WO2012015039A1 - アグロバクテリウム菌を用いた、オオムギ属植物へ遺伝子導入を行う方法およびオオムギ属植物の形質転換植物の作成方法 - Google Patents
アグロバクテリウム菌を用いた、オオムギ属植物へ遺伝子導入を行う方法およびオオムギ属植物の形質転換植物の作成方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- the present invention relates to a method for introducing a gene into a barley plant through an Agrobacterium.
- the present invention further relates to a method for producing a transformed plant of a barley genus plant via an Agrobacterium.
- direct DNA introduction method For transformation of monocotyledonous plants such as barley, wheat, corn and rice, which are the main cereals, physicochemical methods (direct DNA introduction method) such as polyethylene glycol method, electroporation method, particle gun method and agro
- direct DNA introduction method an event that a target gene is introduced in a fragmented manner or introduced in multiple copies frequently occurs. Therefore, a transformant that does not express the target gene or shows weak and abnormal expression (gene silencing) appears frequently.
- the method using protoplasts since the culture period is prolonged, seed sterility or malformation due to culture mutation is likely to occur in the obtained transformant.
- the gene transfer method via Agrobacterium spp. Is introduced by fragmentation introduction of the target gene, keeping the copy number of the target gene low by controlling the expression of genes in the pathogenic region (vir region) of the Ti plasmid. There is little to be done. Therefore, many individuals can be obtained in which the target gene is highly expressed in the obtained transformant, and there is a great advantage that the difference in expression level between individuals is small compared to the direct introduction method.
- the gene transfer method via Agrobacterium is commonly used as a method for transforming dicotyledonous plants.
- the host of the genus Agrobacterium in nature is limited to dicotyledonous plants, and for a long time it has been thought not to infest monocotyledonous plants (Potrykus 2000: Non-patent Document 1).
- Patent Document 2 a high-efficiency transformation method via Agrobacterium was reported for the first time in the main crop of rice.
- Non-Patent Document 5 (1997, Non-Patent Document 5) injures immature embryos with a particle gun before inoculating Agrobacterium.
- Trifonova et al. 2001 Non-Patent Document 12 showed that the treatment of immature barley embryos before inoculation with Agrobacterium by particle gun does not improve the transformation efficiency, almost no injury Processing is not used.
- Callus induction medium includes Murashige & Skoog (MS) inorganic salts (Murashige & Skoog 1962 Non-Patent Document 13), 30 g / l maltose, 1.0 g / l casein hydrolyzate, 350 mg / l myo-inositol, 690 mg / l 1.0 mg / l thiamine hydrochloride, 2.5 mg / l 3,6-dichloro-2-methoxybenzoic acid (dicamba), 1.25 mg / l CuSO 4 5H 2 O (Bartlett et al. 2008 Non-Patent Document 9 and Harwood et al.
- Non-Patent Document 11 3.5 g / l phytagel, pH 5.8 is commonly used (Jacobsen et al. 2006 Non-Patent Document 8, Bartlett et al. 2008 Patent Document 9, Hensel et al. 2008 Non-Patent Document 10, Harwood et al. 2008 Non-Patent Document 11).
- the Agrobacterium suspension used as the inoculum is obtained by shaking culture overnight in a liquid medium.
- the method of inoculating Agrobacterium is a method in which an Agrobacterium suspension is dropped onto a scutellum of an immature embryo (Jacobsen et al. 2006 Non-Patent Document 8, Bartlett et al.
- Non-Patent Document 9 Harwood et al. 2008 Non-Patent Document 11
- a method in which an immature embryo is immersed in an Agrobacterium suspension and then decompressed using a vacuum pump Hensel et al. 2008 Non-Patent Document 10
- the time of inoculation that is, the time from contact of the Agrobacterium suspension with immature embryos until transfer to the coculture medium.
- the inoculation method of either the dropping method or the immersion pressure reducing method takes about 2 hours at the longest from 20 minutes. Inoculation of Agrobacterium to immature embryos is performed on the day of isolation of immature embryos or on the next day after overnight culture.
- dicamba 2.5 mg / l (11.3 ⁇ M) dicamba is used as a plant growth regulator in a solid medium for co-culturing with Agrobacterium (Jacobsen et al. 2006, Non-patent Document 8, Bartlett et al. 2008 Non-patent document 9, “Harwood et al.” 2008 Non-patent document 11).
- a dicamba at a concentration of 2.5 mg / l (11.3 ⁇ M) has been conventionally used to dedifferentiate immature embryo scutellum cells and induce callus with redifferentiation ability (Wan and Lemaux 1994) Non-patent document 14). Co-culture is performed for 2-3 days.
- Non-Patent Document 11 It is subcultured at intervals of about 2 weeks in the same medium until a callus (transformed cell mass) showing clear resistance to the selected drug is obtained. Approximately 4-6 weeks later, the selected drug-resistant callus is transplanted to a transition medium, pre-regeneration medium containing the selected drug, or a regeneration medium containing the selected drug (shoot induction medium). The callus cultured in the pre-redifferentiation culture medium is then transplanted to the regeneration medium containing the selective drug. Further, the regenerated shoots and seedlings are transplanted to a rooting medium containing a selective drug and not containing a plant growth regulator, thereby obtaining a barley transformed plant.
- Transformation efficiency per immature embryo The transformation efficiency per immature embryo that has been reported so far is as follows. For the cultivar Golden Promise, 7% (Tingey et al. 1997 Non-Patent Document 5), 12% (Matthews et al. 2001 Non-Patent Document 15), 9.2% (Murray et al. 2004 Non-Patent Document 16), 36 % (Bartlett et al. 2008 Non-Patent Document 9), 86.7% (Hensel et al. 2008 Non-Patent Document 10). In Non-Patent Documents 9 and 10, although high efficiency is obtained in some cases, such high efficiency is not stably obtained. It is 2% (Hensel et al. 2008 Non-Patent Document 10) for the variety Tafeno, and 2% (Hensel et al. 2008 Non-Patent Document 10) for the variety Helium.
- Non-patent Document 17 Prior art Ke et al. 2002 (Non-patent Document 17) on gene transfer into immature barley embryos by Agrobacterium is to analyze the expression of ⁇ -glucuronidase (GUS) reporter gene after co-culture with Agrobacterium Then, the transfer efficiency of T-DNA to immature embryo cells due to the difference in medium composition for co-culture was evaluated. Using an immature embryo immediately after isolation, inoculating Agrobacterium for 30 minutes and using a co-culture medium in which the basic inorganic salt concentration of the MS medium is equal (x1) or 1/10 (x0.1). Co-cultivation was performed for days.
- GUS ⁇ -glucuronidase
- 0.25 mg / l (1.1 ⁇ M) 2,4-D is low in concentration to promote dedifferentiation and is not commonly used for callus induction from the scutellum (Serhantova et al. 2004 Non-Patent Document 18) .
- X-Gluc 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid
- Non-Patent Document 17 In order to investigate the impact of specific factors on the efficiency of T-DNA introduction into immature embryos, extreme conditions were used in this experiment. Continuing the culture at a 1 / 10-fold concentration condition will have a detrimental effect on the plant, so that a sufficient number of T-DNA transduction events will occur for stable transformation.
- Non-Patent Document 17 in all tests using a medium having a concentration of 1/10 times the concentration of MS inorganic salts, the immature embryo was isolated and the coculture was completed at least 72, regardless of the presence or absence of preculture. A culture period of time (3 days) is applied.
- Patent Document 19 a conventional technique for gene transfer into immature rice corn embryos by Agrobacterium , applied heat treatment to immature embryos of rice and corn before inoculation with Agrobacterium (Patent Documents). Reported that 1) and centrifugation (Patent Document 2) or heat and centrifugation (Patent Document 3) improve the efficiency of gene transfer to immature embryonic scutella, resulting in improved transformation efficiency did. It has also been reported that by using these treatments, transformants could be obtained from varieties that could not be transformed so far.
- Patent Document 4 by applying pressure treatment (Patent Document 4) to immature embryos before inoculation with Agrobacterium, gene transfer efficiency into scutellum cells is improved in the same manner as centrifugation, resulting in improved transformation efficiency. It has been reported to improve. Heat, centrifugation, heat and centrifugation, and pressure treatment are all used to increase the efficiency of gene transfer into immature embryonic scutellum.
- the present invention relates to a method for gene transfer into a plant of the genus Barde (Hordeum), which makes it possible to perform transformation with higher efficiency than the conventionally known Agrobacterium method, and a method for transforming a plant of the genus Barley
- the purpose is to provide a creation method.
- the present inventors have a) an anti-auxin, b) a cytokinin, c) a phenoxy auxin at a concentration of less than 2 ⁇ M, and / or a benzoic system.
- Gene transfer efficiency can be achieved by co-culturing in a co-culture medium that satisfies one or more of the following conditions: auxin is contained at a concentration of less than 5 ⁇ M, or phenoxy auxin and / or benzoic auxin is not contained. I found it to improve. However, when this co-culture medium was used, callus formation after co-culture was suppressed, and a transformed plant could not actually be obtained.
- the inventors have performed centrifugation and / or pressure treatment of immature embryo tissues of barley plants before inoculation with Agrobacterium, and / or In addition, the effect of centrifugation and / or pressure treatment during or after the coexisting step of culturing in the coexisting medium was examined. It was found that callus formation efficiency from embryonic tissue can be improved. As a result, according to the present invention, it has become possible to efficiently transform a barley plant. In addition, the centrifugation process and / or pressurization process to an immature embryo tissue can also be implemented either before inoculating Agrobacterium or after the coexistence step.
- a method for producing a transformed plant of a plant of the genus Hordeum (I) inoculating Agrobacterium into immature embryo tissues of barley, and in the presence of the Agrobacterium, one or more of the following conditions a) to c): a) containing anti-auxin; b) including cytokinin, c) contains phenoxy auxin at a concentration of less than 2 ⁇ M and / or contains benzoic auxin at a concentration of less than 5 ⁇ M or does not contain phenoxy auxin and / or benzoic auxin; In the coexisting medium that satisfies the above, perform the coexistence step of culturing the above tissue, (Ii) before inoculation with Agrobacterium, during the coexistence step and / or following the coexistence step, performing a step of centrifugation and / or pressure treatment of the tissue, (Iii) performing a resting step of culturing the tissue in
- Aspect 12 The method according to any one of aspects 1 to 11, wherein the Agrobacterium is a bacterium selected from the group consisting of LBA4404, EHA101, EHA105, AGL0, AGL1, and 58C1.
- Aspect 13 The method according to any one of embodiments 1 to 12, wherein the plant of the genus Barley is barley (H. vulgare).
- the present invention makes it possible to transform a barley genus plant with high efficiency. This makes it possible to stably obtain a transformed plant body with good reproducibility, and to reduce the cost for obtaining the plant body.
- FIG. 1 is a graph showing the effect of addition of anti-auxins and dicamba to a co-culture medium on gene transfer efficiency into immature barley embryos. Fifteen immature embryos were tested in each group.
- the columns in FIG. 1 are the system from which the plant hormone is not added, the system in which 5 ⁇ M of anti-auxin TIBA is added, the system in which 1.13 ⁇ M of dichroic auxin is added, and the system in which 11.3 ⁇ M of dicamba is added. The results are shown.
- shaft of FIG. 1 shows a GUS expression index.
- Expression of the GUS gene in the scutellum region is 87.5 (expressed in 75% or more of the scutellum), 62.5 (expressed in 50% or more and less than 75% of the scutellum), 37.5 ( Expressed in 25% or more and less than 50% of the scutellum), 17.5 (expressed in 10% or more and less than 25% of the scutellum), 6.5 (expressed in 1% or more and less than 10% of the scutellum), 0.5 (Expressed in less than 1% exceeding 0% of the scutellum) and 0 (no expression) were evaluated, and the average value was used as the GUS expression index.
- FIG. 2 is a graph showing the effect of addition of cytokinins and antiauxins to the co-culture medium on the efficiency of gene transfer into immature barley embryos. Fifteen immature embryos were tested in each group.
- the columns in FIG. 2 are each a system without addition of plant hormones, a system with 5 ⁇ M of 6BA, a system with 5 ⁇ M of 4-PU, a system with 5 ⁇ M of zeatin, a system with 5 ⁇ M of TIBA, and 5 ⁇ M of paclobutrazol. Each added system is shown.
- 6BA, 4-PU and Zeatin are cytokinins
- TIBA and paclobutrazol are antiauxins.
- FIG. 2 shows a GUS expression index.
- the GUS expression index in FIG. 2 was determined in the same manner as described in FIG.
- FIG. 3 is a graph showing the effect of the addition of 2,4-D to the co-culture medium on the efficiency of gene transfer into barley immature embryos. Fifteen immature embryos were tested in each group.
- the columns in FIG. 3 show the results of the system without addition of plant hormones, the system with addition of 1.13 ⁇ M of 2,4-D phenoxy auxin, and the system with addition of 11.3 ⁇ M of 2,4-D, respectively, from the left. Show.
- shaft of FIG. 3 shows a GUS expression index.
- the GUS expression index in FIG. 3 was determined in the same manner as described in FIG.
