Methods for Agrobacterium-mediated transformation Number:6,822,144 from the United States Patent and Trademark Office (PTO) owispatent

Title: Methods for Agrobacterium-mediated transformation

Abstract: The present invention relates to methods for improving the transformation frequency of Agrobacterium-mediated transformation of maize embryos. A preferred method for transforming maize using Agrobacterium comprises the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to said embryo; co-cultivating the embryos with Agrobacterium; culturing the embryos in medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene.

Patent Number: 6,822,144 Issued on 11/23/2004 to Zhao,   et al.

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Inventors:	Zhao; Zuo-Yu (Urbandale, IA); Gu; Weining (Urbandale, IA); Cai; Tishu (Urbandale, IA); Pierce; Dorothy A. (Urbandale, IA)
Assignee:	Pioneer Hi-Bred International, Inc. (Des Moines, IA)
Appl. No.:	963096
Filed:	November 3, 1997

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	788018	Jan., 1997	5981840

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Current U.S. Class:	800/320.1 ; 435/412; 435/419; 435/424; 435/430.1; 435/469; 800/294
Current International Class:	C12N 15/82 (20060101)
Field of Search:	800/294,320.1,300,300.1,301,302,303 435/412,419,424,430,430.1,469

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References Cited [Referenced By]

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U.S. Patent Documents

	5164310		November 1992		Smith et al.
	5177010		January 1993		Goldman et al.
	5384253		January 1995		Krzyzek et al.
	5484956		January 1996		Lundquist et al.
	5569597		October 1996		Grimsley et al.
	5591616		January 1997		Hiei et al.
	5595733		January 1997		Carswell et al.
	5932782		August 1999		Bidney
	5981840		November 1999		Zhao et al.
	6020539		February 2000		Goldman et al.

Foreign Patent Documents

	586 355		Mar., 1994		EP
	604 662		Jul., 1994		EP
	672 752		Sep., 1995		EP
	687 730		Dec., 1995		EP
	2 211 204		Jun., 1989		GB
	4-222527		Aug., 1992		JP
	WO 91/02071		Feb., 1991		WO
	WO 92/06205		Apr., 1992		WO
	WO 92/09696		Jun., 1992		WO
	WO 95/10178		Apr., 1995		WO

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Narasimhulu et al., Early Transcription of Agrobacterium T-DNA Genes in Tobacco and Maize, The Plant Cell, 8: 873-886 (1996). . Ritchie et al., "Agrobacterium tumefaciens-mediated expression of gusA in maize tissues", Transgenic Research, 2: 252-295 (1993). . Schlappi et al., "Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer", The Plant Cell, 4: 7-16 (1992). . Shen et al., "T-DNA transfer to maize cells: Histochemical investigation of .beta.-glucuronidase activity in maize tissues", Proc. Natl. Acad. Sci. USA, 90: 1488-1492 (1993). . Shen et al., "Excision of Ds1 from the genome of maize streak virus in response to different transposase-encoding genes", Plant Molecular Biology, 36: 387-392 (1998). . Songstad et al., "Establishment of friable embryogenic (type II) callus from immature tassels of Zea mays (poaceae)" American Journal of Botany, 79-7: 761-764 (1992). . 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Primary Examiner: Fox; David T.
Attorney, Agent or Firm: Pioneer Hi-Bred International, Inc.

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application U.S. Ser. No. 08/788,018 filed Jan. 24, 1997, now U.S. Pat. No. 5,981,840.

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Claims

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What is claimed is:

1. A stably transformed PHN46 or PHJ90 inbred maize plant wherein said PHN46 or PHJ90 inbred maize plant has been transformed by contacting an immature embryo with Agrobacterium comprising a nucleic acid of interest.

2. A transgenic seed produced by the plant of claim 1.

3. A transformed PHN46 or PHJ90 maize cell produced by a method comprising the steps of: a) contacting at least one immature embryo from a PHN46 or PHJ90 maize plant with Agrobacterium capable of transferring at least one gene to the embryo; b) co-cultivating the embryo with Agrobacterium; c) culturing the embryo in a medium comprising N6 salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene.

4. A transformed PHN46 or PHJ90 make cell produced by a method comprising the steps of: a) contacting at least one immature embryo from a PHN46 or PHJ9 maize plant with Agrobacterium capable of transferring at least one gene to the embryo; b) co-cultivating the embryo with Agrobacterium; c) culturing the embryo in a medium comprising salts other than MS salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium and a selective agent to select for embryos expressing the gene.

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Description

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FIELD OF THE INVENTION

This invention relates to methods for plant tissue culture and plant regeneration and in particular this invention relates to methods for transforming maize using Agrobacterium.

BACKGROUND OF THE INVENTION

Agrobacterium-mediated transformation methods have been used principally in dicotyledonous plants. Agrobacterium-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition, Agrobacterium-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.

