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WO2008148298A1 - Amélioration de la tolérance au froid des plantes à l'aide du gène oscipk03 de la protéine kinase provenant du riz - Google Patents

Amélioration de la tolérance au froid des plantes à l'aide du gène oscipk03 de la protéine kinase provenant du riz Download PDF

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Publication number
WO2008148298A1
WO2008148298A1 PCT/CN2008/001047 CN2008001047W WO2008148298A1 WO 2008148298 A1 WO2008148298 A1 WO 2008148298A1 CN 2008001047 W CN2008001047 W CN 2008001047W WO 2008148298 A1 WO2008148298 A1 WO 2008148298A1
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plant
gene
tolerance
expression
plants
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PCT/CN2008/001047
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WO2008148298A8 (fr
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Yong Xiang
Lizhong Xiong
Yuemin Huang
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Huazhong Agricultural University
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Publication of WO2008148298A1 publication Critical patent/WO2008148298A1/fr
Publication of WO2008148298A8 publication Critical patent/WO2008148298A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • the present invention relates to the field of plant biotechnology, and more particularly, to the isolation, cloning, function identification and use of a DNA fragment (gene) from rice that is associated with cold tolerance in plants.
  • the cold-tolerance of transgenic plants is markedly improved by transferring a complete translation region (Coding sequence) of the gene linked with the constitutive promoter of tobacco mosaic virus (CaMV35S).
  • proteins kinase families in plants such as CDPK % CIPK > MAPK and the like are found to play an important role in the transduction of stress signals and tolerance of plants to the stresses.
  • genes for these protein kinases can be induced or repressed under different environmental stresses, it is believed that these genes are important in the process of response of plants to the stress. Therefore, the separation and identification of these functional genes that contribute to plant response to environmental stresses are of significance in genetic modification and breeding of crops for their tolerance to the stress. Attempts have been made to improve plant tolerance to stress.
  • Transgenic Arabidopsis plants that overexpress DREBlA and DREB2A showed increased tolerance to low temperature, drought and high salinity as compared to their wild type counterparts (Liu Q et al., "Two transcription factors, DREBl and DREB2, with an EREBP/AP2 DNA domains separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.” Plant Cell. 1998, 10: 1391-1406).
  • the research group of Thomashow at Michigan State University also bred plants with enhanced cold tolerance via genetic transformation with Arabidopsis CBFl gene.
  • Rice is one of the most important cereal crops, and rice with improved cold tolerance is of great significance to human. Therefore, there exists an urgent need to identify the functional genes associated with cold tolerance and breed cold and chill tolerant rice lines in order to increase the yield of rice.
  • An object of the present invention is to isolate and clone from rice a DNA fragment containing the complete coding region of a gene associated with cold tolerance, and to use the gene to improve the stress tolerance of rice and other plants, and particularly, to increase the cold tolerance of transgenic plants through gene modification.
  • Structural analysis of this stress-associated gene named OsCIP 'K03, revealed that it belongs to plant CIPK protein kinase family.
  • the present invention relates to the isolation and use of a DNA fragment containing OsCIPK03 gene, which confers plants with enhanced tolerance to stress such as low temperature and the like.
  • Said DNA fragment is as shown in SEQ ID NO: 1, or is a highly homologous DNA sequence substantially equivalent to SEQ ID NO: 1, or is a subfragment functionally equivalent to SEQ ID NO: 1.
  • the gene of the present invention or a homologous gene thereof can be obtained by screening a cDNA or genomic library using a cloned OsCIPK03 gene as the probe.
  • the OsCIPK03 gene of the present invention and any DNA segments of interest or homologous DNA segments thereof may also be obtained by amplification from genomic DNA, mRNA and cDNA using PCR (polymerase chain reaction) technology.
  • the sequence containing OsCIPK03 gene can be isolated using the above methods. By transforming plants with said isolated sequence in any expression vector that can direct the expression of an exogenous gene in plant, transgenic plants with enhanced tolerance to low temperature can be produced.
  • any strong promoter or inducible promoter can be added to the position preceding the transcription initiation nucleotide, or alternatively, an enhancer may be used.
  • an enhancer region can be ATG start code and start code of contiguous regions and the like, provided that the enhancer region is in the same frame as the coding sequence to ensure the translation of a complete sequence.
  • the expression vector bearing the OsCIPKOS gene of the present invention can be introduced into plant cells by conventional biotechnological means such as Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation and the like (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2 nd Edition)).
