JP2000023675A - Transformation of corn with agrobacterium that is applicable over a wide range of varieties - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the transformation efficiency of plants, e.g. nice, corn and the like, and to obtain transformed plants having excellent herbicide- resistant property, pest-resistant property, cold-resistant property or drying- resistant property by cultivating plant tissues inoculated with Agrobacterium bacteria under a specific condition. SOLUTION: The transformed plant is obtained by cultivating plant tissues of corn (premature embryo) or the like inoculated with Agrobacterium bacteria in a culture medium containing a substance (the concentration is preferably 10 μM), e.g. silver nitrate, silver thiosulfate complex salt or the like, which inhibits the effect of ethylene or a substance (the concentration is preferably 0.5-1 μm), e.g. an ACC synthase inhibitor such as aminoethoxyvinyl glycine or the like, which inhibits the biosynthesis of ethylene. Further, transformed cells are preferably prepared by cultivating them in a medium containing carbenicillin (the concentration is preferably 100-250 mg/l).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for transforming maize with Agrobacterium which can be applied to a wide variety of varieties.

[0002]

2. Description of the Related Art As methods for transforming monocotyledonous plants such as maize and rice, which are main cereals, an electroporation method and a particle gun method have been conventionally known. However, these physical gene transfer methods have problems such as the introduction of multiple copies of the gene, the inability to insert the gene in an intact form, and the occurrence of many malformations and sterility in transformed plants.

[0003] A gene transfer method using Agrobacterium is commonly used as a method for transforming dicotyledonous plants. The host of Agrobacterium is limited to dicotyledonous plants and is not expected to infest monocotyledonous plants (De Cleene and De Ley1976; Reference 3), but transforms monocotyledonous plants with Agrobacterium Attempts have been made.

[0004] Grimsley et al., T-DNA of Agrobacterium
Maize streak virus
virus) was inoculated into corn growth points, and it was reported that corn streak virus infection was confirmed. The above observations are interpreted as indicating that Agrobacterium can transfer DNA into corn, since inoculation of corn streak virus DNA alone does not show such infectious symptoms (Grimsley et al. 1987;
Reference 5). However, this result does not indicate that the T-DNA was integrated into the nucleus, since the virus can multiply without being integrated into the nuclear genome. Thereafter, the infection efficiency was highest when inoculated into the corn shoot apex (Grimsley et al. 1988; Reference 6), indicating that the Agrobacterium plasmid VirC gene is essential for infection. (Grimsley et al. 1989; reference 7).

[0005] Gould et al. Wound a corn growth point with a needle, inoculated with a strongly pathogenic Agrobacterium EHA1 having a kanamycin resistance gene and a β-glucuronidase gene (GUS gene), and grown after treatment. When the points were selected with kanamycin, plants showing resistance were obtained. Southern analysis was performed to confirm that the progeny seed had the introduced gene. As a result, the transgene was confirmed in some of the seeds (Gould et al. 1991; Reference 4). This means
This shows that transformed cells and non-transformed cells were mixed in the plants obtained by selecting kanamycin from the growth points treated with Agrobacterium (chimerism).

Have attempted to introduce a kanamycin resistance gene into wheat embryos using Agrobacterium. First, the embryo was treated with an enzyme to damage the cell wall, and then inoculated with Agrobacterium. Of the treated calli, very few kanamycin-resistant calli proliferated, but plants could not be regenerated from this callus. When the presence of the kanamycin resistance gene was confirmed by Southern analysis, structural changes in the transgene were observed in all resistant calli (Mooney et al. 199).
1; Reference 11).

[0007] Raineri et al. After scratching the rice scutellum,
Strongly pathogenic Agrobacterium A281 (pTiBo542)
When treated with eight varieties, tumor-like tissue growth was observed in two varieties, Nipponbare and Fujisaka-5. Furthermore, the kanamycin resistance gene and the β-glucuronidase gene (GUS gene) are added to the Ti plasmid obtained by removing the hormone synthesis gene from T-DNA.
Inoculation of Agrobacterium harboring the plasmid into which rice was inserted into rice embryos resulted in the growth of kanamycin-resistant calli. In the resistant calli, expression of the GUS gene was observed, but no transformed plant could be obtained. From these facts, we interpret that Agrobacterium T-DNA was introduced into rice cells (Raineri et a
l. 1990; reference 14).