Monocotyledons are not a natural host of Agrobacterium. Although Agrobacterium-mediated transformation has been reported for asparagus (Bytebier B., et al. Proc. Natl. Acad Sci. USA 84:5354-5349, 1987) and for Dioscore bublifera (Schafer et al. Nature 327:529-532, 1987), it was generally believed that plants in the family Gramineae could not be transformed with Agrobacterium (Potrykus I. Biotechnology 8:535-543, 1990).

Grimsley et al. (Nature 325:177-179, 1987) reported that cDNA from maize streak virus could be delivered to maize plants by Agrobacterium tumefaciens and that the plants became infected with the virus. The research did not demonstrate that the cDNA reached the maize genome nor did it demonstrate stable integration of streak virus nucleic acid. Later studies demonstrated that Agrobacterium could be used to deliver a kanamycin-resistance gene and a GUS (.beta.-glucuronidase) gene to shoot apices of maize after shoot apex injury (Gould J. et al. Plant Physiol. 95:426-434, 1991 and U.S. Pat. No. 5,177,010 to Goldman et al.). In these studies plants generated from the tissue exposed to Agrobacterium contained both transformed cells and, non-transformed cells suggesting that the method did not uniformly deliver nucleic acid to the maize tissue.

European Patent Application Publication Number 604 662 A1 to Hiei et al. discloses a method for transforming monocotyledons using Agrobacterium. In this method, plant tissues were obtained from the monocotyledon maize and the tissues were exposed to Agrobacterium during the tissue dedifferentiation process. Hiei et al. disclose a maize transformation protocol using maize calli. Saito et al. disclose a method for transforming monocotyledons using the scutellum of immature embryos (European Application 672 752 A1). Ishida et al. also disclose a method specific for transforming maize by exposing immature embryos to A. tumefaciens (Nature Biotechnology, 1996, 14:745-750). The methods were optimized for inbred A188 maize lines. Transformation frequencies ranged from 12% to 30% at their highest for immature embryos from A188 lines that were 1.0-1.2 mm in length. Maize lines derived from crosses of A188 had significantly lower transformation frequencies ranging from 0.4% to about 5.3%. The transformation frequencies using A188 and A188 crosses are summarized in Table 1. A188 is not generally considered a commercially useful line and Ishida et al. failed to obtain recovery of stable transformants in lines other than those containing A188.

A need still exists for a method that will: (a) produce significantly higher transformation frequencies in lines other than those reported by Ishida et al. (supra); and, (b) produce transformed inbred lines other than line A188; including transformed inbreds representing a range of genetic diversities and having significant commercial utility.

SUMMARY OF THE INVENTION

This invention relates to methods for optimizing Agrobacterium-mediated transformation in maize. Significantly higher transformation frequencies for genotypes such as the product of A188 crossed to other inbreds would result in a higher throughput for production of transformed plants. This increased frequency would be useful, for example, to evaluate the efficacy of a larger number of genes in transgenic plants of corn or to generate a larger number of transgenic plants containing a particular foreign gene in a given period of time. Similarly, methods permitting the transformation of a variety of inbred lines would be commercially valuable.

In one aspect of this invention, the invention relates to a method for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating maize plants expressing the gene. In one embodiment the contacting step additionally comprises the step of contacting the immature embryos with Agrobacterium in a medium comprising N6 salts and in another embodiment the contacting step additionally comprises contacting the immature embryos with Agrobacterium in a medium comprising MS salts. Preferably the contacting step takes place in the absence of AgNO.sub.3. In one embodiment the embryos are cultured in a PHI basic media system and in another embodiment the embryos are cultured in a PHI combined media system. The immature embryos used in the method are preferably about 0.3 mm to about 4 mm in length and more preferably about 0.8 mm to about 2.0 mm in length. The Agrobacterium concentration used in the contacting step is preferably about 1.times.10.sup.8 cfu/ml to about 1.5.times.10.sup.9 cfu/ml and more preferably about 0.5.times.10.sup.9 to about 1.0.times.10.sup.9 cfu/ml. The contacting step preferably takes place in a liquid suspension and the co-cultivation step preferably takes place on a solid medium. Preferably, a medium containing MS salts is used in the regeneration step. In a preferred embodiment of this invention the method includes a resting step that comprises culturing the embryos in medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. Preferably the embryos are cultured for about 1 to about 15 days. In one embodiment the antibiotic used is carbenicillin and a preferred concentration of carbenicillin is about 50 mg/l to about 250 mg/l. This method also relates to maize plants transformed by this method and to maize cells transformed by this method.

