KR20020006263A - A method of regenerating and transforming chinese cabbage(Brassica campestris ssp. pekinensis) using hypocotyl segments - Google Patents

Abstract

PURPOSE: A method for regenerating cabbage using hypocotyl and a method for producing cabbage transformed with useful foreign gene are provided to establish a system which is allowed to introduce a foreign gene useful to cabbage. CONSTITUTION: The method for regenerating a cabbage comprises steps of germinating cabbage seed to prepare hypocotyl, isolating the hypocotyl, and regenerating a cabbage using the hypocotyl in the culture media containing silver nitrate(AgNO3). In the method for producing the transformed cabbage, the cabbage seed is firstly disinfected and germinated to prepare hypocotyl. And then, hypocotyl is isolated and pre-cultured. Expression vector comprising a foreign gene(e.g., Agrobacterium) is introduced into the hypocotyl, to allow hypocotyl to transform with the foreign gene. The hypocotyl is used to regenerate a transformed cabbage in the culture media containing silver nitrate, hereby transformed cabbage being produced.

Description

A method of regenerating and transforming chinese cabbage (Brassica campestris ssp.pekinensis) using hypocotyl segments}

The present invention relates to a regeneration method using the cabbage of the cabbage and a method of producing a cabbage transformed with a useful foreign gene.

The occurrence of plants related to cabbage is as follows.

Seed plant development, which occurs as a process of sexual reproduction, begins with the division of single cells called fertilized eggs. Embryos are formed by the initial cell proliferation and differentiation. In plant development, this first stage of development is called embryoogenesis. This embryo is an unfinished body that is in the process of development, and as it is formed, seed shells and udders are formed around it, and seeds are formed. Thus, the finished pear is in ripe seed. The seed belly of the seed plant is composed of four parts: infant, hypocotyl, root, and cotyledon. There are dividing tissues at the tip of infant and root.

The second stage of seed plant development is germination. It is a phenomenon in which a young plant is born by growing an embryo that has undergone a period of dormancy, and organs are formed based on each part of the vessel. More specifically, germination is the formation of leaves and stems by cell proliferation in the meristem of the infant tip and differentiation and growth of proliferated cells, and cell proliferation and proliferation of the proliferated cells in the proximal tip meristem. Roots are formed by growth, and a young plant is formed. In this process, the axis is the base of the stem. After germination, the differentiation and growth of stems, leaves, and roots continues due to the infinite activity of the meristem at the growth point of the stem and root tip.

On the other hand, Chinese cabbage is a cruciferous vegetable that grows well in cool climates and is widely grown in Northeast Asia such as Korea, China, and Japan. Chinese cabbage is divided into a leaf-shaped cabbage that overlaps with a leaf, a dirty cabbage that does not become a bulbous shape, and a half-grain cabbage in its intermediate form. In particular, Chinese cabbage is the basic material for soaking kimchi, and occupies the highest ratio in the production and cultivation area of leaf vegetables grown in Korea. One of the biggest disasters in domestic cabbage cultivation is pests, which include turnip mosaic virus, wart disease, soft disease, and cabbage moth and cabbage butterfly. Recently, researches to overcome the major pests of Chinese cabbage using genetic engineering techniques have been actively conducted. The precedent of such research is to develop an effective method for transgenic cabbage.

To date, there have been reports on the genetic conversion of Chinese cabbage worldwide, including those reported by Jeon et al. (1995) and myself (1997). Because of this, selection of gene converts is quite difficult and the continuous production of reproducible gene converts is difficult.

Therefore, the present inventors have attempted to establish an effective system capable of introducing foreign genes useful for cabbage while solving the above problems.

1 schematically shows a method for producing a cabbage transformed with a foreign gene according to the present invention.

Figure 2 is a schematic diagram of the expression vector (pMOGbar) containing herbicide resistance gene.

3 is a schematic diagram of an expression vector (pGR006) containing a male sterile organic gene.

4A is a photograph of leaves of untransformed cabbages treated with 0.075% and 0.15% basa. 4B is a photograph of pMOGbar-transformed cabbage leaves treated with 0.3% bastard.

Figure 5 is a photograph showing the difference in the surgical development of normal Chinese cabbage and male sterile genetically converted cabbage.

Figure 6 is a electrophoresis picture of the PCR analysis of the transgenic cabbage.

Figure 7 is an electrophoresis picture of genomic southern analysis of transgenic cabbage.

