KR100271136B1 - Manufacturing method of transgenic orchids - Google Patents

Abstract

PURPOSE: Provided is a genetically manipulated orchid, specifically the Cymbidium sp. by the particle-mediated transformation technology. In details, provided is a method of stimulating generation of the meristematic tissue continuously before or after the DNA coated particle projection to transform the protocorm-like body(PLB). The PLB has morphogenesis and differentiation. The method offers a broadly applicable methodology of genetic technique to improve the orchids. CONSTITUTION: The transformation method comprises the steps of: (i) culturing the first PLB in a liquid medium to induce the second PLB; (ii) placing PLB containing the first and second PLB on the surface of target to project microparticles; (iii) projecting microparticles with adding physical acceleration, wherein the microparticles transmit copies of foreign gene structures including one of interested foreign gene and selective marker gene; (iv) inducing projected PLB to make the proliferating PLB which is capable of bringing out shoots, wherein PLB is cultured in a medium containing a selection substance of the selective gene which provides resistance to select transformed PLB in which the interested gene can be expressed; and (v) obtaining clone transformed orchid from the above shoots.

Description

Transformation method of orchids

The present invention relates to genetic engineering techniques of general plants, and more particularly, to a method of transforming orchids using a particle-mediated transformation technique by microparticle projection.

Orchids usually grow all over the world and are the largest family of flowering plants in the genus 800 and over 25,000. Disease and stress resistance, early flowering, induction of various engineered flower colors and morphological changes by genetic engineering of orchids are of commercial importance. The improvement of orchids by the conventional plant breeding method is not effective for practical use due to the long germination-to-flowering period (years), slow seed ripening (months), and difficulty in analyzing seedlings. Therefore, improvement of orchids using genetic engineering techniques is of interest.

Genetic engineering is a way of inserting new genes into selected individuals to get the desired traits. Unlike mating, new genes are added while maintaining the genotype of the good variety. In order to successfully carry out the genetic engineering approach, it is a prerequisite to have an effective gene transfer system into a given species. There are two methods for plant transformation (gene transfer): transfer of DNA to cells using biological vectors, mainly through the soil plant pathogen Agrobacterium, and DNA into plant cells. Direct delivery is mainly through microparticle projection.

One of the most commonly used transformation techniques today is Agrobacterium, a soil plant pathogen, and these bacteria can use their Ti (tumor-inducing) or Ri (root-inducing) plasmids. DNA fragments have the ability to transfer into infected plant cells. However, the successful application of this technique depends on the host range of Agrobacterium, and many economically useful crops have not yet been transformed by this method. In an effort to develop a method for transplanting genes regardless of plant species or genotype, several techniques for delivering DNA directly to plant cells have been studied. These include electroporation and microinjection. ), DNA inhalation using PEG- or liposome-mediated, silicon carbide whiskers and microparticle projection. Microparticulate technology, also known as bioolistics or microprojectiles, is the coating of DNA on small carrier particles and the physical projection of these particles into plant cells. This method has several advantages. First, the removal of the cell wall is not necessary to introduce DNA. Second, DNA can be introduced into differentiated cytoplasm, such as meristem and protoplasts. Third, no special biological carrier manipulation is required. Finally, it does not rely on binding or mutual recognition of host cells and biological vectors to transfer DNA to cell membranes.

Currently, the propagation and breeding of orchids depends on the culture of protoplasts derived from apical meristem of seeds and shoots. The storage organ protocorm is formed from an embryo and has an apical meristem and leaf primodium. Protocorm-like bodies (PLBs), also known as somatic protoplasts, are induced by in vitro culture of apical or axillary bud meristems, functionally and structurally. Similar to the prokaryote of an embryo. Such meristems are suitable for particle projection and for the recovery of transgenic plants. Some herbaceous cells can be transformed in a manner similar to Agrobacterium transformation (Klein et al., Proc. Natl. Acad. Sci. USA, 88, pages 8502-8505, 1988). It has been proven in plants. Soybean germ cells were transformed by this particle projection-mediated transformation technique (McCabe et al., Bio / Technology, 6, pages 923-926, 1988), which is described in US Pat. No. 5,015,580. The technique is described. U.S. Patent No. 5,681,730 also describes a transformation technique of coniferous (gymnosperm) using microparticle projection. The advantage of this approach is that the shoots can be generated directly from the primary and secondary meristems without difficulty of redifferentiation, which reduces phytohormone processing and minimizes somaclonal variation.

