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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)
  • C12N15/8201—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
• • 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
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/008—Methods for regeneration to complete plants
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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)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• • 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

Definitions

• the present invention relates to a method for preparing a transformed Luffa cylindrica Roem, more particularly, relates to a method of preparing a transformed
• Luffa cylindrica Roem. belonging to Cucurbitaceae is originated from the tropical Asia, and have been cultivated widely in Korea.
• Luffa cylindrica Roem. has alternate and palmate leaves comprising petiole. The leaf is 13 and 30 cm in length and width respectively and has the acute-end lobe. It is hairless and has serrated edges.
• the flower of Luffa cylindrica Roem. is yellow and blooms on August-September.
• Luffa cylindrica Roem. is monoecious and the inflorescence of the male flower is a raceme and one female flower exists. Its fruit, a gourd, is green and has a large cylinder-like shape. The outside of the fruit has vertical lines and a reticulate develops inside of the flesh.
• Luffa cylindrica Roem. is annual herb and grows about 12 cm long. The stem is green and pentagonal and grows climbing other physical solid.
• Young fruits are edible and matured fibers are generally used in washing ships and decks and manufacturing slippers or baskets and used as shoes mats, inner cloth of bonnet.
• Luffa cylindrica Roem. has effect on the treatment of fever, enteritis and swell etc..
• the extracts from vines alive are used as an ingredient in cosmetics and medicine.
• the Seed of Luffa cylindrica Roem. containing 40% oil is used as oil sources and oil cake is used as fertilizer and feed.
• Such crops can be used for the preparation of useful proteins; however, most of them are protected by patents. Therefore, there is a limitation in the commercial usage of such crops.
• the present invention has been completed by establishing a germination condition of seeds of Luffa cylindrica Roem. which is usable as a protein-producing host; a coculturing method with Agrobacterium tumefaciens; and an auxin-free composition of a regeneration medium.
• a preparation of a transformed Luffa cylindrica Roem. with Agrobacterium tumefaciens could be done more effectively in shorter time. Accordingly, it is an object of this invention to provide a method for preparing a transformed Luffa cylindrica Roem. using Agrobacterium tumefaciens.
• a method for preparing a transformed Luffa cylindrica Roem which comprises the steps of: (i) inoculating a cotyledon from Luffa cylindrica Roem with Agrobacterium tumefaciens harboring a vector, in which the vector is capable of inserting into a genome of a cell from Luffa cylindrica Roem and contains the following sequences: (a) a replication origin operable in the cell from Luffa cylindrica Roem; (b) a promoter capable of promoting a transcription in the cell from Luffa cylindrica Roem; (c) a structural gene operably linked to the promoter; and (d) a polyadenylation signal sequence; (ii) placing the inoculated cotyledon on a medium containing BAP (6-benzylaminopurine) and culturing the inoculated cotyledon
• Agrobacterium tumefaciens in more efficient manner.
• 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 MA, B5, LS, N6 and White's, energy source and vitamins, but not limited to. Sugars are more preferable as energy sources and sucrose is the most preferable. It is preferred that vitamins for seed germination include nicotinic acid, 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 for seed germination is unlikely to contain plant growth regulators.
• 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 Luffa cylindrica Roem 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: (a) a replication origin operable in the cell from Luffa cylindrica Roem; (b) a promoter capable of promoting a transcription in the cell from Luffa cylindrica Roem; (c) a structural gene operably linked to the promoter; and (d) a polyadenylation signal sequence.
• "operably linked" in the term ⁇ a structural gene operably linked to the promoter” means that the promoter regulates the expression of the structural gene.
• a polyadenylation signal sequence means the sequence regulating the polyadenylation of 3' end of mRNA produced in eukaryotic cells .
• a preferable polyadenylation signal sequence is tumor inducing (Ti) plasmid genes of Agrobacterium, such as the nopaline synthase (NOS) gene and plant genes like the 7s soybean storage protein genes and the pea E9 small subunit of the RuBP carboxylase gene.
• Ti tumor inducing
• NOS nopaline synthase
• 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) .
• 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 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
• cosmetic raw material e.g. albumin, antibiotic peptide
• Inoculation of the explant with Agrobacter ⁇ um tumefaciens involves procedures known in the art. For example, cotyledon with removed growth point is immersed in medium of Agrobacterium tumefaciens to coculture, thereby inoculating the cotyledon with Agrobacterium tumefaciens.
• acetosyringone is employed in the coculturing to promote infection of Agrobacterium tumefaciens into an 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 more preferable as energy sources and sucrose is the most preferable. It is preferred that vitamins in regeneration medium include nicotinic acid, 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 regeneration medium does not contain auxin such as NAA ( ⁇ -naphthalene acetic acid) , indole acetic acid and (2 ,4-dichlorophenoxy) acetic acid, which is those distinguishing the medium of present invention from the conventional one.