[0008] As described above, research reports have been made suggesting that gene transfer by Agrobacterium is possible even in gramineous crops such as rice, corn, and wheat, but all have problems in reproducibility. However, the confirmation of the introduced gene was incomplete, and no persuasive results were shown (Potrykus 1990; Reference 13).

Chan et al., After injury to a rice immature embryo cultured for 2 days in the presence of 2,4-D, a kind of hormone, possessed the npt II gene and the GUS gene in a medium containing potato suspension cultured cells. Agrobacterium was inoculated. When the treated immature embryos were cultured on a G418-supplemented medium, regenerated plants were obtained from the induced calli. When the location of the GUS gene in the regenerated and progeny plants was confirmed by Southern analysis, it was reported that the presence of the transgene was observed in both the regenerated and progeny plants (Chan et
al. 1993; Reference 2). This result supports the transformation of rice by Agrobacterium, but the transformation efficiency is very low at 1.6%, and the regenerated plant showed normal growth with respect to 250 immature embryos tested. Was only one individual. Since enormous effort is required to remove immature embryos from rice, it is hard to say that such a low transformation efficiency is at a practical level.

In recent years, by using a superbinary vector having a part of the virulence gene of a strongly pathogenic Agrobacterium, stable and highly efficient transformation can be performed even in monocotyledonous plants such as rice and corn. (Hiei et al. 1994; Reference 8, Ishida et al.
1996; Reference 9). According to these reports, Agrobacterium-mediated transformation is stable and highly efficient, and the resulting transformed plant has few mutations, the introduced gene has a low copy number, and It has the advantage of many intact forms.

[0011] However, transformation by Agrobacterium using a superbinary vector can provide transformed plants with an efficiency of 5 to 30% in maize. However, adaptable varieties are limited. Transformation of H99, a typical variety, is extremely inefficient, so that it is necessary to improve the method to be applicable to a wide variety of varieties in order to put this method to practical use. In addition, even when using a public corn cultivar A188 that has been successfully transformed using a super binary vector as a material, the transformation efficiency is low with the co-transformation vector, and the transformation efficiency is further reduced with A188. Enhancing is also important for use in raising practical varieties.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to transform corn varieties whose transformation efficiency was extremely low by the conventional Agrobacterium method, and also to transform corn varieties that can be transformed by the conventional method. It is to provide a way to increase efficiency. More specifically, the present invention provides, for the first time, a method for stably producing a transformed plant of H99, which is a public cultivar of maize, by a transformation method using Agrobacterium. The present invention also provides a method for improving the transformation efficiency of A188, which is a public corn variety, by at least 20% as compared with the conventional method. further,
The present invention provides a method capable of performing stable and high-frequency transformation even with A188, which is relatively easy to transform, or a method using a co-transformation vector with low transformation efficiency.

[0013]

DISCLOSURE OF THE INVENTION The present inventors have formed a callus, particularly a type I callus, from an immature embryo after Agrobacterium infection using a corn variety H99 which cannot be transformed by the conventional Agrobacterium method as a material. The media additives were examined using the rate as an index. As a result, callus induced from immature embryos in a medium containing a substance that inhibits the action of ethylene, for example, silver nitrate, is a type I callus, and conventionally, it was considered that there was no apparent effect on the formation of type I callus. It was found that silver nitrate was extremely effective in inducing type I calli in immature embryos infected with Agrobacterium.

[0014] Type I calli are one of the corn dedifferentiated calli, and are relatively compact and have a low degree of dedifferentiation. On the other hand, type II calli are flyable in shape,
The callus is often used in conventional transformation tests because it is suitable for isolating protoplasts, but it is a callus that is difficult to handle because it easily loses its redifferentiation ability.

