KR100447920B1 - Method for Preparing Transformed Cucumis sativus L - Google Patents

Abstract

The present invention relates to a method for preparing a cucumber transformant using Agrobacterium tumefaciens and a cucumber transformant prepared therefrom, and more specifically, to (i) be inserted into the genome DNA of a cucumber cell. Infecting cucumber cotyledon with Agrobacterium tumefaciens , which is embedded with a vector having the following sequence: (a) origin of replication operating in cucumber cells; (b) a promoter capable of promoting transcription in cucumber cells; (c) a structural gene sequence encoding a foreign protein functionally linked to said promoter; And (d) a polyadenylic signal sequence that operates in cucumber cells to form polyadenyls at the 3 'end of RNA; (Ii) resuspending the infected cucumber cotyledons in a medium containing BAP (6-Benzylaminopurine) and NAA (α- Naphtalene acetic acid) to form shoots; And (iii) rooting the re-differentiated shoots in a rooting medium containing NAA to form a transformed cucumber, and a method for producing a cucumber transformant and a cucumber transformant prepared therefrom.

Description

Method for preparing cucumber transformants and transformed cucumbers {Method for Preparing Transformed Cucumis sativus L}

The present invention relates to a method for producing a cucumber transformant, and more particularly, to a method for producing a cucumber transformant using Agrobacterium tumefaciens and a cucumber transformant prepared therefrom.

Cucumis Cucumis ( cucumis sativus L), which belongs to the fruit family, is divided into open-field cultivation and greenhouse cultivation, mainly on the continent of Europe. Cucumbers in Asia are in the south and north of China, South China and Northeast Asia. It is estimated to have been introduced about 1,500 years ago in Korea, and it is used as fresh or processed vegetables and widely used as a raw material for cosmetics such as lotion and packs.

Cucumber cultivation area in Korea is gradually decreasing, but the total cultivation area is increasing due to the rapid increase in facility cultivation area. With the expansion of plant cultivation, modernization of facilities, varieties improvement and development of cultivation technology, the total production in 1995 was twice that of 1980.

The most problematic problem when growing cucumber, an important fruit vegetable, is pest. Cucumber pests have been reported fungal, bacterial and viral diseases. Among the pests, diseases caused by fungi and bacteria can reduce the damage through drug control and continuous management, but the damage caused by viruses is currently impossible to control. In addition, since the damage caused by pests is not so small, the method of controlling them is currently the only means by drugs. In particular, as the plant cultivation area increases, the damage caused by various pests is gradually increasing, but no clear solution has been proposed. The reason is that it takes 5-10 years to breed a trait. Therefore, shortening the breeding period by introducing various traits using genetic engineering methods has been proposed as one method to solve the above problems.

To date, there have been ongoing studies on the regeneration and transformation of cucumbers, for example: Rajasekaran et. Al., Flower formation in vitro by hypocotyl explants of cucumber (Cucumis sativus L.) Ann-Bot. London: Academic Press., 52 (3): 417-420 (1983)), leaves (Chee and Tricoli, Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L. Plant-Cell-Rep. Berlin, 7 (4) 274-277 (1988)), Cotyledon (Cade et. Al. Organogenesis and embryogenesis from cucumber ( Cucumis sativus L.) cotyledon-derived callus.HortScience 22: 1130 (1987); Gambley, -RL; Dodd, -WA) Effect of hypocotyl length on morphogenesis of explants of cucumber (Cucumis sativus L.) in vitro.19 (2): 165-169 (1992)), pollen (Lazarte and Sasser, Asexual embryogenesis and plantlet development in anther culture of Cucumis sativus L Cucumber, regeneration, tissue culture.Hortsci.American Society for Horticultural Science.17 (1): 88 (1982)), protoplasts (Jia, -SR et al. Embryogenesis and plant regeneration from cotyledon protoplast culture of cucumber (Cucumis sativus L.). J-Plant-Physiol. 124 (5): 393-398 (1986); Colijn-Hooymans et al .. Competence for regeneration of cucumber cotyledons isrestricted to specific developmental stages. Plant-cell, -tissue-organ-cult. 39 (4): 211-217 (1994)) and the like have been reported to obtain a plant re-differentiated as a sample.

