KR100363122B1 - A method for the development of transgenic garlic plants by gene manipulation and its transgenic garlic - Google Patents

Abstract

The present invention relates to a method for providing a genetically engineered garlic plant ( Allium sativum ) using a transformation technique by microparticle projection (particle gun). When the growth point of garlic is collected and incubated under appropriate conditions, it is possible to induce the occurrence of callus. These callus are undifferentiated meristems and can be regenerated into complete plants. For efficient transformation, it is necessary to obtain a specific type of callus which is relatively active in cell division and cultured in a specific medium, and to project the callus microparticles coated with recombinant DNA. In addition, for efficient transformation, the transformant screening process using a screening agent after microparticle projection must proceed effectively, and the use of an effective screening agent such as hygromycin is essential in this process. Recombination of genes used for transformation also requires selection of an effective promoter for garlic tissue. Therefore, using the technique described in the present invention, selection of garlic callus with high transformation efficiency, projection of microparticles coated with recombinant DNA, selection of transformants, regeneration and propagation of transformants, identification and purification of transformants The process effectively yields garlic transformants with useful traits.

Description

A method for the development of transgenic garlic plants by gene manipulation and its transgenic garlic}

The present invention relates to a technique for genetically engineering plants, and more particularly, to a method for transforming garlic using a particle bombardment-mediated transformation technique.

Garlic is a representative spice and vegetable crop belonging to the family Liliaceae, and it is the second largest crop of onions and onions. It is cultivated all over the world, and is especially used by Koreans. It is also used as a raw material for health foods or medicines. Garlic is non-degradable due to its physiological characteristics, and because it propagates through clove, it is not breeding, so the number of varieties is extremely limited. In our country, winter temperatures are relatively high and the southern coastal areas are shorter and the warmer areas grown in Jeju Island are colder and colder areas grown in lower and longer winter areas.They are grown in areas like Yecheon Garlic, Uiseong Garlic and Danyang Garlic. The varieties that are adapted to the climate of the country are divided into mountainous regions for convenience.

As such, if the conventional breeding method using pollen cannot be used, the recently developed genetic engineering molecular breeding method can be applied as an alternative. Genetic molecular breeding is a method of transplanting and expressing a new functional gene into the genome of a selected individual to obtain a desired trait. Unlike traditional breeding, breeding of new functional genes while maintaining the genotype of a good breed is possible, enabling efficient breeding in a short time.

In order to successfully perform genetic engineering methods, it is necessary to effectively transplant foreign genes into target cells, obtain appropriate target samples, and regenerate technologies to obtain complete plants from transformed cells. There are two methods of effective plant cell transformation (gene transplantation). The first method is to transfer foreign gene DNA to plant cells using a biological vector, which mainly uses the soil plant pathogen Agrobacterium tumefaciens . And second, a method of projecting DNA microcoated metal microparticles by direct delivery of foreign gene DNA into plant cells (Hansen and Wright, Trends Plant Sci . 4; 226-231, 1999).

The most commonly used plant transformation technology today is the plant pathogen Agrobacterium, which is a part of their Ti (tumor-inducing) or Ri (root-inducing) plasmid DNA. (T-DNA) has the ability to metastasize and transplant into infected plant cells. Therefore, when the target gene is recombined in T-DNA and co-cultivated in a fungal plant having the same, plant cell transformation is possible. However, the critical limitation of this technique lies in the compatibility, including the host range of Agrobacterium and the target cells to be transformed, and ultimately affects the successful regeneration of the transformed cells. Therefore, many economically useful crops have not yet been transformed by this method (Hansen and Wright, Trends Plant Sci . 4; 226-231, 1999).