The use of microparticulate transformation for the transformation of orchids is very limited, and only two studies have reported on the transformation of Dendrobium orchids and their commercial value. For high symbidium species, there are no reports of gene transfer by this method. Kuehnle and Sugii (Plant Cell Reports 11: 484-488, 1992) have obtained transformed dendrovium. However, it did not show the expression of screenable marker genes such as GUS and did not confirm whether the transformed plants were chimeric or not. Chia et al. (Plant Journal 6: 441-446, 1994) have transformed dendrobium orchids using particle projection techniques. Instead of antibiotic selection, they developed another selection that uses the expression of the genes (luciferase) that make up the fireflies fluorescent. The system uses a photon-counting video camera-photomultiplier system and a high magnification analysis microscope to select and isolate luminescent cell populations. The entire selection and separation is repeated until the pure transformed cell line is isolated. Plants are then regenerated in isolated cell lines. This method requires expensive equipment, and because of the time-consuming microscopic selection of transformed cells and the characteristic slow developmental growth of orchids, this technique is not practical for transformation for orchid improvement.

Orchids actually have some characteristics that are different from other plants in their transformation. First, orchid cells have a low growth rate. Second, orchid cells do not respond well to various manipulations in tissue culture. Third, plant regeneration from isolated differentiated cells does not occur in orchids. Fourth, orchid cells are not easy to select using antibiotics such as kanamycin. Orchids are commonly known to be highly resistant to aminoglycosides such as kanamycin, and kanamycin of 500 mg / L or more is usually required for selection of transformed cells (Chia et al., Plant Journal 6: 441). -446, 1994). Fifth, orchid cells contain high amounts of phenolic acid, and their oxides are poisonous to cells. Finally, because of the multicellular structure of the meristem (protoplasts) used to transform orchids, the resulting transgenic plants can sometimes become chimeras with transgenic and non-transforming parts. One method of obtaining homogeneously transformed individuals from granulatively applied meristems is to self-pollinate the treated generation, produce the next generation (F1) and the next generation (F2) and modify the properties added from them. There is a way to select individuals that have them. The other is to insert DNA into the meristem in the early stages of differentiation, and then stimulate continued meristem development during the antibiotic selection. The application of the above methods to transformation of orchids is limited primarily by long generation alternations and low growth in tissue culture. Therefore, no protocol is useful for the practical use of genetic engineering techniques in orchids, especially in Symbidium.

Accordingly, it is an object of the present invention to provide an effective method for transforming orchids, in particular Symbidium genus, by microparticle projection.

More specifically, the present invention provides a method for stimulating the generation of continuous meristems during before or after particle projection for transforming the generated protoplasts, followed by morphogenesis and differentiated protoplasts. It provides a method for transforming a glomerulus and a methodology that has broad applicability in the genetic engineering of orchids that can accelerate orchid improvement.

FIG. 1 shows the effect of three different culture techniques on protoplast proliferation before particle impact.

2 is a schematic of the T-DNA region of the gene carrier pKH200. The GUS-INT gene and NPTII gene-related sites are located between the Matrix Attachment Regions DNA.

3 is a chart showing the effect of two culture methods on protoplast proliferation after microparticle projection.

4 is a diagram showing long-term stable expression of the marker gene (GUS) in three transformed orchid lines.

Figure 5 shows a PCR (Polymerase chain reaction) amplified fragment of the appropriate marker gene (NPTII) introduced from two transformed orchid lines.

The present invention is to produce a genetically engineered orchid using a gene carrier that can be expressed in plants,

1) inducing secondary protoplasts by culturing the primary protoplasts in a liquid medium;

2) placing on the target surface for microparticle projection a protoplast comprising both primary and secondary protoplasts;

3) physically accelerate and project microparticles that are much smaller than the cells of the protoplasts, which deliver at least foreign gene vectors containing the desired external gene and a selectable marker gene;

4) Induce the proliferation of the projected protoplasts to form protoplasts capable of forming shoots in a liquid medium, and transformed from these proliferated protoplasts to express the desired gene. Culturing prokaryotic spheres in a medium containing a selection material to select them; And

5) to provide a method for producing orchids produced by genetic recombination, characterized in that it comprises the step of regenerating the resulting shoots into transformed orchids of the clone.