However, according to the methods developed to date, there is still a problem of low re-differentiation rate, re-differentiated plants develop in abnormal form, early flowering and aging in in-flight culture (Ziv and Gadasi, Enhanced embryogenesis) and plant regeneration from cucumber (Cucumis sativus L.) callus by activated charcoal in solid / liquid double-layer cultures.Plan-Sci. 47 (2): 115-122 (1986); Gambley, -RL; Dodd, -WA Effect of hypocotyl length on morphogenesis of explants of cucumber (Cucumis sativus L.) in vitro.19 (2): 165-169 (1992); Kim JW et al., Plant regeneration through organogenesis and somatic embryogenesis of Cucumber ( Cucumis sativus L. Korean J. Plant Tissue Culture, 25 (2): 125-129 (1998).

Thus, many improvements still remain regarding the regeneration and transformation of cucumber plants.

Throughout this specification, a number of articles are referenced and their citations are indicated in parentheses. The disclosures of the cited articles are hereby incorporated by reference in their entirety, so that the level of the technical field to which the present invention belongs and the gist of the present invention are explained more clearly.

Accordingly, an object of the present invention is to provide a method for producing a cucumber transformant by Agrobacterium tumefaciens .

Another object of the present invention to provide a cucumber transformant prepared by the above production method.

1 is a photograph showing the regeneration of cucumber cotyledon according to the present invention;

2 is a genetic map of the binary vector pRD320 used in the present invention;

3 is a photograph comparing a transformant and a non-transformant according to the present invention;

4 is a photograph showing the effect of agar concentration on rooting in the rooting medium;

Figure 5a is a photograph showing the results of the GUS analysis for identifying the transformants prepared according to the present invention; And

Figure 5b is a gel photograph showing the PCR results confirming the melon transformant of the present invention.

According to one aspect of the present invention, the present invention (i) infects the cotyledon of cucumber with Agrobacterium tumefaciens which can be inserted into the genome DNA of cucumber cells and has a vector having the following sequence: The steps of: (a) origin of replication operating in cucumber cells; (b) a promoter capable of promoting transcription in cucumber cells; (c) a structural gene sequence encoding a foreign protein functionally linked to said promoter; And (d) a polyadenylic signal sequence that operates in cucumber cells to form polyadenyls at the 3 'end of RNA; (Ii) resuspending the infected cucumber cotyledons in a medium containing BAP (6-Benzylaminopurine) and NAA (α- Naphtalene acetic acid) to form shoots; And (iii) rooting the re-differentiated shoots in a rooting medium containing NAA to form transformed cucumbers.

In this study, five different cucumber varieties in Korea were obtained for the regeneration method to induce degeneration from cotyledon slices and to obtain re-differentiated individuals with high frequency, and for the efficient cucumber transformation using Agrobacterium tutoration.

The present invention is described in more detail as follows:

I. Preparation of Transforming Materials

Among the cucumbers that can be used as a transgenic material are leaves, stems, and leaf diseases. Such materials can be obtained from various parts of the plant, but most preferably from seeds. The seed is preferably sterilized beforehand using a disinfectant such as chlorine (especially sodium hypochloride) or the like.

Ⅱ. Seed germination

In a preferred embodiment of the present invention, the medium used for germination of seeds includes, but is not limited to, nutritional basal medium, energy sources and vitamins such as MS, B5, LS, N6 and White's. The energy source is more preferably a saccharide, most preferably sucrose. In addition, the vitamin preferably includes nicotinic acid, thiamine, pyridoxine and the like. Meanwhile, the germination medium of the present invention may further include MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate), which serves to buffer the pH change, and may further include agar as a solid support. The plant growth regulator is not added to the germination medium of the present invention.

III. Preparation of Transgenic Tissue

Tissues that can be used in the present invention can be used all tissues generated from the germination of seeds, preferably cotyledon and hypocotyl, most preferably cotyledon. When cotyledons are obtained from germinated seeds, it is desirable to collect them so that the growth point is completely excluded. In addition, it is preferable that the cotyledons use the whole uncut cotyledons.