- the present invention is a method for introducing a gene into an immature embryo tissue of a plant of the genus Hordeum, (I) inoculating the tissue with Agrobacterium, and in the presence of the Agrobacterium, one or more of the following conditions a) to c): a) containing an antiauxin, b) including cytokinin, c) contains phenoxy auxin at a concentration of less than 2 ⁇ M and / or contains benzoic auxin at a concentration of less than 5 ⁇ M or does not contain phenoxy auxin and / or benzoic auxin; Performing a coexistence step of culturing the tissue in a co-culture medium satisfying (Ii) before inoculation with Agrobacterium, during the coexistence step and / or subsequent to the coexistence step, performing a step of centrifuging and / or pressurizing the tissue.
- a gene transfer method is a method for introducing a gene into an immature embryo tissue of a plant of the
- the present invention is a method for producing a transformed plant of a plant of the genus Barde (Hordeum), (I) inoculating Agrobacterium into immature embryo tissues of barley, and in the presence of the Agrobacterium, one or more of the following conditions a) to c): a) containing anti-auxin; b) including cytokinin, c) contains phenoxy auxin at a concentration of less than 2 ⁇ M and / or contains benzoic auxin at a concentration of less than 5 ⁇ M or does not contain phenoxy auxin and / or benzoic auxin; In the coexisting medium that satisfies the above, perform the coexistence step of culturing the above tissue, (Ii) before inoculation with Agrobacterium, during the coexistence step and / or following the coexistence step, performing a step of centrifugation and / or pressure treatment of the tissue, (Iii) performing a resting step of culturing the tissue in
- the plant from which the plant tissue usable in the present invention is derived is a plant of the genus Barley.
- a plant of the ⁇ barley genus '' in this specification it is not limited, but H. arizonicum, H. bogdanii, H. H. chilense, H. comosum, H. cordobense, H. depressum, H. erectifolium, H. euclaston, H. flexuosum, H. fuegianum, H. guatemalense, H. gussoneanum, H. intercedens, H. jubatum , H. lechleri, H. marinum, H. murinum, H.
- H. secalinum H. stenostachys
- H. tetraploidum H. vulgare (barley).
- barley H. vulgare
- the plant tissue that can be used in the present invention is an immature embryo.
- immature embryo refers to an embryo of an immature seed that is in the ripening process after pollination.
- the stage (ripening stage) of the immature embryo to be used in the method of the present invention is not particularly limited, and it may be collected at any time after pollination, but it is 7 to 21 days after pollination. preferable. Although immature embryos can be used on the day of removal, they may be pre-cultured immature embryos.
- ripe seed refers to a seed that has been matured as a seed after the ripening process after pollination has been completed.
- the gene introduction method and the preparation method of a transformed plant of this invention utilize Agrobacterium bacteria. Except for the particularly specified steps, it can be carried out according to each step of a gene transfer method and a transformation method using known Agrobacterium bacteria.
- Coexistence step In the present invention, a coexistence step is performed in which an immature embryo tissue inoculated with Agrobacterium is cultured in the presence of the Agrobacterium. This step is a step of ensuring the introduction of DNA from Agrobacterium into plant cells by culturing the plant tissue inoculated with Agrobacterium in the presence of Agrobacterium.
- usable plant tissues are used after being isolated and collected from a plant of the genus Barley. Therefore, in the present invention, a tissue (immature embryo) is first isolated and collected from a plant body of the genus Barley, and the isolated and collected tissue is inoculated with Agrobacterium. Alternatively, the isolated and collected tissue may be pre-cultured, and the cultured tissue may be inoculated with Agrobacterium. Preferably, the tissue on the day of collection or the day after collection is inoculated with Agrobacterium. When the tissue is precultured, the period will be described separately.
- the size of the immature barley embryo used in the present invention is not particularly limited, but those having a size of 1.5 to 2.5 mm can be preferably used.
- the immature embryo as described above may be subjected to heat treatment (Patent Document 1) for increasing the transformation efficiency.
- Heat treatment is performed before inoculation with Agrobacterium. The heat treatment for increasing the transformation efficiency will be described in detail later.
- Agrobacterium is inoculated into the tissue of a barley genus plant.
- “inoculation” refers to bringing Agrobacterium into contact with a plant tissue (eg, scutellum), and various Agrobacterium inoculation methods are known in the art. Examples of the method include a method of adding a plant tissue to a suspension in which Agrobacterium is suspended in a liquid medium, a method of directly dropping a suspension of Agrobacterium on a plant tissue on a co-culture medium, Examples thereof include a method of injecting an Agrobacterium suspension into the tissue, a method of immersing the plant tissue in the Agrobacterium suspension, and reducing the pressure. However, the method of inoculating Agrobacterium in the present invention is not limited to these methods.
- various additives such as acetosyringone, surfactant, porous ceramics, etc. are added to the Agrobacterium to improve the transformation efficiency by Agrobacterium. It can be included in the suspension.
- the Agrobacterium that can be used in the present invention is not particularly limited and may be any known Agrobacterium that can be used for the Agrobacterium transformation method.
- examples of Agrobacterium include, but are not limited to, LBA4404, EHA101, EHA105, AGL0, AGL1, or C58C1.
- a super binary vector Non-patent Documents 2 and 3
- a strain having a pathogenic region of Ti plasmid pTiBo542 possessed by Agrobacterium A281 (Non-patent Document 20) is used. It is preferable to use it.
- Agrobacterium has a property of introducing a gene inserted into T-DNA of a plasmid in Agrobacterium into the plant genome. Therefore, Agrobacterium that can be used in the present invention has a plasmid in which a gene intended to be expressed in a plant is inserted into T-DNA.
- a plant can be transformed by inoculating plant tissues with Agrobacterium having the plasmid. Thereby, the preferable character is provided to the plant cell in the tissue.
- Examples of plasmids for Agrobacterium that can be used in the present invention include, but are not limited to, pSB131, pSB134, pNB131, and pIG121Hm.
- the medium used in this step is referred to as “coexisting medium” in this specification.
- the coexisting medium may be one usually used for culturing plant cells, and examples thereof include a medium based on LS inorganic salts (Non-patent Document 21) and N6 inorganic salts (Non-patent Document 22).
- a medium in which the concentration of inorganic salts and the like is lower than that usually used for example, a 1 ⁇ concentration MS medium
- the medium is 1/2 times or less, more preferably 1/5 times or less, and a medium that is 1/10 times or less, for example, 1/10 concentration MS medium (Non-patent Document 17) can be most suitably used.
- the present inventors have conducted intensive research, and as shown in Examples 1 and 2 below, by adding antiauxins and / or cytokinins in the coexisting medium, or by adding phenoxy-type auxin in the coexisting medium and It has been found that gene transfer efficiency is remarkably improved by lowering the benzoic auxin concentration or not adding phenoxy auxin and / or benzoic auxin to the co-culture medium, or by a combination of these conditions. This is one of the most prominent features of the present invention.
- Anti-auxin is a substance having an action of antagonistically inhibiting the action of a compound having an auxin action.
- the antiauxin is not limited, but 2,3,5-triiodobenzoic acid (TIBA), paclobutrazol, 2,4,6-trichlorophenoxyacetic acid (2,4 , 6-T), p-chlorophenoxyisobutyric acid (PCIB), maleic hydrazide, uniconazole P and the like can be preferably added to the co-culture medium.
- TIBA 2,3,5-triiodobenzoic acid
- PCIB p-chlorophenoxyisobutyric acid
- maleic hydrazide uniconazole P and the like
- the cytokinins are not limited, but include 6-benzylaminopurine (6BA), kinetin, N-phenyl-N ′-(4-pyridylurea) (4-PU), zeatin, Thidiazuron and ⁇ -dimethylallylaminopterin (2-ip) can be preferably added to the co-culture medium.
- the concentrations of antiauxin and cytokinin added to the co-culture medium are each preferably 0.1 to 20 ⁇ M, more preferably 0.5 to 10 ⁇ M, and most preferably 2 to 7 ⁇ M.
- Non-patent Document 17 showed that when a 1/10 concentration MS medium without auxin was used as a barley coexisting medium, high gene transfer efficiency was obtained. However, Non-Patent Document 17 does not indicate that a transformed plant is actually obtained. Non-Patent Document 17 also shows that the gene transfer efficiency is lower when auxin is not added to the MS medium at the same concentration than when low concentration 2,4-D is added. Such disclosure in Non-Patent Document 17 suggests that it is disadvantageous in terms of gene transfer efficiency that no auxin is added, and a small amount of 2,4-D is necessary.
- the concentration of the phenoxy auxin in the co-culture medium is 2 ⁇ M or less, preferably 1 ⁇ M or less, more preferably 0.5 ⁇ M or less, more preferably 0.3 ⁇ M or less.
- concentration of the benzoic auxin is 5 ⁇ M or less, preferably 4 ⁇ M or less, more preferably 3 ⁇ M or less, further preferably 2 ⁇ M or less, and most preferably no additive.
- these concentrations of phenoxy auxin and benzoic auxin may be included.
- the phenoxy auxin is not limited, but 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5- T) and the like, and 3,6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), etc. are suitable as benzoic auxins.
- dicamba 3,6-dichloro-2-methoxybenzoic acid
- picloram 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid
- other auxins such as indole-3-acetic acid (IAA) and ⁇ -naphthalene acetic acid (NAA) may be added to the co-culture medium.
- the present inventors actually did not add the plant growth regulator described in Non-patent Document 17, or added 1/10 concentration of 2,4-D at a very low concentration of 0.25 mg / l (1.13 ⁇ M).
- the MS medium was used as a barley co-culture medium, and transformation experiments were conducted. However, even when a sufficient amount of auxin was added in the resting and selection processes, callus formation from immature embryos after co-culture was extremely severe. The callus was hardly obtained.
- Non-Patent Document 17 investigates how various conditions in co-cultivation affect gene transfer efficiency during co-cultivation, and the conditions used in the experiment are directly applied to transformation. It is not supposed to be possible. Actually, in Non-Patent Document 17, “In order to investigate the impact of specific factors on the efficiency of T-DNA introduction into immature embryos, extreme conditions were used in this experiment .... MS Continuing the cultivation at a 1 / 10-fold concentration of inorganic salts will have a detrimental effect on the plant, and therefore a sufficient number of T-DNA transduction events for stable transformation.
- the present inventors considered that callus formation could be performed while maintaining high gene transfer efficiency even if conditions for suppressing dedifferentiation of blastocyst cells were given to the co-culture medium. Repeated.
- the scutellum of immature barley embryos co-cultured in a co-culture medium containing no auxin or containing a low concentration of auxin before the Agrobacterium inoculation, during and / or after the coexistence process
- There is a remarkable effect of improving the callus induction rate by centrifugation treatment to immature embryos, before inoculation of Agrobacterium, during coexistence process and / or pressurization treatment to immature embryos after coexistence process, or combination of these treatments I found out.
- Non-Patent Documents 25 and 26 examples of such additives include silver nitrate (Non-Patent Documents 25 and 26) and cysteine (Non-Patent Document 23).
- “Cultivation” in this step refers to placing a plant tissue on a solidified co-culture medium or in a liquid co-culture medium and growing it at an appropriate temperature, light / dark conditions and period.
- the form of the medium is not particularly limited as long as the medium components are sufficiently supplied to the plant tissue.
- Solidification of the co-culture medium can be carried out by adding a solidifying agent known in the art, and as such a solidifying agent, for example, agarose or the like is known. In the present invention, such a solidified co-culture medium can be suitably used.
- the culture temperature in this step can be appropriately selected, and is preferably 18 ° C-30 ° C, more preferably 25 ° C.
- the culture in this step is preferably performed in a dark place, but is not limited thereto.
- the culture period of this step can also be selected as appropriate.
- the coexistence process is generally performed for 2 to 3 days, whereas the present inventors coexist for 2 to 3 days under the conditions of the present invention for suppressing dedifferentiation and callus formation.
- the culture period in the coexistence step of the present invention is preferably 6 hours to 36 hours, more preferably 12 hours to 24 hours.
- the present inventors proceeded with research and performed pre-culture before inoculation with Agrobacterium, culturing under conditions where phenoxy auxin and / or benzoic auxin was reduced or removed, the same as in the case of the coexistence step described above. Furthermore, the present inventors have found that callus formation from barley immature embryos after the coexistence process is inhibited. Therefore, in the present invention, regardless of whether it is precultured or cocultured, the period during which the phenoxy auxin and / or benzoic auxin is reduced or removed from the isolation of the immature embryo to the end of the coculture. Is preferably 6 hours to 36 hours, more preferably 12 hours to 24 hours.
- the co-culture method in the present invention promotes gene transfer to the scutellum side of immature embryos.
- a method of placing and culturing immature embryos so that the scutellum side faces upward and the hypocotyl side is in contact with the medium can be preferably used.
- the immature embryo is generally placed so that the scutellum side is in contact with the medium downward, and the method is greatly different from the present invention.
- centrifugal treatment and / or pressurization treatment are carried out by carrying out centrifugal treatment of barley immature embryos before, during and / or following the coexistence of Agrobacterium. It was found that even barley immature embryos co-cultured under the suppressive condition have a remarkable effect that callus can be sufficiently induced.
- the conditions for centrifugation in this case may be the same as those described in WO2002 / 012520 (Patent Document 2). Specifically, it is performed in a centrifugal acceleration range of about 100G to 250,000G, 500G to 200,000G, preferably 1000G to 150,000G, and most preferably about 1100G to 110,000G.