• auxin such as NAA ( ⁇ -naphthalene acetic acid) , indole acetic acid and (2 ,4-dichlorophenoxy) acetic acid, which is those distinguishing the medium of present invention from the conventional one.
• the amount of BAP in the regeneration medium ranges from 1 to 4 mg/1. If the amount is less than 1 mg/1 or exceeds 4 mg/1, the regeneration rate tends to drop sharply.
• the amount of BAP ranges 1.0 to 2.5 mg/1 and most preferably 2.0 mg/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
• 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 culture temperature is 25 ⁇ 1 ° C
• light culture time dark culture time is 16 hours: 8 hours.
• Culturing period is changeable, 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 Luffa cylindrica Roem on the medium.
• the transformed Luffa cylindrica Roem 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 more preferable as energy sources and sucrose is the most preferable. It is preferred that vitamins in the rooting medium include nicotinic acid, 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 amount of NAA is in the range of from 0.005 to 0.03 mg/1.
• 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 Luffa cylindrica Roem produced according to the present invention may be confirmed using procedures known in the art. For example, using DNA sample from tissue of transformed Luffa cylindrica Roem, PCR is carried out to reveal exogenous gene incorporated into a genome of Luffa cylindrica Roem 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 Luffa cylindrica Roem which comprises the steps of: (i) inoculating an intact cotyledon from Luffa cylindrica Roem with Agrobacterium tumefaciens harboring a vector, in which the vector is capable of inserting into a genome of a cell from Luffa cylindrica Roem and contains the following sequences: (a) a replication origin operable in the cell from Luffa cylindrica Roem; (b) a promoter capable of promoting a transcription in the cell from Luffa cylindrica Roem; (c) a structural gene operably linked to the promoter; and (d) a polyadenylation signal sequence; (ii) placing the inoculated cotyledon on a medium containing 1.0-2.5 mg/1 of BAP (6- benzylaminopurine) by insert and
• the method of this invention which is developed for producing a transformed Luffa cylindrica Roem, as exemplified and demonstrated in Examples below, exhibits much higher transformation and regeneration efficiency, leading to production of transformed Luffa cylindrica Roem having desirable traits with higher reproducibility.
• Fig. 1 shows regenerated cotyledons from Luffa cylindrica Roem according to this invention
• Fig. 2 represents a genetic map of binary vector pRD320 used in this invention
• Fig. 3 demonstrates comparison between transformant (panel A) of this invention and non-transformant (panel B) ;
• Fig. 4a shows the results of GUS analysis confirming transformant according to this invention.
• Fig. 4b shows the results of PCR elucidating transformant according to this invention.
• Luffa cylindrica Roem' s Seeds 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 media were prepared with a combination of cytokinin (BAP, 6-Benzylaminopurine) and auxin (NAA, ⁇ -naphthalene acetic acid) (See Table 1) .
• the basal media containing MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins) , 500 mg/i of MES (2- (N- Morpholino) ethanesulfonic acid Monohydrate) , 3% sucrose and 0.6% agar were employed for regeneration.
• each of 12 individuals was placed on each medium above and cultured for 30 days at 25 ⁇ 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 regeneration rate and the ability in shoot formation were exhibited in the wide range of 6.67-40% and 0.49-1.0, respectively, depending on the media used.
• the hormone-combination experiment showed that at 1-4 mg/1 concentration, BAP was found to accomplish higher regeneration rate and ability in shoot formation, while at more than 4 mg/1, to decrease regeneration rate.
• panels A and B represent shoots regenerated from cotyledons at 10 and 20 days, respectively from placing on media.
• the medium optimal for transformation contains only BAP (2 mg/1) as cytokinin.
• 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 medium 5 of Example 2 in insertion or placing at full length, followed by determining the regeneration rate in the same manner as Example 2.
• coculturing medium MSB5 medium with 2 mg/1 of BAP
• the Agrobacterium tumefaciens and cotyledons was cocultured for 3 days at 25 ° C under conditions for light culture.
• the cotyledons inoculated with Agrobacterium tumefaciens were cultured for 4 weeks at 25+l ° 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 various concentrations of kanamycin in addition to MS-B5, 0.5 g/1 MES, 3% sucrose and 0.4% phytagel .
• 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 25% of regeneration rate and about 5% of transformation rate, and that with 200 mg/1 of kanamycin revealed about 2.5% 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. In the rooting medium, as NAA concentration became higher, the roots generated were found to be thicker and shorter, and the period required for rooting to be shorter.
• the preferred concentration of NAA in rooting medium is about 0.01 mg/1.
• Example 5 The transformants produced in Example 5 were confirmed as follows :
• 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. 4b) .
• lanes 1, 2 and 3 show molecular marker, PCR product of plasmid carrying pat gene as positive control, PCR product of genomic DNA from non- transformed Luffa cylindrica Roem repectively, and lanes 4-10 show PCR product of genomic DNA from transformed Luffa cylindrica Roem, respectively.

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