JPH11318250A - Fertile transgenic corn - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject fertile plant capable of stably expressing a heritable recombinant DNA integrated into a chromosome by setting regenerable and fertile callus cultures from a plant Zea mays to be transformed and treating the resultant transformed cells under specific conditions. SOLUTION: Callus cultures such as a friable embryo callus culture initiated from a regenerable and fertile immature hybrid embryo are set from a plant Zea mays to be transformed and the set cell cultures are then bombarded with recombinant DNA-coated microprojectiles such as a bacterium dap A gene to thereby transform the cultured cells. The transformed cells are subsequently identified or selected to thereby regenerate a fertile transgenic plant Zea mays capable of stably expressing a heritable recombinant DNA. As a result, the transgenic plant Zea mays is obtained. The callus cultures to be bombarded are preferably groups of 30-80 mg per group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to Zea mays.
Breeding transgenic plants (including
(Often called maize or corn). The invention further relates to the production of fertile transgenic plants by particle bombardment and subsequent selection techniques.

[0002]

BACKGROUND OF THE INVENTION Plant genetic engineering, which requires the isolation and manipulation of genetic material (usually in the form of DNA or RNA) and subsequent introduction of that genetic material into plants or plant cells, is a state of the art agricultural and agricultural technology. Promising considerable potential for improving plant quality. Increased grain value, high yield, feed value, reduced production cost, pest resistance, stress resistance,
Drought resistance, the production of pharmaceuticals, chemicals and biological molecules, and other beneficial features may also be achieved by genetic engineering techniques. As genes are identified, cloned, and further manipulated,
It is still necessary that the resulting plant be fertile and be introduced into the relevant plant in such a way as to transfer the gene to its progeny.

[0003] Various methods have been developed and are currently available for transforming various plants and plant cells with DNA. Generally, such plants are dicotyledonous, but some successful cases using certain monocotyledonous cereals have been reported. However, some species have not been able to be transformed using any method. Thus, prior to the present invention, the introduced recombinant D
No technology has been developed that allows the production of tightly transformed Zea mays plants in which NA is transmitted through at least one complete sexual cycle. This failure in the art has been well documented in the literature and has been extensively reviewed in recent reviews. (I. Potryku
s, Trends in Biotechnolog, 7,269
(1989); Weizing et al., Ann. Rev. of Genetic
s, 22,421 (1988), F. Cooking et al., Science, 236, 1259.
(1987).

Some techniques that have been attempted or planned for introducing DNA into corn cells include electroporation, microinjection, microprojectile bombardment, liposome fusion, Agrobacterium-mediated transfer,
Methods include bolus injection and exposure to bare DNA in solution.

[0005] For example, J. Dewet et al., Experimen.
tal Manipulation of Ovule
Tissue (experimental operation of ovule tissue); Chap
ed., New York Longman (198
5) pages 197-269 and Y. Ohta, PNA
S USA, 83, 715 (1986) describes pollen grains and D
We reported a method of introducing DNA into maize by applying the pollen to the hair of maize after mixing the NA solution. There is no molecular data in these articles confirming that exogenous DNA has been introduced into corn cells.

Arnzen et al., In published European Patent Application No. 275,096, describe that after incubating DNA and maize pollen, maize ears were pollinated to produce seeds. Plants originating from this seed were reported to contain the introduced DNA, but the introduced DNA
There is no indication that is transmitted through the complete estrous cycle.

A. Graves et al. Plant Mo
l. Biol. 3, 43 (1986), Zea
Agrobacterium-mediated transformation of the Mays seedling was reported. The evidence was based on assays that were sometimes unreliable. To date, no further successful examples of pollen and Agrobacterium-mediated transfer techniques have been reported.

[0008] Microprojectile bombardment techniques for generating transformed corn cells have been reported. This technique is based on Sanfor
d et al. Sci. & Techn. , 5
27 (1987) and U.S. application Ser. No. 07 / 151,807 filed Feb. 29, 1988.
C. Sanford et al. Published European Patent Application No. 33
No. 1,855. Klein et al.
lant Physiol. , 91 440 (198
9) describes the production of transformed corn cells using the microprojectile bombardment method. However, the cells used could not be regenerated into plants. Therefore, the culture of some type of renewable maize cells can be performed by bombardment.
No protocol describing the introduction of NA has been published. None of the reported stable gene transfer results from regenerating viable plants and transmitting the transferred gene through at least one sexual cycle after the maize callus bombardment procedure.

D. In published European Patent Application No. 270,356, McCabe et al., Reported how to bombard maize pollen with DNA, apply the pollen to maize hair, and reportedly treat seeds containing exogenous DNA. Disclose the formation. However, D
There is no evidence that NA is transmitted, and no further results have been reported by this study group.

M. E. FIG. Promm et al., Natur
e, 319791 (1986), reported that electroporation of corn protoplasts resulted in transformed cells, but these cells did not produce regenerated plants. Electroporation of corn protoplasts
C. Rhodes et al., Science, 24.
0,204 (1988). In the latter case, the recipient cells can be transmitted and regenerated to the plant, but the plant itself does not propagate. In addition, the method of cell line production used by Rhodes et al. Was not reproducible.

Another obstacle to successful production of a transgenic maize plant that is fertile is that the ability to regenerate the transformed cells (in the case of protoplasts or cultured cells) and the ability to reproduce are not destroyed. In some cases, only a few transformed cells were selected. Since low levels of transformed cells are generally produced by transformation techniques, some sort of selection procedure is often required. However, selection generally requires the use of some toxic agents, such as herbicides and antibiotics that are detrimental to either the ability to regenerate or the ability to reproduce the resulting plant.

On the other hand, it has been found that the protoplasts of untransformed corn cells and calli at least regenerate to form mature plants, and that the resulting plants often have reproductive potential. I have. For example, R.
D. Hillite et al. Bio / Technology
y, 7, 581 (1989); M. Pri
oli et al., Bio / Technology, 7, 58.
9 (1989) discusses a method for producing a protoplast from a cell culture and a method for recovering a fertile plant therefrom. C. A. Rhodes et al., Bio
/ Technology, 5, 56 (1988)
He has reported attempts to regenerate maize plants from protoplasts isolated from embryogenic maize cell cultures.

However, for the exogenous DNA, which maize tissue or cultured tissue is the appropriate recipient, for example, it is easy to accept and safely integrate the exogenous DNA, and at the same time, a part of the germ line, ie, Technical researchers could not determine if they contained an effective number of cells that were part of the cell line leading to the next plant generation.

[0014] Thus, the present technology faces a dilemma. Also, certain transformation techniques have been planned or reported to produce transformed maize cells, and have been proposed as potential recipients because certain cells and tissues are capable of plant regeneration. But with this technology,
No combination of techniques to successfully produce transformed maize plants capable of transmitting the introduced DNA through one complete sexual cycle could be found.

[0015]

Therefore, a stable, transgenic, transgenic, Zea mays
It is an object of the present invention to produce plants and seeds that transfer the introduced gene to progeny. It is also an object of the present invention to produce stable transgenic plants and seeds by bombardment and at least some of the transformed cells by a selection treatment that produces a high level of viability. In addition to maize, it is also an object of the present invention to produce fertile, stable and transgenic plants of other cereals of the grass family.

[0016] The references listed in the following references are incorporated in this.
Armstrong, CL, et al. (1985) 164:
207-214, Callis, J, et al. (1987) G.
enes & Development 1: 1183-1200,
M. (1983) Nuc Acids R
es 11: 36a, Chu, CC. Et al. (1975) S
ci Sin (Beijing) 18: 659-668, Cook
ing, F.S. Et al. (1987) Science 23
6: 1259-1262, DeWet et al. (1985) P.
oc Natl Sci USA 82: 7870-
7873, Freeling, JC, et al. (1976) M
aydica XXI: 97-112, Graves,
A, et al. (1986) Plant Mol Biol
7: 43-50, Green, C, et al. (1975) Cr.
op Sci 15: 417-421, Green,
C, et al. (1982) Maize for Biolog.
icalResearch (maize for biological research), Plant Mol. Biol. Asso
c. , Pp367-372, Gritz, L, et al. (19)
83) Gene 25: 179-188, Guille
y, H, et al. (1982) Cell 30: 763-77.
3, Hallauer, AR, et al. (1988) Corn.
and Corn improvement, 3rd edition, AgronomySo
city of America, pp. 469-56
4, Jefferson, R, et al. (1987) EMBO
J 6: 3901-3907, Kamo, K, et al. (1
985) Bot Gaz 146: 327-334, K
lein, T, et al. (1989) Plant Physi.
ol 91: 440-444, Klein, T, et al. (1
988a) Proc Natl Acad Sci U
SA 85: 4305-9, Klein, T, et al. (19
88b) Bio / Technology 6: 559-
563, Lu, C, et al. (1982) Theor App.
l Genet 62: 109-112, McCab.
e, D, et al. (1988) Bio / Technology.
6: 923-926, Murashige, T, et al. (1962) Physiol Plant 15:47.
3-497, Neuffer, M, (1982) Mai.
ze for BiologicalResearch
(Maize for Biological Research), Plant Mo
l. Biol. Assoc. , Pp19-30, Phi
llips, R, et al. (1988) Corn and C.
orn improvement (Corn and Corn Improvement), 3rd edition, Agronomy Society
of America, pp345-387, Potr
ykus, I (1989) Trends in B
iotechnology (Trends in Biotechnology) 7:26
9-273, Rhodes, Ca, et al. (1988) Sc.
issue 240: 204-7, Sambrook,
J, et al. (1989) Molecular Clonin.
g: Laboratory Manual (Molecular Cloning; Laboratory Manual), 2nd edition, Cold S
spring Harbor LaboratoryPr
ess, Sanford, J, et al. (1987) Part.
Sci & Techn 5: 27-37, Weis
ing, K, et al. (1988) Ann Rev of G.
enetics 22: 421-478, Yanish.
-Perron, L, et al. (1985) Gene 33:
109-119.

[0017]

SUMMARY OF THE INVENTION The present invention relates to a fertile, transgenic Zea mays plant containing recombinant DNA that is passed on to its progeny,
Recombinant DNA integrated into the chromosome is desirable.

The present invention further provides a transgenic Ze.
a mays plants, plant cells, plant parts, as well as all products obtained from seeds. The invention also relates to seeds containing the recombinant DNA, as well as progeny that have inherited the recombinant DNA. The present invention further relates to the improvement of the quality of transgenic plants and the subsequent transfer of the recombinant DNA to Zea ma.
ys plants or lines.

The present invention also relates to a process for producing a fertile Zea mays transgenic plant containing the recombinant DNA. The process is based on microprojectile bombardment, selection, plant regeneration, and traditional backcrossing. The present invention further relates to fertile traits other than Zea mays which are not reliably transformed by routine methods such as electroporation, Agrobacterium, injection methods and previous ballistic methods. It is related to the process of producing converted plants.

The present invention also relates to a transformed embryogenic tissue, a transgenic seed produced therefrom, and RI
And the reproduction of mature fertile maize plants obtained from subsequent generations.

A preferred embodiment of the present invention is a recombinant chimeric gene expressed as a seed storage protein, for example, a 10 kD zein protein, wherein the concentration of at least one amino acid is
A fertile transgenic corn plant that has been tightly transformed so that it is elevated beyond what is present in the parent non-transformed strain. Expression or overexpression of various copies of the above-described genes substantially increases the overall concentration of certain amino acids, such as lysine, methionine, and threonine. As used herein, the phrase "substantially elevated" refers to a particular amino acid being present in a corresponding plant or plant part that has not been transformed with the seed storage protein gene described above.
Means 10-20% more.

In a preferred embodiment, the present invention employs a controlled method for selecting transformed callus strains following microprojectile bombardment of friable embryogenic callus aggregates coated with recombinant DNA. Produces reproductive, transgenic plants.

[0023]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a species of Zea mays ,
Producing fertile transgenic and seed and plants obtained from such transgenic plants,
Aims plant tissues and seeds, as well as the subsequent progeny and Seisanbutsu to be obtained therefrom, transgenic Zea mays plants food or feed value is improved is preferable. The transgenic plants produced here include all plants of this type, including field corn, popcorn, sweet corn, flint corn and dent corn.

"Transgenic" includes whole cells, cell lines, calli, tissues, parts of plants or plants, and genotypes that have been partially altered by the presence of recombinant DNA. Technology of "heterogeneous DN
A "," exogenous DNA ", or" foreign DNA ", wherein the DNA of interest is introduced into the genotype by genetic engineering or is initially treated with the gene of the parent plant. Introduced into a type, which is then transferred to later generations by sexual crossing or asexual reproduction, where "genotype" is the total amount of genetic material within a cell, narrowly, or chromosomally or extrachromosomally. Thus, "transgenic" as used herein refers to Zea mays genotype by a conventional plant breeding method or a naturally occurring event such as randomized cross-production, viral psoriasis or a spontaneous mutation. Partial changes are not allowed.

"Inheritable" means that the DNA is transmitted through at least one complete sexual cycle, that is, transmitted from one plant through its germline to progeny plants.

