Method of producing transgenic maize using direct transformation of commercially important genotypes

Abstract

Methods for transformation of maize with nucleic acid sequences of interest are disclosed. The method involves subjecting immature zygotic embryos or Type I callus to high velocity microprojectile bombardment. The method is capable of producing transformed maize lines of commercial importance and their hybrid combinations.

Description

FIELD OF THE INVENTION

[0002] The present invention relates to the transformation of maize genotypes by microprojectile bombardment.

BACKGROUND OF THE INVENTION

[0003] The use of genetic engineering to introduce new agronomically important traits into maize such as insect resistance will have many commercial benefits. In order to accomplish this in the most expedient fashion it is necessary to have a method of transformation that can be used with maize genotypes that are commercially valuable.

[0004] The majority of instances of maize transformation have used a genotype known as A188, or derivatives of A188. This is because these lines are easily established in vitro as an embryogenic line that forms Type II, or friable, embryogenic callus and suspension cultures. Such Type II cultures have been exclusively preferred as a recipient of introduced genes in transformation methods. Unfortunately, A188 is an inferior inbred for the development of commercially important hybrids. (Hodges et al., Biotechnology, 4:219, 1986). Working with such “model” maize lines as A188 is disadvantageous in that extensive breeding is usually required in order to develop maize lines with a desirable genetic composition. What is needed is a method that can be used with commercially valuable maize lines without the need for reliance on such “model” systems based on Type II or suspension cultures.

[0005] Microprojectile bombardment has been advanced as an effective transformation technique for cells, including cells of plants. In Sanford et al., Particulate Science and Technology, 5: 27-37 (1987) it was reported that microprojectile bombardment was effective to deliver nucleic acid into the cytoplasm of plant cells of Allium cepa (onion). Christou et al., Plant Physiology 87: 671-674 (1988) reported the stable transformation of soybean callus with a kanamycin resistance gene via microprojectile bombardment. Christou et al. reported penetration at approximately 0.1 to 5% of cells. Christou further reported observable levels of NPTII enzyme activity and resistance in the transformed calli of up to 400 mg/L of kanamycin. McCabe et al., Bio/Technology 6: 923-926 (1988) report the stable transformation of Glycine max (soybean) using microprojectile bombardment. McCabe et al. further report the recovery of a transformed R1 plant from an R0 chimeric plant.

[0006] Transformation of monocots and, in particular, commercially valuable maize lines, has been problematic. Although there have been several reports of stable plant transformation utilizing the microprojectile bombardment technique, such transformation has not resulted in the production of fertile, regenerated transgenic maize plants of a commercially valuable genotype—each report used the genotype A188 or its derivatives (Fromm et al, BioTechnology, 8:833, 1990; Walters et al., Pl. Mol. Biol. 18:189, 1992, Gordon-Kamm et al., Plant Cell, 2:603, 1990). There are two reports of maize transformation using commercially valuable lines but both rely on the availability of Type II, friable embryogenic callus as a recipient for gene delivery (Jayne et al., 1991 Meeting of the International Society for Plant Molecular Biology, Abstract #338; Aves et al., 1992 World Congress on Cell and Tissue Culture, In Vitro 28:124A, Abstract #P-1134). Prior to the present invention, successful direct transformation of commercially valuable maize lines has not been achieved using microprojectile bombardment of immature zygotic embryos or Type I embryogenic callus.

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIG. 1 shows the plasmid map of the vector pCIB3064 containing the 35S/bar chimeric gene.

[0008] FIG. 2 shows the plasmid map of the vector pCIB3089 containing the 35S/B-Peru chimeric gene.

[0009] FIG. 3 shows the plasmid map of the vector pCIB4421 containing the PEP Carboxylase promoter fused to the synthetic BT coding sequence.

[0010] FIG. 4 shows the plasmid map of the vector pCIB4430 containing the pollen-specific promoter fused to the synthetic BT coding sequence.

[0011] FIG. 5 shows the plasmid map of the vector pCIB4431 containing the pollen-specific promoter fused to the synthetic BT coding sequence and the PEP Carboxylase promoter fused to the synthetic BT coding sequence.

