JP2008259497A - Method for creating transformant of domestic variety of soybean through agrobacterium and method for acquiring seed of current generation and progeny of transformant in short period of time - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for simply creating a transformant of a Japanese domestic variety of soybean through a bacterium of the genus Agrobacterium and to simultaneously provide a technology for acquiring each seed of the current generation and the progeny of the transformant in a short period of time. <P>SOLUTION: A method for creating the transformant of the soybean comprises utilizing a domestic variety Kariyutaka. The method is characterized as follows: the transformant having prescribed characteristics is created by a culturing step of using a 1/2 cotyledonous part derived from each seed absorbing water as an explant of the soybean, infecting the explant with the bacterium of the genus Agrobacterium, culturing the infected explant in an adventitious bud-inducing culture medium and inducing an adventitious bud, conducting culturing in a shoot-forming culture medium and forming a shoot and subjecting the shoot to rooting, growing and seedling formation. Otherwise, the method for creating the transformant of the soybean is characterized by using the resultant transformant, hybridizing the transformant with an optional variety and creating a variety inheriting the excellent characters from the progeny thereof. The transformant is created by the method and a product is derived from the transformant. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a soybean transformant and a transformant, and more specifically, a method for producing a soybean transformant using the Agrobacterium method using the domestic variety Kariyutaka, A transformant produced by the method and having a characteristic of cultivating in a very short period of time compared to a conventional variety by the conventional method, and mating with an arbitrary variety using the transformant, This is related to a method for creating a variety that inherits the above.

  Soybean transformants are produced by, for example, the particle gun method and the Agrobacterium method. However, the conventionally reported methods still have much room for improvement in order to be used as a general technique. Conventional methods have not yet established a simple method for producing transformants that can be easily used in laboratories having normal culture facilities.

  The present inventors have so far made two methods, “a method using cotyledons in germinating seeds” (see Non-Patent Document 1) and “a method using hypocotyl” (see Non-Patent Document 2), An attempt was made to produce a soybean transformant, but unfortunately, the transformant was not yet obtained.

    In general, the Agrobacterium method has advantages in that the number of introduced copies is small and gene silencing is less likely to occur, and that research is advanced at low cost, as compared with the particle gun method. If there is a method for efficiently obtaining soybean transformants by the Agrobacterium method, it is considered that the development of new soybean seeds will make a significant progress. However, the situation is that a stable system has not yet been established for the production of soybean transformants.

  In addition, it takes a lot of labor and time to produce transformants. Furthermore, soybean transformants are rarely used in transformant generations, and usually it is necessary to repeat selfing and immobilize the transgene. In particular, it takes a lot of time to obtain seeds from the transformant generation. Therefore, it is necessary to establish a method for obtaining seeds of a progeny whose transformant generation and generation have been renewed in a short period of time.

Planta 216: 723-735 (2003) Planta 219: 1042-1049 (2004)

  Under such circumstances, the present inventors, in view of the above-mentioned prior art, have conducted intensive research with the goal of developing a method for efficiently producing soybean transformants by the Agrobacterium method using domestic varieties. As a result of repeated development, by using the cotyledon part of the seed that has absorbed water as the plant part, the degree of Agrobacterium infection is high, the ability to induce adventitious buds and the ability to form shoots, the ability to root from shoots is high, and The present inventors have found that a transformant having the characteristic that the time of arrival can be extremely advanced can be produced, and the present invention has been completed.

  The present invention provides a method for producing a soybean transformant using Kariyutaka of domestic varieties, and a soybean transformant produced by the method and having a characteristic of culling during a short-term culture, as much as possible. An object of the present invention is to provide a labor-saving technique for obtaining seeds of transformant generations and generations whose generations have been renewed in a short period of time.

