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WO2003057889A1 - Methode de preparation de cucumis sativus transforme - Google Patents

Methode de preparation de cucumis sativus transforme Download PDF

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Publication number
WO2003057889A1
WO2003057889A1 PCT/KR2002/001461 KR0201461W WO03057889A1 WO 2003057889 A1 WO2003057889 A1 WO 2003057889A1 KR 0201461 W KR0201461 W KR 0201461W WO 03057889 A1 WO03057889 A1 WO 03057889A1
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Prior art keywords
cucumis sativus
cotyledon
naa
medium
regeneration
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Inventor
Sun Lee
Je-Geun Yoo
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Nexgen Biotechnologies Inc
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Nexgen Biotechnologies Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
    • A01H6/346—Cucumis sativus[cucumber]
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones

Definitions

  • the present invention relates to a method for preparing a transformed Curcumas sativa, more particularly, relates to a method of preparing a transformed Cucumis sativus using Acrobacterium tumefaciens and a transformed Cucumis sativus prepared therefrom.
  • Cucumbers ( Cucumis sativus L.) belonging to Cucurbitaleae are classified into a type of rising outdoors consumed primarily in the Europe and a type of raising greenhouse consumed primarily in the United Kingdom.
  • Cucumbers cultivated in the Asia can be classified into southern part types and northern part types. In Korea, Cucumbers are estimated to have appeared about 1,500 years ago.
  • Cucumbers may be used as fresh or processed vegetable and have been commonly employed as raw material in cosmetics such as facial lotion and pack, contributing to income of farmhouses .
  • the prime consideration matter is damage from various diseases. It has been reported that the diseases found in cucumbers are caused by fungi, bacteria and virus. The occurrence of diseases by fungi and bacteria may be reduced through the prevention with agricultural chemicals and/or continuous management, but the damage by virus cannot be avoided yet. In addition, the damage by noxious insects may be considerable and the treatment with insecticides is recommended as a sole method of prevention. Especially, in parallel with increasing cultivation area using facilities, the damages by noxious insects of various kinds has been on the increase, but the reliable solution thereto has not been provided yet, which is due primarily to problems including that a period of 5-10 years is required in breeding a cultivar having desirable character. Thus, as one of solutions to such problems, the genetic engineering technology capable of introducing a variety of characters into plant has been already recommended, rendering a breeding period shortened.
  • the present inventors have made intensive research to be from the shortcomings of the conventional transformation methods for Cucumis sativus and as a result, we have developed a successful method to ensure preparation of a transformed Cucumis sativus with higher reproducibility. Accordingly, it is an object of this invention to provide a method for preparing a transformed Cucumis sativus using ⁇ grrojbacterium tumefaciens . It is another object of this invention to provide a transformed Cucumis sativus .
  • Fig. 1 shows regenerated cotyledons from Cucumis sativus transformed according to this invention
  • Fig. 2 represents a genetic map of binary vector pRD320 used in this invention
  • Fig. 3 demonstrates comparison between transformant of this invention and non-transformant ;
  • Fig. 4 demonstrates the effect on rooting depending on agar concentration in rooting medium;
  • Fig. 5a shows the results of GUS analysis confirming the occurrence of transformation in Cucumis sativus
  • Fig. 5b shows the results of PCR elucidating transformed Cucumis sativus according to this invention.
  • a method for preparing a transformed Cucumis sativus which comprises the steps of: (a) inoculating a cotyledon from
  • BAP
  • the present inventors have made extensive research with 5 cultivars of Cucumis sativus developed in Korea, and finally developed methods for regeneration with higher frequency and transformation with Agrobacterium tumefaciens in more efficient manner, as exemplified in Examples below:
  • the present invention will be described in more detail as follows:
  • the preferred explants for transformation includes leaf, stem and petiole, but not limited to.
  • the explants may be obtained from several plant organs and most preferably from seed. It is preferred that the seed is sterilized with sterilizing agent such as chlorine and chlorides (e.g., sodium hypochloride) before use.
  • sterilizing agent such as chlorine and chlorides (e.g., sodium hypochloride) before use.
  • the medium for seed germination comprises nutrient basal medium such as B5, LS, N6 and White's, energy source and vitamins, but not limited to. Sugars are useful as energy source and sucrose is the most preferable. It is preferred that vitamins for seed germination include nicotine, thiamine and pyridoxine .
  • the medium for seed germination in this invention may further contain MES (2-
  • the explant for transformation includes any tissue derived from seed germinated. It is preferred to use cotyledon and hypocotyl and the most preferred is cotyledon. It is advantageous to remove growth point completely from cotyledon as explant and to use explant in whole not in dissected one.