The transgenic plant of the present invention can be produced by the following method. (I) To establish a renewable cell culture. A friable embryogenic callus is desirable. (Ii) transforming the above-described cell culture by microprojectile bombardment. (Iii) identifying or selecting transformed cells. (Iv) regenerating transgenic plants capable of reproduction from the transformed cells. Recombination D
In order to develop transgenic elite strains and varieties or commercial hybrid seeds containing NA, some of the plants of the present invention may be transformed using conventional cross-breeding techniques into transgenic plants capable of reproduction. Can also produce transgenic seeds.

I. Plant strains and tissue culture Cells in which cells that have been found to be particularly useful for the production of transgenic maize plants that are capable of reproduction are callus that can be regenerated before and after the selection method detailed below. Cells. Generally, such cells are obtained from meristems containing cells that have not yet finally differentiated. Generally, such tissues of grasses, particularly maize, include tissues found in the roots of young leaves, immature stalks, immature embryos, and cotyledonous nodes. It is desirable to have immature embryos as cotyledons. Methods for preparing or maintaining callus from such tissues and plant types are well known in the art, and details thereof are described in Phillips et al.
8), which is hereby incorporated by reference.

The specific callus used must be able to regenerate fertile plants. During and after selection, the regeneration ability may be significantly reduced,
For the firing / selection process used here to succeed,
The ability to specifically regenerate specific cells is important. Therefore,
It is important to start with cultured cells that are as regenerative as possible. It has been found that calli at the age of about 3 months or more and up to about 36 months have a sufficiently high level of regenerative capacity, and this is desirable. By transferring the sample to a regeneration medium and monitoring the shoots, roots, and seedlings, the regeneration ability of specific cultured cells can be easily measured. The total log of seedlings generated per petri dish or per gram weight of tissue may be used for a crude quantitative estimate of regenerative capacity. Generally, cultures that yield at least one plant per callus tissue are desirable.

Maize callus culture can be initiated from a number of different plant tissues, among which useful cultures are immature immature seeds from ears, when the embryos are about 1-3 mm long. Desirably obtained from maize embryos. Generally, this length is about 9-14 days after pollination. Under sterile conditions, the embryos are placed on conventional solid media with the hypocotyl down (scutellum up). After a few days to a few weeks, callus tissue emerges from the scutellum. After the callus has grown sufficiently, cell proliferation from the scutellum is evaluated for friability and the presence of clearly defined embryos. By "friable stiffness" is meant that the tissue is easily dissipated without damaging the cells. The tissue with this morphology is then transferred to a fresh medium and subcultured on a routine basis about every two weeks. Sieving is used during the subculture period to reduce aggregation and optionally increase cell surface area.

The callus initiation medium is preferably solid. In a preferred embodiment, the start / maintenance medium is generally Armstr
Chu et al. (1975) described by Ong et al. (1985).
Or the MS salt of Murashige et al. (1962). The basal medium is supplemented with sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D). Supplementary components such as L-proline and casein hydrolyzate have been found to improve callus culture initiation frequency, morphology and growth. Cultures are generally kept in the dark, but low light intensity may be used. The concentration of the synthetic hormone 2,4-D required for maintenance and growth is generally about 0.3
３3.0 mg / 1.

In this, transformation and regeneration have been successful using friable embryogenic callus, which is another reproducible transformable plant capable of producing the transgenic plants of the invention. It does not imply that the use of such cells, tissues or organs is not possible. The only practical requirement for transformed cells is that after transformation, the plants containing the recombinant DNA must be able to regenerate after the particular selection or screening procedure actually used.

For example, cells grown in liquid suspension culture may be used. In order to establish such a culture,
After 4-6 months, type II callus is transferred to liquid growth medium. Methods and references relating to the production of renewable suspension cell cultures can be found in C.W. E, Green et al.
e for Biological Research
(Maize for biological research), Plant Mol
ec. Biol. Assoc (1982), 367-3.
On page 72, R.I. Phillips et al., Corn
andcorn Improvement (corn and cone improvement), Agronomy Soc. Ame
r. (3rd edition, 1988) at pages 345-387 and Vasil is Cell Culture an
d Somatic Cell Genetics
f Plants (Cell Culture of Plants and Genetics of Somatic Cells), Volume I, Laboratory Procedure
reand Their Applications
(Lab procedures and their applications), Academic Pre
ss (1984), pages 152-158. In general, a liquid growth medium for suspension culture is similar to the formulation of a solid callus induction medium. ABA (abscisic acid) (10-7M) may be added to the liquid growth medium to enhance the regenerative ability and increase the vitality of the medium. It is desirable not to screen before introduction into the liquid medium.

The culture in the liquid medium is carried out by subculture suitable for vigorous growth and its regenerating characteristics. In a preferred embodiment, the culture is subcultured once a week at a dilution of 1: 8-9 with fresh growth medium.

II. DNA used for transformation "Recombinant DNA", as used herein, is derived or isolated from a source, optionally after a partial scientific modification, and subsequently introduced into Zea mays , DN
Say A. Recombinant DNA "derived" from a source
An example would be a DNA sequence that has been identified as a useful fragment in a particular organism and that is subsequently scientifically synthesized in a substantially pure form. Examples of DNA “isolated” from a source can be further manipulated, eg, amplified, in genetic engineering methodology for use in the present invention.
DNs that are removed or removed from the above sources by chemical means, for example using restriction endonucleases
A sequence.

Thus, "recombinant DNA" includes fully synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA. In general, recombinant DNA is not originally present in the Zea mays genotype, which is the recipient of the DNA, but to enhance the production of certain gene products, such as, for example, storage proteins, certain Zea mays
It is within the scope of the present invention to separate a gene from a genotype, and to subsequently introduce various copies of that gene into the same genotype. Recombinant DNA includes plant genes as well as non-plant genes such as those from bacteria, yeast, animals or viruses, the same or different Z genes.
Examples include, but are not limited to, modified genes, including ea mays genotype genes, gene proteins, and DNAs of chimeric genes.

Among them, the recombinant D used for the transformation
NA may be cyclic or linear, double-stranded or single-stranded. Generally, DNA is a form of chimeric DNA that also contains coding regions, such as plasmid DNA, flanked by regulatory sequences that facilitate the expression of recombinant DNA present in the resulting corn plant.
For example, recombinant DNA itself is either active in Zea mays, or a target for transformation Zea m
Includes or contains a promoter that will use a promoter already present in the ays genotype.

The composition and method of constructing a recombinant DNA capable of transforming a certain plant is well known to those skilled in the art. May be used. DN
The invention is not dependent on the particular recombinant DNA composition used, as the particular composition of A is not critical to the invention.

K. Weissing et al., Ann. Re
v. Genetics, 22, 421 (1988).
Describes suitable DNA components, selectable marker genes, reporter genes, enhancers, introns, and the like, and provides appropriate references for construction therefrom. J. Sambrook et al., Mol
eco Cloning: A Laborat
oryManual (Molecular cloning; laboratory manual), Cold Spring Harbor
Laboratory Press (2nd edition, 198
9) provides an appropriate configuration method. Generally, recombinant DNA is relatively small, to minimize susceptibility to physical, chemical or enzymatic degradation, which has been found to increase as DNA grows.
That is, it is less than 30 kD.

Recombinant DNA suitable for use therein
Include any DNA that provides or enhances the beneficial characteristics of the resulting transgenic corn plant. Food value increase, high yield, pest resistance, disease resistance,
This DNA is thought to encode a protein or antisense RNA transcript to promote herbicide resistance and the like. For example, this DNA, like the danA gene, provides DHDP synthase for increased lysine production and Bacillus thuri for pest resistance.
ngiensis (Bt), δ-endotoxin or protease inhibitors, as well as bacterial EPSP synthase for glyphosate herbicide resistance, and chitinase or glucan endo-1,3-β-glucosidase for fungicidal activity.

Important in improving the value of food or feed are genes encoding proteins containing high concentrations of essential amino acids. For example, corn-soy meal poultry feeds are generally supplemented with synthetic methionine or methionine analogs in order to be nutritionally appropriate and support the optimal growth of chickens. Developing a corn strain that provides high levels of methionine can reduce the need for methionine supplements. The development of such a high methionine corn strain is achieved by introducing a highly expressed gene or genes encoding a high methionine protein into the corn genome.

Examples of genes encoding high methionine proteins include: 1. ) Maize 15k
Gene encoding D coin protein (11% methionine), (Pedersen et al., J. Biol. Ch.
em, 261, 6279 (1986)), 2.) Gene encoding Brazil nut storage protein (18% methionine), (Altenbach et al., Plant M
ol. Biol. , 8; 239 (1987)) and 3. ) Maize 10 kD-zein protein (methionine 2
2.5%), (Kiriha
ra et al., Gene, 71, 359 (1988)). A preferred gene is the 10 kD-zein protein, which
Because this protein product normally accumulates in kernels, methionine is an endogenous maize gene twice as high as the 15 kD-zein protein. To obtain high levels of expression in the seed, the coding sequence may optionally be fused to the highly expressed, regulatory sequences of the seed-specific gene. Alternatively, introduction of an additional copy of the intact, endogenous 10 kD-zein gene into the corn genome can also increase the methionine content of the corn seed.

Lysine, an essential amino acid in the diet of humans and monogastric animals, is among the three largest amino acids in most of the major crops, especially cereals. Consequently, cereal-based diets must be supplemented with synthetic lysine or lysine-containing oilseed protein meal. In addition, most oilseed meals themselves have an inadequate source of lysine, so frequent adjustment of the feed mixture for lysine will result in meals that are too high in other, less desirable nutrients. Therefore, increasing the lysine content of either the cereal and / or oilseed crops, or both, will result in significant nutritional value and a significant savings for end consumers such as pigs and poultry farmers. Will do.

One approach to improving the lysine content of cereals is to break free biosynthetic pathways and accumulate free lysine. The dapA gene of E. coli is
Encodes dihydrodipicolinate synthase (DHDPS), a key regulatory enzyme whose activity in plants is strongly feedback inhibited by lysine. This bacterial enzyme is about 200 times less sensitive to inhibition by lysine. By introducing and expressing the dapA gene in plant cells, free lysine synthesis can be continued even after the original plant DHDPS has been completely inhibited.

Of particular importance in maintaining yields from corn plants and making a significant contribution to the cost control of corn cultivation is protection from pest attack. In the United States, various lepidopteran pests, including larvae that feed on the ear of corn, pest beetle, and corn, as well as Coleoptera, such as Diabrotica species, are among the major corn pests. Protecting corn from pest attack is expensive for growers and requires the use of harmful chemical pesticides that are timely applied. The routine breeding and selection methods did not allow for the development of new corn strains that were substantially resistant to the major pests, and thus introduced or inherited pest resistance genes or sequences into plants in accordance with the present invention. This will reduce grower costs, reduce the use of toxic chemical pesticides, and provide more effective pest management.

An essential element of the present invention is the introduction of the pest resistance gene into corn cells, and the integration of the gene into the whole corn plant, through mitotic and meiotic processes, to the final grandchild of the plant. It is a mitotic replication of a gene that is inherited by the human.

Bacillus thuringien
sis (or "Bt") comprises nearly 20 known subspecies of bacteria that produce endotoxin polypeptides that are toxic when ingested by a wide variety of insect species. The biology and molecular biology of endotoxin protein (Bt protein) and the corresponding gene (Bt gene) are described in R. Whitely et al., Ann. Rev .. Microbiol. , 40,549
H. Hofte et al., Microbiol. R
ev. , 53, 242 (1989). The genes encoding the various Bt proteins have been cloned and sequenced. A segment of the Bt polypeptide is essential for toxicity to various lepidopteran pests, and the first 5
The survey revealed that it was included in 0%. As a result, the truncated B encoded by the truncated Bt gene
The t polypeptide often retains toxicity against many lepidopteran pests. The HD73 and HD1 Bt polypeptides were toxic to important lepidopteran pest larvae of corn plants in the United States, such as larvae that feed on the ears of corn borer, pest beetle, and corn. M. G
eister et al., Gene, 48, 109 (198
6); J. Adang et al., Gene,
36, 289 (1985), HD1 and HD, respectively.
The gene encoding the 73 Bt polypeptide is
Cloned and sequenced, culture collections (eg, Bacillus Gen, Columbus, Ohio)
etic Stock Center, USDA Bt stockcollecti, Peoria, Illinois
on) can be cloned using standard protocols from HD1 and HD73 species.

The DNA encoding the new, previously uncharacterized Bt toxins may be cloned from the host Bacillus bacterium using the protocol previously used for cloning the Bt gene. . This includes D. isolated from Bacillus bacteria on a suitable plasmid or a phage vector that is replicated in a suitable host.
Structure of NA bank and Bt protein or homologous Bt
Includes the use of antibodies raised against DNA separated from the gene sequence. The approximate position of the Bt coding sequence may be first determined using detection assays of the cloned DNA. The exact location of the Bt coding sequence can be determined by sequencing the cloned DNA segment, Bt
By determining the presence of a large open reading frame of this sequence that can encode the protein, and comparing the amino acid sequence obtained from the DNA sequence to that obtained from the partial amino acid sequence of the Bt protein, Are determined using various standard methods, including confirmation of the Bt encoding.