The present invention for solving the above-described problems comprises the following technical means.
(1) A method for producing a transformant of soybean by using Kariyutaka of domestic varieties, 1) using a cotyledon part of a seed that has absorbed water as a plant part of a soybean explant 2) the cotyledon 3) Incubate in adventitious bud induction medium to induce adventitious buds 4) Incubate in shoot formation medium to form shoots 5) Initiate roots, grow and seedling from shoots , To produce a transformant having a predetermined characteristic by the culture process of, or, using the obtained transformant, to cross with any cultivar, to produce a cultivar that inherited an excellent trait from its progeny, And a method for producing a soybean transformant.
(2) Using the obtained transformant, cross with a North American variety excellent in Agrobacterium infectivity, adventitious bud induction ability, and shoot formation ability, or a domestic variety excellent in rooting ability from shoots. The method according to (1) above, wherein a variety that inherits an excellent trait from its progeny is created.
(3) The method according to (2) above, wherein Jack or Thorne is used as the North American variety.
(4) The method according to (1) above, wherein the concentration of Agrobacterium to be infected is OD 0.3 to 0.5.
(5) A method for producing a transformant of soybean using domestic varieties of Kariyutaka, which has a characteristic of being settled at the time of culturing by the culturing step of 1) to 5) described in (1) above. A method for producing a soybean transformant, comprising producing a transformant.
(6) A Kariyutaka transformant produced by the method described in (5) above, which has a characteristic to be settled during culture.
(7) A product derived from a transformant produced by the method according to any one of (1) to (5).
(8) By using the transformant produced by the method according to any one of (1) to (5) above, the transformant generation and / or generation was renewed in a short period of time compared to the ordinary method. How to get progeny seeds.

Next, the present invention will be described in more detail.
The present invention is a method for producing a transformant of soybean, comprising using a cotyledon part of a seed soaked as a plant part of soybean explant, infecting the cotyledon with Agrobacterium, adventitious bud Induction of adventitious buds by culturing in induction medium, shoot formation by culturing in shoot formation medium, creation of transformants by rooting, growing and seedling from shoots The obtained transformant is used to cross with an arbitrary variety and to produce a variety that inherits an excellent trait from its progeny.

  In the present invention, using the obtained transformant, for example, North American varieties excellent in Agrobacterium infectivity, adventitious bud induction ability, and shoot formation ability, or domestic varieties excellent in rooting ability from shoots And producing a cultivar having inherited excellent traits from its progeny is a preferred embodiment. Further, in the present invention, it is a preferable embodiment to use Jack or Thorne as the North American variety. In addition, the present invention is a method for obtaining seeds of a progeny whose transformant generation and / or generation has been renewed in a shorter period of time by using the transformant produced by the above-described method. It has characteristics.

  Next, the method of the present invention will be specifically described. In the present invention, it is important to use absorbed cotyledon seeds as soybean explants. Conventionally, as an explant, there have been various examples of methods for using cotyledons in germinating seeds and methods for using hypocotyls, and the present inventors have examined the method of using cotyledons in germinating seeds. The preparation of explants (especially how to insert a scalpel) was extremely complicated and was considered to exceed the range of experimental levels that can be performed in the laboratory. In addition, the method using the hypocotyl showed extremely high infection efficiency of Agrobacterium, but the induction of adventitious buds from the hypocotyl was lower than the method using cotyledons in the germinated seeds described above. I understood.

  In the present invention, the use of seed cotyledons that have absorbed water as the explant has the great advantage that the preparation of the explant is relatively easy. In addition to this, in the past, domestic varieties had a low degree of Agrobacterium infection, ability to induce adventitious buds and ability to form shoots, and there were no varieties with high rooting ability from shoots. It has been found that all have the advantage that high-performing varieties are obtained in all of these.

  Conventionally, even North American varieties Jack and Thorne, which had been considered to have a high degree of Agrobacterium infection, adventitious bud induction ability and shoot formation ability, did not have cultivars with high rooting ability from shoots. In consideration of this, it has been considered that it is very important for the utilization of domestic varieties to find a line that can establish a variety with high rooting ability from shoots in domestic varieties.

  Next, the process of Agrobacterium infection, adventitious bud induction, shoot formation, rooting from shoots, growth and seedling in the present invention will be specifically described. First, the cotyledons of seeds that have absorbed water are cut out as explants. Next, this is infected with Agrobacterium and cultured in a co-culture medium (CCM) for about 5 days, and then cultured in an adventitious bud induction medium (SIM) for about 14 days to induce shoots. In the meantime, selection with antibiotics and herbicides is used to induce shoots.

  Next, in order to elongate the shoots, subculture is performed in a shoot-forming medium (SEM) about every 14 days until the shoots become 3 to 4 cm while selecting with antibiotics and herbicides. Next, in order to root the shoot, it is cultured in Rooting medium (RM), the root of the shoot is rooted, the rooted shoot is grown by cultivating with aseptic material, and this is transplanted into the horticultural soil and seedling Let In the process of forming a plant body as described above, a commercial medium can be obtained and used as the CCM, SIM, SEM, and RM.

