KR100850525B1 - Method for transforming cactus - Google Patents

Abstract

 The present invention relates to a cactus transformation method. According to the present invention, transformed cactus domains can be effectively obtained by adding the step of inducing the generation of cacti domains before and after introduction of a gene having a desired characteristic in the cactus.

Cactus bulb, transformation

Description

Method for transforming cactus {Method for transforming cactus}

1 is a photograph showing a cactus domain that is not transformed.

Figure 2 is a photograph showing the transformed cactus domains.

Figure 3 shows the results of GUS staining in the transformed and untransformed cactus.

Figure 4 shows the RT-PCR results for confirming the expression of the GUS gene in the cactus.

To date, various plant transformation methods have been reported. Transformation of cells or tissues after introduction of the gene using tissues or cells, genes expressing the target substance without tissue culture step, or traits required for plants, such as genes providing disease resistance to plants There is an on planta transformation. Introducing foreign genes using tissues or cells is the most commonly used method for plant transformation, in which tissues or cells isolated from plants are transformed, followed by tissue culture in a suitable soil or medium to be transformed plants. To obtain. This method is best established in tobacco and petunia. The transformation of tissue or silver cells can be accomplished using Agrobacterium, a soil microorganism, or by means of a gene gun (bollistic transfer), fusion (PEG mediated fusion), electroporation or liposomes. It is carried out by a method such as mediation.

In particular, Agrobacterium co-cultivation method has been conventionally used only for the transformation of dicotyledonous plants, but in recent years also used for transformation of monocotyledonous plants, specifically, the tissue section is co-cultured with Agrobacterium and then an appropriate selective marker ( Transgenic plants are obtained by differentiation in a regeneration medium containing a selection marker. This method requires coculture, removal of Agrobacterium using antibiotics, identification of transformants, etc. As a result, the regeneration ability of plant tissues is impaired during each step, resulting in failure to obtain re-differentiated individuals or The frequency of occurrence is significantly reduced. In order to solve this problem, the proposed method uses a highly pathogenic argobacterium that can preculture plants or improve transformation efficiency, but no fundamental solution has been proposed.

Meanwhile, in addition to Agrobacterium, TMV (tobacco mosaic virus) or CPMV (cow-pea mosaic virus) may be used as soil microorganisms used for transformation. In addition, a method using a gene gun is a method of transforming a plant by coating DNA of a foreign gene on tungsten or gold particles, and then using a gene gun to shoot particles in a plant tissue to introduce DNA into plant cells. This method is mainly used for transforming monocotyledonous plants, including horticulture, which does not fall within the host range of Agrobacterium, but is also used for dicotyledonous plants. In using this method, the optimum conditions under which DNA can penetrate into plant cells vary depending on the type and tissue of the bombarded plant, and the size and density of the DNA, the method of coating and the firing of the DNA-coated particles. Several factors, such as speed and number of shots, can work and establishing optimal conditions for them is very important. In addition, this method is generally applicable to all tissues, but it is advantageous to use tissues with active cell division and ability to differentiate. There have been many reports of successful transgenic plants using tissues such as meristems, young leaves and embryogenic suspensions. As such, the Agrobacterium co-culture method or plant transformation method using particle bombardment requires a regeneration process of the plant. Therefore, there is a disadvantage that it is difficult to apply these methods to plants that have not been well established or have a long regeneration time. In addition, if undesired genetic variation, which occurs during the somatic mutation transformation process in re-differentiated plants, occurs during the tissue culture process, appropriate control mechanisms should be established to find and prevent mutagenesis. Therefore, the need to minimize somatic mutations requires the development of new transformation methods that eliminate or digest tissue culture steps by transferring and regenerating genes into intact tissue sections without going through in-flight culture.

In planar transformation method has been proposed as a method to obtain a transformant without tissue culture and regeneration process. This method obtains transformed seeds or stems from stems that transform cells located in plant growth points or meristems and then re-differentiate from transformed cells. As a method for transforming meristems, transformation methods using vacuum, floral meristem dipping method, and agrobacterium spraying method have been developed. This method is best established in Arabidopsis. Specifically, Agrobacterium is introduced directly into the meristem of the plant in the pollen, and the transformants are selected by culturing seeds produced from the plant in a selection medium. Agrobacterium's T-DNA is inserted into the chromosomes of germ cells and passed on to the next generation to show transformants. On the other hand, a method of obtaining transformed seeds by applying foreign gene DNA to a flower owner of pollinated flowers without using Agrobacterium was reported in 1992 in rice and tobacco. According to this method, a pollen tube pathway is formed through pollen nucleus through pollination of plants, and the transformed seed can be obtained by cutting the stigma of pistil in which the pollen tube is formed and introducing DNA directly. Can be.