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

The method of the present invention can be used to produce transformed orchid plants comprising transformed orchid cells, wherein the transformed orchid cells are heterologous DNA carriers that make up an expression cassette. (Or a vector), the structure comprises a transcriptional initiation region in the 5 'to 3' direction and a peptide coding region located below and below its transcriptional control region from the aforementioned transcriptional initiation region. Genetic engineering techniques of orchids can be achieved through the use of particle projection-mediated plant transformation techniques that transform protoplasts with DNA delivered as microparticles and then select transformants. The culture technique described herein utilizes the regeneration of a relatively large number of meristem cells subjected to particle projection and stimulates the continuous development of projected meristems, resulting in increased stable transformation frequency. Proliferate the projected meristem and select prokaryotic globules in which the introduced genes are expressed. Shoot regeneration is then induced from meristems and produced transformed seedlings. Preferred plants for carrying out the invention are the genus Symbidium.

The objects, advantages and characteristics of the present invention will become more apparent from the following specification and drawings.

It has been found that the general approach of particle projection-mediated transformation of plant tissues in accordance with the present invention can be successfully applied to orchids in tissue culture. Using this method, transformed symbidium orchids could be developed. Since the technique used in the present invention can be applied equally to other orchids, it is now genetically engineered to be resistant to disease of orchids, manipulation of flowering period, manipulation of flower color and form and other horticultural value. Development of transformed orchid plants is possible. This method is applicable to all orchids and in particular to symbiotic orchids.

The term orchid used in the present invention relates to Orchididales in the Spermatophyta. Examples of orchids used in the practice of the present invention include, for example, Asianthus, Airides, Anoectochilus, Ansellia, Aplectrum, and Letterer. Arethusa, Calante, Cattlea, Coelogyne, Corallorhiza, Cymbidium, Cypripedium, Crytopodium, Dendrobium, Epidendrum, Goodyera, Listera, Maccodes, Oncidium, Phalaenopsis, Pholidota ), The Orchidaceae including the genus Rhynchostylis and Vanda. Symbidium is preferred for the practice of the present invention.

The method described herein relates to the introduction of foreign genes into the chromosome of an orchid plant. Such foreign genes are preferably from other organic tissues, ie, DNAs derived from the same or different species, which are introduced into orchid cells through artificial manipulation. Foreign genes usually contain sequences that allow the product to synthesize a transcriptional product of an orchid cell or a protein of interest.

DNA constructs typically contain flanking regulatory sequences that are effective at causing expression of the protein. Examples of flanking regulatory sequences include promoters sufficient to initiate transcription in plant cells, and terminator and / or polyadenylation sequences sufficient to terminate gene products by termination of transcription or translation. There is this. It is also possible to include translational enhancers in transcription around the promoter to aid in the efficiency of gene expression, particularly the expression of protein products. All of these regulatory regions can be engineered to express genes consistently in cells transformed, or to express tissue-specific or inductively by specific elements.

The DNA construct may optionally include flanking DNA sequences contained in chromatin tissue. Examples of such DNA sequences include matrix attachment regions (Spiker and Thompson, Plant Physiol. 110: 15-21, 1996), and transformation booster sequences (Meyer et al., Proc. Natl. Acad. Sci. USA 85: 8568-8572, 1988). The use of the transformation booster sequence resulted in a several-fold increase in the frequency of transformation using bioolistics (Buising and Benbow, Mol. Gen. Genet. 243: 71-81, 1994). The use of lateral substrate attachment regions promoted the expression of genes introduced into poplar (Han et al., Transgenic Research 6, 415-420, 1997).

Various genes can be used for orchids. Phenotypic features include enzymes that provide resistance to disease, manipulation of flowering periods, control of plant color and form, control of plant aromas, plant growth and morphology, and the like. Genes can be obtained from prokaryotes or eukaryotes, bacteria, fungi, yeasts, viruses, plants and mammals or those synthesized in whole or in part thereof.