The present invention has been completed based on the above findings, and inoculates a plant tissue with Agrobacterium having a plasmid containing a gene having a preferable trait, and inhibits the plant tissue from acting on ethylene. This is a method for efficiently obtaining callus of a plant transformed with the above gene by culturing in a medium containing a substance or a substance that inhibits the biosynthesis of ethylene. The callus of the transformed plant thus obtained can be regenerated into a complete plant. Hereinafter, the configuration of the present invention will be specifically described.

Inhibits the action of ethylene or its biosynthesis
That substance Ethylene fruit ripening, flower senescence, growth control of plant morphogenesis, leaf, fruit drop, germination, flowering, epinasty, degradation of chlorophyll, a hormone with physiological effects such as promotion of respiration, enzyme It is involved in many physiological functions of plants through induction and promotion of metabolism. Ethylene is produced when these physiological responses occur, and is induced by various external stress factors such as injury, disease, physical load, low temperature, dryness, flooding, heavy metals, and toxic gases (Hyoto, 1994; reference 21). In higher plants, ethylene is produced by the methionine-ACC pathway, which includes ACC
Two enzymes, synthase and ACC oxidase, play important roles.

Substances that inhibit the action of ethylene used in the present invention include substances that directly inhibit the action of ethylene. For example, silver nitrate is known as a substance that inhibits the action of ethylene, and has been added to the medium of plant tissue culture for the purpose of reducing the damage caused by ethylene generated during culture (Ma et al. 1998; Reference 10, Tiainen et al. 199
2; Reference 17). Even in corn, the addition of silver nitrate to the culture medium increases the induction rate of type II callus (Carvalho e
t al. 1997; Reference 1, Songstad et al. 1991; Reference 16, Vai
n et al. 1989a; Reference 19) or improve the rate of regeneration (Songstad et al. 1988; Reference 15).
Among them, Vain et al. Added that the addition of silver nitrate to the culture medium increased the formation rate of type II callus, but had no apparent effect on the formation of type I callus (Vain et al. 19).
89b; reference 20). As a substance that inhibits the action of ethylene,
In addition to silver nitrate, silver thiosulfato complex (STS), 2,5-norbornadiene (NBD), 1-methylcyclopropene (1-M
CP) are known.

The concentration of the substance which directly inhibits the action of ethylene in the medium is, for example, 0.1 to 1,000 μM, preferably 1 to 100 μM, more preferably 5 to 50 μM in the case of silver nitrate.

Another group of substances which inhibit the action of ethylene include the inhibitors of ethylene synthesis. Ethylene is S-
Adenosyl-L-methionine (SAM) is converted to ACC by 1-aminocyclopropene-1-carboxylate (ACC) synthase, and ACC is converted to ethylene by ACC oxidase. That is, in order to suppress the synthesis of ethylene, the synthesis of SAM to ACC may be suppressed, or the synthesis of ACC to ethylene may be suppressed. In order to suppress the synthesis of ACC from SAM, an ACC synthase inhibitor may be used. In order to suppress the synthesis of ACC to ethylene, an ACC oxidase inhibitor may be used.

Substances that inhibit ethylene biosynthesis include AC
Examples include aminoethoxyvinylglycine (AVG) and aminooxyacetic acid, which are C synthase inhibitors, and cobalt ion and normal-propyl gallate, which are ACC oxidase inhibitors.

The concentration of the ACC synthase inhibitor in the medium is, for example, 0.1 to 10 in the case of aminoethoxyvinylglycine.
μM, preferably 0.5-1 μM. The concentration of the ACC oxidase inhibitor in the medium is, for example, 1-100 μM, preferably 1-10 μM in the case of cobalt ions.

[0022] Plants which may be subject to vaccination methods Agrobacterium transformation method according fungi, including monocots and dicots,
Many plants are included. Preferred plants are monocots. More preferably, corn, which is a major cereal,
Monocotyledonous plants such as rice, particularly corn.

The plant tissue to be inoculated with Agrobacterium is preferably an immature embryo, but may be a type I callus, a type II callus, a suspension cultured cell, a growth point, or the like.