[0048] Chimeric Bt genes useful in the present invention include 5 'DNA sequences, including DNA sequences capable of initiating transcription ("promoter") and downstream translation where the location of the Bt sequence in corn plants has been confirmed. . The chimeric Bt gene is
Also included are 3 'DNA sequences that include sequences derived from the 3' non-coding regions of genes that can be expressed in corn. Most importantly, the chimeric Bt gene is Bacillus
or a DNA sequence encoding a toxic Bt polypeptide produced by T. thuringiensis or a toxic portion thereof or having a substantial amino acid sequence homologous thereto. The Bt coding sequence includes: (I) a DNA sequence encoding a pesticidally effective protein substantially homologous to Bt endotoxin effective against corn pests, such as the HD73 or HD1 Bt sequence; (ii) an insecticide of Bt endotoxin polypeptide DNA sequence coding for an effectively active segment, such as an insecticidally effective HD73 or HD1 polypeptide truncated from the carboxy and / or amino terminus, (iii) a truncated Bt sequence, supplemental such as: A truncated Bt sequence fused in frame with a sequence encoding a polypeptide that provides advantages. (A) a selectable gene, such as a gene that confers resistance to antibiotics or herbicides; (b) a reporter gene, such as luciferase or β-glucuronidase, which facilitates detection or assay of the product; and (c) degradation of Bt protein. A DNA sequence encoding a polypeptide sequence that is used supplementarily for stabilization or that enhances the effectiveness of the Bt protein against pests, such as protease inhibitors, and (d) Bt protein for specific components inside and outside corn cells. A sequence to help direct, eg, a signal sequence.

In order to obtain optimal synthesis of Bt protein in corn, it may be appropriate to adjust the DNA sequence of the Bt gene so that it more closely resembles a gene that is effectively expressed in corn. The method using codons of many Bt genes, including the HD73 and HD1 genes, is more similar to the method used by Bacillus species and differs from the method used by genes expressed in maize. Gene expression can be improved by replacing such rarely used bacillus codons with those more frequently used in maize plants (E.
Murray et al., Nucl. Acid Res. , 1
7, 477 (1989)). Such codon replacement requires base substitution without altering the amino acid sequence of the resulting Bt polypeptide. The Bt polypeptide is identical in sequence to a bacterial gene or a segment thereof. Complete Bt coding sequences, or fragments thereof, with desired maize codon content higher than the original bacterial gene, can also be synthesized using standard chemical synthesis protocols, including site-directed mutagenesis and DNA synthesis. It can be introduced or assembled into the Bt gene using standard protocols such as polymerization and ligation (F. Murray et al., Nucl. A.
cids Res. , 17, 477 (1989)).

Apart from the recombinant DNA sequences which act as transcription units or parts thereof, useful recombinant D
NA performs regulatory or structural functions and is not thought to be transcribed. This DN also serves as a tool for the genes that make up the mutants and, optionally, helps in identification, gene addition, or isolation of corn DNA segments.
A is introduced. See Weising, cited earlier, for additional examples.

To facilitate the identification and selection of transformed cells, the recombinant DNA introduced into the plant cells will generally further contain a selectable marker and / or sceptor gene. Alternatively, the selectable marker is located elsewhere and is used in a co-transformation procedure. Both the selectable marker and the reporter gene are flanked by appropriate regulatory sequences so that they can be expressed in plants. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide resistance genes.

Specific examples of selectable marker genes are given in Wesing et al., Cited earlier. A preferred, selectable marker gene is hygromycin
A phosphotransferase (HPT) coding sequence, obtained from E. coli, confers resistance to the antibiotic hygromycin B. Other selectable markers include the antibiotics kanamycin, neomycin and G14B
Transposon Tn5 encoding resistance to
(AphII) encodes the aminoglycoside phosphotransferase gene and resistance or resistance to glyphosate, 2,2-dichloropropionic acid, methotrexate, imidazolinone herbicide, sulfonylurea herbicide, promoxinil, phosphinothricin and other herbicidal compounds Genes included. These selectable marker genes that confer resistance or resistance to such phytotoxin compounds have commercial utility in the resulting transformed plants.
Selectable marker genes encoding enzymes that convey resistance to phytotoxin compounds are listed in Tables 1 and 2 below.

[0053]

[Table 1]

[0054]

[Table 2]

The reporter gene is used to identify potentially transformed cells and assess the functionality of the regulatory sequences. Reporter genes encoding marker proteins that can be easily assayed are well known in the art. Generally, a reporter gene is a gene that encodes a protein that is not present or expressed in the recipient organism or tissue, and whose expression is manifested by easily detectable properties, such as a phenotypic change or enzymatic activity. Weising et al., Supra, provide examples of such genes. Preferred genes include the chloramphenicol acetyl transferase gene (cat) of E. coli Tn9, the β-glucuronidase gene (gus) of the uidA locus of E. coli, and the luciferase gene of firefly Photinus pyralis. After introducing the DNA into the recipient cells, the expression of the reporter gene is examined at appropriate times. Such a desirable assay includes E. coli β-glucuronidase (GU
S) Gene is required. (R. Jwfferson et al., BNBO J.,
16,3901 (1987)). Maize cells transformed and expressing this gene stain blue when exposed to the substrate 5-bromo-4chloro-3-indolyl-β-D-glucuronide (X-GLUC) in the extracellular medium. I do.

Regulatory sequences useful herein include any constitutive, inducible, tissue or organ-specific, or stage-specific promoter that can be expressed in a particular plant cell. Weising et al., Cited above, have published such suitable promoters. The following is a portion of a list representative of promoters suitable for use in this. Agrob including mannopine synthase, nopalic synthase, octopine synthase
T-DNA regulatory gene of acuteriumtumefaciens, corn alcohol dehydrogenase promoter,
A light-inducible promoter, such as the ribulose-bisphosphate-carboxylase / oxygenase small subunit gene from various species, and the majoa chlorophyll a / b binding protein gene promoter, the cauliflower mosaic virus (CaMV) 35S and 19S promoters; Inducible or structural, including promoters that are developmentally regulated, such as maize wax, zein or bronze promoters, as well as promoters that exhibit organ-specific expression or expression during a particular developmental stage of the plant. Synthetic or other natural promoters.

Other components such as introns, enhancers and polyadenylation sequences are also part of the recombinant DNA. Such components may or may not be necessary for DNA function, but are thought to provide improved DNA expression by acting on transcription, mRNA stability, and the like. Such components can be included as desired to obtain optimal performance for transforming plant DNA. For example, a maize AdhIS first intron is placed between the promoter and coding sequence of a particular recombinant DNA structure. This intron has been shown to increase protein production of maize cells when included in the DNA structure. (J. Callis et al., Genes and Debelop., 1,1183 (198
3)). However, sufficient expression for the selectable marker to perform satisfactorily is often obtained without introns. (T. Klein et al., Plant Physiol., 91, 440 (1989). One example of a suitable alternative intron is Zea mays s.
hrunken-1 first intron. Such other components must be compatible with the rest of the DNA structure.

III. DNA transfer process Recombinant DNA can be introduced into renewable maize cell culture, and is preferably introduced into callus culture via particle bombardment. A general description of suitable particle bombardment devices is provided in Sanford et al. (1987), the contents of which are incorporated herein by reference. In the bombardment of non-renewable maize suspension cultures, the protocol for the use of the device is described by Klein et al. (198a, 1988b and 1989).
However, there are no published protocols on bombardment of callus culture or renewable maize cells.

The microprojectile bombardment process is also referred to as a biolistic process, in which very small particles of biologically inactive material are transformed into callus by recombinant DNA.
Intervenes in the transportation of When an inert particle is coated with DNA and accelerated to a suitable speed, one or more particles are released from the particle and enter one or more cells expressed in the cell. Can be. Some of the recipient cells express the transfected DNA because they retain the transfected DNA tightly.

The particles, referred to as microprojectiles, are generally dense materials such as tungsten or gold. Coat them with the relevant DNA. The microprojectile is then placed on the surface of a large projectile that serves to transport the motive force from a suitable energy source to the microprojectile. After accelerating the large projectiles and microprojectiles to the appropriate speeds, they prevent the large projectiles from continuing their forward course, but allow the DNA-coated microprojectiles to continue, The callus cells are brought into contact with a bombardment device. Such a suitable device can use a motive force such as gunpowder, or an electric shock wave can be fired (P. Christou et al., Plant Physiol., 87, 671 (1919).
88)). Devices in which gunpowder is the motive force are currently preferred and are described in more detail in Sanford et al. (1987), which is hereby incorporated by reference.

The protocol for the use of the explosive device is:
(1988a, b) and includes two important steps. First, in a specified sequence, the tungsten microprojectiles are mixed with DNA, calcium chloride, and spermidine in an aqueous solution. As taught, the concentrations of the various ingredients
It is mixed. The preferred procedure requires exactly the procedure of Klein et al. (1988b), except for doubling the described optimal DNA concentration. Second, the DNA-coated microprojectiles, large projectiles, and recipient cells are placed in place on the device to provide motive force to the large projectiles. Some of this step, which may vary, includes the distance from the end of the barrel to the recipient cells as well as the degree of vacuum in the sample chamber. The recipient-ent tissue is placed 2-15 cm below the stop plate tray, preferably 5 cm.

Among them, the callus culture useful for the regeneration of transgenic plants is generally at about the middle of the transfer period, and therefore must pass the “lag” phase associated with the transfer to a new medium. It must be before the "steady-state" phase is reached, with the extension of the non-subculture period to the medium. This organization has about 30
It is used in the form of ~ 80 mg sections, preferably 40-60 mg.
The agglomerate is placed on a Petri dish or other surface and placed essentially in a manner that confirms the following. (i) The darkest concentration of metal-DNA particles is placed in the center space of the dish, and the tissue placed there is considered to be damaged during the attack. (ii) Since the number of particles reaching the tissue decreases with increasing distance from the center of the explosion to the tissue, tissue away from the center of the dish is considered less likely to be bombarded. The mesh screen is preferably made of metal and is placed on a dish to prevent tissue splashing and draining. An alternative method of presenting the bombardment tissue is to spread the tissue in a thin layer on filter paper. The tissue is bombarded one or more times with metal particles coded with DNA.

IV. Selection Process Bombarding calli with recombinant DNA, containing the recombinant DNA as soon as the DNA has penetrated some cells,
Moreover, it is necessary to identify and select cells that still have sufficient regenerative ability. There are two general approaches that have proven useful in accomplishing this. The first method involves transforming callus or plants regenerated therefrom using various standard methods, including assays for reporter gene expression, if any, or assessment of the phenotypic effects of the recombinant DNA.
Can be screened for. Alternatively, and desirably, when the selectable marker gene is transmitted together with or as part of the recombinant DNA, a selective agent may be used that detects the expression of the selectable marker gene, and The callus cells that have been transformed can be identified.

Since the selection conditions must be selected so that the growth and accumulation of the transformed cells are possible, but at the same time the growth of the non-transformed cells is inhibited, the virtual selection of the transformed cells is based on the transformation treatment. Is a critical part of success.
This situation is complicated by the fact that the vitality of individual cells in a population is often dependent on the vitality of neighboring cells. Also, the selection conditions are less stringent, excluding the ability of callus cells to regenerate plants and the fertility of the resulting plants. Therefore, the effect of the selection agent on cell viability and morphology must be evaluated. This can be accomplished by experimentally generating a growth inhibition curve for a particular selective agent and pre-transforming the tissue. This will establish a concentration range that inhibits growth.

When using a selectable marker gene, callus clumps can be recovered from bombardment on non-selective media or, preferably, transferred directly to media containing the selective agent. Can be.

The screening procedure involves exposure to toxic drugs and may use multiple steps of continuous change and selection of drug concentrations. Specific concentrations and cycle lengths may need to be varied for each particular drug. The currently preferred selection procedure involves using an initial selection step at a relatively low concentration of toxic drug, followed by a higher concentration later selection step. In this way, the selective agent can exert its toxic effect slowly and over a longer period. The concentration of the drug is preferably a concentration that produces a level of about 5-40% growth inhibition as initially determined from a growth inhibition curve. The effect is that transformed cells can grow and divide preferentially, but block non-transformed cells, but not to the extent that growth of non-transformed cells is disrupted. Several individual transformed cells
As soon as a sufficient number of divisions have occurred, the tissue is transferred to a higher concentration of toxic drug, killing substantially all non-transformed cells. Changes to higher concentrations also reduce the likelihood that untransformed cells will adapt to the drug. Higher concentrations are desirable, within a growth inhibition range of about 30-100%. The length of the initial selection cycle is about 1 to 4 weeks, preferably about 2 weeks. The subsequent selection cycle is about 1-12 weeks, preferably about 2-10 weeks. Virtually transformed maize cells are generally identified as proliferative sector regions of tissue in an environment of non-proliferating cells. At various times during the entire selection procedure, calli are also cultured in non-selective media.

As soon as the callus sector is identified as a hypothetically transformed cell, phenotypic and / or genotypic analysis can confirm the transformation. When using a selection agent, one example of a phenotypic assay is to measure the increase in the weight of a recent transformed cell when compared to various concentrations of a control. Other assays used depend on the function of the recombinant DNA. For example, if the DNA encodes an enzyme or protein, an enzymatic or immunological assay specific to the particular enzyme or protein is used. Other gene products may be assayed using appropriate bioassays or chemical assays. Other such techniques are well known in the art and will not be repeated here. The presence of the gene can be confirmed by a conventional procedure, such as Southern blotting or polymerase chain reaction (PCR).