Expression plasmids, which are usually used in plants, contain one or more cassettes, the products of which can act independently or in combination. When various cassettes are used, they can be in the same or different plasmids. When cassettes are present in other plasmids, these plasmids may be performed by the same microprojectile or by different microparticles, and these microparticles may mix with each other and be projected onto the target tissue.

The expression cassette can be engineered to include a translocation stop codon followed by a transcription initiation region, initiation codon, coding sequence of the gene, and transcriptional termination region. The direction is 5'-3 'which is the transfer direction. Cassettes are usually about 10 kilobases (kb) or less in length, often about 6 kb or less, with a minimum length of usually about 1 kb. Expression cassettes are also provided in a DNA carrier having at least one replication system. For convenience, it is common to have a functional replication system in Escherichia coli.

In this method, after each manipulation step, the resulting carrier is cloned to confirm correctness of the manipulation through sequencing. In addition, a wide range of host range replication systems can be used in E. coli replication systems. In addition, in replication systems, at least one marker gene is often present and useful for one or more hosts, or different marker genes are used for individual hosts. That is, one marker gene is used for selection in prokaryotic hosts, while the other marker gene is used for selection in eukaryotic hosts, especially orchid hosts. Label genes confer resistance to antibiotics, toxins, heavy metals or biocide such, and provide a visible phenotype through the production of new compounds in transgenic plants. Exemplary genes used were neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrile and nitrile. Gentamicin resistance genes.

Examples of marker genes suitable for plant selection include beta-glucuronidase to provide indigo production, luciferase to produce visible light, kanamycin resistance or G418 There are NPTII, which provides resistance, HPT, which provides hygromycin resistance, and a mutated aroA gene, which provides glyphosate resistance. Various fragments, including various DNA constructs, expression cassettes, label genes and the like, are introduced continuously by cleavage by appropriate restriction enzymes and insertion of specific carriers or fragments into available sites. After ligation and cloning, the DNA carrier is separated for later manipulation. All these techniques are exemplified in detail in the literature and are specifically exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1987.

Several instruments or devices for introducing carrier particles into plant cells are known to those of ordinary skill in the art. Biolistic cell transformation apparatus can be used in the practice of the present invention. An example of the device is a device known as the Biolithic Particle Acceleration Device (Model Biolistic PDS-1000 / He) and is currently available from Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 (tel. 510-741-1000). . The device has a particle projection chamber that is divided into two separate compartments by a height adjustable stop plate. The plate on which the top plate is installed and the fine particles are coated is quickly pushed to the stationary plate by pressurized helium. When the fine particles are delivered to the stop plate, the plate coated with the fine particles is stopped and only the fine particles are fired onto the target plate. The microparticles penetrate the cell walls of the cells on the target plate and transfer the DNA carriers into the cells of the target tissue. Microparticles include metals, glass, silica, ice, polyethylene, polypropylene, polycarbonates and carbon compounds (eg graphite, diamond). At present, metal particles are preferred. Examples of suitable metals include, but are not limited to, tungsten, gold and iridium. The particle projection chamber is partially evacuated before being used to prevent excessively reducing the propulsion force of the microprojectile due to the inflow of air. The chamber applies partial vacuum so that the target tissues do not separate during their projection. A vacuum between 400 and 800 mm Hg is suitable.

Target tissues capable of the next regeneration of shoots are used to practice the present invention. The choice of specific tissue will vary depending upon the clonal propagation system available and the specific species that is most suitable will be transformed. Examples of target tissues include embryos, callus tissues, stems, existing meristems (eg apical and root meristems) and induced meristems (eg prokaryotes, prokaryotic glomeruli). . Preferably it is a tissue selected from the kind consisting of meristems (both present and induced) and tissues capable of induction into meristems. More preferably, they are prokaryotic spheres. The projections are directed to the target tissue so that the microparticles are introduced into one or more cells and transformed.