- the time for the centrifugal treatment is appropriately selected according to the centrifugal acceleration, but it is usually preferable to carry out the treatment for 1 second or longer. There is no particular upper limit on the centrifugation time, but the object can usually be achieved in about 10 minutes. In addition, when the centrifugal acceleration is large, the gene transfer efficiency can be significantly improved even when the centrifugal acceleration is very short, for example, 1 second or less. On the other hand, when the centrifugal acceleration is small, it is preferable to perform the centrifugation process for a long time. Appropriate centrifugation conditions can be easily set through routine experiments. Such centrifugation may be performed either before or after excision of the hypocotyl described later.
- the centrifugation treatment may be performed before or after the co-culture, or may be taken out during the co-culture and centrifuged, but is preferably performed before and / or after the co-culture.
- the point which performs the centrifugation process of plant material is the most remarkable characteristic in this invention.
- Example 3 in a system cultured in a co-culture medium not added with a plant growth regulator, centrifugation to barley immature embryos before inoculation with Agrobacterium and / or immature embryos after the coexistence step It was found that the centrifugal treatment also has a remarkable effect of improving the callus induction rate from barley immature embryos.
- Example 4 it was found that even in a system cultured in a co-culture medium supplemented with anti-auxin, good callus formation was observed by centrifugation.
- the pressure treatment of immature embryos before inoculation with Agrobacterium and / or the pressure treatment of immature embryos after the coexistence process also increases the callus induction rate from barley immature embryos, similar to the above-mentioned centrifugation. There is a remarkable effect to improve.
- the pressure treatment can be performed using, for example, a method described in WO2005 / 018169 (Patent Document 4).
- the pressure treatment is not limited, but is preferably performed in the range of 1.7 to 10 atm, more preferably in the range of 2.4 to 8 atm.
- the pressure treatment time can be appropriately selected according to the magnitude of the pressure, but is preferably 0.1 second or more and 4 hours, and more preferably 1 second to 30 minutes. Further, the pressure treatment may be performed either before or after excision of the hypocotyl.
- the pressure treatment may be performed before or after the co-culture, and may be performed during the co-culture, but is preferably performed before and / or after the co-culture.
- the point which pressurizes a plant material is also the most remarkable characteristic in this invention.
- the combination of the above centrifugal treatment and pressure treatment can be suitably performed.
- “before inoculation with Agrobacterium” means that the treatment is performed before the step of inoculating Agrobacterium before co-culture.
- in the coexistence process means that the treatment is performed during the co-cultivation.
- after the coexistence step means that the treatment is performed in the resting step performed after the co-culture.
- “perform the centrifugation and / or pressure treatment of the tissue before inoculation with Agrobacterium, during the coexistence step and / or following the coexistence step” 1) A mode in which centrifugation and / or pressure treatment is performed before inoculation with Agrobacterium; 2) A mode in which, after inoculation with Agrobacterium, centrifugation and / or pressure treatment is performed during co-cultivation; 3) A mode in which centrifugation and / or pressure treatment is performed after the coexistence step and before the resting step; 4) A mode of carrying out centrifugal treatment and / or pressure treatment in the resting step; and 5) A mode of carrying out centrifugal treatment and / or pressure treatment in a plurality of stages 1) to 4) above. . All of these embodiments are encompassed by the present invention.
- the plant tissue is cultured in a resting medium after the coexistence process.
- This step is a step of removing the Agrobacterium from the plant cell and growing the plant cell after the coexistence step.
- the medium used in this step is referred to as “resting medium” in this specification.
- the resting medium may be one usually used for culturing plant cells, and examples thereof include a medium based on LS inorganic salts (Non-patent Document 21) and N6 inorganic salts (Non-patent Document 22).
- the resting medium in this step preferably contains an antibiotic. Unlike the antibiotics for selection used in the following selection process, the antibiotics contained in the resting medium are intended to eliminate Agrobacterium. Although not limited, cefotaxime and / or carbenicillin can be preferably used as an antibiotic.
- the resting medium in this step preferably contains a plant growth regulator.
- plant growth regulators benzoic auxins and / or phenoxy auxins can be preferably used. Since auxins generally have an action to dedifferentiate plant tissues, most or all of the plant tissues become dedifferentiated tissues (callus) in this step and the subsequent selection step.
- the term “dedifferentiated tissue” or “callus” is used by culturing a part (explant) of a differentiated plant tissue in a medium containing a plant growth regulator such as auxin or cytokinin. The resulting tissue refers to an amorphous and undifferentiated cell mass that does not have the form of the original plant tissue.
- “Cultivation” in this step means that a plant tissue is placed on a solidified resting medium or in a liquid resting medium, and allowed to grow at an appropriate temperature, light / dark conditions and period.
- the form of the medium is not particularly limited as long as the medium components are sufficiently supplied to the plant tissue.
- the solidification of the resting medium can be carried out by adding a solidifying agent known in the art, and as such a solidifying agent, for example, agarose or the like is known.
- the culture temperature in this step can be appropriately selected, and is preferably 20 ° C-35 ° C, more preferably 25 ° C.
- the culture in this step is preferably performed in a dark place, but is not limited thereto.
- the culture period in this step can also be appropriately selected, and is preferably 1 to 20 days, more preferably 10 days.
- the selection process and the redifferentiation process described below are generally performed in the plant transformation method using Agrobacterium.
- this selection step is not essential.
- the transformation improvement treatment described later when the transformation improvement treatment described later is performed, the target transformant can be obtained without going through the selection step.
- the following description is for illustration and this invention is not limited by the following description.
- This step is a step of selecting a transformant from the tissue obtained in the above step based on the presence or absence of gene transfer.
- the medium used in this step is referred to as “selection medium” in the present specification.
- Examples of the medium that can be used as the selection medium include media based on LS inorganic salts (Non-Patent Document 21) and N6 inorganic salts (Non-Patent Document 22).
- auxins are added to the selective medium.
- the selection medium contains a plant growth regulator.
- the auxin used in the selection process is not particularly limited, but is preferably dicamba and / or 2,4-D. Furthermore, various additives can be added as necessary.
- the selection of the transformed plant can be performed, for example, by culturing the plant that has undergone the coexistence step and / or the resting step in a selection medium containing an appropriate selection agent, and determining whether or not there is resistance to the selection agent.
- the selective drug that can be used in this step those usually used in the technical field can be used.
- antibiotics or herbicides can be used as selective drugs.
- the antibiotic for example, hygromycin, chloramphenicol, G418, kanamycin, or blasticidin S can be used.
- the herbicide for example, phosphinoslysin, bialaphos or glyphosate can be used.
- the DNA inserted into the T-DNA in Agrobacterium may contain not only genes intended to be expressed in plants but also, for example, resistance genes for selected drugs. is necessary. Resistance genes for such selective drugs are known in the art.
- this step for example, when selection is performed in a selection medium containing hygromycin, it is necessary that the plant be introduced with a hygromycin resistance gene in addition to the gene intended to be expressed in the plant.
- selection of transformed plants can be performed based on the sugar requirement of plant cells.
- Sugars that can be used by plant cells include sucrose and glucose, but it is known that mannose cannot be used. Therefore, when plant tissues are cultured in a medium containing mannose as the main carbon source, the plant tissues die off or cease to grow because there is little or no available sugar.
- Selection based on sugar requirement utilizes this principle. That is, in order to use this selection method, DNA inserted into T-DNA in Agrobacterium is not only a gene intended to be expressed in plants, but also phosphorylated mannose isomerase (PMI). ) It is necessary to include a gene. Here, the plant cell introduced with the PMI gene can use mannose as a carbon source.
- PMI phosphorylated mannose isomerase
- Non-Patent Document 24 Such a method can also be performed for other sugars. For example, since a plant cell into which a xylose isomerase gene has been introduced can use xylose as a carbon source, it can be applied to such a method.
- This step can be repeated several times by changing the component composition of the medium.
- the concentration of the selected drug is increased in each selection step, thereby increasing the certainty of drug selection and increasing the possibility of obtaining transformed plants.
- This selection process is preferably performed at least once, more preferably twice.
- the transformed tissue is efficiently obtained by cutting off the proliferated portion of the tissue cultured in the medium containing the selected drug and subjecting only the proliferated portion to the next selection step. It is also possible to do.
- “Cultivation” in this step means that a plant tissue is placed on a solidified selection medium or in a liquid selection medium and grown at an appropriate temperature, light and dark conditions and period.
- the form of the medium is not particularly limited as long as the medium components are sufficiently supplied to the plant tissue.
- the selection medium can be solidified, for example, with agarose or the like.
- the culture temperature in this step can be appropriately selected, and is preferably 20 ° C-35 ° C, more preferably 25 ° C.
- the culture in this step is preferably performed in a dark place, but is not limited thereto.
- the culture period of this step can also be selected as appropriate.
- the selection process is performed twice, the primary selection is performed for 2 weeks and the secondary selection is performed for 2 weeks, for a total of 4 weeks.
- the entire selection process is preferably performed for 3-8 weeks, more preferably 4-6 weeks.
- Redifferentiation step After the tissue cultured in the resting medium is selected if necessary, a step of redifferentiation in the redifferentiation medium is performed.
- the medium used in this step is referred to as “regeneration medium” in the present specification.
- the regeneration medium does not contain auxins.
- barley transformation may use pre-regeneration culture medium (transition medium, pre-regeneration medium).
- This medium usually contains auxins (Jacobsen et al. 2006, Non-Patent Document 8, Bartlett et al. 2008, Non-Patent Document 9, Harwood et al. 2008, Non-Patent Document 11).
- the pre-redifferentiation culture medium may contain a selective drug. The tissue cultured in the pre-redifferentiation culture medium is transferred to the regeneration medium and cultured.
- the regeneration medium may contain a selective drug.
- the selective drugs that can be used in this step are the same as those defined in the selection step. However, in this step, it is not always necessary to use the same selected drug as the selected drug used in the selection step. In that case, it is necessary for the plant to be introduced with resistance genes for two or more selected drugs from Agrobacterium.
- Redifferentiation in the present invention means that a plant tissue that has been completely or partially dedifferentiated again acquires the properties of the original plant tissue or plant body.
- auxins are used during the coexistence process and / or resting process and / or selection process, all or part of the plant tissue is dedifferentiated. Therefore, by subjecting to this step, the dedifferentiated tissue is redifferentiated and a completely transformed plant body can be obtained.
- “Cultivation” in this step refers to placing a plant tissue on a solidified regeneration medium or in a liquid regeneration medium and growing it at an appropriate temperature, light / dark conditions and period.
- the form of the medium is not particularly limited as long as the medium components are sufficiently supplied to the plant tissue.
- the regeneration medium can be solidified using, for example, agarose.
- the culture temperature in this step can be appropriately selected, and is preferably 20 ° C-35 ° C, more preferably 25 ° C.
- the culture in this step is preferably performed under illumination for 16-24 hours / day, but is not limited thereto.
- the culture period in this step can also be appropriately selected, and is preferably 7 days to 21 days, more preferably 14 days.
- transformation improvement treatment refers to a treatment for achieving an improvement in transformation efficiency.
- Such a transformation improving process is not limited, but includes, for example, the following or a combination thereof.
- Non-Patent Document 26 2003: Non-Patent Document 26
- Heat treatment Reference: WO1998 / 054961: Patent Document 1
- c) treatment of inoculating Agrobacterium in the presence of powder see: WO2007 / 069643: Patent Document 5
- g) Treatment of adding cysteine to the co-culture medium Treatment of adding cysteine to the co-culture medium.
- heat treatment and powder addition are both treatments for improving gene transfer efficiency, and addition of silver nitrate has an effect of improving the callus induction rate.
- Non-Patent Document 25 Zhao et al. 2001
- Ishida et al. 2003 Non-Patent Document 26
- Silver nitrate can be added to the co-culture medium at a concentration of 1 ⁇ M to 50 ⁇ M, preferably 1 ⁇ M to 10 ⁇ M, for example.
- the heat treatment can be performed using, for example, a method described in WO1998 / 054961 (Patent Document 1).
- the plant material is treated at 33 ° C. to 60 ° C., preferably 37 ° C. to 52 ° C., for 5 seconds to 24 hours, preferably 1 minute to 24 hours before contacting with Agrobacterium.
- the treatment of inoculating Agrobacterium in the presence of powder can be performed using, for example, the method described in WO2007 / 069643 (Patent Document 5). Specifically, for example, it is performed by mixing Agrobacterium suspension and powder and inoculating the plant material, or mixing plant and powder and inoculating it with Agrobacterium.
- the powder is not limited, but is a porous powder, glass wool, or activated carbon, preferably porous ceramic, glass wool, or activated carbon, more preferably hydroxyapatite, silica gel, or glass wool.
- cysteine can be added to the co-culture medium at 10 mg / l to 1 g / l, preferably 50 mg / l to 750 mg / l, more preferably 100 mg / l to 500 mg / l.
- a preferable transformation improving treatment is a treatment of adding AgNO 3 to the co-culture medium, a heat treatment, a treatment of inoculating Agrobacterium in the presence of powder, a treatment of adding cysteine to the co-culture medium, or a combination thereof. is there.
- the means for “physically / chemically damaging one or more sites selected from the radicle, bud, and hypocotyl” is not particularly limited, and various physical Processing and chemical processing are included.
- the physical treatment includes, but is not limited to, for example, excision or injury with a sharp blade (for example, a scalpel), removal or injury with an instrument having a sharp tip (for example, tweezers), and the like.
- the chemical treatment includes, for example, treatment with an agent such as an acidic or alkaline substance that loses or reduces the function of plant cells, or a herbicide component that is toxic to cells.
- the embryo is a part that will become a plant in the future, and includes the roots, shoots, and hypocotyls.
