JP2019110860A - Method for producing transgenic plant body - Google Patents

Abstract

To provide a convenient and quick transgenic technique that can be applied to various plants including the plant species and varieties for which tissue culture is difficult.SOLUTION: There is provided a method for producing a transgenic plant body, including inoculating a shoot apex of a seed embryo with Agrobacterium to grow the seed embryo.SELECTED DRAWING: None

Description

  The present invention relates to a method of producing a genetically modified plant by Agrobacterium using an in planta transformation method, and a method of in planta transformation of a plant by Agrobacterium.

  Conventional gene transfer methods using Agrobacterium, which have been widely used in plants until now, require techniques for plant regeneration after infection with Agrobacterium, and in plants for which regeneration techniques have not been established, Agrobacteria No gene transfer method was available. Similarly, the direct gene transfer method by the gene gun method or the whisker method requires regeneration technology by tissue culture, and has been a great hindrance to fostering new breeds that become materials of new industries. For this reason, there has been a need for a method of easily regenerating a plant and introducing a nucleic acid directly into a plant tissue, and directly introducing a gene into a tissue which develops into a plant without using a special plant regeneration technology.

  In planta transformation method by inoculating Agrobacterium tumefaciens with a scratch on kenaf sprout as a method of obtaining a transformed plant without using tissue culture (Patent Document 1), rice seed Implanta transformation method by inoculating Agrobacterium tumefaciens by wounding the sprouting part of the embryo (Patent Document 2), Agrobacterium in the shoot apical meristem of sorghum (Sorghum bicolor (L.) Moench) seedlings An in planta transformation method (non-patent document 1) by inoculating Um. Tumefaciens is known.

  However, there is a need for additional implanter transformation methods that can be applied to various types of plants.

Patent No. 4170078 Patent No. 4754968

Journal of Genetics, Vol. 94, No. 3, September 2015, pp. 425-434

  The present invention aims to establish a simple and rapid transformation technique that can be applied to various types of plants, including plant species and varieties that are difficult to culture tissue.

  The present inventors diligently studied to solve the above-mentioned problems. As a result, it has been surprisingly found that a transformant can be obtained without going through tissue culture by combining the method of generating individual plants directly from the shoot apex of the plant seed embryo and the Agrobacterium method. , Completed the present invention.

That is, the present invention
[1] A method for producing a transformed plant, which comprises inoculating an Agrobacterium to the shoot apex of a seed embryo and growing the seed embryo.
[2] The method according to [1], wherein the top of the stem is wounded and the wound site is inoculated with Agrobacterium;
[3] A method for in planta transformation of a plant, comprising inoculating the Agrobacterium at the shoot top of a seed embryo,
[4] The method according to [3], wherein the shoot apex is wounded and the wound site is inoculated with Agrobacterium, and [5] any one of [1] to [4] wherein the plant is a dicotyledonous plant Provide the method described in one. Hereinafter, they are collectively referred to as "the method of the present invention".

  According to the method of the present invention, a genetically modified plant individual can be obtained directly from a shoot top site transfected with Agrobacterium. Since the method of the present invention does not require tissue culture, it also applies to plant species and varieties for which plant regeneration techniques from tissue pieces by tissue culture have not been established, and plant species and varieties which can not regenerate plants by tissue culture. be able to. Furthermore, in the method of the present invention, a transgenic plant is obtained under a non-sterile growth environment which can not be achieved by the conventional tissue culture method, because the gene is introduced into the shoot apex of the seed embryo and the plant individual is regenerated directly from the site. It is possible to obtain transformed plants in a short period of time as compared with the conventional method. Thus, the present invention provides a simpler and more efficient method for plantlet plant transformation.

