JP2005198600A - Recombinant theaceous tree and method for preparing the same tree - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient preparation method for a recombinant in a theaceous tree, particularly tea tree in which preparation of a recombinant by a conventional method was difficult, and to provide the recombinant tree and to provide a method for selecting the recombinant. <P>SOLUTION: The method for preparing the recombinant theaceous tree comprises introducing a gene, particularly a gene containing a gene encoding a fluorescent protein localized in an organelle into an embryo callus of the theaceous tree. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing a genetically modified tree using embryo callus in a camellia tree, particularly a tea tree, and a genetically modified camellia tree. The present invention also relates to a method for producing a genetically modified camellia tree and a genetically modified camellia family tree, including a transformant selection step for non-destructively measuring a fluorescent protein localized in an organelle. .

  In general, transgenic plants in which new traits have been given to herbaceous plants such as barley, wheat, rice, tobacco, etc. by genetic recombination techniques have been prepared. Production of recombinant trees has been reported. However, the production of genetically modified plants in tea trees while being useful as economic plants has so far been conducted in 2001 by a group such as Mondal of India using cotyledons and gene introduction using the Agrobacterium method, and GUS (β- This is only a report (Non-Patent Document 1) in which a transformed plant was selected using a glucuronidase) system. Less transformation in the tea tree is suitable for gene transfer necessary for the production of efficient genetically modified plants, and there is no way to select plant culture cells with differentiation-inducing ability and subsequent efficient transformation plants. This is considered to be sufficient. The group of Mondal et al. Introduces genes using cotyledons. However, only two cotyledons in a tea tree can be obtained from one seed, which is never an efficient method for producing a genetic recombinant.

In 1996, Kato reported that germination could be induced with embryo callus derived from buds instead of conventional tissue fragments (Non-patent Document 2), but so far, genetic recombination using this embryo callus has been reported. There is no. Furthermore, in order to obtain a gene recombinant efficiently, it is desirable that a method for selecting a transformant is simple and differentiation can be induced as it is after selection. The creation of recombinants such as Mondal et al. Uses the GUS (β-glucuronidase) reporter system as a method for selecting transformants, but this requires cell immobilization and is therefore used for induction into subsequent germination. It cannot be used, and it is difficult to say that it is an efficient method for obtaining a genetic recombinant. In general, a method for selecting a transformant using a green fluorescent protein GFP derived from Aequorea jellyfish as a fluorescent marker is well known as a simple and non-destructive method for selecting a transformant (Non-patent Document 3). However, camellia trees such as tea trees have autofluorescence that overlaps with the fluorescence wavelength of GFP, and it has been clarified that GFP cannot be used as a marker by a commonly used method (FIG. 2).
TK Mondal, A. Bhattacharya, PS Ahuja and PK Chand, (2001) Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos.Plant Cell Rep 20: 712 -720. M. Kato, (1996) Somatic embryogenesis from immature leaves of in vitro grown tea shoots.Plant Cell Rep 15: 920-923. M.-J.Cho, HWChoi, D.Okamoto, S.Zhang and PGLemaux, (2003) Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures.Plant cell Rep 21: 467-474. Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H. (1999) Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants.Plant J. 18: 455 -463.

  The present invention solves the above-mentioned problems of the prior art in which genetic recombination has been difficult, and provides an efficient method for producing genetically modified trees in camellia trees, particularly tea trees, and genetically modified camellia trees Is. In addition, the present invention solves the problems of the prior art in which a genetically modified camellia tree that uses a fluorescent protein as a marker could not be selected, and provides an efficient selection method.

  The present invention relates to a method for producing a genetically modified camellia tree, characterized by introducing a gene into embryo callus of the camellia tree.

  The present invention relates to a method for producing a genetically modified camellia tree in which the camellia tree is a tea tree.

  The present invention relates to a method for producing a genetically modified camellia tree, wherein the embryo callus is an embryo callus that is generated from the tip of a leaf generated by bud culture and from the middle assistant part.

  The present invention relates to a method for producing a genetically modified camellia tree, the gene of which comprises a gene that encodes a fluorescent protein.

  The present invention relates to a method for producing a genetically modified camellia tree in which the fluorescent protein is the green fluorescent protein GFP derived from Aequorea or its mutant sGFP (S65T).

  The present invention relates to a method for producing a genetically modified camellia tree in which a fluorescent protein is localized in the organelles of the camellia tree.