- the hypocotyl is a cylindrical portion that becomes the axis of the embryo, and buds are generated from the upper end and radicles are generated from the lower end.
- the radicle, bud, and hypocotyl are understood as meanings commonly used in this technical field.
- one or more sites selected from radicles, buds, and hypocotyls refers to larvae, shoots, hypocotyls. Mean all combinations selected from one, two, or three sites. Specifically, the combinations are as follows: 1) larvae, 2) larvae, 3) hypocotyls, 4) larvae and larvae, 5) larvae and hypocotyls, 6) buds and embryos Axes, 7) Roots and shoots and hypocotyls.
- “before inoculation with Agrobacterium” means that the treatment is performed before the step of inoculating Agrobacterium before co-culture.
- in the coexistence process means that the treatment is performed during the co-cultivation.
- after the coexistence step means that the treatment is performed in the resting step performed after the coexistence engineering culture.
- the immature embryo tissue is selected from larvae, buds, and hypocotyls before inoculation of Agrobacterium to the immature embryo tissue, during and / or after the coexistence step 1 "Performs a process of physically / chemically damaging more parts" 1) A mode in which the site is damaged before inoculation with Agrobacterium; 2) Aspect of damaging the above site after inoculation with Agrobacterium; 3) A mode in which the site is damaged after the coexistence step and before the resting step; 4) A mode of damaging the site in the resting process; and 5) A mode of damaging the site in any one of steps 1) to 4). All of these embodiments are encompassed by the present invention.
- “high transformation efficiency” means that a target gene is introduced into plant cells with high efficiency, that transformed callus is induced with high efficiency from immature embryos, etc., and that high efficiency is obtained from transformed callus. It is a concept that includes re-differentiation.
- “transformation efficiency is improved” means that the introduction efficiency of a target gene into a plant cell is improved, the rate of induction of transformed callus from immature embryos, etc. is improved, This is a concept including improvement of regeneration efficiency.
- Whether or not a gene has been introduced into a plant tissue can be determined by various known methods. For example, by using a gene to be transformed as a reporter gene such as GUS ( ⁇ -glucuronidase) gene, luciferase gene or GFP gene, the presence or absence of transformation can be confirmed by visualizing the expression site of these reporter genes by a simple and known method. It is possible to confirm about.
- GUS ⁇ -glucuronidase
- GFP luciferase gene
- plant cells can be cultured in a medium containing antibiotics or herbicides, or antibiotic solutions or herbicide solutions can be added to plants. It is also possible to confirm the presence or absence of transformation using the resistance expression as an index.
- Example 1 Effect of co-culture medium composition on gene transfer efficiency (anti-auxin, auxin) Materials and Methods Immature embryos (size 1.5-2.0 mm) from which the hypocotyl of barley (variety: Golden Promise) was removed were aseptically collected and submerged in 2 ml microtubes containing 1 ml sterile water. In order to increase gene transfer efficiency, the tube containing the immature embryo was heat-treated at 43 ° C. for 5 minutes in a water bath.
- Immature embryos were washed once with a liquid callus induction medium CIMT (Tingay et al. 1997 Non-Patent Document 5) excluding plant growth regulators (hereinafter collectively referred to as anti-auxin, cytokinin, and auxin).
- An immature embryo inoculated with Agrobacterium was mixed with 100 ⁇ M acetosyringone in barley co-culture medium (1/10 concentration of MS inorganic salt and MS vitamin, 10 g / l glucose, 0.5 g / l MES, 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 , PH 5.8 , 8 g / l agarose) and placed so that the scutellum side faces upward.
- test was implemented in 4 test areas which added two types of plant growth regulators to the co-culture medium. That is, there were 4 test groups of hormone-free without plant growth regulator added, 5 ⁇ M TIBA (antiauxin), 1.13 ⁇ M dicamba (auxin), 11.3 ⁇ M dicamba (auxin).
- Immature embryos cultured for 24 hours at 25 ° C. in the dark were treated with CIMT resting medium supplemented with 0.5 mg / l 2,4-D and 1.25 mg / l CuSO 4 .5H 2 O (Tingay et al. 1997 It was transplanted to Patent Document 5).
- the resting medium was supplemented with 250 mg / l carbenicillin 100 mg / l cefotaxime for Agrobacterium sterilization.
- immature embryos were immersed in 0.1 M phosphate buffer (pH 6.8) containing 0.1% Triton X-100. And left at 37 ° C.
- a phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc) and 20% methanol was added. After overnight treatment at 37 ° C., the expression of GUS gene was examined.
- GUS gene in the scutellum region is 87.5 (expressed in 75% or more of the scutellum), 62.5 (expressed in 50% or more and less than 75% of the scutellum), 37.5 ( Expressed in 25% or more and less than 50% of the scutellum), 17.5 (expressed in 10% or more and less than 25% of the scutellum), 6.5 (expressed in 1% or more and less than 10% of the scutellum), 0.5 Evaluation was made in 7 stages (exceeding 0% and less than 1% of the scutellum) and 0 (no expression), and the average value was expressed as a GUS expression index. The number of immature embryos tested was 15 in each group. The number of immature embryos tested was 15 in each group.
- Example 2 Effect of co-culture medium composition on gene transfer efficiency (cytokinin, anti-auxin) Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Agrobacterium inoculation methods are the same as in Example 1. In addition, 6 test sections to which 5 types of plant growth regulators were added were used as the co-culture medium. That is, hormone-free without plant growth regulator, 5 ⁇ M 6BA, 5 ⁇ M 4-PU, 5 ⁇ M Zeatin (above, cytokinin), 5 ⁇ M TIBA, 5 ⁇ M paclobutrazol (above, anti-auxin) 6 The test area was designated. CIMT resting with immature embryos cultured at 25 ° C.
- Example 2 in the dark for 24 hours, supplemented with 0.5 mg / l 2,4-D and 1.25 mg / l CuSO 4 .5H 2 O as in Example 1. It was transplanted to a medium (Tingay et al. 1997 Non-Patent Document 5). The resting medium was supplemented with 250 mg / l carbenicillin 100 mg / l cefotaxime for Agrobacterium sterilization. After culturing in the dark at 25 ° C. for 2 days, the expression of the GUS gene was investigated in the same manner as in Example 1, and the result was expressed as a GUS expression index. The number of immature embryos tested was 15 in each group.
- Example 3 Effect of Centrifugal Treatment on Compact Callus Formation Materials and Methods Materials and Agrobacterium inoculation methods are the same as in Example 1. The co-culture medium was free from plant growth regulators.
- the immature embryo co-cultured for 24 hours at 25 ° C. in the dark was transferred to a 2 ml microtube containing 1 ml of sterilized water and centrifuged at 25 ° C. and 1,500 rpm (20,000 ⁇ g) for 10 minutes. Thereafter, transplanted to the same CIMT resting medium (Tingay et al. 1997 Non-Patent Document 5) supplemented with 0.5 mg / l 2,4-D and 1.25 mg / l CuSO 4 .5H 2 O as in Example 1. did.
- the resting medium was supplemented with 250 mg / l carbenicillin 100 mg / l cefotaxime for Agrobacterium eradication.
- the scutellum of each immature embryo was divided into 4 parts. Divided sections were transplanted to a medium having the same composition. Thereafter, the culture was further continued for 8 days under the same culture conditions. After the culture, the shape of the callus formed on the scutellum divided section was observed. In barley, only callus with a compact and embryogenic shape can be subcultured and has the potential to re-differentiate in the future. On the other hand, spongy callus containing a lot of water is often formed, but such callus has lost redifferentiation ability. Therefore, the number of scutellum sections that formed compact callus was counted and used as the callus formation rate.
- the gene introduction region can be used even if resting culture is performed without any treatment. It was revealed that compact callus formation from blastocyst cells is strongly suppressed. On the other hand, 80% of the test plots subjected to the centrifugal treatment formed compact callus, suggesting the possibility of obtaining transformed callus at a certain frequency by subsequent selection with hygromycin. In addition, the present inventors have found that a similar callus formation promoting effect is also obtained when immature embryos before inoculation with Agrobacterium are subjected to centrifugal treatment or pressure treatment.
- Example 4 Effect of Centrifugal Treatment on Hygromycin-resistant Callus Formation Materials and Methods Materials and Agrobacterium inoculation methods, heat treatment conditions, coexistence steps, centrifugation treatment conditions and resting steps are the same as in Example 3.
- 5 ⁇ M TIBA which is an antiauxin, was added to the co-culture medium. The resting process was continued for 10 days after dividing the scutellum into 4 parts. After the resting process, each divided scutellum section was further divided into 4 parts (16 parts per immature embryo), and CIMT selection medium (Tingay et al.) Supplemented with 50 mg / l hygromycin B and 1.25 mg / l CuSO 4 .5H 2 O. al.
- Example 5 Effect of co-culture medium composition on gene transfer efficiency (hormone free, 2,4-D) Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Methods Materials and Agrobacterium inoculation methods are the same as in Example 1. That is, a co-culture medium containing 1/10 concentration of MS inorganic salt was used. As test plots, three types were used: a hormone-free co-culture medium containing no plant growth regulator, and a co-culture medium supplemented with 1.13 ⁇ M 2,4-D and 11.3 ⁇ M 2,4-D. The immature embryo co-cultured for 24 hours in the dark at 25 ° C. was subjected to CIMT-less addition of 0.5 mg / l 2,4-D and
- Ting medium (Tingay et al. 1997 Non-Patent Document 5) was transplanted.
- the resting medium was supplemented with 250 mg / l carbenicillin 100 mg / l cefotaxime for Agrobacterium sterilization.
- the expression of the GUS gene was investigated in the same manner as in Example 1, and the result was expressed as a GUS expression index.
- the number of immature embryos tested was 15 in each group.
- Example 6 Effect of Centrifugal Treatment Before Agrobacterium Inoculation and After Co-culture on Compact Callus Formation Materials and Methods Preparation of materials and Agrobacterium inoculation method are the same as in Example 1. Heat treatment to immature embryos was performed in the same manner. Note that 5 ⁇ M of TIBA was added to the co-culture medium.
- Example 7 Preparation of a comparative material for transformation efficiency and inoculation with Agrobacterium using a conventional coexisting medium containing daikanba and a coexisting medium with 1/10 concentration of MS inorganic salt are the same as in Example 1. The heat treatment was performed in the same manner.
- Test plot (1) An immature embryo inoculated with Agrobacterium was obtained from Harwood et al. The scutellum was placed downward in the co-culture medium described in (2008 Non-Patent Document 11). Co-culture was performed at 25 ° C. in the dark for 3 days. Centrifugation was not performed in this test section.
- Test group (2) The isolated immature embryo was transferred to a 2 ml microtube containing 1 ml of sterilized water and centrifuged at 15,000 rpm (20,000 ⁇ g) for 10 minutes at 25 ° C. Thereafter, the cells were cocultured in the same manner as in the test group (1).
- Test plot (3) After co-culture as in test plot (1), immature embryos were transferred to a 2 ml microtube containing 1 ml of sterile water and 10 ° C. at 25 ° C. and 15,000 rpm (20,000 ⁇ g). Centrifuged for minutes.
- Test plot (4) immature embryos inoculated with Agrobacterium, barley co-culture medium containing 100 ⁇ M acetosyringone (1/10 concentration of MS inorganic salt and MS vitamin, 10 g / l glucose, 5 ⁇ M TIBA, 0.5 g / l MES, 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 , pH 5.8, 8 g / l agarose) and placed so that the scutellum faces upward. The cells were cultured at 25 ° C. in the dark for 24 hours. Centrifugation was not performed in this test section.
- Test group (5) The isolated immature embryo was transferred to a 2 ml microtube containing 1 ml of sterilized water and centrifuged at 15,000 rpm (20,000 ⁇ g) for 10 minutes at 25 ° C. Thereafter, the cells were cocultured in the same manner as in the test group (4).
- Test plot (7) Excluding TIBA, which is a plant growth regulator, from the co-culture medium of test plot (4), after co-cultivation was carried out in the same manner as in test plot (4), immature embryos were placed in 2 ml containing 1 ml of sterile water. It was transferred to a microtube and centrifuged at 15,000 rpm (20,000 ⁇ g) for 10 minutes at 25 ° C.
- the immature embryos obtained in the test groups (1), (2), (3), (4), (5), (6), and (7) were 0.5 mg / l 2,4-D, respectively. And transplanted to a CIMT resting medium (Tingay et al. 1997 Non-Patent Document 5) containing no selection drug supplemented with or.
- the resting medium was supplemented with 250 mg / l carbenicillin 100 mg / l cefotaxime for Agrobacterium sterilization. After culturing in the dark at 25 ° C. for 7 days, immature embryos were divided into 4 parts, and CIMT resting medium without selection drug (Tingay et al.
- Non-Patent Document 5 After resting culture for 10 days in the dark at 25 ° C., the 4 immature embryo sections were further divided into 3 to 6 sections (12 to 24 sections per immature embryo), and 0.1 mg / l 2,4-D, It was plated in 1.5 [mu] M CuSO 4 and 75 mg / l hygromycin CIMT selection medium supplemented with (Tingay et al.1997 non-Patent Document 5). And it culture
- the obtained hygromycin-resistant callus was placed on a transition medium (Harwood et al. (2008 Non-Patent Document 11)) containing no hygromycin, and only one per divided section. For about 2 weeks.