In an effort to develop a method for transplantation of genes regardless of plant species or genotype, several techniques for delivering DNA directly to plant cells have been studied. These include electroporation and microinjection. microinjection, PEG- or liposome-mediated DNA inhalation, silicon carbide whiskers, and microparticle projection. Among these, microparticle projection technology known as bioolistics or microprojectiles is the coating of recombinant DNA into small carrier metal particles and the physical projection of these particles into plant cells (Klein). et al ., Nature 327; 70-73, 1987: Sanford, Plant Physiol . 79; 206-209, 1990). This method has several advantages. First, the removal of the cell wall is not necessary to introduce DNA. Second, DNA can be easily introduced into cells of meristems and differentiated tissues. Third, no special biological carrier manipulation is required. Finally, the transfer of DNA into the cell does not depend on the binding or mutual recognition of the transgenic target cell and the biological vector. Examples of transformation with these particle projection methods include corn (Fromm et al ., 1990 Biotechnology 8; 833-839, 1990), rice (Christou et al ., Biotechnology 9; 957-962, 1991), barley (Wan and Lemaux, Plant Physiol . 104; 37-48, 1994), wheat (Vasil et al ., 1992 Biotechnology 10; 667-674, 1992), soybean (McCabe et al ., Biotechnology 6; 923-926, 1988), candy It has been proven in important crops such as sorghum (Bower and Birch, Plant J. 2; 409-416, 1992). The advantage of this method is that shoots that are re-differentiated in transformed cells can be generated directly from primary and secondary meristems, thus reducing plant hormone processing and minimizing somatic mutations.

Breeding of new varieties, including pest-resistant garlic, odorless garlic, muchia garlic, and pharmacologically enhanced garlic by genetic engineering, is of commercial importance. Due to the physiological characteristics of nutrient breeding garlic, it is almost impossible to improve varieties by conventional plant breeding methods such as mating. Therefore, improvement of garlic using genetic engineering techniques is of interest. It is also of academic importance for the study of nonflammable mechanisms such as garlic.

Garlic actually has several different characteristics compared to other plants in its transformation. First, garlic has a very large genome of about 10 ¹² base pairs. Second, garlic cells do not respond well to various manipulations in tissue culture and transformation processes such as protoplast culture. Third, the regeneration of plants from cultured differentiated cells is difficult. Fourth, garlic cells are not easy to select for transgenic cells using antibiotics such as kanamycin. As shown in the results of this experiment, garlic is resistant to kanamycin of 500 mg / L or more. Finally, because of the multicellular structure of the meristem (calus) used for garlic transformation, the resulting transgenic plant can sometimes be a chimera with transgenic and nontransgenic parts.

Therefore, none of the experimental methods reported to date are useful for the practical application of garlic's genetic engineering techniques, and thus no successful cases of transgenic garlic have been reported worldwide. Barandiaran et al. (Barandiaran et al. , Plant Cell Rep . 17; 737-741, 1998) attempted to transform several tissues of garlic by microparticle projection but only reported transient expression of marker genes in some tissues. No garlic transformants with recombinant DNA genes have been obtained worldwide. In the case of onions belonging to the same genus, Eady et al obtained transformants using Agrobacterium this year (Eady et al. 2000. Annual Meeting of the International Society for Plant Molecular Biology, Quebec). They used jellyfish green fluorescence protein genes as marker genes (Eady et al ., Plant Cell Rep . 19; 376-381, 2000).

It is an object of the present invention to provide an effective method of transforming garlic by microparticle projection. More specifically, to provide a suitable medium composition and culture conditions in order to make the resulting callus suitable for transformation, to induce the generation of continuous meristem before particle projection or after particle projection to increase the efficiency of foreign gene transplantation, and then By providing a method of effectively screening morphogenesis and regenerating callus to obtain a regeneration plant, the present invention provides a method of broad application of genetic engineering technology that can accelerate the improvement of garlic properties. For example, herbicide-resistant transformed garlic plants were produced using herbicide-resistant genes as target genes.

1 is a picture of the selection and transformation efficiency assay of a specific callus effective for fine particle projection,

Figure 2 is a photograph of the susceptibility of non-transformed garlic callus to the selector hygromycin,

Figure 3 is a photograph of the expression control efficiency of the various promoters in garlic callus,

4 is a schematic of the recombinant genes pC1301-ALSGUS and pCambia1201 used in this experiment,

5 is a depressed medium culture photograph for selecting a transgenic callus,

6 is a photograph of shoots re-differentiated from callus identified as a transformant,

Figure 7 is a photograph of the long-term stable expression of the marker gene (GUS) in the transformed garlic shoots,

FIG. 8 is a diagram showing a Southern blot of a corresponding DNA fragment after amplifying a selected gene (HPT) and herbicide resistance target gene (ALS) transplanted to identify transformed garlic by PCR (polymerase chain reaction).