[0012] FIG. 6 shows the plasmid map of the vector pCIB4433 containing the pith-preferred promoter fused to the synthetic BT coding sequence and the 35S/bar chimeric gene.

[0013] FIG. 7 shows the plasmid map of the vector pCIB4436 containing the 35S/C1 chimeric gene.

SUMMARY OF THE INVENTION

[0014] The present invention is drawn to the stable transformation of maize with nucleic acid sequences of interest, the regeneration of fertile transgenic maize plants and their subsequent use for the creation of commercially valuable lines and hybrids created with those lines. In the invention, immature zygotic embryos are subjected to high velocity microprojectile bombardment as a means of gene delivery within about 14 days after excision from the plant. After initiation of embryogenic callus and selection for transformed cells, stably transformed plants may be regenerated which express the foreign genes of interest. Alternatively, callus derived from immature zygotic embryos having the Type I character may also be employed as a recipient for gene delivery. The method is applicable to any genotype of maize, especially commercially important ones. In this manner, the method produces transformed maize lines of commercial importance and their hybrid combinations.

DETAILED DESCRIPTION OF THE INVENTION

[0015] A method for the transformation of any maize line and the regeneration of transgenic maize plants is provided. The method involves the delivery of nucleic acids, or particularly, genes of interest, directly to immature zygotic embryos. Alternatively, said nucleic acids or genes of interest may be delivered to serially-propagated Type I embryogenic callus obtained from immature zygotic embryos. Stably transformed cells are obtained and are regenerated into whole, fertile plants that express the foreign gene(s). Furthermore, the fertile transformed plants are capable of producing transformed progeny that express the foreign gene(s). The method provides for the direct transformation of commercially important maize genotypes, for obtaining transformed inbreds and for their hybrid combinations.

[0016] Embryogenic callus of maize is obtained by the process of somatic embryogenesis. Somatic embryogenesis is a process by which fully or partially-formed embryos arise from somatic tissue. This is in contrast to zygotic embryogenesis whereby embryos form from gametic tissue. Somatic embryogenesis may be induced from several viable tissues of maize, including leaf bases (Conger et al., Pl. Cell Rep. 6:345, 1987), tassel primordia (Rhodes et al., Pl. Sci. 46:225, 1986) and immature embryos (Green et al., Crop Science 15:417, 1975). In the present invention, immature embryos are the preferred source of embryogenic callus.

[0017] Immature zygotic embryos of maize can be obtained by pollinating an ear with viable pollen then removing the ear from the plant at some later time. Typically, immature embryos for use as sources of embryogenic callus are in the size range of about 0.5 mm to about 3.0 mm, more particularly about 1.0 to about 2.5, especially preferred from 1.5 to 2.0. The immature embryos may, for example, be removed from the ear individually by dissection or in bulk by “creaming” the kernels. Isolated immature embryos are placed with the zygotic embryo axis side in contact with an appropriate nutrient medium (Green et al., supra, 1975). Embryogenic callus is typically observed on the immature embryo within about 14 days of placement on the medium. This initial period is called the “initiation” step. After the initiation step, the embryogenic callus is generally transferred to a different medium for establishment and maintenance of serially-propagatable embryogenic callus (called the “maintenance” medium), although the same medium as that for “initiation” may also be used. Incubation of cultures typically takes place at 25 C in the dark or under low light. Embryogenic callus can be obtained in this way from a wide variety of maize genotypes.

[0018] Two main types of embryogenic callus have been described in the scientific literature. Type I embryogenic callus has been defined as “translucent, convoluted and compact callus” (Tomes et al., Theor. Appl. Genet. 70:505-509, 1985) or as “compact, morphologically complex” (Armstrong et al., Planta, 164:207-214 (1985)). Type II embryogenic callus has been defined as “friable and fast growing [callus] with well defined somatic embryos with suspensor-like structures” (Tomes et al., supra, 1985) or as “friable, embryogenic” (Armstrong et al., supra, 1985). It is within the scope of this invention that either or both types of embryogenic callus may be obtained from the immature zygotic embryos, or other viable tissue such as tassel primordia, leaf bases or meristems which may also be used as a source of embryogenic callus.

