plants (L3) and 2~ ,9crl p.ics plants. On the contrary. the leaves of SIPC'-p5c.s plants still remained green with a high rate of growth. The average fresh shoot weight and fresh root weight are shown .in Table I1 (bottom half). These values indicated that SIPC': p~cs plants (L~ and L7) grew significantly larger (P<0.01: t = 6.03 to 7.79) under salt-stress conditions than Acrl p.5cs plants (L T) and' control plants (L3). in spite of the firidin~
that the proline level was lower in .SIPC'p.>cs plants: Of the two SIPC.'h.5c.s lines. Li was the better one: In conclusion. stress-inducible transgene expression in h.5cs plants shows significant advanta~~es over constitutive expression of the p.5cs-trans~~ene in ~~row-th of rice plants under salt- and water-stress conditions.
WO 99/66785 Ptr'T/US99/1-4336 Table 2. Growth performance of transgenic plants in soil antler water-stress or satt-stress conditions __ _ 1-resh Shout Vvt hrcsh Rout V t t Valve' in V'atcr-Stress Expt:
Rice l.inc Prumowr Imgi plant) (mei plant) Comparison Shoot 11~t Root Wt .151 Inducihl~3t)U20 t 9()t2U ( 1.1:L3 9.Sd 3.21 It) (l.3) IUU) IU01 JS1U2 IL Cunstituticr~~t?t6U 13Ut2U ( 1.3:L3 ).i.22 8.05 I ) ( 183) I-L1) JSI12(LS) Inducible9.(UtIOU(310122Ut301?3-t)L7:L3 1.97 6.22 .IS1121L71 Inducibtc730t6U(2.13)170120(189) LI:LS 7.fi.l 5.88 1 J fresh )hoot M~t I=rcsh Rout 11'1 t Value~ in VaCI-Stress Gxpt.
hransernic l.in c I'rumutrr (me i plant) (me i plant) (:'ompuri,un Shoot Wt Root Wt J5110(1.31 Inducihlc32Ut.1t)tlUl!)70tIU(IUU) l.I:L3 5.68 d.18 )~IU3IL1)Constitutive8UtIUtltl8l)IIUt30(!37) 1.1:L3 6.03 7.79 J1112 (l.~lInducihlrlU3Utl-tU ?.ll)t3U1.i.13)1.3:L3 11.7? 11.9?
(33?) .151121L7) Inducihlc87t)tl3U(272)180t3U(257) 1.7:L3 7.83 7.67 * As compared to the t values ofSmclenr'.s clisrrihmiur~ table.
t".~;t"=b!=2.33 and to.ot(n=6)=3.17.
All values higher than 3.17 are significant.
Fresh shoot and root weir=hts are in mg/plant. Mteans~SE represents the averages of 6 plants ( Wt). Values in parentheses are the percentages ofp.5r.s-transgenic plants compared to control plants (L3). represented by 100. The spread of data within each set of 6 plants was rather small. For example. the actual values for the fresh shoot wt of six JS110 (L3) plants in the water-stress experiment (top half of table) were: ?80. 282. 288. 31 ~. 320 and 325: the actual values for the fresh shoot wt of'six JS112 (L6) plants «-ere: 8=10. 846. 860.
1025. 1045 and 100.
Although the invention has been described in detail for the purpose of illustration. it is understood that such detail is solely for that purpose.
and variations can be made therein by those skilled in the art without departin~~ from the spirit and scope of the invention which is defined by the following claims.
TOLERANT TRANSGENIC CEREAL PLANTS
This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/090.61. filed June 2~. 1998.
FIELD OF THE INVENTION
The present invention relates to transgenic cereal plants which are transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant and a method of increasin~.~ or conferring=
water stress or salt stress tolerance to a cereal plant.
BACKGROUND OF THE INVENTION
Environmental stresses. such as drought. increased salinity of soil. and extreme temperature. are major factors in limiting plant growth and productivity. The worldw°ide loss in yield of three major cereal crops. rice. maize (corn). and wheat due to water stress (drought) has been estimated to be over ten billion dollars annually. Salt stress and drou~=ht stress are the two most important abiotic stresses. Of the 4.870 million hectares of agricultural land in the world. 930 million ( 19% of total) are salt-affected areas (FAO Quarterly Bulletin of Statistics. Vol. 9'/a (1996)). Moderate levels ofsalt content in the soil (such as ~0 mM) cause a substantial decrease in the yield of crops.
High levels of salt in the soil (higher than 100 or I ~0 mM) are not at all suitable for planting most cereal crops. Approximately 5.2% of the agricultural lands are under drought stress (FAO Quarterly Bulletin of Statistics. Vol. 9 e/4 ( 1996)). and the loss of crop yield is also very significant.
In practical terms. rice is the most important crop because a high percentage of the worlds population depends on it for their staple food.
To~~ether with wheat and corn. these three cereal crops constitute the major source of food and calories to feed the people. With an increase in population and a decrease in arable land. there is a real possibility of a food shorta~~e by the year 2030. Therefore, it is essential to frilly utilize plant biotechnolo~~v to improve plants and produce more food.
Breeding of stress-tolerant crop cultivars represents a promisin~~ strate~~y t~
tackle these problems (Epstein et al.. "Saline Culture of Crops: A Genetic Approach."
WO 991bb785 PCT/US99/14336 Science. 210:399-404 ( 1980)). However. conventional breeding is a slow process for generatin~~ crop varieties with improved tolerance to stress conditions.
Limited ~~ermplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species are additional problems encountered in conventional breeding.
s Recent progress in plant ~Tenetic transformation and availability of potentially useful genes characterized from different sources make it possible to generate stress-tolerant crops using transgenic approaches (Tarczynski et al.. "Stress Protection of Transgenic Tobacco by Production of the Osmolyte Mannitol." Science. 29:508-~ 10 (1993):
Pilon-Smits et al.. "Improved Performance of Trans~~enic Fructan-Accumulating Tobacco Under Drought Stress.' Phvsioi. Plant. 107:1?-130 (1990). Transformation of cereal plants with a~~ronomicallv useful genes that increase tolerance to abiotic stress is one important way to minimize yield loss. For example. it would be highly desirable to produce trans~?enic rice plants that can give reasonable yield when grown in marginal or waste lands that contain relatively high levels of' salt. such as 100-1 ~0 mM.
in the soil.
Characterization and cloning of plant genes that confer stress tolerance remains a challenge. Genetic studies revealed that tolerance to drought and salinity in some crop varieties is principally due to additive gene effects (Akbar et al..