- the obtained green cell mass or shoot was placed on a regeneration medium (Harwood et al. (2008 Non-Patent Document 11)) containing 30 mg / l hygromycin, 0.2 mg / l indolebutyric acid (IBA) and 5 ⁇ M CuSO 4 . Then, the cells were cultured for about 2 weeks under light conditions at 25 ° C.
- phosphate buffer pH 6.8 containing 0.1% Triton X-100 (registered trademark), and 1% at 37 ° C. Let stand for hours. After removing the phosphate buffer, a phosphate buffer containing 1.0 mM mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc) and 20% methanol was added. After treatment at 37 ° C. for 24 hours, the expression of the GUS gene was examined. In addition, the GUS gene expression was investigated on a single leaf from a maximum of one plant per slice of immature embryo divided.
- test group 4 in which the immature embryo was not centrifuged, a compact callus having redifferentiation ability was not formed, and no transformant was obtained.
- no transformants could be obtained from immature embryos cultured in the conventional coexisting medium containing dicamba after centrifugation in Test Group 2. This is because T-DNA was introduced into a portion of an immature embryo in which callus was not formed later by performing centrifugation before co-culture.
- expression of the GUS gene was detected near the center of the immature embryo on the hypocotyl side.
- test group 6 in which 5 ⁇ M TIBA, which is an anti-auxin, was added to the co-culture medium showed a higher transformation efficiency than the hormone-free test group 7.
- Example 8 Genetic analysis of the transgene in the next generation of self-breeding transformants
- Example 6 Two hygromycin-resistant and GUS-positive transformed plants obtained from different immature embryos in the test section (6) were cultivated in a greenhouse. From the immature seeds obtained, 1.5 to 2.0 mm immature embryos were aseptically collected. The immature embryo was placed on a regeneration medium containing 5 ⁇ M CuSO 4 (Harwood et al. (2008 Non-Patent Document 11)) and cultured under light conditions at 25 ° C. for about 2 weeks.
- T1 self-fertilizing next generation
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Abstract
Description
1)未熟胚の単離とアグロバクテリウムの接種
未熟胚の直径が1.5-2.0mmに育ったオオムギの穂から未熟種子を採取し、次亜塩素酸ナトリウム溶液で殺菌し、無菌的に未熟胚を取り出す。得られた未熟胚から胚軸を切除し、オオムギのカルス誘導培地に胚盤を上向きに置床する。カルス誘導培地には、Murashige & Skoog (MS) 無機塩類(Murashige & Skoog 1962 非特許文献13)、30g/l マルトース、1.0g/l カゼイン加水分解物、350mg/l ミオ-イノシトール、690mg/l、1.0mg/l 塩酸チアミン、2.5mg/l 3,6-ジクロロ-2-メトキシ安息香酸(ダイカンバ)、1.25mg/l CuSO45H2O(Bartlett et al. 2008 非特許文献9およびHarwood et al. 2008 非特許文献11でのみ添加)、3.5 g/l フィタゲル、pH5.8が共通して用いられている(Jacobsen et al. 2006 非特許文献8, Bartlett et al. 2008 非特許文献9, Hensel et al. 2008 非特許文献10, Harwood et al. 2008 非特許文献11)。 接種源に用いるアグロバクテリウム懸濁液は、液体培地で一晩振盪培養することにより得る。アグロバクテリウムの接種方法は、未熟胚の胚盤上にアグロバクテリウム懸濁液を滴下する方法(Jacobsen et al. 2006 非特許文献8, Bartlett et al. 2008 非特許文献9, Harwood et al. 2008 非特許文献11)と、未熟胚をアグロバクテリウム懸濁液中に浸漬した後、真空ポンプを用いて減圧を行う方法が行われている(Hensel et al. 2008 非特許文献10)。接種が行われる時間、すなわち、アグロバクテリウム懸濁液と未熟胚が接触してから共存培養の培地への移植を行うまでの時間については、これらの報告中には、具体的な記述がないが、滴下法も浸漬減圧法のどちらの接種方法も20分から長くても2時間程度であると考えられる。なお、未熟胚へのアグロバクテリウムの接種は、未熟胚を単離した当日、または、一晩培養した翌日に行われている。
アグロバクテリウムを滴下法または浸漬減圧法を用いて接種した後、未熟胚は、共存培養用の培地へ移動される。未熟胚は、胚盤側を下向きに培地に接するよう置床されるのが一般的である(Jacobsen et al. 2006 非特許文献8, Bartlett et al. 2008 非特許文献9, Hensel et al. 2008 非特許文献10, Harwood et al. 2008 非特許文献11)。未熟胚を置床する際の向きについて、Hensel et al. (2008 非特許文献10) は、胚盤側を下向きに置床して共存培養を行った場合、29%の形質転換効率であったのに対し、胚盤を上向きにした場合には、約7分の1に当たる4.1%の形質転換効率に止まったことを報告している。
共存培養後、未熟胚は、上述のカルス誘導培地にアグロバクテリウムを除菌するための抗生物質(例としては、160 mg/l チメンチン)と50 mg/l ハイグロマイシンなどの選抜薬剤を添加した培地へ置床される。カルスを誘導するための植物成長調節物質には、2.5 mg/l (11.3μM)ダイカンバが共通して用いられている(Jacobsen et al. 2006 非特許文献8, Bartlett et al. 2008 非特許文献9, Hensel et al. 2008 非特許文献10, Harwood et al. 2008 非特許文献11)。選抜薬剤に対して明瞭な耐性を示すカルス(形質転換細胞塊)が得られるまで、同培地で2週間程度の間隔で継代される。およそ4-6週間後、選抜薬剤耐性カルスは、選抜薬剤を含む再分化前培養培地(transition medium, pre-regeneration medium)もしくは、選抜薬剤を含む再分化培地(シュート誘導培地)に移植される。再分化前培養培地で培養したカルスは、その後、選抜薬剤を含む再分化培地に移植される。さらに、再分化したシュートおよび幼植物体は、選抜薬剤を含み植物成長調節物質を含まない発根培地に移植され、オオムギ形質転換植物体が得られる。
これまでに報告されている1未熟胚当たりの形質転換効率は、以下の通りである。
品種Golden Promiseでは、 7% (Tingey et al. 1997 非特許文献5)、12%(Matthews et al. 2001 非特許文献15)、9.2% (Murray et al. 2004 非特許文献16)、36%(Bartlett et al. 2008 非特許文献9)、86.7% (Hensel et al. 2008 非特許文献10)。なお、非特許文献9や10においては、場合によって高効率ではあるものの、このような高い効率が安定的に得られているものではない。品種Tafenoでは、2%(Hensel et al. 2008 非特許文献10)、品種Heliumでは、2%(Hensel et al. 2008 非特許文献10)である。
Ke et al. 2002(非特許文献17)は、アグロバクテリウムとの共存培養後におけるβ-glucuronidase (GUS)レポーター遺伝子の発現を解析することで、共存培養用の培地組成の違いによる未熟胚細胞へのT-DNAの転移効率を評価した。単離直後の未熟胚を用い、30分間アグロバクテリウムを接種、MS培地の基本無機塩類濃度を等倍(x1)または1/10倍(x 0.1)とした共存培地を用いて、3日間共存培養を実施した。オーキシン作用を有する植物成長調節物質である0.25mg/l(1.1μM)2,4-ジクロロフェノキシ酢酸(2,4-D) を添加または無添加の共存培地を用いた。0.25mg/l (1.1μM)2,4-Dは脱分化を促すには濃度が低く、胚盤からのカルス誘導には通常使用されていない(Serhantova et al. 2004 非特許文献18)。共存培養後における5-ブロモ-4-クロロ-3-インドリル-β-D-グルクロン酸(X-Gluc)を用いたGUSの組織化学的観察の結果、MS無機塩類を1/10倍濃度とした培地試験区で、胚盤細胞への遺伝子導入効率が高く、MS無機塩類を1/10倍濃度でかつ植物成長調節物質を無添加とした培地試験区でさらに効率が高まることを示した (Ke et al. 2002 非特許文献17)。
Hiei et al. (2006 非特許文献19)は、アグロバクテリウムを接種する前のイネおよびトウモロコシの未熟胚に対し、熱処理(特許文献1)や遠心処理(特許文献2)あるいは熱および遠心処理(特許文献3)を行うことにより、未熟胚胚盤への遺伝子導入効率が向上し、結果的に形質転換効率が向上することを報告した。これらの処理を用いることにより、これまで形質転換できなかった品種で形質転換体を得ることができたことも報告している。また、アグロバクテリウムを接種する前の未熟胚に加圧処理(特許文献4)をすることにより、遠心処理と同様に胚盤細胞への遺伝子導入効率が向上し、結果的に形質転換効率が向上することが報告されている。熱、遠心、熱および遠心、加圧処理は、何れも、未熟胚胚盤への遺伝子導入効率を上げるために用いられている。
[態様1]
オオムギ属(Hordeum)の植物の、未熟胚組織へ、遺伝子導入を行う方法であって、
(i)アグロバクテリウム菌を上記組織へ接種し、該アグロバクテリウム菌の存在下で、以下のa)からc)の1つまたは複数の条件
a)アンチオーキシンを含む、
b)サイトカイニンを含む、
c)フェノキシ系オーキシンを2μM未満の濃度で含むおよび/またはベンゾイック系オーキシンを5μM未満の濃度で含むか、あるいは、フェノキシ系オーキシンおよび/またはベンゾイック系オーキシンを含まない;
を満たす共存培地の中で、上記組織を培養する共存工程を行う、
(ii)アグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記組織を遠心処理および/または加圧処理する工程を行う、
ことを含む、上記方法。
[態様2]
オオムギ属(Hordeum)の植物の、形質転換植物の作成方法であって、
(i)アグロバクテリウム菌をオオムギの未熟胚組織へ接種し、該アグロバクテリウム菌の存在下で、以下のa)からc)の1つまたは複数の条件
a)アンチオーキシンを含む、
b)サイトカイニンを含む、
c)フェノキシ系オーキシンを2μM未満の濃度で含むおよび/またはベンゾイック系オーキシンを5μM未満の濃度で含む、あるいは、フェノキシ系オーキシンおよび/またはベンゾイック系オーキシンを含まない;
を満たす共存培地の中で、上記組織を培養する共存工程を行い、
(ii)アグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記組織を遠心処理および/または加圧処理する工程を行い、
(iii)上記組織をレスティング培地で培養するレスティング工程を行い、そして、
(iv)上記組織を再分化培地で再分化させる工程を行う、
ことを含む、上記方法。
[態様3]
前記共存工程開始後6時間ないし36時間以内に、レスティング工程を開始する、態様1または態様2に記載の方法。
[態様4]
前記共存工程開始後12時間ないし24時間以内に、レスティング工程を開始する、態様3に記載の方法。
[態様5]
未熟胚を単離後6時間ないし36時間以内に、共存工程を終えレスティング工程を開始する、態様1または態様2に記載の方法。
[態様6]
未熟胚を単離後12時間ないし24時間以内に、共存工程を終えレスティング工程を開始する、態様5に記載の方法。
[態様7]
上記未熟胚組織に対するアグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記未熟胚組織において幼根、幼芽、および胚軸から選択される1またはそれ以上の部位を物理的/化学的に損傷する工程を行う態様1ないし態様6のいずれか1に記載の方法。
[態様8]
前記共存培養において前記未熟胚組織を、胚盤側を上向きにし、かつ、胚軸側を前記共存培地に接するように置床して培養する、態様1ないし態様7のいずれか1に記載の方法。
[態様9]
以下の形質転換効率向上処理のうち、少なくとも1つを行う、態様1ないし態様8のいずれか1に記載の方法。
a)熱処理;
b)硝酸銀の共存培地への添加;
c)粉体の存在下でアグロバクテリウムを接種する処理;
[態様10]
上記(iii)レスティング工程と、(iv)再分化工程の間に薬剤選抜工程を含む、態様1ないし態様9のいずれか1に記載の方法。
[態様11]
(iii)レスティング培地、および/または、薬剤選抜工程の選抜培地が、植物成長調節物質を含む、態様1ないし態様10いずれか1に記載の方法。
[態様12]
前記アグロバクテリウム菌が、LBA4404、EHA101、EHA105、AGL0、AGL1、および58C1からなる群から選択される菌である、態様1ないし態様11のいずれか1に記載の方法。
[態様13]
オオムギ属の植物がオオムギ(H. vulgare)である、態様1ないし態様12のいずれか1に記載の方法。
(i)アグロバクテリウム菌を上記組織へ接種し、該アグロバクテリウム菌の存在下で、以下のa)からc)の1つまたは複数の条件
a)アンチオーキシンを含む、
b)サイトカイニンを含む、
c)フェノキシ系オーキシンを2μM未満の濃度で含むおよび/またはベンゾイック系オーキシンを5μM未満の濃度で含むか、あるいは、フェノキシ系オーキシンおよび/またはベンゾイック系オーキシンを含まない;
を満たす共存培地の中で、上記組織を培養する共存工程を行う、
(ii)アグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記組織を遠心処理および/または加圧処理する工程を行う、
ことを含む、遺伝子導入方法を提供する。
(i)アグロバクテリウム菌をオオムギの未熟胚組織へ接種し、該アグロバクテリウム菌の存在下で、以下のa)からc)の1つまたは複数の条件