Preferred clonal propagation methods for the practice of the present invention are those described below. This method consists of several steps; 1) treatment with solid protoplast derived media for a time sufficient to induce the formation of protoplasts in the desired apical meristem; 2) transfer the treated tissue to the liquid protoplasty growth medium to stimulate the continuous development of meristem; 3) the protoplasts are treated in shoot regeneration medium for a time sufficient to induce the formation of shoots; 4) The treated shoots are transferred to the nutrient medium until the roots grow. In this way it causes the production of transformed plants and allows them to grow to complete plants in the soil.

The tissue culture system described above provides a method of increasing the frequency of transformation by allowing a greater number of cells in the tissue to be injected to be present at a particular phase of a well-transformed cell division cycle. Therefore, the more shock is applied to more parts of the cell at this phase in the tissue, the higher the probability of obtaining stable expression in the affected tissue. The first and second sub-epidermal cells of the target tissue are treated in a liquid protoplasty growth medium for a minimum time sufficient to induce the formation of surface meristem, followed by the use of a DNA construct. do. In addition, the tissue is projected onto a DNA carrier prior to transfer to shoot regeneration medium to produce shoots.

DNA coated with small carrier particles delivered to the cell enters the genomic DNA of any part of the projected cell. After proper selection, the entire tissue or organism is generally regenerated from the transformed cells. Applying the general process of particle projection-mediated transformation of plant tissue to obtain transformed plants involves the regeneration of chimeric transgenic plants containing both transformed and non-transformed cells. It is important to avoid. It is generally important to obtain non-chimeric transgenic plants by projecting genes into differentiated tissues, such as meristems, which are then described in US Pat. No. 5,015,580 that can be directly regenerated into seedlings. The present invention stimulates the continuous generation of meristem by culturing protoplasts in a liquid medium before particle projection, and allows the meristem to proliferate even after particle projection. In another aspect, the present invention is to obtain an orchid plant stably transformed by culturing the meristem projected by a combination of liquid and solid medium.

The use of good starting genes is an essential part of the transformation method. Important factors for the effective selection of transformed cells are the type of selectable marker gene, expression level, and the time and effect of selection after transformation (Angenon et al., Review, Angenon et al., In Plant Molecular Biology Manual C1). : 1-13, Kluwer Academic Publishers, Boston, Gelvin and Schilperoort, Eds, 1994). Marker genes usefully include genes encoding antibiotics, antagonists and pesticide resistance and confer resistance to the toxic levels of amino acids or analogs. Neomycin phosphotransferase II (NPTII) is the most widely used selectable marker gene for plant transformation. The sensitivity of plant cells to selectable marker genes depends on the phenotype and tissue culture status, such as physiological status, plant size and type. Therefore, the minimum level of selection agent that can completely inhibit the growth of untransformed cells is determined for each transformation and regeneration system. Kanamycin is generally used at a concentration of 50-200 mg / L. As a selection agent that effectively inhibits the growth of non-transformed cells while minimizing the toxicity of dead cells to adjacent transformed cells, there is a period sufficient to ensure the growth of projected cells.

In contrast, screenable marker genes are genes that encode products that are easy to screen for searched and transformed tissue. Useful screening genes are beta-glucuronidase genes for use in GUS or conventional colorometric assay systems. In general, the use of introns containing the GUS gene is preferred because the gene with the intron is expressed only in eukaryotic (plant-like) cells and not other contaminated microorganisms. GUS expression can observe both transient gene expression following particle-mediated transformation and stable gene expression from virtually all plant tissues after reselection. Once the plant or seedling appears to have been transformed, the plant is grown. Such transformed plants can be regenerated repeatedly by the use of cell and tissue cultures.

The following examples are described for a better understanding of the invention and are not intended to limit the scope of the invention. The description of the key terms used in the following specific examples are as follows: meristem—plant tissue in which new cells have been developed and have not been differentiated from them; Protocorm-a small reservoir derived from an embryo containing apical meristem and leaf primordium; Protocorm like body (PLB)-a tissue structure derived from asexual tissue, resembling a prokaryotic body; Primary protoplasts-protoplasts induced by aseptic culture of apical meristem; Secondary protoplasts-protoplasts formed on the surface of the primary protoplasts in culture; Proliferative Prokaryotic Spheres-Prokaryotic globules that proliferate on the surface of protoplasty graft, including primary and secondary prototypical glomeruli.