  In the method of the present invention, the above-mentioned medium, culture conditions, and the like can be arbitrarily adjusted according to the degree of shoot induction, elongation, shoot rooting, and growth. In the present invention, using the cotyledons of water-absorbed seeds as explants, and examining the difference between the varieties of the degree of Agrobacterium infection, the degree of infection of the North American varieties Jack and Thorne is high, It has been found that even domestic varieties Kariyutaka can establish varieties with a high degree of infection similar to those of Jack and Thorne.

  Next, we tested and examined the varietal differences in rooting ability from shoots. When not infected with Agrobacterium, North American varieties cannot establish varieties with high rooting ability from shoots. However, it was found that the domestic variety Kariyutaka can establish a variety with higher rooting ability from shoots than the North American variety. In addition, when Agrobacterium is infected (when a gene is actually introduced), it is possible to produce soybean transformants with high shoot-forming ability from domestic varieties.

  This is a cross between North American varieties (Jack, Thorne, etc.) with high Agrobacterium infection, adventitious bud inducing ability and shoot-forming ability and low rooting ability from shoots, and domestic varieties with high shoot rooting ability. This suggests that by producing and cultivating cultivars that inherited the superior traits of both varieties from the progeny of mating, it is possible to produce soybean transformants that inherited the superior traits.

  For example, the Agrobacterium method has the advantage that the introduced copy number is small and gene silencing is less likely to occur than the particle gun method, and that the Agrobacterium method can be implemented at low cost and at low cost. As for soybean, various methods for transforming soybean using various explants have been tried, but it is a fact that a stable system has not yet been established. For example, cotyledons in germinated seeds are used as explants. So far, no transformant has been obtained by the method used or the method using the hypocotyl.

  On the other hand, in this invention, the transformant which has the outstanding character can be obtained by using the cotyledon of the seed which absorbed water as an explant. Also crossed with North American varieties with high shoot-forming ability (Jack, Thorne, etc.), or with domestic varieties with high rooting ability from shoots (Kariyutaka, Toyosu, etc.) It is possible to create and cultivate varieties that inherited the superior traits of both varieties from their progenies, thereby establishing and providing a method for producing soybean transformants using domestic varieties. Is.

  The transformation method of soybean via Agrobacterium not only requires a very complicated operation, but also takes a lot of time and labor to produce a transformant. Furthermore, transgenes are rarely evaluated in transformant generations, and analysis is usually performed after the transgene is fixed. Therefore, if seeds can be obtained from the transformant generation in a short period of time, the time required for immobilization can be greatly shortened. The present inventors have found that transformants of domestic varieties Kariyutaka have extremely characteristic culture characteristics.

  In the transformant of Kariyutaka, adventitious buds derived from cotyledons on a medium containing cytokinin form flower buds after about one month of culture under a constant culture condition of 26 ° C. for 16 hours. After rooting these adventitious adventitious buds, they will settle in about one month when transplanted to the soil. If it is a normal variety, it will begin to arrive about 2-3 months after transplanting to the soil. In addition, it became clear that the Agrobacterium transformation method using cotyledons of seeds that had absorbed water was suitable for Kariyutaka. This method using Kariyutaka can be used suitably as a tool for gene function analysis in soybean because seeds of the transformant generation can be obtained in a very short time.

The present invention has the following effects.
(1) To provide a method for producing a soybean transformant that enables the production of a soybean transformant by omitting complicated operations by using cotyledons of seeds absorbed by the domestic variety Kariyutaka. it can.
(2) It is possible to provide domestic varieties having excellent culture characteristics similar to those of US varieties Thorne and Jack.
(3) A method for producing a new transformant of soybean domestic varieties by the Agrobacterium method can be established and provided.
(4) The domestic cultivar Kariyutaka according to the present invention has a flower at the time of cultivation and can shorten the period until harvesting by about 3 months.
(5) It is possible to provide a method for producing a new transformation system in which the transformant is bred with North American varieties or domestic varieties to produce progeny transformants.
(6) consequently, it becomes feasible to obtain the next generation (T ₂ generation) subsequent generations updated progeny seeds of transformants in a short period of time.
(7) Using domestic varieties (Kariyutaka), it is possible to produce a transformant having the characteristics of culling during culture.
(8) By using the transformant, seeds can be harvested about 3 months earlier than in the case of North American variety (Thorne).

  EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.

