KR20130052194A - Transformation method using by seed of orchids - Google Patents

Abstract

PURPOSE: A method for transforming seeds of orchids is provided to shorten time for producing a transgenic plant without a process of inducing protocorm or a PLB(protocorm-like body). CONSTITUTION: A method for transforming seeds of orchids comprises a step of transfecting a seed with an agrobacterium containing a recombinant gene. Orchids are selected among Acianthus genus, Aerides genus, Anoectochilus genus, Ansellia genus, Aplectrum genus, Arethusa genus, Calanthe genus, Cattleya genus, Coelogyne genus, Corallorhiza genus, Cymbidium genus, Cypripedium genus, Crytopodium genus, Dendrobium genus, Epidendrum genus, Goodyera genus, Listera genus, Macodes genus, Oncidium genus, Phalaenopsis genus, Pholidota genus, Rhynchostylis genus, and Vanda genus. The seed is processed by disinfecting and cutting in a disk shape.

Description

Transformation method using by Seed of Orchids}

The present invention relates to a method for transforming orchids using Agrobacterium directly on seeds without tissue culture.

In addition to traditional breeding techniques, 'transformation methods' have been proposed as methods for improving plants. Therefore, the development of efficient and rapid transformation methods is a very important core technology in this field. To date, plant transformation methods can be broadly classified into direct transformation methods and indirect transformation methods.

Examples of direct transformation methods are electroporation (see Shimamoto K. et al., Nature, 338: 274-276, 1989; and Rhodes CA et al., Science, 240: 204-207, 1989), microparticle projection methods. (See Christou P. et al., Bio / Technology, 9: 957-962, 1991) and polyethylene glycol (PEG) method (see Datta, SK et al., Bio / Technology, 8: 736-740, 1990). . As an indirect transformation method, the Agrobacterium mediated transformation method (henceforth Agrobacterium transformation method) is mentioned. Agrobacterium is a phytopathogenic bacterium. Agrobacterium infects plants and has the property of introducing T-DNA regions present on the plasmids (eg, Ti plasmids or Ri plasmids) to their own plants. The Agrobacterium transformation method uses introduction into plants in this T-DNA region as a means of introducing genes into plants. Simply plant is infected with Agrobacterium containing the desired recombinant gene. After infection, the desired recombinant gene is introduced into the plant genome from Agrobacterium.

Among the direct transformation methods, microparticle projection for plant transformation was attempted mainly on monocots, including orchids, which did not have good Agrobacterium method, but there were many problems in terms of stability of transformants. As the transformation by bacterium was successful and the transformation by Agrobacterium was common in other monocotyledonous plants, transformation method using Agrobacterium in plants became the standard method. The Agrobacterium transformation method is well established in dicotyledonous and monocotyledonous plants, and a large number of stable transgenic plants expressing the desired recombinant genes are currently cultivated.

In the transformation of turbulence, microparticle projection was successful in the early stages (Anzai H et al., 1996 plant Tissue Culture letters 13: 265-271; Kuehnle AR et al., 1992 plant cell report 11: 484-488 Chia TF et al., 1990 Plant journal 6: 441-446). There was room for improvement in stability and efficiency. Subsequent Agrobacterium transformations were successful in turbulence such as phalaenopsis eggs, symbidium and dendrobium (Belarmino and Mii, 2000 Plant Cel Reports 19: 435-442; Knapp et al., 2000 Plant Cel Reports 19: 893-898; Yu et al., 2001 Plant Cel Reports 20: 301-305; Chai et al., 2002 Scientia Horticulturae 96: 213-224; Liau et al., 2003 Plant Cel Reports 21: 993-998; Men et al., 2003 Plant Cel Reports 21: 592 -598; Misiba et al., 2005 Plant Cel Reports 24: 297-303; Chan et al., 2005 Transgenic research 14: 279-288). To date, the transformation of orchids has been carried out using prokaryotic globules derived from apical meristem of shoots. Protocorm-like bodies (PLBs) are induced by in vitro culture of apical or axillary bud meristems, and are functionally and structurally similar to embryonic protoplasts. Therefore, in order to transform with this material, organic and in-flight cultivation of PLB has to be made in advance, and it takes a lot of time and effort, and has problems such as the generation of mutants and the possibility of chimera during the culturing process, and the transformation efficiency is also poor. .