9 is a diagram confirming the RNA transcript fragments of the selected gene (HPT) and herbicide resistance gene (ALS) transplanted and expressed in the transformed garlic by Northern blot,

10 is a photograph of a transformed garlic plant growing in the soil,

11 is 4 ppm herbicide GLEAN ^? To assay herbicide resistance of transformed garlic plants ^. This figure was cultured in a medium containing (chlorsulforan, DuPont).

The present invention is to produce a genetically engineered garlic using recombinant genes expressible in plants,

1) cultivating callus, which is easy to transform and active, is cultured under specific conditions;

2) concentrating the reprogrammable callus cells on the surface of the target for microparticle projection;

3) transplanting foreign genes by physically accelerating projection of fine metal particles coated with recombinant foreign genes containing at least the desired foreign gene and transformant selection gene;

4) selectively propagating the transformed cells in an effective selection medium;

5) obtaining transgenic garlic plants in regeneration medium;

6) to provide a method for producing genetically engineered garlic, characterized in that it comprises the step of massively multiplying the transformed garlic using polycythenic growth method.

Hereinafter, the present invention will be described in more detail.

The invention can be used to produce a transformed garlic plant comprising transformed garlic cells, which transformed garlic cells contain foreign genes. This garlic genetic engineering technique can be achieved through the use of plant transformation technology by particle projection, which transplants foreign gene DNA delivered as fine particles into callus, selects transformed cells and obtains transformed plants through regeneration. have. The culture technique described herein induces the proliferation of a relatively large number of meristem cells subjected to particle projection, and through effective selection of transformed meristems, proliferates the projected meristems and expresses the callus in which the introduced genes are expressed. Make effective selection. It then induces shoot development from meristem to produce transformed plants. Garlic samples used to carry out the invention are Danyang and Euclinic regional garlic.

The objects, advantages and characteristics of this invention will become more apparent from the following specification and drawings. In this invention, it was found that the plant transformation method using the microparticle projection method can be successfully applied to garlic transformation, and the herbicide-resistant transformed garlic could be developed using this method. Since the technology used in the present invention can be applied to transform various target genes into garlic in the future, it is possible to use genetically engineered insecticide-resistant garlic, odorless garlic, muchia garlic, garlic with enhanced pharmacological ingredients, etc. It is possible to develop varieties of valuable transformed garlic.

The methods described herein relate to the implantation of foreign target genes into the genome of garlic plants through artificial manipulation. Such foreign genes are preferably all of gene DNA derived from other species such as animals, plants and microorganisms, and foreign genes generally include a sequence capable of synthesizing a specific RNA transcript or a protein of interest.

DNA vectors typically contain regulatory sequences that are effective at causing expression of the protein. Examples of expression control sites include a promoter sufficient to control transcription in plant cells, and a polyadenylation sequence sufficient to normally terminate transcription or translation to obtain an intact gene product and / or Or a terminator. It is also possible to include transcriptional and translational efficiency enhancing sites around the promoter to enhance the efficient expression of the gene, especially the expression of the protein product. In particular, if the promoter is appropriately selected, the gene can be constitutively expressed in transformed cells, or tissue-specific or inducible by specific factors can be manipulated.

Various target genes can be used for garlic transformation. Enzyme protein genes that can provide resistance to pests and herbicides, enzyme protein genes that regulate metabolism to enhance pharmacological components or weaken odors, genes involved in plant growth and morphology Etc. can be very diverse. Genes can be used in any prokaryote or eukaryote, that is, any gene derived from microorganisms, plants and animals such as bacteria, fungi, yeast, viruses.

Recombinant plasmids, which are used for plant transformation by microparticle projection, contain one or more gene cassettes, the gene products of which can be interacted with independently. When various cassettes are used, they may be located separately in different plasmids, unlike the same plasmid or Agrobacterium method. When separate cassettes are present in different plasmids, these plasmids may be coated on the same microprojectile, may be coated on different microparticles, or may be mixed with each other and mixed with each other to project onto a target tissue. You may.

Expression cassettes include a promoter that regulates transcription, a transcription initiation portion, a translation initiation codon, an amino acid coding region of a protein, and a transcriptional terminator that terminates transcription following a translation stop codon. Are arranged towards The expression cassette is also recombined like a plasmid vector with at least one origin of replication. For convenience, it is common to have a origin of replication acting in E. coli.