  In this example, an attempt was made to establish a transformation system for soybean by the Agrobacterium method.

1) Method In the culture experiment, two cultivars, US and Jack, were used. The transformation method tried improvement based on the conventional method [Planta (2003) 216: 723-735]. An infection experiment was conducted on two varieties using LBA4404, EHA101, and EHA105, which are 3 strains of Agrobacterium. As the expression plasmid DNA, pIG121-Hm (having a hygromycin selectable marker gene) having a GUS gene which is one of reporter genes downstream of the CaMV35S promoter was used. The degree of infection was determined by the degree of GUS staining of the tissue piece.

2) Preparation of explants to inoculate Agrobacterium The seedlings on the 5th day of germination were used as explants. The seeds were cut into two lengths, and the base of the extending hypocotyl was cut off with a scalpel. Concentrating on the part from which the epicotyl was removed (part considered to have high redifferentiation ability), 7 or 8 wounds having a depth of about 0.5 mm were made.

3) Agrobacterium inoculation and co-cultivation When 1/2 (half) of the cotyledon was cut off, it became easier to contact the co-culture medium, resulting in an increase in the degree of Agrobacterium infection. As a result of the infection experiment, EHA100 stained the best with respect to the degree of GUS staining immediately after co-culture. When an infection experiment was performed using EHA105, no difference was observed between the two varieties, Jack and Thorne. Since the explants turned back during co-cultivation and were not in contact with the culture medium, when the explants were pressed against the culture medium using tweezers only once on the third day of co-culture, the degree of staining was significantly improved. .

4) Shoot induction and selection To confirm how much selection pressure needs to be applied, select from the beginning of the shoot induction culture on a medium containing hygromycin in an unexposed Jack explant. A test was conducted. Although thick shoots may appear at the beginning of the culture, none of these shoots were transformed. Presumably, the transformation was thought to be contained in shoots emanating from the shoots derived from the injured location. This difference in the way the shoots come out is thought to be due to the way the scratches are made when preparing the explants.

  The shoots seen in the early stage of culture are thought to have an adverse effect on the subsequent formation of multiple buds, and if the wounds can be accurately damaged and the tissue around them can be divided, thick shoots can be obtained. Can be prevented to some extent. There was no significant difference in the rate of shoot regeneration between Jack and Thorne. Moreover, as a result of conducting the selection test at the time of shoot elongation culture in the same manner as the shoot induction culture, it was considered that selection with about hymg: 10 mg / l was appropriate.

  As described above, how to damage the explant has a great influence on the frequency of multi-bud formation, and the transformation frequency is dramatically increased by bringing the explant into close contact with the medium during co-culture (for that purpose, In the case of Jack and Thorne, Agrobacterium strain is most frequently infected with EAA105, and in Jack, Agrobacterium is infected in the part where the hypocotyl is removed. However, it was suggested that by improving a little, it was possible to have the same degree of infection as Thorne, and that it was possible to select effectively by advancing the selection time. I understood.

  In this example, the establishment of the Agrobacterium infection method, the establishment of transformant selection conditions, and the examination of the culture conditions (using thidiazuron (TDZ) instead of BAP) were attempted.

1) Examination of Agrobacterium Infection Method Agrobacterium infection method was examined in the same manner as in Example 1 above. As a result, among the 3 strains of LBA4404, EHA101, and EHA105, the one with the best infection was EHA105. There was no difference in the degree of infection between the two varieties of Jack: Thorn, which are plant materials subjected to the experiment. Variations in the degree of Agrobacterium infection were observed. The culturing time of Agrobacterium used for the infection experiment was extremely shortened, and by adding acetosyringone (4 mg / L) as an inducer at the time of infection, the variation could be remarkably improved. Thus, the conditions at the time of infection were established.

2) Examination of selection conditions for transformants Regarding conventional methods, a) the selection time was advanced and the concentration of hygromycin as a selection agent was changed according to the culture stage, b) a drug that suppresses the growth of Agrobacterium itself. The modification was attempted by adding 25 mg / L of meropenem (meropen).

  Immediately after infection with Agrobacterium, selection was performed with 5 mg / L hygromycin, and after 2 weeks, the cells were transplanted to a 10 mg / L medium. Thereafter, subculture selection was performed with a 10 mg / L medium every two weeks (note that 25 mg / L meropen is contained in all the mediums). The presence of the gene-transferred cells was also confirmed in the explants 53 days after infection by GUS staining. Furthermore, shoots considered to be hygromycin resistant were observed in the cultures on day 65 after infection. These results suggest that the transfected cells can be selectively cultured. The appearance rate of shoots that seemed to be resistant to hygromycin was 1.9%.

