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  • 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
  • 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
  • A01H4/002—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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
  • A01H6/342—Citrullus lanatus [watermelon]
• • C—CHEMISTRY; METALLURGY
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  • 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
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  • 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 Ci trullus vulgaris, more particularly, relates to a method of preparing a transformed Ci trullus vulgaris using Acrobacterium tumefaciens and a transformed Ci trullus vulgaris prepared therefrom.
• Watervulgarisns ( Ci trullus vulgaris L.) belonging to Cucurbitaceous, are cultivated primarily in the North Africa and the Southeast Asia such as Korea, Japan, and China. The China has a cultivation area for Watervulgarisns which amounts to more than 60-70% of that of the whole world. Thus, the China has become a major target in export of watervulgarisn seed.
• Watervulgarisns have been consumed chiefly from early summer to early autumn, but recently, as a result of developed technologies for cultivation using facilities starting from late in the 1980s, a production of watervulgarisns throughout the year has been permitted and consumption thereof has been made in all seasons.
• the total cultivation area of watervulgarisns is 40,000 ha which reaches to 11.9% of Korean vegetable cultivation area in 1999, and its continuous increase will be expected.
• the yield of watervulgarisns in raising outdoors is 260 thousand tons and that in raising by facilities is 670 thousand tons, and the total yield reaches to 930 thousand tons in 1999.
• a breeding of watervulgarisn has been made by routine and conventional breeding methods which has some problems: (a) having technological obstacles in enhancement of low- temperature pollen elongation, a construction of seed- gathering system by male sterilities, an improvement of sugar content, a development of seedless breeds, a an enrichment of storage and transport, a quality control of productive seeds and the like; (b) requirement for wider cultivation areas, higher cost and longer period resulting in incapable of satisfying consumer demand rapidly; and (c) difficulty in selection and fixation of cultivar with desirable traits .
• the genetic engineering technologies have been requested to develop novel breeds of watervulgarisns.
• the present inventors have made intensive research to resolve the need in the art and as a result, we have completed the present invention by establishing a novel method for transformation of Cucumis melo such as a germination condition of seeds, a coculturing method with Agrobacterium tumefaciens and a unique composition of a regeneration medium.
• a preparation of a transformed Cucumis melo with AsrroJbacterium tumefaciens could be done more effectively in shorter time.
• Agrobacterium tumefaciens Agrobacterium tumefaciens .
• Fig. 1 represents a photograph demonstrating variation of regeneration ability of cotyledon from Cucumis vulgaris depending on germination time;
• Fig. 2 represents a genetic map of binary vector pRD400 used in this invention
• Fig. 3 represents a photograph showing in vi tro growth and rooting patterns of Cucumis vulgaris according to this invention.
• Fig. 4 represents a gel photograph showing the results of PCR elucidating transformed Cucumis vulgaris according to this invention.
• a method for preparing a transformed Cucumis vulgaris which comprises the steps of: (a) inoculating a tissue of cotyledon or hypocotyl from Cucumis vulgaris with Agrobacterium tumefaciens harboring a vector, in which the vector is capable of inserting into a genome of a cell from Cucumis vulgaris and contains the following sequences: (i) a replication origin operable in the cell from Cucumis vulgaris; (ii) a promoter capable of promoting a transcription in the cell from Cucumis vulgaris; (iii) a structural gene operably linked to the promoter; and (iv) a polyadenylation signal sequence; and
• a method for preparing a transformed Cucumis vulgaris which comprises the steps of: (a) germinating a seed of Cucumis vulgaris in a germination medium by dark culture for 2-6 days and successive light culture for 12-30 hours; (b) inoculating a tissue of cotyledon from Cucumis vulgaris formed by germination with Agrobacterium tumefaciens harboring a vector, in which the vector is capable of inserting into a genome of a cell of cotyledon from Cucumis vulgaris and contains the following sequences: (i) a replication origin operable in the cell from Cucumis vulgaris; (ii) a promoter capable of promoting a transcription in the cell from Cucumis vulgaris; (iii) a structural gene operably linked to the promoter; and (iv) a polyadenylation signal sequence; and (c) regenerating the inoculated tissue from Cucumis vulgaris in a medium containing 6.0-1.0
• the present invention will be described in more detail as follows: I . Preparation of Starting Material for Transformation
• the preferred explant for transformation includes leaf, stem and petiole, but not limited to.
• the explant 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
• the medium for seed germination comprises 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 for seed germination include nicotine, thiamine and pyridoxine .
• the medium for seed germination in this invention may further contain MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate) as buffering agent for pH change and agar as solid support. The medium is unlikely to contain plant growth regulators.
• the period for dark culture is critical for seed germination.