There are several problems with other plants in transforming orchids with PLB. First, orchid cells slow down their growth. 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. Seeds of orchids exist in embryos without embryos and cannot germinate under normal circumstances. They develop after incubation on nutrient media and grow into protozoa. In 2005, Mishiba et al. (Plant Cell reports 24: 297-303) used a protocorm derived from seeds to transform the PLB. One protoplast was studied, and the protoplasts or precultured seeds were not transformed because they did not survive the treatment of hygromycin or kanamycin used as selection markers during transformation. In general, orchid seeds are immature and free of endosperm. Therefore, they have little germination capacity and can be germinated only in a finely tuned nutritional medium on board by interaction with commensal bacteria in nature (Joseph Arditti, 1967, The Botanical Review 33: 1-97). Therefore, in order to perform transformation by directly infecting orchid seeds with Agrobacterium, a method to overcome the fundamental problems of these orchid seeds must be developed.

The present invention is intended to solve the above problems. An object of the present invention is to provide an improvement of the Agrobacterium transformation method of orchid plants.

The present invention includes a step of directly infecting orchid seeds with Agrobacterium containing a desired recombinant gene for the transformation method of orchid plants. In this method, the seed refers to infecting the pod in the form of a disk cut in order to overcome the biologically weak problem of orchid seeds. Seeds provided for infection with Agrobacterium are transformed monocots, preferably the Gramineae family, more preferably Phalaenopsis L.

According to the method of the present invention, it is possible to generate a transformed plant more efficiently and more quickly than the conventional Agrobacterium transforming method. The method by this method does not require treatment, such as redifferentiating the plant sample to be transformed.

Conventional methods usually require an induction period of 3 to 4 weeks to induce PLB before infection with Agrobacterium. In contrast, the method of the present invention does not require a process for inducing protocom or PLB, and it is possible to shorten the time required for producing a transgenic plant. Moreover, according to the method of the present invention, it is possible to shorten the period requiring selection compared to the conventional method, and to reduce the influence of culture variation.

Figure 1 (A) is a photograph showing the aging phalaenopsis pods used for transformation.
Figure 1 (B) is a photograph showing a state in which the pod cut the eggplant to effectively infect Agrobacterium.
Figure 2 (A) is a photograph showing the germination of the phalaenopsis germination of the phalaenopsis obtained according to the method of the present invention about 50 days after Agrobacterium infection.
Figure 2 (B) is a transformant in the state of migrating to the conception after about 120 days after Agrobacterium infection.
3 is a result of verifying the transformation by PCR using the GUS gene region.
4 is a result of verifying the transformation using tissue staining method by GUS gene expression.

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

The present invention relates to a method for transforming an orchid plant, comprising a step of infecting an original seed with Agrobacterium containing a desired recombinant gene. The seed is in a pod state after fertilization, in particular, the seed is processed into sections cut into discs after disinfection with a pod.

The 'plant' to which the method of the present invention is applied is an orchid plant. The most preferred plant to which the method of the invention is applied is a phalaenopsis.

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

It has been found that the orchid seeds can be directly applied for transformation according to the present invention. This method could be used to develop a transgenic phalaenopsis. 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 traits. Development of converted orchid plants is possible. This method is applicable to all orchids and especially phalaenopsis orchids.

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

The method described herein relates to introducing foreign genes into the genome 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.