In another aspect of this invention, the invention relates to a method for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to said embryo in a medium comprising N6 salts; co-cultivating the embryo with Agrobacterium in a medium comprising N6 salts; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene in a medium comprising MS salts. Preferably, the medium of the contacting step lacks AgNO.sub.3 and the medium of the co-cultivating step includes AgNO.sub.3. Preferably the Agrobacterium concentration used in the contacting step is about 1.times.10.sup.8 cfu/ml to about 1.5.times.10.sup.9 cfu/ml. Preferably, the contacting step takes place in a liquid and the co-cultivating and culturing steps take place on a solid medium. In one embodiment of this method, the method additionally comprising the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. Preferably the antibiotic is carbenicillin. This invention also relates to maize plants and to maize cells transformed by this method.

In yet another aspect of this invention, a method is disclosed for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to said embryo in a medium comprising N6 or MS salts; co-cultivating the embryo with Agrobacterium in a medium comprising MS salts; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene in a medium comprising MS salts. Preferably the medium of the contacting step lacks AgNO.sub.3 and the method of the co-cultivating step includes AgNO.sub.3. Also preferably, the contacting step takes place in a liquid and the co-cultivating and culturing steps take place on a solid medium. In one embodiment of this method, the method additionally comprising the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of Agrobacterium.

This invention also relates to a method for optimizing the production of transgenic maize plants of a first genotype using Agrobacterium-mediated transformation comprising the steps of: isolating immature embryos from maize; separating the embryos into treatment groups; incubating each treatment group separately in a medium comprising N6 or MS salts and in a suspension of Agrobacterium at concentrations ranging from about 1.times.10.sup.8 cfu/ml to about 1.times.10.sup.10 cfu/ml; co-cultivating the embryos with Agrobacterium on a solid medium; culturing the embryos in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos transformed by Agrobacterium; identifying the treatment group with the highest transformation frequency ; and using the concentration of Agrobacterium generating the highest transformation frequency to transform other embryos from the first genotype. In one embodiment of this method, the medium of the incubating step and the co-cultivating step is a medium comprising N6 salts and in another embodiment of this method, the medium of the incubating step is a medium comprising MS salts and the medium of the co-cultivating step is a medium comprising N6 salts. In yet another embodiment, medium of the incubating step is a medium comprising N6 salts and the medium of the co-cultivating step is a medium comprising MS salts. The method also preferably includes the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. Preferably the antibiotic is carbenicillin and preferably, the combined length of the co-cultivating step and the resting step is at least three days. Where a resting step is used, the length of the resting step is from more than 0 to about 10 days. In a preferred embodiment, the length of the resting step is about 3 to about 5 days.

In another aspect of this invention, the invention relates to transformed maize plants produced by a method comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene.

In yet another aspect of this invention, the invention relates to transformed maize cells produced by a method comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; and culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene: and identifying embryos expressing the gene.

In a preferred aspect of this invention, the invention relates to a method for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; culturing the embryo in a medium containing salts other than MS salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C provide a diagram illustrating the construction of a preferred vector of this invention. FIG. 1(a) diagrams the exemplary gene segments incorporated into the exemplary vectors used in a preferred method of this invention. FIG.1(b) illustrates plasmid pPHP8904 incorporating the exemplary gene segments. FIG. 1(c) illustrates plasmid pPHP10525.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The development of maize hybrids requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations. Breeding programs combine the genetic backgrounds from two or more inbred lines or various other broad-based sources into breeding pools from which new inbreds are developed by selfing and selection of desired phenotypes. The new inbreds are crossed with the inbred lines and the hybrids from these crosses are evaluated to determine which of those have commercial potential.

Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all of the desired characteristics, other sources can be included in the breeding population. A single cross hybrid maize variety is the cross of two inbred lines, each of which has a genotype that complements the genotype of the other. The hybrid progeny of the first generation is designated F.sub.1. In the development of hybrids, only the F.sub.1 hybrid plants are sought. Preferred hybrids are more vigorous than their inbred parents. This hybrid vigor, or heterosis, can be maintained in many polygenic traits, including increased vegetative growth and increased yield.

The development of a hybrid maize variety involves three steps: (1) selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, that, although different from each other, are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrid progeny (F.sub.1). Inbred lines produced in such a breeding program naturally fall into what are termed different heterotic groups. Maximal heterosis, or hybrid vigor, is typically produced by crossing two inbreds, each from a different heterotic group. At least several distinct heterotic groups can be identified, with numerous inbreds belonging to each heteotic group. A significant amount of research and development goes into the identification and recovery of inbred lines of commercial importance. For example, some 400-500 new inbred lines may be proposed by a single seed corn corporation each year as a result of over 2,000,000 pollinations. Of those proposed new inbreds, fewer than 50 and more commonly fewer than 30 are selected for commercial use. Those inbred lines that are used in commercially important hybrids are considered to be "elite" inbred lines. Not only is there a need to directly transform inbred lines that are commercially important for the hybrid corn market but there is also a need for those inbreds to cover a wide range of genetic diversity.