  The present invention relates to a method for producing a genetically modified camellia tree in which organelles are mitochondria and plastids.

  The present invention makes it possible to produce an efficient genetically modified camellia tree in a camellia tree, particularly a tea tree, which has been difficult with the prior art. In addition, the present invention enables efficient selection of genetically modified camellia trees, which is difficult with conventional techniques.

  Examples of camellia trees in the present invention include camellia, sasanqua, tea, and the like, and tea trees are particularly preferable.

  The embryo callus according to the present invention can be prepared from the tip of the leaf and the middle part produced by culturing the bud, unlike the callus generated from adult tissue pieces such as roots, stems, leaves and flowers. Embryonic callus of camellia trees generally accompanies autofluorescence.

  Genes used for the introduction of embryo callus in the present invention are, for example, genes used in general gene recombination techniques such as plasmids, binary vectors, drug resistance genes, genes involved in gene expression control, genes involved in selection markers, etc. And other genes related to cold resistance, antibacterial resistance, disease resistance, catechin content, etc. include plasmids, binary vectors, antibiotic resistance genes, promoter genes, polyadenine addition signal sequence genes, organelle targeting signals Useful genes such as sequences and fluorescent protein marker genes are preferred.

  In the present invention, the drug resistance gene includes an ampicillin resistance gene, a chloramphenicol resistance gene and the like, and a kanamycin resistance gene, a carbenicillin resistance gene and a hygromycin resistance gene are preferable.

  In the present invention, promoters involved in gene expression control include cauliflower mosaic virus 35S promoter, nopaline synthase gene promoter, rice actin promoter, Arabidopsis thaliana CAB2, Arabidopsis thaliana RD29A promoter, tobacco EF1α promoter, and polyadenine addition signal sequence. Include a polyadenine addition signal sequence of nopaline synthase gene, a polyadenine addition signal sequence of cauliflower mosaic virus, and the like, and a cauliflower mosaic virus 35S promoter and a polyadenine addition signal sequence of nopaline synthase gene are preferable.

  In the present invention, examples of the fluorescent protein marker gene include GFP, CFP, YFP, and RFP, and sGFP (S65T) is preferable.

  In the present invention, organelles in which fluorescent proteins are localized in cells include cell membrane, cell wall, nucleolus, smooth endoplasmic reticulum, rough endoplasmic reticulum, vacuole, peroxisome, ribosome, nucleus, Golgi vesicle, lysosome , Golgi, protein granule, endoplasmic reticulum, mitochondria or plastid, and the like, preferably mitochondrion or plastid. In order to localize the fluorescent protein to the organelle, for example, there is a method of adding an organelle targeting signal sequence to the fluorescent protein by a well-known gene recombination technique.

  The organelle targeting signal sequence used to localize fluorescent protein markers in the mitochondria or plastids in the present invention is not limited to this, but for mitochondria targeting, the N of F1 ATPase gamma-subunit of Arabidopsis thaliana A terminal 78 amino acid residue, ribulose 1,5 diphosphate carboxylase / oxygenase small-subunit transit peptide is preferred for plastid targeting.

  In the present invention, useful genes such as a promoter gene for expressing a fluorescent protein marker, a polyadenine addition signal sequence gene, an organelle targeting signal sequence, and a fluorescent protein marker gene are reconstructed using 35Ω-sGFP (S65T described in Examples). ) It is preferably carried out in a plasmid, but it may also be carried out with a suitable commercially available plasmid such as pBR322, pUC, pBluescript, etc.

  In the present invention, useful genes such as promoter gene, polyadenine addition signal sequence gene, fluorescent protein marker gene, etc. are commercially available EGFP (Clontech), cauliflower mosaic virus 35S promoter (Clontech), nopaline synthase gene polyadenine addition signal sequence (Clontech) May be used after being modified by PCR or the like.

  The binary vector used for introducing the useful gene reconstituted in the present invention into embryo callus has a gene (T-DNA) capable of infecting plant cells, an appropriate restriction enzyme site and a drug resistance gene Any binary vector may be used, such as pGA binary vector and pBI121 binary vector, and pIG121HM binary vector and pBCH1 binary vector are preferred.