V. Plant Regeneration and Seed Production The cell line that is apparently transformed must then be regenerated into a plant and the resulting plant's reproductive capacity measured. Transformants that test positive by genotype and / or phenotype analysis
Place on medium that promotes tissue differentiation and plant regeneration. Regeneration is performed according to standard procedures known in the art. This procedure usually involves reducing callus growth and reducing auxin levels that promote somatic embryo development or other tissue differentiation. One example of a regeneration procedure is described in Green et al. (19
82). In the growing room or green room, the plants are grown to maturity and appropriate sexual crossing is performed, as described by Neuffer (1982).

Regeneration is important to the present invention, but can be done in a conventional manner. If the selectable marker has been transformed into the cell, a selective agent is incorporated into the regeneration medium to further confirm that the regenerated plant has been transformed. Because regeneration techniques are well known and not critical to the invention, any technique that accomplishes regeneration and produces fertile plants may be used.

VI. Analysis of R1 progeny Plants regenerated from the transformed calli were subjected to R0 generation or R0 generation.
Called a plant. Seeds produced by various sexual crosses of R0 generation plants are referred to as RI progeny or R1 generation. When R1 seeds germinate, the resulting plant is also referred to as the R1 generation.

Through one complete sexual cycle, recombinant D
To confirm satisfactory transmission and inheritance of NA, the R1 generation must be analyzed to confirm the presence of transforming DNA. Given that plants and plant parts are used in place of callus, they may be analyzed in any of the ways described above for analyzing the evidence that bombarded calli have been transformed.

[0072] Recombinant DNA may exhibit different types of inheritance patterns for different transformants. For example, recombinant DNA is passed on to progeny according to Mendelian rules of inheritance. This type of heritability involves the transmission of DNA by both male and female germ cells, and the maize nuclear DN
With stable assembly or incorporation into A. Another type of inheritance pattern is material inheritance, in which recombinant DNA is transmitted primarily or exclusively through female germ cells. Analysis of the offspring of the various sexual crosses confirms the genetic pattern of the particular transformed cell.

VII. Recombination D in other maize varieties
Establishment of NA Next, fertile, transgenic plants are used in a conventional maize breeding program to incorporate the introduced recombinant DNA into the desired strain or variety. Methods and references for corn improvement are:
(1988), incorporated herein by reference. The conversion process (return mating) is among the approaches used in conventional breeding programs. Briefly, the first fertile transgenic plant is crossed to an elite inbred strain for exchange. Some plants have recombinant DNA,
Like some plants, the offspring of this cross separate. Plants with the DNA are then recrossed to elite inbred strains, resulting in offspring progeny. The original elite inbreeding is modified D
This backcrossing process is repeated until it is converted to a strain containing NA, but nevertheless has all the important characteristics originally found in the parent. Generally, this method requires about 6-8 generations. A separate backcross program is generally used for each elite strain that is converted to a genetically derived elite strain.

Generally, if the recombinant DNA is incorporated into many different hybrid combinations, the commercial value of the transgenic corn produced therein will be maximized. Maturity, farmers generally cultivate several hybrids based on differences in maturity, tolerance, or other agricultural management characteristics. Crossbreeds that fit in one area are generally not fit in other areas due to differences in characteristics such as maturity, disease and pest resistance, so farmers must select hybrids based on their geographic location. No. Therefore, the recombinant D is used to produce many hybrid combinations containing the desired heterologous DNA.
It is necessary to incorporate NA into large parent strains.

The techniques and techniques required for breeding corn and transferring genes from one strain or variety to another are well known to those skilled in the art. Therefore, through these breeding procedures,
Introduction of the recombinant DNA into other strains or variants can be accomplished.

VIII. Use of Transgenic Plants The transgenic plants produced therein are expected to be useful for various commercial or research purposes. Features that are beneficial to growers (eg, agricultural management features such as pest resistance, herbicide resistance or increased yield),
Features that are beneficial to consumers of kernels harvested from plants (eg, improved nutritional content in human food and animal feed);
Alternatively, transgenic plants are created for use in traditional agriculture with features that are beneficial to food processors (eg, improved processing characteristics). When used in this way, the plants are generally grown for using the grains for human or animal food. However, other parts of the plant, including stalks, husks, growing parts, etc., also have utility, such as part of animal silage or for ornamental use. Often, corn and other cereal chemical components (eg, oils and starches) are extracted for food or industrial use, thus increasing the level of such components or creating transgenic plants with modifications. is there.

Transgenic plants are sometimes used for commercial production of proteins or other molecules, in which case the relevant molecules are extracted or purified from plant parts, seeds and the like. To produce such molecules, plant cells and tissues may be cultured, grown in vitro, or fermented.

The transgenic plants may be used in commercial breeding programs, in other words, crossed or improved to related cereal seed plants. The improvement encoded by the recombinant DNA may be transferred, for example, from corn cells to another species, such as by fusion of a protoplast.

Transgenic plants may be studied or investigated, including the creation of new mutant plants by insertional gene induction, to identify beneficial mutants that can later be created by routine mutations and selection. The improvement has many uses. One example would be the introduction of a transposable element-encoding recombinant DNA sequence used to effect genetic changes. The methods of the present invention are also used to create plants with unique "signal sequences" or other marker sequences that can be used to identify patented strains or varieties.

The following non-limiting examples are illustrative of the present invention. They well illustrate the general procedure used to prepare fertile Zea mays plants of the present invention that expresses recombinant DNA and transmits that DNA to progeny. Unless otherwise specified, all components and percentages are by weight. The probability of emerging specific transformation events is a function of the amount of material that has undergone the transformation procedure. It is therefore necessary to repeat the procedure when the individual situations arising in the procedure described therein do not produce a transformant.

Example I: Reproduction from callus strain AB11
Transgenic plant Zea mays capable

I. Initiation and maintenance of maize cell cultures that retain the ability to regenerate plants immature hybrids produced by pollination of inbred B73 plant pollen (University of Iowa) and inbred A188 plant (University of Minnesota Cereal Improvement Association) From the embryo, easy to break,
Embryogenic maize callus culture was started. Embryo length 1.5 ~
Ears were harvested when they reached 2.0 mm. 50% v / v for each ear
The surface was sterilized with a commercial bleach (2.63% v / v sodium hypochlorite) at room temperature for 20 minutes. The ears were then washed with sterilized, distilled and deionized water. Immature embryos were aseptically separated and placed in a nutrient start / maintenance medium along with the root / sprout axis exposed to the medium. The start / maintenance medium (hereinafter referred to as "F medium") contains 2% (w / v) sucrose, 1.5 mg / 1 2,4-dichlorophenoxyacetic acid (2,4-D), 6 mM proline, and 0.25% Gel
N6 basic medium (Chu, 19) containing rite (San Diego, Kelco)
75). Prior to autoclaving, the pH was adjusted to 5.9. Unless otherwise stated, all tissue culture procedures were performed under aseptic conditions. Immature embryos at 26 ° C
Incubated in the dark. Cell proliferation from the scutellum of immature embryos was evaluated for friability and the presence of well-defined somatic embryos. After starting plate culture of immature embryos
On day 10-14, the tissue with this morphology was transferred to fresh medium. The tissue was then subcultured on a routine basis every 14-21 days. 70-80 mg of tissue was harvested from a section of tissue that reached approximately 1 g in size and transferred to fresh medium. Subcultures were always carefully and visually monitored to ensure that the correct morphology was maintained. The presence of somatic embryos ensured that the plants had developed in culture under appropriate conditions. The cell culture named AB11 used in this example was such a culture and was started about one year before the bombardment procedure.

II. Plasmid Plasmid, pHYGI1, is a standard recombinant DNA
A 3.2 kb circular plasmid assembled into vector-pBS + (Stratgene, San Diego, CA) using the technique. 553 bp Bcl-Bam containing the Maze AdhIS first intron (Callis et al., 1987).
The HI fragment was replaced with a CaNY 35S promoter,
It was inserted between PCHN1-1 and the hygromycin coating sequence assembled according to Example V. 1 and 2 show maps of pHYGI1. A sample of pHYGI1 was prepared on March 16, 1990 in the American type of Leakbilly, Maryland, USA, in accordance with the provisions of the Budapest Treaty.
Deposited with the Culture Collection and assigned accession number 40774.

PBII221 was flanked at the 5 'end by the CaMV 35S promoter and at the 3' end by the nos polyadenylation sequence.
Contains glucuronidase coding sequence.
This plasmid was assembled by inserting a maize AdHIS first intron between the 35S promoter and the coding sequence of pBII221 (Jefferson et al., 1987). 3 and 4 show maps of pBII221. Plasmids were introduced into embryogenic callus cultures AB11 by the microprojectile challenge method.

III. DNA transfer process Seven to twelve days before the microprojectile challenge method, the embryogenic maize callus strain AB11 was subcultured. AB11 was prepared as follows for the bombardment technique. Five agglomerates or callus, each about 50 mg wet weight, were placed in the center of a 60 x 15 mm sterile Petri plate (Falcon 1007) in a cross-shaped manner. The plate was stored in a closed container with wet paper towels throughout the bombardment process.

M-10 tungsten particles (Biolistics) exactly as described by Klein et al. (1988b) except as described below.
Was coated with the plasmid. (i) Two times the recommended amount of DNA was used. (ii) DNA precipitation on the particles was performed at 0 ° C. (iii) The test tube containing the DNA-coated tungsten particles was stored on ice throughout the bombardment process.

All tubes contain 1 mg / ml plasmid D
It contained 25 μl of 50 mg / ml M-10 tungsten in water, 225 μl of 2.5 M CaCl 2 and 100 mM aspermidine, along with a total of 5 μl of NA. When using both plasmids,
2.5 μl each. One test tube contains plasmid pB
II221 only, and T.I. E. FIG. A buffer (see Table 2 below) was included.

All tubes were incubated on ice for 10 minutes. The particles were pelleted by centrifugation at room temperature for 5 seconds in an Eppendorf centrifuge, and 25 μl of the supernatant was discarded. The test tubes were stored on ice throughout the bombardment process. Each preparation was used for up to five shells.

The large projectiles and stop plate were from Biolisti
c (Ithaca, New York). They were sterilized as described by the supplier. The microprojectile attack device was obtained from Biolistic.

The stop plate was placed in the slot closest to the cylinder and the sample plate tray was placed 5 cm below the bottom of the microprojectile attack device stop plate tray. A plate of callus tissue prepared as described above,
Placed in the center of the sample plate tray and removed the lid of the Petri dish. In order to retain the tissue throughout the bombardment,
3 x 3mm mesh made of iron with direct current electricity.
A hard wire mesh of X7cm2 was placed on the open plate. Biolis
The tungsten / DNA preparation was sonicated as described by tic and a 2.5 μl suspension was pipetted on top of the large projectile at each bombardment. The device was operated as described by the manufacturer. Table 2 summarizes the firing methods used.

[0091]

[Table 3]

Two callus bombardment plates containing pBII221 were used as F medium plates (without hygromycin)
Was transferred to S., and the calli were cultured in the dark at 26 ° C. Seven days later, the callus was washed with GUS assay buffer (5-
1-bromo-4-chloro-3-indolyl-β-D-glucuronide (Research Organic), 100 mM sodium phosphate pH 7.0, 5 mM each of ferricyan potassium and ferrocyan potassium, 10 mM EDTA and 0.065%
Transferred to 35 × 10 mm Petri dishes (Palcom 1008) containing Triton X-100. Incubate at 37 ° C for 3 days, then count the blue cells to get 2
The transient GUS-expressing cells found in the two plates
A total of 313 and 335, along with the other plates bombarded,
It is suggested that the NA transmission process was also taking place. Because the GUS assay is destructive, the plates of the tissue used for the GUS assay were discarded after counting.

When the medium had been cooled to 45 ° C., an appropriate amount of 100 mg / ml sterile filtered hygromycin B dissolved in water was added and the hygromycin B (Calbio) was added before pouring the plate.
chem) was incorporated into the medium.

After bombarding all samples, p
All calli and T. cerevisiae samples on plates treated with HYGI1 / pBII221 E. FIG. Two of the plates were transferred to F medium containing 15 mg / 1 hygromycin B (10 pieces of callus per plate). This is called the round 1 selection plate. T. E. FIG. Calli from the treated plates were transferred to F medium without hygromycin. Since this tissue was subcultured every 2-3 weeks on a non-selective medium, this is called a non-selective control callus.

After 14 days of selection, substantially identical tissues appeared on both selective and non-selective media. 7 pHYG
All calli on the I1 / pBII221 plate, E. FIG. One callus of the treated plate was transferred from a round 1 selection plate to a round 2 selection plate containing 60 mg / 1 hygromycin. Each round 2 selection plate contained 10 calli of 30 mg pieces per plate, resulting in a swelling of the total number of plates.

After 21 days on the round 2 selection plate, 60 mg
All material was transferred to a round 3 selection plate containing / 1 hygromycin. 75 days after the bombardment,
Several sets of round 3 selection plates were checked for viable callus sectors. One of the sectors was growing on pHYGI1 / pBII221 treated plates from an environment of necrotic tissue. This sector is PH3
This was transferred to F medium without hygromycin.

After 19 days on F medium without hygromycin, PH3 medium was added to F medium containing 60 mg / 1 hygromycin.
Was transferred. Growth could be sustained through multiple subcultures in the presence of 60 mg / 1 hygromycin.