[0019] In order to obtain embryogenic callus the isolated immature maize embryos must be cultured on an appropriate medium. Many types of medium have been shown to be useful for the establishment of embryogenic callus from a variety of genotypes, including some commercially important ones (Hodges et al., Bio/Technology 4:219, 1986; Duncan et al., Planta 165:322, 1985). In practice, a preferred medium must be found experimentally for each genotype. Typically in such an experimental procedure a selection of basal media, sucrose concentrations, and growth regulator types and concentrations are combined in a factorial arrangement. Immature embryos from each genotype to be tested are placed onto medium representing each factorial combination. Initiation frequencies are scored for each medium and the ones producing the highest scores are used in a second round of experimentation. In this second round, the selected media combinations are further optimized for the individual genotypes by fine-tuning the growth-regulator type and concentration, and sucrose concentration. For example, Table I below indicates the preferred medium for the initiation of embryogenic callus for several of the genotypes disclosed in this invention.

TABLE I
Genotype   Initiation Medium
LH51       MS basal medium, G10 amendments, 6% sucrose, 5
           mg/L dicamba
CG00716    JMS basal medium, G5 amendments, 9% sucrose, 5
           mg/L dicamba
CG00526    D basal medium, G8 amendments, 2% sucrose, 5
           mg/L chloramben
CG00642    JMS basal medium, G5 amendments, 10% sucrose, 4
           mg/L dicamba
LH82       LM basal medium, G6 amendments, 4% sucrose, 0.2
           mg/L 2,4-D
CG00689    MS basal medium, G1 amendments, 6% sucrose, 0.5
           mg/L 2,4-D
CGNC4206   MS basal medium, G1 amendments, 6% sucrose, 0.5
           2,4-D
CG00629    D basal medium, G8 amendments, 2% sucrose, 5
           mg/L chloramben
(H99×FR16) JMS basal medium, G5 amendments, 2% sucrose, 1
xPa91      mg/l dicamba
Hi II      JMS basal medium, G5 amendments, 2% sucrose, 10
           mg/L silver nitrate, 5 mg/L dicamba

[0020] The basal media formulas used for the initiation of embryogenic callus of the genotypes in Table I may be found in the following, citations and is herein incorporated by reference: “D”, (Duncan et al., Planta 165:322, 1985); “KM”, (Kao et al., Planta 126:105, 1975); “LM”, (Litvay et al., Plant Cell Reports 4:325, 1985); “MS”, (Murashige et al, Physiologia Plantarum 15:473, 1962). The basal medium “JMS” is the medium known as “SH” (Schenk et al., Can. J. Bot. 50:199, 1972) modified by replacing the inorganic nitrogen compounds with those found in “MS” (Murashige et al., supra, 1962).

[0021] The formulas of the amendments used for the initiation of embryogenic callus of the genotypes in Table II are found in the following table. The amendments used with the “KM” basal medium were as described in Kao et al., supra, 1975).

	TABLE II
	Amendment Formulas,
	per Liter of Medium
	G1	G5	G6	G8	G10
	Component	mg
	Nicotinic Acid	0.5	5.0	0.5	0.2	0.5
	Pyridoxine-HCl	0.5	0.5	0.1	0.2	0.5
	Thiamine-HCl	0.1	5.0	0.1	0.5	0.1
	Glycine	2.0	—	—	—	2.0
	myo-Inositol	100	100	100	—	100
	Choline HCl	—	—	—	0.1	—
	Riboflavin	—	—	—	0.1	—
	Biotin	—	—	—	0.1	—
	Folic Acid	—	—	—	0.5	—
	CaPantothenate	—	—	—	0.1	—
	Cyanocobalamin	—	—	—	0.014	—
	Casein hydrolysate	100	100	100	—	—
	Proline	2800	2800	2800	—	—

[0022] Often, the preferred “maintenance” medium must be determined experimentally, as is done for “initiation”. Table III describes the “maintenance” medium found to be useful for several of the genotypes disclosed in the present invention.