  In the present invention, the method for introducing a gene into an embryonic callus cell of a binary vector is not limited to this, but is preferably an Agrobacterium method or a particle gun method, and the Agrobacterium method includes, for example, Agrobacterium EHA105 strain and embryo callus. By co-culturing, the particle gun method can be carried out by using, for example, a particle gun (PDS-1000 / He) manufactured by BIO-RAD.

  In the method for producing a genetically modified camellia tree of the present invention, it is preferable to perform selection by an antibiotic based on the introduced antibiotic resistance gene used in a general genetic recombination technique. Although not limited, kanamycin, carbenicillin and hygromycin are preferable, and it can be carried out by culturing antibiotic-resistant gene-transferred cells in a medium containing kanamycin, carbenicillin and hygromycin.

  In the present invention, the selection of genetically modified trees is possible with the presence of fluorescent protein markers in living cells and is equipped with a spectral imaging system capable of resolving fluorescence spectra, such as Spectral Unmixing from Applied Spectral Imaging. By using SPECTRAVIEW of Spectral Imaging Inc., etc., it can be confirmed separately from autofluorescence of camellia tea tree callus callus. In the method for producing the recombinant camellia tree of the present invention in which the fluorescent protein is localized in the organelle, the fluorescence is enhanced by confocal laser microscopy (localization of the fluorescent protein in the organelle) ( For example, it is possible to select a recombinant camellia tree by distinguishing it from the autofluorescence of the camellia tea tree callus callus by measuring using MRC1024) of Biorad.

  In the method for producing a genetically modified camellia tree of the present invention, differentiation induction by a plant hormone is preferably performed, and IBA (indole-3-butyric acid) BA (N6-benzyladenine) and GA (gibberellin) are used as plant hormones. Preferably, it can be carried out by culturing the gene-transferred cells in a medium containing IBA, BA, and GA as described in Examples below.

Construction of sGFP (S65T) binary vector for mitochondria and plastid localization Processing of DNA fragments and binding of DNA fragments, cloning of DNA fragments, confirmation of DNA sequence, treatment of Escherichia coli etc. (transformation, culture, extraction of plasmid) Etc.) were performed according to the method described in Molecular Cloning 3rd edition, Cold Spring Harbor Laboratory Press, etc. unless otherwise specified.

  Mitochondrial sGFP (S65T) was obtained by digesting Arabidopsis thaliana F1 ATPase gamma-subunit cDNA with the restriction enzyme HaeIII, and then containing a DNA fragment (MAMAFRFRREG RRLLPSIAAR PIAAIRSPLS SDQEEGLLGV RSITQKKVVRMRMKVVRM 35Ω-sGFP (S65T) plasmid (Chui et al.) Containing a cauliflower mosaic virus 35S promoter digested with SalI and NcoI, blunt-ended, sGFP (S65T) gene, and polyadenine addition signal sequence of nopaline synthase gene (Niwa et al.).

  From the obtained plasmid, a chimeric gene comprising an N-terminal 78 amino acid residue of F1 ATPase gamma-subunit of Arabidopsis thaliana, a fusion gene of sGFP (S65T) and a polyadenine addition signal sequence of nopaline synthase gene was digested with EcoRI and then blunt-ended. Further, XbaI digestion was performed to obtain a 50 ng DNA fragment. This was digested with the restriction enzymes XbaI and SmaI and 100 ng of the binary vector pBCH1 (Ito et al. This was obtained by excising the GUS gene of pIG121HM (Ohta et al.,) With XbaI, SacI, A ligation reaction was performed to bind a binary vector containing a linker DNA containing XhoI and KpnI recognition sequences, followed by transformation into Escherichia coli to prepare the target sGFP (S65T) binary vector for mitochondria.

  The plastid sGFP (S65T) is derived from a plasmid containing the Arabidopsis ribulose 1,5 diphosphate carboxyxylase / oxygenase small-subunit 1A gene, including a transit peptide region (MASSMLGSSAT MVASPAQAM VAPFNGLKSS AAFPATRKAN NDITSITSNG GRVNC sequence; The fragment was excised with BfaI, SphI, blunt-ended, introduced into 35Ω-sGFP (S65T) plasmid that had been blunt-ended after digestion with SalI, NcoI (Chui et al.).