(Production of plant expression vector)

In order to introduce a desired recombinant gene into a plant, an appropriate plant expression vector containing the desired recombinant gene should be prepared. Such a plant expression vector is produced by using a well-known gene combination technique. For the construction of a plant expression vector for use in the Agrobacterium transformation method, for example, pCAMBIA-based vectors are preferably used, but not limited thereto. Recombinant gene refers to any DNA that you wish to introduce into a plant. In the present invention, the desired recombinant gene is not limited to natural or isolated ones, but also includes synthetic DNA. Synthetic DNA may include, for example, a product synthesized or modified by a person skilled in the art by a gene having a known sequence. In this case, it can be effectively used in the case of gene expression suppression gene of small size, such as SiRNA or miRNA. In the present invention, a desired recombinant gene is desired to be expressed in a transformed plant, and genes that modify disease resistance, stress resistance, flower shape or color with any DNA that is endogenous or exogenous to the plant, and cause the same. Transcriptional regulators, and the like. In addition, reporter genes such as glucuronides (GUS) or green fluorescent protein (GFP) can be used to determine whether or not to transform or promoter properties.

When intended for expression in a plant, the desired recombinant gene includes a form in which its own promoter (ie, the promoter in which the gene is operably linked) is operable. In addition, when it is requested to further include a promoter other than the magnetic promoter in the case where it does not include the magnetic promoter, it is operatively connected with any suitable promoter. Examples of promoters that can be used include structural promoters and promoters that are selectively expressed in plant parts and inducible promoters. In plant expression vectors, moreover, various regulatory elements are linked to host plants in a state capable of operating in cells. Regulatory elements preferably include selectable marker genes, plant promoters, terminators, and enhancers. The type of plant expression vector to be used and the type of regulatory elements can be obtained by converting them in accordance with the purpose of transformation, which is well known among those skilled in the art. 'Plant promoter' refers to a promoter that is operably linked to a selectable marker gene and that is expressed in a plant. Examples of such promoters include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter and the nopalline synthetase promoter.

 The 'terminator' is located downstream of the region encoding the protein of the gene and arranges for the termination of transcription and addition of the poly A sequence when DNA is mRNA transcribed. Examples of terminators include, but are not limited to, CaMV35S terminator and Nopalline Synthetase Terminator (Tnos).

'Enhancer' is used to increase the expression efficiency of the gene of interest. As an enhancer, it is preferable to include an enhancer in an upstream region in the CaMV35S promoter. Enhancers can use multiple enhancers in a single plant expression vector.

'Selection marker gene' can be used to facilitate the selection of transgenic plants. Drug resistance genes such as the hygromycin phosphotransferase (HPT) gene to confer hygromycin resistance, and the neomycin phosphotransferase II (NPTII) gene to confer ganamycin resistance can be used as appropriate. It is not limited to these.

(Transplantation of plants)

Agrobacterium strains used for plant transformation are specific cell lines, such as LBA4404, EHA105 and C58c1, isolated and modified from Agrobacterium tumefaciens. Agrobacterium is used for plant infection after transformation with plant expression vectors containing the desired recombinant genes. By infecting the seed with the transformed Agrobacterium, the desired recombinant genes are introduced into the plant. The introduced recombinant gene is present in the genome in the plant (integrated).

 Seeds are harvested after mating, harvested pods, surface sterilized, cut into 2 mm thick discs, and infected with transformed Agrobacterium under aseptic manipulation. During the period of Agrobacterium infection (co-culture), the seeds are incubated approximately 2 to 5 days, preferably 2 days, under cancer conditions. At this time, the temperature is generally 26 ~ 28 ℃, preferably 28 ℃. Seeds are then treated with appropriate fungicides (eg carbenicillin, cytotoxin) to remove Agrobacterium in the medium, and screening markers (eg, hygro) to select only transformed individuals. Drug resistance such as mycin resistance).