"Breeding For Soil Stress.~~ In Pro~~ress in Rainfed Lowland Rice. International Rice Research Institute.
manila. Philippines, pp. 263-272 (1986): Akbar et al.. "Genetics of Salt Tolerance in Rice.' In Rice Genetics. International Rice Research Institute. Manila.
Philippines. pp.
399-409 ( 1986)). However. the underlying molecular mechanism for the tolerance has never been revealed. Physiological and biochemical responses to high levels of ionic or nonionic solutes and decreased water potential have been studied in a variety of plants.
Based on accumulated experimental observations and theoretical consideration.
one 2~ su~~gested mechanism that may underlie the adaptation or tolerance of plants to osmotic stresses is the accumulation of~compatible. low molecular weight osmolytes such as sugar alcohols. special amino acids. and glycine hetaine (Greenway et al..
"Mechanisms of Salt Tolerance in Nonhalophytes." A1111t1. Rev. Plant Pl3vsiol.. 31: 149-190 ( 1980): Yancey et al.. "Livin~~ With Water Stress: Evolution of Osmolyte System." Science. 217:
1214-12~~
( 1982)). In particular, proline level is know to increase in a number of plants and bacteria under drought or salt stress. Recently. a trans~~enic study has demonstrated that accumulation of the su''ar alcohol mannitol in trans~~enie tobacco conferred protection against salt stress fTarczvnski et al.. "Stress Protection of Trans~wnic Tobacco by WO 99/66785 PCT/US99/~4336 - J
Production of the Osmolye Mannitol." Science. ?~9:~0$-~ 10 ( 1993)). Two recent studies using a trans~~enic approach have demonstrated that mmabolic en~~ineerin~~ of the ~~lvcine betaine biosynthesis pathway is not only possible but also may eventuallw lead to production of stress-tolerant plants (Holmstrom et al.. "Production of the E.schcmichicr coli Betaine-Aldehyde Dehydrogenase, An Enzyme Required for the Synthesis of the Osmoprotectant Glycine Betaine, in Transgenic Plants." Plant .1.. 6:749-7~$ ( 1994):
Rathinasabapathi et al., "Metabolic Engineerin~~ of Glvcine Betaine Synthesis:
Pfant Betaine Aldehyde Dehydro;~enases Lacking Typical Transit Peptides are Tar~~eted to Tohacco Chloroplasts Where they Confer Betaine Aldehvde Resistance." Planta.
193:1 ss-162 ( 1994)).
In addition to metabolic changes and accumulation of low- molecular wei~Yht compounds, a large set of genes is transcriptionallv activated which leads to accumulation of new proteins in vegetative tissue of plains under osmotic stress conditions. including the late embryogenesis abundant (L1:A? family.
dehvdrines. and 1 ~ COR47 (Skriver et al., "Gene Expression in Response to Ahscisic Acid and Osmotic Stress." Plant Cell. 2:503-512 ( 1990), Chandler et al.. "Gene L:xpression Re~~ulated by Abscisic Acid and its Relation to Stress Tolerancc.~~ AI71111. Rev: Plant Phvsiol. Plant Mol.
Biol.. :~~:1 13-141 ( 1994)). The expression levels of a Illllllhel' of ~~enes hale been reported to be correlated with desiccation. salt. or cold tolerance of ditterent plant varieties of the same species. It is generally assll117ed that sh'tSS-111dlICed prOle111S n 7l~~ht play a role in tolerance. but the functions of many stress-responsive ~.~enes are unknown.
Elucidatin~~ the fiu~ction of these stress-responsive ~~enes and enzymes involved in the biosynthesis of stress-induced osmolytes will not only advance the understanding of plant adaptation and tolerance to environmental stresses. but also may 2~ provide important information for designing new strate~_ies for crop improvement (Chandler et al.. "Gene Expression Regulated by Abscisic Acid and its Relation to Stress Tolerance." Annu. Rev. Plant Phvsiol. Plant Mol. Biol., =t~:1 13-141 ( 1994)).
Several genes that encode key enzymes involved in the biosynthesis of specific osmolvtes (such as mannitol, proline. or glycine betain e) have been introduced into tobacco cells. The regenerated transgenic tobacco plants showed partial tolerance to drought and to salt stress (Tarezynski et al.. "Stress Protection of~Trans~~elzic Tobacco by Production of Osmotic Mannitol," Science. ?s9:~0$-~ 1 U ( 1993 ): Kishor et al..
..~'.~1't\pl'eSSloll Of ~1~-tyrroline-3-carboxvlate Svnthetaw Increases Praline Production WO 99/66785 PCT/US99/1d336 _:
and Confers Osmotolerance in Transgenic Plants." Plant Physiol.. 108:1387-1394 ( 199 1:
Lilies et al.. "Enhanced NaC1 stress tolerance in trans~~enic tobacco expressing bacterial choline dehydrogenase."' Biotech., 14:177-180 ( 1966)). I-Iowever. only transgenic tobacco was used for these studies. and similar work on producing stress-tolerant cereal S crop plants has not been carried out. It is not clear whether these genes in transgenic cereal plants will enhance salt or drought tolerance since the physiology of dicot plants.
such as tobacco. is very different from monocots. such as cereal plants. Thus.
only experimentation using cereal crop plants can provide the answer.
The present invention is directed to overcoming the above-noted deficiencies in the prior art.
SUMMARY OF THE INVENTION
The present invention relates to a trans~~enic cereal plant transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or 1 ~ salt stress tolerance to the plant.
The present invention also relates to a cereal plant cell or protoplast transformed with a nucleic acid encoding an enzyme For proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant regenerated from said cereal plant cell or protoplast.
Another aspect of the present inyen lion is a method of conferrin~~. water stress or salt stress tolerance to a cereal plant including.: transforming, a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis.
The present invention also relates to a method of increasin'.! tolerance of a cereal plant to water stress or salt stress conditions. the method includin~~
increasing 2~ levels of an enzyme for proline biosynthesis in the cereal plant.
The present invention allows the production of cereal plants with increased tolerance to water stress (drought) and salt stress. Thus, an enzy°tne for proline biosynthesis can be used as a molecular tool for ~~enetic crop improvement by conterrin~~
stress tolerance.
WO 99/66785 PCT/US99/t4336 DETAILED DESCR11'TION OF THE INVENTION
The present invention relates to a trans~~enic cereal plant transformed with a nucleic acid encodiny~ an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant.