  From the resulting plasmid, the cauliflower mosaic virus 35S promoter, the Arabidopsis ribulose 1,5-diphosphate carboxylase / oxygenase small-subunit 1A transit peptide and sGFP (S65T) fusion gene and the nopaline synthase gene polyadenine addition signal sequence The chimeric gene consisting of was digested with EcoRI, blunt-ended, and further digested with XbaI to obtain a 50 ng DNA fragment. This was combined with a 100 ng binary vector pBCH1 (Ito et al.) Digested with restriction enzymes XbaI and SmaI by a ligation reaction, transformed into Escherichia coli, and the target sGFP (S65T) binary vector for plastids was obtained. Created (FIG. 1).

  Purification of the sGFP (S65T) binary vector for mitochondria and plastids from E. coli was performed with the QIAfilter Plasmid Midi Kit (Qiagen).

  The binary vector pBCH1 has been transferred.

Introduction of binary vector into Agrobacterium The sGFP (S65T) binary vector for mitochondria and plastids was introduced into Agrobacterium EHA105 strain by electroporation. Agrobacterium suspension prepared for electroporation (Agrobacterium EHA105 strain was cultured at 30 ° C. in 100 mL of LB liquid medium until OD600 was 0.7, cooled on ice, and collected by centrifugation. Washed once with sterile water and twice with 10% glycerin solution and finally suspended in 0.5 mL of 10% glycerin solution and adjusted to 40 μL and sGFP for mitochondria and plastids prepared as described above (S65T ) After placing 10 ng of binary vector DNA in an electroporation cell, it was made under conditions of voltage; 1.8 kV, condenser; 25 μF, resistance; 200Ω under ice cooling. After electroporation, Agrobacterium EHA105 strain was selected by hygromycin resistance on LB medium containing 50 mg / L hygromycin. Hygromycin resistant Agrobacterium strain EHA105 was cultured in LB medium containing 50 mg / L of hygromycin for subsequent co-culture with tea tree callus.

Transformation of tea tree callus About 0.02 g of buds from Indian black tea seedlings were taken and cultured in 30 ml of MS medium supplemented with 4 mg / l of BA and 2 mg / l of IBA. Somatic embryos differentiate from the part. These somatic embryos (0.001 g) are cultured in the same medium to obtain embryo callus. This was used as a material (Kato, 1996).

  0.1 g of this embryo callus was immersed in 30 ml of MS liquid medium containing 0.001 g of hygromycin-resistant Agrobacterium for 5-10 minutes. Thereafter, the cells were transferred to a 9 cm petri dish containing MS medium supplemented with 4 mg / l BA and 2 mg / l IBA, and placed in dark conditions at 25 ° C. for 3 days for co-culture.

  For gene transfer by particle gun, 0.005 g of embryo callus is uniformly spread to about 2 cm in diameter on MS medium supplemented with BA 4 mg / l and IBA 2 mg / l, and 0.5 μg of sGFP (S65T) binary for mitochondrion and plastid Vector DNA was introduced at PDS-1000 / He, BIO-RAD (gold particles: 1 μm, rupture disk: 1100 psi, vacuum: 28 inches Hg, distance to embryo callus: 6 cm).

Protocol for selection of transformed tea tree callus In the Agrobacterium method, after co-cultivation, the cells were cultured for 8 weeks in 30 ml of MS medium supplemented with BA 4 mg / l, IBA 2 mg / l, kanamycin 25 μg / ml, carbenicillin 300 μg / ml, Embryos were grown in 30 ml of MS medium supplemented with 0.4 mg / l BA, 2 mg / l IBA, 25 μg / ml kanamycin, and 300 μg / ml carbenicillin to obtain buds. Alternatively, culture was also performed in a medium supplemented with 5 mg / l GA3, 3 mg / l BA, 0.5 mg / l IBA, 25 μg / ml kanamycin, and 300 μg / ml carbenicillin.

  In the particle gun method, 0.005 g of embryo callus after gene introduction is cultured at 25 ° C. for 2 weeks in light conditions, and then MS medium supplemented with BA 4 mg / l, IBA 2 mg / l, kanamycin 25 μg / ml, carbenicillin 300 μg / ml Transfer to 30 ml. The subsequent course is similar to Agrobacterium.