A188 is a useful genotype for the development of corn transformation methods, since it is known to be highly responsive in producing a friable type of embryogenic callus that lends itself to tissue culture (Phillips, R. L. et al. "Cell/Tissue Culture and In Vitro Manipulation" pp. 345-387, in Corn and Corn Improvement, G. F. Spraque and J. W. Dudley, eds., American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc. 1988 and Armstrong, C. L. et al., Maize Genet. Coop. News Letter 59:92-93, 1985). However, A188 is no longer generally considered to be a useful inbred parent of commercial hybrid corn. A188 is not used directly in any commercial hybrid and is a poor starting material for backcrossing into inbreds used as parents of commercial hybrids. Use of non-elite starting material for back crossing will in some cases delay release of the final commercial hybrid by one to two years. In addition, with non-elite starting material, there is a higher risk of negative genetic effects on the final hybrid product. Therefore, the ability to transform only A188-containing lines is of limited value to the commercial hybrid corn market.

Although Hiei et al. and Ishida et al., both supra, were successful in using Agrobacterium to transform A188-containing maize lines, non-A188 inbred lines could not be transformed using this method (Ishida et al. Nature Biotechnology 14:745-750, 1996). Some of the non-A188 inbreds tested included lines that were useful for hybrid field corn breeding. A significant need still exists for methods to transform non-A188 inbred lines, including inbred lines that are commercially important for the hybrid corn market. The commercial corn market includes a wide range of hybrids with different genetic backgrounds and successful inbred breeding programs need to cover a wide range of genetic diversity.

High efficiency transformation of maize is important in analyzing the usefulness of any of a variety of genes in transgenic corn plants. High efficiency transformation of maize is also important because large numbers of transgenic plants are needed to study the effect of a particular gene within a given period of time. The ability to directly transform agronomically important inbreds at a usable frequency and across a wide range of genetic diversity is important for the development of commercial hybrid seed products with improved traits including, but not limited to, insect resistance, disease resistance, herbicide resistance, increased yield, increased tolerance to environmental stresses (such as drought, heat, etc.), enhanced seed quality (such as increased or modified starch, oil and/or protein content), and the like.

Although non-Agrobacterium-mediated transformation methods are known, including, but not limited to, particle bombardment, electroporation and silicon carbide fiber-mediated transformation (Songstad, D. D., et al. Plant Cell, Tissue and Organ Culture 40:1-15, 1995), the utility of these methods is limited because the methods produce low transformation frequencies and/or because the methods may only be useful for a restricted number of genotypes. For example, transformation frequencies by bombardment have been reported to be less than 2% for the "Hi-II" genotype used in the present invention (Songstad, D. D. et al. In Vitro Cell. Dev. Biol. Plant 32:179-183, 1996), even though Hi-II is one of the more responsive and efficient maize genotypes in tissue culture. Protoplast systems have also been used for transformation studies including electroporation and polyethylene glycol (PEG)-mediated methods for nucleic acid uptake. Reports indicate that maize protoplast systems can show good transformation frequencies (Donn, G., in Abstracts of the VIIth International Congress on Plant Cell and Tissue Culture, IAPTC, A2-38, p. 53, 1990). The protoplast genotype used in this study was a specially derived complex synthetic maize genotype, He/89, that demonstrated good regeneration capability and had low rate of plant abnormalities (Morocz, S., et al. Theoretical and Applied Genetics 80:721-726, 1990). However, aside from the specialty lines such as HE89, most genotypes, including agronomically important genotypes are not very amenable to the use of protoplast targeted transformation methods. Consequently, Wilson et al. (in Transformation of Plants and Soil Microorganisms, Wang, Herrera-Estrella et al. eds. Cambridge University Press, p. 65-80, 1995) conclude that "genotype constraints and the reduced vigor and fertility of plants regenerated from protoplasts probably outweigh the benefits of protoplasts as recipients for the integration of foreign DNA." Only a limited number of genotypes are amenable to the use of protoplasts for transformation and the quality of the plant produced from protoplast culture is often not as good as the quality of the plant produced from other transformation systems.

Ishida et al. (supra) discuss a method for transforming inbred A188 embryos and F.sub.1 embryos derived from crosses of A188 with other inbred lines through the co-cultivation of the embryos with Agrobacterium tumefaciens using superbinary vectors. Table 1 of that publication summarized the frequencies for maize transformation obtained in that study. Maximum transformation frequencies reached 30.6% for A188 but the transformation frequency only reached 5.3% for embryos derived from crosses of A188 and the average transformation frequency for A188 was about 15%. Non-A188-containing lines could not be transformed by these methods. The transformation frequencies using embryos derived from A188 and F.sub.1 embryos from crosses of A188, as reported in Table 1 of the Ishida et al paper, are summarized in Table 1 below. The transformation frequencies of F.sub.1 embryos ranged from 0.4-5.3% and was defined as the proportion of total embryos which produced GUS expressing (GUS+) plants. In this table, and as used herein, the term "GUS+" refers to transgenic events where GUS gene expression can be detected.