Example 1 Preparation of Symbiotic Protoplasts for Particle Impact

The apical part of the symbiotic orchid is isolated under sterile conditions, and Paek et al., Proc. Induced to form protoplasts as described in NIOC, Nagoya, Japan (1997). In primary protoplasts less than 12 months old, 5% sucrose, 2 mg / L alpha-naphthalene acetic acid, 0.1% charcoal and 0.7% agar (pH 5.4) were added to the induced protoplasts. Induced and maintained on Knudson Orchid medium (Duchefa Biochmie BV, Izaak Enchedeweg 40, 2031 CS Haarlem, The Netherlands). Before applying the particle impact, the protoplasts were incubated in a liquid medium using a low speed stirrer (50-100 rpm) for 7-10 days and then 5% sucrose and 0.7% in a 60 mm Petri dish to be served as a target surface. Place in a circle of 1 to 2 cm on Nudeson orchid medium with agar. Control groups (tissues without impact) are similarly plated. Incubation of all plant material is carried out at 25 ± 2 ° C with incubation time of 16 hours under 25 to 45 uE / m sup 2 seconds using incandescent lamps.

Example 2 Measurement of the Effect of Liquid Culture on Proliferation of Protoplasts Before Particle Impact

Three different cultures with Murashige and Skoog (MS) and 15 Physiologia Plantarum 31 (1962) in medium with 3% sucrose without phytohormone; Liquid culture, fixed liquid culture and solid culture through agitation are used to induce primary protoplasts to form secondary protoplasts before particle impact. In solid culture, 40 protoplasts are cultured in 90 mm Petri dishes containing 25 ml MS medium with 0.7% agar added. In liquid culture, 40 protoplasts are incubated at 50-100 rpm with or without stirring in a 250 ml Earlymeyer-flask containing 50 ml MS liquid medium. Control groups (tissues without impact) are cultured similarly. Cultured protoplasts are weighed for production of secondary protoplasts.

Figure 1 shows the data obtained from this procedure. The y-axis represents the ratio of proliferation (number of proliferative glomeruli proliferated to protoplasts), and the x-axis represents the days cultured before particle impact. Slowly stirred liquid culture resulted in the proliferation of the highest prokaryotic glomeruli.

Example 3 Gene Expression Vector

The plant gene expression vector used in this example is pKH200, and is disclosed in Fig. 2 and Han et al., Transgenic Research, 1997. The vector contains two different plant expression cassettes for two different genes that can be expressed in plant cells, and the antibiotic resistance marker gene (Kanamycin resistance) has been added for use in bacterial hosts. One of the plant gene expression cassettes contains the gene NPT-II, which shows resistance to the antibiotic kanamycin. Kanamycin offers utility as selectable markers in several plant species. The NPT-II expression cassette comprises a nopaline synthase promoter and an octopine synthase polyadenylation sequence flanking the coding sequence. The second expression cassette encodes a beta-glucuronidase enzyme or GUS and is used as a screenable marker that expression can be retrieved by conventional histochemical analysis. The GUS gene has a cauliflower mosaic virus 35s promoter (CaMV35s) at 3 'and a nofarin synthase polyadenylation region at 5'. Two expression cassettes flank the matrix attachment region (MAR) fragments obtained from the genome clones of tobacco (Hall et al., Proc. Natl. Acad. Sci. USA, 1991).

Example 4 Conditions of Microprojectile Particle Projection

The values in which the actual particle acceleration method is used are calculated as described in the manual provided by the operator based on the use of the model Biostick PDS-1000 / He (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547). Stop plates, helium accelerator plates, and microparticles are supplied by Bio-Rad Laboratories. Plasmid DNA is precipitated in the presence of microparticles, as described in Klein et al., 327 Nature 70 (1987). Microparticles are made from gold particles about 0.6 μm in diameter. Each impact ejects about 500 μg of particles associated with 0.42 μg of plasmid DNA in a 6 μl volume. The particle projection chamber creates a vacuum at 28 inches of mercury pressure. All particle projection is performed by pressurizing with helium within the range of 1,100 to 1,550 psi, approximately 60 mm from the stop plate to the surface of the target tissue.