8 is a schematic diagram of a pBt 21 expression vector.

9 is a schematic diagram of the pHGCTB expression vector.

10 is a schematic diagram of a pGR008 expression vector.

11 is a schematic diagram of a pGR008 expression vector.

12 is a chart showing the transformation efficiency of various genes using the embryonic axis of Seoul cabbage.

Figure 13 is a chart showing the transformation efficiency of the various genes using the embryonic axis of three strikes.

Figure 14 is a chart showing the transformation efficiency of various genes using the axis of the chapter book.

Figure 15 is a chart showing the transformation efficiency of various genes using the axis of the jangwon model.

The present invention is to provide a method for reproducing using the cabbage axis of the cabbage and a method for producing a cabbage transformed with a useful foreign gene.

To produce cabbage transformed with a useful foreign gene, a process of transforming the cells of cabbage with foreign DNA and regenerating the transformed cabbage from the transformed cells is required. Figure 1 schematically shows a preferred method for producing cabbage transformed with a foreign gene according to the present invention.

First, in the present invention, transgenic embryos were transformed with foreign genes and embryonic embryos were selected as reproducible target cells. Embryo is the part that grows like a stem at the lower part of cotyledon and growth point in seedlings before the normal leaves emerge after about 5-6 days of seed germination. The embryonic axis grows about 5-10cm after 5 days of seed germination. If this is cut to 1cm size, 5-10 tissues can be obtained from one seed, so more samples can be obtained for transformation than other tissues. have. In addition, explantation is good for plant regeneration and infecting Agrobacterium during transformation is much easier than other tissues. Because the embryos are not bulky when cut, and when inoculated with Agrobacterium solution, only 200-300 embryos can be put into the solution at one time, so the transformation experiment can be performed much more easily than cotyledon tissues that must be infected one by one. have. And, cabbage is very difficult to obtain a transformant when using a tissue other than the axis.

Since most of the transformation experiments are performed aseptically in a container, the seeds of the cabbage must be disinfected.

Explantation is prepared by artificially incubating the cabbage seed in the dark or light state. Most cabbages are too short and poor in normal mining, making them difficult to harvest and use for transformation.

The period of cutting the cabbage from the cabbage is preferably about 5 days after sowing. When too young, 3 to 4 days after sowing, almost no axial axis is formed and the tissue is so fragile that it is easy to die when the transformation experiment is performed to obtain a transformed tissue. On the other hand, the older embryonic axis is much longer, so much more embryonic fragments can be obtained, but the ability to regenerate plants rapidly decreases, making it difficult to obtain regenerating plants when the desired genes are transformed. About 5 days after sowing cabbage seeds, the cotyledon fully develops, and the length of the ventral axis is about 4-5 cm.

On the other hand, in order to transform the embryonic embryonic cells into foreign genes, an expression vector containing the foreign genes should be introduced into the embryonic embryonic cells.

Methods of introducing expression vectors include Agrobacterial-mediated transformation, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and the like. The most widely used method of introducing an expression vector into a plant is a method of using the natural transformation system of Agrobacteria and a method of directly injecting DNA using a gene gun. Agrobacteria have Ti or Ri plasmids, which have genes that can be transformed into plants by foreign genes. Agrobacterium vector systems and methods of gene transfer through Agrobacteria are described in Gruber et al., Supra, Miki et., Supra, and Moloney et al., Plant Cell Reports 8: 238 (1989).

Foreign genes that may be introduced into cabbage include herbicide-tolerant genes and male sterile genes.

On the other hand, the expression of foreign genes in the host cell cabbage is characterized by regulatory elements such as promoters, polyadenylation sequences, tissue-specific regulatory elements and enhancers. Under the control of). Foreign genes are functionally linked to promoters, which include constitutive promoters, inducible promoters, and tissue-specific promoters.

Artificially induced axle is weaker than a normally axed axle, so when directly inoculated with Agrobacterium bacteria with foreign genes, it is easy to die and cannot obtain a transformed cabbage. Therefore, pretreatment is necessary before vaccination. Cut embryos are precultured in MS solid medium or MS liquid medium.

In order to perform the pretreatment process more efficiently and conveniently, it is preferable to pretreat the liquid redifferentiation medium by suspension culture (suspension culture) at 100rpm rather than pretreatment in the solid medium. In the case of pretreatment in solid medium, the operation of inoculating the sample into the microsuspension should be carried out when inoculating Agrobacteria, whereas in case of pretreatment in liquid medium, the agrobacterial suspension is added to the liquid medium containing the embryo and shaken together. This is because the vaccine can be given.