Ⅳ. Infection with Agrobacterium Tumation

Transformation of cucumber cells is carried out with Agrobacterium trimers incorporating Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983) ). More preferably, a binary vector system is used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986)). pBin19, pRD400, pRD320 and the like.

Binary vectors used in the present invention are basically (a) origin of replication operating in cucumber cells; (b) a promoter capable of promoting transcription in cucumber cells; (c) a structural gene sequence encoding a foreign protein functionally linked to said promoter; And (d) a polyadenylic signal sequence that operates in cucumber cells to form polyadenyls at the 3 'end of RNA. In addition, the vector preferably contains an antibiotic gene (eg, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) as a selection marker. Meanwhile, the vector may additionally include a gene encoding a reporter substance (eg, luciferase, β-glucuronidase, etc.). Promoters that can be used in the binary vector include, for example, the cauliflower mosaic virus 35S promoter, the 1 'promoter, the 2' promoter or the nopaline synthase (nos) promoter. Meanwhile, the structural gene sequence encoding the foreign protein is determined according to the desired trait to be obtained. For example, herbicides (glyphosate, sulfonylureas, etc.) resistance genes, viral resistance genes, pest resistance genes (e.g. Bt genes), adverse condition (drought, temperature, salinity) resistance genes, genes for quality improvement (e.g. sugar content) Increase, delay in ripening, etc.), genes related to medical protein production (EGF, antigens or antibodies of various diseases, insulin, etc.), genes related to functional cosmetic production (albumin, antimicrobial peptides, etc.).

Infection of plant cells by Agrobacterium incorporating the above-described vectors may employ conventional methods known in the art. For example, by co-culturing the cotyledons collected so that growth points are excluded, Agrobacterium is infected with cotyledons. Co-culture fluids preferably include acetosyrigon, which is intended to help transform Agrobacterium into plants.

Ⅴ. subdivision

Regeneration of plant tissues transformed with Agrobacterium should be carried out in a regeneration medium containing strictly controlled components and component amounts.

According to the present invention, the regeneration medium includes, but is not limited to, nutritional basal medium, energy sources and vitamins such as MS, B5, LS, N6 and White's. The energy source is more preferably a saccharide, most preferably sucrose. The vitamin preferably includes nicotinic acid, thiamine, pyridoxine and the like. On the other hand, the re-differentiation medium of the present invention may further include MES to serve as a pH buffer, and may further include agar as a solid support.

The regeneration medium of the present invention includes plant growth regulators. Cytokinins contained in plant growth regulators include, but are not limited to, 6-benzylaminopurine (BAP), kinetin, zeatin, isopentyl adenosine, and the like, most preferably 6-benzylaminopurine. In addition, the redistribution medium of the present invention must essentially include auxin-based growth regulators such as α-naphthalene acetic acid (NAA), indole acetic acid, (2,4-dichlorophenoxy) acetic acid, and most preferred auxin-based growth regulators. Is α-naphthaleneacetic acid.

The amount of cytokinin contained in the regeneration medium, particularly the amount of BAP, is preferably 1-4 mg / l, and the regeneration rate is greatly reduced when the content is less than 1 mg / l and more than 4 mg / l. More preferably the amount of BAP is 1.5-2.5 mg / l, most preferably 2.0 mg / l.

The amount of the auxin-based growth regulator, in particular NAA, is preferably 0.001-0.08 mg / l, and the regeneration rate is greatly reduced when the content is less than 0.001 mg / l and 0.08 mg / l. More preferably, the amount of NAA is 0.005-0.03 mg / l, most preferably 0.01 mg / l.

On the other hand, the regeneration medium of the present invention does not contain CuSO ₄ or casein hydrolyzate, which is known to enhance regeneration of gourds and crops, and shows excellent regeneration rate and shoot formation ability. This is very surprising and shows the superiority of the regeneration medium of the present invention.

According to a preferred embodiment of the present invention, it further comprises antibiotics (eg, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) to select the transformed tissue. For example, when kanamycin is used as an antibiotic, the amount is preferably adjusted to 70-150 mg / l. If the content is less than 70 mg / l, the regeneration rate is high but the final transformation rate is almost 0%. There is a problem approaching to, and if it exceeds 150 mg / L, there is a problem that the regeneration rate is greatly reduced.