FIG. 1 shows the preparation of plants for use in the shoot top implanter method. FIG. 2 shows the structure of pCA23_GFBSD2. FIG. 3 is a photograph showing a shoot apex portion of a plant where two weeks of GFP gene expression was observed after in planta transformation. The upper part shows the result of stereomicroscope observation under visible light, and the lower part shows the result of stereomicroscope observation under fluorescence. FIG. 4 is a photograph showing the state of regenerated plants 4 to 6 weeks after the planta transformation. FIG. 5 is a photograph showing regeneration of a 4- to 6-week-old transformed plant after in planta transformation. A shows the result of the stereomicroscope observation under visible light, B shows what expanded the stem and leaf part of A. FIG. The left of B shows the result of stereomicroscope observation under visible light, and the right of B shows the result of stereomicroscope observation under fluorescence. FIG. 6 shows the regeneration of 6 to 8 week old transgenic plants after in planta transformation. The left shows the result of stereomicroscope observation under visible light, and the right shows the result of stereomicroscope observation under fluorescence. FIG. 7 shows the results of electrophoresis for confirming T-DNA insertion in regenerated plants obtained by in planta transformation. Fig. 8 shows the structure of the genome editing vector pGEgP237_IAA9 (Fig. 8a) and the target sequence of the tomato IAA9 gene (Fig. 8b). In FIG. 8a, GFBSD2 is a sequence (Pro 2x35S) containing the 2x cauliflower mosaic virus 35S gene promoter (SEQ ID NO: 6) and the omega translation enhancer (SEQ ID NO: 2) and the Arabidopsis heat shock protein 18.2 kDa gene terminator (Ter 18.2). Fig. 7 shows a fusion gene GFBSD2 expression cassette (SEQ ID NO: 7) of the GFBSD2 gene and blasticidin resistance gene expressed under the control of (SEQ ID NO: 8). U6 represents the Arabidopsis thaliana U6 snRNA gene promoter (SEQ ID NO: 11). gRNA indicates a guide RNA containing the target recognition sequence (SEQ ID NO: 12). Cas9 shows Cas9 containing a 3x nuclear localization signal (SEQ ID NO: 14). NPTII shows the kanamycin resistance gene NPTII (SEQ ID NO: 3) expression cassette which is expressed under the control of 2x cauliflower mosaic virus 35S gene promoter and cauliflower mosaic virus 35S gene terminator. In FIG. 8 b, lower case letters indicate the PAM sequence of Sp (Streptococcus pyogenes) Cas9. FIG. 9 shows mutation analysis in pGEgP237 IAA9-introduced regenerated plants by the in planta transformation method. FIG. 9a shows the appearance of pGEgP237 IAA9 introduced tomato plants on day 13 (left) and day 30 (right) after the implementation of the planta transformation method. The upper part shows the result of stereomicroscope observation under visible light, and the lower part shows the result of stereomicroscope observation under fluorescence. FIG. 9 b shows mutation analysis by PCR-RFLP method. Open triangles indicate AccI-cleaved fragments derived from wild type, and black triangles indicate mutagenized fragments not subjected to AccI cleavage as a result of mutagenesis, or as a result of mutagenesis, the electrophoretic mobility is different from that of wild-type derived The cleavage fragment is shown. FIG. 10 shows the results of analysis of mutant sequences introduced on the tomato IAA9 gene. The underline indicates the target sequence and the box indicates the SpCas 9 PAM sequence. A hyphen (-) indicates a base deletion. On the right side of the figure, the number of inserted bases is indicated by + and the number of deletions is indicated by-.

  The method of the present invention can be applied to seed plants and can be applied to any angiosperm and gymnosperm including dicotyledonous and monocotyledonous plants. Therefore, examples of target plants of the present invention include dicotyledonous plants such as, but not limited to, tomato, tobacco, soybean, cotton, cotton, sugar beet, azuki bean, buckwheat, cucumber, melon, rape, radish, lettuce, Alfalfa, pea, sweet potato, potato etc .; monocotyledonous plants such as, but not limited to, rice, corn, wheat, barley, buckwheat, sorghum, sugar cane, banana, pineapple etc; and gymnosperms such as However, pine, cedar, ginkgo, saotetsu etc. may be mentioned. Preferably, dicotyledonous plants are used.

  The Agrobacterium used in the method of the present invention is not particularly limited as long as it is a bacterial species and strain capable of infecting a plant. For example, but not limited to, Agrobacterium tumefaciens, Agrobacterium vitis, Agrobacterium rhizogenes, Agrobacterium radiobacter, And their various strains.

  When Agrobacterium infects a plant, it has the ability to integrate the T-DNA region within its own Ti plasmid or Ri plasmid into the plant chromosome. The Agrobacterium method is a transformation method utilizing this ability of Agrobacterium. Therefore, the Agrobacterium to be used in the method of the present invention is a Ti plasmid or Ri plasmid in which a foreign gene to be introduced into a plant is incorporated into the T-DNA region, or a T-incorporated foreign gene to be introduced into a plant. It may contain a binary vector having a DNA region. Such Agrobacterium can be prepared by methods known in the art. For example, various intermediate vectors for the integration of foreign genes into Ti or Ri plasmids are known. In addition, many binary vectors have been developed in the art, and these known vectors can be used in the present invention. The introduction of the vector into Agrobacterium may be carried out by a known method, such as a heat shock method, an electroporation method, a triparental mating method and the like.