Suitable nucleic acids encoding an enzyme for proline biosynthesis include the P.SC ~S ~~ene of mothbean and a feedback-inhibition insensitive mutant.
P~C,S-129A. of the PLC'S gene. The sequence of the PLC'S gene can be found in Kishor et al..
''Overexpression of 0~-pyrroline-~-carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Trans~,enic Plants." Plant Phvsiol., 108:1387-1394 (1990.
which is hereby incorporated by reference. and the sequence of the PSC'S-129A
mutant gene can be found in Zhan~~ et al.. "Removal of Feedback Inhibition of P.SC',S
in Plants."
J. Bioi. Chem.. 270:20491-20496 (1990. which is hereby incorporated by reference.
Cereal which can be transformed in accordance with the subject invention are members of the family Gr~crinir7ecre (also known as Pocrc~eoc~). and include rice (~~enus Or;yvu). wheat. maize (corn). barley. oat. sorghum. and millet. Preferably.
the cereal is rice. w=heat. or corn. and most preferably the cereal is rice. Many species of cereals can be transformed. and within each species the numerous subspecies and varieties can be transformed. For example. within the rice species is subspecies lndica rice (W
y=a .smiou ssp. Indica). which includes the varieties IR36. IR64. IR72. Pokkali. Nona Bokra.
KDML10~. Suponburi 60. Suponburi 90. Basmati 38~. and Pusa Basmati 1. Another rice subspecies is Japonica. which includes Nipponbere. Kenfeng and Tainun~~ 67.
Examples of suitable maize varieties include A188. B73. VA?2. L6. L9. K1. 509. X922, 482. HNP.
and 1GES. Examples of suitable wheat varieties include Payon. Anza. Chris.
Coker 983.
FLA301. FLA302. Fremont and Hunter.
2~ Having identified the cereal plant of interest. plant cells suitable for transformation include immature embryos. calli. suspension cells. and protoplasts. It is particularly preferred to use suspension cells and immature embryos.
These cereal plant cells are transformed with a nucleic acid. which could be RNA or DNA and which is preferably cDNA. encodin~~ an enzyme for proline biosynthesis. The nucleic acid can be biolo~~icallv isolated or synthetic. In the followin~~
Examples, a key enzyme for proline biosynthesis. ~~-pyrroline-~-carboxylate synihase (PSCS). is encoded by the I'~C'S'~ene of mothbean. Howeewr. other genes encoding an etlzyme for proiine biosynthesis. includin~~ a feedback-Illhlbltl(lil lnSeI1S1t1Ve n7uta17t ot: the P~C:S ~~ene. P.SC'.S-129A. can also be utilized.
Transformation of plant cells can be accomplished by using a plasmid.
The plasmid is used to introduce the nucleic acid encoding an enzyme for proline ~ biosynthesis into the plant cell. Accordingly. a plasmid preferably includes DNA
encodin~~ an enzyme for proline biosynthesis inserted into a unique restriction endonuclease cleavage site. Heterologous DNA, as used herein. refers to DNA
not normally present in the particular host cell transformed by the plasmid. DNA
is inserted into the vector using standard cloning procedures readily known in the art.
This generally involves the use of restriction enzymes and DNA li~~ases. as described by Sambrook et al.. Molecular Clonin~~: A Laboratory Manual. 2d edition. Cold Sprin~~ Harbor Laboratory Press. Cold Sprin~~ Harbor. New York ( I 989). which is hereby incorporated by reference. The resulting plasmid which includes nucleic acid encoding an enzyme for proline biosynthesis can then be used to transform a host cell. such as an A~nohucter°ium and/or a plant cell. (See generally. Plant Molecular Biolo~~v Manual. 2nd Edition. Gelvin et al., Eds.. Kluwer Academic Press. Dordrecht. Netherlands ( 1994). which is hereby incorporated by reference).
For plant transformation. the plasmid preferably also includes a selectable marker for plant transformation. Commonly used plant selectable markers include the hy';romvcin phosphotransferase (hp!) gene. the phosphinothricin acetyl transferase ~~ene (hur). the ~-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS). neomycin ~'-O-phosphotransferase gene (np/ II). or acetolactate svnthase ~~ene (ALS).
Information on these selectable markers can be found in Bowers. "Markers for Plant Gene Transfer" in Trans:.~enic Plants. Kung et al.. Eds.. Vol. I . pp. 89-123. Academic Press, NY ( 1993).
2~ which is hereby incorporated by reference.
The plasmid preferably also includes suitable promoters for expression of the nucleic acid encoding an enzyme for proline biosynthesis and for expression of the marker ~~ene. The cauliflower mosaic virus 3~S promoter is commonly used for plant transformation. as well as the rice actin 1 ~~ene promoter. In plasmid p,1S102 used in the followin~~ Examples. the nucleic acid encodin~~ an enzyme for proline biosynthesis is under the control of the constitutive rice actin 1 ;gene promoter and the marker ~_ene (hur) is under control of the cauliflower mosaic virus 3sS promotr-r. Other promoters useful for plant transformation with an enzyme for proline hiosvnthesis include those from the genes encodin~~ ubiquitin and proteinase inhibitor II (PINII ). as well as stress-induced promoters (such as the HVA1 gene promoter of barley. an abscisic acid (ABA)-induciblc promoter. such as ABRCI from barley linked to a rice Act-100 miniri~ai promoter. and a HVA22 intron).
The plasmid designated p.IS 112 has been deposited pursuant to. and in satisfaction of. the requirements of the Budapest Treaty on the International Reco~~nition of the Deposit of Microorganisms for the Purposes of Patent Procedure. with the American Type Culture Collection (ATCC). i 0801 University Boulevard, Manassas. VA
201 10-2209. under ATC C Accession No. on .tune 17. 1999.
For plant transformation. the plasmid also preferably includes a nucleic acid molecule encodin~~ a 3' terminator such as that from the 3~ 11011-COdln~.! re~~ion of genes encoding a proteinase inhibitor. actin. or nopaline svnthase (nos).
Other suitable plasmids for use in the subject invention can be constructed.
For example. ~~enes encoding an enzyme for proline biosynthesis other than the PSC:f 1 S gene of mothbean could be ligated into plasmid ,1S 109 after use of restriction enzymes to remove the P~C :f gene. Other promoters could replace the actin 1 ~~elle promoter present in plasmid .15102. Alternatively. other plasmids in general containing genes encodin~~ an enzyme for proline bioswthesis under the control of a suitable promoter. with suitable selectable markers. can be readily constructed usin~~ techniques well known in the art.