It is most preferable to insert the cotyledon on the regeneration medium into the medium surface. Unlike leaf slices, cotyledons have scars on the edges and then lie flat on the media and rarely re-differentiate and callus formation is extremely low.

According to the above conditions, when the transformed cucumber tissue is cultured in a regeneration medium, it is re-differentiated through a callus state to form shoots.

Ⅵ. Rooting

Redifferentiated tissue is rooted in the rooting medium of the present invention to obtain transgenic cucumbers. Rooting medium of the present invention includes, but is not limited to, a nutritional basal medium, energy sources and vitamins such as MS, B5, LS, N6 and White's. The energy source is more preferably a saccharide, most preferably sucrose. The vitamin preferably includes nicotinic acid, thiamine, pyridoxine and the like. On the other hand, the re-differentiation medium of the present invention may further include MES to serve as a pH buffer, and may further include agar as a solid support.

In the rooting medium of the present invention, cytokinin is rarely used as a plant growth regulator, and auxin is mainly used. NAA, indole acetic acid, (2,4-dichlorophenoxy) acetic acid, etc. can be used as said auxin type growth regulator, Most preferably, it is NAA. In the rooting medium of the present invention, the preferred content of NAA is 0.001-0.08 mg / l, and if the content is less than 0.001 mg / l, the rooting time is relatively long, and there are many problems in that a long thin root without root hair occurs. In case of exceeding 0.08 mg / l, there is a problem in that the stem in contact with the medium is enlarged while it is not rooted.

In addition, when the rooting medium of the present invention contains agar as a solid support, a content of 0.2-0.7% (w / v) is preferable. When the content of agar is less than 0.2%, it does not function as a solid support, and when it exceeds 0.7%, root elongation is delayed, short and coarse roots are formed, and problems appearing in the rooting out of the medium.

Iii. Identification of transformants

Cucumber transformants prepared by the present invention can be carried out by methods known in the art. For example, by performing PCR using DNA samples isolated from tissues of transformed cucumbers, foreign genes inserted into the genome of cucumbers by transformation can be easily identified. Transformants can also be identified by Southern blotting and Northern blotting as disclosed in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). On the other hand, when the vector used for transformation contains the β-glucuronidase gene, a part of the re-differentiated cotyledon tissue is replaced with X-gluc (5-Bromo-4-Chloro-3-Indole-β-D By immersing in a substrate solution such as -Glucuronic Acid), the color development can be easily confirmed by visually observing the color development.

According to the most preferred embodiment of the present invention, the present invention is (i) cleavage of cucumbers with Agrobacterium tumefaciens , which can be inserted into the genome DNA of cucumber cells and embedded with a vector having the following sequence: Infecting the entire non-foliar cotyledon: (a) origin of replication operating in cucumber cells; (b) a promoter capable of promoting transcription in cucumber cells; (c) a structural gene sequence encoding a foreign protein functionally linked to said promoter; And (d) a polyadenylic signal sequence that operates in cucumber cells to form polyadenyls at the 3 'end of RNA; (Ii) The infected cucumber cotyledons were implanted into medium containing 1.5-2.5 mg / L of BAP (6-Benzylaminopurine) and 0.005-0.03 mg / L of NAA (α- Naphtalene acetic acid) and re-differentiated to form shoots. Making a step; And (iii) rooting the re-differentiated shoots in a rooting medium containing NAA 0.005-0.03 mg / l and agar 0.2% -0.7% to form a transformed cucumber. do.

According to another aspect of the present invention, the present invention provides a cucumber transformant produced by the method of the present invention described above.

As described above, the production method of the cucumber transformant of the present invention is very high in transforming efficiency and regeneration efficiency, and excellent in reproducibility of preparing the cucumber transformant.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1: Preparation of Plant Tissue

Five varieties of cucumbers (long-grilled cucumbers, baekbong chopped cucumbers, chopped cucumbers, samnamcheongjang cucumbers, and strong samcheok cucumbers) were used for the experiment. First, the seeds were disinfected in 5% NaOCl solution for 15 minutes, and then washed three times for 15 minutes with sterile distilled water. Subsequently, germinated medium containing 1/2 MSMS (Murashige & Skoog medium including Minimal Salts), 2% sucrose and 0.6% agar was incubated for 4 days at 25 ± 1 ° C. under dark conditions. Then, only seedlings without main leaves were selected, cotyledon sections and hypocotyls were collected and used for the following regeneration conditions establishment experiments.