GFP monitoring GFP fluorescence was measured using a spectral imaging system SPECTRAVIEW (Spectral Imaging) with 0.05 g of embryonic callus cells and 0.005 g of transgenic embryos and 0.05 g of embryo callus cells and 0.005 g of nontransfected embryos as controls. Inc.), and only the fluorescence of the expressed GFP introduced by eliminating the autofluorescence of the tea tree by spectrally resolving individual fluorescence by spectral unmixing (Applied Spectral Imaging) was measured. In addition, observation of GFP fluorescence in mitochondria and plastids was similarly performed by using 0.05 g of embryo callus cells and 0.005 g of transgenic embryo cells and 0.05 g of embryo callus cells and 0.005 g of leaves not transfected as a control. The sample was placed on a confocal laser microscope (excitation light: 488 nm Kr / Ar laser light, fluorescence: 506-538 nm bandpass filter, Bio-Rad MRC1024) and observed.

PCR and RT-PCR
Genomic DNA and total RNA were extracted from cells and leaves of transformed embryo callus. mRNA was obtained using Oligotex ™ -dT30Super mRNA Purification Kit (Takara, Japan). Insertion and expression of sGFP (S65T) cDNA into the genome was confirmed by PCR and RT-PCR. Oligonucleotide primer GFP1 chemically synthesized from the sequence of sGFP (S65T) (SEQ ID NO: 1); 5′AAGCTGACCCTGAAGTTCATC3 ′ (130th to 150th sequence of SEQ ID NO: 1, SEQ ID NO: 2) and GFP2; 5′TTTACTTGTTACAGCTCGTCCA3 '(The 707th to 727th complementary strand of the sequence of SEQ ID NO: 1, SEQ ID NO: 3) was used for PCR and RT-PCR. PCR was performed in 35 cycles of 1 μg genomic DNA and 5.0 μM primer, 1 cycle 94 ° C., 30 seconds denaturation, 60 ° C., 1 minute annealing, 70 ° C., 1 minute extension reaction. The mRNA of sGFP (S65T) cDNA was 2 μg of mRNA, 5.0 μM primer, mix reaction Qiagen onestep RT-PCR (Qiagen), reverse transcription reaction (50 ° C., 30 minutes), first PCR activation step ( 95 ° C., 15 minutes), 94 cycles, 1 minute denaturation, 50 ° C., 1 minute annealing, 70 ° C., 1 minute extension reaction. The amplified DNA was confirmed by agarose electrophoresis and stained with ethidium bromide.

Results FIG. 2 shows fluorescence images of non-recombinant and mitochondrial localized GFP recombinant tea leaves. A strong fluorescent signal (shown in black) can be seen in the central part of the leaf (leaf vein). This fluorescent signal appears to be stronger in the recombinant tea leaves at first glance, but it is simply due to the difference in the thickness of the leaf veins. There is no difference.

  Table 1 shows the results of recombination of tea tree embryos performed by the Agrobacterium method (A) and the particle gun method (B). Embryos transformed with GFP for mitochondrial localization by the Agrobacterium method and particle gun method show efficient secondary embryo formation, and localization of sGFP (S65T) to the mitochondria It was shown that the formation was not affected. It was also shown that localized sGFP (S65T) on the plastid has no effect in the particle gun method.

  Table 2 shows the growth of embryos on different media. When cultured at BA 0.4 mg / l and IBA 2 mg / l, a small amount of germination was observed. When cultured at 5 mg / l GA3, 3 mg / l BA, 0.5 mg / l IBA, germination was observed at higher levels, indicating differentiation induction from transformed tea tree callus calligraphy.

  FIG. 3 shows a non-transformed tea tree embryo callus (A) using a spectral imaging system (SPECTRAVIEW (Spectral Imaging Inc.), Spectral Unmixing (Applied Spectral Imaging)), and a trait by Agrobacterium method or particle gun method. It is the photograph of germination (C) from a conversion tea tree callus (B) and a transformation tea tree callus. The green fluorescence of sGFP (S65T) is observed only in the transformant without being lost to autofluorescence. Furthermore, fluorescence confined to mitochondria was observed by a confocal laser scanning microscope.

  In addition, PCR and RT-PCR were performed in order to confirm the integration of sGFP (S65T) into the tea tree embryo genome and the expression in the recombinant tea leaves (598 bp size fragment). Amplification in recombinant tea tree embryos by particle gun method was weak (Fig. 4, lanes 3, 4 and 5), but amplification in recombinant tea tree embryos by Agrobacterium method was very strong and integrated into the genome Was confirmed. Furthermore, expression in the recombinant tea leaves was confirmed by rt-PCR (FIG. 5, lane 3).