In addition to the origin of replication, the recombinant gene used for transformation is composed of various gene expression cassettes such as a selectable gene, a marker gene, and a target gene. Select genes confer resistance to antibiotics, toxins, heavy metals, or such biocides, such as NPTII (neomycinphosphotransferase), HPT (hygromycin phosphotransferase), CAT (chloramphenicol acetyltransferase), nitrilease (nitrilase) And gentamicin resistance genes. At least one selection gene is present in one plasmid because different selection genes are used for individual hosts. That is, one selection gene is used for selection in prokaryotic cell hosts such as Escherichia coli, and the other selection gene is used for selection in eukaryotic cell hosts.

Examples of suitable marker genes that provide a visual phenotype through the production of new compounds in transgenic plants include GUS (β-glucuronidase), which produces indigo, CAT, which produces acetylchloramphenicol, and visible light. Luciferase which is used for opening, and green fluorscent protein (GFP) that emits green fluorescence are used.

Target gene cassettes are useful genetic recombinants capable of conferring a desired trait in a transformed plant. The purpose of molecular breeding by transformation is to transplant expression of this target gene. Thus the uniqueness of the transgenic plant can be determined by this target gene.

Recombination and cloning techniques of various genes and vectors are illustrated in detail in the literature. (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1987)

Various particle acceleration devices can be used in the practice of the present invention to effectively implant DNA-coated fine particles into plant cells. An example of this device is available commercially from Bio-Rad Laboratories, USA as a Bioistic particle accelerator (model Biolistic PDS-1000 / He). The fine particles may be metal, glass, silica, ice, polyethylene, polypropyline, polycarbonate, and carbon compounds (eg graphite, diamond) and the like, but metal particles are preferred at present. There are no limitations on the size and type of suitable metal particles, but the experiment includes tungsten, gold and iridium particles with a diameter of 0.5-3 μm. The microparticles coated with DNA are quickly driven by pressurized helium. As the microparticles penetrate the cell wall of the cells on the target plate, the DNA coated on the particles is implanted into the cells of the target tissue. At this time, the particle projection chamber is partially vacuumed before the projection is carried out in order to prevent excessive inferior of the propulsion force of the microprojectile due to the inflow of helium air. The chamber applies partial vacuum so that the target tissues do not separate during their particle projection.

Examples of target tissues include meristems of embryos, stems, apical and roots and induced meristems such as callus. The choice of specific tissue can vary depending on the clonal proliferation methods available and the regeneration capacity of the tissue.

Preferred clonal propagation methods for the practice of this invention are described below. This method consists of several steps; 1) incubating the callus in a specific medium for a sufficient time to obtain a callus suitable for transformation at the growth point of the garlic scales; 2) transfer to fresh growth medium at appropriate times before microparticle projection to stimulate vigorous development of meristems; 3) propagating the transformed cells after projecting the recombinant gene DNA; 4) treating the regeneration medium for a time sufficient to induce the formation of shoots by regeneration; 5) Regenerated shoots are cultured in nutrient medium until roots grow and grow into seedlings; 6) Purify the transformed garlic plants and transplant them into the soil to grow into complete plants.

Callus of garlic forms a viscous substance on the surface during cultivation and becomes an obstacle in the transformation process of transplanting foreign genes. Therefore, in order to minimize the formation of the viscous material to increase the transformation efficiency, the experiment was to adjust the composition of callus growth medium and subculture before the projection so that foreign genes can be effectively inserted into the genome in garlic callus. The reason why the target callus is transferred to a new medium before the microparticle projection as described above is that the larger number of cells in the tissue to be particled are present within a specific cycle of well-transformed cell division, resulting in an effect of increasing the frequency of transformation. do. In other words, the more cells in a given cycle in a tissue being targeted, the higher the probability of transformation. Therefore, grow them in a new callus growth medium for the time required to form surface meristems of the first and second layers of the target tissue, and then perform particle projection.