Example 2: Establishment of a Medium Composition Suitable for Regeneration of Plant Tissues

BAP (6-Benzylaminopurine), Zeatin (Keattin) and Kinetin (cytokinin) and NAA (α- Naphtalene Acetic Acid) as auxin growth regulators Ten media were prepared (see Table 1). The basic medium composition for regeneration was MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins), 500 mg / L MES (2- (N-Morpholino) ethanesulfonic acid Monohydrate), 3% sucrose and 0.6% agar.

division Cytokine (mg / ℓ) NAA (mg / L) BAP Kinetin Zeatin Badge 1 2.00 - - - Badge 2 2.00 - - 0.01 Badge 3 2.00 - - 0.10 Badge 4 4.00 - - 0.01 Badge 5 4.00 - - 0.10 Badge 6 - 2.70 - 5.00 Badge 7 - 3.00 - 0.20 Badge 8 - 5.00 - 1.00 Badge 9 - - 1.00 0.10 Badge 10 - - 2.00 0.20

Subsequently, 20 individuals were placed on the medium and photocultured at 26 ± 1 ° C. and 8,000 Lux for 16 hours / 8 hours (day / night), and then cultured for 30 days. . The regeneration rate was obtained by converting the number of subdivided sections into percentages of the total number of damaged sections, and the average number of shoots was obtained by converting the total number of shoots generated as percentages with respect to the number of sections subdivided. The results are shown in Table 2 below.

badge kind Fall Long agitation Powerful samcheok Cupronickel Samnam Office Regeneration rate Average new seconds Regeneration rate Average new seconds Regeneration rate Average new seconds Regeneration rate Average new seconds Regeneration rate Average new seconds Badge 1 0 0 0 0 0 0 0 0 0 0 Badge 2 30 1.2 23 1.1 15 1.5 90 2.4 25 1.5 Badge 3 25 1.0 20 1.2 13 1.2 65 3.2 15 1.5 Badge 4 25 1.0 13 1.0 5 1.1 85 1.3 15 1.0 Badge 5 20 1.2 10 1.1 10 1.1 25 1.2 0 1.2 Badge 6 13 1.6 15 1.2 25 1.2 0 0 13 1.2 Badge 7 15 1.1 10 1.4 10 1.1 10 1.0 0 0 Badge 8 - - - - - - 15 1.1 15 1.2 Badge 9 25 1.2 25 1.2 15 1.5 75 1.9 35 1.5 Badge10 15 1.4 10 1.1 13 1.2 65 2.1 45 1.3

As can be seen in Table 2, the cucumbers of the five varieties used in the experiment showed a variety of re-differentiation rate of 0-90% and shoot bud formation ability of 1.0-3.1, depending on the type, the re-differentiation rate and shoot formation ability of Baekbong dalgi Was the highest.

In addition, among cytokinin, BAP showed higher re-differentiation rate and shoot formation ability compared to zeatine and kinetin, while BAP 2 mg / l showed high re-differentiation rate and shoot-forming ability, whereas re-differentiation rate was higher than 4 mg / l. It tended to decrease. In addition, when NAA was not added or when added at a lower concentration (0.01 mg / l) than when added at a high concentration (0.1 mg / l), it showed high regeneration rate and shoot formation ability. In particular, BAP 2 mg / l and NAA 0.01 mg / l showed the highest regeneration rate in the medium treated with co-treatment, the average shoot number was very high at 2.5 per section, and the minimum number of days required for regeneration was 20 days. .

The results of the above experiments were for cotyledons. In the case of hypocotyls, the regeneration rate was very low compared to cotyledons, almost no shoots were formed, and most of the treatments did not re-differentiate, but callus formed and died or stopped growing. It was.