• the dark culture is performed for 2-6 days and the light culture for 12-30 hr. More preferably, the period for dark culture is 3-5 days and that for light culture is 20-28 hr. Most preferably, the period for dark culture is 4 days and that for light culture is 24 hr. Regeneration of explant is largely dependent on the period for dark culture as demonstrated in Example.
• the illumination intensity for light culture is usually 3000- 5000 lux. It is preferred that seed germination is performed at the temperature of 25 ⁇ 1 ° C.
• 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.
• the binary vector useful in this invention carries: (i) a promoter capable of forming RNA operating in the cell from Cucumis vulgaris; (ii) a structural gene operably linked to the promoter; and (iii) 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, 1' promoter, 2' promoter and promoter nopaline synthetase (nos) 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., ver
• Inoculation of the explant with Agrobacterium tumefaciens involves procedures known in the art. Most preferably, the inoculation involves dissecting cotyledon with growth point removed and immersing these sections in culture of Agrobacterium tumefaciens to coculture, thereby inoculating the cotyledon with Agrobacterium tumefaciens .
• the Acrrojbacterium tumefaciens is infected through dissected side. Such method is developed to shorten a coculturing time remarkably. This effect may be accomplished using only two sections of cotyledon. In the present invention, the period for coculturing is 1 hr-5 min, more preferably 20-7 min.
• 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.
• the auxin for example, 1-naphthalene acetic acid, indole acetic acid, (2 , 4-dichlorophenoxy) acetic acid
• the amount of cytokinin in the regeneration medium is 4.0-1.5 mg/1, the most preferably 2.0 mg/1.
• the amount of the auxin is preferably 0.02-0 mg/1, the most preferably 0 mg m/1.
• 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
• the culture in regeneration medium is performed under the following conditions: 25 ⁇ 1 ° C; 16 hr:8 hr (light culture : dark culture) .
• the period necessary for culture varies widely, preferably about 3-6 weeks.
• Culturing according to the conditions described above allows successfully a regeneration of shoots through callus formation from the transformed explant of Cucumis vulgaris on the medium.
• the transformed Cucumis vulgaris 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
• 1-naphthalene acetic acid, indole acetic acid and (2,4- dichlorophenoxy) acetic acid and the most preferable is indole acetic acid.
• antibiotics to select transformed Cucumis vulgaris are not contained in the rooting medium of this invention.
• the transformed Cucumis vulgaris 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 vulgaris, PCR is carried out to elucidate exogenous gene incorporated into a genome of Cucumis vulgaris 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) .
• a method for preparing a transformed Cucumis vulgaris which comprises the steps of: (a) germinating a seed of Cucumis vulgaris in a germination medium by dark culture for 3-5 days and light culture for 20-28 hours, in which the germination medium contains a nutrient basal medium selected from the group consisting of MS, B5, LS,
• the method of this invention which is developed for producing a transformed Cucumis vulgaris, as exemplified and demonstrated in Examples below, exhibits much higher transformation and regeneration efficiency with shorter period for manipulation, giving rise to production of transformed Cucumis vulgaris having desirable traits with higher reproducibility.
• EXMPLE 1 Preparing of Explants 4 cultivars of Cucumis vulgaris (Apollo, SacheolKul, SuperKeumcheon and Keumbo) , which have been developed in Korea, were employed in regeneration and transformation experiments. Seed coats from seeds of 4 cultivars kept at 4 ° C were removed with physical method, sterilized with occasional agitation in 4% NaOCl solution for 30 min and washed 4 times with DW. The sterilized seeds were placed on germination media containing 1/2 MSMS, 1.0% sucrose and 0.6% agar and then cultured to germinate seed for 4 days at 25 ⁇ 1°C under dark culture condition. Thereafter, the resulting cotyledons or hypocotyls were used as samples.
• EXAMPLE 2 Regeneration of Explant Tissue To prepare a suitable medium composition for regeneration of cotyledon or hypocotyl, the cotyledon and hypocotyl obtained above were placed on 4 types of media containing ingredients described in Table 1, and cultured for 4 weeks at 25 ⁇ 1 ° C 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 basal media containing MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins), 500 m /# of MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate), 3% sucrose and 0.6% agar were employed for regeneration.
• hypocotyl the regeneration rate was much lower, shoots were rarely regenerated, and callus formed were finally dead.
• the medium optimal for transformation contains BAP (2 mg/1) without auxin.