  • the expression vector comprising the OsCIPKOi gene of the present invention can be used to transform a host that is selected from a wide variety of plants including rice, so as to cultivate cold tolerant plant lines.
  • the gene of the present invention is expressed by induction of stress, and therefore its promoter is an inducible-type promoter.
  • its promoter is an inducible-type promoter.
  • SEQ ID No: 1 in the Sequence Listing shows the sequence of the DNA fragment isolated and cloned in accordance with the present invention, comprising the OsCIPKOi gene encoding region.
  • Fig. 1 is the flow chart of isolation and identification of the OsClPKOi gene.
  • Fig. 2 shows the expression levels of the OsCIPKOS gene at different time points under different stresses including drought, high salinity, cold, PEG and ABA, as measured by Northern hybridization.
  • Fig. 3 shows the expression vector used for genetic transformation of rice.
  • the vector over- expressing OsCIPKOS is obtained by replacing the portion from attRl to attR2 as shown in the figure with the target gene OsCIPKOS through a LR recombination reaction.
  • Fig. 4 shows that over-expression of the OsCIPKOS gene in rice enhances the cold tolerance of the plant.
  • D Survival rate of the transgenic plants and the wild type control after low temperature stress (4°C stress for 4 days and normal growth for 7 days).
  • Fig.5 shows that the over-expression of the OsCIPKOS gene increases the proline content in the plants.
  • the plants were grown to the 4-leaf stage and were subjected to low temperature stress at 4 0 C and samples were taken at day 0, 1, 3, 6 to determine the proline content.
  • Fig. 6 shows the determination of the expression amounts of genes related to proline synthesis and transportation in the plants over-expressing OsCIPK03.
  • a cDNA clone designated EI101L12 derived from the rice variety MingHui No. 63 was obtained.
  • This cDNA is the full-length cDNA of the OsCIPK03 gene, a gene associated with stress tolerance.
  • the association of the gene with stress tolerance is evidenced by the following facts. Firstly, it was observed upon an analysis using a cDNA chip technique that the expression amount of the cDNA clone EI 10 ILl 2 in the rice variety "ZhongHan No. 5" (a publicly used rice variety available from Shanghai Academy of Agricultural Sciences, China) was increased by 2.5 times after a drought stress treatment for 15 days.
  • Example 1 Isolation and cloning of a DNA fragment containing the OsCIPK03 gene segment Analysis on the expression profiles of drought inducible genes of the rice variety "ZhongHan No. 5" (a publicly used rice variety available from Shanghai Academy of Agricultural Sciences, China) led to the finding of an EST (expression sequence tag) that is strongly induced by drought, the expression amount of which was increased by 2.5 times or above at the later stage of drought stress.
  • EST expression sequence tag
  • the rice variety "Zhonghan No. 5" was used as the material and treated at 3-leaf stage with drought stress, cold stress, high-salinity stress, abscisic acid (ABA) and polyethylene glycol (PEG) respectively.
  • the drought stress treatment was conducted by stopping the supply of water to rice seedlings and sampling at 0 h, 3 h, 6 h, 12 h and 24h.
  • the cold stress treatment was conducted by placing the rice seedlings in a cold chamber at 4°C and sampling at 0 h, 3 h, 6 h, 12 h and 24h.
  • the high-salinity stress treatment was conducted by immersing the seedling root in the 200 mM/L NaCl solution and sampling at 0 h, 5h, 14h and 24h.
  • the ABA treatment was conducted by immersing the seedling root in the 100 ⁇ M/L ABA solution and sampling at 0 h, 3 h, 6 h, 12 h and 24h.
  • the PEG treatment was conducted by immersing the seedling root in the 20% PEG6000 solution and sampling at 0 h, 3 h, 5 h and 12 h.
  • the total RNA of the leaves were extracted (Trizol reagent, purchased from Invitrogen), then subjected to RNA membrane transfer and finally to Northern hybridization using OsCIPK03 as the probe according to the experimental methods described in Joseph Sambrook, "Molecular Cloning", Science Press, Peking, 1999.
  • Example 3 Construction and transformation of a vector over-expressing OsCIPK03 gene
  • Example 2 showed that expression of the OsCIP K03 gene of the present invention can be induced by drought, cold, high-salinity, abscisic acid (ABA) and polyethylene glycol (PEG). In order to better illustrate the function of this gene, it was over-expressed in rice and the phenotype of transgenic plants was characterized.
  • ABA abscisic acid
  • PEG polyethylene glycol
  • Steps were the following: amplifying exogenous fragments by PCR using the positive clone pGEM-OsCIPK03 obtained in Example 1 as the template and using the primers and conditions in Example 1; ligating the exogenous fragments into the intermediate vector pENTR/D-TOPO (purchased from Invitrogen, refer to the instruction of the kit for specific steps); ligating the gene of interest into the genetic transformation vector pCB2004H bearing the tobacco mosaic virus promoter (CaM35S) via LR recombination reaction (refer to the instruction of LR recombination reaction kit from Invitrogen); transforming E. coli. strain DHlO ⁇ (purchased from Invitrogen); and screening positive clones to obtain the transformed vector.