In general, when the cotyledons of 4-5 days old in cancer cultures are placed on the regeneration medium, callus is formed around the medium contact surface after about 7 days, and shoots are re-differentiated from the callus enlarged after about 15 days. Candles began to form (see FIG. 1). In FIG. 1, A panel shows cotyledons 10 days after media placement and B panel shows shoots re-differentiated at the base of 20-day cotyledons.

As a result, the cotyledons are excellent materials for regeneration of cucumbers, and the regeneration conditions are most optimal when a medium containing BAP (2 mg / L) in cytokinin and NAA (0.01 mg / L) in auxin growth regulator is used. It can be seen that the condition of.

Example 3 Investigation of Regeneration Rate According to Condition of Plant Tissue Samples

In order to investigate the regeneration rate according to the condition of the lineage tissue sample, the cotyledons selected as the most suitable sample and the cotyledons cut about 2 mm above the growth point were placed on medium 2 selected as the most suitable medium. The regeneration rate and the average number of shoots were examined in the same manner as in Example 2.

As a result, the regeneration rate of the whole cotyledon section was high and the average number of shoots was high. Therefore, in order to re-differentiate the shoots of cucumber cotyledons, it was found that it is preferable to use the whole cotyledons without removing the upper part of the cotyledons.

Example 4 Investigation of Regeneration Rate According to the Dental Procedure

In order to investigate the redifferentiation rate according to the dentifrice method, the medium 2 of Example 2 by inserting the whole cotyledon fragment shown to the highest regeneration rate in Example 3 and the cotyledons lying on the medium surface The re-differentiation rate was investigated in the same manner as in Example 2, which was placed on the base.

As a result, the cotyledon slices, unlike the leaf slices, had almost no re-differentiation and very low callus formation rate when the wounds were injured at the edge and laid down on the media surface. These results are the opposite of the case of re-differentiating the leaf sections, it was confirmed that the teeth of the cotyledon sections were inserted into the teeth.

Example 5 Investigation of the Effect of Trace Elements on the Regeneration Rate

CuSO ₄ and casein hydrolysate, known to enhance regeneration of gourds and crops, were treated in different concentrations of medium 2 of Example 2 to investigate the effect on regeneration rate.

As a result, CuSO ₄ and casein hydrolyzate did not promote regeneration of cucumber cotyledons. These results are in contrast to previous research results in the art.

Example 6: Establishment of Transformation Conditions

In order to establish the most suitable transformation conditions, MSB5 (Murashige & Skoog medium including Gamborg B5 vitamins) medium containing Bamgapgi selected from the above example and Bmg 2 mg / L and NAA 0.01 mg / L The transformation was attempted using different co-culture methods, incubation periods, and treatment temperatures during co-culture.

First, the seeds of Baek-dae cucumber were germinated in the same manner as in Example 1 to obtain cotyledon slices obtained therefrom. P RD320 vector with gus :: npt II fusion gene and pat (phosphinithricin acetyl transferase) gene shown in FIG. 2 (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 Mol Biol Int J. Mol Biol Biochem Genet Eng 21 (3): 415-428 transformed with (1993)) Agrobacterium tyumeo Patience ( Agrobacteriumtumefaciens ) GV3101 (Mp90); Plant-cell-rep. 15 (11): 799-803 (1996)) were incubated for 18 hours in a super broth: 37 g / L Brain heart infusion broth (Difco), 0.2% sucrose, pH 5.6). The cotyledon sections were immersed and mixed for 10 minutes.

Then co-cultivated in coculture medium (Murashige & Skoog medium including Gamborg B5 vitamins) medium containing BAP 2 mg / L and NAA 0.01 mg / L) under photoculture conditions at 25 ° C. for 2 days After culturing, Agrobacterium tumerification and cotyledons were co-cultured at low temperature (4 ° C.) for 4 days. Then, unlike the conventional method, the cotyledons were not subjected to a clean up step, and cotyledons were added to the medium based on MS-B5, MES 0.5 g / L, 3% sucrose, and 0.4% pitagel. / l, NAA 0.01 mg / l, carbenicillin 500 mg / l and kanamycin 100 mg / l were added and cultured at 26 ± 1 ° C. and 8,000 lux, 16 hours / 8 hours (day / night) for 4 weeks It was.