3) Examination of culture conditions Conventionally, BAP was used for shoot induction after Agrobacterium infection, but thidiazuron (TDZ), which is said to have an effect of inducing more shoots, was used. TDZ is a plant hormone classified into the same cytokinin as BAP. TDZ (2 μM, 4 μM, 10 μM, 20 μM) was added to the shoot induction medium instead of BAP, and the induction rate of shoots was examined. However, in this experiment, Agrobacterium infection was not performed and only the culture was performed.

  As a result, it was found that TDZ induces many small shoots compared to BAP. Although the number of small shoots induced by TDZ could not be counted, as a result of observation, the induction rate of small shoots was 2 μM <4 μM> 4 μM> 10 μM> 20 μM. When the explants in which small shoots were induced were placed on a shoot elongation medium, shoot elongation was observed, so it was considered possible to use TDZ instead of BAP.

  The appearance rate of shoots that are considered to be resistant to hygromycin is about 1 per explant 50, but in order to increase the formation rate of shoots, increase the number of acquired shoots by using thidiazuron (cytokinin-like substance). Tried. It is necessary to confirm the introduction of the HPT gene (hygromycin resistance gene) by PCR together with GUS staining as needed for shoots considered to be hygromycin resistance.

  In this example, in order to establish a transformation system of soybean by the Agrobacterium method, examination of Agrobacterium infection conditions, examination of adventitious bud induction conditions, and examination of transformation methods were attempted.

1) Examination of Agrobacterium infection conditions Agrobacterium infection conditions were examined in the same manner as in Example 1. As a result, the concentration of Agrobacterium to be infected was most preferably OD 0.3 to 0.5. Infecting Agrobacterium for 20 hours and then co-culturing for 5 days significantly increased the infection efficiency. Better results were obtained in the culture in the coexisting medium when cotyledons in germinated seeds were used for the explants than in the coexisting medium in the past reported example (Planta 2004, 219: 1042-1049). In the case of co-cultivation, the inoculation efficiency could be increased by placing the explants in a state of penetrating the medium vertically.

2) Examination of adventitious bud induction conditions By culturing cotyledons infected with Agrobacterium under strong light conditions (140-180 μmoles / s / m ² ), adventitious bud induction ability could be remarkably improved. . The adventitious bud induction rate was 75% in the case of strong light, whereas it was 12.5% under low light conditions (50 to 80 μmoles / s / m ² ). After infection with Agrobacterium, adventitious bud inducibility can be increased by culturing in a shoot induction medium used in a transformation method using a cotyledon in germinated seeds.

  The adventitious bud formation rate in the embryonic-tip medium was 28.0%, and the adventitious bud formation rate in the cotyledon node medium was 40.0%. When the effect of cytokinin was investigated, the effect of BAP (benzyladenopurine) was more effective than TDZ (thidiazuron). In Jack and Thorne, the difference between cultivars was clearly recognized, and it was revealed that Jack has higher ability to induce adventitious buds in the culture method [BAP (1 μM) ... Jack: 62.5%, Thorne: 28.5%].

3) Examination of transformation method (cotyledon: present invention, hypocotyl: reference example)
When embryonic-tip was cultured, it was found that the explant was greatly curved with its elongation, and the subsequent handling became extremely difficult. Embryonic-tips are extremely small to handle as explants, so care must be taken with tweezers. In order to solve these problems, we examined the use of the hypocotyl about 5 days after seeding as an explant, and the infection time was different from embronic-tip, and was co-cultured for 5 days after 30 minutes of infection. The degree of infection was good. In the method of placing explants in co-cultivation, the infection efficiency could be increased by placing the explants in a state of penetrating the medium vertically, similarly to embryonic-tip. By using the above method, it was possible to induce adventitious buds as well as embryonic-tip.

  From the above results, it was suggested that the transformation method using embryonic-tip is partly improved and the hypocotyl part is used as an explant, so that it can be used as a simpler method than the conventional method.

  In this example, varietal differences in the degree of Agrobacterium infection, varietal differences in adventitious bud induction ability and shoot formation ability, and varietal differences in rooting ability from shoots, and traits using Jack and Thorne A conversion experiment was conducted.