To show that PH3 callus has acquired the hygromycin resistance gene, according to Example V
Genomic DN from PH3 callus and control callus
A was separated and analyzed by Southern blotting. D isolated
NA (10 μg) was digested with BamHI (NEB),
Electrophoresis was performed on a 0.8% w / v agaros gel in E (40 mM Tris acetate, pH 7.6, 1 mM EDTA) buffer at 15 V for 16 hours. This DNA was transferred to a Nytran membrane (Schleicher and Schuell). Transfer, hybridization and washing conditions were performed according to the manufacturer's recommendations.

According to the supplier's instructions, Oligo Labelling
With any primer labeled with Kit (Pharmacia),
α-92P-dctP (ICN Radiochemicals), 32P
A labeled probe was prepared. The template DNA used was pH
The 1055 bp BamHI fragment of YGI1
Contains the entire coding sequence.

While strengthening the screen, the membrane was exposed to Kodak X-OMAT AR film in the X-OMATIC cassette. A band of PH3 callus was confirmed at the predicted position of 1.05 kb, indicating the presence of HPT-encoded callus. No bands were found in the control callus. To demonstrate that the hygromycin gene was incorporated into the high molecular weight DNA, undigested DNA from PH3 callus and control callus was electrophoresed and blotted and hybridized as described above. At the same mobility as the uncut DNA, only undigested PH3 DNA showed hybridization to the probe. No hybridization was observed in the control callus DNA. The result is an intact pHYGI
As with one or a small non-chromosomal plasmid, PH3
It demonstrates the absence of the HPT coding sequence in the calli. This result is consistent with the incorporation of the hygromycin gene into high molecular weight DNA.

VI. Plant regeneration and seed production From a plate containing 60 mg / 1 hygromycin, thiamine hydrochloride 0.5 mg / 1. 2,4-D 0.75 mg / 1, sucrose 50 g /
1. RM5 basic salt supplemented with asparagine 150mg / 1, Gelrite 2.5g / (Sanjego, Kelco) (Murashige et al., 1
962), a part of the PH3 callus was transferred to the RMS medium.

After 14 days in RM5 medium, most of the PH3 and non-selective control calli were transferred to R5 medium (RM5 medium except for 2,4-D). The plates were incubated for 7 days in the dark at 26 ° C., irradiated for 14 hours at 26 ° C., and transferred to a dark light management mode for 10 hours. In this regard, R5
1 quart canned jar containing 100 ml of medium (B
All), the formed seedlings were transferred. Plants were transferred from jars to vermiculite for 7-8 days before transplanting to solids and growing to maturity.
PH3 produced a total of 45 plants, and control calli produced a total of 10 plants.

The inbred Zea mays MBS501 (M
ike Brayton Seeds) FR4326 (Illinois Foundation R)
esearch) and the patent registered inbred strain LM1112
Controlled pollination of mature PH3 plants was performed by standard techniques. Harvest seeds 45 days after pollination, 1-2
Let dry for days.

VII. Analysis of R1 Progeny The presence of HPT and GUS gene sequences in R1 plants was tested by PCR analysis. The expression of the HPT gene
HPT activity was measured by an enzyme assay. Table 3 summarizes the data. These data demonstrate that recombinant DNA is transmitted and expressed through parents and are consistent with Mendelian inheritance. In the original callus, Southern plot evidence that the HPT coding sequence is integrated into high molecular weight DNA, combined with genetic data, suggests that the foreign DNA sequence is integrated into the chromosome. The presence of the GUS gene in the R1 progeny demonstrates co-transformation and inheritance of the non-selected gene.

[0105]

[Table 4]

[0106]

[Table 5]

[0107]

[Table 6]

The HPT enzyme assay is based on the following method. SKDatta et al., Bio / Technology, 8,736 (1990),
M. Stabell et al., Anal. Bioshem., 185, 319 (1990), E. Caban.
es-Bastos, Gene, 77, 169 (1989).

Root samples (0.2 g) are cut from 7-10 day old seedlings and snap frozen in liquid nitrogen (N2). Use disposable pestle and test tube, alumina and 400μ1 EB
(50 mM Tris-HCl, 10% v / v glycerol, 0.1 mM PM
(SF, pH 7.0) for 30-60 seconds and then centrifuged for 10 minutes in an Eppendorf microcentrifuge. Certric
Transfer the supernatant to the on 30 filter unit and use the Beckman G
Desalting was performed by centrifugation at 5,000 rpm and 4 ° C. for 30 minutes using a fixed-angle rotor of an RP centrifuge. Use 1 ml of EB for washing each filter unit,
Spin for minutes. Retentate was collected and stored at -70 ° C. Transgenic callus tissue and control samples (0.2 g) were ground as described above and the supernatant was used as positive and negative controls.

Bradford, Anal. Bioshem., 72, 248 (1976)
The protein was quantified with the BioRad kit using the method described above. The protein concentration of the root extract ranged from 0.2 to 2 μg / μl. 20m
M Tris-maleic acid, 13 mM MgCl2, NH4Cl, 0.5 m
MDDT, 50 μM ATP, 0.61 μg / μl hygromycin B, 25 μg / μl bovine serum albumin and 12
Root extract (4 μg total protein) was added to the reaction mixture containing μ Ci / μl γ32P-ATP. The reaction volume was 32 μl. The reaction mixture was incubated at 37 ° C. for 30 minutes.

1 μl of the reaction mixture was mixed with polyethyleneimine
Cellulose thin-layer chromatography plates (Sigma
Chem). Developed with 50 mM formic acid pH 5.4, air dried and exposed to Kodak XAR-5 film. The reaction product hygromycin phosphate migrates near the solvent front under these conditions.

To perform the PCR assay, a 0.1 g sample was taken from plant tissue and frozen in liquid nitrogen. next,
0.1 M Tris-HCl 200 μl, 0.1 M NaCl, 20 mM EDTA, 1
Samples were ground with 120 grit carborundum at 4 ° C. in% Sarkosyl, pH 8.5. After the phenol / chloroform extract and ethanol and isopropanol precipitation, T.I. E. FIG. Samples were suspended and analyzed by polymerase chain reaction. (KB Mullis, U.S. Patent No.
4,683,202). 50 mM KCL, 10 mM Tris-HCl pH 8.
4,3mM MgCl2,100μg / ml gelatin, 0.25μM / ml, each
0.25M of appropriate primers, each with 0.2mM deoxynucleotide triphosphate (dATP, dCTP, dGTP)
TP, dTTP), 2.5 units of Tag DNA polymerase (Cetus), and 10 μl of the DNA preparation in a volume of 1
PCR was performed with 00 μl. Place mineral oil on the mixture and heat at 94 ° C
Heat up to 55 ° C for 1 minute, 72 ° C for 1 minute, 94 ° C for 1 minute
Amplified for 35 cycles. Next, the mixture was heated at 50 ° C. for 2 minutes.
Incubation was further performed at 72 ° C. for 5 minutes. PCR product 1
0 μl was electrophoresed in an agarose gel and stained with ethidium bromide to make it visible.

For analysis of the presence of the HPT gene, one PCH primer complementary to the 35S promoter,
One PCH primer complementary to the T coding sequence was used. Therefore, in order to produce a properly sized PCR product, the HPT template DNA must include the adjacent 35S promoter region, Adh1 intron, and 5 'protein coding sequence region.

For analysis of the presence of the GUS gene, a PCH primer complementary to a sequence in the GUS protein coding region was used. If the template DNA contains an intact coding region between the two primers, a 797 bp PCR product is expected.

Example II: Encoding a seed storage protein
Breeding from callus strain AB63S containing recombinant DNA
The 10 kD zein storage protein of transgenic plants capable of being produced in maize kernel endosperm is one of a representative class of proteins referred to as “seed storage proteins”. This protein contains very high concentrations of methinion (22.5%) and is encoded by the ZpS10 / (22) gene (MS Benner et al., Tfeor. App.
l. Genet., 78, 761 (1989); in which the gene referred to as z10). Therefore, increased expression of the z10 gene can be used to increase corn methionine content. Fertile transgenic corn containing the chimeric z10 gene can be achieved by the procedure of Example I with some minor modifications. Example II also demonstrates the introduction of recombinant DNA using a different callus strain than that used in Example I.

I. Tissue Culture Strains The embryogenic maize callus strain, named AB63S, used in this example was generated by crossing between the elite inbred strains A188 and B73 by the initiation and maintenance procedures described in Example I. Made from immature embryos. The callus stock was launched seven months before the bombardment role. Eleven weeks prior to bombardment, the tissue was sieved, forced through a 1.9 mm screen, and plated on N6 maintenance medium.
After 1-2 weeks of growth, friable, embryogenic calli were selected from the resulting plants, transferred to fresh medium and grown for 1.5-3 weeks before re-sieving. This cycle of sieving and recovery of friable callus was performed a total of three times before bombarding the tissue.

Plasmid pH as described in Example I
YGI1 and pBII221 were used in this example. In addition, plasmid pZ27Z10 was included in the DNA / tungsten preparation. By standard recombinant DNA techniques
A 3.2 kb circular plasmid vector-constructed using pUC118 (J. Vieira et al., Methods Enzymol., 153, 3 (1987)).
pZ27Z10 is a maize 10 kD zein gene (Kirihara
Et al., Gene, 7,359 (1988) referred to as z10 in this document) and the 27 kD zein gene of the maize (DEGeraght) located immediately adjacent to the coding sequence and the 3 'non-coding sequence.
y, ph. D. Thesis, University of Minnesota (1987)). z27 regulatory gene, z10 coding and 3 '
The sequence combination is referred to herein as the chimeric z27-z10 gene. The polyA signal of the CaMY genomic region flanking the 35S promoter positions 3 'of the z10 gene sequence of pZ27Z10. FIG. 9 and FIG. 10 show maps of this plasmid. A sample of the plasmid pZ27Z10 was prepared according to the provisions of the Budapest Treaty by the American Type Culture Collection, Leakbilly, MD.
Was deposited under accession number ATCC 40938.

III. DNA transfer process Three weeks prior to microprojectile bombardment, tissues of the maize embryogenic callus strain AB63S were subcultured. The tissue was prepared for bombardment by sieving through a 1.9 mm screen. The sieved tissues were plated on sterile, approximately 500 mg thin filters per filter, 5.5 cm Whatman No. 1 filter discs. A total of 6 disc tissues were prepared. Prior to microprojectile bombardment, the filter disks containing the tissue were transferred to empty Petri plates. The plate was left uncovered in a laminar flow hood for 20 minutes to dry the tissue slightly. Plates were stored in a humid environment until used for bombardment to prevent further drying of the tissue. The DNA used for bombardment consisted of the plasmids pHYGI1, pZ27Z10, and pBII221, 1: 1: 1 (by weight). DNA was precipitated on tungsten particles as described in Example I.

Bombardment was performed as described in Example I, except that the Biolisti ™ PDS1000 (DuPont) device was used, and that no screen was placed over the tissue. Six filter discs containing callus tissue were processed as follows. Two filters were bombarded twice with DNA / tungsten preparation (potential positive treatment 2X), three filters were bombarded once with DNA / tungsten preparation (potential positive treatment 1X), One filter is a tungsten / water suspension using the same weight as the tungsten used for DNA / tungsten bombardment.
Bombarded twice (negative treatment).

IV. Selection of Transformed Callus After bombardment, filter disks containing callus tissue treated with 2X DNA were transferred to round 1 selection plates, F medium containing 15 mg / l hygromycin.
Two filters with IX DNA treatment as well as filters with negative control treatment were also transferred to the round 1 selection plate. A third filter of 1X DNA treatment was transferred to F medium without hygromycin for use as a non-selected control callus. Since this unselected control callus is potentially positive, it is only maintained during the first two rounds of selection for comparison purposes. Thereafter, non-bombarded AB63S callus maintained in F medium without hygromycin was used as hygromycin selection control callus.

After 5 days, small clumps (about 25 mg) of callus were transferred from filter discs to fresh round 1 selection medium. The plate culture density was 10 callus clumps per plate. By this time, the callus had increased about twice in weight. In the same way, unselected control calli were transferred to F medium without hygromycin. Fifteen days after bombardment, all tissues were transferred from round 1 selective medium to round 2 selective medium (F medium containing 60 μg / hygromycin).
In this transfer, the plate culture density was 14 callus clumps per plate. In the 14 days since the last transfer, the callus had increased about three times in volume. Twenty-three days later, all tissues were transferred from round 2 selection medium to round 3 selection medium containing 60 μg / hygromycin.

Five days after the bombardment, five living sectors were found surrounded by necrotic callus clumps. The five sectors were considered to have been derived from only one callus aggregate from the round 2 selection, as they appeared adjacent to one another on successive plates from the previous transfer. This sector resulted from 2X DNA treatment and was called callus strain Metl.

The callus strain Me was added to an F medium containing 60 μg / ml of hygromycin and an F medium containing no hygromycin.
tl was transferred. After incubation for 22 days, there was no apparent difference in the growth and appearance of the tissue on the two media. This callus strain Met1 grew rapidly and did not appear to be blocked by the presence of hygromycin in the medium. Metl callus was fairly brittle and embryogenic in both media and could not be visually distinguished from control AB63S callus grown on F media without hygromycin.