Subsequently, the regenerated shoots were transferred to a rooting medium containing a certain amount of NAA (0, 0.01 and 0.1 mg / l), kanamycin 100 mg / l and a certain amount of agar (0.4%, 0.6% and 0.8%). And 8,000 Lux, 16 hours / 8 hours (day / night) incubated in the conditions of the subject was estimated as a transformant was analyzed using the method of Example 7 below.

division Co-culture conditions Intercept subdivision Transformation Number of individuals % Differentiation Number of individuals Transformation rate (%) Primary Room temperature culture 200 40 20.0 10 5.0 Low temperature treatment 200 12 6.0 3 1.5 Secondary Room temperature culture 100 21 21.0 4 4.0 Low temperature treatment 100 7 7.0 2 2.0 Sum Room temperature culture 300 61 20.3 14 4.7 Low temperature treatment 300 19 6.3 5 1.7

As can be seen in Table 3, when co-culturing Agrobacterium turmeric and cucumber cotyledons, the treatment group co-cultured for 4 days at low temperature (4 ℃) than co-culture for 2 days at room temperature (25 ℃) Regeneration and transformation rate were significantly lower. In particular, in the case of low-temperature treated cotyledons, necrosis did not re-differentiate during the growth process but rather necrotic or sulfided as it was.

Comparison of cotyledons and normal transformants that are necrotic as yellowing without being rooted in rooting medium as non-transformants is shown in FIG. 3. In Figure 3 A panel is normal transformants, B panel non-transformants, respectively.

division Kanamycin concentration (mg / L) Intercept subdivision Transformation Number of individuals % Differentiation Number of individuals Transformation rate (%) Primary 50 40 35 87.5 - - 100 40 12 30.0 2 5.0 200 40 2 5.0 0 0.0 Secondary 100 100 31 31.0 14 3.1 200 100 3 3.0 0 0.0

As can be seen in Table 4, when the kanamycin concentration of the selection medium was 50 mg / l, the regeneration rate was high, but the transformation rate was nearly 0%, and the kanamycin concentration was 100 mg / l. In the case of about 30% regeneration rate and about 5% transformation rate, and when the kanamycin concentration of 200 mg / L, about 3% regeneration rate and 0% transformation rate. Therefore, it can be seen that the concentration of kanamycin in the selection medium is about 100 mg / l.

On the other hand, as the concentration of NAA increases in rooting medium, root hairs develop in the thick and short roots, and the rooting days are short. Also observed. On the other hand, when NAA was not treated, the rooting time was relatively long and many thin roots without root hairs were observed. Thus, it can be seen that a suitable concentration of NAA in the rooting medium is about 0.01 mg / l.

In addition, the lower the agar concentration in the rooting medium (0.4% and 0.6%), the easier rooting and normalization, while the agar concentration above 0.8% delays root elongation, forms short and thick roots, and roots out of the medium. (See FIG. 4). In FIG. 4, the A panel shows rooting patterns in the medium to which 0.6% agar is added, and the B panel shows the medium to which 0.8% agar is added.

Example 7: Identification of transformants

Identification of the transformant in Example 6 was carried out as follows:

Example 7-1. GUS Analysis

To measure β-glucuronidase activity, a part of the redifferentiated cotyledon tissue was immersed in an X-gluc (5-Bromo-4-Chloro-3-Indole-β-D-Glucuronic Acid) solution, After incubation at 37 ° C. for 24 hours, the transformants were identified by visually observing the color development (blue) (see FIG. 5A).

Example 7-2. PCR analysis

Isolation of plant genome DNA for PCR analysis was extracted by Edwards method ( Nucleic Acids Research , 19: 1349 (1991)). The primers used in the PCR analysis correspond to the nucleotide sequence of the pat gene of the vector in the Agrobacterium tumerification, with the forward primer 5'-AGA CCA GTT GAG ATT AGG CCA G-3 'and the reverse primer 5 '-GCC TCA TGC AAC CTA ACA GA-3'. PCR reaction was carried out using Taq polymerase, total denaturation at 96 ° C. for 2 minutes, denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and polymerization at 72 ° C. for 2 minutes 35 times. The reaction was extended for 10 minutes at 72 ° C., and the reaction product was analyzed on a 1.0% agarose gel (see FIG. 5B). In Fig. 5b, PCR product of lane positivity containing a 1 kb leather, lane 1, pat gene, lane 2, PCR product based on chromosomal DNA of wild cucumber, and lane 3 of lane 3 were selected for transformation of cucumber. PCR products were loaded with the sieve DNA as a template. As can be seen in Figure 5b, 0.5 kb DNA band corresponding to the pat gene is observed in the lane for the transformant of the present invention, it can be confirmed that the transformation.