Agrobacterium is inoculated into a plant tissue, containing a plasmid having a gene for imparting a desirable trait to a plant inserted into T-DNA. This allows
Transform plant cells in the tissue. Methods for this are well known and are described, for example, in Grimsley (Reference 5). Preferred methods are described in Hiei et al. (8) and Ishida et al.
This is a method using a super binary vector described in (Reference 9). The super binary vector is a binary vector in which a part of the Vir region of a strongly pathogenic bacterial system having a high infectivity among Agrobacterium tumefaciens bacterial systems is arranged in a T-DNA-containing plasmid.
For example, LBA4404 / pTOK233, LBA4404 / pSB131 and the like can be mentioned. A method using a co-transformation vector (WO95 / 16031 and Reference 29) can also be used. Ko
Transformation vectors are two unconnected T-DNs in one Agrobacterium.
This is a vector in which A is placed. For example, LBA4404 / pSB424, L
BA4404 / pSB324 and the like.

Agrobacterium having a superbinary vector or a vector for co-transformation is placed in a suitable liquid medium (referred to herein as a bacterial suspension medium) at about 1 × 10 ⁸ to 1 × 10 ⁹ cfu / It is preferable that the suspension is carried out to a concentration of ml and the suspension is brought into contact with plant cells.

Agrobacterium inoculation is performed by a method other than the method using a super binary vector or a co-transformation vector, if the gene for transformation is incorporated into the genome of the plant tissue cells. Is also good.

The Agrobacterium which can be used for inoculation may be any strain which is commonly used in such a method, for example, Agrobacterium rhizogenes and Agrobacterium tuberculosis. Mefaciens (Agrobacterium tumefac
iens), preferably Agrobacterium tumefaciens.

Culture of inoculated cells Plant cells are cultured in the presence of Agrobacterium to ensure inoculation. The medium for that is referred to herein as a "co-culture medium". This medium may be one commonly used for maintaining plant cells. The culture temperature may be appropriately selected. Usually, the cells are cultured in a dark place.

Subsequently, the plant cells are transferred to a callus induction medium to form callus. Substances that inhibit the action of ethylene need to be added to the callus induction medium. As a result, the callus formation rate is greatly improved, and the callus formed is a type I callus. Formation of type I callus can be confirmed by observation with the naked eye or a stereoscopic microscope. Type I calli are preferred because they have less loss of regeneration potential than type II calli and can be easily regenerated into a desired transformed plant.

Further, an antibiotic is added to the callus induction medium in order to remove Agrobacterium. For example, cefotaxime is often added to the culture medium to select for transformed cells from plant tissues after Agrobacterium infection or to regenerate plants from transformed cells to remove Agrobacterium attached to the tissues. Antibiotics to be added. Many reports of plant transformation by Agrobacterium use cefotaxime for eradication (Chan et al. 1993; Reference 2, Hiei et al. 1994; Reference
8, Ishida et al. 1996; Reference 9, Uze et al. 1997; Reference 1
8) However, Orlikowska et al. Found that the addition of cefotaxime reduced the rate of plant regeneration from rose leaf pieces (Orlikows
ska et al. 1996; reference 12). Carbenicin is also a kind of antibiotic like cefotaxime, and can be used for removing Agrobacterium after co-culture. Use of carbenicin in the method of the present invention is particularly preferred because it further enhances callus formation efficiency.

In practicing the present invention, the medium to be used is not particularly limited as long as it does not lose or reduce the effect of inhibiting the action of ethylene or the effect of inhibiting the biosynthesis of ethylene. White, M
S (LS), B5, Nitsch-Nitsch, Schenk-Hildebrandt, a medium containing an inorganic salt such as N6 (References 22 to 2)
8). Among these media, preferred media are White, M
A medium containing an inorganic salt such as S (LS), B5, and Schenk-Hildebrandt can be mentioned. A particularly preferred medium is MS
Examples of the medium include an inorganic salt of (LS). These media include carbon sources such as sucrose; various vitamins; various amino acids; boiled liquids or extracts of potatoes, coconut, etc .; auxins, cytokinins, abscisic acid, gibberellins or auxins for inducing cell division and morphogenesis. If necessary, a growth regulator or the like having an action similar to these may be included.