V. Confirmation of transformed calli Inhibition tests were performed using Metl callus and control AB63S calli. Before you start an interdiction test,
2 Metl callus in F medium without hygromycin
Grow for one day. Met1 callus and control A
B63S calli were transferred to plates of F medium containing 0, 15, 60, 100 and 200 mg / l hygromycin. For each concentration of hygromycin, three plates of each callus strain were prepared. Approximately 50mg per plate
5 to 6 callus pieces (total callus per plate was about 300 mg).

After incubation for 28 days, the callus weight on each plate was measured. The average weight obtained at each concentration of hygromycin was divided by the average weight of callus obtained at 0 mg / l hygromycin to determine the growth inhibition rate. The results show that at hygromycin concentrations of 15, 60 and 100 mg / l, M
The growth of et1 calli was shown to be completely inhibited.
Met on a medium containing 200 mg / l hygromycin
The growth of one callus was inhibited by about 20%. In contrast, AB6
3S callus growth was inhibited at all hygromycin concentrations, and at 200 mg / l hygromycin, AB63S callus was inhibited by about 90%. From these results, it was confirmed that the callus strain Met1 exhibited resistance to hygromycin present in the growth medium.

Southern blot assays were performed to confirm the presence of the hygromycin resistance gene in the Met1 callus DNA and to determine whether the callus also acquired the chimeric z27-z10 gene. HP
For detection of the T coding sequence, DNA samples separated from the Met1 callus and the control callus were digested with the restriction enzymes BamHI, HindII1 and BstEII. The digested DNA sample was electrophoresed on a 0.8% agarose gel and blotted onto a Nytran membrane as described in Example I. Visual inspection of the ethidium bromide stained gel before blotting shows that BamHI digestion appears to be complete, but HindII1 is also BstEII.
Did not cleave the DNA to any significant extent. Biotin-labeled HPT coding sequence 1.
The blot was probed with the 05 kb BamHI fragment.
Conditions used for hybridization, washing and detection were as suggested by the BRL Photogene ™ kit used in this experiment.

After hybridization, BamHI digested Met
About 2.7, 4.5 and 6.5 kb in the lane containing 1 DNA
In addition to the size, a labeled band having the expected size of 1.05 kb was confirmed. The results indicated that the HPT coding sequence was present in the DNA of the callus strain Metl. The other 2.7, 4.5 and 6.5 kb bands indicated incomplete digestion of the restriction enzymes or the presence of multiple rearranged copies of the HPT sequence. HindI
In the case of I1 and BstEII digestion, hybridization signals could be seen at the position of undigested DNA in both lanes. This result indicates that the HPT coding sequence is Met1
It was shown to be integrated into the high molecular weight DNA of the genome. No dehybridization signal was observed in any lane containing a control callus DNA digest.

For detection of the chimeric z27-z10 gene,
D isolated from Met1 callus and control callus
Southern blot assays were performed using NA samples. The DNA sample was prepared using the restriction enzymes BamHI and Eco.
Digested with RI. BamHI digestion releases the 2.76 kb fragment containing the z10 coding sequence and the three non-coding sequences of the pZ27Z10 structure used for this transformation. The 7.26 kbp Z27Z10 plasmid contains:
There is only one EcoRI site. Photogene kit (BR
Using the conditions described in L), the generated blot was hybridized with a biotin-labeled probe containing the entire z10 coding sequence.

In the case of BamHI digestion, a 9 kb lane containing both Met1 and control callus DNA was used.
And at about 15 kb, the endogenous z10 sequence showed a hybridization signal. A complementary band was observed at about 3.5 kb in the Metl sample, but not in the control DNA sample. No strong hybridization signal was observed at the expected 2.76 kb size of Met1 DNA. Met
The absence of a labeled 2.76 kb band in BamHI digestion of 1 DNA indicated incomplete digestion of the DNA or rearrangement of the introduced DNA.

In the case of EcoRI digestion, hybridization signals of the endogenous z10 sequence were shown at about 12 kb and 16 kb in both Mer1 and control callus DNA. A complementary band was observed at about 14 kb in the Met1 sample, but not in the control DNA sample. This result indicated that a new z10 sequence was present in the Metl callus DNA, which was not present in the control callus DNA sample.

The above results were confirmed by the second Southern blot test in which Met1 and control callus DNA were digested with BamHI, NcoI and NsiI. M
Samples of undigested DNA of et1 and control callus were also included. The filters were probed with a 32P-labeled z10 coding sequence probe. All of the digests showed a new z10 hybridization signal not present in the negative control callus DNA sample in the Metl callus DNA. From the above results, it was confirmed that a new z10 coding sequence was present in the Met1 callus. A hybridization signal was observed in the lane containing the undigested DNA at the position of the undigested DNA of Met1 and control callus. These results indicated that the z10 sequence introduced into the chromosomal DNA of Met1 callus was accumulated.

VI. Plant regeneration and production of transgenic seeds As described in Example I, calli of the Metl strain and the control AB63S strain were plated on RM5 medium.
After incubation for 14 days in the dark at 26 ° C., the calli were transferred to R5 medium as described in Example I. Incubate at 26 ° C. in the dark for 7 days, then transfer to light under the conditions described in Example I. After 14 days in a bright place, Mag containing 100 ml of RS medium
The seedlings were transferred to an enta box (Sigma Chemical). After 7-14 days in this medium, the seedlings were transferred to vermiculite for 7 days and then transferred to soil, where they grew to maturity. From the Met1 callus, a total of 49 plants (Met
l-1 to mMet1-49), and a total of 25 plants were regenerated from the control AB63S callus. To confirm that the plants regenerated from the Metl callus retained the introduced DNA, PCR analysis was performed on DNA samples prepared from leaf tissue. One set of six oligodeoxyribonucleotides was used for this purpose. Table 7 below summarizes the sequences, orientations and relative positions within the chimeric z27-z10 gene structure shown in Figure 8 for these oligonucleotides.

[0133]

[Table 7]

Oligonucleotides (or primers)
To one z27 oligonucleotide (z27
5 ′ or z27mid) and one z10 oligonucleotide (z10mid or z103 ′) using a pair of oligonucleotides, rather than from the endogenous maize z227 or z10 gene,
The design was such that fragment amplification occurred only from the 7-z10 gene. The appearance of an amplified fragment of the expected size during a PCR reaction using these z27-z10-specific oligonucleotide pairs is a feature indicating the presence of the chimeric z27-z10 gene in a particular callus or plant DNA sample. is there.

A pair of z27 oligonucleotides (z27mid having z275 ′ or z273 ′) or z10 oligonucleotides (z1mid having z10mid)
05 'or z103'), the control AB
635 callus DNA, Met1 callus DNA or pz
PCR performed on 27z10 plasmid DNA
When used in a reaction, amplification of fragments representing the z27 regulatory sequence or z10 coding sequence, respectively, occurs. These reactions are specific callus or plant DN
Serves as a positive control for amplification of the endogenous maize gene sequence from the A sample.

Leaf DNA was prepared from two Met1 RO plants (designated Met1-1 and Met1-2) and one control AB63SRO plant, as described below.
PCR reactions were performed on these DNA samples using oligonucleotide pairs specific for the HPT coding sequence, the chimeric z27-z10 gene, the z27 regulatory sequence and the z10 coding sequence. From the gel analysis of the PCR reaction product, the amplification product of the expected size
Obtained for all reactions using -1 DNA and Met1-2 DNA. Control AB63S DNA was negative for the HPT coding sequence and the chimeric z27-z10 gene, but positive for the endogenous z27 regulatory sequence and the z10 coding sequence. Based on these results, the Met1 RO plants tested showed that the introduced HPTDN
A and the chimeric z27-z10 DNA.

As a solid evidence that the characteristic z27-z10 PCR reaction product contained both the z27 regulatory sequence and the z10 coding sequence, Southern blot analysis was performed on the PCR reaction products. Southern blot filters were prepared from two agarose gels loaded with the same sample. The sample contains z27 as described above.
Regulatory sequence, z10 coding sequence, and chimeric z2
Includes PCR reaction products obtained by amplification of the 7-z10 gene sequence. One blot was hybridized with the z10 coding sequence probe and the other blot with the z27 regulatory sequence probe.
As a result, the z10 coding sequence probe was converted to the z10 coding sequence PCR reaction and chimera z27-z10
While hybridized with the amplified fragment from the gene PCR reaction, it did not hybridize with the amplified fragment from the z27 regulatory sequence PCR reaction. Similarly, the z27 probe is the z27 regulatory sequence PC
It hybridized with the amplified fragment from the R reaction and the chimeric z27-z10 gene PCR reaction, but did not hybridize with the amplified fragment from the z10 coding sequence PCR reaction. From these results, it was found that the fragment amplified in the PCR reaction contained the expected gene sequence, and that chimera z2
The characteristic PCR amplification product for the 7-z10 gene was shown to contain both the z27 regulatory sequence and the z10 coding sequence.

Inbred lines of corn A100, B73,
Pollination regulation of mature Met1 RO plants was performed for A654 and H99. In addition, several self- and sib pollinations were performed. Met1 RO plants were used as both male pollen donors and female pollen recipients for a total of 130
Was pollinated. Mature seeds were harvested 45 days after pollination and dried for 1-2 weeks.

VII. Analysis of R1 progeny The presence of the HPT sequence and the chimeric z27-z10 gene
P of DNA from R1 plant tissue for three sets of progeny
Evaluated by CR analysis.

The first set of R1 progeny analyzed consisted of four immature tuft-seed from the RO plant Met1-7. These tuft-seed were obtained through open pollination of silk grown from Met 1-7 tufts. About 18 days after pollination, the tuft-seed was removed from the plant and the surface was disinfected. Inner milk was cut out from each seed, frozen on dry ice, and stored at -70 ° C until used for DNA separation.

Embryos from various offspring were transferred to R5 medium and germinated. After 10 days, the seedlings were subcultured to vernmiculate for 7 days, and then transferred to soil for growth and ripening. PC for DNA isolated from each endosperm using a set of oligonucleotides specific for the HPT coding sequence, the chimeric z27-z10 gene and the maize Adh-1 gene
An R analysis was performed. Three of the four endosperm DNA samples were found to be positive for all of the above gene sequences. The fourth DNA sample was negative for the HPT coding sequence and the chimeric z27-z10 gene sequence, but positive for the endogenous Adh-1 sequence. From these results, the HPT sequence and chimera z27-z1
It was found that all 0 genes were transmitted to the R1 progeny of Met1-7. A similar analysis was performed on A65
This was performed on 24 seeds obtained from the 4XMet1-1 cross. 24 days after pollination, the seeds were removed from the ears, and the surface was sterilized and disinfected. Endosperm and embryos were separated and processed as described above. By PCR analysis of endosperm DNA samples,
Nine of the 24 progeny contained the HPT sequence and the chimera z27-
It was found that both z10 genes were received.
The remaining 15 offspring did not carry any of the introduced gene sequences. The third set of R1 progeny analyzed was R
28 plants 2 derived from self-pollination of O plants Met1-6
It was prepared from the leaf tissue of a week-old seedling.

Analysis of the leaf DNA samples indicated that 24 of the R1 progeny contained both the HPT sequence and the chimeric z27-z10 gene. The remaining four R1 descendants (proge
ny) did not carry any of the introduced genes.
The results of the above analysis are summarized in Table 8, which demonstrates that a significant portion of the R1 progeny of the Metl plant is inherited by the introduced DNA.

[0143]

[Table 8]

Z27z10 / HPT- from a distantly crossed group
The ratio of z27z10 + / HPT + plants to plants is consistent with a 1: 1 segregation ratio at a single locus. In addition, z27z10 / MPT plants from the self-pollinated group
The ratio of 10 + / HPT + plants is consistent with a 3: 1 segregation ratio of a single locus. Data also includes introductory HPT and z27-z
This shows that 10 sequences are linked.

Example III: Set encoding insecticidal proteins
Fertility transcript from callus AB12 containing recombinant DNA
Proteins encoded by various Bacillus thuringiensis genes in sgenic plants have been shown to be useful in many pesticidal applications. Production of a transgenic corn plant by a particular hetero-Bt gene will enhance the plant's resistance to certain pests. Recombinant Bt
Fertile transgenic plants containing the gene are made according to Example 1 with some minor modifications, as shown below.

At the same time, the callus AB11 of Example 1 is initiated to produce another callus. AB12 is produced from an isolated cross of plants having the same parental genotype (A188XB73). This example is AB11 and AB63S
In addition, it shows that the fertile transgenic plant regenerates from the third callus line, and that the production of the fertile transgenic plant is not due to properties unique to the single callus line alone.

The plasmid pHYGI1 described in Example 1 was used, but pMS533, a plasmid containing the insecticide Bacillus thuringiensis endotoxin (BT) gene fused in frame with the neomycin phosphotransferase (NPTII) gene, was also used. The segment of DNA from the CaMV and nopaline synthase promoter is located 5 'from the fusion gene. At the 3 'position from the fusion gene is the polyA region of the nopaline synthase gene and a fragment of DNA derived from the tomato protease inhibitor I gene.

The callus is carried out as described in Example I, only the DNA used in the tungsten / DNA preparation containing the plasmids pHYGII and pMS533 is different. One tube contains only 5 μl TE (10 mM Tris-
Hcl pH 8.0, 1m MEDTA).

The following implantations were made: 11 × pHY
GII / pMS533 (possible for positive treatment) and 3XTE preparation (control treatment).

