JPH07507445A - transformed wheat - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] transformed wheat field of invention The present invention allows foreign DNA to be taken up through pollen tubes, thereby leading to mature transformation. Transformation, including obtaining transformed embryos that can develop into transformed plants. A method for producing wheat plants is provided.

The invention also provides transformed wheat plants.

Background of the invention and prior art The advent of recombinant DNA technology and its application in the transformation of various organisms One of the main objectives is to provide general and agricultural This technique is useful for plant transformation in commercially important crops, e.g., especially wheat. It is the application of techniques. Such transformation results in improved rhizome tissue, weed control New traits that provide certain growth benefits, such as chemical resistance and insect resistance. The aim is to produce transformed plants with the following.

With the steady increase in the world's population, the yield of grain crops, especially important crops like wheat, of such plants with growth benefits such as those mentioned above. An urgent need to do this by developing new varieties It has long been thought that gender exists.

Various methods have been developed for transforming plants. This kind of method Preparation of protoplasts from plant tissues, in vitro characterization of protoplasts transformation and subsequent transformation of the transformed protoplasts into transformed wheat plants. Including the reproduction of et al.

terium tumefaciens) system, and Kono system. By injecting recombinant T-DNA from Ti or Ri plasmids into plant cells. inserted into the nuclear DNA of For transformation of plant tissues using exogenous DNA Yet another method involves bombardment of plant cells using a carrier particle projectile coated with DNA. This bombardment causes these projectiles to penetrate the plant cells and introduce foreign D into them. Insert NA.

Microinjection of foreign DNA into plant organs, especially reproductive or regenerative organs Various other methods of injection are also used for the purpose of producing transformed plants. ing.

All of the above methods are mainly used for dicotyledonous (Dicotyledon OUS) It has been developed for the transformation of plants and is well adapted to such plants. There is. However, these are monocotyledons (Mon.

. . It is not very applicable for the transformation of tyledonous) plants, especially wheat plants. It has been found that there is no such thing.

One of the reasons that the aforementioned methods cannot be applied to many monocots is that during tissue culture It is extremely difficult to put those grown cells into a complete plant and regenerate them. The fact is that

Transformation of wheat plants with uptake of plasmid DNA during pollen tube development A method for “Proc eedings of the 5eventh 1 international Wheat Genetics Symposium, Vol, l, p, 7 79-781”, Miller and Koebner Eds SCamb ridge, england.

13 19 July, 1988. These authors Phosphodoransph, an aminoglycoside that confers kanamycin resistance when plasmid containing the chelase II gene (NPTII) and the NPTII gene. Hypothetical wheat chromosome replication origins to facilitate gene and foreign DNA integration reported the use of a plasmid containing this point. These plasmids are First remove the tip of the shrunken stigma that has grown to the point where the powder tube is exposed. and then by adding a solution containing the plasmid to that pollen tube. , and incorporated into exposed pollen tubes. This plasmid DNA is grown The pollen tube migrates with the male nucleus toward the pollen tube, and then enters the ovule during fertilization. It seems that it was As a result, about 1% of the seeds obtained were kanamycin. resistant, which is caused by the presence of kanamynon-containing cultures in their seeds. and the ability of such germinating plants to germinate and grow in 100 Mature small plants in soil constantly soaked with water containing mg/I kanamycin. Explained by its ability to grow into wheat plants. Using NPTII gene probe Further extracted from kanamycin resistant plants by Southern blot analysis The DNA most frequently contains NPTII sequences with plasmids containing the wheat origin mentioned above. It was revealed that it contains. Furthermore, when self-crossing, the result The second generation of plants (F2) produced by this process are all kanamycin resistant; indicates that the NPT11 sequence has been transferred to the second generation. wheat genome What is the difference between true integration into NPTII and hypothetical NPTII-bearing endophytes? No differences were shown.

However, since the aforementioned publication, this claimed transformation procedure in wheat It must be pointed out that there have been no subsequent announcements confirming the success of the law. above The various presentations and conferences that followed the presentation enabled researchers in the field to: In fact, it is impossible to reproduce the above method and it is impossible to transform wheat. We are revisiting various other techniques described herein to It should be noted that this has become clear. The above-mentioned technology can be applied to wheat In a recent review of current procedures for transforming target cereal plants, (Potrykus, 1990, Bio/Techno) 8.535-542), pollen tube treatment methods are further discussed in this review. However, it was concluded that there was little chance of success. In the author's words, "the pollen tube circuit. The transformed plants do not regenerate at all and are probably There's not much of a future. ”.

In addition, the above-mentioned treatment method is different from wheat even though it is a monocotyledonous plant. Success has been achieved only in rice plants with different scientific aspects (Luo and W. u, 1989, Plant Molecular Bio4ogy Report It should also be noted that ter 7, 69-77).

Additionally, all published wheat plants transformed by any of the preceding methods Among these, any wheat strain is obtained from a commercially available crop variety, i.e., a wheat crop. It has not become an agricultural cultivar that is bred on a large scale for this purpose. transform Many of the hypothetical wheat plants are of little use for agricultural applications, at best For example, commercially available wheat lines transformed by backcrossing and crosses with other varieties. It can be used as a starting point for trial acquisition.

Summary of the invention Growing transformed wheat plants, especially commercial crop cultivars, i.e. wheat crops Transformation is one of many cultivars used around the world for It is an object of the present invention to provide a method for producing wheat plants. be.

It is another object of the present invention to provide commercially available varieties of transformed wheat plants.

Suitable recombinant DNA molecules for the purpose of producing transformed wheat plants as described above. It is yet another object of the present invention to provide.

All other objects of the invention will be apparent from the following description and claims. It seems to be the case.

According to the above objectives of the invention: (a) remove all pollinating plant rot before it matures (and thereby After emasculating the florets of the plant, the stigmas of the florets are then planted with wheat of the same or a different variety. Introducing synchronized pollination by pollinating with rot of mature male pollinating plants from plants. (b) applying a drop of an aqueous DNA solution to one or more pollinated stigmas; Then, pollen tubes begin to grow into the style within the pollinated stigma. There is a post-pollination period in which the ovaries are present but have not yet reached the ovary. , the DNA solution is foreign to the plant and desired within the plant. at least one gene capable of inducing expression of a desired trait, and optionally comprising a DNA vector carrying an additional marker gene; (C) A drop of the DNA solution is applied once the DNA vector reaches and enters the ovule. the column head in a humid environment for a period of time to ensure that (d) the treated plants of step (c) are kept in a protect the plant from additional insemination by (e) selection of transformed wheat plants; growing the seeds under modified conditions for the purpose of A method is provided for producing a transformed wheat plant comprising:

Sometimes referred to herein as "aspects of truncation" According to one embodiment of the present invention, the stigma is truncated after pollination, and further treated with an aqueous DNA solution. Apply the drops on the truncated stigma. Sometimes referred to herein as "untreated stigma embodiment" In accordance with other aspects of the referenced invention, a drop of aqueous DNA solution is placed on an untreated column. application, followed by a period of time after pollination.

The above-mentioned truncation preferably cuts the stigma tip between 1/2 and 21/2 hours of pollination, more preferably. Preferably, pollination is carried out by decapitation for 1-2 hours. Cultivar ATI R(CNOS”/PJ62/4/GLL/3/T.