• Example 2 With the cotyledon showing the most excellent regeneration ability evaluated in Example 2, a relationship between a harvest time of cotyledon and a regeneration ability was examined as follows: First, seeds of SuperKeumcheon were treated in the same manner as described in Example 1, then cultured for 1, 2, 3, 4 and 5 days, respectively, at 25 ° C+1 ° C under dark condition and additionally cultured at 25°C ⁇ 1 ° C and 4,000 lux for 1 day under light condition. Thereafter, the cotyledons were taken and placed on the medium containing 2 mg/1 of BAP representing the most outstanding regeneration rate in Example 2 , followed by culturing for 4 weeks to measure the regeneration rate and the number or regenerated shoots .
• panels A, B, C and D represent regeneration patterns of cotyledon of Cucumis vulgaris dark-cultured for 1 day, 2 days, 4 days and 5 days, respectively.
• the group cultured for 1-2 days had some deviation among sections and showed to generate a multitude of shoots, and the group cultured for 4 days gave rise to 2-3 regenerated shoots which were derived from callus generated in dissected region.
• the group dark-cultured for 5 days showed the decrease by less than half in regeneration ability. Therefore, the most suitable germination period is 4 day-dark culture.
• Agrobacterium tumefaciens (Agrobacterium tumefaciens GV3101(Mp90) ; Plant-cell -rep. , 15(11)799-803(1996)) transformed with binary vector pRD400 (Fig. 2) were cultured in super broth (37 g/1 brain heart infusion broth (Difco) , 0.2% sucrose, pH 5.6) containing 200 ⁇ M of acetosyringone for 18 hrs, the resulting medium was diluted with Infection broth containing 1 mg/1 of 2,4-D and ingredients of Table 3 and DMSO solution to the ratio of 1:37:2.
• super broth 37 g/1 brain heart infusion broth (Difco) , 0.2% sucrose, pH 5.6
• LB and RB represent left and right border of T-DNA, respectively
• MCS represents multiple cloning site
• Tnos and Pnos represent termination sequence and promoter sequence of nos, respectively
• nptll represents neomycin phosphotransferase II sequence.
• the cotyledon was placed in a coculturing medium containing 2 mg/1 of BAP (4.04 g/1 MSB5, 3.0% sucrose, 0.5 g/1 MES, 0.6% agar, pH 5.6) and cultured at 4,000 lux under 16 hour-light culture condition at 25 ° C ⁇ 1 ° C for 2 days.
• Cultured cotyledon was placed in the regeneration medium of Table 3 containing solely 500 mg/1 of carbenicillin and pre-cultured at 25 ° C ⁇ 1 ° C for 7 days to induce generation of shoots. Then, the shoots induced were cultured in the selection medium of Table 3 containing 200 mg/1 of kanamycin for 4 weeks followed by fixing the regenerated shoots selected with kanamycin.
• Agrobacterium tumefaciens began to form a multiple of callus after 10 days , and to form a multiple of shoots after 2-3 weeks. However, all shoots formed were not found to be transformed and in the case of subculturing the shoots regenerated in selection ' medium or rooting medium, most shoots with no transformation were likely to be whitened or withered. On the other hand, the transformed shoots were found to form roots after about 2-3 weeks and the rooting was also observed even in several subculture (Fig. 3) . In Fig.
• panel A is a photograph of transformed Cucumis vulgaris which was grown in vi tro (means that Cucumis vulgaris transformant can fix roots normally and proliferate on media containing antibiotics)
• panel B is a photograph showing rooting patterns on selection media containing antibiotics.
• Genomic DNA for PCR analysis was obtained from transformant selected in Example 4 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 nptll gene (encoding neomycin phosphotransferase II) of the vector in Agrobacterium tumefaciens : forward primer, 5' -GAT GGA GTG CAC GCA GGT-3' and reverse primer, 5'-TCA GAA GAA CTC GTC AAG-3'.
• the mixture consisting of 2.5 ⁇ l of lOx reaction buffer (Boeringher anheim, containing 2.5 roM Mg 2+ , pH 7.5), 2.0 ⁇ l of mixture of dNTPs (10 nM) , 1 ⁇ l of template DNA and 0.25 ⁇ l of Taq polymerase (5 U/ ⁇ l) per 25 ⁇ l of total reaction solution was used.
• the PCR was performed in such a manner that pre-denaturation at 94 ° C for 1 minute and denaturation at 94 ° C for 1 minute were done consecutively, and total 35 cycles were done in which each cycle is composed of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute and extension at 72 ° C for 2 minute, followed by final extension at 72 °C for 10 minute.
• the PCR product was subject to electrophoresis on 1.0% agarose gel
• lane M shows 1 kb ladder
• lanes 1-5 show PCR products of Cucumis vulgaris transformed according to this invention
• lane 6 shows PCR products of Cucumis vulgaris not transformed.
• novel Cucumis vulgaris with desirable traits can be obtained with higher regeneration and transformation rate.
• the method of the present invention can shorten a transformation time remarkably.

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