On the other hand, after infection with a foreign gene using the embryonic cells of the cabbage, in order to produce a transformed cabbage, an effective regeneration system of cabbage is required. That is, transformed cells prepared from embryonic axis should be regenerated into cabbage through somatic embryogenesis, and introduced foreign genes should be stably expressed later.

In the present invention, the following culture medium was used to efficiently re-differentiate cabbage tissue using the cabbage of the cabbage. Embryogenic redistribution medium was MS basic medium [ammonium nitrate 1650mg / l, boric acid 6.20mg / l, calcium chloride 332.20mg / l, cobalt chloride hexahydrate 0.025mg / l, cupric sulfate 0.025 Mg / l, Disodium EDTA 37.26mg / l, Ferrous Sulfate 27.8mg / l, Glycine 2.0mg / l, Magnesium sulfate 180.7mg / l, Manganese sulfate 16.9 Mg / l, Myo-inositol 100mg / l, nicotinic acid 0.5mg / l, potassium nitrate 1900mg / l, potassium phosphate 170mg / l, pyridoxine HCL 0.5mg / l, sodium molybdate ) 0.25 mg / l, thiamine HCL 0.1 mg / l, zinc sulfate 8.6 mg / l, 1 mg / l NAA (Naphthaleneacetic acid), 3 mg / lBA (Benzylamino purine)] and silver nitrate. Silver nitrate is present in the regeneration medium for embryo reproduction, which shows a remarkably high plant regeneration rate (see Experimental Example 1). Therefore, AgNO ₃ should be added to the selection medium when the transgenic plant is induced from the cabbage of the cabbage. Preferred concentrations of silver nitrate are 1-5 mg / l.

Further, the method of re-differentiating the embryonic explant is as follows: incubating in re-differentiation medium under 25-27 ° C. light conditions; Completely leaf-developed individuals (replanted leaves of more than 1 cm in length) were transferred to root organic media (MS base medium, 8 g / l agar, 300 mg / l cell taksim, pH 5.8). After transfer, the roots are organic and well-organized green plants are transferred to pots and grown in greenhouses.

The reason for the reorganization of roots during embryonic regeneration is that if the roots are not artificially reorganized, the transformed cabbage will not grow normally and will die when transferred to the soil.

Meanwhile, in order to select transformed cells, the expression vector should include at least one genetic selection marker. In Chinese cabbage, the use of hygromycin as an antibiotic during transformation is much easier to reliably acquire the transformed plant than when using other selection antibiotics. At this time, the selection medium should be prepared so that only cells transformed with foreign genes can survive using antibiotics.

The explant transformed according to the present invention is reproduced as a transgenic plant in which the gene is uniformly transferred so that the foreign gene is delivered to the offspring. Transformed cells usually have one or several nearby cells that can be transformed, and the cells continue to grow and grow to complete plants. On the other hand, when the transformed plant emerges directly from the selection medium, it is most ideal, but usually the callus is organic and the plant is differentiated from it. Therefore, it is desirable to obtain a plant from which the callus is selected and transferred to a fresh medium. At this time, it is preferable to subculture the antibiotic-resistant callus or young re-differentiated cabbage plants to the selection medium having the same components every four weeks. If four weeks have not been passaged, it is likely that Agrobacterium will grow or callus will die on the selection medium.

It is desirable to organic the roots when regenerating the embryonic explants. If the roots do not form normally, the cabbage cannot continue to grow.

Expression of foreign genes in plants suspected of being transformed can be confirmed by PCR, Northern blot, Southern blot and the like.

Transformed plants can hybridize with untransformed plants to introduce foreign genes into a new genetic environment.

In addition, the transformed plant can self-pollinate to produce offspring with homozygous genes.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. However, the present invention is not limited to the following examples and experimental examples.

Experimental Example 1. Effect of Silver Nitrate on Chinese Cabbage Cattle Regeneration>

After incubating for 5 days in Seoul cabbage, the embryonic axis is cut at intervals of 1 cm and added to the basic MS medium medium 1mg / lNAA, 3mg / lBA, 8g / l agar, pH 5.8, as shown in Table 1 below addition of AgNO ₃ in 0~5㎎ / ℓ, respectively, and examined the effect on hypocotyl regeneration when AgNO _3.