a)アンチオーキシンを含む、
b)サイトカイニンを含む、
c)フェノキシ系オーキシン2μM未満の濃度で含むおよび/またはベンゾイック系オーキシンを5μM未満の濃度で含むか、あるいは、フェノキシ系オーキシンおよび/またはベンゾイック系オーキシンを含まない;
を満たす共存培地の中で、上記組織を培養する共存工程を行い、
(ii)アグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記組織を遠心処理および/または加圧処理する工程を行い、
(iii)上記組織をレスティング培地で培養するレスティング工程を行い、そして、
(iv)上記組織を再分化培地で再分化させる工程を行う、
ことを含む、上記方法を提供する。
本発明の遺伝子導入方法及び形質転換植物の作成方法は、アグロバクテリウム細菌を利用する。特に明記する工程以外は、公知のアグロバクテリウム細菌を利用した遺伝子導入方法、形質転換方法の各工程に従って行うことが可能である。
本発明において、アグロバクテリウム菌を接種した、未熟胚組織を、該アグロバクテリウム菌の存在下で培養する、共存工程を行う。本工程は、アグロバクテリウム菌を接種した植物組織を、アグロバクテリウム菌の共存下にて培養することにより、アグロバクテリウム菌から植物細胞へのDNAの導入を確実にする工程である。
本発明において、アグロバクテリウム接種前、共存工程中および/または共存工程に次いでのオオムギ未熟胚の遠心処理を行うことにより、脱分化およびカルス形成を抑制する条件で共存培養したオオムギ未熟胚であっても、十分にカルスを誘導することのできるという顕著な効果があることを見出した。この場合の遠心処理の条件は、WO2002/012520(特許文献2)に記載の条件と同様でよい。具体的には、通常100G~25万G、500G~20万G、好ましくは1000G~15万G、最も好ましくは1100G~11万G程度の遠心加速度範囲で行われる。また、遠心処理の時間は、遠心加速度に応じて適宜選択されるが、通常1秒間以上行うことが好ましい。遠心時間の上限は特にないが、通常、10分間程度で目的を達成することができる。また、遠心処理時間は、遠心加速度が大きい場合にはごく短い時間、例えば1秒以下でも遺伝子導入効率を有意に向上させることができる。一方、遠心加速度が小さい場合には、遠心処理を長く行うことが好ましい。なお、適切な遠心処理条件は、ルーチンな実験により容易に設定することができる。このような遠心処理は、後に述べる胚軸の切除の前後のいずれで行ってもよい。
1)アグロバクテリウムの接種前において遠心処理および/または加圧処理をする態様;
2)アグロバクテリウムの接種後、共存培養中に遠心処理および/または加圧処理をする態様;
3)共存工程後、レスティング工程の前に遠心処理および/または加圧処理をする態様;
4)レスティング工程において遠心処理および/または加圧処理をする態様;および
5)上記1)ないし4)のいずれかの複数の段階において遠心処理および/または加圧処理をする態様、を意味する。それらの態様は全て本発明に包含される。
本発明の形質転換植物の作成方法においては、上記共存工程の後にさらにレスティング工程、再分化工程を経て、形質転換植物を作成する。
以下に記載する選抜工程および再分化工程は、アグロバクテリウム菌による植物の形質転換方法において一般に行われている方法である。なお本発明の形質転換植物の作成方法において、この選抜工程は必須のものではない。例えば、後に述べる形質転換向上処理をした場合には、選抜工程を経なくても目的とする形質転換体を得ることができるからである。なお選抜工程を行う場合、以下の記載は例示のためのものであり、本発明は以下の記載により限定されるものではない。
レスティング培地で培養した組織を、必要ならば選抜した後に、再分化培地で再分化させる工程を行う。本工程で使用される培地は、本明細書中では「再分化培地」という。再分化培地は、オーキシン類は含まない。
また本発明の遺伝子導入方法と形質転換植物の作成方法において、上記で述べた遠心処理と加圧処理に加えて、以下に述べる形質転換向上処理を行ってもよい。本明細書において「形質転換向上処理」とは、形質転換効率の向上を達成するための処理をいう。このような形質転換向上処理としては、限定されるものではないが、例えば以下のようなものあるいはこれらの組み合わせが含まれる。
a)硝酸銀の共存培地への添加(参照:AgNO3(Zhao et al. 2001:非特許文献25、Ishida et al. 2003:非特許文献26)、
b)熱処理(参照:WO1998/054961:特許文献1)、
c)粉体の存在下でアグロバクテリウムを接種する処理(参照:WO2007/069643:特許文献5)、ならびに、
g)共存培地にシステインを添加する処理(Frame et al. 2006:非特許文献27)。
本発明においては、アグロバクテリウムの接種前、共存工程中および/または共存工程に次いで、未熟胚組織において幼根、幼芽、および胚軸から選択される1またはそれ以上の部位を物理的/化学的に損傷する処理を行うことができる。
1)アグロバクテリウムの接種前において上記部位を損傷する態様;
2)アグロバクテリウムの接種後、上記部位を損傷する態様;
3)共存工程後、レスティング工程の前に上記部位を損傷する態様;
4)レスティング工程において上記部位を損傷する態様;および
5)上記1)ないし4)のいずれかの複数の段階において上記部位を損傷する態様、を意味する。それらの態様は全て本発明に包含される。
本発明の遺伝子導入方法と本発明の形質転換植物の作成方法により、安定的に高い効率でオオムギ属植物の形質転換を行うことができる。よって植物の形質転換効率の向上が達成される。
共存培地組成が遺伝子導入効率に及ぼす効果(アンチオーキシン、オーキシン)
材料および方法
オオムギ(品種:Golden Promise)の胚軸を除去した未熟胚(大きさ1.5-2.0mm)を無菌的に採取し、1ml滅菌水を入れた2mlマイクロチューブに沈めた。遺伝子導入効率を高めるため、未熟胚の入ったチューブを43℃で5分間ウォーターバスにて加熱処理を行った。100μMアセトシリンゴンを含むMG/L液体培地(非特許文献28)にハイグロマイシン耐性遺伝子を有するアグロバクテリウム菌株 EHA101(pIG121Hm)(非特許文献2)を懸濁し、一晩(約20時間)28℃で振盪培養(180rpm)を行い接種源とした。菌濃度は、O.D.値 = 1.0(660nm)に調整した。熱処理した未熟胚に接種源を加え、真空ポンプを用いて500mbarで10分間の減圧処理を行った。植物成長調節物質(以下、アンチオーキシン、サイトカイニン、およびオーキシンを総称して植物成長調節物質という)を除いた液体カルス誘導培地CIMT(Tingay et al. 1997 非特許文献5)で一度未熟胚を洗浄した。アグロバクテリウムを接種した未熟胚を100μMアセトシリンゴン含有のオオムギ共存培地(1/10濃度のMS無機塩およびMSビタミン、10g/lグルコース、0.5g/l MES、5μM AgNO3および5μM CuSO4、pH5.8、8g/lアガロース)上へ移植し、胚盤側が上向きになるように置床した。なお、共存培地には植物成長調節物質を2種類添加した4試験区で試験を実施した。すなわち、植物成長調節物質無添加のホルモンフリー、5μM TIBA(アンチオーキシン)、1.13μM ダイカンバ(Dicamba)(オーキシン)、 11.3μM ダイカンバ(Dicamba)(オーキシン)の4試験区とした。
未熟胚を用いたアグロバクテリウムによるオオムギの形質転換方法で最も用いられている11.3μM ダイカンバ(Tingay et al. 1997 非特許文献5、Jacobsen et al. 2006 非特許文献8、 Bartlett et al. 2008 非特許文献9、 Hensel et al. 2008 非特許文献10、 Harwood et al. 2008 非特許文献11)では、最も遺伝子導入効率が低かった(図1、第4カラム)。1.13μM ダイカンバの添加(図1、第3カラム)では、植物成長調節物質を添加しなかった区(図1、第1カラム)と同程度の遺伝子導入効率が観察された。このことは、Ke et al. (2002 非特許文献17)が1/10濃度のMS培地共存培地に用い、2,4-Dを添加して得られた結果と類似している。なお、アンチオーキシン(5μM TIBA)の添加は、オオムギ未熟胚胚盤への遺伝子導入効率を顕著に高めた(図1、第2カラム)。
共存培地組成が遺伝子導入効率に及ぼす効果(サイトカイニン、アンチオーキシン)
材料および方法
材料およびアグロバクテリウムの接種方法は、実施例1と同一である。なお、共存培地には植物成長調節物質を5種類添加した6試験区を供試した。すなわち、植物成長調節物質無添加のホルモンフリー、5μM 6BA、5μM 4-PU、5μM ゼアチン(Zeatin)(以上、サイトカイニン)、5μM TIBA、5μM パクロブトラゾール(Paclobutrazol)(以上、アンチオーキシン)の6試験区とした。25℃、暗黒下で24時間培養した未熟胚を、実施例1と同じ、0.5 mg/l 2,4-Dおよび1.25 mg/l CuSO4・5H2Oを添加したCIMTレスティング培地(Tingay et al. 1997 非特許文献5)に移植した。 なお、本レスティング培地にはアグロバクテリウム除菌用に250mg/l カルベニシリン 100mg/l セフォタキシムを添加した。25℃暗黒下で2日間培養した後、実施例1と同様にGUS遺伝子の発現を調査し、GUS発現インデックスとし数値化した。供試した未熟胚数は各区15個とした。
植物成長調節物質を添加しなかった区で最も遺伝子導入効率が低く、インデックスは15前後を示した(図2、第1カラム)。その他のサイトカイニン類、アンチオーキシン類は30以上の数値を示した(図2、第2から第6カラム)。このことから、共存培地へのサイトカイニン類、アンチオーキシン類の添加により、オオムギ未熟胚胚盤への遺伝子導入効率が顕著に高まることが初めて明らかとなった。
遠心処理がコンパクトなカルス形成に及ぼす効果
材料および方法
材料およびアグロバクテリウムの接種方法は、実施例1と同一である。なお、共存培地は植物成長調節物質を無添加とした。
遠心処理を実施した試験区からは、80%の効率でコンパクトなカルスが形成されたのに対し(表1、下段)、遠心処理を行っていない試験区では、その1/4以下の17.5%がコンパクトなカルスを形成するにとどまった(表1、上段)。Ke et al. (2002 非特許文献17)において、植物成長調節物質を含まない共存培地で遺伝子導入効率が一定程度向上することが示されていたが、同文献中の「1/10濃度のMS培地で培養を継続すると、植物材料に有害な影響がある。」および「安定的形質転換には、十分な数の遺伝子導入受容細胞が再分化能を維持していると同時に、十分な数のT-DNA導入イベントを植物材料中に生じさせるという優れたバランスを見出す必要がある。」などの記載にあるとおり、そのまま何の処理も加えないでレスティング培養を実施しても、遺伝子導入領域である胚盤細胞からのコンパクトなカルスの形成が強く抑制されることが明らかとなった。一方、遠心処理を実施した試験区では、80%がコンパクトなカルスを形成し、その後のハイグロマイシンによる選抜で形質転換カルスを一定頻度で得られる可能性を示唆した。なお、本発明者らは、アグロバクテリウム接種前の未熟胚に遠心処理や加圧処理を実施した場合も同様なカルス形成促進効果があることを見出している。さらに、本実験系では、アグロバクテリウム接種前の未熟胚に遠心処理および加圧処理を実施しても、共存培養後の未熟胚への遺伝子導入効率は向上せず、あくまでコンパクトなカルスの形成促進に効果が見られた。
遠心処理がハイグロマイシン耐性カルスの形成に及ぼす効果
材料および方法
材料およびアグロバクテリウムの接種方法、熱処理条件、共存工程、遠心処理条件およびレスティング工程は、実施例3と同一である。なお、共存培地にはアンチオーキシンである5μM TIBAを添加した。胚盤を4分割したのち10日間レスティング工程を継続した。レスティング工程後、各分割胚盤切片をさらに4分割(未熟胚当たり16分割)し、50mg/l ハイグロマイシンBおよび1.25mg/l CuSO4・5H2Oを添加したCIMT選抜培地(Tingay et al. 1997 非特許文献5)に移植した。本選抜培地にはアグロバクテリウム除菌用に250mg/l カルベニシリンを添加した。なお各胚盤切片は、由来する未熟胚ごとに培地上に区分けして置床した。25℃暗黒下で 2週間培養した後、水分を多く含むスポンジ状のカルスではなく、コンパクトなカルスを選んで、同組成の2次選抜培地へ移植し、同条件でさらに3週間培養した。培養後、選抜培地上で増殖したハイグロマイシン耐性のコンパクトなカルスをカウントした。なお、同じ未熟胚由来の胚盤切片から複数の耐性カルスが得られた場合があっても、未熟胚当たりでは最大で1つのハイグロマイシン耐性カルスまでをカウントの対象とした。
修正Harwood et al.(2008 非特許文献11)のプロトコルによる従来法では、共存培養終了後、2日目の遺伝子導入効率は、遠心処理なし試験区および遠心処理試験区に比べて、遺伝子導入効率(GUS発現)は低かった(データ非表示)。しかしながら、レスティング工程においてコンパクトなカルスは問題なく形成された。ハイグロマイシンによる選抜により生き残ったカルスは、遠心処理試験区に比べると少なく、結果的に2次選抜までで33.3%の効率にとどまった(表2、上段)。共存培養後に遠心処理を実施しなかった未熟胚では遺伝子導入効率は高かったものの、コンパクトなカルスがほとんど形成されず、選抜培地で生き残るカルスを見出すことはできなかった(表2、中段)。共存培養後に遠心処理を実施した形質転換系では、コンパクトなカルスが数多く形成され、2次選抜までで80%の未熟胚からハイグロマイシン耐性カルスを得ることができた(表2、下段)。この結果は、アンチオーキシンTIBAにより高効率の遺伝子導入を生じた未熟胚胚盤細胞について、遠心処理をしない場合には通常のカルス形成に至らないが、遠心処理をした場合には通常のカルス形成とほぼ同様にコンパクトなカルスが形成され、選抜工程でも従来法と同様に選抜カルスを得られることを示している。
共存培地組成が遺伝子導入効率に及ぼす効果(ホルモンフリー、2,4-D)
材料および方法
材料およびアグロバクテリウムの接種方法は、実施例1と同一である。すなわち、1/10濃度のMS無機塩を含む共存培地を用いた。試験区としては、植物成長調節物質を含まないホルモンフリーの共存培地、1.13μM 2,4-Dならびに11.3μM 2,4-Dを添加した共存培地の3種類を供試した。25℃、暗黒下で24時間共存培養した未熟胚を、実施例1と同じ、0.5 mg/l 2,4-Dおよび1.25 mg/l CuSO4・5H2Oを添加したCIMTレスティング培地(Tingay et al. 1997 非特許文献5)に移植した。 なお、本レスティング培地にはアグロバクテリウム除菌用に250mg/l カルベニシリン 100mg/l セフォタキシムを添加した。25℃暗黒下で2日間培養した後、実施例1と同様にGUS遺伝子の発現を調査し、GUS発現インデックスとし数値化した。供試した未熟胚数は各区15個とした。
植物成長調節物質を添加しなかったホルモンフリーの共存培地で最も遺伝子導入効率が高かった(図3、第1カラム)。これに対し、11.3μM 2,4-Dを添加した共存培地を用いた場合には、遺伝子導入効率は最も低い値を示した(図3、第3カラム)。1.13μM 2,4-Dの添加(図3、第2カラム)でも、ホルモンフリーの共存培地(図3、第1カラム)と比較すると遺伝子導入効率は1/6程度と非常に低い値を示した。実施例1で、2,4-Dと同じオーキシン1.13μM ダイカンバ添加区がホルモンフリー区と同等の遺伝子導入効率を示した結果とは大きく異なった(図1第1カラムおよび図1第3カラム)。このことは、フェノキシ系オーキシンの2,4-Dとベンゾイック系オーキシンのダイカンバとの間で、それらのオーキシン濃度が遺伝子効率に及ぼす影響が異なっていることを示しており、それは、両者の作用性がやや異なることに由来すると考えられる。
アグロバクテリウム接種前と共存培養後における遠心処理がコンパクトなカルス形成に及ぼす効果
材料および方法
材料の調製およびアグロバクテリウムの接種方法は、実施例1と同様である。未熟胚への熱処理も同様に実施した。なお、共存培地にはTIBAを5μM添加した。