  FIG. 6 is a photograph of a bud regenerated from a recombinant tea tree embryo produced according to the present invention.

  According to the present invention, a genetically modified camellia tree can be efficiently produced. Moreover, according to the present invention, genetically modified camellia trees can be selected efficiently. As a result, the present invention is used in the case of breeding for the purpose of imparting useful properties such as cold resistance, antibacterial activity, disease resistance, catechin content to genetically modified camellia trees, preferably tea trees. This makes it possible to efficiently develop new varieties of camellia trees.

References・ Y. Niwa, T. Hirano, K. Yoshimoto, M. Shimizu, and H. Kobayashi: Non-invasive quantitative detection and applications of nontoxic-, S65T-type green fluorescent protein in living plants.Plant J. (1999 ) 18: 455-463.
・ Yukihiro Ito, Mitsugu Eiguchi, Nori Kurata. (2001) KNOX homeobox genes are sufficient in maintaining cultured cells in an undifferentiated state in rice.Genesis 30: 231-238
・ Ohta S, Mita S, Hattori T, Nakamura K. (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence.Plant Cell Physiol. 31: 805-813.
・ WI. Chiu, Y. Niwa, W. Zeng, T. Hirano, H. Kobayashi, and J. Sheen: Engineered GFP as a vital reporter in plants.Current Biology (1996) 6, 325-330.
・ M. Kato, (1996) Somatic embryogenesis from immature leaves of in vitro grown tea shoots.Plant Cell Rep 15: 920-923.

T-DNA region diagram of sGFP (S65T) expression vector for mitochondria and plastids. Fluorescent image of non-recombinant and mitochondrial localized GFP recombinant tea leaves. Left: non-recombinant tea leaves, right: mitochondrial localized GFP recombinant tea leaves. Fluorescence image capture is by FluorImager (Amersham Pharmacia Biotech). Uptake conditions: excitation light: 488 nm, detection condition: 530 DF30 (515-545 nm) filter used, pixel size: 200 microns, digital resolution: 8 bits, detection sensitivity: normal, PMT: 800V. Photographs of germination from untransformed tea tree callus (A) and transformed tea tree callus (B) and transformed tea tree callus by spectral imaging system SPECTRAVIEW (Spectral Imaging Inc.). Bar indicates 10 μm. PCR of genomes of recombinant tea tree by particle gun and recombinant tea tree by Agrobacterium infection. Lane 1: molecular weight markers (TaKaRa, λHindIII), lane 2: (plasmid), lane 3: (particle gun recombinant embryo), lane 4: (particle gun recombinant embryo), lane 5: (particle gun recombinant embryo) Lane 6: (Agrobacterium recombinant embryo), Lane 7: (Agrobacterium recombinant embryo), Lane 8: (Agrobacterium recombinant embryo). The target 598 bp size band is shown. RT-PCR of recombinant tea leaves. Lane 1: molecular weight markers (TaKaRa, λHindIII), lane 2: GFP plasmid, lane 3: recombinant tea leaf extracted RNA RT-PCR, lane 4: recombinant tea leaf extracted RNA PCR. Photograph of buds regenerated from recombinant tea tree embryos.

Claims (12)

  A method for producing a genetically modified camellia tree, characterized by introducing a gene into an embryo callus of a camellia tree.   The production method according to claim 1, wherein the camellia tree is a tea tree.   The production method according to claim 1 or 2, wherein the embryo callus is an embryo callus that is generated from a tip of a leaf generated by culturing of a bud and a middle assistant part.   The production method according to claim 1, 2 or 3, wherein the gene contains a gene encoding a fluorescent protein.   The production method according to claim 4, wherein the fluorescent protein is a green fluorescent protein GFP derived from Aequorea or its mutant sGFP (S65T).   The production method according to claim 5, wherein the fluorescent protein is localized in the organelle of a camellia tree.   The preparation method according to claim 6, wherein the organelles are mitochondria and plastids.   A genetically modified camellia tree produced by the method according to any one of claims 1 to 7.   A genetically modified camellia tree introduced with a gene encoding a fluorescent protein.   The camellia tree according to claim 9, wherein the camellia tree is a tea tree.   The camellia tree according to claim 9 or 10, wherein the fluorescent protein is localized in an organelle.   The camellia tree according to claim 11, wherein the organelles are mitochondria and plastids.