TABLE II
Genotype   Maintenance Medium
LH51       MS basal medium, G10 amendments, 3% sucrose,
           0.25 mg/L 2,4-D
CG00716    N6 basal medium, 25 mM proline, 100 mg casein
           hydrolysate, 2% sucrose, 1.5 mg/L 2,4-D
CG00526    D basal medium, G8 amendments, 2% sucrose, 0.5
           mg/L 2,4-D
LH82       LM basal medium, G6 amendments, 3% sucrose, 3
           mg/L chloramben
CG00689    MS basal medium, G10 amendments, 2% sucrose,
           1.5 mg/L 2,4-D
CGNC4206   D basal medium, G8 amendments, 2% sucrose, 1.2
           2,4-D
(H99×FR16) D basal medium, G4 amendments, 2% sucrose, 0.5
xPa91      mg/l 2,4-D
Hi II      N6 basal medium, G5 amendments, 2% sucrose, 1
           mg/L 2,4-D

[0023] In Table III, “N6” basal medium refers to that described in Chu et al. Scientia Sinica, XVIII:659, 1975. The formulas for the amendments used in “maintenance” medium may be found in Table II.

[0024] According to the present invention, nucleic acid sequences or genes of interest are delivered to the immature embryos within the “initiation” step of the development of an embryogenic callus, i.e., within about 14 days of the placement of the immature embryos on a nutrient medium capable of supporting the intiation and development of embryogenic callus. After gene delivery and initiation of embryogenic callus, the embryogenic callus is transferred to a “maintenance” medium for subculture in either the presence or absence of a selection agent. In another embodiment of the invention, Type I callus developed using the methods above may also be used as a recipient of the nucleic acid sequences or genes of interest. In this instance, after delivery of the nucleic acid of interest, the Type I embryogenic callus is normally transferred to fresh “maintenance” medium, with or without a selection agent.

[0025] There are available many types of microprojectile bombardment devices, all working on essentially the same principle of accelerating micrometer size particles sufficient to cause penetration into the target tissues and cells. Since it is known that bombardment devices based on gunpowder do not work for the present invention, the preferred devices are those that use some form of gas expansion for the accelerating force for the microprojectiles such as air, carbon dioxide, nitrogen, water vapor or helium. The most preferred device for use in the claimed method is one based on compressed helium such as the DuPont PDS-1000/He.

[0026] For the bombardment of Type I embryogenic callus, the callus must be subdivided into smaller pieces. This can be achieved by chopping, maceration, dissection or other mechanical means. It is also possible to subdivide the callus through enzymatic means. Enzymes that digest the cell wall or cell-wall components may be used to reduce the integrity of the callus mass. Such enzymes include cellulases, macerases, pectinases, hemi-cellulases and others well known in the art. After the callus is subdivided, it is rinsed several times with liquid culture medium.

[0027] In preparation for gene delivery by microprojectile bombardment, either the immature embryos or Type I embryogenic callus may optionally be pre-treated with an osmotically-active agent to plasmolyze the cells for a period from about 1 to about 24 hours, preferably from about 1 to about 12 hours, most preferably from about I to about 6 hours. Typically, the recipient material is treated with a concentration of sucrose that produces an osmotic pressure in the medium that is greater than that in the recipient material. The concentration,of sucrose may range from about 2 to about 18%, preferably from about 6 to about 12%. It is also within the scope of this invention that other osmotically-active agents may be used in concentrations sufficient to cause the plasmolysis of the cells of the recipient material, such as sorbitol, glucose, mannitol and various molecular weight ranges of polyethylene glycol. The recipient material is kept in the presence of the osmotically-active agent after gene delivery for a period of about 1 to about 24 hours, preferably from about 3 to about 18 hours, most preferably from about 10 to about 18 hours.

[0028] In a preferred embodiment of the invention, the set-up and use of the microprojectile bombardment device, as well as the targeting of the recipient material, is described below. Other arrangements are also possible.