Selection of Transformed Callus In the method of the present invention, callus which has obtained the target gene by transformation is selected as necessary. Selection of transformed plants can be performed by adding an appropriate antibiotic to a medium for forming callus and determining whether or not the medium has resistance. In this case, as a selection marker, a commonly used marker can be used by a conventional method. For example, an antibiotic resistance gene such as hygromycin, phosphinothricin (PPT), or kanamycin is exemplified. At this time, it is preferable to introduce into the vector, in addition to the desired gene, an antibiotic-resistant gene added to the medium. A test for confirming the production of the desired protein from the adjusted host cells
It can be performed as follows. That is, a reporter gene may be introduced into the host cell prepared as described above at a desired gene site of the vector, a protein serving as a reporter may be produced from the host cell, and the protein may be detected.
Here, as the reporter gene used for the desired gene site, chloramphenylchol acetyltransferase (CAT), β-glucuronidase (GUS), luciferase gene, green fluorescence protein (GFP)
And the like. The callus selection may be repeated a plurality of times, in which case the timing of adding the substance that inhibits the action of ethylene to the medium is preferably at the initial selection stage.

[0033] In reproducing the present invention differentiation and complete plants of callus, callus obtained by adding a substance that inhibits the action or the biosynthesis of ethylene in the medium sequentially cultured in regeneration medium and rooting medium Then, it can be easily differentiated and regenerated into a complete plant.

[0034]

According to the method of the present invention, the efficiency of transformation of plants such as rice and corn is improved. Also,
Maize varieties, which were extremely low in transformation efficiency by conventional methods, can also be transformed. Therefore, it is possible to easily and quickly improve the plant variety. Preferred traits imparted to plants include, but are not limited to, herbicide resistance, pest resistance, cold resistance, drought resistance, and the like.

[0035]

The present invention will be described in more detail with reference to the following examples. Examples 1 to 4 are examples showing that the formation rate of type I callus is improved by the method of the present invention, and Examples 5 and 6 are examples in which callus transformed with the target gene was also selected. is there. The compositions of the various media used in the examples are summarized in Table 5 below.

Example 1 Immature embryo of maize variety H99 (size 1.0-1.2 mm)
Was aseptically removed, infected with Agrobacterium tumefaciens super binary vector LBA4404 (pSB131) (Reference 9) suspended in a liquid medium at a concentration of about 1 x 10 ⁹ cfu / ml, and placed on a co-culture medium, followed by 25 The cells were cultured in the dark at ℃ for 3 days. Cultured immature embryos with 10 μM silver nitrate (AgNO ₃ ) and 250 m
The plate was placed on a callus induction medium containing either g / l cefotaxime or 100 mg / l carbenicillin (a composition obtained by removing phosphinothricin (PPT) from the selection medium). 25 ° C,
After culturing in the dark for 10 days, the type I callus formation rate was examined.

The results are shown in FIG. When the antibiotic added to the medium for removing Agrobacterium bacteria after co-culture was cefotaxime, the callus formation rate from H99 immature embryos was about 10%. Addition of silver nitrate at a concentration of 10 μM to a medium of the same composition improved the callus formation rate to 30% or more.
When carbenicillin was used as the antibiotic, the callus formation rate was 30% or more even when silver nitrate was not added. Furthermore, when silver nitrate was added to the carbenicillin-supplemented medium, the callus formation rate was improved to nearly 60%. Thus, by replacing the conventional cefotaxime and silver nitrate-free medium with carbenicillin and silver nitrate-containing medium, the callus formation rate was about 5%.
Carbenicillin and silver nitrate were found to be effective for callus formation from H99 immature embryos infected with Agrobacterium.

Example 2 Similar to silver nitrate, silver thiosulfate complex (silver thiosulfate) used as an inhibitor of ethylene in tissue culture and the like
ate, STS) on callus formation. H
99 immature embryos were cultured under the same conditions as in Example 1 using LBA4404 (pSB131).
After infection and co-culture, 5-30 μM STS or 10 μM
The plate was placed on a callus induction medium containing silver nitrate and 100 mg / l carbenicillin. After culturing at 25 ° C in the dark for 2 weeks, type I
The callus formation rate was investigated.

FIG. 2 shows the results. The callus formation rate in the medium without STS or silver nitrate was about 40%. On the other hand, in the medium supplemented with STS, 8
It showed a callus formation rate of 0% or more, and was found to have the same effect as silver nitrate.