Havin~~ identified the plasmid. one technique of transforming cereal plant cells with a ~~ene which encodes for an enzyme for proline biosynthesis is by contactin«
the plant cell with an inoculum of an A~t,>rvhucnc~rium bacteria transformed with the plasmict comprisin~~ the ~~ene that encodes far the enzyme for proline biosynthesis.
Generally. this procedure involves inoculatin~~ the plant cells with a suspension of the 2~ transformed bacteria and incubating the cells for ~8 to 72 hours on re~~eneration medium without antibiotics at 25-28°C.
Bacteria from the genus A~>mohucncrirun can be utilized to transform plant cells. Suitable species include A~mohcrrterium ~tmrc~Jecicn.s and A~oohcrclenim~i rlri=o~c~nc.s. A~rohur~crirm7lumcyac~icns (e.~~., strains LBA4404 or EHAIOS) is particularly useful due to its well-known ability to transform plants.
In inoculating the cells of cereal plants with .-t ~>ruhocvc~rirrm accordin~~
to the subject invention. the bacteria must be transtormcd with a vector which includes a ~~ene encoding for an enzyme for praline biosynthesis.
WO 99/66785 PCT/US99/~4336 _g-Plasmids. suitable for incorporation in ;a,slrnhcrwerirrnr. which include a gene encoding for an enzyme for proline biosynthesis. contain an origin of replication for replication in the bacterium E5L'l1L'1'IC'ylr(I c'Ull. all Orl~.jlll Of replication for replication in the bacterium fl~,~rohuc~enirrm lumefacic~ns. T-DNA ri~~ht border sequences for transfer of genes to plants. and marker genes for selection of transformed plant cells.
Particularly preferred is the vector pBII?1 which contains a low-copy RK2 origin of replication. the neomycin phosphotransferase (nptll) marker gene with a nopaline synthase (NOS) promoter and a NOS 3' polyadenylation signal. T-DNA plasmid vector pBI121 is available from Clonetech Laboratories. Ine.. 4030 Fabian Wav. Palo Alto.
California 94303. A gene encoding for an enzyme for proline biosynthesis is inserted into the vector to replace the beta-;~lucuronidase (GUS) gene.
Typically. A~~robucleritrnT spp. are transformed with a plasmid by direct uptake of plasmid DNA after chemical and heat treatment. as described by Holsters et al.
"Transfection and Transformation of ~~~rvhae~e~rimn mme~f~rcic.~ns.'" Mol.
Gen. Genet..
1 ~ 163:181-187 ( 1978), which is hereby incorporated by reference: by direct uptake of plasmid DNA after electroporation. as described by Shen et al.. "Efficient Transformation ofA~nrnhucJc~ritrnt spp. by High Voltage Electroporation."' Nucleic Acids Research.
17:838 ( 1989). which is hereby incorporated by reference: by triparental conju4~ational transfer of plasmids from Esc~herichiu coli to .4~ruhcrctc~ritu~~ mediated by a Tra+ help strain as described by Ditta et al.. "Broad I-lost Range DNA Clonin<~ System for Gram-negative Bacteria: Construction of a Gene Bank of Rhcohilmr molilo~i."' Pror.
Natl. Acad.
Sci. USA. 77:7347-73~ 1 ( 1981 ). which is hereby incorporated by reference;
or by direct eonjugational transfer from E.scherichiu cvli to A~I'Ohuclc'!'11u71 as described by Simon et al.. "A Broad Host Range Mobilization System for i» rims Genetic En;~ineering:
2~ Transposon Muta~~enesis in Gram-negative Bacteria." Biotechpolo~y. 1:784-791 ( 198?).
which is hereby incorporated by reference.
Another method for introduction of a plasmid containing nucleic acid encodin~~ an enzyme for proline biosynthesis into a plant cell is by transformation of the plant cell nucleus. such as by particle bombardment. As used throughout this application.
particle bombardment (also known as biolistic transformation) of the host cell can be accomplished in one of several ways. The first involves propelling inert or biolo~~ically active particles at cells. This technique is disclosed in U.S. Patent Nos.
4.9~1~.Oi0.
6.036.006. and 5.100.79?. all to Salltot'd et al.. which are hereby incorporated by reference. Generally. this procedure involves propellin~~ inert or biologicaily active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the plasmid can be introduced into the cell by coating the particles with the plasmid containing the heterologous DNA. Alternatively, the target cell can be surrounded by the plasmid so that the plasmid is carried into the cell by the wake of the particle.
Biologically active particles (e.g.. dried bacterial cells containing the plasmid and heterologous DNA) can also be propelled into plant cells.
A further method for introduction of the plasmid into a plant cell is by transformation of plant cell protoplasts (stable or transient). Plant protoplasts are enclosed only by a plasma membrane and will therefore take up macromolecules like heterolo~~ous DNA. These engineered protoplasts can be capable of regenerating whole plants. Suitable methods for introducing heterolo~,ous DNA into plant cell protoplasts include electroporation and polyethylene glycol (PEG) transformation. As used throughout this application. electroporation is a transformation method in which.
generally. a high concentration of plasmid DNA (containing heterologous DNA) is added to a suspension of host cell protoplasts and the mixture shocked with an electrical field of 200 to 600 V/cm. Following electroporation. transformed cells are identified by growth on appropriate medium containing a selective agent.
As used throughout this application. transformation encompasses stable transformation in which the plasmid is integrated into the plant chromosomes.
In the Examples which follow. rice has been transformed usin~~ biolistic transformation. Other methods of transformation have also been used to successfully transform rice plants. including the protoplast method (for a review, see Cao et al..
2~ "Regeneration of Herbicide Resistant Transgenic Rice Plants Following Microprojectile Mediated Transformation of Suspension Culture Cells." Plant Cell Rep.. 11:586-( 1992). which is hereby incorporated by reference). and the ,~lgrv6crcterium method (Hiei et al.. "Efficient Transformation of Rice (Ori_cr .suliru L.) Mediated by Agrobcrclerirrnr and Sequence Analysis of the Boundaries of the T-DNA.' The Plant .iournaf.
6:271-?82 ( 1994). which is hereby incorporated by reference). Biolistic transformation has also been used to successfully transform maize (for a review. see Mackey et al..
"Transgenic Maize," /r~ Transsenic Plants. Kung et al.. Eds.. vol. ?. pp. ? 1-33 ( 1993 ).
which is hereby incorporated by reference) and wheat (see U.S. Patent No. ~.~0~.76~ to Vasil et al.. which is herehv° incorporated by reference).