B//JAR″S”/CRESPO, Hazera 5eed Co,, Is rae I), BAGULA S” (CM 59123, CIMMYT, Mexico), CIANO79 (CM31678, CIMMYTSMexic o), Genaro81 (VEERY #3 “S”, CIMMYT, Mex i co), KAUZ″S-(CM67458, CIMMYTSMex ic o), KEA″S” (CM21335, CIMMYT, Mexico) 1, MILAN (231BWSN37, CIMMYT, Mexico), OPAT AM85 (CM40038, CIMMYTSMexico), Papag’o M86 (0M52359, CIMMYTS Mexico), PAVON76 (CM8399, CIMMYT, Mexico), 5eri82 (VEERY #5 “S”, CIMMYT, Mex i co), and Sha f i r (SON64A/TZPP//NA160/3/FA, Hazera 5 eed Co1, l5rael), spring bread -breadX, aestivum L,) cultivation All cultivars were tested according to the invention, but in the case of these cultivars the truncation was Preferably, this is done at the 11/2 hour mark. This favorable between pollination and truncation The time periods may also be applicable to pond wheat species and cell variety plants.

Generally, when practicing the truncated aspect of the invention, between the pollination and the truncated tip of the stigma. The interval should be selected according to the wheat genotype and environmental conditions used. example For example, wheat variety and environmental conditions may influence relatively rapid pollen deposition on stigmas and It seems to be advantageous for the subsequent development of pollen tubes and growth into the stigma and style. In some cases, this interval can be in the range of 1-11/2 hours. other For example, wheat cultivars and environmental conditions may result in slower, pollen deposition. and in cases where it is advantageous for subsequent pollen tube development, this spacing may be 11/1 It can be in the range of 2-2 hours.

As described above, the wheat phenotype and environmental conditions used previously were used to develop this product. In some cases, pollination and DNAfi fabrics may also be used when performing the Ming untreated stigma aspect. It should be executed at the above-mentioned timing between .

According to both embodiments, the DNA solution applied in step ('b) of the method The droplets of DNA vectors transported by the growing pollen tube reach the ovule. The pollinated stigma (stage (C) ) to be kept in proper condition on top. A suitable period of time is a short period (at least 12 hours, e.g. I found that it was about 12-14 hours.

The invention also provides recombinant DNA vectors for use in the above methods. provided.

The DNA vector of the present invention is capable of inducing the expression of a certain trait in plants. It is desirable to transfer the gene into the plant. . Such genes include, for example, genes expressing nitrogen fixing structural proteins, The herbicide can be a gene that expresses a protein that confers herbicide tolerance on the plant. . Furthermore, the vector can also be used in suitable conditions, e.g. Reporter genes such as the uidA gene encoding β-glucuronidase This gene can also serve as a marker of successful transformation. , this means that if the substrate (X-Glue) for the expressed enzyme is provided, In this case, cells expressing this gene convert this substrate into a detectable product. This is because it is possible.

The DNA vector can be used for example with antibiotic resistance, such as kanamycin resistance. also include a plant selectable marker gene, which can be a gene encoding a is preferred. This type of information used in experiments conducted within the framework of the present invention A non-limiting example of a gene is tubalin (nopa Ilne) synthase (NO3) promoter and polyadenylation of TL-DNA (gene 4) APH (3') II (NPTI I) gene linked to the conversion signal sequence (Gielen.

J. et al., 1984, Koncz et al., 1989).

This API (3°) I I (NPTI +) gene is an enzyme aminoglycone It encodes a phosphotransferase, so it cannot be transformed. confers kanamycin resistance to plants.

In order to obtain a large amount of DNA vector necessary for preparing the DNA solution. Other bacterial selectable marker genes, such as Amp', are also present in bacteria. It can be included within the vector for the purpose of propagating the NA vector.

Such NA bases used in experiments conducted within the framework of the present invention. Non-limiting examples of vectors include plasmids pPcV702nifD and pPC V702GUS, and both of Kowara carry the above-mentioned API (3') II gene. Contains, and in pPcV702n i fD, Klebsiella Klebsiella pneumoniae (Klebsiella pneumoniae) n1fD The coding region is the 358 promoter and NOS terminator of CaMV. and in pPCV702GUS, β-glucuronidase (GUS) coding region also contains the CaMV 353 promoter and the NO Connected to S terminator. Therefore, by the method of the present invention, these plastics can be Wheat plants transformed with either Sumid are kanamycin resistant and/or Alternatively, the GUS enzyme can be expressed.

The invention also provides transformed wheat plants that are cultivars of agricultural commercial crops.

In accordance with the present invention, transformed wheat plants of several commercial crop cultivars have been obtained for the first time. As a result, a long-standing request was fulfilled. Transformed microorganisms obtained according to the present invention Wheat plants can be grown for five generations without losing the foreign genes that transformed the source plants. It grew over time.

Brief description of the drawing The figures in the accompanying drawings illustrate what is shown below, and FIG. Schematic representation of PcV702n±fD, FIG. 2 shows plasmid pPcV702 Figure 3a is a schematic representation of GUs, as described in Example 4. Wheat varieties, plants transformed according to the method of the invention, and plasmids Collected from pPCV702ni, BamHI and H4ndlTI system NPTII gene template for limited endonuclease digested DNA samples. Figure 3b shows the results of Southern blot hybridization of the lobe. As described in Example 4, Southern plot results (Figure 3a%Figure 4a Transformation designated TAU89107-1 determined from s and Figure 5) Related restriction fragments of pPCV702nifD integrated into the wheat genome in the body is a schematic restriction map illustrating endonuclease sites; Figure 4a shows the wild-type wheat variety and the present invention as described in Example 4. Therefore, Pstl NPIII probe on DNA samples digested with restriction endonucleases The results of southern plot-hybridization of B. Figure 4b shows the Southern blot results (Figure 3a, pP integrated into the wheat genome as determined from Figures 4a and 5). Schematic diagram illustrating the restriction endonuclease sites for cV702n±fD. As described in Example 4, FIG. 5 is a NPTI! Fragments and and the remainder of the 702 u±fD vector, wild-type wheat plants, and plus By selfing of the first generation plants transformed with mido pPcV702n i fD. The second generation (F2) obtained by The results of Southern blot-hybridization with the same DNA sample are shown. the law of nature, FIG. 6 shows the NPTII fragment and transformation as described in Examples 4 and 5. Transformed wheat plants obtained by selfing of transgenic TAU89107-1 Second generation (F2) BamHI and HindllI digested DNA sample It is the result of Southern plot hybridization with FIG. 7 shows the NPT III fragment as described in Examples 4 and 6. and transformed plants obtained by selfing of plants from family F2 of TAU89107. BamHI and 14i ndIII digestion of third generation (F3) of mature wheat plants This is the result of Southern blot hybridization with the DNA sample, Figure 8a shows the APH present in transformed wheat plants of the 5hafir cultivar. (3”) Agarose of DNA sample obtained by PCR amplification of II gene The results of gel electrophoresis are shown. DNA samples were prepared as described in Example 7. The plant originates from the transformant TAU89107-1 and is the original of each generation of the plant. Extracted from F2, F3, and F resulting from self-pollination, Figure 8b shows transformed cells obtained by selfing as described in Example 7. Using total DNA extracted from the F4 progeny of TAtJ89-107-1, PCR amplification of the PI (3°) II gene and its gene 4 terminator The results of agarose gel electrophoresis of DNA samples obtained by Figure 9 shows gene 4 terminator fragments and 5hafir wheat plants and transformations. Southern blot with DNA samples of F4 generation descendants of body TAU89107-1 Figure 10a shows the results of hybridization, as shown in Example 9. TAU8910 produces normal germination when grown in medium containing kanamycin F5 progeny CC44 of 7-1 transformed cells (TAU89107-1-J 18-9 -IB-4A) was grown under the same growth conditions as a wild type Sha f that turned white. shown in comparison with ir plants, FIG. 10b shows CC44 transformants, as shown in Example 9, or NPT II technology using DNA templates extracted from plants of wheat variety 5hafir. Gel of PCR amplification product of ile (51bp) and its gene 4 terminator Figure 11 shows the separation analysis of pPcV702 as shown in Example 10. A commercially cultivated plant transformed with GUs and searched with the same plasmid. First generation (Fl) Southern of wheat plants of varieties Pavon76 and 5eri82 The plot shows the results of hybridization, and these DNA samples are Digested with EcoRI and Hindll, FIG. DNA extracts from F, transformants of cultivar 5eri82 as shown in API (3°) II from The results of agarose gel electrophoresis of NA samples are shown, and further, FIG. 13 shows Streptomyces hygroscopica as shown in Example 12. BA genetics of pix (Streptmyces hygroscopicus) Transformation with plasmid p PAT-NPT I 1100.1-28 carrying offspring Agarose of 0.6 kb PCR product obtained from transformed F, generation transformants. The electrophoresis pattern on the gel is shown.