TABLE 1 Transformation of A188 and crosses by Ishida et al The results in this Table were reported in Ishida et al., (Nature Biotechnology 14:745-750, 1996). GUS+ plants are those which showed positive staining for expression of the GUS gene. Number of immature embryos Callus Plants growing regen- Experi- Inoc- on erated GUS+ Fre- ment ulated herb- on herb- plants quency Variety No. (A) icide icide (B) B/A.sub.1 %) A188 1 44 28 9 6 13.6 2 52 33 10 7 13.5 3 51 46 13 7 13.7 4 70 56 26 14 20.0 5 76 30 12 9 11.8 6 369 200 71 44 11.9 7 121 46 33 20 16.5 8 27 15 8 5 18.5 9 36 26 18 11 30.6 10 77 38 32 16 20.8 W117xA188 1 112 36 8 4 3.6 2 114 26 10 6 5.3 W59ExA188 1 104 44 1 1 1.0 A554xA188 1 247 46 7 5 2.0 W153RxA188 1 284 69 2 1 0.4 H99xA188 1 219 18 4 3 1.4

The present invention, while complementing the work of Ishida et al., provides an improved method for generating a significant increase in Agrobacterium-mediated transformation frequency for A188-containing lines and for successfully transforming non-A188 inbreds across a wide range of genotypes. In this invention, methods described by Ishida et al. (including the same source of A188 and the same vector) were used to transform A188 lines to demonstrate that the Ishida et al. methods could be reproduced in a different laboratory, on the same A188 genotype. The results of these initial transformation studies are provided in Example 2 and the data are summarized in Table 3. The results of these experiments produced transformation frequencies similar to those reported by Ishida et al. and ranged from about 9% to about 18% for A188 transformation in four separate experiments.

The methods of Ishida et al. were then used to transform a genotype termed Hi-II. This provided a baseline for transformation frequencies that could be used as a comparison with the transformation protocols of this invention. Hi-II is similar to the A188 .times. inbred crosses used by Ishida et al. (i.e., those listed in Table 1) to the extent that Hi-II is derived from both A188 and a non-A1 88 inbred, B73 (Armstrong et al. Maize Genetics Cooperation Newsletter 65:92-93, 1991). Details on the derivation of Hi-II and the results of the Hi-II transformation studies using the Ishida et al. method, are provided in Example 3. The data are summarized in Table 4. The results of these experiments show that transformation frequencies obtained for Hi-II, using the protocols of Ishida et al., ranged from 0.8 to 7.1% and were also in the same general range of transformation frequencies as those obtained by Ishida et al. for A188 inbred crosses. The results reported in Example 2 and Example 3 demonstrated that the method of Ishida et al. was reproducible by others. These results allow comparisons to be made between the new Agrobacterium-mediated transformation methods of this invention and those reported in the literature.

The preferred Agrobacterium-mediated transformation process of this invention differs from that of Ishida et al. and Hiei et al. in several respects and can be broken into several steps.

As will be discussed in more detail below, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium (step 1; the infection step). In this step the immature embryos are preferably immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2; the co-cultivation step). Preferably the immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Preferably the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4; the selection step). Preferably, the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5; the regeneration step) and preferably calli grown on selective medium are cultured on solid medium to regenerate the plants.

Infection Step

As a first step for practicing this invention, immature embryos are isolated from maize and exposed to Agrobacterium. Immature embryos are an intact tissue that is capable of cell division to give rise to callus cells that can then differentiate to produce tissues and organs of a whole plant. Immature embryos can be obtained from the fertilized reproductive organs of a mature maize plant. Exemplary methods for isolating immature embryos from maize are described by Green and Phillips (Crop Sci. 15:417-421, 1976). Maize immature embryos can be isolated from pollinated plants, as another example, using the methods of Neuffer et al. ("Growing Maize for genetic purposes." In: Maize for Biological Research W. F. Sheridan, Ed., University Press, University of North Dakota, Grand Forks, N.Dak. 1982.). Another method is provided in Example 4. The immature embryos are preferably used at approximately 6 days to about 20 days after pollination, more preferably about 7 days to 18 days after pollination, still more preferably about 8 days to 16 days after pollination, and in a particularly preferred embodiment about 9 days to about 12 days after pollination. Preferably, the embryos exposed to Agrobacterium range from about 0.3 to 4 mm in size, more preferably about 0.6 to 3.0 mm, still more preferably about 0.8 to 2.0 mm and in a particularly preferred embodiment about 1.0 mm to about 1.2 mm in size. Immature embryos are preferably aseptically isolated from the developing ear and held in sterile medium until use.