  The foreign gene is not particularly limited, and may be a gene encoding any protein or peptide. In addition to the target foreign gene to be introduced into a plant, a marker gene for selecting transformants may be inserted into the T-DNA region. Examples of the marker gene include, but are not limited to, kanamycin resistant gene, hygromycin resistant gene, antibiotic resistant gene such as blasticidin resistant gene, and fluorescent protein (GFP) gene. Marker genes capable of determining the introduction can be used.

  In the present invention, “seed embryo” refers to an embryo present in the seed of a target plant, ie, an embryo in a state of being enclosed in a seed coat. The seeds may be either immature or full ripe, and may be either before or during germination. Here, "in germination" refers to a process from the state where sprouting has started but the stem top of the embryo is still wrapped in the seed coat until the seedling completely breaks the seed coat and appears. In the present invention, seed embryos at any stage during germination may be used. In the method of the present invention, preferably, seed embryos of seeds before germination are used. Therefore, in the present invention, it is not necessary to subject the seeds to a germination induction treatment in advance.

  In the present invention, in order to inoculate Agrobacterium, seed embryos are removed from the seeds. The method for removing the seed embryo from the seed is not limited and can be appropriately selected by those skilled in the art. For example, the seed embryo may be removed by cutting the outer surface of the seed coat with a scalpel and peeling the seed coat from the incision using a forceps. Preferably, then, the shoot apex of the seed embryo is made to project by excising the cotyledon of the taken-out seed embryo.

  In the present invention, the shoot apex of the seed embryo may be injured prior to inoculation with Agrobacterium, and the wound site may be inoculated with Agrobacterium. Preferably, the growing point of the shoot apex of the seed embryo is injured. The shape of the wound is not particularly limited, and examples thereof include perforations and incisions. Preferably, one or more incisions are made using a scalpel, razor, cutter knife, needle-like tool with sharp tip, or the like.

  The method for inoculating Agrobacterium to the shoot apex of the seed embryo is not particularly limited as long as Agrobacterium can infect the plant through the shoot apex by inoculation. For example, a method of immersing a shoot apex of a seed embryo in a suspension of Agrobacterium, a method of applying a suspension of the Agrobacterium to a top of a shoot of a seed embryo using a cotton swab soaked in the suspension, And a method of injecting a suspension of Aumobacterium into the shoot top of the seed embryo, or a method of spraying a suspension of Agrobacterium onto the shoot top of the seed embryo, and the like. When the top of the stem of the seed embryo is immersed in a suspension of Agrobacterium, the infection efficiency of Agrobacterium can be further improved by performing a reduced pressure treatment in the immersed state. A suspension of Agrobacterium is prepared by suspending Agrobacterium in a liquid medium. The concentration of cells in the suspension may be selected appropriately for infection depending on the method of inoculation. As the liquid medium, a known liquid medium may be used, and depending on the inoculation method, for example, MS liquid medium, B5 liquid medium, AB liquid medium, LB liquid medium, 1% -10% sucrose solution, etc. are used Be done.

  After inoculation of Agrobacterium into the seed embryo, the seed embryo is grown under conditions suitable for each target plant. For example, Agrobacterium-inoculated seed embryos are placed on an agar medium and cultured at 20 to 30 ° C. For culture of seed embryos after Agrobacterium inoculation, a special culture medium for tissue culture is not required as in tissue culture. For example, a known culture medium suitable for plant culture such as MS medium, B5 medium, Hyponexus medium, etc. It may be used. The grown plants are grown to the desired growth stage, with appropriate transplantation. According to the method of the present invention, an adult plant can be obtained in a short period of time as compared with the conventional transformation method using tissue culture, since the plant is regenerated directly from the top of the shoot of the seed embryo without tissue culture. For example, in the case of tomato, the conventional transformation method using tissue culture takes about six months to obtain an adult plant, while the method of the present invention gives an adult plant in about three to four months. In order to further shorten the period, plant hormones that promote plant growth may be administered to the Agrobacterium inoculation site for a short period of time, for example, 2 to 30 days. Examples of the plant hormone include, but are not limited to, zeatin, kinetin, benzyladenine, thidiazuron, indoleacetic acid, naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid and the like.