Description of specific implementations The invention will now be described in more detail in the examples described below and the accompanying drawings. Explained in detail. These examples are to be considered as detailed illustrations of the invention. and should not be considered as a limitation.

Example 1 - Plasmid Two plasmids, pPcV7, both derived from vector pPcV702. 02 mice and pPcV702GUs in Figures 1 and 2, respectively. were prepared as shown (Koncz and Sche I, 1976; an d Koncz et al., 1989). In both plasmids, a Encodes minoglycoside (kanamycin) phosphotransferase The APH (3') II or NPTII gene is NO3 (Tubarin (n opa l1ne) synthase) promoter, and serves as a selective marker. <TL-DNA> The upper 2 gene (gene 4) is the boria demolization signal, Po1 It was connected to yA (terminator). When this gene is expressed in plants, , confers kanamycin resistance.

Plasmid pPcV702ni contains the CaMV 35S promoter and NO Klebshera pneumoniae unit located between the S terminator Contains a recording area.

Plasmid pPcV702GUF contains the CaMV 35S promoter and NO β-glucuronidase C, a reporter gene, linked to the S terminator Contains the coding region of the GUS) gene.

The above plasmid was dissolved in 10mM Tris, 1mM EDTA, pH 8. Mixtures of similar coiled and relaxed DNA types are described hereinafter. It was used to prepare the DNA solution to be applied to the stigma in the transformation method described above. .

Example 2: Preparation of wheat plants and administration of DNA Several commercially available wild type springs Wheat plants selected from Bread wheat cultivars are grown in thermostatically controlled temperature systems using supplemental light. They were grown in pots indoors (21±2°C). In addition, the same type of wheat was planted as before. A second group of plants was grown in net houses on sandy loam soil. child These groups of wheat plants were planted at the beginning of the experiment, with additional plantings at 10-day intervals. to ensure a constant supply of pollen when needed for pollination. wheat When the plants reached growth state 57 (appearance of 3/4 panicles), the panicles of the experimental plants were i.e. cut each spikelet at half its height and cut off the two outer florets. The middle floret was removed leaving the . The immature stamens are separated from the two remaining florets (spikes). (wins) were removed from each. Cut all spikelets on one side of the flower axis into a resistant I marked the base with a water-based marker. Then, cover the ears with a glassine paper bag. It was.

Each pistil of the two florets of the central spikelet attached to the treated panicle mentioned above. When the stigmas of these become feathered and begin to open outwards (4-5 days after emasculation), i.e. When the pistil shows signs of maturity, run a glassine paper bag across the apex. Cut open and halve the spikelets to allow the mature stamens to protrude outward from the spikelet. Cut the panicle of a male pollinating plant (of the same or a different cultivar) into pieces on the paper. I inserted it into the bag. Leave the pollinating plant ears in the paper bag for 20-30 minutes. The stamens were allowed to grow and the pollen was released. Plants for endowment The panicle was twisted several times to ensure that the pollen was distributed to all the florets on the panicle. after that , the ears of pollinating plants were removed and a glassine paper bag was closed with a clip.

Pollinate between 1/2 hour and 2 hours later, usually between 1.5 hours and 2 hours later. Pollen tubes form within the panicle and begin to grow (however, pollen tubes begin to grow beyond the style). At the stage (which has not yet reached the ovary), the tip of the feather-like stigma is sharpened. Cut with fine-tipped scissors to expose the pollen tube described above, and then remove 10 μm of DNA. Add a drop of the solution (2-3 μg of plasmid DNA - Example 1) to a microtip swab. Use as an adjunct to pre-label and cut off half of the panicle. It was applied to the tip of the stigma. Half without tltjm used as control A 10 μm thick layer of water was applied to the florets attached to the panicles. The above-mentioned pollination treatment method It should be noted that pollen tube formation within the pollen tubes starts in a synchronized manner. be. Furthermore, the aforementioned period between pollination and cutting of the pollinated stigma tip is approximately 1 -11/2 hour period allows for relatively rapid pollination contact and subsequent intrastylar and It is optimal for conditions that favor the development of pollen tubes into the flower style, and is During the 2-2 hour period, the aforementioned pollen contact and pollen tube development are slower. The environmental conditions and the wheat used are optimal under the given circumstances. Varies depending on plant species.

Open the treated glassine bag by pulling down to fully expose the ears. The sealed plants are placed in a humid chamber inside a temperature-controlled greenhouse. In this greenhouse, a water sprayer provided a fine mist for 12 to 14 hours. there was. For plants grown in net houses, temperatures must drop below 20°C. The treatment was carried out in the afternoon. After application of the DNA, the plant is supplied with high humidity. The pollen tubes are covered overnight in a plastic bag to allow pollen tube growth into the ovary and subsequent reception of female gametes. or, where appropriate, exposed to dew. I did it like that. After exposure to fog or dew, the glassine paper bag is lifted up and processed. The treated panicle was covered and closed with a clip. The detailed operation treatment method is explained for each treated ear. We recorded about each.

A similar experiment was conducted according to the procedure described above, but in this experiment Without cutting off the tip of the stigma, a DNA solution was applied to the tip of the uncut pollinated stigma. . The results of both experiments are shown in Table 1, as referenced in Example 3 below. It was shown to.

Example 3: Bioassay (a) APH(3')11 expression: pPcV702 was obtained by the transformation method of Example 2 using the vector. The obtained seeds were soaked in a low concentration of chlorite for 5 minutes, washed with distilled water, and then , transferred to a water-agarose Petri dish containing 150 μg/m+ kanamycin. , into a growth chamber at 25°C. After 10 days, all seeds were placed on MS culture. After transferring to the ground, the decolorization of the shoots and the number and length of roots were recorded. Scores were recorded for each item. In one experiment, seedlings were treated with kanamycin. Stored in a growth chamber for 4 days in a medium containing medium, then an additional 4 days for MS growth. They were transferred to a growth medium and scored as described above.