The Agrobacterium used to transform the embryos is modified to contain a gene of interest. Preferably the gene is incorporated into a gene vector, to be delivered to the embryo. A variety of Agrobacterium species are known and Agrobacterium species employed for dicotyledon transformation can be used. A number of references review Agrobacterium-mediated transformation in monocots and dicots. These include, among others, Hooykaas, P. J. (Plant Mol. Biol., 13:327-336, 1989); Smith, R. H. et al. (Crop Science, 35:301-309, 1995); Chilton, M. O. (Proc. Natl. Acad Sci. (USA), 90:3119-3210, 1993); and Moloney et al. In: Monograph Theor. Appl. Genet., N.Y., Springer Verlag 19:148-167, 1993).

Many Agrobacterium employed for the transformation of dicotyledonous plant cells contain a vector having a DNA region originating from the virulence (vir) region of the Ti plasmid. The Ti plasmid originated from Agrobacterium tumefaciens. Nucleic acid containing a gene encoding a polypeptide to be expressed in maize can be inserted into this vector. Alternatively, the gene can be contained in a separate plasmid which is then inserted into the Ti plasmid in vivo, in Agrobacterium, by homologous recombination or other equivalently resulting processes. A vector has also been developed which contains a DNA region originating from the virulence (vir) region of Ti plasmid pTiBo542 (Jin et al., 1987, J. Bacteriol. 169:4417-4425) contained in a super-virulent Agrobacterium tumefaciens strain A281 showing extremely high transformation efficiency. The plasmid containing the gene of interest was incorporated into the virulent Agrobacterium tumefaciens strain A281 since strain A281 is known to have a high transformation efficiency (see Hood, E. E. et al., 1984, Bio/Tech 2:702-709; Komari, T. et al., 1986, Bacteriol 166:88-94). This type of vector is known in the art as a "superbinary vector" (see European Patent Application 0 604662A1 to Hiei et al.).

Superbinary vectors are preferred vectors for the transformation methods of this invention. Exemplary superbinary vectors useful for introducing nucleic acid encoding polypeptide for expression in a maize plant via Agrobacterium-mediated transformation methods include the superbinary pTOK162 (as disclosed in Japanese Laid-Open Patent Application no. 4-222527). This vector includes regions that permit vector replication in both E. coli and A. tumefaciens. The plasmid includes a T-DNA region, characteristic of Ti plasmids. Nucleic acid containing a gene encoding a polypeptide to be expressed in maize is inserted in the T-DNA between the T-DNA borders. Other superbinary vectors are known and these vectors can similarly be incorporated into Agrobacterium (see e.g., Komari, T., Plant Cell Reports 9:303-306, 1990 for pTOK23).

Examples of genes useful for expression in transformed plant cells are known in the art. Exemplary genes include, but are not limited to, Bt genes or patatin genes for insect resistance; the Hm1 gene and chitinase genes for disease resistance; the pat, bar, EPSP syntase gene or ALS genes for herbicide resistance; genes encoding proteins with altered nutritional properties; genes encoding enzymes involved in starch or oil biosynthetic pathways; down-or up-regulatory sequences for metabolic pathway enzymes; and the like. As those of ordinary skill in the art will recognize, this is only a partial list of possible genes that can be used with the transformation method of the present invention. Furthermore, as those of ordinary skill in the art will also recognize, regulatory sequences including promoters, terminators and the like will also be required, and these are generally known in the art. Example 1 discloses the construction of a preferred superbinary vector pPHP10525. This vector contains virb, virC and virG genes isolated from superviral strain A281. The vector includes 35Sbar and ubi/GUS plant expression cassettes inserted between the T-DNA borders. Plant expression cassettes preferably comprise a structural gene to which is attached regulatory DNA regions that permit expression of the gene in plant cells. The regulatory regions consist at a minimum of a promoter capable of directing expression of a gene in a plant cell. The promoter is positioned upstream or at the 5' end of the gene to be expressed. A terminator is also provided as a regulatory region in the plant expression cassette and is capable of providing polyadenylation and transcription terminator functions in plant cells. The terminator is attached downstream or at the 3' end of the gene to be expressed. Marker genes, included in the vector, are useful for assessing transformation frequencies in this invention.

The nucleic acid encoding a polypeptide for expression in maize is inserted into the T-DNA region of the superbinary vector using suitable restriction endonuclease recognition sites, by homologous recombination, or the like. General molecular biological techniques used in this invention are provided, for example, by Sambrook, et al. (eds.) (Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the use of homologous recombination to incorporate nucleic acid into plasmids contained in Agrobacterium tumefaciens is disclosed by Herrera-Esterella, L. et al. (EMBO J. 2:987-995, 1983) and Horsch R. H. et al., (Science 223:496-498, 1984). The recombinant plasmid is selected in Agrobacterium based on the use of a selectable marker incorporated into the plasmid. Generally these markers are nucleic acid encoding proteins that typically confer antibiotic resistance.