アグロバクテリウム接種前遠心処理を実施した試験区(試験区1、表3上段)と共存培養後遠心処理を行った試験区(試験区2、表3下段)では、ほぼ同様の高い効率で遺伝子導入領域である胚盤細胞からのコンパクトなカルスの形成が行われた。すなわち、遠心処理をアグロバクテリウム接種前に行っても、共存培養後に行っても、カルスの形成は促進された。なお、滅菌水中で7,100hPaの強さで5分間加圧した未熟胚でも同様なカルスの形成に対する効果が見られ、アグロバクテリウム接種前でも共存培養後でもその効果に違いは見られなかった。アグロバクテリウム接種前に遠心処理および加圧処理を行っても、遺伝子導入の効率は特に向上しなかった。なお、本実施例6で用いた共存培地を用いて、共存培養期間を48時間(2日間)または72時間(3日間)とさらに長くした試験を実施した。しかしながら、カルス形成が強く抑制され、上記強度の遠心処理をアグロバクテリウム接種前または共存培養後に実施しても、レスティング培地で未熟胚から再分化能を有するコンパクトなカルスはほとんど形成されなかった。
ダイカンバを含む従来法共存培地とMS無機塩を1/10濃度とした共存培地による形質転換効率の比較
材料の調製とアグロバクテリウムの接種については実施例1と同様である。熱処理も同様に実施した。
試験区(1):アグロバクテリウムを接種した未熟胚をHarwood et al.(2008 非特許文献11)に記載の共存培地に胚盤を下向きに置床した。共存培養は、25℃、暗黒下で3日間実施した。この試験区においては遠心処理を行わなかった。
試験区(2):単離した未熟胚を1ml滅菌水が入った2mlマイクロチューブに移し、25℃、15,000rpm (20,000 xg)で10分間遠心処理した。その後は、試験区(1)と同様に共存培養した。
試験区(3):試験区(1)と同様に共存培養を実施したのち、未熟胚を1ml滅菌水が入った2mlマイクロチューブに移し、25℃、15,000rpm (20,000 xg)で10分間遠心処理した。
試験区(4):アグロバクテリウムを接種した未熟胚を100μMアセトシリンゴン含有のオオムギ共存培地(1/10濃度のMS無機塩およびMSビタミン、10g/lグルコース、5μM TIBA、0.5g/l MES、5μM AgNO3および5μM CuSO4、pH5.8、8g/lアガロース)上へ移植し、胚盤が上向きになるように置床した。25℃、暗黒下で24時間培養した。この試験区においては遠心処理を行わなかった。
試験区(5):単離した未熟胚を1ml滅菌水が入った2mlマイクロチューブに移し、25℃、15,000rpm (20,000 xg)で10分間遠心処理した。その後は、試験区(4)と同様に共存培養した。
試験区(6):試験区(4)と同様に共存培養を実施したのち、未熟胚を1ml滅菌水が入った2mlマイクロチューブに移し、25℃、15,000rpm (20,000 xg)で10分間遠心処理した。
試験区(7):試験区(4)の共存培地から植物成長調節物質であるTIBAを除き、試験区(4)と同様に共存培養を実施したのち、未熟胚を1ml滅菌水が入った2mlマイクロチューブに移し、25℃、15,000rpm (20,000 xg)で10分間遠心処理した。
オーキシンのダイカンバを含む従来法共存培地を用いた形質転換では、未熟胚当たりの形質転換効率は試験区1(遠心処理なし)で30%、試験区3(共存培養後遠心処理)で20%であった(表4)。これに対し、未熟胚に遠心処理しMS無機塩類を1/10濃度とした共存培地を用いた3種類の試験区、試験区5(アグロバクテリウム接種前遠心処理)では90%、試験区6(共存培養後遠心処理)では100%、試験区7(ホルモンフリー共存培地および共存培養後遠心処理)でも80%という非常に高い形質転換効率を示した。未熟胚に遠心処理を施さなかった試験区4では、再分化能を有するコンパクトなカルスが形成されず、形質転換体は全く得られなかった。また、試験区2の遠心処理をした後ダイカンバ含有の従来法共存培地で培養した未熟胚からも、全く形質転換体を得ることができなかった。これは、共存培養前に遠心処理を行うことによって、後にカルスが形成されない未熟胚の部分にT-DNAが導入されたことによるものである。実際に、共存培養後のGUSアッセイで、未熟胚の胚軸側の中心付近にGUS遺伝子の発現が検出された。また、共存培地にアンチオーキシンである5μM TIBAを添加した試験区6がホルモンフリーの試験区7より形質転換効率が高い傾向を示した。
形質転換体の自殖次世代における導入遺伝子の遺伝解析
実施例6試験区(6)の異なる未熟胚から得られた2個体のハイグロマイシン耐性かつGUS陽性の形質転換植物を温室において栽培し、得られた未熟種子から1.5~2.0mmの未熟胚を無菌的に採種した。当該未熟胚を、5μM CuSO4を含む再分化培地(Harwood et al.(2008 非特許文献11))に置床し、25℃明条件下で約2週間培養した。得られた自殖次世代(T1)植物体の根の一部を切り取り、1.0 mM 5-ブロモ-4-クロロ-3-インドリル-β-D-グルクロン酸(X-gluc)および20%メタノールを含むリン酸緩衝液に浸漬した。37℃で24時間処理した後、切断根におけるGUS遺伝子の発現を調査した。また、T1植物体を30mg/lハイグロマイシンと5μM CuSO4を含む再分化培地(Harwood et al.(2008 非特許文献11))に置床し、25℃明条件下で約10日間培養することにより、ハイグロマイシン耐性を調査した。
根でGUSの発現が観察されたT1個体は、すべてハイグロマイシン耐性を示した(表5)。根でGUSの発現が認められなかったT1個体は、すべて30mg/lハイグロマイシン含有の再分化培地で褐変し枯死した(表5)。これは、用いた形質転換系統が2系統とも同様であった。また、導入遺伝子の発現個体と非発現個体の比率は、1因子分離の予測値3:1に適合していた(表5)。この結果は、GUS遺伝子とハイグロマイシン耐性遺伝子を含有するT-DNAが、アグロバクテリウム菌株EHA101/pIG121Hmを介してオオムギゲノムに組み込まれ、メンデルの法則に従い自殖次世代に遺伝子したことを明確に示している。
Claims (13)
- オオムギ属(Hordeum)の植物の、未熟胚組織へ、遺伝子導入を行う方法であって、
(i)アグロバクテリウム菌を上記組織へ接種し、該アグロバクテリウム菌の存在下で、以下のa)からc)の1つまたは複数の条件
a)アンチオーキシンを含む、
b)サイトカイニンを含む、
c)フェノキシ系オーキシンを2μM未満の濃度で含むおよび/またはベンゾイック系オーキシンを5μM未満の濃度で含むか、あるいは、フェノキシ系オーキシンおよび/またはベンゾイック系オーキシンを含まない;
を満たす共存培地の中で、上記組織を培養する共存工程を行う、
(ii)アグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記組織を遠心処理および/または加圧処理する工程を行う、
ことを含む、上記方法。 - オオムギ属(Hordeum)の植物の、形質転換植物の作成方法であって、
(i)アグロバクテリウム菌をオオムギの未熟胚組織へ接種し、該アグロバクテリウム菌の存在下で、以下のa)からc)の1つまたは複数の条件
a)アンチオーキシンを含む、
b)サイトカイニンを含む、
c)フェノキシ系オーキシンを2μM未満の濃度で含むおよび/またはベンゾイック系オーキシンを5μM未満の濃度で含むか、あるいは、フェノキシ系オーキシンおよび/またはベンゾイック系オーキシンを含まない;
を満たす共存培地の中で、上記組織を培養する共存工程を行い、
(ii)アグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記組織を遠心処理および/または加圧処理する工程を行い、
(iii)上記組織をレスティング培地で培養するレスティング工程を行い、そして、
(iv)上記組織を再分化培地で再分化させる工程を行う、
ことを含む、上記方法。 - 前記共存工程開始後6時間ないし36時間以内に、レスティング工程を開始する、請求項1または請求項2に記載の方法。
- 前記共存工程開始後12時間ないし24時間以内に、レスティング工程を開始する、請求項3に記載の方法。
- 未熟胚を単離後6時間ないし36時間以内に、共存工程を終えレスティング工程を開始する、請求項1または請求項2に記載の方法。
- 未熟胚を単離後12時間ないし24時間以内に、共存工程を終えレスティング工程を開始する、請求項5に記載の方法。
- 上記未熟胚組織に対するアグロバクテリウム菌の接種前、共存工程中および/または共存工程に次いで、上記未熟胚組織において幼根、幼芽、および胚軸から選択される1またはそれ以上の部位を物理的/化学的に損傷する工程を行う請求項1ないし請求項6のいずれか1項に記載の方法。
- 前記共存培養において前記未熟胚組織を、胚盤側を上向きにし、かつ、胚軸側を前記共存培地に接するように置床して培養する、請求項1ないし請求項7のいずれか1項に記載の方法。
- 以下の形質転換効率向上処理のうち、少なくとも1つを行う、請求項1ないし請求項8のいずれか1項に記載の方法。
a)熱処理;
b)硝酸銀の共存培地への添加;
c)粉体の存在下でアグロバクテリウムを接種する処理; - 上記(iii)レスティング工程と、(iv)再分化工程の間に薬剤選抜工程を含む、請求項1ないし請求項9のいずれか1項に記載の方法。
- (iii)レスティング培地、および/または、薬剤選抜工程の選抜培地が、植物成長調節物質を含む、請求項1ないし請求項10いずれか1項に記載の方法。
- 前記アグロバクテリウム菌が、LBA4404、EHA101、EHA105、AGL0、AGL1、および58C1からなる群から選択される菌である、請求項1ないし請求項11のいずれか1項に記載の方法。
- オオムギ属の植物がオオムギ(H. vulgare)である、請求項1ないし請求項12のいずれか1項に記載の方法。
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AU2011283474A AU2011283474B2 (en) | 2010-07-29 | 2011-07-29 | Method for gene introduction into hordeum plant using agrobacterium, and method for production of transformed plant of hordeum plant |
EP11812628.3A EP2599382B1 (en) | 2010-07-29 | 2011-07-29 | Method for gene transfer into plant belonging to genus hordeum using agrobacterium bacterium, and method for production of transgenic plant of plant belonging to genus hordeum |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014003976A (ja) * | 2012-05-31 | 2014-01-16 | National Agriculture & Food Research Organization | 植物成長阻害ホルモンを用いた植物形質転換方法 |
US11180770B2 (en) | 2017-03-07 | 2021-11-23 | BASF Agricultural Solutions Seed US LLC | HPPD variants and methods of use |
US11371056B2 (en) | 2017-03-07 | 2022-06-28 | BASF Agricultural Solutions Seed US LLC | HPPD variants and methods of use |
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US8806599B2 (en) * | 2012-06-11 | 2014-08-12 | Symantec Corporation | Systems and methods for implementing multi-factor authentication |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998054961A2 (en) | 1997-06-02 | 1998-12-10 | Novartis Ag | Plant transformation methods |
WO2002012521A1 (fr) | 1999-06-04 | 2002-02-14 | Japan Tobacco Inc. | Procede permettant d'ameliorer l'efficacite du transfert de genes dans des cellules vegetales |
WO2002012520A1 (fr) | 1999-06-04 | 2002-02-14 | Japan Tobacco Inc. | Procede permettant d'ameliorer l'efficacite du transfert de genes dans des cellules vegetales |
WO2005017169A1 (ja) | 2003-08-13 | 2005-02-24 | Japan Tobacco Inc. | 植物材料への遺伝子導入を行う方法 |
WO2007069301A1 (ja) * | 2005-12-13 | 2007-06-21 | Japan Tobacco Inc. | 粉体を用いて形質転換効率を向上させる方法 |
WO2008105509A1 (ja) * | 2007-02-28 | 2008-09-04 | Japan Tobacco Inc. | 選抜工程を経ないアグロバクテリウム菌による形質転換植物の作成方法 |
WO2009122962A1 (ja) * | 2008-03-31 | 2009-10-08 | 日本たばこ産業株式会社 | アグロバクテリウム菌による形質転換植物の作成方法 |
WO2011013764A1 (ja) * | 2009-07-29 | 2011-02-03 | 日本たばこ産業株式会社 | アグロバクテリウム菌を用いた、コムギ属の植物へ遺伝子導入を行う方法、コムギ属の植物の形質転換植物の作成方法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BRPI0510189A (pt) * | 2004-05-13 | 2007-10-02 | Basf Ag | misturas fungicidas para combater fungos nocivos fitopatogênicos, agente, processo para combater fungos nocivos fitopatogênicos, semente, e, uso dos compostos |
JP4631689B2 (ja) | 2005-12-14 | 2011-02-16 | ダイキン工業株式会社 | イオン伝導体 |
WO2008105508A1 (ja) * | 2007-02-28 | 2008-09-04 | Japan Tobacco Inc. | 3,6-ジクロロ-o-アニシン酸を含む共存培地で植物組織を培養する共存工程を含む、植物の形質転換効率を上昇させる方法 |
DE102010013474B4 (de) | 2010-03-30 | 2015-08-13 | Thiele Gmbh & Co. Kg | Kettenschloss |
-
2010
- 2010-07-29 JP JP2010170871A patent/JP2013212051A/ja not_active Withdrawn
-
2011
- 2011-07-29 CA CA2806822A patent/CA2806822A1/en not_active Abandoned
- 2011-07-29 AU AU2011283474A patent/AU2011283474B2/en active Active
- 2011-07-29 EP EP11812628.3A patent/EP2599382B1/en active Active
- 2011-07-29 WO PCT/JP2011/067493 patent/WO2012015039A1/ja active Application Filing
- 2011-07-29 US US13/812,412 patent/US9284567B2/en active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998054961A2 (en) | 1997-06-02 | 1998-12-10 | Novartis Ag | Plant transformation methods |
WO2002012521A1 (fr) | 1999-06-04 | 2002-02-14 | Japan Tobacco Inc. | Procede permettant d'ameliorer l'efficacite du transfert de genes dans des cellules vegetales |