The seeds obtained from the previous transformation procedure represent the F1 generation, which was Water-agarose treated with hypochlorite and containing 150 μg/ml kanamycin At the same time, the parental control was placed in a Petri dish without kanamycin. I put it in. Transfer germinated seeds with normal roots and unbleached primary leaves to pots. and placed at 18°C (14 hours light/10 hours dark). These F, seedlings are and the seeds obtained therefrom represent the F2 generation, which is subsequently , sown without selective forcing to increase seeds. seeds obtained from it The child represents F.

APTII activity, i.e., the vector used to transform the primary parent plant. To investigate the expression of the more conserved NPTII gene, F3 generation seedlings were germinated on water-agarose containing 150 μg/mI of kanamycin. Ta. AP The TI+ gene is shown to be present and expressed. these green saplings The leaf DNA was transferred to a greenhouse and subjected to Southern blo sorting and PCR for specific sequences. Analyzed by amplification (see Examples 4 to 11 below).

The above kanamycin selection method is applicable to T cells up to and including Fs (5th) generation. All successive generations of the transformed plant line designated AU89107-1 This further indicates that the previous transformation is stable. are doing.

(b) GUS expression: axenic from originally transformed plants transformed with pPcV702GUs. β in roots cut from Fl seedlings grown in conditions (i.e. isolated) - Expression of glucuronidase was demonstrated using X-G Iuc as substrate.

The appearance of blue color due to the specific cleavage of the 1-4μ bond of X-Glue showed the activity of GUS incorporated into (Jef Ferson et al. , 1987) o In 40% of plants expressing GUS activity (Table 1), The analysis of the related exogenous genes integrated into the genome (so-called GUS) ±dA) (Example 10 below and Figure 11) ). This GUS assay was performed as described in Example 2 above. For example, a severed stigma (i.e. without a stigma tip) or an uncut stigma. Plants transformed with a DNA solution containing pPcV702GUs applied to Comparisons were also made between F. seedlings originating from F.

Surprisingly, the "uncut stigma" group showed better results than the "cut stigma" group14. 6% vs. 5.8%), this result indicates that the growing pollen tube was physically exposed to the DNA solution. This contradicts previously held beliefs that (See references in the Background section above).

Table 1 Evaluation of GUS activity in roots of germinating F1 transformantsJefferson Using the histochemical assay described by et al., (1987), The position where the blue color of the X-Gluc cleavage product appeared was confirmed.

Example 4 Insertion of NPT II [APtl (3°) II] into the wheat genome - Southern plot hybridized transformed wheat, i.e. the above-mentioned Kana The presence of foreign DNA in mycin-resistant plants is shown in Figures 3-7 and 9. This was confirmed by Southern protein analysis. Hypothetical transformants and wild Total DNA (15 pg) extracted from leaves of type plants was subjected to standardization using various restriction enzymes. Digested using standard methods and separated on a 0.8% agarose gel, followed by plotted on a Ron filter. isolated from pPcV702GUs, and Apal/Hindl containing all NPTII genes labeled with 32p The ll fragment was used to detect the presence of integrated foreign DNA within the wheat genome. It was used as a probe. Hybridization with this probe is possible under austerity conditions. , that is, x5 Denhardt's solution, x4 5SC 101% SDS, at 60°C. Then, at the same temperature, x4 5SC10,1% SDS I rinsed it 4 times inside.

As shown in Figure 3a, total DNA was extracted from the wild-type wheat cultivar Genaro8. 1 (VEERY #3 “S”, CIMMYTSMexi co), Ser　 i82 (VEERY#5″S″, CIMMYT, Mexico), and Sha　f　ir　　(SON64A/TZPP//NAl60/3/FA) , and transformed with plasmid pPcV702n, TAU89 104-3 and kanamycin resistance (Km” designated TAU89107-1). ), that is, extracted from a hypothetical transformant expressing APTII activity. Lottery All released DNA was digested with restriction endonucleases using standard methods previously described. BamHI and HindIll and then run on an agarose gel. and transferred onto a nylon filter. As a positive control, plasmid Two DNA samples from pPcV702GUS were also prepared, one with the enzyme One was digested with BamH■ and HindIII, and the other was digested with enzymes Apal and H1. ndll+ and run these samples on the same agarose gel as described above. and transferred onto a nylon filter. DN on nylon filter The A sample was converted to the above NPTII Apal by the above hybridization method. /HindlII fragment. The hybrid shown in Figure 3a The results of the cell formation showed that the transformant TAU89107-1 had a polymer of approximately 10.5 kb. NPTII probe on JIBamHI/HindrII DNA fragment It has been proven that they carry foreign DNA that hybridizes with. wild None of the type DNA fragments can hybridize with the NPTII probe. This indicates that this gene is not present in these genomes. are doing. The schematic restriction map shown in Figure 3b shows the restriction map of the protein into the wheat plant genome. Possible method for inserting a portion of lasmid PPCV702ni (Figure 1) ``genome'' refers to this genomic DNA. This map is shown in Figure 3a and Based on the hybridization results shown in Figure 4a and plasmid Known restriction enzyme sites from pPcV702n engineering (Figure 1) and transformants It was confirmed that the n1fD sequence does not exist in the DNA of TAU89107-1. It was created based on.

As shown in Figure 4a, total DNA was extracted from the same wild cells as described for Figure 3a. DNA samples extracted from type cultivars and transformant wheat plants are the two plasmid vectors in the NPTII gene and the amp” gene. It was digested only with the restriction endonuclease Pstl, which cuts each. Digested D NA samples were separated on an agarose gel and subsequently transferred onto a nylon filter. In addition, as before, NPTII Apal/Hindlll DN Hybridization was performed with the A fragment probe. Depending on the result of hybridization, the shape The transformant TAU891.07-1 was able to hybridize with the probe. It is said that it has two Pstl DNA fragments (approximately 6.9 and 0.8 kb). It became clear that The schematic limit map shown in Figure 4b is similar to that of Figure 3b. , which is the same as shown in Fig. 5 in Figs. 3a and 4a. , total DNA of wild-type wheat cultivars 5haf]r and TAU89107-1. Nine F2 transformants of F1 plants (1, 2, 3, 4, 5, 6, 7, 8, and 9) (see Figures 3a and 4a)). These DNA samples was digested with restriction endonucleases Psl and BamHI as before, It was separated on an agarose gel and transferred to a nylon filter. nylon fi DNA samples on the router were subjected to probe NP formation as previously described.

According to this result, second generation transformants 4.5.6.8 and 9, in particular, Genomic DNA that is capable of hybridizing to the probe and is wild type Pstl/BamHI DNA fragment that does not seem to exist in A (approximately 2 5 and 3.5 kb).

Example 5: Southern hybrid formation of F2 DNA amHI and Hind 10.5 kb fragment in F2 plants of TAU89107-1 digested with llI The presence of pieces is shown in FIG.

Lines 1 and 2 represent the F2 transformant TAU89107-1-J18; Line 4 is the F of TAU89107-1, F2TAU89 derived from the white value of the plant. 107-1-KIO transformant and further described above in Example 4. It is something. The probes used for these analyzes are shown below: Hi ndIII-Δpal NPTII fragment or PCR-synthesized gene 4 (described in Example 8), both of which are As a result, the same /N hybrid formation pattern was shown.