(1) Agrobacterium infection and rooting from shoots Seeds were immersed in sterilized water overnight, the cotyledons were cut out, infected with Agrobacterium, and cultured in a co-culture medium (CCM) for 5 days. Next, in order to induce shoots, cotyledons infected with Agrobacterium were cultured for 14 days in adventitious bud induction medium (SIM), and selection with antibiotics and herbicides was performed to induce shoots. Next, in order to extend until the shoot became 3-4 cm, subculture was performed in SIM medium every 14 days. Next, in order to root the shoots, they were cultured with Root medium (RM), then cultured with aseptic materials, grown into seedlings, and transplanted to horticultural soil.

(2) Examination of differences between cultivars 1) The degree of infection of Agrobacterium in each cultivar by GUS staining was compared. Table 1 shows the results. The degree of Agrobacterium infection is as follows: 0 ^* : explants that were not stained at all, 1 ^* : explants that were only partially stained, 2 ^* : overall stained Although the color of the explant was 3 ^* : the explant was dyed as a whole, and its color was dark.

2) In order to compare the adventitious bud inducing ability and the shoot-forming ability between cultivars, the adventitious bud inducing ability and the shoot formation rate were compared. Table 2 shows the results. The adventitious bud inducing ability and shoot-forming ability of each variety are as follows: 0 ^* : explant in which no adventitious bud induction was observed, 1 ^* : outside in which adventitious bud induction was observed only in part Explants, 2 ^* : explants in which adventitious buds were induced overall but less in amount 3 ^* : explants in which adventitious buds were induced overall and evaluated in large amounts . The induction rate was calculated by the following equation: number of explants with induction rate = 1 ^{* to} 3 ^* / number of all explants × 100.

  3) In addition, in order to compare varietal differences in rooting ability from shoots, rooting ability from shoots in each variety was compared. Table 3 shows the results. Regarding Tachiyutaka, since there were few extended shoots, the comparison of rooting ability was not performed. The rooting rate was calculated by rooting rate = number of shoots rooted / number of shoots transferred to rooting medium × 100.

(3) Transformation experiment The GUS reporter gene can very clearly identify the site of infection by its tissue staining. However, since the stained tissue pieces die, it is impossible to continue the subsequent culture. Therefore, since it was confirmed that infection was possible to some extent, the reporter gene was changed to sGFP (green jellyfish-derived green photoprotein).

  Using an expression vector containing a CaMV355S pro-sGFP-NosT region derived from pMpC123 as an expression vector, a transformation experiment was performed by glyphosate (6 mg / L) selection. In Jack, one transformed adventitious bud was obtained from 50 explants. I was able to get it. According to the literature (Plant Cell Rep., 25: 206-213, 2006), the transformation rate in the cultivar Thorne is reported as (4/70 = 5.7%). Then, when a transformation experiment centering on Thorne (explant number of about 150) was performed, it was found that the method using the cotyledons of the seed that absorbed water was the most effective.

  In this example, the Agrobacterium transformation method using cotyledons as explants, which was established in the laboratory of the present inventors, was applied to Kariyutaka. In addition, the expression vector of FIG. 1 was constructed using a green fluorescent protein (GFP) reporter gene as a transgene for transformation experiments (FIG. 1). We succeeded in producing a transformant having GFP fluorescence from Kariyutaka. It was found that the frequency of appearance of shoots having GFP fluorescence of Kariyutaka was much higher than that of Thorne. However, the number of transformation shoots with rooting was relatively small. As a result, the transformation efficiency of Thorne was 0.85%, whereas the transformation efficiency of Kariyutaka was 0.77%.

  Similarly, in the transformation experiment, anchoring was observed during the culture in the shoot elongation medium. Furthermore, after rooting of the transformed shoots, potting was performed, and after 2 months, wrinkles were observed. Moreover, although the number of seeds obtained from the transformant generation is about several grains, since GFP fluorescence was observed, it was considered that the transgene was inherited in the next generation.

  As shown in FIG. 2, Kariyutaka was found to have characteristics that settled during the culture compared to the culture characteristics of the American variety Thorne, and it was found that the timing of the landing was extremely advanced. This is a phenomenon seen under conditions where cytokinin is present for 16 hours in day length (constant at 26 ° C.). As a result, it was revealed that Kariyutaka can shorten the time required for harvesting by about 3 months compared to Thorne.

  Below, the detail of the test method implemented in the present Example is demonstrated.