[0029] The DNA is prepared for microprojectile bombardment by chemical precipitation in the presence of micrometer size gold, essentially according to the published procedure of DuPont. In addition to gold, other dense particles of micrometer size may be used, such as tungsten or platinum. In one modification of the DuPont procedure, the particles themselves are first prepared by suspending them in water and sonicating. The sonicated particles are then pelleted by centrifugation and resuspended in an aqueous solution of 50% glycerol. Particles prepared in this way are then aliquoted into individual tubes containing approximately 3 mg of gold particles per tube in a volume of 50 ul. DNA is added to each tube in varying amounts, depending upon the number of plasmids to be used, their sizes and the final concentration of DNA desired. For example, in a typical preparation to include four plasmids for use in the present invention, 2 ug of pCIB3089, 2 ug pCIB4436, 3 ug pCIB4433 and 4 ug pCIB4430 are added to each tube of aliquoted gold particles. Next, about 50 ul of 2.5 M CaCl2 and about 20 ul of 1M spermidine are added, in that order, to each tube while vortexing for about 3 minutes. The DNA/gold complex is then gently centrifuged. The supernatant is removed, the particles are washed once with 250 ul of absolute ethanol, pelleted again and then resuspended in about 75 ul of fresh absolute ethanol. Each tube prepared in this way is enough of the DNA/gold complex for six “shots” with the PDS-1000/He. Ten ul of the well-suspended DNA/gold complex is pipetted onto the macrocarrier sheet in a vibration-free environment.

[0030] In the PDS-1000/He device, a burst of helium is released by rupture of a plastic disk which is available in different pressure grades. For example, single disks, or combinations of disks, can be obtained which rupture at 200, 450, 650, 900, 1100 1350, 1550, 1800, 2000 and 2200 pounds per square inch of helium. This burst of gas propels the macrocarrier sheet, which is stopped by a stainless steel screen. The screen may be of different mesh sizes, such as 10×10, 16×16, 24×24, etc. Other settings are the macrocarrier flight distances, gap distance, and particle flight distance. These settings are described in detail in the manufacturer's user's manual. Typically, a gap distance of about 5.5 mm, a macrocarrier flight distance of about 10 mm and a particle flight distance of about 6 to 9 cm is used. In addition, a screen or baffle may be inserted within the particle flight distance between the stopping screen and the target plate. Such a screen or baffle disturbs the shock wave from the expanding gas thereby reducing damage to the target. In one example, stainless steel screens with an opening of about 100 um is used. Other opening sizes and material composition may be used.

[0031] The immature embryos or Type I embryogenic callus may be arranged on the target plate in different patterns. Through a series of experiments, optimized patterns were developed for immature embryos. In one optimized pattern, the immature embryos are arranged in a circular pattern, the circle being about 2 cm in diameter. The immature embryos are placed on the periphery of the circle. Approximately 36 immature embryos are placed onto each target plate. Furthermore, the target plate may be angled relative to the microcarrier launch assembly. This ensures maximum saturation of the basipetal portion of the immature embryo by the particle spread. It is the basipetal portion of the immature embryo that gives rise to the embryogenic response.

[0032] In one example of the bombardment of Type I embryogenic callus, the callus is placed on the periphery of a circle about 1 cm diameter on a nutrient medium. The mechanical settings of the bombardment device may be placed at positions similar or identical to the settings recited above for the bombardment of immature embryos.

[0033] It should be noted that the target pattern and gun settings are interrelated. In other words, the use of other mechanical settings on the microprojectile bombardment device can produce other optimal arrangements of the recipient tissue on the target plate. Other combinations of mechanical settings and target patterns are within the scope of the invention.