Example 6: Southern hybridization of F, DNA amHI and Hind F of the TAU89107-1 transformant digested with lll, 9 contained in the plant, The presence of 8 kb and 10.5 kb fragments is shown in FIG. Wild type 5hafir? Their DNA is shown in line 1. Lines 2.3 and 4 are F3 families shown below. , respectively, TAU89107-1-029-21 and TAU89107-1 -17-14, and TAU89107-1-18-9. southern block The method was previously described in Example 4. Pros used for these analyses. The tube was as previously described in Example 5, namely HindIll-Apal. fragment or a PCR-synthesized gene 4 terminator fragment, both of which are Both methods showed the same hybridization pattern.

Example 7: PCR amplification In Figure 8(a), standard conditions for DNA fragments obtained after PCR method are shown. Electrophoretic pattern on agarose gel at the bottom, i.e., transformant 5 ha fir TAU89107 IKm” subfamily F2, F8, and F4 Results of PCR amplification of the inserted NPTII coding sequence contained in the substitute plants. (See also Example 11 below for PCR methods).

The DNA templates used in the PCR method are shown below, and they are Line No. 1 = Control sample, no DNA template present in reaction, Line No. 2 = control sample, non-transformed, wild type 5hafir DNA, Line No. 3 = Transformant sample, F2 generation, 5 hafir “I’AU8 9107-1-B19 DNA.

Line No., 4 = transformant sample, F2 generation, 5hafir TAU891 07-1-J20 DNA.

Line No., 5 = transformant sample, F8 generation, 5hafir TA0891 07-1-J18-X DNA.

Line No., 6 = transformant sample, F8 generation, 5hafir TAU891 07-1-J18-9 DNA, line No. 7-transformant sample, F4 generation 5hafir TAU89107-1-J18-9-IB DNA, line No. 8 = transformant sample, F4 generation, 5hafir TAtJ89107 -1-J18-9-IK DNA, line No., 9=Hae l l I restriction error DNA fragment from ΦX174 bacteriophage cleaved with endonuclease For size markers,

Therefore, the F2, F3, and F4 generations of transformants ShafirTALI 89107-IKm” (see also Table 2 of Example 11 below) The presence of foreign DNA within the Unambiguously verified by PCR amplification. Contains NPTII coding sequence In all such transformants, the 790 bp fragment was 3, F4) was synthesized from the template.

Figure 8(b) shows the transformant TAU89107-1Km' obtained by selfing. APR (3°) I gene and its The results of PCR amplification of the gene 4 terminator adjacent to this are shown. For PCR reaction The primers used in this are shown below, and they are: Primer NO31 covers the 21st part of the coding region of NPTII as shown below. (equivalent to 26 nucleotides from the 47th nucleotide to the 47th nucleotide), which is = 5-GCACGCAGGTTCTCCGGCCGCTTGGG-3° , Primer No. 2 covers 74 of the coding region of NPTlt as shown below. (equivalent to 31 nucleotides from the 4th to the 775th nucleotide), that teeth, = 5'-CCCGATTCGCAGCGC8TCGCCTTCding^TCG C-3', Primer No. 3 is a reverse primer and contains 4 genes as shown below. represents the 3゛ end of the terminator, which is =5゛-^TTATAC8TAACA CGCACA-3° (total of 19 nucleotides) It is.

The DNA template and primers used in PCR amplification are shown below. And that is, Line No. 1 = BstEll digested bacteriophage λ DNA size marker, Line No. 2 = control template, non-transformed, wild type 5hafir DNA and , primers 1 and 3, Line No., 3 = transformant sample, F4 generation, 5hafir TAtJ89 107-1-829-21-IA2 and primers 1 and 3 (approximately 1.1 5kb), Line No., 4 = transformant sample, F4 generation, 5hafir TAU891 07-1-J 18-9-IB and primers 1 and 3, line No. 5 = Transformant sample, F4 generation, 5hafir TAU89107-1-J 18-9-IB and primers 2 and 3 (about 0.5 kb), Line No., 6 = transformant sample, F4 generation, 5hafir TAU891 07-1-J 18-X-IB and primers 1 and 3, line No. 7 = Transformant sample, F4 generation, 5hafir TAU89107-1-J 18-X-IB and primers 2 and 3, line No. 8=Hae III DNA of bacteriophage ΦX174 digested with I.

It is.

Linkage between the NPTII coding region and the adjacent gene 4 terminator The knot has been maintained through four generations of selfing in the TAU89107-IKm’ family. Ta.

Example 8: Stability of NPTII sequences The stability of the NPTII sequence in different plant organs was determined using TAU89107-1. A variety of F4 transformants were evaluated.

The presence of a 10.5 kb fragment was detected in Southern blot analysis of representative plants from three families. As shown, this was demonstrated in F4 plants (Fig. 9).

F. Total DNA extracted from plants was digested with BamHI and Hindll. , and a gene 4 (polyA) terminator fragment obtained by PCR amplification. explored (Figures 1 and 2). The DNA template is shown below, which is: Line No. 1 = control template, non-transformed, wild type 5hafir DNA, line No, 2 = transformant sample, F4 generation, 5hafir TAU89107- 1-H29-21-IAIC (C=associated with the main tiller from which DNA was extracted) ), Line No. 3 = Transformant sample, F4 generation, 5hafir TAU891 07-1-H29-IA2C (sourced from the same F3 panicle as in line N092) main saplings from other plants), Rai: /No, 4 = transformant sample, F4 generation, 5hafir TAU89107-1-J 18-9-IBC (DNA Extracted main saplings), Rai: /No, 5 = transformant sample, F4 generation, 5h afir TAtJ89107-1-318-9-IBR (R=DNA extracted associated with the roots of plants), Line N096-transformant sample, F4 generation, 5 hafir TAU89] , 07-1-J 18-X-IBC (main young tree from which DNA was extracted), line No. , 7 = transformant sample, F4 generation, 5hafir TAtJ89107-1 -J18-X-IBR (plant root from which DNA was extracted).

10 present in F4 plants, as shown by the results displayed in Figure 8b. ．． The 5kb fragment is a terminator that connects to the coding region of NPTII. - (poly A sequence of gene 4) should also be included. Therefore, termine Southern blonot analysis performed using the data sequence revealed that the incorporated sequences were autologous. It was demonstrated that the virus was stably maintained through four generations of breeding. Approximately 10kb Two adjacent bands were observed in the F and F4 generations.

Example 9 Kanamycin resistant F, plant Resistance to kanamycin was determined by F germinated on 150 μg/ml kanamycin. , expressed in plant seedlings. Representative F. plants were grown from wild-type 5hafir. It is shown in Figure 10a together with kanamycin-sensitive seedlings.

F, plant (CC44) is a descendant of F3 family of TAU89107-1-J 18-9 It is. Mature F5 kanamycin-resistant 5hafir transformant plants have a phenotypic (plant height, leaf, and panicle shape) of untransformed wild type 5 ha Similar to ir. This representative FS plant was divided into Fl, F5, and F3 generations. We investigated resistance to kanamycin at the seedling stage; Seed propagation via selfing without kanamycin was carried out in F2 and F4 generations. Wata.

Coding of NPTII “tail J (51bp) and gene 4 (polyA) The presence of a link to the terminator was determined using primers 2 and 3 in Figure 8b. Verified by CR amplification (Example 7). Kanamainone-resistant transformant CC44 and kanamycin-sensitive 5hafir PCR products were digested with Haell. on a 12% agarose gel with the DNA of bacteriophage Φx174. was subjected to electrophoresis.