Example 3 The effect of aminoethoxyvinylglycine (AVG), an inhibitor of ACC synthase, on callus formation was examined. HBA immature embryos were cultured under the same conditions as in Example 1 using LBA4404 (pSB
131), coculture with 0.5-3 μM AVG or 10
The plate was placed on a callus induction medium containing μM silver nitrate and 250 mg / l cefotaxime. After culturing at 25 ° C. in the dark for 2 weeks, the type I callus formation rate was examined.

FIG. 3 shows the results. The callus formation rate in the medium without AVG or silver nitrate was about 30%. In contrast, in the medium supplemented with 0.5 and 1 μM AVG,
It showed a callus formation rate of about 60%, and it was clarified that it had an effect of increasing the callus formation rate like silver nitrate.

Example 4 The effect of cobalt ions, which are inhibitors of ACC oxidase, on callus formation was examined. H99 immature embryos were infected with LBA4404 (pSB131) under the same conditions as in Example 1, cocultured, and then placed on a callus induction medium containing 10 μM cobalt chloride hexahydrate or silver nitrate and 250 mg / l cefotaxime. . After culturing at 25 ° C in the dark for 10 days, the type I callus formation rate was examined.

FIG. 4 shows the results. The callus formation rate in the control medium without cobalt chloride or silver nitrate was
It was less than 10%. On the other hand, the culture medium to which cobalt chloride was added exhibited a callus formation rate of 30% or more, and it was clarified that there was an effect of increasing the callus formation rate as in the case of silver nitrate.

Example 5 The immature embryos of the maize public varieties A188 and H99 were subjected to LBA4404 (pSB131) or LBA4404 (pSB
624) (the structure of which is shown in FIG. 5) was infected and co-cultured. The immature embryos after co-culture were placed on a selection medium containing 10 μM silver nitrate, 100 mg / l carbenicillin and 5 mg / l phosphinothricin (PPT). The H99 immature embryos were cultured in a medium of the same composition without PPT for 3-4 days before being placed on the selection medium, and then placed on a selection medium containing PPT. After culturing for 2 weeks, the immature embryos were placed on a medium having the same composition containing 10 mg / l PPT and cultured for 3 weeks. The grown calli were cut into small pieces having a size of 1-2 mm, placed on a medium having the same composition, and cultured for 3 weeks. All cultures on the selection medium were performed at 25 ° C. in the dark. The grown calli were again cut into small pieces, placed on regeneration medium containing 250 mg / l cefotaxime and 5 mg / l PPT, and
The cells were cultured at 2 ° C. under illumination for 2 weeks. The redifferentiated seedlings were transferred to a rooting medium and then potted. Cut off leaf pieces of young plant
X-gluc was used to investigate the expression of the GUS gene.

The transformation results when A188 was infected with LBA4404 (pSB131) are shown in Table 1. While the transformation efficiency of the conventional method was 15% on average, the transformation by this method in which silver nitrate was added to the selection medium and cefotaxime was replaced with carbenicillin resulted in an average of 20% or more higher efficiency of 40.2%. Converted plants were obtained. In addition, according to this method, it was revealed that transformation was performed stably at a high efficiency of 25% or more throughout all tests.

Table 2 shows the transformation results when A188 was infected with the co-transformation vector LBA4404 (pSB624). Nearly 400 immature embryos were infected by the conventional method, but 9 transformed plants were obtained, with an efficiency of 2.3.
% Was low. On the other hand, in this method to which silver nitrate and carbenicillin were added, more than 40 transgenic plants were obtained from a total of 190 immature embryos. The average efficiency was 21.6%, which was higher than the average when LBA4404 (pSB131) was transformed by the conventional method. The efficiency of each test was stable at 10 to 20% or more.

Table 3 shows the results of the transformation of the cultivar H99 by this method.
It was shown to. In the conventional method, no regenerated plants were obtained at all, even if the transformation test was performed more than ten times using H99 as a material, whereas in the present method, a total of seven independent transformed plants were obtained in four tests. Obtained. The average transformation efficiency was 3.8%, but in some tests, the transformation efficiency was 15%, which was as high as the transformation efficiency of A188 according to the conventional method.