  Based on the binary vector pMDC (Curtis and Grossnikulaus 2003), it was modified for this study. Further, EHA105 (Hood et al. 1993) was used as the Agrobacterium strain.

  pMDC123 contains Bar (herbicide glufosinate resistance gene) as a selection marker gene for plant transformants. For this test, the restriction enzyme HindIII site to EcoRI site were deleted.

  Based on pMDC123, pMDC123GFP was constructed in which a cauliflower mosaic virus 35S promoter derived from pIG121-Hm (Akama et al. 1992), a terminator of nopaline synthase and a sGFP gene (Niwa et al. 1999) were inserted (FIG. 1). .

(2) Agrobacterium culture and infection medium EHA105 strain having pMDC123GFP was cultured at 28 ° C. for 2 days in YEP medium (An et al. 1988) containing 50 mg / l kanamycin (Wako Pure Chemical Industries, Ltd.). A single Agrobacterium clone was inoculated into liquid YEP medium containing 50 mg / l kanamycin and cultured at 28 ° C. overnight at 160 rpm. Then, a part of the solution was transferred to 200 ml of YEP medium and cultured overnight at 28 ° C. to OD ₆₅₀ = 0.3−0.5.

The bacterial solution cultured overnight on the day of infection was centrifuged at 3,500 rpm for 10 minutes to collect bacteria, and 7.5 μM 6-benzylaminopurine (BAP), 0.7 μM gibberellic acid (GA ₃ ), 20 mM. MES [2- (N-morpholino) ethanesulfonic acid], resuspended in 1/10 B5 medium containing 3% sucrose and 200 μM acetosyringone. The bacterial solution concentration was adjusted to OD ₆₅₀ = 0.7-0.8 using a spectrophotometer.

(3) Adjustment of explant change and infection Sterilized ripe seeds were immersed in sterilized pure water for about 16 hours. Cut the cotyledons by cutting with a scalpel along the navel of the seed. Further, a female was put in the horizontal direction to obtain 1/2 cotyledons. About 50 explants including part of the hypocotyl were transferred to a 100 mm × 20 mm petri dish containing Agrobacterium suspension. These explants were lightly stirred every 10 minutes and infected for 30 minutes. After infection, five explants were placed on a co-culture medium spread with filter paper, 5 pieces per petri dish with the flat side facing the medium.

The co-culture medium was B5 salt, B5 vitamins, 7.5 μM BAP, 0.7 μM GA ₃ , 20 mM MES, 3% sucrose, 200 μM acetosyringone, 400 mg / l cysteine, 154 mg / l dithiothreitol, pH 5. 4. Contains 0.425% agar. The coculture was performed at 26 ° C. for 5 days with a 16-hour day length (140-180 μmoles / s / m ² ).

(4) Culture conditions and regeneration of plant body After co-cultivation, explants were induced to adventitious buds (SI) medium [B5 salt, B5 vitamins, 3% sucrose, 3 mM MES, 5.0 μM BAP, 25 mg / l meropen (Dainippon Sumitomo Pharma Co., Ltd., pH 5.7).

Furthermore, 5 pieces of explants were placed on a Petri dish on a SI medium (SIM) containing 0.7% agar with the flat surface facing the medium. The explants were cultured at 26 ° C. for 2 weeks at a day length of 16 hours (140-180 μmoles / s / m ² ).

For selection, explants were transplanted into SIM medium containing 6 mg / l glufosinate and cultured for 2 weeks. Furthermore, culture was carried out in the same manner for another 2 weeks. Thereafter, MS salt, B5 vitamins, 3% sucrose, 3 mM MES, 1 mg / l zeatin riboside, 0.5 mg / l GA ₃ , 0.1 mg / l IAA, 100 mg / l DL-pyroglutamic acid, 50 mg / It was transferred to a shoot formation medium (SEM) containing 1 L-asparagine, 25 mg / l meropen (Dainippon Sumitomo Pharma), 6 mg / l glufosinate, pH 5.7, 0.7% agar.

  When transplantation is repeated in SEM medium every two weeks, shoot elongation is observed. In particular, Kariyutaka is recognized as having arrived at the stage of shoot elongation compared to other varieties, and may lead to flowering. Among them, the shoots extending to 2.5 cm or more were cut with a scalpel and then aseptically immersed in a 1 mg / l indole butyric acid solution for about 1 minute. These shoots were transplanted to rooting medium (RM) containing MS salt, B5 vitamins, 2% sucrose, 3 mM MES, pH 5.6, 0.7% agar. After 2-3 weeks of rooting treatment, the seedlings were removed from the medium, washed with water, and then potted. After acclimatization treatment for one week, they moved to a closed greenhouse with a daytime condition of 16 hours and trained.