  • the intermediate vector pENTR/D-TOPO purchased from Invitrogen, refer to the instruction of the kit for specific steps
  • ligating the gene of interest into the genetic transformation vector pCB2004H bearing the tobacco mosaic virus promoter (CaM35
  • the transformed vector was introduced into the rice variety "ZhongHua No. 11" (a publicly used rice variety available from Institute of Crop Science, Chinese Academy of Agricultural Sciences) using a rice genetic transformation system mediated by Agrobacterium.
  • a transgenic plant was obtained through precultivation, infestation, co-cultivation, screen of the callus with hygromycin resistance, differentiation, rooting, seedling establishment and transplanting.
  • One of the obtained transgenic rice plants was designated as T35.
  • the present applicants obtained in total 23 independent transgenic rice plants.
  • the genetic transformation system of rice (japonica rice subspecies) mediated by Agrobacterium, established by National Key Laboratory of Crop Genetic Improvement where the present applicants work, was used as the genetic transformation system of rice.
  • the genetic transformation was conducted as follows.
  • phytohormones used in culture media of the present invention were as follows: 6-BA (6-Benzylaminopurine); CN (Carbenicillin); KT (Kinetin); NAA (Napthalene acetic acid); IAA (Indole-3 -acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); CH (Casein Enzymatic Hydrolysate); HN (Hygromycin B); DMSO (Dimethyl Sulfoxide); N6mac (N6 macroelements solution); N6mic (N6 microelements solution); MSmac (MS macroelements solution); MSmic (MS microelements solution).
  • VitaminB 1 (Thiamine HCl) 0.1 g
  • VitaminB ⁇ (Pyridoxine HCl) 0.1 g
  • Distilled water was added to dissolve the compounds and the resulting solution was brought to 1000 ml and stored at 4°C for use.
  • NAA 100 mg NAA was weighed and dissolved in 1 ml 1 N potassium hydroxide for 5 minutes, then 10 ml distilled water was added for complete dissolution. The resulting solution was brought to 100 ml and stored at 4 0 C for use.
  • 125 g glucose was weighed and dissolved with distilled water. The resulting solution was brought to 250 ml, sterilized and stored at 4°C for use.
  • Vitamins stock solution ( 100X) 10 ml
  • Distilled water was added to a volume of 900 ml, and the pH value was adjusted to 5.9 with 1 N potassium hydroxide.
  • the resulting mixture was boiled and brought to 1000 ml.
  • the resulting medium was dispensed into 50 ml Erlenmeyer flasks (25 ml/flask), and the flasks were sealed and sterilized.
  • Vitamins stock solution ( 100X) 10 ml
  • Distilled water was added to a volume of 900 ml, and the pH value was adjusted to 5.9 with 1 N potassium hydroxide.
  • the resulting mixture was boiled and brought to 1000 ml.
  • the resulting medium was dispensed into 50 ml Erlenmeyer flasks (25 ml/flask), and the flasks were sealed and sterilized.
  • Vitamins stock solution ( 100X) 2.5 ml
  • Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 5.6 with 1 N potassium hydroxide. The resulting medium was sealed and sterilized.
  • the medium Prior to use, the medium was heated to dissolve and 5 ml glucose stock solution and 250 ⁇ l AS stock solution were added. The resulting medium was dispensed into the culture dishes (25 ml/dish).
  • Vitamins stock solution ( 100X) 2.5 ml
  • Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 5.6 with 1 N potassium hydroxide. The resulting medium was sealed and sterilized.
  • Vitamins stock solution (100X) 1 ml
  • Distilled water was added to a volume of 100 ml, and the pH value was adjusted to 5.4.
  • the resulting medium was dispensed into two 100 ml Erlenmeyer flasks and the flasks were sealed and sterilized.
  • Vitamins stock solution (100X) 2.5 ml
  • Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 6.0.
  • the resulting medium was sealed and sterilized.
  • the medium Prior to use, the medium was dissolved and 250 ⁇ l HN and 400 ppm CN were added. The resulting medium was dispensed into the culture dishes (25 ml/dish).
  • Vitamins stock solution (100X) 2.5 ml
  • Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 5.9 with IN potassium hydroxide. The resulting medium was sealed and sterilized.
  • the medium Prior to use, the medium was dissolved and 250 ⁇ l HN and 200 ppm CN were added. The resulting medium was dispensed into the culture dishes (25 ml/dish).
  • Vitamins stock solution (100X) 10 ml
  • Distilled water was added to a volume of 900 ml, and the pH value was adjusted to 6.0 with IN potassium hydroxide. The resulting mixture was boiled and brought to 1000 ml. The resulting medium was dispensed into 50 ml Erlenmeyer flasks (50 ml/flask), and the flasks were sealed and sterilized.
  • Vitamins stock solution (100X) 5 ml
  • the bright yellow, compact and relatively dry embryogenic callus was selected, put onto the secondary culture medium, and cultured in darkness for 2 weeks at 25 ⁇ 1°C.
  • the compact and relatively dry embryogenic callus was selected, put onto the pre-culture medium, and cultured in darkness for 2 weeks at 25 ⁇ 1°C.
  • Agrobacterium EHA 105 (a strain commercially available from Cambia) was pre-cultured on the LA culture medium with corresponding resistance selection at 28°C for 2 days;
  • the callus was transferred onto a sterilized filter paper and dried, and then put onto the co-culture medium for 3-day culture at 19-2O 0 C.