The foreign recombinant gene DNA coated on the fine particles delivered to the cell is inserted into the genome of the projected cell. After selection of the transformed cells under appropriate conditions, the plants are re-differentiated. When transforming plant tissues by particle projection to obtain transformed plants, it is important to avoid the development of chimera transformed plants containing both transformed and non-transformed cells. In other words, it is important to obtain non-chimeric transgenic plants by projecting gene DNA into undifferentiated meristem such as callus. Once the transformed shoots come out through the sure selection in the callus step, it is possible to mass-proliferate the shoots by polycythenic propagation method (Korean Patent No. 0134140). The efficiency of transformation is increased and a large number of stably transformed garlic plants can be obtained through a redifferentiation step.

The selection of good selection genes is an essential part of the transformation method. Important factors for the effective selection of transformed cells are the structure of the selection gene, the level of expression and the selection of the selection agent (Wilmink and Don, Plant Mol. Biol. Rep. 11; 165-185, 1993). Selection genes include genes encoding proteins that confer resistance to antibiotics, metabolites and herbicides, and may also confer resistance to the toxicity of amino acids or homologues. NPTII is most widely used as a selection gene for plant transformation. The sensitivity of a plant cell to a selection gene depends on the physiological state, the size of the plant and the tissue of origin and tissue culture. Therefore, the minimum level of selection agent that can completely inhibit the growth of untransformed cells is determined in consideration of the efficiency of transformant selection and regeneration. Subculture periods are determined to efficiently inhibit the growth of untransformed cells while minimizing the toxicity of dead cells to adjacent transgenic cells.

Marker genes are genes that encode enzymes that form easily labeled products in transformed tissues and plants. A useful marker gene is GUS, which can effectively observe both the transient expression of genes following particle projection and the stable expression of genes in various tissues of re-differentiated plants after selection.

The present invention will be described in more detail with reference to the following examples. However, the following examples are described to help the understanding of the present invention and are not intended to limit the scope of the present invention.

Example 1 Induction of Callus Suitable for Transformation

Early callus derived from the growth point of garlic scales is a slow growth rate in the medium, the metabolites of saccharides are secreted around the cells and a viscous membrane is formed, making it difficult to use as a tissue for transformation. Therefore, in the present invention, by adjusting the media composition and the passage culture time appeared to affect the callus proliferation and re-differentiation, the recombinant foreign gene by particle projection is effectively inserted into the genome of garlic callus and re-differentiated.

Danyang and Uiseong local varieties of garlic were collected and surface peels were removed and the surface of the scales was sterilized in 70% ethanol for 2 minutes and bleach (0.5% sodium hypochlorite solution; NaOCl) for 10 minutes. After washing three times with sterile water, the growth point (0.3 mm or less) existing on the root of the sample was extracted under a microscope and 0.1 ppm (2,4-D (2,4-dichlorophenoxy acetic acid), a plant growth regulator, Callus was induced by incubating for 3-4 months in MS solid medium containing mg / l) (Murashige and Skoog; Murashige and Skoog, Plant Physiol . 15; 473-497, 1962).

Among the various inorganic nutrients in the medium used for plant tissue culture, nitrogen is generally the most influential element in the growth and regeneration of cells. Therefore, this experiment focused on the content of nitrogen source in the composition of the medium. MS medium commonly used in plant tissue culture contains 1650 ppm of ammonium nitrate (NH ₄ NO ₃ ) and 1900 ppm of potassium nitrate (KNO ₃ ) as nitrogen sources. When the concentration of each of these nitrogen sources was changed while maintaining the other components and contents of the MS medium, callus proliferation to be used for transformation resulted in the best growth when the ammonium nitrate concentration was lowered to half the level (825 ppm) of MS medium. In the case of potassium nitrate, callus growth was best when raised to 2 times the level of MS medium (3800 ppm) (Table 1). On the other hand, as the callus grows faster, the secretion of viscous substances on the surface is significantly reduced.

Garlic callus culture medium composition for transformation Nitrogen source Ammonium Nitrate Potassium Nitrate Nitrogen source concentration (relative to MS) 1/4 1/2 One 2 4 1/4 1/2 One 2 4 Nitrogen Source Concentration (PPM) 412.5 825 1650 3300 6600 475 950 1900 3800 7600 Callus growth rate (%) 266 332 301 233 221 257 263 298 340 301

Therefore, the concentration of ammonium nitrate is halved, and potassium nitrate is repeated in subculture for 6 months to 1 year at twice the concentration of MS medium (modified MS medium) to multiply callus to give viscous, high transformation efficiency and high regeneration rate. Secured.