  After culture of seed embryos inoculated with Agrobacterium, selection of transformants may be performed by a conventional method. For example, it is possible to confirm whether the Agrobacterium-derived T-DNA region has been incorporated into the genome of the obtained plant body by PCR method, Southern hybridization method, Northern hybridization method or the like. Alternatively, an Agrobacterium comprising a Ti or Ri plasmid in which a marker gene such as an antibiotic resistance gene or a fluorescent protein (GFP) gene is incorporated into a T-DNA region, or a binary having a T-DNA region incorporating the marker gene By using Agrobacterium containing a vector, transformants can be easily selected using antibiotic resistance or fluorescent protein expression as an index. For example, for selection of effective transformants, Agrobacterium-inoculated with an antibiotic corresponding to a marker gene introduced into the plant via Agrobacterium for a short time, for example, 2 to 30 days There is a method of selecting a living plant as a transformant by administering to a site.

  Therefore, the present invention further provides a method for in planta transformation of a plant, which further comprises the steps of excising the cotyledon from the seed embryo and projecting the top of the stem, and inoculating the top of the stem with Agrobacterium. A method of producing a genetically modified plant is provided, which comprises the step of growing an embryo. Still further, the present invention provides a plant implanta comprising the steps of excising a cotyledon from a seed embryo and projecting a shoot apex, wounding the shoot apex, and inoculating the shoot apex with Agrobacterium. A method for producing a genetically modified plant is provided, which comprises a transformation method and a step of further growing the seed embryo. Furthermore, the present invention further comprises the steps of: excising a cotyledon from a seed embryo and projecting a shoot apex, inoculating the shoot apex with an Agrobacterium, growing the seed embryo, and producing the trait generated from the shoot apex Provided is a method for producing a genetically modified plant, which comprises the step of obtaining a converted plant. The method of the present invention can also be used, for example, for genome editing of plants.

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

Example 1 Preparation of tomato transformant (1) Preparation of tomato plant The growth conditions of the tomato plant were: illumination 30,000 lux, temperature 20-25 ° C., and photoperiod: 16 hours light / 8 hours dark. The seeds of tomato (Solanum lycopersicum cv. Ailsa Craig) were surface-sterilized with hypochlorous acid, soaked in sterile water for 10 hours, and then the seed coat was removed using a knife and tweezers. The cotyledons of the seed embryos removed from the seeds were removed with a scalpel or razor, and used as test material for inplanta transformation. (Figure 1)

(2) Preparation of Agrobacterium tumefaciens In this example, the Agrobacterium tumefaciens GV 2260 strain was used. Agrobacterium tumefaciens was transformed using the binary plasmid vector pCA23_GFBSD2 (FIG. 2). The T-DNA of pCA23_GFBSD2 expresses kanamycin under the control of rice translation elongation factor gene promoter (Pro oef 1) (SEQ ID NO: 1) and rice heat shock protein 16.9 kDa gene terminator (Ter 16.9 B) (SEQ ID NO: 4) Sequences containing the resistance gene NPTII expression cassette (SEQ ID NO: 3), 2x cauliflower mosaic virus 35S gene promoter (SEQ ID NO: 6) and omega translation enhancer (SEQ ID NO: 2) (Pro 2x35S) and Arabidopsis heat shock protein 18.2 kDa gene terminator (Ter 18.2) (SEQ ID NO: 8) + GFP gene and blast expressed under control of cauliflower mosaic virus 35S gene terminator (Ter 35S) (SEQ ID NO: 5) Fusion gene GFBSD2 expression of discoidin resistance gene containing cassette (SEQ ID NO: 7). The integration of TCA of pCA23_GFBSD2 into the plant genome enables drug selection with kanamycin and visible selection with GFP. Agrobacterium tumefaciens carrying pCA23_GFBSD2 was cultured on LB agar medium containing 50 mg / L kanamycin at 28 ° C. for 24-48 hours, selected, and used for in planta transformation. Agrobacterium tumefaciens used for the imprinta method is cultured on LB agar medium containing 50 mg / L kanamycin at 28 ° C. for 18 hours, and then the culture solution is collected to obtain 50 μM acetosyringone, 0.05% silhouette L. -77 (Biomedical), suspended in MS liquid medium containing 3% sucrose, and the final cell concentration was 0.01 OD (600 nm).