(3) Confirmation of transgene of transformant Total DNA was extracted from a part of the leaves of the planted young plant, and the transgene was confirmed by PCR. Moreover, the transformant derived from Kariyutaka was able to obtain fully ripe seeds in 2-3 months after potting. This indicates that harvesting was possible about three months earlier than other varieties (eg, Thorne).

In this example, the generation test of the progeny whose generation of the transformant using Kariyutaka was renewed was conducted. A seedling of a transformant derived from Kariyutaka produced by the same method as in Example 5 above was taken out from the medium, washed with water, then potted and acclimatized for 1 week, and then 16 hours long. This was transplanted and cultivated in soil of a closed greenhouse under a constant culture condition of 26 ° C. After potting, we were able to harvest the ripe seeds in about 2-3 months (T ₁ seed).

Next, trained and seeded soil closed greenhouse the harvested seeds (T ₁ seed) to 26 ° C. certain culture conditions for 16 hours day length from transformants Kariyutaka was ripe in about two and a half months seeds (T ₂ seed) and training were seeded in the soil of a closed system greenhouse under the same conditions, we were able to harvest the ripe seeds in about two months (T ₃ seed). Similarly, the ripe T ₃ seeds were trained were seeded in soil closed greenhouse above conditions, it was possible to harvest of T ₄ seeds ripe in about 3 months. The results are summarized in Table 4. According to this example, the soybean transformant produced by the method of the present invention was renewed in a very short period of about 2-3 months compared to the normal variety, and the generation of seeds of the transformant generation in the same period. It has been demonstrated that progeny seeds can be obtained.

  As described in detail above, the present invention relates to a method for producing a transformant of soybean domestic varieties via Agrobacterium and a method for obtaining the seed of the transformant in a short period of time. It is possible to provide a method for producing a new soybean transformant capable of easily producing a soybean transformant. In particular, when a normal variety is used, it takes about 9 months to obtain seeds of the transformant generation, whereas using Kariyutaka, it can be shortened by about 3 months. The time required for immobilization of the introduced gene can be greatly shortened. According to the present invention, a soybean transformant having the property of culling during culture can be produced and provided. The present invention has a high technical significance as providing an epoch-making technique capable of obtaining a seed of a progeny that has been renewed in generations and generations of a transformant in a very short period of time compared to a normal variety.

The structure of an expression vector having a GFP reporter gene used in Example 5 is shown. The comparison of the period until the arrival and harvest of Thorne and Kariyutaka is shown.

Claims (8)

  A method for producing a transformant of soybean using domestic varieties Kariyutaka, (1) using a cotyledon part of the seed soaked as a plant part of soybean explant, (2) Infect Agrobacterium, (3) Culturing in adventitious bud induction medium to induce adventitious buds, (4) Culturing in shoot formation medium to form shoots, (5) Rooting, growth, seedling from shoots Create a transformant with the specified characteristics by the culture process, or use the resulting transformant to cross with any cultivar and create a cultivar that inherits superior traits from its progeny A method for producing a soybean transformant,   Using the resulting transformant, cross with North American varieties with excellent Agrobacterium infectivity, adventitious bud inducing ability, and shoot-forming ability, or with domestic varieties with excellent rooting ability from shoots. The method according to claim 1, wherein a cultivar having inherited an excellent trait is produced.   The method according to claim 2, wherein Jack or Thorne is used as the North American variety.   The method according to claim 1, wherein the concentration of Agrobacterium to be infected is OD 0.3 to 0.5.   A method for producing a transformant of soybean using domestic varieties of Kariyutaka, wherein the transformant has a characteristic of being settled at the time of culturing by the culturing step of (1) to (5) according to claim 1 A method for producing a soybean transformant, characterized by producing a body.   A transformant of Kariyutaka, which is produced by the method according to claim 5 and has a characteristic of being settled during culture.   A product derived from the transformant produced by the method according to any one of claims 1 to 5.   By using the transformant produced by the method according to any one of claims 1 to 5, it is possible to obtain seeds of a progeny whose transformant generation and / or generation has been renewed in a shorter period of time compared to a normal method. Method.