In order to confirm the performance of the callus thus obtained, three days after the particle projection was performed using the plasmid pCambia1201 (MRC, UK) recombined with the GUS gene, the results of assaying the expression of the GUS gene showed a remarkable difference as shown in FIG. 1. . The left side of the figure is a callus cultured for 4 months in MS medium and the right side is a callus cultured under conditions optimized by the present invention, the transient expression efficiency of the projected foreign gene GUS showed a difference of 50-100 times.

Example 2 Selection of Effective Screening Agent

In the step of selecting transformants, various antibiotics or herbicides were used as screening agents. Garlic has not been reported to be resistant to various screening agents. Therefore, the resistance of garlic callus using antibiotics kanamycin, hygromycin, and herbicide PPT (phosphinothricin), which are useful for the selection of transgenic plants such as tobacco and rice, were examined. All of the nontransgenic callus died when 50 ppm of hygromycin was included in the growth medium (left) and 25 ppm of hygromycin was included in the regeneration medium (right). In the case of kanamycin and PPT, no viable screening effects could be observed, including live callus at 500 and 50 ppm, respectively. For the purpose of screening transformants, kanamycin is generally used at concentrations of up to 500 ppm and PPT up to 50 ppm for plants other than garlic. Therefore, hygromycin was selected as a selection agent for garlic transformant selection, and the hygromycin resistance gene was recombined as a selection gene, coated on gold or tungsten particles, and particle projection was performed.

Example 3 Particle Projection Conditions of Particle Guns

The target callus, which had been stabilized for at least 6 months after induction at the growth point, was transferred to a new solid medium to induce vigorous cell division and particle projection for 7-15 days. This results in the effect of increasing the frequency of transformation by allowing a greater number of cells in the tissue to be injected into a specific cycle of well-transformed cell division. In other words, the more cells in a given cycle in a tissue, the more likely it is to be transformed. Therefore, it is necessary to grow particles in a new callus growth medium for the time required to form surface meristems of the first and second layers of the target tissue.

Recombinant plasmid DNA used for transformation is mixed with microparticles and precipitated by adding 250 mM calcium chloride and 100 mM spermidine according to the method of Klein et al . (Klein et al ., Nature 327; 70-73, 1987). Microparticles were used with gold particles of about 0.6 μm in diameter or tungsten particles of 0.7 μm and were purchased from Bio-Rad Laboratories, USA with a particle gun (Biolistic PDS-1000 / He). The actual particle projection conditions were carried out by adjusting the transformation efficiency to the maximum by repeated experiments. In one projection, about 750 μg of particles coated with 10 μg of plasmid DNA were ejected to a volume of 7 μl. As a result of transient GUS marker gene expression analysis after the projection, the vacuum inside the particle gun chamber was 25-30 inch Hg, the helium pressure was 1000-1500 psi, the particle movement distance was 3-5 inches, and the baffle was 63-212 μm. It was determined that the implementation was most effective. Table 2

Fine Particle Projection Conditions Vacuum degree inside chamber (inch Hg) 25-30 Helium pressure (psi) 1000-1500 Particle Travel Distance (inch) 3-5

Example 4 Recombinant Promoter Selection

Several gene expression control sites, or promoters, which regulate the effective expression of foreign genes transplanted into plants have been reported. In general, in the case of monocotyledonous plants, it is known that the promoter isolated from the monocotyledonous plant is effective and the efficiency increases several to tens of times when intron is added (McElory et al., Plant Cell, 2; 163-171). , 1990). In the case of garlic, such promoter activity has not been reported, and the expression of the marker gene was investigated by recombining the marker gene GUS with various promoters and then performing particle projection (FIG. 3). The promoter used was 35S (35S gene promoter of Cauliflower mosaic virus), 35S-INT to which intron of corn alcohol dehydrogenase gene ( Adh1 ) was added, promoter Adh of corn alcohol dehydrogenase gene including intron, rice Actin gene ( Act-1 ) promoter ACT isolated, and the corn ubiquitin gene ( ubi-1 ) promoter Ubi etc. are mentioned. As shown in FIG. 3, unlike the other monocotyledonous transgenic plants reported so far, garlic showed that the 35S promoter was about 8 times more effective than the promoter isolated from monocotyledonous plants such as rice, especially in the case of 35S without intron. It was about three times higher than 35S-INT with intron. Therefore, in subsequent experiments, the 35S promoter was used by recombination with the target gene.