AgNO ₃ concentration (mg / L) 0 One 2 3 4 5 Shoot Differentiation Rate (%) 6.3 81.3 83.8 78.3 75 77.5 Root Differentiation Rate (%) 93.8 6.3 3.8 6.7 5 7.5

As shown in Table 1 above, as a result of tissue culture using the axillary of semi-binding cabbage (Seoul cabbage), silver nitrate must be added to the embryonic regeneration medium (MS basic medium, 1mg / ℓNAA, 3mg / ℓBA). ), It can be seen that only a remarkably high plant regeneration rate is achieved.

Example 1 Gene Conversion Technology Using Cabbage Axis

1. Plant Sample

The Chinese cabbage varieties were used as Korean cabbage (half cabbage), Samjin cabbage (cabbage cabbage), Olympic cabbage (cabbage cabbage), Jangwon cabbage (cabbage cabbage), and a copy (cabbage order).

2. Seed disinfection

The seeds of the Chinese cabbages were soaked in 70% ethanol for 1 minute and 50% Rox solution for 20 minutes, shaken at 150 rpm for 20 minutes, and then washed 3-4 times with sterile water. Seeds of sterile cabbage were germinated in germination medium (MS base medium, 3% sucrose, 0.3% gelatin, pH 5.8). Seeds were sown about 40 tablets per bottle, and after 5 days after sowing, the cotyledons were fully developed and cultured until the length of the ventral axis was about 4-5 cm.

At this time, other varieties other than Seoul cabbage should be grown enough because most of the short axis. Therefore, these short-growing varieties were cultivated under dark or weak light.

3. Shaft Organics for Gene Conversion of Chinese Cabbage

Explants were harvested from seedlings of Chinese cabbage 5 days old and cut to about 1 cm in length. At this time, the growth point is excluded because it is likely that division may not be performed normally due to the strong division.

4. Preculture

The cut embryos were preincubated for 1 to 2 days in MS liquid medium, and approximately 200 explants were implanted in 20 ml of liquid medium, and cultured by shaking at 150 rpm when using the liquid medium.

5. Plasmid Vector Preparation

(1) Expression vector (pMOGbar) in which foreign gene is herbicide-resistant gene

As shown in FIG. 2, pMOGbar is a plant expression carrier, which includes a bassta herbicide resistance gene (bar) functionally connected with the 35S promoter and a hygromycin resistance gene (Hyg ^R ) functionally connected with the 35S promoter. have. Here, the hygromycin resistance gene is an antibiotic selection marker. pMOGbar is a terminator containing T _nos and T _i7 , respectively.

(2) Expression vector where the foreign gene is male sterile organic gene (pGR006)

As shown in FIG. 3, pGR006 was produced to induce male infertility by functionally connecting a barnase gene that inhibits pollen formation to P _TA29 , a promoter specifically expressed in the plant's surgical tissue, tappetum tissue. Low hygromycin resistance gene. pGR006 also contains the Barstar gene, and the Barstar gene is expressed only in prokaryotic cells and not in eukaryotic cells, acting as an inhibitor of Barnase enzyme activity.

When the enzyme barnase, which destroys RNA, is formed during the formation of tapitium tissue during surgery, it can artificially block the formation of pollen by destroying the RNA.

6. Agrobacteria Agrobacterium ) And culture of plasmid

Bacteria were harvested from the permanently preserved Agrobacterial solution (containing Agrobacteria LBA 4404 and their respective pMOGbars, pGR006) and streaked to YEP solid media supplemented with 50 mg / l of kanamycin. Incubation was carried out in the dark. One colony was selected and incubated for 2 days while shaking at 200 rpm in a 5 ml YEP liquid medium.

Then, 1 day before the Chinese cabbage transformation, 200 µl of solution was taken from these seed cultures, transferred to 20 ml of YEP liquid medium, and incubated with shaking for one day.

7. Agrobacterial Infections

The precultured embryos were placed in a conical tube containing 20 ml of MS liquid medium, 2 ml of agrobacterial solution was added, followed by shaking at 200 rpm for 20 minutes to apply germs to the entire embryo. Then, the MS liquid medium was poured out and the MS solution buried in the hypocotyl was absorbed with sterile filter paper.