[0048]

Table 1 Transformation results of LBA4404 (pSB131) (variety A188) Test Number of immature embryos Number of redifferentiations Number of redifferentiations (%) GUS + number GUS + (%) 1 21 10 47.6 6 28.6 2 24 16 66.7 8 33.3 3 47 28 59.6 23 48.9 Total 92 54 58.7 37 40.2 -------- -------------------------------------------------- -------- Conventional method 923 232 25.1 139 15.1 Conventional method: A188 transformation results by Ishida et al. (1996)

[0049]

Table 2 Transformation results of LBA4404 (pSB624) (variety A188) Test Number of immature embryos Redifferentiation number Redifferentiation (%) GUS + number GUS + (%) Improvement method 1 45 21 46.7 13 28.9 2 50 17 34.0 7 14.0 3 95 43 45.3 21 22.1 ------------ -------------------------------------------------- ------ Total 190 81 42.6 41 21.6 Conventional method 1 123 1 0.8 1 0.8 2 133 17 12.8 7 5.3 3 132 4 3.0 1 0.8 ------------------ -------------------------------------------------- Total 388 22 5.7 9 2.3

[0050]

Table 3 Transformation results of LBA4404 (pSB131) (variety H99) Test Number of immature embryos Number of redifferentiations Number of redifferentiations (%) GUS + number GUS + (%) 1 63 1 1.6 1 1.6 2 41 3 7.3 1 2.4 3 59 3 5.1 2 3.4 4 20 3 15.0 3 15.0 -------- -------------------------------------------------- -------- Total 183 10 5.5 7 3.8

Example 6 A maize cultivar A188 immature embryo was prepared under the same conditions as in Example 1.
LBA4404 (pSB131) was infected and co-cultured. The immature embryos after co-culture were placed on a selection medium containing 10 μM silver nitrate, 100 mg / l carbenicillin and 5 mg / l PPT, respectively, and a medium having the same composition without silver nitrate (primary selection). After culturing for 2 weeks, immature embryos were placed on a medium of the same composition containing 10 mg / l PPT and a medium of the same composition without silver nitrate, respectively, and cultured for 3 weeks (secondary selection). The grown calli were cut into small pieces of 1-2 mm in size, placed on a medium of the same composition containing 10 mg / l PPT, and cultured for 3 weeks (third selection). All cultures on the selection medium were performed at 25 ° C. in the dark. The grown calli were again cut into small pieces, and 250 mg / l cefotaxime and 5 mg / l
l The cells were placed on a regeneration medium containing PPT and cultured at 25 ° C under illumination for 2 weeks. After transferring the regenerated seedlings to the rooting medium,
I raised the pot. GUS using X-gluc
The gene expression was investigated.

The results are shown in Table 4. The transformation efficiency of immature embryos cultured in a medium containing no silver nitrate in both primary and secondary selection and immature embryos cultured in a medium containing no silver nitrate only in the primary selection was as low as 0-5%, Transgenic plants were obtained at a high rate of about 30% in the immature embryos cultured in the medium containing silver nitrate from the primary selection. From this, it became clear that the addition of silver nitrate to the medium at the initial stage of selection is more effective in increasing the transformation efficiency.

[0053]

[Table 4] Table 4 Effect of silver nitrate at each selection stage AgNO _{3 in} selection medium (μM) Primary secondary immature embryos Redifferentiation number Redifferentiation (%) GUS + number GUS + (%) 0 0 20 1 5.0 1 5.0 0 10 20 2 10.0 0 0 10 0 20 10 50.0 6 30.0 10 10 22 9 40.9 6 27.3

[0054]

[Table 5] Bacterial suspension medium LS inorganic salt, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine HCl, 1.0 mg / l thiamine HCl, 100 mg / l myo-inositol, 1.0 g / l casamino acid, 1.5 mg / l l 2,4-D, 68.5 g / l
Sucrose, 36.0 g / l glucose, 100 μM acetosyringone, pH 5.2