  • Transformation of cells derived from Cucumis sativus is carried out with Agrobacterium tumefaciens harboring Ti plasmid (Depicker, A. et al . , Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York (1983)). More preferably, binary vector system such as pBinl9, pRD400 and pRD320 is used for transformation (An, G. et al . , Binary vectors" In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York(1986) ) .
  • the binary vector useful in this invention carries: (i) a replication origin operable in the cell from Cucumis sativus ; (ii) a promoter capable of promoting a transcription in the cell from Cucumis sativus; (iii) a structural gene operably linked to the promoter; and (iv) a polyadenylation signal sequence.
  • the vector carries antibiotics- resistance gene as selective marker, e.g. carbenicllin, kanamycin, spectinomycin and hygromcin.
  • the vector may alternatively further carry a gene coding for reporter molecule (for example, luciferase and ⁇ -glucuronidase) .
  • Examples of the promoter used in the binary vector include but not limited to Cauliflower Mosaic Virus 35S promoter,
  • the structural gene in the present vector may be determined depending on traits of interest.
  • Exemplified structural gene may include but not limited to genes for herbicide resistance (e.g. glyphosate, sulfonylurea) , viral resistance, vermin resistance (e.g., Bt gene), resistance to environmental extremes (e.g. draught, high or low temperature, high salt cone), improvement in qualities (e.g. increasing sugar content, retardation of ripening) , exogenous protein production useful as drug (EGF, antigen or antibody to various diseases, insulin) or cosmetic raw material (e.g. albumin, antibiotic peptide) .
  • herbicide resistance e.g. glyphosate, sulfonylurea
  • viral resistance e.g., vermin resistance (e.g., Bt gene)
  • resistance to environmental extremes e.g. draught, high or low temperature, high salt cone
  • improvement in qualities e.g. increasing sugar content, retardation of ripening
  • exogenous protein production useful as drug EGF, antigen or
  • Inoculation of the explant with Agrobacterium tumefaciens involves procedures known in the art.
  • the most preferred explant, cotyledon with removed growth point is immersed in medium of Agr ojbac e ium tumefaciens to coculture, thereby inoculating the cotyledon with Agrobacterium tumefaciens .
  • acetosyringone is employed in the coculturing to promote infection of Agrobacterium tumefaciens into explant cell .
  • the regeneration medium of this invention may contain nutrient basal medium such as MS, B5 , LS, N6 and White's, energy source and vitamins, but not limited to. Sugars are useful as energy source and sucrose is the most preferable. It is preferred that vitamins in regeneration medium include nicotine, thiamine and pyridoxine.
  • the regeneration medium may further contain MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate) as buffering agent for pH change and agar as solid support.
  • the medium must contain plant growth regulators.
  • Cytokinin as plant growth regulator may include but not limited to 6-benzylaminopurine (BAP) , kinetin, zeatin and isopentyladenosine and BAP is the most preferable cytokinin.
  • BAP 6-benzylaminopurine
  • the regeneration medium contains, as essential ingredient, auxin such as NAA ( ⁇ -naphthalene acetic acid), indole acetic acid and (2 , 4-dichlorophenoxy) acetic acid, and the most preferable is NAA.
  • the amount of BAP in the regeneration medium ranges from 1 to 4 mg/1, more preferably from 1.5 to 2.5 mg/1 and most preferably 2.0 mg/1. If the amount is less than 1 mg/1 or exceeds 4 mg/1, the regeneration rate tends to drop sharply.
  • the preferable amount of NAA in the regeneration medium is in the range of from 0.001 to 0.08 mg/1, more preferably from 0.005 to 0.03 mg/1 and most preferably 0.01 mg/1. If the amount is less than 0.001 mg/1 or exceeds 0.08 mg/1, the regeneration rate is very likely to drop sharply.
  • the regeneration medium of this invention removes the necessity of CuS0 or casein hydrolysate which is generally used in the art for regeneration of plants belonging to Cucurbitaleae .
  • the medium of the invention exhibits improved regeneration rate and ability in regenerated shoot formation of Cucumis sativus without CuS0 4 or casein hydrates, which clearly demonstrates excellency of the medium.
  • the medium further contains antibiotics (e.g. carbenicllin, kanamycin, spectinomycin or hygromcin) for selection of transformed explant .
  • antibiotics e.g. carbenicllin, kanamycin, spectinomycin or hygromcin
  • 70-150 mg/1 is a preferred amount. If the amount is less than 70 mg/1, the final transformation rate becomes 0 while the regeneration rate is higher; and if exceeding 150 mg/1, the regeneration rate is very likely to dive dramatically.