(3) In planta transformation of tomato plants by Agrobacterium tumefaciens The cut end of the top of the stem top of the plant from which the cotyledon was removed from the tomato seed embryo prepared in (1) above was cut using a scalpel . Next, the injured plant is immersed in the 0.01 OD (600 nm) Agrobacterium tumefaciens bacterial solution prepared in (2) above, and subjected to reduced pressure treatment (-0.025 MPa, 10 minutes) to obtain a wound. Agrobacterium tumefaciens inoculation was carried out at the top of the marked stem. After completion of infection, excess Agrobacterium tumefaciens bacterial solution was wiped off from the plants, transferred to MS agar medium containing 3% sucrose, and coculture was performed for 5 days in the dark at 23 ° C. in the dark.

  After 5 days of co-culture, the plant is washed several times with sterile water containing 12.5 mg / L meropenam (manufactured by Wako Pure Chemical Industries, Ltd.), and after wiping off excess water from the plant, 3% sucrose is removed. The cells were re-transplanted into MS agar medium containing. After transplantation, an aqueous solution containing 1.5 mg / L zeatin and 100 mg / L kanamycin was applied to the shoot apex of the plant. The application was performed about 5 times every 2 to 3 days. The top of the shoot where expression of the transgene GFP was observed after applying zeatin and kanamycin solution is shown in FIG. The plants in which GFP expression was observed in the shoot apex were about 80% of the plants treated with inplanta.

  After 2-3 weeks of planta transformation, the plants are transferred to a magenta box containing MS agar medium containing 3% sucrose to promote plant regeneration and observe GFP expression of stems and leaves regenerated from the top of the shoot. , Visible selection of transformed plants. The state of the regenerated plant after 4 to 6 weeks after the planta transformation is shown in FIG. Among the plants at the growth stage shown in FIG. 4, those plants in which GFP gene expression was observed in the entire stem and leaf part are shown in FIG. 5 and FIG. As apparent from FIG. 6, GFP expression was observed not only in stems and leaves but also in flower buds and fruits. In about 10% of the planters treated with the planta, plant individuals in which gene transfer occurred in whole plant tissue as shown in FIGS. 5 and 6 were obtained.

(4) Confirmation of introduced gene by PCR method A plant obtained by the planta transformation method of the above (3) according to a protocol provided by the manufacturer using NucleoSpin (registered trademark) Plant II kit (Takara Bio Inc.) Genomic DNA was prepared from the leaves of the body.

  Design the omega F1: 5'-GTATTTTTACAACAAATTACCAACAAACAAC- 3 '(SEQ ID NO: 9) in the vicinity of the omega translation enhancer sequence (SEQ ID NO: 2) located downstream of the 2x cauliflower mosaic virus 35S gene promoter located on T-DNA of pCA23_GFBSD2. Also, GFP_R100: 5'-CGCCGGACACGCTGAACTTGT-3 '(SEQ ID NO: 10) was designed in the region located 100b downstream from the translation initiation codon of the GFBSD2 gene, and a primer for amplifying the GFBSD2 gene fragment by PCR was synthesized.

  The PCR reaction was performed using TaKaRa Ex Taq (registered trademark) Hot Start Version (manufactured by Takara Bio Inc.) according to the reaction protocol recommended by the manufacturer. The obtained PCR fragments were separated by 1.5% agarose gel electrophoresis to confirm the presence or absence of gene transfer. Although the PCR fragment of 171 bp is amplified by the above designed PCR primers, as shown in FIG. 7, amplification of the expected 171 bp fragment was obtained from all of the regenerated plants observed for GFP expression (Line # 1) ~ 6).

  Furthermore, in the same manner, a rio grande cultivar, which is a tomato showing poor tissue culture, was subjected to in planta transformation. As a result, it was confirmed that a transformed plant was obtained.

