WO2006019205A1

WO2006019205A1 - Method for preparing transformed luffa cylindrica roem

Info

Publication number: WO2006019205A1
Application number: PCT/KR2004/002745
Authority: WO
Inventors: Sun Lee; Jae Geun Yoo
Original assignee: Nexgen Biotechnologies, Inc.
Priority date: 2004-08-17
Filing date: 2004-10-28
Publication date: 2006-02-23
Also published as: KR20060016313A; KR100711144B1

Abstract

The present invention relates to a method of 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 to obtain regenerated shoots; and (iii) culturing the regenerated shoots on a rooting medium to obtain the transformed Luffa cylindrica Roem.

Description

METHOD FOR PREPARING TRANSFORMED LUFFA CYLINDRICA ROEM

FIELD OF THE INVENTION

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 using Acrobacterium tumefaciens and a transformed Luffa cylindrica Roem prepared therefrom.

BACKGROUND OF THE INVENTION

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. In oriental medicine, 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.

Gene manipulation of crops through plant-gene engineering has been progressed remarkably and at present, it enables us to culture the tissues of most crops in flask and to transform them with foreign genes.

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.

Although the transformation of other crops of Cucurbitaleae such as watermelons, melons and cucumbers has been reported (Choi, P. S. et al . , Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens. Plant Cell Rep. 13:344-348(1994) ; Dong J. Z. et al . , Transformation of melon{Cucumis MeIo L.) and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Biotechnology 9:858-863(1991) ; and Lee K. W. et al . , Expression of TMV Coat Protein Gene in Transgenic Tobacco and Cucumber Plants Using Binary Vector System of Agrobacterium tumefaciens. Korean Journal of Plant Tissue Culture 23(4)205-210(1996)) , the transformation of Luffa cylindrica Roem. has not been developed yet.

Therefore, the present inventors made a research on Luffa cylindrical in order to develop commercially useful crop and established tissue-culturing condition for Luffa cylindrica Roem. Finally transformed Luffa cylindrica Roem. has been prepared through the regeneration and transformation of Luffa cylindrica Roem..

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. According to the present method, 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.

It is another object of this invention to provide a transformed Luffa cylindrica Roem. prepared with the Agrobacterium tumefaciens. DETAILED DESCRIPTION OF THE INVENTION

In one aspect of this invention, there is provided 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 to obtain regenerated shoots; and (iii) culturing the regenerated shoots on a rooting medium to obtain the transformed Luffa cylindrica Roem.

The present inventors have made extensive research with Luffa cylindrica Roem for developing an efficient transforming method and finally established conditions for regeneration with higher frequency and transformation with

Agrobacterium tumefaciens in more efficient manner.

The present invention will be described in more detail as follows. I . Preparation of Starting Material for Transformation

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.

II . Seed Germination According to a preferred embodiment of this invention, 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. In addition, 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.

III . Preparation of Plant Tissue for Transformation

In this invention, 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.

IV. Inoculation with Agrobacterium tumefaciens

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. In the present invention, "operably linked" in the term ^λΛa structural gene operably linked to the promoter" means that the promoter regulates the expression of the structural gene.

In the present invention, the term "a polyadenylation signal sequence" means the sequence regulating the polyadenylation of 3' end of mRNA produced in eukaryotic cells .

According the preferable embodiment, 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.

In addition to this, it is preferred that 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 β- 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) .

Inoculation of the explant with Agrobacterϊum 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. Preferably, acetosyringone is employed in the coculturing to promote infection of Agrobacterium tumefaciens into an explant cell.

V. Regeneration

It is necessary that explant tissue, which is transformed with Agrobacterium tumefaciens, be regenerated in regeneration medium with strictly controlled ingredients and quantities thereof. 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. In addition, 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.

Furthermore, the regeneration medium does not contain auxin such as NAA (α-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.

Preferably, 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.

More preferably, the amount of BAP ranges 1.0 to 2.5 mg/1 and most preferably 2.0 mg/1.

According to a preferred embodiment of this invention, the medium further contains antibiotics (e.g. carbenicllin, kanamycin, spectinomycin or hygromcin) for selection of transformed explant .

In the case of using kanamycin as selective marker, 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 most preferably, 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.