As described above, various transformation methods have been used for the transformation of plants, but the method that can be applied according to the characteristics of the plant is limited, so that successful transformation is achieved only for a relatively limited species of plants.

On the other hand, in the case of cactus, which has recently increased in value for ornamental use, many researchers have attempted transformation, but it is known that the effective transformation cannot be performed by conventional methods.

Cactus is a fleshy perennial herbaceous plant, a tropical plant, and the plant belonging to the cactus family has about 2,000 species all over the world, and it is divided into three groups of tree cactus, cactus, and white hair cactus. Cactus can be cultured in large quantities by seed propagation, by asexual propagation of aliquots, cuttings or grafting, or as a sexual propagation. Recently, the value of cactus as an ornamental plant is increasing, especially grafted cactus is gaining popularity. However, viral infections in cacti are a serious problem. Virus infections in large trees, such as deltas, reduce graft activity, poor growth, assortment fades and poor quality. Currently, three types of cactus viruses have been found in Korea, including CVX (Cactus Virus X). In particular, many cases of such viral infection have been reported in grafted cactus, which is valuable for ornamental use, and prevention of viral infection has become an important problem for farmers developing ornamental grafted cactus. Virus-infected cactuses have a thick pale mosaic pattern. Cactus grafted to the larvae infected with the virus, such as sulphides and malformations, severe death. In the early stages of infection, the disease does not appear externally to the extent that it is difficult to visually identify, but over time, it causes mosaicism, malformations, atrophy, and necrosis. In flowers, they form mosaic patterns on the petals. CVX attaches to hands and instruments during grafting, and contaminated juice is transmitted through the wound. Currently, there are about 3 kinds of cactus viruses isolated in Korea, and these types are known to have no mediated insects, and they are mainly transmitted through the juice of roots and the wounds of the roots in soil. . Viruses are more damaging in combination infections of two or more than single infections.

In addition, the most damaging to cacti species, stem disease is a disease that is difficult to control when the disease occurs. Dark brown to black irregular lesions are formed from the branch of the stem and when the lesions progress, the lesions expand and rot. It is known to be caused by fungi such as Fusarium. The growth temperature ranges from 6 to 38 ° C, but the growth temperature is 25 to 28 ° C. Most fungi invade through wounds, so planting should ensure that the plants are intact. It spreads soil and starts to rot from the ground due to fungi. During trophic breeding, it is spreading by cutting and air spreading by scattering of conidia. As it progresses, it fades black and eventually the whole plant is rubbed. In addition, Rhizoctonia is the cause of the rot, bacterial soft disease, black spot disease, soot disease, white dog disease.

The present inventors studied the transformation method of the cactus as part of an effort to introduce disease resistance to the cactus or to develop new varieties of cactus, and as a result, the transformation of the existing plant transformation method is difficult. The present invention was completed by successfully performing transformation of known cacti.

An object of the present invention is to provide a method for transforming a cactus.

It is also an object of the present invention to provide a novel cactus of transformed.

In order to achieve the above object, the present inventors introduced a gene having a characteristic suitable for the purpose to be introduced into the cactus by using a plant transformation vector, and confirmed the expression. Agrobacterium was used for plant transformation. Specifically, the cactus is transformed by using a pin-prickle method or a vacuum-infiltration method, or a combination of both methods, at the site where the domain of the grafted cactus is to be formed, and the expression of the GUS gene is expressed. It was confirmed. After the expression vector containing the GUS gene was transformed into Agrobacterium, it was applied to YEP solid medium and incubated for 2 days in an incubator, and then Agrobacterium was grown to form a colony and used for transformation. In the pin-prickle transformation method, a region of the grafted cactus is formed by puncturing a wound 5 to 10 times with 0.1 to 0.3 mm of tungsten pins, and then immersed in a transformation buffer to agrobacterium. Was performed by inoculating the wound site. In the vacuum-infiltration method, the prepared transformation buffer is placed in a glass beaker, the grafted cactus is upside down to contain the domains, and then it is put in a vacuum container at a constant pressure (10 cmHg to 50 cmHg) for a predetermined time. After the vacuum was applied, the vacuum pressure was abruptly removed so that the transformation buffer could effectively penetrate. On the other hand, in order to induce the production of magnetic domains in the cactus infected with Agrobacterium, about the top of the cactus was cut using a cutter knife.

From the resulting domains, the expression of GUS protein was confirmed by X-Gluc staining. As shown in FIG. 1, the non-transformant was discolored in white, but the transformant was confirmed to be dyed blue by GUS staining.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto. The documents cited in the detailed description of the invention are incorporated herein by reference.