Example 2 Preparation of genome-edited tomato plants by the planta transformation method As one form of the embodiment of the planta transformation method, tomato transformed plants are prepared by the planta transformation method using a genome editing vector, and the genome editing is performed. It has been demonstrated that mutation can be introduced into target genes by

(1) Structure of genome editing vector A genome editing vector shown in FIG. 8a was constructed using the CRISPR / Cas9 system as genome editing. The Aux / IAA transcription factor IAA9 gene was selected as the target sequence and the mutation introduced into the sequence, and the target sequence 5'-GAGCTCAGGCTCGGTCTAcctgg-3 '(lower case indicates the PAM sequence of SpCas9) shown in FIG. Number 13). The target sequence was artificially synthesized and used as a guide RNA shown in FIG. 8a to complete the binary vector pGEgP237_IAA9.

(2) Preparation of a genome-edited tomato plant by the planta transformation method The constructed binary vector was introduced into the Agrobacterium tumefaciens GV 2260 strain, and the tomato seed embryo by the planta transformation method described in Example 1 The gene was introduced at the top of the stem of the plant to produce a regenerated transformed plant. The resulting tomato transformant is shown in FIG. 9a.

  Among the plants into which pGEgP237_IAA9 was introduced, genomic DNA was prepared from true leaves of plants in which GFP fluorescence was observed. Preparation of genomic DNA was performed using a NucleoSpin (registered trademark) Plant II kit (manufactured by Takara Bio Inc.) according to a protocol provided by the manufacturer. A recognition sequence for restriction enzyme AccI is present in the target sequence of IAA9, and as a result of genome editing, if a mutation is introduced, the AccI recognition site disappears, or the length of AccI cleaved fragment changes. The mutation analysis which arose in the target sequence of IAA9 was performed by PCR-restriction enzyme length polymorphism (RFLP) analysis using AccI using this. Using the prepared genomic DNA as a template, the vicinity of the target sequence of the IAA9 gene was amplified using TaKaRa Ex Taq (registered trademark) Hot Start Version (manufactured by Takara Bio Inc.). When the PCR fragment was digested with AccI using restriction enzymes, the transformed plant-derived PCR fragment into which pGEgP237_IAA9 had been introduced had a sequence that was not cleaved by AccI as a result of mutagenesis introduced into the IAA9 target sequence, or a cleaved fragment It was found that sequences of varying length were included (Figure 9b). When the mutation sequence was further analyzed by sequence analysis for plants for which mutation introduction was confirmed, it was found that a base insertion type, a base deletion type, or a base insertion and a base deletion immediately after the PAM sequence on the target sequence of IAA9. It became clear that mutations containing combinations were introduced (FIG. 10).

  According to the present invention, a simple and efficient plant plant transformation method is provided. Therefore, according to the present invention, transformed plants having economic and agricultural added value can be obtained conveniently and efficiently.

SEQ ID NO: 1; Rice translation elongation factor gene promoter (Pro osef 1)
SEQ ID NO: 2; Tobacco mosaic virus omega translation enhancer sequence
SEQ ID NO: 3; NPTII gene sequence
SEQ ID NO: 4; Rice heat shock protein 16.9 kDa gene terminator (Ter 16.9 B)
SEQ ID NO: 5; Cauliflower mosaic virus 35S gene terminator (Ter 35S)
SEQ ID NO: 6; 2X Cauliflower mosaic virus 35S gene promoter
SEQ ID NO: 7; Fusion gene sequence of GFP gene and blasticidin resistant gene (GFBSD2)
SEQ ID NO: 8; Arabidopsis heat shock protein 18.2 kDa gene terminator (Ter 18.2)
SEQ ID NO: 9; PCR primer omega_F1
SEQ ID NO: 10; PCR primer GFP_R100
SEQ ID NO: 11; Arabidopsis U6 snRNA gene promoter
SEQ ID NO: 12; gRNA sequence
SEQ ID NO: 13; Target sequence on tomato IAA9 gene
SEQ ID NO: 14; Streptococcus pyrogenes Cas9 nucleotide sequence (comprised Nuclear localizing signal)

Claims (5)

  A method for producing a transformed plant, which comprises inoculating an agrobacterium into a shoot apex of a seed embryo and growing the seed embryo.   The method according to claim 1, wherein a wound is applied to a shoot apex and the wound site is inoculated with Agrobacterium.   A plant implanter transformation method comprising inoculating an Agrobacterium into the shoot apex of a seed embryo.   4. The method according to claim 3, wherein the top of the stem is wounded and the wound site is inoculated with Agrobacterium.   The method according to any one of claims 1 to 4, wherein the plant is a dicotyledonous plant.