The DNA template and primers used in PCR amplification are shown below. Yes, that is, Line No. 1 = bacteriophage Φx174 digested with Hae I II DNA.

Rai: /No, 2 = control template, non-transformed, wild type 5hafir DNA, and and primers 2 and 3, Line N03 = Kanamycin resistant FsShafir TAU89107-1-J 18-9-IB-4A plant (=CC44) and primers 2 and 3 Search (approximately 0.5 kb).

Example 10: GUS reporter gene integration vector pPCV702GUS The presence of other foreign DNA sequences incorporated by plating may result in Southern hybridization. demonstrated this in F1 plants. This result is shown in Figure 11. The figure shows F, extracted from wheat plants transformed with pPCV702GtJS, and , shows total DNA probed with the same plasmid. In this Southern Brosoto 5hafir (line N01) and transformant DNA sample (line N01). Samples 2 and 3) contained approximately 5 μg of total kenome DNA. These samples Restriction endonucleases EcoRI and HindIII .

In the integrated DNA, 35S promoter-GUS coding region-N Approximately 10 ng of vector was used to prove the presence of 2.9 kb including the OS terminator. vector pPC~'702GUS was digested with the same enzymes and analyzed by Southern blot analysis. (denoted as 702GUS).

Line N011 - represents wild type S+1afir control, line No. 2=F, small Representing the wheat transformant 5eri82 TAU90-56-13, and further Rai:/N o, 3 = F + transformant Pavon76 TAtJ90 -84-7.

The previously observed results are consistent with the high yielding commercial wheat varieties Pavon76 and 5eri8. In the F1 transformants of 2, there was an integrated 2.9 kb GUS fragment. This serves as evidence that this is the case.

In a similar method, the 5er182 transformant TAU90GUS/NPTII F, subtribe (see Table 2 of Example 11 below).Transformant TAU9055- 9 and TAU9056-13, pPcV702GUs vector. (transformed by a vector) directly as shown in FIG. Evaluation of the presence of GUS, NPTII, or both by PCR I did this. This figure shows the standard DNA fragments obtained after the PCR method. It represents the electrophoresis pattern on agarose gel under the following conditions. F1 of several types of Mari, 5eri82 TAU90 GUS/NPTII transformants Inserted NPTII or GtJS coding contained within generation subfamily The results of PCR amplification of the sequences are shown. Two DNA templates used in the PCR method and their amounts are shown below, which are: Line No. 1 - control sample, GUS primers (primers N03 and 4 , see Table 2 of Example 11) was added to the reaction, but no DNA template was added. Line No. 2 = control sample, NPTII primer (primer No. 1 and and 2, see Table 2 of Example 11) were added to the reaction, but no DNA template was present. addition, Line No. 3 = control sample, GUS primer and 5eri8 as template 2 Added wild type DNA (500n gDNA), line No. 4 = control sample , added NPTII primer and 5eri82 wild-type DNA as template (5 00ng DNA), line No., 5 = transformant sample, GUS primer and and added TAU9055-9 DNA as a template (50 ng DNA), line No, 6 = transformant sample, NPTII primer and TAU as template Added DNA of 9055-9 (50 ng DNA), line No. 7 = transformant TAU9054-10 DNA as sample, GUS primer and template Addition (50 ng DNA), line No., F3 = transformant sample, GUS protein Add TAU9054-11 DNA as a primer and template (50 ng DNA A) (synthesis not observed), Line N019 - Transformant sample, NPTII primer and as template Add DNA of TΔU9056-13 (200ngDNA), line No. 1 0 = Φx174 bacteriophage cleaved with Haell + restriction endonuclease size markers for DNA fragments from the DNA of the sample; It is.

All prior transformants are part of the transformants shown in Table 2. It is necessary to keep this in mind (Example 11).

Therefore, as shown in FIG. 12, 1170bp GUS and 790bp The NPTII fragment of was synthesized by transformant TAU9055-9 (respectively lines 5 and 6). The GUS fragment was also present in TAU9054-10 transformants. It was confirmed that it is included (line 7), but it is not included in TAU9054-11. (line 8). Insertion of the NPTll coding sequence also demonstrated in U9056-13 transformants (line 9). This transformation The body is 2. It is also shown in Figure 11 where a 9kb GUS fragment is observed. Ru.

All transformants verified by Southern blot and PCR amplification were showed positive GUS expression in the area (Table 1).

Example 11: Transformation of different wheat varieties with two plasmid vectors (engineering a) estivum L, )) Variety Genaro81 (VEERY #3-3”, CIMMYT, Mex i co), KEA″S-(CM21335, CIMM YT, Mexi co), Papango M86 (CM5235.9, CI MMYT, Mex i co), Pavon76 (CM8399, CIMM YT, Mex i co), Se r i 82 (VEERY #5 “S”, CIMMYT, Mexico), and 5hafir (SON64A/TZP P//NAl60/3/FA, Hazera 5eed Co, , l5rae l) (See also Example 2-4 above) Transformation by the administered exogenous DNA We investigated the conversion ability. The previous wheat flavor was treated as described in Example 2 above. However, the exogenous DNA administered to these plants , details are described in Example 1 above. After the transformation process, The treated plants were subjected to the bioassay method detailed in Example 3 above. used and further tested by PCR assay performed as described below. The possibility and frequency of transformation were determined.

In transformants containing the NPTII coding sequence, the NPTI■ fragment is PC using the total DNA of the transformant as template and the primers shown below. Synthesized by R.

Primer No. 1 (5' region of NPTI I coding sequence): = 5 '-GCACGCAGGTTCTCCGGCCGCTTGGG-3' Primer -No, 2 (Reverse block of 3' region of NPTI I coding sequence = 5-GAA GGCGAGCGCTGCGAATCGGG-3'GUS coding sequence. In transformants containing the GUS sequence, the total DNA of the transformant as a template or and by PCR using the primers shown below.

Primer No. 3 (5° region of GUS) = 5'-GAT to GTGG 8A-cho CAGCGTTGGTGGG-3 "Primer No. 4 (GUS 3" region reverse primer).

= 5-GCCAGTGGCGAAATATTCCCCGTGC-3” for 2 cycles Transformations that occurred in different wheat varieties during the experiment were determined by the previous two assays. The characterization is summarized in Table 2.

In Table 2 above, various indications in "Presence and expression" are as shown below. Km' = the transformant expresses the NPTII gene. i.e. water containing 150 μg/ml kanamycin (Km) - Kanamai, as observed from green seedlings that are capable of growth on agarose. (see Example 3 above).

NPTI I"' = Directly detected in transformants by using PCR method The presence of an NPTII fragment (the PCR method presented hereinafter) ).

GUS” = observed in bioassay using X-Gluc as substrate The transformants exhibit GUS activity in roots as shown in Example 3 above. Shine on).

GUSPe” = directly detected in transformants by using PCR method. presence of a GUS fragment (see PCR method presented hereinafter) Yo).

Umbrella = kanamycin resistance in seedlings (NPTll) or GUS development in roots Evaluation was conducted according to the current (GUS).