Example

Example 1 Agrobacterium Transformation

The pBI121 vector (Clontech, USA) was used as the plant transformation vector for the transformation test. To detect the transformation, a β-glucuronidase (GUS) gene expressed by the Cauliflower Mosaic Virus (CaMV) 35S promoter was introduced into the T-border of the vector. Agrobacterium was used for transformation, and for this purpose, a recombinant vector containing the GUS gene was transformed into Agrobacterium. Agrobacterium transformation was performed using the Freeze-thaw method. Agrobacterium was used as LBA4404 (Invitrogen, CA), and 10 µl of the prepared pBI121 vector was added to 100 µl of Agrobacterium, and reacted on ice for 30 minutes. The cells were then rapidly frozen in liquid nitrogen for 1 minute and immediately reacted for 5 minutes at 37 ° C heat-block. After completion of the reaction, 1 ml of liquid YEP medium (Yeast extract 1%, NaCl 0.5%, Bactopepton 1%) was added, and the diluted Agrobacterium was incubated at 28 ° C. and 200 rpm for 4 hours. After the incubation was completed, the supernatant was removed by centrifugation at 3,000 rpm for 2 minutes, and the diluted Agrobacterium was diluted again in 100 µl of liquid YEP medium. Diluted Agrobacterium was applied to YEP solid medium to which kanamycin was added at a concentration of 25 mg / L and incubated for 2 days in a 28 ° C. incubator to select agrobacterium grown in colonies and used for transformation.

Example 2: Transformation of Cactus

(1) Agrobacterium culture

Agrobacterium was incubated in a liquid YEP medium (Yeast extract 1%, NaCl 0.5%, Bactopepton 1%) until a value of 0.7-0.8 was measured in a turbidity incubator at 28 ° C and 250 rpm. It was used for the experiment. After culturing Agrobacterium in 500 ml of YEP medium in total, Agrobacterium was recovered by centrifugation at 3,000 rpm for 15 minutes, and the recovered Agrobacterium was transformed into a transformation buffer (1/2 MS salt, 50 g / L). Diluted in 50 ml of sucrose, 0.5 g / l MES, and 200 μl / l silwet (pH 5.7)) was used.

(2) Pin-prickle transformation

Grafting cactus ( Notocactus scopa, Sungjung, obtained from: Cactus Research Institute, Gyeonggi-do Agricultural Research Institute) Agrobacterium was inoculated at the wound site by dipping into a transformation buffer.

(3) Vacuum-infiltration method

Place the prepared transfection buffer in a glass beaker, turn up the grafted cactus ( Notocactus scopa, Sungjung, obtained from Gyeonggi-do Institute of Agricultural Research and Development, Cactus Research Institute) to hold the mole region, and put it in a vacuum container for a period of time (5 to 30 minutes). ) Vacuum was applied at a constant pressure (10 cmHg ~ 50 cmHg), and the vacuum pressure was rapidly removed to allow the transformation buffer to effectively penetrate. In addition, the experiment was performed by mixing or treating the gastric vacuum-infiltration method and the pin-prickle method alone.

(4) Stabilization of Agrobacterium inoculated individuals

Cactus inoculated with Agrobacterium was used to remove excess buffer using paper towels and incubated for 24 hours in a 28 ℃ cow.

(5) induction of domain

In order to induce the production of magnetic domains in the cactus infected with Agrobacterium, about the top of the cactus was cut using a cutter knife. The cactus cut off at the top was incubated for 24 hours in the dark at 28 ℃. Thereafter, the individual was transferred to soil and planted. After about 4 weeks, the spines of the cactus began to form, and when the diameter was about 1 cm, the mother was separated from the mother and verified for transformation. As a control group, the group that did not induce the generation of the magnetic domains was used, and the expression of the GUS gene was examined from those generated in the control group and compared with the group that induced the generation of the magnetic domains. As a result, as confirmed in Table 1, it was confirmed that the GUS gene was generated only in the group inducing autogenogenesis before and after transformation.

Example 3: Transformation Verification (GUS Staining)

In order to verify the transformation, the expression of the GUS protein was confirmed by X-Gluc staining using the GUS gene inserted into the pBI121 plant transformation vector. First, the induced cactus glomerular tissue was placed in pre-cooled 90% acetone and fixed in ice for about 20 minutes, and then acetone remaining on the epidermis was removed using a paper towel. Acetone-free plants were placed in X-Gluc staining solution (0.1% Triton-X, 50 mM NaPO ₄ , 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 10 mM EDTA, 2 mM X-Gluc) and stained completely onto tissue. The vacuum was maintained for about 30 minutes using a vacuum pump to allow this to soak. The tissue was sufficiently moistened in the dyeing solution, and then placed in the dyeing solution and reacted for about 8 hours in a 37 ° C incubator. After staining, 70% alcohol stained porcine tissue was added and the tissues of the non-transformed domains were decolorized to become completely white and verified by comparison with the transformants. As shown in FIGS. 1 and 2, the non-transformant was discolored in white, but the transformant was confirmed to be dyed blue by GUS staining.