After implantation, calli from the pHYGII / pMS533 treatment were placed on round 1 selection plates (selection plates), F medium containing 15 mg / l hygromycin as 10 pieces per plate.
The same was done for two of the plates that had been implanted with the TE preparation (select control callus). One plate of callus with the TE preparation was placed on F medium without hygromycin; this callus was retained as a source of control tissue (non-selective control callus) throughout the experiment.

After 18 days, the calli were transferred from the round 1 selection plate to a round 2 selection plate containing 60 mg / l hygromycin as 10 30 mg pieces per plate. After 21 days of selection on round 2 selection plates, callus was completely inhibited. The entire callus was transferred from round 2 selection plates to round 3 containing 60 mg / l hygromycin.
Transferred to selection plate.

On the round 3 selection plate, 42
After a period of days, six sectors were observed to have propagated from the surrounding gangrene tissue, all of which were pHYGI1 /
From pMS533 treated samples. Callus lines were transferred to F medium. After 24 days, one callus line, designated CB2, has grown considerably. Part of each callus system is (i)
It was transferred to F medium containing 15 mg / l hygromycin or (ii) F medium containing 60 mg / l hygromycin. Control calli were transferred to F medium containing 15 mg / l hygromycin.

Only one of the six callus lines, CB2, was able to continue growing on a separate medium in the presence of 60 mg / l hygromycin. DNA was released from a portion of this callus and the presence of the Bt gene was determined by Southern blot (Southern b.
lot) confirmed by analysis.

DNA was digested with a restriction enzyme combining BamHI and XhoI. BamHI cuts at the 5 'end of the Bt coding sequence and XhoI cuts at the 3' end of the coding region. The probe labeled A 32P is 1.8 kb of pMS533.
Made from BamHI / XhoI restriction fragment. The expected 1.8 kb band was confirmed after mating and radiography. No bands were present in DNA from untransformed calli.

Plants are regenerated from CB2, as described above.
Control pollination was performed to produce the R1 generation. R1 plants are PCR
The analysis confirmed the presence of the HPT and Bt gene sequences and the enzyme assay as described in Example I confirmed the HPT gene expression. PCR analysis was performed on DNA isolated from root tissue of R1 seedlings as described in Example I. Each DNA sample is PC
To test for suitability for R, PCR analysis was performed using primers specific to the native maize 10Kd zein gene. Primers used for PCR analysis of the Bt gene were those corresponding to the 35S promoter and those corresponding to the Bt coding sequence. Primers used for HPT PCR analysis are the same as those described in Example I. The results of this analysis are as shown in Tables 9 to 14. The results indicate transmission of the transfected HPT and Bt genes from male and female parents, and the observed frequencies are consistent with Mendelian genetic rules. The results are consistent with the insertion of the HPT and Bt genes into chromosomal DNA. In addition, inheritance in a linked state of the HPT and Bt genes was confirmed, indicating that the two genes were inserted close to the same chromosome.

[0156]

[Table 9]

[0157]

[Table 10]

[0158]

[Table 11]

[0159]

[Table 12]

[0160]

[Table 13]

[0161]

[Table 14]

Example IV: Through Two Complete Sexual Cycles
From Callus Line AB12 Transmitting Recombinant DNA by Induction
Transgenic plants

I. Plant Lines and Tissue Culture Callus line AB12 was used and is described in Example III.

II. Plasmids The plasmids pDII221 and pHYGI1 described in Example I were used.

III. DNA callus implantation was performed as in Example I except that the DNA used in the tungsten / DNA preparation was different. All tubes contained 25 μl 50 mg / ml M-10 tungsten in water, 25 μl 2.5 M CaCl 2 and 10 μl 10 M
Contains a total of 5 μl 1 mg / ml total plasmid with 0 mM spermidine. One tube contains no plasmid and 5 μl TE buffer.

The following implants were made: 2XPBII221
Preparations (for transient expression); 7XpHYGII preparations (with positive treatment); and 3XTE preparations (negative control treatment).

After performing all the injections, PBII2
Calli from 21 treatments were transferred to F medium as five 50 mg pieces per plate. Two days later, the calli were placed in GUS buffer as in Example I. The count of the number of calli temporarily expressed was 686 and 84.
Five GUS-positive cells were found, suggesting that the particle release process is also taking place in other implantation plates.

IV. After selection of transform callus, calli from pHYGII treatment were transferred to round 1 selection plates, ie 1 plate per plate.
Zero 25 mg pieces were placed in F medium containing 15 mg / l hygromycin. The same was done for two of the plates implanted with the TE preparation (select control callus). One plate of callus bombarded with the TE preparation was placed on F medium without hygromycin; this callus was retained throughout the experiment as a source of control tissue (unselected control callus).

After 13 days, the calli on the Round 1 selection plate were indistinguishable from the unselected control calli. All calli were transferred from the round 1 selection plate to the round 2 selection plate containing 60 mg / l hygromycin. An approximately 5-fold increase in the number of plates has occurred. The callus on the Round 2 selection plate gained substantial weight after 23 days, at which point it appeared necrotic. All calli were transferred from the round 2 selection plate to the round 3 selection plate containing 60 mg / l hygromycin.

[0170] At day 58 post-implantation, three live sectors were observed to grow from surrounding dead tissue. 3
All strains were from pHYGII treatment and 24
C, 56A and 55A.

After 15 days on the maintenance medium, growth of each line was observed. Line 24C grew well, but 55A and 56
A's growth was slower. 60 mg / l for all three strains
Was transferred to F medium containing hygromycin. Non-selective control calli from the maintenance medium were also plated on F medium containing 60 mg / l hygromycin.

After 19 days on 60 mg / l hygromycin, the growth of line 24C appeared to be completely inhibited, whereas the control showed about 80% of the weight gain of 24C. Line 56A was completely necrotic and line 55A was very close to necrosis. Lines 24C and 55A were re-transferred to F medium containing 60 mg / l hygromycin in the same manner as control tissues. After 23 days on 60 mg / l hygromycin, the 24C line again appeared completely uninhibited. The 55A strain has a negative control callus of 60 mg / l.
It was completely killed like a negative control callus on a second exposure to F medium containing hygromycin.

V. Confirmation of Transform Callus A Southern blot was prepared from BamHI-cleaved DNA from the 24C line and probed with the HPT probe described in Example I. As shown in FIG. 8, the band observed for the 24C line was observed at the expected size of 1.05 kb, indicating that the 24C line contains the HPT coding sequence. No bands were observed in DNA from control tissues. The callus line 24C was renamed PH2.

The hygromycin gene has a high molecular weight DNA
To demonstrate that they had been incorporated into DNA, the DNA isolated from the PH2 callus and the control callus was (i) without restriction enzymes,
(ii) BamHI as described above, or (iii) HPT
The plasmid pHYGI1 only once within the coding sequence
Was digested with PstI. Samples were blotted and probed with the HPT coding sequence as previously described.

Only the uncleaved PH2 DNA is uncleaved DN
Hybridization of the position A to the probe is shown, demonstrating that the hygromycin gene has been incorporated into high molecular weight DNA. The expected band of 1.05 kb for PH2 DNA cut with BamHI was observed as indicated above. PH2 DNA cut with PstI
For 5., if the hygromycin gene is present on the uncleaved intact pHYGI1 plasmid, 5.
You should see a 9kb band. If the gene is incorporated into genomic DNA, two or more bands of various sizes (depending on the site of action of PstI in the host DNA) would be expected. Molecular weight is about
Two bands of 6.0 and 3.0 kb were observed. These data are consistent with the incorporation of the hygromycin gene into high molecular weight DNA. No hybridization was observed for DNA from control calli by any of the above treatments.

These results demonstrate that the HPT coding sequence is not present in PH2 calli as an intact pHYGI1 or small non-chromosomal plasmid. These are also consistent with the incorporation of the hygromycin gene into high molecular weight DNA. Southern blot analysis further demonstrated that the HPT coding sequence was also in close proximity to the 35S promoter sequence.

VI. Plant regeneration and seed production The PH2 line was transferred to RM5 medium to regenerate plants as in Example I, along with the non-selective control callus. After 25 days on R5 medium, the plantlets were transferred to the R5 bird position and allowed to grow for 20 days. At this point, plantlets for a week
They were transferred to vermiculite and then to soil where they were grown to sexual maturity. Then, controlled pollination was performed using inbred line B73.

VII. Analysis of R1 Progeny A. Ten R1 progeny plants from the PH2 B73XPH2.6 cross as pollen donors were assayed for HPT expression using both root elongation and yellow leaf assays. One positive plant was identified. The presence of the HPT gene in positive plants was determined as described in Example I by HP
Confirmed by Southern blot analysis using a T probe.

For the bioassay of root elongation, a commercial bleach diluted 1: 1 with water and sterilized in 0.1% Alconex for 20 minutes in a 125 ml Erlenmeyer flask and three times in sterile water. Rinse. 50mg / ml seed
Water was absorbed overnight in sterilized water containing captan while stirring at 150 rpm.

After absorbing the water, the solution was transferred from the flask, and contained a flow box (Flow Labor) containing three germinated papers saturated with H2O.
atories). A fourth piece of water-saturated germinated paper was placed on top of the seed. Seeds were allowed to germinate for 4 days.

After the seeds germinate, the tip of the main root is about 1 cm
Were cut from each seedling, plated on medium containing MS salt, 20 g / l sucrose, 50 mg / l hygromycin, 0.25% gellite and incubated at 26 ° C for 4 days in the dark.

The roots were evaluated for abundance of root hairs and root shoots. When the root has root hairs and root shoots, it is classified as transgenic (hygromycin receptive),
When there were few root shoots, no transformation was performed (hygromycin sensitivity).

After cutting off the tip of the root, one PH2
The sprouting of the panicle and one control panicle was transferred to a wet vermiculate and then grown for 5 days in the dark. At this point, a 1 mm section was cut from the tip of the cotyledon sheath to perform the yellow leaf bioassay, the surface was sterilized for 10 seconds, and MS basal salts,
Plated on 2.5 g / l gellite with either g / l sucrose, zero (control) or 100 mg / l hygromycin and incubated at 26 ° C for 18 hours in the dark. Each plate contains duplicate sections of each shoot. Plates were then incubated for 48 hours at 26 ° C. in a 14 hour light, 10 hour dark and graded on a scale from 0 (all brown) to 6 (all green) for percentage of green in leaf tissue. When shoots were at the zero stage they were classified as untransformed (hygromycin sensitive) and at 3 or higher as transformed (hygromycin resistant).

B. PH2 as a Pollen Recipient 80R1 progeny plants from a PH2 plant named PH2.23XB73 cross were assayed for HPT expression by root elongation and 26 were positive. Expression of the HPT gene was further confirmed for all 26 by direct exposure of seedlings to hygromycin-containing medium in a flow box. Two of the resistant plants were analyzed by PCR to confirm the presence of the HPT sequence.

VII. Analysis of R2 progeny Transgenic R1 with PH2 as pollen parents
The plants were grown to maturity and used to pollinate FR4326 plants. R2 seeds from this cross were germinated and tested for the presence of the HPT sequence by PCR.
The expression of the HPT gene was determined by the HPT enzyme assay described in Example I.

Three of the 19 descendants tested were HP
It had a T gene sequence and also expressed HPT enzyme activity. The rest did not contain the HPT sequence and showed no enzymatic activity. This result is due to pollen transmission and DN by genetic engineering.
A demonstrates that A is expressed over two generations, indicating that the gene is stable.

Example V: The friable, embryogenic maize callus culture AB12 described in Example III was used.

Using standard recombinant DNA techniques, 3.2 k
vector-PBS + (Stratagen
e, Inc., SanDiego, CA).
LUC-1 was assembled. pCHN-1 is Aqrobacterium tu
Contains the hygromycin B phosphotransferase (HPT) coding sequence from E. coli (Gritz et al. 1983) to which the nopaline synthase (nos) polyadenylation sequence of mefaciens (Chilton and Barnes 1983) is attached at the 3 'end. Expression is with cauliflower mosaic virus (CaMV) 3 located upstream from the HPT coding sequence.
Driven by the 5S promoter (Guilley et al. 1982). Plasmids pBII221 and pHYGI1 were also used in this example.

PLUC-1 contains a firefly luciferase coding sequence (Dewet et al. 1987), which has a CaMV35S promoter attached at the 5 'end and a nos polyadenylation sequence attached at the 3' end. doing. This plus is used primarily as a negative control DNA. Plasmids were released by microprojectile bombardment
It was introduced into B12. 26 plates were prepared in the same manner as described in Example I. One tube contains only plasmid pBII221; two tubes contain pHY
Contains both GI1 and pBII221 plasmids; and one tube contains only plasmid pLUC-1.
The implants performed are summarized in Table 15.

[0190]

[Table 15]

Two plates of calli bombarded with pBII221 were assayed for transient GUS expression. When the number of blue cells was counted, 291 were counted in two plates.
And 479 transient GUS-expressing cells, suggesting that the DNA release process was taking place for the other implantation plates.

Immediately after all the samples were implanted, p
HYGII / pBII221, pCHN1-1 / pBII22
Calli from all of the plates treated with 1 and 3 of the plates treated with pLUC-1 were added to round 1 selection plates, F medium (5 pieces per plate) containing 15 mg / l hygromycin B per plate. Moved. Calli from the fourth plate treated with pLUC-1 were transferred to F medium without hygromycin. This tissue was used as a non-selective control callus.