Example 12 Integration of the -bar- gene into two different wheat varieties High yield, semi-cured Springbread wheat cultivar 5eri82 (VEERY #5 “S”, CIMMYT, Mexico), and 5haf ir (SON64A/ TZPP//NAl60/3/FASHazera 5eed Co,, l5r ael), Streptomyces hygroscopicus (Streptomyces hygroscopicus) Plasmid p carried on the bar offspring of S hygroscope Tatsudan Transformed with PAT-NPT11 100.1-28 (D’eBlock etc.

, 1987, White et al., 1990): (7) Genes are Cetylation allows the herbicides bialaphos and Glufosinate can be inactivated. It encodes acetyl-CoA) transferase.

This gene is derived from the CaMV 35S promoter and polyA (terminator) Ita is linked to the sequence and acts as a marker gene within the plant. Plasmi) 'pPAT-NPTI 1 100.1-28 is a puc-derivative (To pfer et al., unpublished personal communication), this is the NPTII translation initiation site ( Christopher, Maas and J.

5che l　l, personal communication) The NI'T11 gene, which has the first intron of the nken gene (sllll), is also keeping.

This bar gene is 0 , electrophoresis of a 6 kb PCR product on an agarose gel under standard conditions. As shown in FIG. 13, which is a pattern, in the F1 generation transformants, Detected by PCR amplification. Corresponds to the first block.

The transformation initiation site is underlined.

Asterisks indicate incorrect nucleotide substitutions that form the Ndel and BamH1 sites. and shows two additional deoxycytidines added to the 5° end.

The second primer was as described below by White et al., (1990 ) with two stop signals and downstream 16 nucleotides, and an additional From nucleotide 533 of the coding region, consisting of two deoxycytidines A reverse primer that is complementary to the sequence up to nucleotide 548.

5'-CCGGATCCCCCGGGTCATCAGATCTCGGTGAC GGGC (37mer) Regarding Figure 13, the DNA template used for the PCR process was as shown below. ri, that is, Line 1 = transformant sample, F8 generation, TAU92P4SHF9-2, PC A fragment reamplified after gel purification of a 0.6 kb fragment synthesized by R. Line 2 = transformant sample, F1 generation, TAU92P4SHF14-1, Line 3 - Transformant sample, F1 generation, TAU92P4SHF14-1, A 0.6 kb fragment obtained by initial PCR synthesis (indicated in line 2) Fragment re-amplified after gel purification of 100%, line 4 = control sample, non-transformed , wild-type 5hafir DNA.

Line 5 = control sample, non-transformed, wild type 5eri82 DNA, line 6 = control sample, no template added in PCR reaction, line 7 = control sample, template added Plasmid pPAT-NPT11 101.1-28 (30 ng) as type , and line 8 = ΦX174 bacterium cleaved by HaeII A size marker for DNA fragments from phage DNA.

NPTI contained in transformants as shown in Examples 7-12 The above-described direct detection of l5GUS and bar gene fragments can be performed using standard P This was done using the CR process (Cetus Corp.

/Hoffmann-La Roche AG).

As shown in Examples 3-12 hereinbefore and the accompanying figures. These findings support the integration of foreign DNA into the wheat genome by the method of the present invention. are doing. The insertion frequencies are as follows for the NPTII and GUS/NPTII genes: , is somewhat higher, ranging from 2.6 to 4.3% according to DNA searches, and even more According to GUS assay over 2 years, it reached 15.5%.

Integration and expression are possible using 3 different promoters and terminators. different genes and different microorganisms with diverse genetic backgrounds. Shown for wheat cultivars. The wheat cultivars used in this study were: All are high yielding modern commercial spring sown wheats, and some of these is grown over vast areas in developed and developing countries. Pigeon The bioclimatic characteristics and fecundity of most transformants are the same regardless of generation (Fl to Fs). , which was comparable to that of the first cultivar.

Therefore, the method of the present invention can stably transform transformed wheat plants, especially foreign DNA. For commercial agricultural use, it is possible to integrate and pass this DNA on to the next generation. It can be concluded that it is possible to prepare something that is a variety Ru.

References 1. DeBlock, M.Y., Bonerman, J., Vand Wiele M,, Dockz, Y, Thoen, @C,.

Gosseie,\’,, Mowa, C,, Thompson4 M, V an Montagu, M., and Leemaus, J. (1987 ), Engine death herbicid resistance = in plants by expression of a detoryfyin g enzyme, The EMPOJ, 6.2513-25182, 0iel en, J,, De Beuckele IIr, M,; 5eurinck, J, ; Deboeck, F, ; De freve.

H, ; LemmerS, M, ; van MOnla2u, M,, and 5 Chell, J., 1984. The α + complete Nucleotide 5equence of the TL-DNA ofTheAgrobacr eriumrume, 1aciens plasmid pTiAcbi Th e EMEOJ, 3:835-846゜3, Jefferson, R,A , Kavanagh T.A., and Bevan M.W., 198 7. Gus Fus Amashishi ■ β-glucuroniOa5e as a 5 intensive and v ersatile gene fusion marker@in higi r plants, EMEOJournal Vat, 6.3901 -3907゜4,KonczC,,hiartiniN,,hiaverhof erR,,Koncz-KalmanZ,,KOrberH,.

Reciei G, and 5chell J, 1989. High f reauency T-DNA-me mountain ajed gen.

tagging in plants, Proc, Natl, Acad, Sci, (U, S, A,), % 8467-84715, Koncz C., and 5chell J., 1986. Tne promotion n of T2-DNA gene 500 ntrolls the tissue e-specific expression of chimeric ge nes carri■п@by a novel type of AgroDtu:rerium binar y vector, Mo1. Gen, Gen, Vo1204, 386-396 6, Lu, Znon=-xun and RWu, 1989. A si mple method for the+rausforma+ion of rice via the pollen = electric ube pathway, Pl am Molecule≠■ Biology Reponer 7: 69-777, Pic2Jd E, , J., M., Jacquemin, F., Granier, M., Bob in and P, Forge shield sword B 1988, Genetic transformation of whea t (Tri+icum aes+ivum) ofplasmid DNA uptake during pollen tube hirmina+ion , Proc, III International Wheat Gen=t ics Symposium, Vol, 1:779-781Cami+r idge, Ll, 10-12 July, 1988S, Po+ry kus, I, 1990. Gen: Transrer to Cere als me(n me’, ssessmen soot B BiolT=rIJIoiogy F, 535-5a29, TopEer, R,, %1aas C,, Horici; e-Granopierre, 5 cell and Sl:1nois sword A H,H.