8. Cocultivation

Infection with agrobacteria in MS co-culture medium (MS base medium, 3 mg / lBA, 1mg / lNAA, 4mg / lAgNO ₃ , 8g / l plant agar, pH 5.6) with sterile 9 cm filter paper The embryonic axis of the Chinese cabbage was placed about 50-100, and incubated with pMOGbar and pGR006 for 2 days, respectively. After the inoculation, the excretion was washed three times with sterile water to which 300 mg / l of cefotaxim was added, and then all excess solution was removed with sterile filter paper and transferred to the selection medium.

9. Transgenic Cabbage Selection

In order to select cells transformed with the foreign genes in the pMOGbar, pGR006 during the coculture, the selection medium [MS base medium, 3 mg / l BA , 1 mg / l NAA , 4 mg / l AgNO ₃ , 8 g / l plant agar, pH 5.8, 10mg / l hygromycin, 300mg / l cefotaxim] were incubated at 20 sections, and hygromycin-resistant callus or callus Young re-differentiated cabbage plants were subcultured to the same selection medium.

10. Root Organics

Among leaflets that were re-differentiated in the hygromycin selection medium, transformed individuals with fully-developed leaves (re-divided leaf length of 1 cm or more) were added to the root organic medium (MS basic medium, 8g / ℓ agar, 300mg / l cell). Taksim, 10 mg / l hygromycin, pH 5.8), and then the roots were organic. Plants whose roots are not well-organized are cleanly cut out of the callus tissue formed at the ends of the regenerated plants and transplanted into new root organic media.

Well-rooted green plants are planted in a 4.4 cm plastic pot containing vermiculite, perlite, and peat moss (2: 1: 1), purified for 1 week, and then transferred to a large pot of soil in a greenhouse. Incubated.

11. Obtaining seed

Transgenic cabbages transformed into useful genes were cold-treated for 30 to 40 days in a low temperature room at 4 ° C., and then transferred to a greenhouse to induce germ differentiation and progeny seed.

Experimental Example 2. Bioassay of Genetically Converted Chinese Cabbage

When comparing the morphological characteristics of the leaves after basa treatment of the cabbage transformed with herbicide-resistant genes and the untransformed cabbage according to Example 1, the unconverted leaves were found to have leaf periphery even under 0.075% batha treatment. Was completely white and died, but the leaves of the transformed cabbage were alive with almost no change of leaves even in the bastard treatment of more than 0.3% (see Fig. 4).

Experimental Example 3. Bioassay of Genetically Converted Chinese Cabbage

When comparing the morphological characteristics of the flowers with respect to the cabbage transformed with the male sterile gene according to Example 1 and the non-transformed Chinese cabbage, as shown in Figure 5, the flowers converted to male sterile gene is a flower surgery It was hardly developed and pollen was not formed, but the normal cabbage flower was normally formed with stamens and pollen (FIG. 5). Therefore, it was found that the transformed male infertility gene had an inhibitory effect on pollen formation.

Experimental Example 4 PCR Analysis of Transgenic Chinese Cabbage

To identify cabbages transfected with expression vectors with foreign genes and hygromycin selection markers, primers of the hygromycin gene were used as 5'-AgC CTg ACC TAT TGC ATC TCC-3 'and 5'-TgT CCg TCA ggA PCR reactions were performed using CAT TgT Tgg-3 '. Genomic DNA extracted from each plant prepared in Example 1 was used as a template of the PCR reaction. Lanes 5-12 are the Chinese cabbages that have been transformed with herbicide-resistance gene (pMOGbar) and regenerated cabbages. It is about. On the other hand, a vector containing hygromycin as a positive control group was used as a PCR template (lanes 2 and 3). Then, genomic DNA of Chinese cabbage that was not genetically converted to the negative control group was used as a PCR template (lane 4).

After electrophoresis of the PCR reaction results, the insertion of the gene was assayed with or without a 367bp band. Lane 1 electrophoresed DNA fragments of known size so as to know the size of the PCR product.

As shown in Figure 6, the transgenic cabbage was able to identify the PCR band.

Experimental Example 5. Southern Analysis of Genetically Converted Chinese Cabbage

In order to determine whether the transgenic Chinese cabbage more accurate, Southern analysis was performed as follows. The genomic DNA was isolated by phenol and chloroform extraction from 1 g of the transgenic cabbage leaves and the non-converted cabbage leaves, respectively. Meanwhile, Southern analysis was performed using a probe labeled with ³² P of a 0.6 kb herbicide-tolerant gene fragment cut with the restriction enzyme sma I as a probe.