  • the cotyledon placing is performed by insertion on the regeneration medium. Unlikely to leaf section, placing cotyledon at full length on the medium results in negligible regeneration and callus formation.
  • Culturing according to the conditions described above allows successfully a regeneration of shoots through callus formation from the transformed explant of Cucumis sativus on the medium.
  • the transformed Cucumis sativus plantlet is finally produced on rooting medium by rooting of regenerated shoots.
  • the rooting medium of this invention may contain nutrient basal medium such as MS, B5 , LS, N6 and White's, energy source and vitamins, but not limited to. Sugars are useful as energy source and sucrose is the most preferable. It is preferred that vitamins in the rooting medium include nicotine, thiamine and pyridoxine.
  • the rooting medium may further contain MES (2- (N- Morpholino) ethanesulfonic acid Monohydrate) as buffering agent for pH change and agar as solid support.
  • auxin is predominantly employed in the rooting medium.
  • the auxin useful includes NAA, indole acetic acid and (2 , 4-dichlorophenoxy) acetic acid, and the most preferable is NAA.
  • NAA indole acetic acid
  • the amount of NAA in the rooting medium is in the range of from 0.001 to 0.08 mg/1. If the amount is less than 0.001 mg/1, the period necessary for rooting is relatively longer and thinner/longer roots without root hairs are frequently induced. If the amount exceeds 0.08 mg/1, the stem in contact with medium tends to be diverent leading to no occurrence of rooting.
  • the rooting medium contains agar as solid support
  • 0.2-0.7% (w/v) is preferable amount. If the amount is less than 0.2%, the support function is largely diminished; if exceeding 0.7%, the elongation of roots is remarkably retarded, thicker/shorter roots are formed and rooting is likely to occur out of medium.
  • the transformed Cucumis sativus produced according to the present invention may be confirmed using procedures known in the art. For example, using DNA sample from tissue of transformed Cucumis sativus, PCR is carried out to reveal exogenous gene incorporated into a genome of Cucumis sativus transformed. Alternatively, Northern or Southern Blotting may be performed for confirming the transformation as described in Maniatis et al . , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) .
  • the tissue from cotyledon regenerated is immersed in substrate solution such as X- gluc (5-Bromo-4-Chloro-3-Indole- ⁇ -D-Glucuronic Acid) so that 'colorimetric reaction may be observed to confirm the occurrence of transformation.
  • substrate solution such as X- gluc (5-Bromo-4-Chloro-3-Indole- ⁇ -D-Glucuronic Acid)
  • a method for preparing a transformed Cucumis sativus which comprises the steps of: (a) inoculating an intact cotyledon from Cucumis sativus with Agrobacterium tumefaciens harboring a vector, in which the vector is capable of inserting into a genome of a cell from Cucumis sativus and contains the following sequences: (i) a replication origin operable in the cell from Cucumis sativus; (ii) a promoter capable of promoting a transcription in the cell from Cucumis sativus; (iii) a structural gene operably linked to the promoter; and (iv) a polyadenylation signal sequence; (b) placing the inoculated cotyledon on a medium containing 1.5-2.5 mg/1 of BAP (6-benzylaminopurine) and 0.005-0.03 mg/1 of NAA ( ⁇ -naphthalene acetic acid) by insert and
  • the method of this invention which is developed for producing a transformed Cucumis sativus, as exemplified and demonstrated in Examples below, exhibits much higher transformation and regeneration efficiency, leading to production of transformed Cucumis sativus having desirable traits with higher reproducibility.
  • EXAMPLE 1 Preparing of Explants 5 cultivars of Cucumis sativus ( anghyongNakhap, BagbongDadaki , IbchuNakhap, SamnamCheongjang and KangryeokSamcheok) , which have been developed in Korea, were employed in transformation experiments. Seeds from 5 cultivars were sterilized for 15 min. in 5% NaOCl solution and washed 3 times for 15 min. with sterilized DW. The sterilized seeds were placed on germination media containing 1/2 MSMS (Murashige & Skoog medium including Minimal Salts), 2% sucrose and 0.6% agar and then cultured to germinate seed for 4 days at 25 ⁇ 1 ° C under dark condition. Thereafter, seedlings without true leaf were selected and cotyledon and hypocotyl were taken therefrom.
  • MSMS Middle & Skoog medium including Minimal Salts
  • a suitable medium composition for regeneration of cotyledon or hypocotyl 10 types of media were prepared with a combination of cytokinin (BAP, zeatin and kinetin) and auxin (NAA) . See Table 1.