Improved 5ets or expression vectors For high 1evel gene express Mr. Amakushi@1n ciico+yledonous anci monocoτyledonou s plants (unpubiisned, pmi rs shield lion≠■ communica+1n) L, EMEOJ, 4.1373-138011, White, J,, ChaJlg, S, Y,, Bibb M, J, and Bibb M, (1990), A Cm5 Tang■狽狽■ cont+ining the bar gene of Strepromy ces hygroscopicus: a select≠b attack■ marker for plant transformation, Nuc l:ic Ac1ds Re5search B 1062゜W-1+T-1,7 0201 WII [T-1,702D + pnI-)・NPTIIm gene (8pnI-Hind [l fragment) W-■[T-2, 107-1,702D Shiragiku) Probe: NPTn fragment, 702D vector TarFig, 5 Fig, 7 Fig, 8α Copy and translation of amendment) Submission (Article 184-8 of the Patent Law) Around September 1, 1994 (

Claims (35)

[Claims] 1. 1. A method for producing a transformed wheat plant, comprising: (a) florets of a wheat plant comprising: Remove florets from wheat plants by removing anthers from all pollinating plants before maturity. Males and then insemination of adult males from wheat plants of the same or different varieties. Inducing synchronous pollination by pollinating the stigma of the floret with the anther of the pollen plant. , (b) applying a drop of an aqueous DNA solution to one or more pollinated stigmas; Afterwards, the pollen tubes in the stigma begin to mature into the style, but are still in the ovary. There is a certain period after pollination during which the DNA solution has not reached the , foreign to the plant, and the development of desirable traits within the plant. at least one gene and optionally one additional gene capable of inducing (c) containing an appropriate DNA vector carrying an additional marker gene; A drop of the DNA solution is added to the ovule so that the DNA vector reaches and enters the ovule. on the head of the column in a moist environment for a period of time to ensure that (d) retaining the plants treated in step (c) on any adjacent plants; protect the plant from additional pollination by (e) collecting the seeds that have grown in the growing wheat plants under modified conditions for selection; The above method including. 2. 2. The method according to claim 1, wherein the step of adding a drop of the aqueous DNA solution The above method, wherein the pollinated stigma is an untreated stigma. 3. 2. The method of claim 2, wherein the DNA solution is added at about 1-2 hours after pollination. The DNA solution was added to the stigma in about 12 days. The above method of leaving in contact for 14 hours. 4. The method according to claim 1, wherein the tip of the pollinated stigma is grown while growing. Make sure that a pollen tube is inside the style but still in front of the ovary. After an appropriate period of time for the DNA to straighten, the stigma is truncated and the DNA solution is applied to the truncated stigma. Apply the above method. 5. 4. The method of claim 4, wherein the truncated tip of the pollinated stigma is removed by about 1 hour after pollination. /2-21/2 hours a day and add a drop of the DNA solution at about 12-14 hours. The above method, in which the cut stigma remains in contact with the cut stigma for a period of time. 6. The method of claim 5, wherein the truncated tip of the stigma is cut about 1-2 hours after pollination. Do the above method. 7. A method according to any of claims 1-6, wherein the DNA vector is , the above method comprising a nitrogen fixation structural gene. 8. 8. The method of claim 7, wherein the gene is derived from Klebsiella pneumoniae ( The above nifD gene of Klebsiella pneumoniae) Method. 9. A method according to any of claims 1-6, wherein the DNA vector is , containing one reporter gene that serves as a marker of successful transformation. How to write. 10. 9. The method of claim 9, wherein the reporter gene is β-glucuronin. The above method of encoding Dase. 11. 7. The method of any of claims 1-6, comprising: is capable of conferring herbicide tolerance when expressed in transformed plants. The above method includes one gene sequence. 12. 12. The method of claim 11, wherein the gene sequence encodes the bar gene. The above method is to convert the data into 13. 13. The method of any of claims 1-12, comprising: The above method also includes a selectable marker gene. 14. 14. The method of claim 13, wherein the marker gene confers antibiotic resistance. The above method is one that can be done. 15. 15. The method of claim 14, wherein the marker gene is expressed in a encodes an aminoglycoside phosphotransferase that confers mycin resistance. The APH (3') II (NPTII) gene is nopaline synthase (NOS) promoter and terminus TL-DNA ipt gene (gene 4) that acts as a inator The method described above, wherein the method is linked to an oxidation signal sequence. 16. A method according to any of claims 1-15, comprising: vector DNA has the restriction map shown in FIG. Klebsiella nucleus linked to the S promoter and NOS terminator Monie nifD coding region, NOS promoter and TL- The APH (3') II (NPT) containing the DNA gene 4 terminator II) The above method, which is a plasmid pPCV702nifD containing the gene. 17. A method as claimed in any of claims 1-15, comprising: The vector DNA has the restriction map shown in FIG. β-glucuronider linked to 35S promoter and NOS terminator GUS coding region, NOS promoter and TL-D The APH(3')II (NPTI) containing the NA gene 4 terminator I) The above method, wherein the plasmid pPCV702GUS contains the gene. 18. Production of transformed wheat plants as described in any of Examples 1-17 A useful DNA vector for co-coding the A subunit of nitrogenase. containing one gene that encodes a nitrogen-fixing structural gene, and the gene is a nitrogen-fixing structural gene. The above DNA vector. 19. The DNA vector according to claim 18, wherein the gene is The above DNA vector is the nifD gene of Klebsiella pneumoniae. 20. A DNA vector according to claim 18 or 19, wherein the additional The above-mentioned DNA vector, which contains one reporter gene. 21. A DNA vector according to claim 20, comprising the reporter gene The above DNA vector, wherein the gene encodes β-glucuronidase. 22. A transformed wheat plant according to any of claims 1-17. A DNA vector useful for production, which allows production in transformed plants. The above-mentioned DNA comprises a DNA sequence capable of conferring herbicide resistance when expressed. vector. 23. A DNA vector according to claim 22, wherein the DNA sequence is The above DNA vector encoding the bar gene. 24. A DNA vector described in any of claims 18-23. The above DNA vector also contains a marker gene. 25. The DNA vector according to claim 24, wherein the marker gene is , which encodes one product that can confer antibiotic resistance, The above DNA vector. 26. The DNA vector according to claim 25, wherein the labeled gene The offspring contains an aminoglycoside phosphorus that confers kanamycin resistance when expressed. The APH(3') II (NPTII) gene encodes a transferase. The gene is linked to the nopaline synthase (NOS) promoter and the nopaline synthase (NOS) promoter. TL-DNA (gene 4) polyadenylation signal that acts as a stimulator The above DNA vector is linked to the sequence. 27. Plasmid pPCV702ni with the restriction map shown in Figure 1 fD. 28. Plasmid pPCV702GU with the restriction map shown in Figure 2 S. 29. Transformation prepared by any one of the methods of claims 1-17 wheat plant. 30. A DNA vector as described in any one of claims 18-26. A transformed wheat plant transformed by a vector. 31. Plasmid pPCV702ni with the restriction map shown in Figure 1 Transformed wheat plants transformed with fD. 32. Plasmid pPCV702GU with the restriction map shown in Figure 2 Transformed wheat plants transformed with S. 33. Transformed wheat plants of commercial crop varieties. 34. 34. The transformed wheat plant according to claim 33, wherein T. beauty salon The transformed wheat plant is of the species T. aestivum. 35. The transformed wheat plant according to claim 33, comprising ATIR ( CNO “S”/PJ62/4/GLL/3/TOB//JAR “S”/CRES PO, Hazera Seed Co. , Israel), BAGULA “S” (CM59123, CIMMYT, Mexico), CIAN079 (CM31 678, CIMMYT, Mexico), Genaro81 (VEERY#3" S”, CIMMYT, Mexico), KAUZ “S” (CM67458, CI MMYT, Mexico), KEA “S” (CM21335, CIMMYT, M exico), MILAN (231BWSN37, CIMMYT, Mexic o), OPATAM85 (CM40038, CIMMYT, Mexico), P apagoM86 (CM52359, CIMMYT, Mexico), PAVO N76 (CM8399, CIMMYT, Mexico), Seri82 (VEE RY#5 “S”, CIMMYT, Mexico) and Shafir (SO N64A/TZPP//NA160/3/FA, Hazera Seed Co ．． , Israel).