As a positive control group, plasmid DNA (lane 2) having the same herbicide resistance gene as in Example 1 was used. Transformation was confirmed by the presence or absence of the band at the same size position as the DNA band shown in the positive control group. Lane 3 also electrophoresed the DNA of cabbage that was not genetically converted to the negative control group. Lane 1 is the electrophoresis of the size marker ladder. Lanes 6 to 14 were electrophoresed using HindIII digested cabbage DNA after transforming with herbicide resistance gene (pMOGbar) using the cabbage of Seoul cabbage.

The DNA fragment bound to the probe in the positive control group (lane 2) was 2.0 kb. In the transformed cabbage (lane 6,9,10,14), the desired band size of 2.0 kb was confirmed, but in the unconverted cabbage (lane 3), this band could not be confirmed (FIG. 7).

Example 2

Chinese cabbage was transformed in the same manner as in Example 1 except for using the expression vector pBt 21 (abbreviated as Bt) instead of pMOGbar and pGR006.

As shown in FIG. 8, pBt 21 is composed of artificially synthesized insecticidal gene (cryIAC) and hygromycin resistance gene (Hyg).

Example 3

Chinese cabbage was transformed in the same manner as in Example 1 except for using the expression vector pHGCTB (abbreviated as CTB) instead of pMOGbar and pGR006.

As shown in Figure 9, pHGCTB is composed of cholera vaccine B fragment (CTB) and hygromycin resistance gene (Hyg).

Example 4

Chinese cabbage was transformed in the same manner as in Example 1 except for using the expression vector pGR008 instead of pMOGbar and pGR006.

As shown in FIG. 10, pGR008 is composed of herbicide-tolerant gene (bar) and Dtx-A (diphtheria toxin gene), a male infertility gene expressing tapitium specificly isolated from tobacco.

Example 5

Chinese cabbage was transformed in the same manner as in Example 1 except for using the expression vector pGR009 instead of pMOGbar and pGR006.

As shown in FIG. 11, pGR009 is composed of Dtx-A (diphtheria toxin gene), a surgical-specific expression male infertility gene isolated from cabbage to herbicide resistance gene.

Experimental Example 6. Transformation Efficiency Measurement

12 to 15, □ Callus represents the efficiency of selecting the transformed cells, ■ Shoots represents the efficiency of obtaining a complete transformed cabbage plants, that is, shoot transformation efficiency in the previously selected cells.

The efficiency of selecting the transformed cells (Callus) was calculated by the equation (1).

shoot transformation efficiency (Shoots) was calculated by the equation (2) .

As can be seen in Figures 12 to 15, even if different varieties of Chinese cabbage can be transformed various genes by the present invention.

Experimental Example 7. Subsequent Test of Genetically Converted Chinese Cabbage

Table 2 below analyzes the results obtained by obtaining the successive seedlings of some of the cabbage obtained herbicide resistance by transformation in accordance with Example 1 to investigate the separation ratio of hygromycin resistance to these individuals.

After seeding and disinfecting the seed of each transgenic cabbage, the seeds were sown in MSO medium containing 30 g / l of hygromycin, and the plants (hygromycin resistance) and the dying plants (surviving in the hygromycin-added medium) The ratio of hygromycin susceptibility) was investigated to determine the separation ratio.

On the other hand, the theoretical ratio is usually separated by 3: 1 according to Mendel's genetic law when one foreign gene is transferred, which is calculated as the theoretical separation ratio. When two foreign genes are entered, the later theoretical separation ratio is 15: 1.

On the other hand, X ² is a goodness-of-fit test that examines the frequency of each category according to the criteria of the classification and tests whether the proportion of the whole fits the proportion of the population, or the independence test of the independence between two different categories of criteria. It is a statistical value used for frequency analysis that analyzes discrete frequencies such as tests. In this experimental example, X ² was used to probabilistically determine whether the expected frequency calculated from the theoretical basis and the observed frequency obtained from the experiment were the same as the population. X ² value was calculated by the equation (3).

If Table 2 of the three Seoul cabbage ^{X 2 = (16-15) 2/} 15 + (4-5) is ² /5=0.266. Since the value of 0.266 is X ² 0.05 (1) = 3.84 when the degrees of freedom (df) = separation coefficient (2) -1 in the X ² -distribution table, X ² = 0.266 is less than X ² = 3.84 We accept the hypothesis that it is a group that divides by 3: 1.