  • the callus was transferred onto a sterilized filter paper and dried; (4) The callus was transferred onto the selective medium and selectively cultured for 2-3 times, 2 weeks for each time. (The screening concentration of hygromycin was 400 mg for the first culture and 250 ppm for the latter cultures).
  • the resistant callus obtained was transferred to the pre-differentiation medium, and cultured in darkness for 5-7 weeks;
  • step (2) The callus was then transferred to the rooting medium, and cultured at 26°C under light for 2-3 weeks, with the culture conditions same as those of step (2) in 4.7.
  • the residual medium on the roots of the callus was washed off, and the seedlings with good roots were transferred into the greenhouse.
  • the greenhouse was maintained moisturized in the first few days.
  • Example 4 Cold tolerance screening of the OsCIPK03 gene transgenic T2 family at seedling stage
  • the expression of the OsCIPK03 gene in the transgenic rice plants was detected using Northern hybridization technology (Fig.4A shows the results of Northern hybridization, the hybridization method was the same as used in Example 2), and part of the families of T2 generation plants of the present invention was screened for the cold tolerance. Particular steps were as followings.
  • the seeds of the T2 generation families were germinated in the rooting media containing 50 mg/ml hygromycin for 5 days. Seedlings having substantially the same level of germination were transplanted into small red pails, and wild type control plants were also cultivated.
  • Example 5 Determination of proline content in the OsCIPK03 gene transgenic T2 family
  • Proline content in plants increases significantly under stresses. Content of proline in plants reflects to some extent the stress tolerance of the plant. Increase of proline content in plant tissues under low temperature enhances the cold tolerance of the plant.
  • the transgenic rice plants of the present invention when grown to the 4-leaf stage, were subjected to low temperature stress at 4 0 C, and samples were taken at day 0, 1, 3, 6 to determine the proline content.
  • Determination of proline content is based on the following principle. Extraction of plant samples with sulfosalicylic acid releases the proline into the solution of sulfosalicylic acid. Treatment of the solution by acidic ninhydrin under heat turns the solution red. All the pigments are transferred into the toluene phase by extraction with toluene, with the shade of the pigment being a representation of the content of the proline.
  • the proline content is determined by colorimetry at 520 nm, followed by reference to the standard curve or calculation using the regression equation.
  • the proline content was determined as follows:
  • the materials for determination were the leaves of the transgenic rice plants of the present invention and the leaves of the non-transgenic rice plants.
  • Acidic ninhydrin solution 1.25 g ninhydrin was added into 30 ml of glacial acetic acid and 20 ml of 6mol/L phosphoric acid, and heated (7O 0 C) under stirring to dissolve. The resulting solution was stored in a refrigerator for use; 2. 3% sulfosalicylic acid: 3 g sulfosalicylic acid was dissolved in distilled water and then the volume was brought up to 100 ml; 3. Glacial acetic acid; 4. Toluene.
  • the proline content in the 2 ml test sample solution was calculated by using the regression equation (or referring to the standard curve), and the percentage of proline in the sample was thereby calculated.
  • the proline content ( ⁇ g/g sample) was calculated by the following formula:
  • Example 6 Determination of the expression amounts of genes related to proline synthesis and transportation in the wild-type and OsCIPK03 gene transgenic family
  • the increase in proline content may be due to the change in the expression amounts of genes related to proline synthesis and transportation. Therefore we used real-time PCR to determine the expression amounts of two genes related to proline synthesis and two genes related to proline transportation in the wild-type and OsCIPK03 gene transgenic family. Experiment results showed that the expression amounts of these four genes were increased to a different degree (5- to 10-fold) in the OsCIPK03 over-expressing plants. Experiment results as shown in Fig. 6 indicate that the over-expression of OsCIPK03 increased the expression of genes related to proline synthesis and transportation, thereby leading to the increase in proline content and hence to the enhancement of cold tolerance of the transgenic plants. The experiment was performed on an ABI 7500 Realtime PCR apparatus.
  • the reaction system was 10 ⁇ l of 2 ⁇ SYBR Green Master Mix Reagent (Applied Biosystems), 1.0 ⁇ l of cDNA templates, 200 nM gene-specific primers, with a total volume of 20 ⁇ l.
  • the reaction conditions were: the first step, 95 0 C 3 min; the second step, 95 0 C 30 sec, 60 0 C 30 sec and 72 0 C 1 min, for 40 cycles.
  • the GeneBank accession numbers for these four genes are: AK102633, AKl 01230, AK067118, AK0666298, respectively.
  • the primers used in the real-time PCR detection were respectively: for AK102633, S'-CTCAAATCAAGGCGTCAACTAAGA-S' and 5'-TTTGTCAATATATACGTGGCATATACCA-S', for AK101230, 5'-CGCCCCTCCCCGTATCT-3' and 5'-AGGAATGCGGCAACAAGTG-S', for AK067118, 5 I -AGGGACGATGGAGTTCTAAAGCT-3 I and 5'-GGGATTCCAAAGGCAAAAAGA-S', and for AK0666298,