Tissue culture studies for the development of high quality cucumbers have been conducted and there have been many reports of cucumber regeneration. Nevertheless, there were still many problems such as low rate of plant regeneration and survival, abnormal morphogenesis, early flowering. From the results of Examples 6 and 7, when the experiment on the cotyledonary cotyledons under the most suitable regeneration conditions and transformation conditions according to the present invention, the regeneration rate of about 20-30% and the transformation rate of about 4% As can be seen, this is a better result than the existing regeneration rate and transformation rate.

The present invention provides a method for producing a cucumber transformant by Agrobacterium tumefaciens and a cucumber transformant prepared therefrom. The method for producing a cucumber transformant of the present invention has a very high transformation efficiency and regeneration efficiency, and has excellent reproducibility of preparing a cucumber transformant.

Claims (12)

A method for preparing a cucumber transformant comprising the following steps: (Iv) infecting cucumber cotyledon with Agrobacterium tumefaciens embedded in the genome DNA of cucumber cells and embedded with a vector having the following sequence: (a) working in cucumber cells Replication origin; (b) a promoter that operates in cucumber cells; (c) a structural gene sequence linked to said promoter that encodes a foreign protein whose expression is to be regulated; And (d) polyadenylic signal sequences operating in cucumber cells; (Ii) resuspending the infected cucumber cotyledons in a medium containing BAP (6-Benzylaminopurine) and NAA (α- Naphtalene acetic acid) to form shoots; And (Iii) rooting the re-differentiated shoots in a rooting medium containing NAA to form transgenic cucumbers. The method of claim 1, wherein the cotyledons of step (iii) are whole uncut cotyledons. The method of claim 1, wherein the tooth in the step (ii) is carried out by inserting the entire cotyledon on the medium surface. 2. The method of claim 1, wherein the amount of BAP in step (ii) is 1-4 mg / l. 5. The method of claim 4, wherein the amount of BAP in step (ii) is 1.5-2.5 mg / l. The method of claim 1, wherein the amount of NAA in step (ii) is 0.001-0.08 mg / L method of producing a cucumber transformant. 7. The method of claim 6, wherein the amount of NAA in step (ii) is 0.005-0.03 mg / L. The method of claim 1, wherein the amount of NAA in the rooting medium of step (iii) is 0.001-0.08 mg / l method for producing a cucumber transformant. The method of claim 8, wherein the amount of NAA in the rooting medium of step (iii) is 0.005-0.03 mg / L method of producing a cucumber transformant. The method of claim 1, wherein the rooting medium of step (iii) further comprises 0.2% -0.7% agar. A method for preparing a cucumber transformant comprising the following steps: (Iii) infecting the whole uncut cotyledons of cucumbers with Agrobacterium tumefaciens , which can be inserted into the genome DNA of cucumber cells and contains a vector having the following sequence: (a) Cucumber Origin of replication operating within the cell; (b) a promoter that operates in cucumber cells; (c) a structural gene sequence linked to said promoter that encodes a foreign protein whose expression is to be regulated; And (d) polyadenylic signal sequences operating in cucumber cells; (Ii) The infected cucumber cotyledons were implanted into medium containing 1.5-2.5 mg / L of BAP (6-Benzylaminopurine) and 0.005-0.03 mg / L of NAA (α- Naphtalene acetic acid) and re-differentiated to form shoots. Making a step; And (Iii) rooting the re-differentiated shoots in rooting medium containing NAA 0.005-0.03 mg / L and agar 0.2% -0.7% to form a transformed cucumber. Cucumber transformant prepared by the method of any one of claims 1-11.