Example 5 Recombinant Gene for Transformation

Recombinant gene for garlic transformation used in this example was used pC1301-ALSGUS and pCambia1201 (MRC, UK) and are shown in FIG. Each vector contains a common expression gene hpt (hygromycin phosphotransferase) and a marker gene GUS (β-glucuronidase) cassette that can be expressed in plant cells. . One of the plant gene expression cassettes, hpt confers resistance to the antibiotic hygromycin, which consists of a 35S promoter (P35S) and a 35S polyadenylation / terminator (T35S). The second expression cassette, GUS, encodes β-glucuronidase enzyme and its expression can be detected by histochemical analysis. 35S promoter (P35S) or ubiquitin promoter (PUbi) and nofarin synthase polyadenylation / It consists of a terminator (Tnos). The herbicide tolerance gene ALS cassette is GLEAN ^? A 35S promoter that encodes a mutant acetolactate synthase enzyme (Haughn et al., Mol. GenGenet. 211; 266-271, 1988) that is resistant to sulfonyluria herbicides such as (DuPont, USA) (P35S) and nofarin synthase polyadenylation / terminator.

[Example 6] Transformant screening after particle projection

Callus projecting microparticles for transformation was incubated in solid medium containing no hygromycin for 7-14 days and transferred to solid medium containing 50 ppm hygromycin at 6 week intervals, followed by incubation. Were screened for 4-6 months. The untransformed callus turned dark yellow and died, whereas the transformed cells continued to divide and grow into new translucent white callus.

Alternatively, a new solid medium containing 50 ppm hygromycin after one week in 50 ppm hygromycin medium is poured into the callus in a semi-solid state before hardening, and hardened to cover the callus surface for 3-4 months while the callus is indented. The method was more effective (FIG. 5). This method was able to selectively culture transformed cells 5 times more efficiently in a short time than when selected on solid medium surfaces.

Transformant selection efficiency according to selection culture method. Culture method Projection Callus Number Hygromycin-resistant callus number (%) Number of rediform transformants (%) Surface culture 1000 40 (4) 8 (0.8) Depression culture 900 4 (0.4) 4 (0.4)

Example 7 Transformant Regeneration

Transformed callus selected with hygromycin was re-differentiated in a modified MS medium (redifferentiation medium) containing 2.5 ppm benzylaminopurine. That is, after incubation for two months in a regeneration medium without addition of hygromycin, the cells were transferred to a regeneration medium containing 20 ppm of hygromycin and cultured. At this time, when the shoots were formed, the callus was divided into 2-5 shoots and transferred to a new medium, followed by incubation for 4 months. The shoots of 2 cm or more were cultured by removing the plant growth regulator and transferring to modified MS medium with the sugar concentration increased to 40 g / L. As shown in Figure 6 it was confirmed that the regeneration of vigorous shoots in the medium with the selection agent.

Example 8 Transformant Analysis by GUS Staining

In order to confirm the transformation, the screened and redifferentiated sample was stained with 2.0 mM X-Glu (5-bromo-4-chloro-3-indoyl-β-D-glucuronic acid) solution for 24-48 hours, followed by blue GUS. Activity was analyzed (FIG. 7). GUS-specific dark blue staining reactions throughout the plant showed that the transformants were systemic and not chimeric.

Example 9 Transformant Analysis by PCR Amplification

Further, genomic DNA was isolated from the sample that was screened and re-differentiated, and PCR amplification and Southern blot were used to determine whether the recombinant gene was transplanted. PCR amplification using HPT and ALS-specific primers in the transformants, and Southern blots of HPT and ALS-specific probes showed no signal in wild, but HPT in plants transplanted with pCambia1201. In the plant transplanted with the pC1301-ALSGUS gene, it was confirmed that both the HPT and ALS genes were transplanted as expected (FIG. 8).