(Example 5) tissue culture after particle projection

After particle administration, tissue culture is divided into two phases, proliferative protoplasty induction and selective culture; 1) Induction of proliferative protoplasts-Murashige and Skoog, Physiologia Plantarum, 1962, ½ times intensity, free of phytohormones and 3% sucrose to promote proliferative protoplasts Two (liquid versus solid) culture techniques are used in one medium. In solid culture, 40 impacted protoplasts are plated in 90 mm Petri dishes containing 25 ml MS medium with 0.7% agar. In solid culture, 40 projected protoplasts are incubated in a 250 ml early flask containing 50 ml MS liquid medium with or without stirring (50-100 rpm). Cultured protoplasts are weighed for production of proliferative protoplasts. Table 1 shows the results of this experiment. Slowly stirred liquid cultures stimulate the development of more prototypical protoplasts than solid cultures. Do not use kanamycin selection during liquid culture. Culture techniques will measure the production of proliferative protoplasts. 2) Selective Culture-90 mm Petri dishes containing 40 ml of secondary protoplasts with multiple proliferative protoplasts on the surface with 25 ml MS medium supplemented with 0.7% agar and 100-200 mg / L kanamycin. Move to. The culture is maintained on a weekly basis with fresh medium. The culture room state is the same as used in Example 1.

Effect of Two Different Cultures (Solid vs. Liquid) on Proliferation of Protoplasts After Particle Impact Culture method ¹ Protoplast growth rate ² Liquid 7.72 + 0.44 solid 4.20 + 0.14

¹ Liquid culture: 40 projected protoplasts were prepared using Murashige and Skoog (MS) medium at 25 ml 1.5-fold intensity, Physiologia Plantarum 31 (1962), 0.7% agar, medium added with 3% sucrose and proliferative source. Plate in 90 mm Petri dishes containing phytohormone-free medium to promote glioblastoma development. For liquid cultures, the 40 impacted protoplasts are incubated in a 250 ml early flask containing 50 ml MS liquid medium with or without agitation (50-100 rpm).

² Cultured protoplasts are weighed for production of proliferative protoplasts. Proliferation is calculated by dividing the total number of generated spheroids by the initial number of spontaneous globules. Data was collected from three independent experiments.

Example 6 Essay of Particle Impacted Tissue for GUS Activity

A collection of protoplasts is sampled over time to determine beta-glucuronides (GUS) activity. Protoplasts are cultured in substrate solution, 2.0 mM 5-bromo-4-chloro-3-indoyl-beta-D-glucuronic acid (5-bromo-4-chloro-3-indoyl-beta- D-glucuronic acid; X-gluc), 0.1 M NaPO ₄ sub 4 buffer, pH 7.0, 0.01 M Na sub 2 EDTA, pH 7.0, 0.5 mM K sub 4 Fe (CN) sub 6 3H sub 2 O, pH 7.0 and Substrate solution containing 0.5 mM K sub 3 Fe (CN) sub 6 and 0.1% Triton X-100 was incubated at 37 ° C. for 18 hours. The number of GUS foci with prokaryotic spheres is checked under analytical stereomicroscope.

Figure 3 is a graphical representation of the results of repeatedly performing the analysis of protoplasts spheroidized in the analysis for transient GUS expression.

Example 7 Selection and Analysis of Transformed Protoplasts

To screen the results of fusion that is relatively less stable from transient activity, kanamycin selection is used. As described in Example 5, only the protoplasts cultured in the liquid medium for 10 to 45 days were added with 0.7% agar and 100-200 mg / L kanamycin for selecting the transformed protoplasts. Transfer to the same medium except. Cultures in kanamycin are passaged in fresh medium every week to minimize the number of deviations from mitigating kanamycin toxicity. Transformed protoplasts are initially identified because of their ability to grow in kanamycin media. Stable fusion and expression are then confirmed by PCR analysis and GUS expression analysis, respectively. Indeed, hundreds of protoplasts are produced through continuous proliferative proliferation. More than 160 such prokaryotic glomeruli survived the kanamycin selection. More than 92 kanamycin resistant plants were obtained from protoplasts. Of these, 52 were selected and subjected to GUS analysis. As a result, 36 expressed the GUS gene. 3 shows GUS expression from projected protoplasts. In essence, it was confirmed that long-term stable expression of the introduced foreign gene was achieved. Based on past experience in other plants, the inserted genes are normally isolated normally in the next generation and inherited through the Mendelian law. In general, the process can regenerate transformed protoplasts that can occur in whole orchid plants.