Once a cereal plant cell or protoplast is transformed in accordance with the present invention. it is re;~enerated to form a transgenic cereal plant.
Generally.
regeneration is accomplished by culturing transformed cells or protoplasts on medium containing the appropriate gro~-th regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of ~l~rrobcrc~enium or other comaminants and to select for the development of transformed cells or protoplasts. Following shoot initiation. shoots are allowed to develop in tissue culture and are screened for marker ;gene activity.
In suitable transformation methods. the cereal plant cell to be transformed can be in ri~ro or in vioo. i.e. the cereal plant cell can be located in a cereal plant.
The invention also provides seed produced by the transgenic cereal plant.
The invention is also directed to seed. which upon germination. produces the transgenic cereal plant.
Also encompassed by the present invention are transgenic cereal plants transformed with fragments of the nucleic acids encoding an enzyme for proline biosynthesis of the present invention. Suitable fragments capable of conferring water stress or salt stress tolerance to cereal plants can be constructed by using appropriate restriction sites. A fragment refers to a continuous portion of the encodin~,~
molecule for an enzyme for proline biosynthesis that is less than the entire molecule.
Non-essential nucleotides could be placed at the ~- and/or 3~ ends of the fragments (or the full length molecules encodin~~ an enzyme for proline biosynthesis) without affecting the functional properties of the fra~~ment or molecule (i.e.
in increasing water stress or salt stress tolerance). For example, the nucleotides encoding an enzyme for proline biosynthesis may be conjugated to a signal (or leader) sequence at the N-terminal end (for example) of the enzyme for proline biosynthesis which co-translationally or post-translationally directs transfer of the enzyme for proline biosynthesis. The nucleotide sequence may also be altered so that the encoded enzvrne is ~0 conju;~ated to a linker or other sequence for ease of synthesis.
purification. or identification ofthe enzyme.
The present invention also relates to a cereal plant cell or protoplast transformed with a nucleic acid encodin~~ an enzyme for proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant ryenerated from said cereal plant cell or protoplast. Unce transformation has occurred. the cereal plant cell or protoplast can be regenerated to form a transgenic cereal plant.
Preferably. the nucleic acid encodin~~ an ellzvllle for proline biosynthesis ~ is controlled by a strong promoter to effect maximum expression of an enzyme for proline biosynthesis. or by a stress-induced promoter to eftect induction of the promoter in response to stress conditions. In one embodiment. the trans~~enic cereal plant cell or protoplast or plant is transformed with the nucleic acid encodin~z the promoter. such as the rice actin 1 gene promoter. by providing a plasmid w '111011 II1CILIdeS DNA
encoding an enzyme for proline biosynthesis and the promoter.
The transgenic cereal plant cell or protoplast or plant cato also be transformed with a nucleic acid encoding a selectable marker. such as the hcn-~.~ene. to allow for detection of transformants. and with a nucleic acid encoding the caulitlower mosaic virus 3iS promoter to control e~cpression of the hur '~enc. Other selectable markers include ~~enes encoding EPSPS. nptIl. or ALS. Other promoters include those from ~_enes encoding actin 1. ubiquitin, and PINII. These additional nucleic acid sequences can also be provided by the plasmid encoding the enzyme for praline biosynthesis and its promoter. Where appropriate. the various nucleic acids could also be provided by transformation with multiple plasmids.
While the nucleotide sequence refs-:rred to herein encodes an enzyme for proline biosynthesis. nucleotide identity to previously sequenced enzymes for proline biosynthesis is not required. As should be readily apparent to those skilled in the art.
various nucleotide substitutions are possible which are silent mutations (i.e.
the amino acid encoded by the particular codon does not chan~~e). It is also possible to substitute a nucleotide which alters the amino acid encoded by a particular codon. where the amino acid substituted is a conservative substitution (i.e. amino acid "homology' is conserved).
It is also possible to have minor nucleotide and/or amino acid additions.
deletions. andlor substitutions in the enzyme for proline biosynthesis nucleotide and/or amino acid sequences which have minimal influence on the properties. secondary structure.
and hvdrophilic/hvdrophobic nature of the encoded enzymes for proline biosynthesis. These variants are encompassed by the nucleic acid encodin~~ an enzyme for proline bi~,svnthesis accordin~~ to the subject invention.
WO 99/66785 PCT/US99/~4336 _ 1~ -The present invention is also directed to a transgenic cereal plant regenerated from the transgenic cereal plant cells or protoplasts: as well as to seed produced by the trans~,~enic cereal plants.
Another aspect of the present invention is a method of conferring water S stress or salt stress tolerance to a cereal plant includin~~ transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis.
In a preferred embodiment, the method further includes regenerating the transformed cereal plant cell or protoplast to form a transgenic cereal plant.
The present invention also includes seed produced by the trans'~enic cereal plant.
The present invention also relates to a method of increasing tolerance of a cereal plant to water stress or salt stress conditions. the method including increasin~~
levels of an enzyme for proline biosynthesis in the cereal plant.
In a preferred embodiment, the plasmid is desi~~nated p.IS102. p.IS107. or pJS112 (See Examples 1 and 2).
EXAMPLES
Example 1 - Construction of the p.ISI07 Plasmid for Plant Transformation Plcrvmicl C'ons~rtrclim p.lS 107 was constructed by isolating a 2.4 kb .Sell fra~~ment containin~~
mothbean (1~~l~rilCl LlC'UI?III'IJIIU L.) P.iC:ScDNA from the plasmid pUbiP~CS
(Hu et al.. "<A
Bifunctional Enzyme (0~-pyrroline-~-carboxylate synthetase) Catalyzes the First Two Steps in Pro Biosynthesis in Plants." Proc. Natl. Acad. Sci. I1SA. 89:934-938 (1992).
which is hereby incorporated by reference). This DNA fragment was blunted with Klenow DNA polymerise and subcloned into the .Smcrl site of~ the expression vector pJS104 (Su et al.. "Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants.'" Plant Phvsiol.. 1 17:913-922 ( I 998). 'which is hereby incorporated by reference) to create pJS107. In p.1S107. the P~C'.S-codiiy~
sequence was downstream of a stress-inducible promoter complex (desi~~aoated as AIPC-ABA
inducible promoter complex). AIPC includes a 49 by ABA-responsive element from the barley Hru?2 gene (Shen et al.. "Functional Dissection Of An Abscisic Acid (ABA)-Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containin~~ A G-Boy And A Novel C'r.s-ACtlll~~ Element.' Plant Cell. 7:?9i-3U7 ( 199;). which is hereby incorporated by reference). a 180 by minimum rice actin ;.gene promoter (Su et al..
"Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants." Plant Physiol.. 117:913-9?2 ( 1998). which is hereby incorporated by reference).
and a Hrcr?? intron (Shen et al.. "Functional Dissection Of An Abscisic Acid (ABA)-Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containing A G-Boa And A Novel C'i.s-Acting Element." Plant Cell. 7:295-307 ( 199;).
which is hereby incorporated by reference). p,lS 107 also contains the bur cassette.
which was used for selection of transgenic calli and plants in the presence of the herbicide.
Bialaphos.
I0 Ti-crrrsformmion vjRice C.'e!ls ~-irh cr rLlolhheun P.iC;S cD;VA (Zhu et al.. "Overexpression Of A 'P~C',S' Gene And Analysis Of Tolerance To Water And Satt Stress In Transgenic Rice." Plant Science 139:1-48 (1998). which is hereby incorporated by reference) The procedure and media used for the establishment of suspension cells 1 S was according to a previously described method (C ao et ai., "Assessment Of Rice Genetic Transformation Techniques. In Rice Biotechnolo~_v. Toenniessen et al.. Eds..
CAB
International. Oxon. UK, pp. 175-198 ( 1991 ): Cao et al.. "Regeneration Of Herbicide Resistant Transgenic Rice Plants Following Microprojectile-Mediated Transformation Of Suspension Culture Cells."' Plant Cell Rep.. 1 1:586-191 ( 1992). which are hereby 20 incorporated by reference). Dehusked rice seeds (l)rwcr su river L. var.
Nipponbare) were used for callus induction. Following growth in suspension cultures. pJS107 was introduced into suspension culture cells by the biolistic method. The cells were cultured and selected in KPR medium (Can et al., "Assessment Of Rice Genetic Transformation Techniques. In Rice Biotechnolo~~v. Toenniessen et al.. Eds.. CAB
International. Oxon.
?5 UK. pp. 175-198 (1991 ). which is hereby-incorporated by reference) containing 8 mg per liter Bialaphos. The resistant calli were transferred to MS regeneration medium to regenerate into plants. Plants regenerated from the same resistant callus were regarded as clones of the same line. Re~~enerated plants were transferred into soil and grown in the greenhouse (32°C day/2?°C night. with supplemental photoperiod of 10 hours).
30 Plasmid p.lS 107 (ABRC 1 /Act-100 promoter/Hva22 intron/P.SCS
cDNA/Pin2 3'//~SS promoter/hur/Nos 3") was introduced into rice suspension cells usin~~
the biolistic-mediated transformation method.
Rcy~cnc~rcuion cmcJAnulu~~i.s of Truns~~cnir Plums A number of transgenic plants ((O you smivu L.) were generated. and four lines with relatively low transgene copy number were analyzed. The results are shown in Table I , below.
Table 1. Analysis of Transgenic Rice Plants Transformed With a Moth bean P~CS
cDNA.
Southern PSCS Proline pg/gShoot Fresh Plant Blot Activitya _ Fresh LeafWeight (g)h Copy Number Control 0 - 27 0.80 N22 5 ++ 45 1.22 N~2 3 48 0.82 N60 2 +++ 71 1. ~ 1 N70 2 ++++ (g 1.90 a P~CS activity was assayed based on the conversion of [~''C] glutamate to [~'C]
proline: TLC separation.
b Eight-week-old plants (4-10 per line) were stressed with no water for 6 days.
then water 1 day. Four cycles (28 days).
Thus, this data indicated that transgenic rice plants produced an increased level of the PSCS enzyme activity as well as proline content (measured by using a colorimetric method) in leaves.
Example 2 - Transformation of Rice Calli with a Moth bean PSCS cDNA and Comparison of an Inducible vs. Constitutive Proline Synthesis in Transgenic Rice Plusmicl Construction Three plasmids were constructed. The components of these plasmids are:
pJS102 (with a constitutive promoter): Rice actin 1 promoter/P~C:ScDNA/Pin 2 3'//355 promoter/hur/Nos 3";
pJSI 12 (with a stress-inducible promoter): ABRC4/Actl-100 promoter/Hmr??
intron/P~C:ScDNA/Pin2 3'//35S promoter/hur/Nos 3': and p,lS 1 10 (with a constitutive promoter and all components as in pJS I 1 ~.
except that a uicl,-1 reporter gene is used in place of the PAC S cDNA in pJS I 12).
_ l; _ Ti-crrzcfirrnnrtiura u~'Ricc~ C'crlli vrirh a ~Llathhcorr~ I',5C :S cD,~~:a The preparation of rice calli. transformation procedure. and regeneration of~
plants were similar to those described in Example 1.
:9ncrlt:si.s ol~Trcrn.s.Qer~ic Plcrnls:
Gro~rth Llr9Gl SIr'C'.S'S' Ti-eutment.s of Plank in Soil Refined and sterilized field soil was used to grow the rice plants in the greenhouse. R~ seeds were germinated in 1/2 MS medium for 7 days. and the 7-day-old seedlings were transplanted into soil in small pots (8x8 inches) with holes in the bottom (4 to 6 plants per pot). The pots were kept in flat-bottomed trays containing water. The seedlin~~s were grown for an additional 2 weeks. and within the third week.
they were tested for Basta resistance. Two Basta-resistant plants with the same plant height per pot were selected for stress treatments. Stress treatments were carried out as follows.
In the first round of stress treatment, water was withheld from the trays for 1 ~ 7 days. and. then, the stressed plants were resupplied with water for 2 days. One or three additional rounds of stress treatments were imposed on the plants. For salt stress. 3-week-old plants were transferred to trays containing.: 300 mM NaC l solution for 20 days. The NaCI solution was changed every 3 days to maintain a constant concentration of NaCI in the soil. The pots containin;~ stressed plants were transferred back to trays containing tap water to allow the stressed plants to recover and grow without stress for 10 days. flfter the I 0 days of recovery. a second round of salt stress was imposed by using the same concentration of NaCI solution for 10 days. Liquid fertilizer (Peters Excel.
N:P:K =
I ~:~: I ~. Scotts Professional Co.) mixed with tap water or NaCI solution was applied to the plants weekly.