Example 10 Transformant Analysis by Northern Blot

In addition, it was confirmed that the foreign genes introduced through the Northern blot analysis after the separation of the total RNA from the selected samples were stably expressed in the transformant. Northern blots of HPT and ALS-specific probes showed no signal in the wild, but HPT and ALS genes in plants transplanted with pCambia1201 and HPT and ALS genes in plants transplanted with pC1301-ALSGUS. All were stably expressed (Fig. 9).

Example 11 Transformant Mass Proliferation and Soil Transplantation

The shoots identified as transformants through the transformant analysis process were grown by inducing polycytheria according to the seed garlic growth method (Korean Patent No. 0134140). After 4-8 weeks storage at 4 ℃ transferred to a modified MS medium (sphere formation medium) adjusted to a sugar concentration of 90 g / L and incubated for 2-4 months to form a globule. Since the globules germinated in the soil to obtain a transformed garlic plants (Fig. 10).

Example 12 Herbicide Resistance Assay

To test herbicide resistance of garlic transformed with herbicide-resistant genes, the herbicide GLEAN ^? It was incubated for 2 months in a medium containing 4 ppm of this. In the case of the non-transformed garlic used as a control, it did not survive, but in the case of transformants, it was observed to grow well at 4 ppm concentration (FIG. 11).

The effect of this invention is to provide an effective method for transforming garlic plants by transplanting recombinant genes by microparticle projection. More specifically, it provides conditions for culturing and screening meristems before or after particle projection to transform the resulting callus, and then obtaining conditions for transforming garlic plants through an effective re-differentiation and propagation process. It ultimately provides a methodology with broad applicability of genetic engineering techniques to transplant and express recombinant genes in garlic.

Claims (12)

In the genetically engineered method of producing a transformed garlic plant, In order to produce a callus suitable for transformation, subcultured in a specific medium for at least 6 months to produce target cells with high efficiency of transformation and regeneration. Physically accelerating the microparticles to the target cell callus, the microparticles deliver recombinant genes containing at least the foreign target gene of interest and a selection gene that can be used for selection, As an effective condition for physically transplanting a foreign recombinant gene into a cell, a specific screening substance, hygromycin, is selected to effectively select and proliferate a callus expressing a foreign gene after projection such as the type and size of metal particles, injection pressure, and distance from a sample. Use, The solid medium containing the selection agent is added in a semi-solid state, and the callus is cultured in a recessed state, Induces transformed shoots in selected callus, Thereafter, the resulting shoots are regenerated with garlic transformed by the polycythenic growth method. The method of producing a transformed garlic plant ( Aliumium sativum L.), comprising the steps of: The method of claim 1, wherein the garlic cells belong to the genus Lilium genus (transgenic garlic) manufacturing method (transgenic garlic) characterized in that. The method of claim 1, wherein the callus tissue comprises a meristem, transformed garlic plant production method characterized in that the tissue capable of induction into the meristem and re-differentiation as a plant or a seedling. The method of claim 1, wherein the primary callus comprises a meristem derived from the growth point of the garlic scales, the callus is a tissue capable of re-differentiation into plants, the transformation is characterized in that the induction into proliferative callus and tissue capable of plant re-differentiation Method for preparing garlic plants. The method of claim 1, wherein the concentration of ammonium nitrate in the medium composition to obtain a high conversion efficiency cleavable callus is half of the MS medium and potassium nitrate twice the MS medium production of the transformed garlic plants Way. According to claim 1, wherein the external gene construct is a selectable marker gene, including the GUS gene, its expression can be searched in callus and re-differentiated shoots, characterized in that it is possible to confirm the characteristics of the transformants survived in the selection Method for producing a conversion garlic plant. The method of claim 1, wherein the selection method includes a hygromycin resistance gene as a selection gene. The method of claim 1, wherein the microparticles comprise metal particles having a diameter of about 0.5 to 3 μm. The method of claim 1, wherein a plurality of the microparticles are provided, and the microparticles having the external gene construct are projected onto the plant target tissue. According to claim 1, In order to effectively select and multiply the callus expressing the foreign genes after the projection in a solid medium, after the addition of a solid medium containing a selection material in a semi-solid state, the callus is cultured in a recessed state transformants Method for producing a transformed garlic plant, characterized in that the selection. The method of claim 1, further comprising the process of the generation of roots from the transformed plant tissue. Transgenic garlic prepared according to the genetic recombination method of claim 1.