The effect of the present invention is to provide an effective method for transforming orchids, in particular Symbidium, by microparticle projection. More specifically, the present invention provides a method for stimulating the generation of continuous meristem before or after particle projection for transforming the generated protoplasts, and then having a protoplast having a tissue that performs morphogenesis. It provides a method for transforming spheres and a methodology with broad applicability of genetic engineering techniques of orchids that can accelerate orchid improvement.

Claims (12)

In a method for producing genetically engineered orchids, the transformed orchid cells contain a DNA construct consisting of an expression cassette, the structure of which is in the 5 'to 3' direction, the transcription initiation region and the transcription. Comprising a peptide coding region downstream from the initiation region and below its transcriptional control region, 1) incubating the primary protoplasts of orchids in a liquid medium to induce secondary protoplasts; 2) having a protoplast containing both primary and secondary protoplasts on the target surface; 3) physically accelerating the microparticles to the protoplasts, the microparticles delivering copies of the foreign gene construct comprising at least one of the foreign gene of interest and a selectable marker gene; 4) Induces the projected protoplasts to form proliferative protoplasts capable of forming shoots in the liquid medium, which are transformed into prototypic glomeruli which are transformed throughout the induction process and express the gene of interest. Culturing prokaryotic globules in a medium containing a selection material of a selection gene that provides resistance to select; And 5) a method for producing transgenic orchids, characterized in that it comprises the step of regenerating the resulting shoots into transformed orchids of the clone According to claim 1, wherein the orchid cells are genus Acianthus, Aerides genus, Anoectochilus genus, Anselia genus, Aplectrum genus Genus Arethusa, Calantthe, Cattlea, Coelogyne, Corallorhiza, Cymbidium, Cypripedium , Crytopodium genus, Dendrobium genus, Epidendrum genus, Goodyera genus, Listera genus, Codes genus, Oncidium genus Of transgenic orchids, characterized in that it is selected from the genus, genus Phalaenopsis, genus Pholidota, genus Rhynchostylis, and genus Vanda. Manufacturing method The method of claim 2, wherein the orchids are Cymbidium genus. The method according to claim 1, wherein the protoplasts include meristem, and the tissues are capable of inducing into meristem and regenerating into plants or seedlings. The method according to claim 4, wherein the primary protoplasts include meristems derived from the apical meristem of orchids, and the secondary protoplasts are capable of induction into secondary protoplasts and regeneration into plants or seedlings. Method for producing a transformed orchid, characterized in that it comprises a tissue derived from the primary protoplasts of the orchid, the tissue capable of induction into proliferative protoplasts and regeneration to plants or seedlings The method of claim 1, wherein the proliferative protoplasts comprise meristems derived from protoplasty grafts including both primary and secondary prototypical globules of orchid, and regeneration to plants or seedlings Method for producing transformed orchids, characterized in that the tissue possible The method of claim 1, wherein the external gene construct comprises a plasmid or screenable marker gene, its expression is detectable in prokaryotic spheroids, and can be confirmed by the characteristics of the transgenic protoplasts surviving the selection. Method for producing a transgenic orchid, characterized in that The method of claim 1, wherein the selectable marker gene encodes the expression of antibiotic resistant properties. The method of claim 1, wherein the fine particles comprise metal particles having a diameter of about 0.5 to 3 μm. The transformation according to claim 1, wherein a plurality of said microparticles are provided, each microparticle with said external gene construct is immobilized thereon, and each said microparticle is propagated to said plant target tissue. Method of manufacturing orchids The method according to claim 1, further comprising the process of the generation of roots from the transformed plant tissue. Transgenic orchids prepared according to the gene recombination method of claim 1