Gnomnh Per f mn7crnec~ o~~Trcrnske~nie Plar~ts~ Urtclen ~'~Cllc'r' .S/r'L'.S',S C.'vnditions Since there was no significant difference in ~~rowrth performance between NT plants and uicl.9 plants in seedlings tested. the uicL-1 plants (L3) were chosen as more suitable control plants for the following experiment because Lhey also contained hew and the same promoter cassette as the p.5rs-transgenic plants.
Before initial water stress. all the 3-week-old plants includin~~ the L3 control plants. were tested for Basta resistance. Healthy. Basta-resistant plants with similar plant hei!~ht were selected for analvzin~_ ~~rownh performance. Under non-stress conditions in soil. no significant differences were observed between logs-containing transgenic plants and SIPC'-uiclA control plants in their grow rth performance during the entire period of the experiment. Upon withholding water from the trays. the absolute water content in the soil decreased from 35% to 1?% after 7 days water stress.
Following 2 cycles of the water stress, the leaves of SIPC'-uicl.-1 control plants started to turn yellow.
and the Actl ~.irs plants showed low-growth rate. whereas the SIPC'-p~rs plants with a stress-inducible promoter showed healthy growth. After 4 cycles of water stress. more severe symptoms, such as leaf chlorosis (in both control and Ac~l p.5cs plants) or death of leaf tips (in control plants only), were found. The SIPC'-p.5cs plants still showed a high rate of growth and less-severe leaf chlorosis. Data in Table 2 (top half) show the avera~Te fresh shoot weight and fresh root weight of the plants after 4 cycles of 7 days water stress.
The results indicated that under water stress. the SIPC' p.5cs plants (L~ and L7), which contained a stress-inducible promoter to drive the p~e.s expression, grew much faster as compared to Acrl-p~c~.s plants (L I ). which contained a constitutive promoter for driving theh.ics expression. The difference between using a stress-inducible promoter and a constitutive promoter was highly significant (P<0.01: t = 5.88 to 7.64).
Gruwlh Perprnurnce orTrcrnsgeraic Rice Plcrr~~.s L'r~clc~r S'crll .~rrc~.ss C'ondirion.s To create high soil salinity, 300 mM NaCI solution was added to the trays in which the pots were placed. At an early sta~~e ( 10 d after the initial stress). the control plants (L3) started to wih and the leaves began to turn yellow. whereas the lots transgenic plants still showed healthy growth. After 20 days of NaCI stress. the Acrl-PSC'S
plants (L I ) also started to wilt. Following l0 days of watering to allow recovery and an additional 10 days of 300 mM NaCI stress. more severe damage occurred in both control plants (L3) and 2~ ,9crl p.ics plants. On the contrary. the leaves of SIPC'-p5c.s plants still remained green with a high rate of growth. The average fresh shoot weight and fresh root weight are shown .in Table I1 (bottom half). These values indicated that SIPC': p~cs plants (L~ and L7) grew significantly larger (P<0.01: t = 6.03 to 7.79) under salt-stress conditions than Acrl p.5cs plants (L T) and' control plants (L3). in spite of the firidin~
that the proline level was lower in .SIPC'p.>cs plants: Of the two SIPC.'h.5c.s lines. Li was the better one: In conclusion. stress-inducible transgene expression in h.5cs plants shows significant advanta~~es over constitutive expression of the p.5cs-trans~~ene in ~~row-th of rice plants under salt- and water-stress conditions.
WO 99/66785 Ptr'T/US99/1-4336 Table 2. Growth performance of transgenic plants in soil antler water-stress or satt-stress conditions __ _ 1-resh Shout Vvt hrcsh Rout V t t Valve' in V'atcr-Stress Expt:
Rice l.inc Prumowr Imgi plant) (mei plant) Comparison Shoot 11~t Root Wt .151 Inducihl~3t)U20 t 9()t2U ( 1.1:L3 9.Sd 3.21 It) (l.3) IUU) IU01 JS1U2 IL Cunstituticr~~t?t6U 13Ut2U ( 1.3:L3 ).i.22 8.05 I ) ( 183) I-L1) JSI12(LS) Inducible9.(UtIOU(310122Ut301?3-t)L7:L3 1.97 6.22 .IS1121L71 Inducibtc730t6U(2.13)170120(189) LI:LS 7.fi.l 5.88 1 J fresh )hoot M~t I=rcsh Rout 11'1 t Value~ in VaCI-Stress Gxpt.
hransernic l.in c I'rumutrr (me i plant) (me i plant) (:'ompuri,un Shoot Wt Root Wt J5110(1.31 Inducihlc32Ut.1t)tlUl!)70tIU(IUU) l.I:L3 5.68 d.18 )~IU3IL1)Constitutive8UtIUtltl8l)IIUt30(!37) 1.1:L3 6.03 7.79 J1112 (l.~lInducihlrlU3Utl-tU ?.ll)t3U1.i.13)1.3:L3 11.7? 11.9?
(33?) .151121L7) Inducihlc87t)tl3U(272)180t3U(257) 1.7:L3 7.83 7.67 * As compared to the t values ofSmclenr'.s clisrrihmiur~ table.
t".~;t"=b!=2.33 and to.ot(n=6)=3.17.
All values higher than 3.17 are significant.
Fresh shoot and root weir=hts are in mg/plant. Mteans~SE represents the averages of 6 plants ( Wt). Values in parentheses are the percentages ofp.5r.s-transgenic plants compared to control plants (L3). represented by 100. The spread of data within each set of 6 plants was rather small. For example. the actual values for the fresh shoot wt of six JS110 (L3) plants in the water-stress experiment (top half of table) were: ?80. 282. 288. 31 ~. 320 and 325: the actual values for the fresh shoot wt of'six JS112 (L6) plants «-ere: 8=10. 846. 860.
1025. 1045 and 100.
Although the invention has been described in detail for the purpose of illustration. it is understood that such detail is solely for that purpose.
and variations can be made therein by those skilled in the art without departin~~ from the spirit and scope of the invention which is defined by the following claims.
Claims (32)
1. A transgenic cereal plant transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance to the plant.
2. A transgenic cereal plant according to claim 1, wherein said cereal plant is a rice plant.
3. A transgenic cereal plant according to claim 1, wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.
4. A transgenic cereal plant according to claim 3, wherein the mutant mothbean P5CS gene is P5CS-129A:
5. A transgenic cereal plant according to claim 1, wherein said transgenic cereal plant includes a nucleic acid encoding a promoter, wherein expression of said nucleic acid encoding said enzyme for proline biosynthesis is controlled by said promoter.