After cultivation under appropriate sterilization and selection conditions, the transformed seed selected is transferred to an appropriate medium (eg, MS medium) and incubated for a suitable period of time. To regenerate the plant, the regenerated transformants are transferred to a rooting medium (eg, MS medium that does not contain plant regulators). After confirming root development, the transformants are transplanted into pollen. The desired recombinant gene introduced into the plant serves for the desired purpose in the plant (for example, overexpression of a desired new trait gene or suppression of expression of this endogenous gene). Whether the desired recombinant gene has been introduced into the plant can be confirmed by methods such as PCR, Southern blot or Northern blot, using well-known techniques of those skilled in the art. On the other hand, if the expression of the endogenous gene is desired in the plant according to the introduction of the desired recombinant gene, the expression of the target endogenous gene is compared, for example, using the Northern blot or qRT-PCR. Test it by

(Example)

Hereinafter, the present invention will be described in detail through examples. This embodiment does not limit the invention. Materials, reagents, and the like used in the examples are those available from commercial sources unless otherwise specified.

(Intersection)

For the transformation of phalaenopsis plants according to the method of the present invention, the pods were harvested 140 days after crossing the Taisuco Mushadian and Sogo Musadian. Sterilize by shaking for a minute. After enough washing with autoclaved water three times, the pod is cut into 2mm thickness vertically using a scalpel to make a section.

(Vector production for plant expression)

As a plant expression vector for transforming Agrobacterium, the Agrobacterium C58c1 strain is transformed using pCAMBIA1301, a plasmid linking a GUS gene containing a 35S promoter and a hygromycin resistance gene.

Agrobacterium infection

The pods cut into 2 mm thick sections were immersed in the transformed Agrobacterium suspension for 5 minutes, followed by 1 / 2MS medium (30 g / L sucrose, 1 g / L casamino acid, 100 mM / L acetosyringone, 2 g / g). Transfer to L gellan gum, pH 5.8) and incubate at 28 ° C. for 2 days under dark conditions to coexist.

(Sterilization and screening)

After completion of coculture, wash the Agrobacterium three times from the seed using 200 ml of wash solution (10 mM MgSO4, 600 mg / L carbenicillin, 1% Tween20) and transfer to selective medium.

Screening medium: 3 g hyponex, 1.5 g peptone, 100 g potato, 10 g sucrose, 8 g agar, 0.5 g charcolnicillin (300 mg / L) and hygromycin (25 mg / L)

(subdivision)

Seeds germinated after selection are regenerated in shoot and root-induced medium.

Regeneration medium: Place selected seeds on (3 g hyponex, 1.5 g peptone, 100 g potato, 10 g sucrose, 8 g agar, 0.5 g charcolnicillin (300 mg / L) and hygromycin (25 mg / L)) Incubate at 27-32 ° C. for 2 weeks.

(Planting into pots)

The re-differentiated transformant is transplanted into the rooting medium (MS medium without hormone supplemented with 25 mg / L of hygromycin), and the growth of the root is confirmed, and then the transformant is transferred to the pollen using fertility.

[Industry availability]

According to the present invention, there is provided an improved seed transformation method of an Agrobacterium mediated orchid plant. In the method of the present invention, the native seed of the plant that induces transformation is infected with Agrobacterium containing the desired recombinant gene. The use of the present invention makes it possible to develop transgenic plants at high efficiency and at a rapid rate.

Claims (5)

A method for transforming orchid plants, comprising the step of infecting an original seed with Agrobacterium containing a desired recombinant gene. According to claim 1, The orchids are genus Acanthus, 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 A method for seed transformation of orchids, characterized in that it is selected from the genus, genus Phalaenopsis, genus Pholidota, genus Rhynchostylis and genus Vanda. The method of claim 1, wherein the seed is in the form of a mature pod after fertilization. The method of claim 3, wherein the seed is disinfected with pods and then processed into sections cut into discs. The method of claim 1, wherein the orchids are genus Phalaenopsis.

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