VI . Rooting 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. In addition, the rooting medium may further contain MES (2- (N- Morpholino) ethanesulfonic acid Monohydrate) as buffering agent for pH change and agar as solid support.

As plant growth regulator, 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. Preferably, 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 corpulent leading to no occurrence of rooting.

More preferably, the amount of NAA is in the range of from 0.005 to 0.03 mg/1.

Alternatively, where 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.

VII . Confirmation of Transformation 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) . Where the vector harbored in Agrobacterium tumefaciens contains a gene encoding β-glucuronidase, the tissue from cotyledon regenerated is immersed in substrate solution such as X-gluc (5-Bromo-4-Chloro-3-Indole-β-D-Glucuronic Acid) so that colorimetric reaction may be observed to confirm the occurrence of transformation. According to the most preferred embodiment of this invention, there is provided 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 culturing the inoculated cotyledon to obtain regenerated shoots; and (iii) culturing the regenerated shoots to obtain the transformed Luffa cylindrica Roem on a rooting medium containing 0.005-0.03 mg/1 of NAA.

In another aspect of the present invention, there is provided a transformed Luffa cylindrica Roem prepared by the methods of this invention described above.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

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; and

Fig. 4b shows the results of PCR elucidating transformant according to this invention.

Examples

Example 1: Preparing of Explants

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. EXAMPLE 2 : Construction of Medium Composition for Regeneration

To construct a suitable medium composition for regeneration of cotyledon or hypocotyl, media were prepared with a combination of cytokinin (BAP, 6-Benzylaminopurine) and auxin (NAA, α-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.

Table 1

Thereafter, 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 results are summarized in Table 2.

Table 2

As shown in Table 2, 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.

In the media containing 2 mg/1 of BAP and No NAA, the highest regeneration rate were exhibited, the average number of shoot regenerated was found to be much larger (1.0 per section) and the period necessary for regeneration was found to be excellent (approx. 20 days) .

All results above were obtained from cotyledon. As to hypocotyl, the regeneration rate was much lower, shoots were rarely regenerated, and callus formed were finally dead. Cotyledons, which were in dark cultivation for 4-5' days, were found to form callus at the region in contact with media, which appeared from about 7 days of culture in the regeneration media; and with the lapse of 15 days, the regenerated shoots emerged from corpulent callus, finally leading to a multitude of shoots (See Fig. 1) .

In Fig. 1, panels A and B represent shoots regenerated from cotyledons at 10 and 20 days, respectively from placing on media. As revealed from the results, as explant for transformation of Luffa cylindrica Roem, cotyledon is the most preferable, the medium optimal for transformation contains only BAP (2 mg/1) as cytokinin.

EXAMPLE 3 : Evaluation of Regeneration Rate Depending on Explant

In an effort to evaluate regeneration rate depending on explant, the cotyledons cut off at 2 mm above from seedlings were placed on the medium 5 which was determined optimal and the regeneration rate and average shoot number thereof were examined in the same manner as Example 2.

From the results, placing with the cotyledon in whole gave rise to higher regeneration rate and average shoot number. Therefore, for regeneration of cotyledon from Luffa cylindrica Roem.regeneration, the cotyledon in whole is preferred than one with upper part removed. EXAMPLE 4: Evaluation of Regeneration Rate Depending on Placing Method

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.

From the results, it can be understood that unlikely to leaf section, placing cotyledon at full length on the medium results in negligible regeneration as well as poor formation of callus. Contrary to regeneration with leaf section, insertion on medium is the most efficient placing method for regeneration of cotyledon from Luffa. cylindrica Roem.

EXAMPLE 5 : Construction of Conditions for Transformation

In order to construct the optimal conditions for transformation, the optimal procedures, periods and temperature for coculturing were examined by use of MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins) media containing 2 mg/1 of BAP.

Firstly, the seeds from the Luffa cylindrica Roem were germinated in the same manner as Example 1 and cotyledons were taken. Agrobacterium tumefaciens GV3101 (Mp90) (Plant- cell-rep. 15 (11) :799-803 (1996) ) harboring pRD320 vector

(Omirulleh, -S. et al. , Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize. Plant. MoI. Biol. Int. J. MoI. Biol. Biochem. Genet. Eng. 21(3) : 415-428(1993)) with gus::nptH fusion gene and pat (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.