Plasmids are introduced into Agrobacterium using methods known in the art, including the triple-cross method disclosed by Ishida et al. (supra) and in a preferred embodiment the plasmid is introduced into Agrobacterium using the method of Example 1.

Agrobacterium containing the plasmid of interest are preferably maintained on Agrobacterium master plates with stock frozen at about -80.degree. C. As used in this invention the term "Agrobacterium capable of transferring at least one gene" refers to Agrobacterium containing the gene of interest, generally in a plasmid that is suitable for mediating the events required to transfer the gene to the cells to be infected. In a preferred embodiment, the master plates are used to inoculate agar plates to obtain Agrobacterium which is then resuspended in media for use in the infection process as described in Example 2. Alternatively, bacteria from the master plate can be used to inoculate broth cultures that are grown to logarithimic phase prior to transformation.

The concentration of Agrobacterium used in the infection step and co-cultivation step can affect the transformation frequency. For example, while Agrobacterium can transform immature embryos of maize, very high concentrations of Agrobacterium may also damage the immature embryos and result in a reduced callus response. To optimize the transformation protocol for a particular maize line, immature embryos from the maize line can be incubated with various concentrations of Agrobacterium. Using the protocols provided in Examples 2-6, the level of marker gene expression and the transformation efficiency can be assessed for various Agrobacterium concentrations preferably within the concentration range of about 1.0.times.10.sup.8 cfu/ml to about 1.times.10.sup.10 cfu/ml. Table 6 in Example 4 demonstrated the effect of varying Agrobacterium concentration to optimize the Agrobacterium concentration for transformation. Using these methods, and those known in the art, concentrations of Agrobacterium in the infection and co-cultivation step that maximize the transformation frequency for a particular maize line can be identified without undue experimentation.

Preferably, Agrobacterium is used for transformations in a concentration range of about 1.times.10.sup.8 cfu/ml to about 1.times.10.sup.10 cfu/ml, more preferably within the range of about 1.times.10.sup.9 cfu/ml to about 1.5.times.10.sup.9 cfu/ml and still more preferably at about 0.5.times.10.sup.9 cfu/ml to about 1.0.times.10.sup.9 cfu/ml. Those skilled in the art will recognize that optimum Agrobacterium concentration ranges may vary for particular maize genotypes and for the particular Agrobacterium strain.

The isolated embryos are added to the Agrobacterium suspension in a liquid contact phase. Preferably the Agrobacterium concentration is selected based on methods disclosed herein to optimize transformation efficiencies. Example 4 provides a preferred method for contacting the embryos with Agrobacterium in a liquid. The contact phase facilitates maximum contact of the immature embryos with the suspension of Agrobacterium. Preferably the embryos are contacted with the suspension of Agrobacterium for a period of at least 5 minutes and preferably between 5 to 15 minutes and more preferably for about 10 minutes.

Preferably the liquid contact phase of the infection step takes place in a liquid solution that includes the major inorganic salts and vitamins of N6 medium referred to herein as "N6 salts" (Chu C. C. Proc. Symp. Plant Tissue Culture, Science Press Peking. pp.43-50, 1987). As used herein, medium containing "N6 salts" includes medium containing about 400-500 mg/l ammonium sulfate and preferably about 463.0 mg/l ammonium sulfate; about 1.0-2.0 mg/l boric acid and preferably about 1.6 mg/l boric acid; about 100-140 mg/l calcium chloride anhydrous and preferably about 125 mg/l calcium chloride anhydrous; about 20-50 mg/l Na.sub.2 -EDTA and preferably about 37.25 mg/l Na.sub.2 -EDTA; about 20-40 mg/l ferrous sulfate.7H.sub.2 O and preferably about 27.8 mg/l ferrous sulfate.7H.sub.2 O; about 80-100 mg/l magnesium sulfate and preferably about 90.37 mg/l magnesium sulfate.H.sub.2 O, about 1.5-7 mg/l manganese sulfate.H.sub.2 O and preferably about 3.33 mg/l manganese sulfate; about 0.4-1.6 mg/l potassium iodide and preferably about 0.8 mg/l potassium iodide; about 1,500-3,500 mg/l potassium nitrate and preferably about 2,830 mg/l potassium nitrate; about 200-600 mg/l potassium phosphate monobasic and preferably about 400 mg/l potassium phosphate monobasic; and, about 1.0-2.5 mg/l zinc sulfate.7H.sub.2 O and preferably about 1.25-1.75 mg/l zinc sulfate.7H.sub.2 O. Other equivalent liquid suspensions can be used and, as summarized in Table 2, media containing MS salts was also successfully used in the infection step. MS salts include about 1,650.0 mg/l ammonium nitrate, about 6.2 mg/l boric acid, about 332.2 mg/l calcium chloride anhydrous, about 0.025 mg/l cobalt chloride.6H.sub.2 O, about 0.025 mg/l cupric sulfate.5H.sub.2 O, about 37.26 mg/l Na.sub.2 -EDTA, about 27.8 mg/l ferrous sulfate.7H.sub.2 O, about 180.7 mg/l magnesium sulfate.H.sub.2 O, about 16.9 mg/l manganese sulfate.H.sub.2 O, about 0.83 mg/l potassium iodide, about 1,900.0 mg/l potassium nitrate, about 170.0 mg/l potassium phosphate monobasic, and about 8.6 mg/l zinc sulfate.7H.sub.2 O. Three different media, PHI-A, PHI-G and PHI-I, were tested in the infection step and these formulations are provided in the Examples.