6. A transgenic cereal plant according to claim 5, wherein said promoter is the rice actin 1 gene promoter.
7. A transgenic cereal plant according to claim 1, wherein said transgenic cereal plant includes a nucleic acid encoding a selectable marker.
8. A seed produced by the transgenic cereal plant of claim 1.
9. A seed which upon germination produces the transgenic cereal plant of claim 1.
10. A cereal plant cell or protoplast transformed with a nucleic acid encoding an enzyme for proline biosynthesis that confers water stress or salt stress tolerance on a cereal plant regenerated from said cereal plant cell or protoplast.
11. A cereal plant cell or protoplast according to claim 10, wherein said cereal plant cell or protoplast is derived from a rice plant.
12. A cereal plant cell or protoplast according to claim 10. wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.
13. A cereal plant cell or protoplast according to claim 12. wherein the mutant mothbean P5CS gene is P5CS-129A.
14. A cereal plant cell or protoplast according to claim 10, wherein said cereal plant cell or protoplast includes a nucleic acid encoding a promoter, wherein expression of said nucleic acid encoding said enzyme for proline biosynthesis is controlled by said promoter.
15. A cereal plant cell or protoplast according to claim 14, wherein said promoter is the rice actin 1 gene promoter.
16. A cereal plant cell or protoplast according to claim 10. wherein said cereal plant cell or protoplast includes a nucleic acid encoding a selectable marker.
17. A transgenic cereal plant regenerated from the cereal plant cell or protoplast of claim 10.
18. A seed produced by the transgenic cereal plant of claim 17.
19. A method of conferring water stress or salt stress tolerance to a cereal plant comprising:
transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis under conditions effective to impart water stress or salt stress tolerance to cereal plants.
transforming a cereal plant cell or protoplast with a nucleic acid encoding an enzyme for proline biosynthesis under conditions effective to impart water stress or salt stress tolerance to cereal plants.
20. A method according to claim 19, wherein said cereal plant cell or protoplast is derived from a rice plant.
21. A method according to claim 19. wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.
22. A method according to claim 21, wherein the mutant mothbean P5CS gene is P5CS-129A.
23. A method according to claim 19, wherein said transforming comprises:
propelling particles at said cereal plant cell under conditions effective for the particles to penetrate the cell interior; and introducing a plasmid comprising the nucleic acid encoding an enzyme for proline biosynthesis into the cell interior.
propelling particles at said cereal plant cell under conditions effective for the particles to penetrate the cell interior; and introducing a plasmid comprising the nucleic acid encoding an enzyme for proline biosynthesis into the cell interior.
24. A method according to claim 23, wherein the plasmid is associated with the particles, whereby the plasmid is carried into the cell or protoplast interior together with the panicles.
25. A method according to claim 19, wherein said transforming comprises:
contacting tissue of the monocot plant with an inoculum of a bacterium of the genus Agrobacterium wherein the bacterium is transformed with a plasmid comprising the gene that increases tolerance to salt stress and drought stress.
contacting tissue of the monocot plant with an inoculum of a bacterium of the genus Agrobacterium wherein the bacterium is transformed with a plasmid comprising the gene that increases tolerance to salt stress and drought stress.
26. A method according to claim 19 further comprising:
regenerating the transformed cereal plant cell or protoplast to form a transgenic cereal plant.
regenerating the transformed cereal plant cell or protoplast to form a transgenic cereal plant.
27. A transgenic cereal plant produced by the method of claim 26.
28. A seed produced by the transgenic cereal plant of claim 27.
29. A method of increasing tolerance of a cereal plant to water stress or salt stress conditions, said method comprising:
increasing levels of an enzyme for proline biosynthesis in said cereal plant.
increasing levels of an enzyme for proline biosynthesis in said cereal plant.
30. A method according to claim 29, wherein said cereal plant is a rice plant.
31. A method according to claim 29. wherein said nucleic acid encoding an enzyme for proline biosynthesis is the P5CS gene of mothbean or a mutant mothbean P5CS gene which is insensitive to feedback inhibition by proline.
32. A method according to claim 31 wherein the mutant mothbean P5CS gene is P5CS-129A.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9056198P | 1998-06-24 | 1998-06-24 | |
| US60/090,561 | 1998-06-24 | ||
| PCT/US1999/014336 WO1999066785A1 (en) | 1998-06-24 | 1999-06-24 | Water stress or salt stress tolerant transgenic cereal plants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2335522A1 true CA2335522A1 (en) | 1999-12-29 |
Family
ID=22223329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002335522A Abandoned CA2335522A1 (en) | 1998-06-24 | 1999-06-24 | Water stress or salt stress tolerant transgenic cereal plants |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU4716499A (en) |
| CA (1) | CA2335522A1 (en) |
| WO (1) | WO1999066785A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7385106B2 (en) | 2000-01-24 | 2008-06-10 | Ramot At Tel Aviv University Ltd. | Plants tolerant of environmental stress conditions, methods of generating same and novel polynucleotide sequence utilized thereby |
| JP2002369634A (en) * | 2001-06-08 | 2002-12-24 | Hitachi Ltd | Gramineous plant having high proline-accumulating ability and method for producing the same |
| KR100440725B1 (en) | 2002-06-20 | 2004-07-15 | 주식회사 그린진 바이오텍 | A Method for Increasing an Abiotic-Resistance in Monocot Plants |
| EP2716654A1 (en) * | 2005-10-24 | 2014-04-09 | Evogene Ltd. | Isolated polypeptides, polynucleotides encoding same, transgenic plants expressing same and methods of using same |
| WO2012099528A1 (en) * | 2011-01-18 | 2012-07-26 | Swetree Technologies Ab | Drought resistant plants and methods for making the same using transcriptional regulators |
| CN112514793A (en) * | 2020-12-21 | 2021-03-19 | 扬州大学 | Screening method of salt-tolerant rice variety |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5639950A (en) * | 1992-09-29 | 1997-06-17 | The Ohio State University Research Foundation | Nucleotide sequence encoding for bifunctional enzyme for proline production |
-
1999
- 1999-06-24 CA CA002335522A patent/CA2335522A1/en not_active Abandoned
- 1999-06-24 WO PCT/US1999/014336 patent/WO1999066785A1/en active Application Filing
- 1999-06-24 AU AU47164/99A patent/AU4716499A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999066785A1 (en) | 1999-12-29 |
| AU4716499A (en) | 2000-01-10 |
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