Table 1. Transformation Efficiency

Example 4: Transformation Verification (GUS Staining)

The same method as in Example 3 was carried out, but the mother cells generated at the GUS staining site of the mother were identically stained by transforming the mothers transformed with the domains induced by the method described in Example 2- (5). By confirming the stained form was confirmed the transfer of the gene and the expression of the transferred gene. In FIG. 3, the left side shows the GUS staining results in the untransformed cactus, and the right side shows the GUS staining results in the cactus which had been transformed.

Example 5: Transformation Verification (RT-PCR)

RT-PCR was performed to confirm whether the GUS gene is stably expressed in the transformed cactus through molecular biological methods. First, total RNA of cactus was extracted by hot-extraction method. The tissue of interest was flash frozen in liquid nitrogen and ground finely in a mortar and transferred to a 2 ml micro centrifuge tube. To this was added 500 μl of extraction buffer [phenol: 0.1 M LiCl, 100 mM Tris-HCl, pH = 8.0), 10 mM EDTA, 1% SDS (1: 1)] heated to about 80 ° C., followed by stirring. 250 μl chloroform-isoamylalchol (24: 1) was added and stirred again. After centrifugation for 5 minutes at 12,000 rpm, the supernatant was transferred to a new tube. Then, an equal amount of 4 M LiCl was added and reacted at room temperature for 14 hours, followed by centrifugation at 12,000 rpm for 10 minutes to remove the supernatant and to obtain a precipitate. The precipitate was dissolved in 150 μl of diethyl pyrocabonate (DEPC) -treated distilled water, and added 1/10 volume of 3 M sodium acetate and twice the total volume of 100% ethanol in a -20 ° C. freezer. The reaction was carried out for 3 hours. Next, the precipitate was obtained by centrifugation at 15,000 rpm 4 ° C. for 30 minutes, dissolved in 50 μl of DEPC-treated distilled water, and stored in a -70 ° C. freezer. The concentration of purified total RNA was measured through a spectrometer, and diluted in a 0.5 ml microcentrifuge tube after diluting to a total volume of 10.5 μl in DEPC treated distilled water so that the total RNA contained 5 μg. Next, 3.0 μl of 10 pM oligo-dT was added, heated at 70 ° C. for 10 minutes with a PCR thermocycler (PTC-0100, MJ Research), cooled to 4 ° C., and then 5 times with 6.0 μl of 2.5 mM dNTPs. 5.0 μl of concentrated reaction buffer was added. After reheating to 37 ℃ to react for 10 minutes and cooled to 4 ℃ again. Then, 0.5 U of 200 U / μl reverse transcriptase was added and reacted at 37 ° C. for 1 hour. Subsequently, cDNA was synthesized by reacting at 70 ° C. for 10 minutes and then stored at 4 ° C. Then, to confirm the expression of the desired gene, 3.0 μl of synthesized cDNA, 1.0 μl of 5 ′ and 3 ′ regions of the cDNA synthesized for amplification of the target gene, respectively, 2.5 μl of 2.5 mM dNTPs and 10 μl of sterile distilled water. PCR was performed using a PCR thermocycler (PTC-0100, MJ Research) by adding 2.0 μl of 10-fold concentrated reaction buffer and 0.5 μl of taq polymerase. PCR was carried out i) 10 minutes at 95 ° C, ii) 30 seconds at 94 ° C, iii) 30 seconds at 56 ° C, iv) 30 seconds at 72 ° C, and then iv) steps 30 times in step ii). After the completion of about 10 minutes at 72 ℃. In FIG. 5, the expression of the GUS gene was not confirmed through RT-PCR in the non-transformed cactus. However, the expression of the GUS gene was confirmed in the transformed mother and the generated sperm. In Figure 4, the first column shows the DNA size marker, the second row shows the RT-PCR result in the untransformed cactus, the third row shows the RT-PCR result in the transformed cactus mother, and the fourth row shows the transformed cactus mother. RT-PCR results are shown for each domain generated in

As such, according to the present invention, it is possible to easily transform a cactus known as a plant species, which is not easily transformed. Therefore, by transforming the cactus with the transformation method according to the present invention, it is possible to develop a new breed of cactus having enhanced disease resistance or antiviral resistance.

Claims (4)

An expression vector containing a foreign gene to be introduced is introduced into a cactus locus-producing region by a pin-prickle method, vacuum-infiltration method, or a combination of the two methods. Doing; And Cactus transformation method comprising the step of inducing autogenogenesis by cutting a portion of the cactus before and after the introduction of the expression vector.  The method of claim 1, wherein the expression vector is introduced into the cactus by Agrobacterium. delete 3. The cactus transformation method according to claim 1 or 2, characterized in that the vacuum-filtration method rapidly removes the vacuum pressure after applying a vacuum of 10 cmHg to 50 cmHg for 5 to 30 minutes.

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