Preferred media used in this step is provided in Example 4. In addition, the media in the infection step preferably excludes AgNO.sub.3. AgNO.sub.3 is preferably included in the co-cultivation, resting (when used) and selection steps when N6 media is used.

Those skilled in the art will recognize that although this method is disclosed for embryos isolated from maize, the method can also be used to transform maize cell suspensions. Therefore, the term "plant cells" as used in this invention can refer to isolated maize cells, including suspension cultures as will as to cells in an intact tissue, such as maize embryos.

Co-cultivation Step

In a next step of a preferred transformation protocol of this invention, the immature embryos are co-cultivated with the Agrobacterium on a solid medium. The embryos are preferably positioned axis down on the solid medium and the medium preferably includes AgNO.sub.3 at a range of about 0.85 to 8.5 mg/l, although 0.01 to 200 mg/l can also be used. The embryos are preferably cocultivated with the Agrobacterium for about 1-30 days, preferably about 2-20 days and more preferably about 3-10 days.

Two media regimes have been identified as useful in the methods of this invention: PHI basic medium and PHI combined medium. A summary of the media regimes used is provided in Table 2. The PHI basic medium contains N6 salts and is used in one embodiment of this invention, in the infection, co-cultivation optional resting and selection steps of this invention; MS salts are preferably used in the regeneration step. The PHI combined medium contains either N6 or MS salts in the infection step, MS salts in the co-cultivation step, N6 salts in the optional resting step and in the selection step and preferably MS salts in the plant regeneration step (Table 2). As illustrated in Examples 4-6, both basic media, containing N6 salts (for example, PHI-B), and the combined medium, using MS salts (for example, PHI-J), in the co-cultivation step demonstrated improved transformation efficiencies.

Preferably, where embryos are incubated on solid media containing N6 salts, the embryos remain on media containing N6 salts through the selection step. For embryos incubated in the co-cultivation step on MS containing medium, the embryos are preferably incubated in N6 salt-containing medium for the optional resting and the selection step. The preferred media combinations of this invention are summarized in Table 2.

Although Saito et al. and Hiei et al. cite the use of N6 salts for rice, Saito et al. specifically cite the use of LS salts rather than N6 salts in the examples for maize. Ishida et al. tested both LS and N6 salts for maize, but failed to obtain any stable transformants with N6 salts. Ishida et al. considered LS salts to be "superior to N6-based media" (supra) and used LS salts for every step of the process in all further experiments. Therefore, whenever the protocol of Ishida et al. is cited in the present application, it is understood that LS salts are included in that protocol. The macro and micro salts in MS medium are identical to the macro and micro salts in LS medium, but the two media differ in the composition of some of the vitamins and other components (Skirvin R. M., In: Cloning Agricultural Plants Via In Vitro Techniques, B. V. Conger, ed., CRC Press, Knoxville, Tenn., pp. 51-140, 1981).

Optional Resting Step

Following the co-cultivation step, the embryos are optionally transferred to a second plate of solid medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. This resting phase is performed in the absence of any selective pressures to permit preferential initiation and growth of callus from embryos containing the heterologous nucleic acid. Preferably, the antibiotic used to inhibit Agrobacterium is carbenicillin and the preferred concentrations of carbenicillin are about 50 mg/l to about 250 mg/l carbenicillin in the solid media, more preferably about 100-125 mg/l carbenicillin. A particularly preferred concentration of carbenicillin is about 100 mg/l. Other antibiotics can be used that inhibit the growth of Agrobacterium and these include for example Cefotaxime, timetin, vancomycin, and the like. Those of ordinary skill in the art of monocot transformation will recognize that the concentration of antibiotic can be optimized for a particular transformation protocol without undue experimentation. The resting phase cultures are preferably allowed to rest in the dark at 28.degree. C. for about 3 to about 5 days, but about 1 to about 15 days can also be used. Example 4 uses a 3-5 day resting period. A preferred resting step medium is PHI-C as provid