Analysis of Subsequent Hygromycin Resistance Isolation Ratio of Chinese Cabbage Transformed with Herbicide Resistant Gene kind Transformed Cabbage System Hygromycin Resistance Hygromycin sensitivity Resistance: Susceptibility Theoretical ratio X ² value Seoul Cabbage 12 ^* 34 361617 1843 3: 10.075: 14: 15.6: 1 3: 11: 13: 13: 1 00.2850.2661.066 Samjin cabbage 12 1823 27 9: 13.2: 1 15: 13: 1 1.0120.341

* Natural pollination (open pollination)

As can be seen in Table 2, in three of the four strains transformed in Seoul cabbage, all three offspring plants were resistant to hygromycin at a ratio of 3: 1, indicating that one copy of the gene was transformed. In one strain, the cabbage was not transformed and the natural pollination resulted in a separation ratio of 1: 1. In other words, the gene of one copy was transformed and it was found that the ratio was 1: 1 when assayed.

One of two herbicide-tolerant lines of Samjin cabbage approached the separation ratio of 15: 1, which means that two copies of the gene were converted. Lose. The X ² values of the transgenic cabbages obtained from the above-mentioned Seoul cabbage Nos. 1, 3, 4 and Samjin Cabbage Nos. ² are the degrees of freedom (df) = separation class (2) -1 day in the X ² -distribution table. When X ^{2 is} _less than _{0.05 (1)} = 3.84, there is no significance and it can be said that it is a 3: 1 division. And X ² value of the time Seoul cabbage 2 system and striking the cabbage 1 line diagram of a X ² distribution table X ² _{0.05 (1)} = less than 3.84 is 1, each expected value: through the X ² test is suitable for 1: 1 and 15 I could confirm it.

As a result of performing the cabbage gene conversion experiment according to the present invention, the cabbage expressing insecticidal genes, herbicide resistance genes, male infertility genes, etc. was obtained, and the gene was stably introduced into the cabbage by PCR and Southern blot analysis. I could confirm it. In addition, we obtained genetically modified cabbage with herbicide resistance significantly higher than that of non-converted cabbage, and obtained transgenic cabbage with little pollen formation in the flowers of Chinese cabbage converted to male infertility. Could.

According to the present invention, an efficient gene conversion technology of Chinese cabbage has been developed, which can be used to transform new and useful genes into cabbage more easily and stably, as well as to produce cabbages having new traits different from existing cabbage varieties. Can breed varieties of cabbage.

Claims (12)

In an in vitro method of regenerating Chinese cabbage, Germinating the seeds to prepare for defecation; Separating the hypocotyl; And And regenerating the cabbage from the embryonic axis in a culture medium containing silver nitrate (AgNO ₃ ). The method of claim 1, wherein the seed germination step is in vitro regeneration of cabbage, characterized in that the culture in the dark or light state. The in vitro regeneration method of the Chinese cabbage, characterized in that the time to separate the embryonic axis is 5 days after sowing. The in vitro regeneration method of cabbage according to claim 1, wherein the concentration of silver nitrate is 1-5 mg /. In the method of producing a transformed cabbage, Germinating the seeds to prepare for defecation; Separating the hypocotyl; Introducing foreign DNA into the isolated hypocotyl via an expression vector; And Regenerating the transformed cabbage from the embryonic axis in a culture medium containing silver nitrate. The method of claim 5, wherein the expression vector is introduced into the embryonic axis by Agrobacterium-mediated transformation. 6. The method of claim 5, further comprising the step of pre-culturing the cut axial axis after separating the axial axis. The method of producing a transformed cabbage according to claim 7, characterized in that the embryonic culture is precultured in a liquid regeneration medium. The method of claim 5, wherein the regenerating the transformed cabbage Culturing in a regeneration medium containing silver nitrate under 25-27 ° C. light conditions; And A method for producing a transformed cabbage, comprising the step of organically rooting in completely leaf-developed individuals of the re-differentiated transformants. The method according to claim 5, wherein the expression vector comprises a hygromycin resistance gene as a selection marker, and the step of introducing a foreign DNA followed by selecting a transformant in a selection medium containing hygromycin. Method for producing a transformed cabbage, characterized in that it further comprises. 6. The method of claim 5, further comprising the step of self-pollinating the transformed cabbage to produce homozygous transformed progeny. 6. The method of claim 5, further comprising the step of incorporating the transformed cabbage with other cabbage to introduce the foreign DNA into a different genetic environment.