  • BAP cytokinin
  • NAA auxin
  • the basal media containing MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins), 500 g/ll of MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate), 3% sucrose and 0.6% agar were employed for regeneration.
  • each of 20 individuals was placed on each medium above and cultured for 30 days at 26 ⁇ 1 ° C and 8,000 lux under the condition of 16 hrs/8 hrs (light/dark) , followed by examination of regeneration rate and average number of regenerated shoots .
  • the regeneration rate was calculated from percentage of ratio of the number of regenerated section to total number of section placed and the average number of regenerated shoot was calculated from percentage of ratio of the number of regenerated shoot to the number of regenerated section.
  • the results are summarized in Table 2.
  • the regeneration rate and the ability in shoot formation were exhibited in the wide range of 0-90% and 1.0-3.1, respectively, depending on cultivar used.
  • BagbongDadaki showed the most excellent regeneration rate and ability in shoot formation.
  • BAP is the most suitable in view of regeneration rate and ability in shoot formation.
  • NAA at relatively low cone. (0.01 mg/1) , was found to give rise to higher regeneration rate and ability in shoot formation compared to the results from no addition and high cone. (0.1 mg/1) .
  • the medium optimal for transformation contains BAP (2 mg/1) as cytokinin and NAA (0.01 mg/1) as auxin.
  • Example 3 In an effort to evaluate the effect of placing method on regeneration rate, the cotyledons in whole, which exhibited the highest reproduction rate in Example 3, were placed on media 2 of Example 2 in insertion or placing at full length, followed by determining the regeneration rate in the same manner as Example 2.
  • CuS0 4 and casein hydrolysates which are generally used in the art for enhancing a regeneration of plants belonging to Cucurbitaleae .
  • Media 2 in Example 2 containing various concentrations of CuS0 and casein hydrolysates were prepared and the regeneration rate was determined in the same manner as Example 2.
  • the regeneration method according to this invention may avoid the use of CuS0 4 and casein hydrolysates in regeneration media.
  • phosphinithricin acetyl transferase gene was cultured for 18 hrs in super broth (37 g/i Brain heart infusion broth (Difco) , 0.2% sucrose, pH 5.6) and the cotyledons were immersed into the media followed by mixing for 10 min.
  • coculturing with Agrobacterium tumefaciens and cotyledons was carried out for 4 days at 4 ° C.
  • the cotyledons inoculated with Agrobacterium tumefaciens were cultured for 4 weeks at 26 ⁇ 1 ° C and 8,000 lux under 16 hrs/8 hrs (day/night) condition in media containing 2 mg/1 BAP, 0.01 mg/1 NAA, 500 mg/1 carbenicillin and 100 mg/1 kanamycin in addition to MS-B5, 0.5 g/1 MES, 3% sucrose and 0.4% phytagel .
  • the regenerated shoots were transferred to the rooting medium containing NAA (0. 0.01 or 0.1 mg/1), 100 mg/1 kanamycin and agar (0.4%, 0.6% and 0.8%) and further cultured at 26+l ° C and 8,000 lux under 16 hrs/8 hrs (day/night) condition to obtain final transformants with roots, which were determined as Example 7 ' .
  • the coculture for 2 days at room temperature (25 ° C) represented much higher regeneration and transformation rate than the coculture for 4 days at lower temperature (4 ° C) . Furthermore, the cotyledons cocultured at lower temperature were not regenerated and became necrotic with etiolation.
  • Fig. 3 demonstrates the difference between successful transformant and non-transformant which was found not to generate root and become necrotic with etiolation.
  • panels A and B represent transformant and non- transformant , respectively.
  • the regeneration rate appeared high but the transformation rate was found to be about 0. Moreover, the selection medium containing 100 mg/1 of kanamycin showed about 30% of regeneration rate and about 3% of transformation rate, and that with 200 mg/1 of kanamycin revealed about 3% of regeneration rate and about 0% of transformation rate. Consequently, it can be understood that the preferred concentration of kanamycin is about 100 mg/1 in the selection medium.
  • the preferred concentration of NAA in rooting medium is about 0.01 mg/1.
  • the rooting medium with relatively low agar (0.4% and 0.6%) was likely to result in normal roots with feasibility; but in the case of the rooting medium containing above 0.8% agar, the elongation of roots was remarkably retarded, thicker/shorter roots were formed and rooting was likely to occur out of medium (See Fig. 4) .
  • panels A and B represent rooting patterns in media with 0.6% and 0.8% agar, respectively.