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Abstract

La présente invention concerne l'isolation d'un clone, la confirmation de la fonction et l'utilisation du gène OsCIPK03 de riz relatif à la tolérance des plantes au froid. Le gène correspond à (a) la séquence d'ADN telle qu'indiquée dans 1-1707 bp de la SEQ ID n°: 1, ou (b) une séquence d'ADN qui code pour une protéine sensiblement équivalente à la protéine codée par (a).
PCT/CN2008/001047 2007-06-01 2008-05-29 Amélioration de la tolérance au froid des plantes à l'aide du gène oscipk03 de la protéine kinase provenant du riz WO2008148298A1 (fr)

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CNA2007100523555A CN101200724A (zh) 2007-06-01 2007-06-01 利用水稻蛋白激酶基因OsCIPK03提高植物耐冷能力
CN200710052355.5 2007-06-01

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP3173485A1 (fr) 2015-11-27 2017-05-31 Kws Saat Se Plante rustique

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CN102234325B (zh) * 2010-04-29 2013-11-13 中国农业大学 植物低钾敏感型相关的蛋白AtLKR1及其编码基因与应用
CN116536286B (zh) * 2023-05-12 2023-11-10 南京农业大学 水稻OsCTK1蛋白及其编码基因的应用

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WO2006134162A2 (fr) * 2005-06-17 2006-12-21 Basf Plant Science Gmbh Polypeptides lies au stress du type lectine d'une proteine kinase et ses methodes d'utilisation dans des plantes

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WO2006134162A2 (fr) * 2005-06-17 2006-12-21 Basf Plant Science Gmbh Polypeptides lies au stress du type lectine d'une proteine kinase et ses methodes d'utilisation dans des plantes

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DATABASE GENBANK [online] 25 October 2003 (2003-10-25), KIKUCHI S. ET AL.: "Oryza sativa Japonica Group cDNA clone: 001-015-H02", Database accession no. (AK111929) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3173485A1 (fr) 2015-11-27 2017-05-31 Kws Saat Se Plante rustique
WO2017089601A2 (fr) 2015-11-27 2017-06-01 Kws Saat Se Plante tolérante au froid
US11345923B2 (en) 2015-11-27 2022-05-31 KWS SAAT SE & Co. KGaA Cold-tolerant plant

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