WO2002012520A1 (fr) | 1999-06-04 | 2002-02-14 | Japan Tobacco Inc. | Procede permettant d'ameliorer l'efficacite du transfert de genes dans des cellules vegetales |
WO2005017169A1 (ja) | 2003-08-13 | 2005-02-24 | Japan Tobacco Inc. | 植物材料への遺伝子導入を行う方法 |
WO2007069301A1 (ja) * | 2005-12-13 | 2007-06-21 | Japan Tobacco Inc. | 粉体を用いて形質転換効率を向上させる方法 |
WO2007069643A1 (ja) | 2005-12-13 | 2007-06-21 | Japan Tobacco Inc. | 粉体を用いて形質転換効率を向上させる方法 |
WO2008105509A1 (ja) * | 2007-02-28 | 2008-09-04 | Japan Tobacco Inc. | 選抜工程を経ないアグロバクテリウム菌による形質転換植物の作成方法 |
WO2009122962A1 (ja) * | 2008-03-31 | 2009-10-08 | 日本たばこ産業株式会社 | アグロバクテリウム菌による形質転換植物の作成方法 |
JP2011120478A (ja) * | 2008-03-31 | 2011-06-23 | Japan Tobacco Inc | アグロバクテリウム菌による形質転換植物の作成方法 |
WO2011013764A1 (ja) * | 2009-07-29 | 2011-02-03 | 日本たばこ産業株式会社 | アグロバクテリウム菌を用いた、コムギ属の植物へ遺伝子導入を行う方法、コムギ属の植物の形質転換植物の作成方法 |
Non-Patent Citations (31)
Title |
---|
BARTLETT, J. G.; ALVES, S. C.; SMEDLEY, M.; SNAPE, J. W.; HARWOOD, W. A.: "High-throughput Agrobacterium-mediated barley transformation", PLANT METHODS, vol. 4, 2008, pages 1 - 12 |
CHENG ET AL.: "Invited review: Factors influencing Agrobacterium-mediated transformation of monocotyledonous species", IN VITRO CELL. DEV. BIOL. PLANT, vol. 40, 2004, pages 31 - 45, XP008053780, DOI: doi:10.1079/IVP2003501 |
CHENG ET AL.: "Invited review: Factors influencing Agrobacterium-mediated transformation of monocotyledonous species", IN VITRO CELL., DEV. BIOL. PLANT, vol. 40, 2004, pages 31 - 45, XP008053780, DOI: doi:10.1079/IVP2003501 |
CHENG, M.; FRY, J. E.; PANG, S.; ZHOU, H.; HIRONAKA, C. M.; DUNCAN, D. R.; CONNER, T. W.; WAN, Y.: "Genetic transformation of wheat mediated by Agrobacterium tumefaciens", PLANT PHYSIOL., vol. 115, 1997, pages 971 - 980, XP002918248 |
CHU, C.-C.: "The N6 medium and its applications to another culture of cereal crops", PROC. SYMP. PLANT TISSUE CULTURE. PEKING: SCIENCE PRESS, 1978, pages 43 - 50 |
FRAME ET AL.: "Agrobacterium protocols", vol. 343, 1, 2006, HUMANA PRESS INC., article "Maize (Zea mays L.) Methods in Molecular Biology", pages: 185 - 199 |
GARFINKEL, D. J.; NESTER, E.W.: "Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism", JOURNAL BACTERIOLOGY, vol. 1144, 1980, pages 732 - 43 |
HARWOOD, W. A.; BARTLETT, J. G.; ALVES, S. C.; PERRY, M.; SMEDLEY, M. A.; LEYLAND, N.; SNAPE, J. W.: "Method in Molecular Biology, Transgenic Wheat, Barley and Oats", vol. 478, 2008, HUMAN PRESS INC., article "Barley transformation using Agrobacterium-mediated techniques", pages: 137 - 147 |
HENSEL G. ET AL.: "Efficient generation of transgenic barley: The way forward to modulate plant-microbe interactions", J.PLANT PHYSIOL., vol. 165, 2008, pages 71 - 82, XP022393011 * |
HENSEL, G.; VALKOV, V.; MIDDLEFELL-WILLIAMS, J.; JOCHEN KUMLEHN, J.: "Efficient generation of transgenic barley: The way forward to modulate plant-microbe interactions", JOURNAL OF PLANT PHYSIOLOGY, vol. 165, pages 71 - 82, XP022393011, DOI: doi:10.1016/j.jplph.2007.06.015 |
HIEI Y. ET AL.: "Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection wit Agrobacterium tumefaciens", PLANT CELL TISSUE ORGAN CULTURE, vol. 87, 2006, pages 233 - 243, XP019454849 * |
HIEI, Y.; ISHIDA, Y.; KASAOKA, K.; KOMARI, T.: "Improved frequency of transformation of rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens", PLANT CELL TISSUE AND ORGAN CULTURE, vol. 87, 2006, pages 233 - 243, XP019454849, DOI: doi:10.1007/s11240-006-9157-4 |
HIEI, Y.; OHTA, S.; KOMARI, T.; KUMASHIRO, T.: "Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA", THE PLANT JOURNAL, vol. 6, 1994, pages 271 - 282 |
ISHIDA, Y.; SAITO, H.; HIEI, Y.; KOMARI, T.: "Improved protocol for transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens", PLANT BIOTECHNOLOGY, vol. 20, 2003, pages 57 - 66, XP009070864 |
ISHIDA, Y.; SAITO, H.; OHTA, S.; HIEI, Y.; KOMARI, T.; KUMASHIRO, T.: "High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens", NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 745 - 750, XP002046364, DOI: doi:10.1038/nbt0696-745 |
JACOBSEN ET AL.: "Agrobacterium protocols", vol. 343, 1, 2006, HUMANA PRESS INC., article "Barley (Hordeum vulgare L.) Methods in Molecular Biology", pages: 171 - 183 |
KE ET AL.: "Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat", EUPHYTICA, vol. 126, 2002, pages 333 - 343 |
LINSMAIER, E.; SKOOG, F.: "Organic growth factor requirements of tobacco tissue culture", PHYSIOL. PLANT., vol. 18, 1965, pages 100 - 127, XP000617350 |
MATTHEWS, P. R.; WANG, M-B.; WATERHOUSE, P. M.; THORNTON, S.; FIEG, S. J.; GUBLER, F.; JACOBSEN, J. V.: "Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector", MOLECULAR BREEDING, vol. 7, 2001, pages 195 - 202, XP002464592, DOI: doi:10.1023/A:1011333321893 |
MURASHIGE, T.; SKOOG, F.: "A revised medium for rapid growth and bio assays with tobacco tissue cultures", PHYSIOL PLANT, vol. 15, 1962, pages 473 - 497, XP002577845, DOI: doi:10.1111/j.1399-3054.1962.tb08052.x |
MURRAY, F.; BRETTELL, R.; MATTHEWS, P.; BISHOP, D.; JACOBSEN, J.: "Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes", PLANT CELL REPORTS, vol. 22, 2004, pages 397 - 402 |
NEGROTTO, D.; JOLLEY, M.; BEER, S.; WENCK, A. R.; HANSEN, G.: "The use of phosphomannose-isomerase as a selection marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation", PLANT CELL REPORTS, vol. 19, 2000, pages 798 - 803 |
POTRYKUS, I.: "Gene transfer to cereals: an assessment", BIO/TECHNOLOGY, vol. 8, 1990, pages 535 - 542, XP008080605, DOI: doi:10.1038/nbt0690-535 |
See also references of EP2599382A4 * |
SERHANTOVÁ, V.; EHRENBERGEROVÁ, J.; OHNOUTKOVÁ, L.: "Callus induction and regeneration efficiency of spring barley cultivars registered in the Czech Republic", PLANT, SOIL AND ENVIRONMENT, vol. 50, 2004, pages 456 - 462 |
TINGAY, S.; MCELROY, D.; KALLA, R.; FIEG, S.; WANG, M.; THORNTON, S.; BRETTELL, R.: "Agrobacterium tumefaciens-mediated barley transformation", PLANT J., vol. 11, 1997, pages 1369 - 1376, XP009080096, DOI: doi:10.1046/j.1365-313X.1997.11061369.x |
TRIFONOVA, A.; MADSEN, S.; OLESEN, A.: "Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions", PLANT SCIENCE, vol. 161, 2001, pages 871 - 880, XP002958562, DOI: doi:10.1016/S0168-9452(01)00479-4 |
WAN, Y.; LEMAUX, P. G.: "Generation of large numbers of independently transformed fertile barley plants", PLANT PHYSIOLOGY, vol. 104, 1994, pages 37 - 48, XP002407772 |
WATSON ET AL.: "Plasmid required for virulence of Agrobacterium tumefaciens", J. BACTERIOL., vol. 123, 1975, pages 255 - 264 |
ZHAO, Z.-Y.; CAI, T.; TAGLIANI, L.; MILLER, M.; WANG, N.; PENG, H.; RUDERT, M.; SCHOEDER, S.; HONDRED, D.; SELTZER, J.: "Agrobacterium-mediated sorghum transformation", PLANT MOL. BIOL., vol. 44, 2000, pages 789 - 798, XP002533153, DOI: doi:10.1023/A:1026507517182 |
ZHAO, Z.-Y.; GU, W.; CAI, T.; TAGLIANI, L.; HONDRED, D.; BOND, D.; SCHROEDER, S.; RUDERT, M.; PIERCE, D.: "High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize", MOL. BREED., vol. 8, 2001, pages 323 - 333, XP001100034, DOI: doi:10.1023/A:1015243600325 |
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CA2806822A1 (en) | 2012-02-02 |
US20130125266A1 (en) | 2013-05-16 |
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EP2599382B1 (en) | 2017-07-19 |
JP2013212051A (ja) | 2013-10-17 |
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US9284567B2 (en) | 2016-03-15 |
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