Co-culture medium LS inorganic salt, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine HCl, 1.0 mg / l thiamine HCl, 100 mg / l myo-inositol, 700 mg / l L-proline, 1.5 mg / l 2 , 4-D, 20 g / l
Sucrose, 10 g / l glucose, 500 mg / l MES, 1
00 μM acetosyringone, 8 g / l agar, pH 5.8

Selection medium LS inorganic salt, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine HCl, 1.0 mg / l thiamine HCl, 100 mg / l myo-inositol, 700 mg / l L-proline, 1.5 mg / l 2 , 4-D, 20 g / l
Sucrose, 500 mg / l MES, 8 g / l agar, 250 mg / l
Cefotaxime or 100 mg / l carbenicillin, 5
-10 gm / l PPT, pH 5.8

Regeneration medium LS inorganic salt, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine HCl, 1.0 mg / l thiamine HCl, 100 mg / l myo-inositol, 700 mg / l L-proline, 5.0 mg / l Zeatin, 20 g /
l Sucrose, 500 mg / l MES, 8 g / l agar, 250 mg
/ l cefotaxime, 5 mg / l PPT, pH 5.8

Rooting medium LS inorganic salt (main salt reduced to 1/2), 0.5 mg / l nicotinic acid,
0.5 mg / l pyridoxine HCl, 1.0 mg / l thiamine HCl, 10
0 mg / l myo-inositol, 20 g / l sucrose, 50
0 mg / l MES, 8 g / l agar, 250 mg / l cefotaxime, p
H 5.8

[0059]

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[Brief description of the drawings]

FIG. 1 is a graph showing the effect of silver nitrate and carbenicillin on callus formation.

FIG. 2 is a graph showing the effect of silver thiosulfate on callus formation.

FIG. 3 is a graph showing the effect of AVG on callus formation.

FIG. 4 is a graph showing the effect of cobalt ions on callus formation.

[Fig. 5] Vector LB for co-transformation
It is a figure showing the structure of A4404 (pSB624).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1:91)

Claims (15)

[Claims] 1. A method for obtaining a transformed plant by culturing a plant tissue inoculated with Agrobacterium fungi in a medium containing a substance that inhibits the action of ethylene or a substance that inhibits the biosynthesis of ethylene. 2. The method according to claim 1, wherein the substance that inhibits the action of ethylene is silver nitrate or silver thiosulfato complex (STS). 3. The concentration of silver nitrate or silver thiosulfato complex salt added to the medium is 0.1 to 1,000 μM, preferably 1 to 1,000 μM.
-The method according to claim 2, wherein the amount is 100 μM, more preferably 10 μM. 4. The method according to claim 1, wherein the plant tissue inoculated with Agrobacterium is corn. 5. The method according to claim 4, wherein the plant tissue inoculated with the Agrobacterium is a corn immature embryo. 6. The method according to claim 1, wherein the transformed cells are obtained by culturing using a medium containing carbenicillin. 7. The method according to claim 6, wherein the concentration of carbenicillin added to the medium is 50-500 mg / l, preferably 100-250 mg / l. 8. The substance inhibiting the biosynthesis of ethylene is AC
The method according to claim 1, which is a C synthase inhibitor. 9. The method according to claim 8, wherein the ACC synthase inhibitor is aminoethoxyvinylglycine. 10. The concentration of aminoethoxyvinylglycine added to the medium is 0.1-10 μM, preferably 0.5-1 μM.
The method according to claim 9, which is μM. 11. A substance that inhibits ethylene biosynthesis,
2. The method according to claim 1, which is an ACC oxidase inhibitor. 12. The method according to claim 11, wherein the ACC oxidase inhibitor is a cobalt ion. 13. The method according to claim 12, wherein the concentration of cobalt ions added to the medium is 0.1-100 μM, preferably 1-10 μM. 14. A method comprising at least one step of selecting transformed cells, wherein the selection medium used in the initial selection step contains a substance that inhibits the action of ethylene or a substance that inhibits the biosynthesis of ethylene. 14. The method according to any one of 1 to 13. 15. The method according to any one of claims 1 to 14, wherein the selection medium is a medium containing an inorganic salt of MS (LS).