  • Example 7-1 GUS Analysis
  • PCR For template in PCR, the isolation of genomic DNA from plant was performed using the method described by Edwards K., et al . (Nucleic Acids Research, 19: 1349(1991)).
  • the primers for PCR were designed to have complementary sequence to pat gene of the vector in Agrobacterium tumefaciens : forward primer, 5' -AGA CCA GTT GAG ATT AGG CCA G-3' and reverse primer, 5' -GCC TCA TGC AAC CTA ACA GA-3'.
  • the PCR using Taq polymerase was performed in such a manner that pre-denaturation at 96 ° C for 2 min. and denaturation at 94 ° C for 1 min.
  • PCR product was subject to electrophoresis on 1.0% agarose gel (Fig. 5b) .
  • lanes M, 1, 2 and 3 show 1 kb ladder, PCR product of plasmid carrying pat gene as positive control, PCR product of genomic DNA from non- transformed Cucumis sativus and PCR product of genomic DNA from transformed Cucumis sativus, respectively.
  • the PCR product of genomic DNA from Cucumis sativus transformed according to this invention exhibited 0.5 kb DNA band corresponding to pat gene, which demonstrates the successful transformation.

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Abstract

La présente invention concerne une méthode de préparation de Cucumis sativus transformé au moyen d'Acrobacterium tumefaciens, notamment, une méthode de préparation consistant à (a) inoculer un cotylédon à partir de Cucumis sativus avec Acrobacterium tumefaciens hébergeant un vecteur approprié, (b) à placer le cotylédon inoculé sur un support contenant BAP (6-benzylaminopurine) et NAA (acide acétique d'α-naphthalène) et à cultiver le cotylédon inoculé pour obtenir des pousses régénérées et, (c) à cultiver ces pousses régénérées sur un milieu d'enracinement de manière à obtenir Cucumis sativus.
PCT/KR2002/001461 2002-01-09 2002-08-01 Methode de preparation de cucumis sativus transforme Ceased WO2003057889A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2006019205A1 (fr) * 2004-08-17 2006-02-23 Nexgen Biotechnologies, Inc. Methode de preparation de luffa cylindrica roem transforme
CN103098714A (zh) * 2013-02-28 2013-05-15 上海交通大学 提高黄瓜子叶节离体再生及转化率的方法
CN113412786A (zh) * 2021-06-17 2021-09-21 宁波市农业科学研究院 一种促进葫芦子叶再生的培养基及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020031139A (ko) * 2002-03-28 2002-04-26 임학태 무 형질전환 방법과 그에 의해 생산된 만추대성 무품종 육성

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988002399A1 (fr) * 1986-10-01 1988-04-07 The Plant Cell Research Institute, Inc. Procedes de regeneration regulee de plantes de l'espece cucumis in vitro a partir de tissus d'explants
WO1990003725A1 (fr) * 1988-10-11 1990-04-19 The Upjohn Company Transformation de concombre par agrobacterium tumafaciens et regeneration de plantes de concombre transformees
JPH05276844A (ja) * 1992-03-31 1993-10-26 Saitama Genshiyu Ikuseikai:Kk 形質転換されたキュウリ種植物の作出方法
EP0952224A2 (fr) * 1998-04-14 1999-10-27 Korea Institute Of Science And Technology Procédé de préparation de concombre transgénique contenant de hauts niveaux de superoxyde dismutase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988002399A1 (fr) * 1986-10-01 1988-04-07 The Plant Cell Research Institute, Inc. Procedes de regeneration regulee de plantes de l'espece cucumis in vitro a partir de tissus d'explants
WO1990003725A1 (fr) * 1988-10-11 1990-04-19 The Upjohn Company Transformation de concombre par agrobacterium tumafaciens et regeneration de plantes de concombre transformees
JPH05276844A (ja) * 1992-03-31 1993-10-26 Saitama Genshiyu Ikuseikai:Kk 形質転換されたキュウリ種植物の作出方法
EP0952224A2 (fr) * 1998-04-14 1999-10-27 Korea Institute Of Science And Technology Procédé de préparation de concombre transgénique contenant de hauts niveaux de superoxyde dismutase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 199347, Derwent World Patents Index; AN 1993-373516 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006019205A1 (fr) * 2004-08-17 2006-02-23 Nexgen Biotechnologies, Inc. Methode de preparation de luffa cylindrica roem transforme
CN103098714A (zh) * 2013-02-28 2013-05-15 上海交通大学 提高黄瓜子叶节离体再生及转化率的方法
CN113412786A (zh) * 2021-06-17 2021-09-21 宁波市农业科学研究院 一种促进葫芦子叶再生的培养基及其应用

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