After two weeks of selection, the tissues looked essentially the same on both selective and non-selective media. pHYGI1 / pBII221 and pH
Eight plates from each of the N1-1 / pBII221 treatments and all calli from two plates of control calli on selective media were transferred from round 1 selection plates to round 2 selection plates containing 60 mg / l hygromycin. Moved to Round 2 selection plates each have 30 calli per plate
Contains 10 mg pieces, which results in an increase in the total number of plates.

The remaining tissues on the selective medium, ie, pHYGI1 / pBII221 and pCHN1-
Two plates of 1 / pBII221-treated tissue and one of the control KS Ruth were placed in GUS assay buffer at 37 ° C. to determine if blue cell clusters were observed two weeks after implantation. . After 6 days in assay buffer, the tissues were counted for GUS expression. The results are summarized in Table 16.

[0195]

[Table 16]

After 21 days on round 2 selection plates, all surviving portions of the material were
Transferred to round 3 selection plates containing 60 mg / l hygromycin. Round 2 selection plates containing only apparently dead cells were preserved. Round 2 and 3 selection plates were periodically monitored for viable growth sectors.

After 35 days on round 3 selection plates, the live sectors of callus on both round 2 and 3 selection plate sets were examined. For these two sectors, growth from the background of dead tissue on plates treated with pHYGI1 / pBII221 was observed.

The first sector, designated 3AA, was obtained from round 3 groups of plates. Both lines were then transferred to F medium without hygromycin. After 19 days on F medium without hygromycin, the 3AA line grew very little while the pH1 line grew rapidly. Both lines were again transferred to F medium for 9 days. 3AA
And the pH1 series were then transferred to F medium containing 15 mg / l hygromycin for 14 days. At this point, the 3AA line was of very poor quality and slow growing. But p
The H1 line grew rapidly on F medium containing 15 mg / l; this line was then subcultured to F medium without hygromycin.

After 10 days in F medium, a pH1 series inhibition test was started. Hygromycin B 1, 10, 30,
Calli at pH 1 were transferred onto F medium containing 100 and 250 mg / l. Five callus plates of each concentration were made, each plate containing 10 pieces of callus of about 50 mg. One plate of unselected control tissue was made for each concentration of hygromycin.

The pH1 series includes hygromycin 0, 1
It was found to be capable of sustaining growth over 9 passages at 0, 30, 100 and 250 mg / l concentrations. Supplementary sectors were collected from round 2 and 3 selection plates at various time points. None of these could be successful in the presence of hygromycin in multiple rounds, ie, two or three subcultures.

To demonstrate that the pH1 callus had taken up the hygromycin resistance gene, DNA was made from the pH1 isolate and a non-selective control callus was prepared by freezing 2 g of callus in liquid nitrogen and adding 6 ml of it. Extraction buffer (7M urea,
250 mM sodium chloride, 50 mM Tris-HCl pH
Transferred to a 30 ml Oak Ridge tube containing 0.0, 20 mM EDTA pH 0.0, 1% chalcosine) to make a fine powder. To this, 7 ml of phenol: chloroform 1: 1 was added, and the tube was shaken and cultured at 37 ° C. for 15 minutes. Sample at 4 ° C
For 10 minutes at 8K. The supernatant was pipetted through Miracloth (Calbiochem 475855) into a disposable 15 ml tube (American Scientific product C3920-15A) containing 1 ml of 4.4 M ammonium acetate, pH 5.2. 6 ml of isopropanol
Was added, the tubes were shaken, and the samples were incubated at -20 ° C for 15 minutes. DNA was pelleted at 4 ° C. for 5 minutes at maximum speed in a Beckman TJ-6 centrifuge. The supernatant is discarded and the pellet is washed with 500 μl of TE-10 (10
mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0)
For 15 minutes at room temperature. The sample was transferred to a 1.5 ml Eppendorf tube and 100 μl of 4.4 M ammonium acetate,
pH 5.2 and 700 μl of isopropanol were added.
This was cultured at −20 ° C. for 15 minutes, and the DNA was pelletized by microcentrifugation (12,000 rpm) in Eppendorf for 5 minutes. The pellet was washed with 70% ethanol, dried, and dried with TE-1 (10 mM Tris-HCl pH 8.0, 1 mM E
DTA).

[0202] 10 µl of the separated DNA was added to RAmHI (N
ER) and analyzed using the HPT probe as described in the Examples. As shown in FIG. 5, a band of pHI callus was observed at the expected 1.05 kb position, indicating the presence of the HPT coding sequence. No bands were found in the control calli.

The hygromycin gene has a high molecular weight DNA
DNA isolated from the pHI callus and the control callus is not treated with restriction enzymes, ii) treated with BamHI as described above, to demonstrate that
Or iii) the plasmid p in the HPT coding sequence
It was treated with PstI, which cuts only HYGI1. Samples were blotted and verified with the HPT coding sequence as described above. Only undigested pHI DNA hybridized to the probe at the position of the uncleaved DNA, demonstrating that the hygromycin gene was incorporated into the high molecular weight DNA. PHIDN digested with BamHI
The expected 1.05 kb band of A was observed as previously shown. If the hygromycin gene is intact pHYG
If present in the I1 plasmid, one would expect the appearance of a 5.9 kb band of pHI DNA digested with PstI. If the gene is incorporated into high molecular weight DNA, the appearance of two or more bands of variable size (the size depends on the location of the attachment [Frankinda] PstI in the host DNA) Should be expected. Three types of bands having molecular weights of about 12, 5.1 and 4.9 kb were observed. This result demonstrates that the hygromycin gene has been incorporated into high molecular weight DNA. The intensity of the 4.9 kb band is approximately twice as strong as the other two bands, suggesting a potential for partial digestion or double-headed repeats of the HPT gene. No hybridization was observed with DNA from control calli by any of the treatments described above.

These results indicate that the HPT coding sequence is not present in pHI calli, as in intact pHYGI1 or a small non-chromosomal plasmid. They consist of the hygromycin gene incorporated into high molecular weight DNA.
In addition, Southern blot analysis revealed that the HPT coding sequence was adjacent to the 35S promoter sequence. The pHI calli were transferred from all the concentrations of hygromycin used in the inhibition test on RMS medium and the plants were regenerated as described in the examples. 65
Of the plants were produced from pHI and 30 were produced from control calli.

To demonstrate that the introduced DNA was retained in the RO tissue, three randomly selected pHI ROs were
Southern blots as described above were applied to BamHI-digested leaf DNA from plants. Blots were probed with the HPT probe as in Example I. As shown in FIG. 6, a 1.05 kb band was observed in all three plants.
The presence of the PT coding sequence was indicated. No bands were seen for DNA from control plants.

The controlled pollination of mature pHI plants was carried out by syngeneic Zea mays lines A188, B73 and Oh4.
3. Performed according to standard method. The seeds were collected on the 45th day after pollination and dried for an additional 1-2 weeks.

The presence of hygromycin resistance properties in RI progeny was assessed by a root elongation bioassay, a yellow leaf bioassay and a Southern blotting procedure. Regenerated pHI
And two ears each from control plants were selected for analysis. The results are shown in FIG. 7 and Tables 17 and 18. The presence of the HPT gene was confirmed in two R2 progeny obtained from maternal parents of pHI.

Plant pHI3-1 (Tables 17 and 18)
Pollinated with OH43 pollen. Nine species from this cross were germinated and DNA was produced from four leaves of progeny plants and analyzed by Southern blotting using HPT coding sequence probes. Two of the four plants tested contained HPT sequences at high copy numbers. The HPT gene was present in two plants, but its expression was not observed in the yellow leaf analysis.

[0209]

[Table 17]

[0210]

[Table 18]

The plant pHI 10.8 (Tables 17 and 18)
Used to pollinate B73 and was self-pollinated. Fifty of the offspring from allogeneic control were tested for HPT expression in both root and leaf analysis. No expression was observed. Similarly, Southern blotting performed on DNA from eight offspring did not detect the HPT gene. For self-pollinated progeny, no evidence of gene presence was obtained from leaf analysis of nine progeny or from Southern blots of DNA from four of these plants.

Recombinant DNA was shown to be passed on to progeny only when transformed plants (both RO and RI) were used as females. This is the pHI recombinant D
It suggests maternal inheritance of NA.

[0213] All the publication contents and patent documents cited herein are included in the literatures here. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many changes and modifications may be made while remaining within the spirit and scope of the invention.

[Brief description of the drawings]

FIG. 1. Plasmid vector-pH used in Example I
3 shows a map of YGI1.

FIG. 2 depicts the relevant portion of linearized pHYGI1 including the HPT coding sequence and related regulatory components. This base pair number starts at the 5 'nucleotide of the indicated restriction enzyme recognition sequence and starts at the EcoRI site at the 5' end of the CaMV 35S promoter.

FIG. 3. Plasmid vector-pB used in Example I
11 shows a map of II221.

FIG. 4. Linearized pBII221, eg, HindI
The region from the II cleavage site to the EcoRI cleavage site is depicted.

FIG. 5. Southern blotting of DNA isolated from PH1 and non-transformed control callus strains,
FIG. 2 is an illustration of the pHYGI1 probe used in the assay.

FIG. 6. R regenerated from PH1 and untransformed callus
1 is an illustration of a pHYGI1 probe used for Southern blotting and assay of leaf DNA isolated from O plants.

FIG. 7 is an illustration of the pHYGI1 probe used for Southern blotting and assay of DNA of leaves isolated from progeny of PH1 RO plants and untransformed RO plants.

FIG. 8: Southern blotting of DNA isolated from PH2 callus strain and untransformed control callus strain,
FIG. 2 is an illustration of the pHYGI1 probe used in the assay.

FIG. 9: Plasmid vector-pZ used in Example II
27 shows a map of 27Z10.

FIG. 10 depicts the linearized plasmid pZ27Z10 containing the z10 coding sequence and related regulatory elements.

FIG. 11 is an illustration of the location of PCR primers within the chimeric z27-z10 gene.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Walters, David A United States 55437 Minnesota, Bloomington, Kel Road 11424 (72) Inventor Kilihara, Julie A United States 55425 Minnesota, Bloomington, Sixteenth Avenue South 9745

Claims (20)

[Claims] 1. A recombinant DN integrated and expressed on a chromosome.
A. A fertile, transgenic Zea mays plant comprising A, wherein the recombinant DNA is transmitted to its progeny via the plant's full sexual cycle. 2. The plant of claim 1, wherein said recombinant DNA comprises a promoter. 3. The plant of claim 1, wherein said recombinant DNA encodes a protein. 4. The plant of claim 3, wherein the expressed transgenic recombinant gene encoding the seed storage protein has an increased amount of effective amino acids. 5. The recombinant gene encodes a 10 kD corn seed protein, and the total nuclear methionine concentration is the corresponding Z in the absence of the recombinant gene.
5. The plant of claim 4, wherein said recombinant gene is expressed such that it is substantially increased above the total nuclear methionine concentration in the ea mays plant. 6. The recombinant gene of claim 1, wherein said recombinant gene expresses a selectable marker gene or reporter gene.
Plants. 7. The plant of claim 6, wherein said selectable marker gene confers resistance or tolerance to a herbicide. 8. A compound selected from the group consisting of hygromycin, 2,2-dichloropropionic acid, phosphinothricin, glyphosate, imidazolinone herbicide, sulfonylurea herbicide and triazoloprimidine herbicide. 7. The plant of claim 6, wherein the possible marker gene confers resistance or resistance. 9. The plant of claim 8, wherein said selectable marker gene confers resistance or resistance to hygromycin. 10. The plant according to claim 6, which expresses a reporter gene. 11. The method according to claim 11, wherein the recombinant DNA is a bacterial dap A.
Gene, Bt-endotoxin gene, protease inhibitor gene, bacterial ESPS synthase gene, chitinase gene, glucan-endo-1, 3-β glucosidase gene, structural DNA, regulatory sequence, discriminating sequence and sequence encoding non-transcribed RNA Done D
2. The plant of claim 1, consisting of an NA sequence. 12. A seed produced by the plant according to claim 1, 4, 6, or 11, which inherits the recombinant gene. 13. An RI and a later generation derived from the plant of claim 1, 4, 6 or 11. 14. The recombinant DNA of claim 1, wherein said recombinant DNA was introduced into said plant or an ancestor of said plant at an embryonic stage.
Plants. 15. The plant of claim 14, wherein said recombinant DNA has been introduced into said plant by microprojectile bombardment. 16. The plant of claim 15, wherein said recombinant DNA has been introduced by callus culture microprojectile bombardment. 17. A method for producing a fertile, transgenic Zea mays plant that stably expresses a chromosomally integrated heritable recombinant DNA, comprising: i) a Zea mays plant to be transformed. Ii) transforming the cells of said culture by bombarding said cells with a recombinant DNA-coated microprojectile; iii) transforming said transformed callus culture Identifying or selecting cells; and iv) a fertile, transgenic Zea mays that stably expresses the recombinant DNA that is inheritable.
Regenerate the plant; a method comprising the steps. 18. The method of claim 17, wherein said cell culture is a fragile embryo callus culture initiated from an immature hybrid embryo. 19. The method of claim 18, wherein said callus culture subjected to bombardment is in a group of about 30 to 80 mg per group. 20. The callus is initiated in a solid medium,
The method of claim 19.