Then, in 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. Following coculturing, without performing clean up step, 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 .

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 25±1^°C and 8,000 lux under 16 hrs/8 hrs (day/night) condition to obtain final transformants with roots, which were determined as Example 6. Fig. 3 demonstrates the difference between successful transformant and non-transformant which was found not to generate root and become necrotic with etiolation. In Fig. 3, panels A and B represent transformant and non-transformant, respectively.

Table 3

^x)number of regenerated cotyledons, and ²⁾number of transformant

As indicated in Table 3, in the case of using the selection medium containing 50 mg/1 of kanamycin, 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. In the rooting medium containing 0.1 mg/1 of NAA, it was observed that the stem in contact with medium tended to be corpulent leading to no occurrence of rooting. In the case of no treatment with NAA, the period necessary for rooting was relatively longer and thinner/longer roots without root hairs were frequently induced. Therefore, it can be known that the preferred concentration of NAA in rooting medium is about 0.01 mg/1.

EXAMPLE 6: Confirmation of Transformant

The transformants produced in Example 5 were confirmed as follows :

Example 6-1: GUS Analysis

To measure activity of β-glucuronidase, the tissues from cotyledons regenerated were immersed in X-gluc (5-Bromo-4- Chloro-3-Indole-β-D-Glucuronic Acid) solution and incubated for 24 hrs at 37^°C . The developed colorimetric reaction (blue) was observed with naked eye to confirm the occurrence of transformation (Fig. 4a) . Example 6-2: PCR Analysis

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. were done consecutively, and total 35 cycles were done in which each cycle is composed of denaturation step at 94^°C for 1 min. , annealing at 55^°C for 1 min. and extension step at 72^°C for 2 min., followed by final extension at 72^°C for 10 min. The PCR product was subject to electrophoresis on 1.0% agarose gel (Fig. 4b) . In 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. As shown in Fig. 4b, the PCR product of genomic DNA from Luffa cylindrica Roem transformed according to this invention, exhibited 0.5 kb DNA band corresponding to pat gene, which demonstrates the successful transformation. Even though there are many researches on tissue culture of Cucurbitaceae, no reports, which are directed to regeneration of Luffa cylindrica Roem, have been made. Referring to the results from Examples 5 and 6, it can be fully understood that the most suitable regeneration and transformation conditions of the present invention provides about 20-30% of regeneration rate and about 5% of transformation rate by use of cotyledon of Luffa cylindrica Roem as explant . In conclusion, successful regeneration and transformation of Luffa cylindrica Roem can be obtained by using the present invention.

Claims

What is claimed is:

1. 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, (ϋ) placing the inoculated cotyledon on a medium containing BAP (β-benzylaminopurine) and culturing the inoculated cotyledon to obtain regenerated shoots; and (iii) culturing the regenerated shoots on a rooting medium to obtain the transformed Luffa cylindrica Roem.

2. The method according to claim 1, wherein the cotyledon of step (i) is in whole not dissected one.

3. The method according to claim 1, wherein placing of step (ii) is performed in such a manner that the total parts of the cotyledon are inserted into the medium layer.

4. The method according to claim 1, wherein an amount of BAP in step (ii) is 1.0-4.0 mg/1.

5. The method according to claim 4, wherein the amount of BAP is 1.5-2.5 mg/1.

6. The method according to claim 1, wherein an amount of NAA in the rooting medium of step (iii) is 0.001-0.08 mg/1.

7. The method according to claim 6, wherein the amount of NAA in the rooting medium of step (iii) is 0.005-0.03 mg/1.

8. A method for preparing a transformed Luffa cylindrica Roem, which comprises the steps of:

(i) inoculating an cotyledon in whole 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 cylϊndrica 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 culturing the inoculated cotyledon to obtain regenerated shoots; and (ϋi) culturing the regenerated shoots to obtain the transformed Luffa. cylindrica Roem on a rooting medium containing 0.005-0.03 mg/1 of NAA.

9. A transformed Luffa cylindrica Roem prepared by the method according to any one of claims 1-8.