[0034] The recombinant DNA molecules of the invention also can include a marker gene to facilitate selection in recombinant plant cells. Examples of markers include resistance to a biocide such as an antibiotic, e.g. kanamycin, hygromycin, chloramphenicol, paramomycin, methotrexate and bleomycin, or a herbicide such as imidazolones, sulfonylureas, glyphosate, phosphinothricin (PPT), glufosinate, or bialaphos. Marker genes are well known in the art. In one embodiment of the invention the selection agent phosphinothricin is used in conjunction with the selectable marker gene known as bar, which encodes for the enzyme phosphinothricin acetyltransferase. This enzyme acetylates the phosphinothricin molecule, thereby rendering it non-toxic to plant cells. The bar gene may be operably linked to a constitutive promoter such as the CaMV 35S promoter; the CaMV 19S promoter; A. tumefaciens promoters such as octopine synthase promoters, mannopine synthase promoters, nopaline synthase promoters, or other opine synthase promoters; ubiquitin promoters, actin promoters, histone promoters and tubulin promoters. Other promoters may also be used. In one embodiment of the present invention, the bar coding sequence is operably linked to the CaMV 35S promoter.

[0035] Selection of transformed cells in vitro is accomplished by including the selection agent of interest in the medium used to induce and support the establishment of embryogenic callus. Only cells in which the selectable marker is integrated into the chromosome and is expressed, i.e., are transformed, will survive the selection agent. Over time, the cells will grow into an embryogenic callus in the presence of the selection agent eventually reaching a mass sufficient for regeneration of whole, fertile plants. Typically, such embryogenic callus can be maintained for long periods of time in the presence of selection agent and still retain its ability to produce whole, fertile plants. The minimum time required to obtain an embryogenic callus of sufficient mass under selection pressure can range from about 2 weeks to about 24 weeks, more preferably about 4 weeks to about 20 weeks, most preferably about 8 weeks to about 12 weeks.

[0036] Transformed cells may also be selected in vitro through visual means. In order to accomplish this, a scorable marker is generally used. Examples of scorable markers would be the regulatory or structural genes controlling anthocyanin biosynthesis, GUS (beta-glucuronidase), luciferase, opine synthetases, thaumatin, beta-galactosidase, unique synthetic epitopes designed for easy detection by ELISA, phycobiliproteins and various fluorigenic substances. In a particular embodiment of the present invention the use is made of coding sequences for the anthocyanin regulatory genes known in the art as C1 and B-Peru (Goff et al., EMBO Journal, 9: 2517, 1990). Such coding sequences, operably linked to one or more of the several constitutive promoters listed above, can be used to isolate transformants on the basis of the red pigmentation of cells transformed with such genes.

[0037] Fertile transformed plants may be regenerated from isolated transformed embryogenic callus by several means. In general, the transformed embryogenic callus is transferred to a nutrient medium devoid of an auxin-type phytohormone, or is passaged through a series of nutrient media with diminishing concentrations of phytohormone. Other phytohormones may be used during the regeneration step, such as cytokinins (both natural and synthetic) and gibberellins. In some instances, inhibitors of phytohormone action may also be used, such as silver nitrate, ancymidol or TIBA. Other amendments to the nutrient medium for regeneration such as activated charcoal and various gelling agents are also known in the art.

[0038] In one example of the present invention, embryogenic callus was removed from maintenance medium containing PPT after 12 weeks and placed on regeneration medium containing MS basal medium, 3% sucrose, 0.25 mg/L 2,4-D and 5 mg/L benzyladenine. After 2 weeks the embryogenic callus began to regenerate and was transferred to MS basal medium with 3% sucrose. Plantlets are obtained during incubation under light. Such plants may be transferred to a greenhouse environment after sufficient root mass has developed.

[0039] In another embodiment of the invention, transformed embryogenic callus is transferred from maintenance medium containing phosphinothricin to regeneration medium consisting of MS medium, 3% sucrose and also containing phosphinothricin. Regeneration under such selective conditions also produces plantlets during incubation under light, which again may be transferred to a greenhouse environment after sufficient root mass has developed.

[0040] As will be evident to one of skill in the art, now that a method has been provided for the stable transformation of maize according to the claimed method, any gene of interest can be used in the methods of the invention. For example, a maize plant can be engineered to express disease and insect resistance genes, genes conferring nutritional value, genes to confer male and/or female sterility, antifungal, antibacterial or antiviral genes, and the like. Likewise, the method can be used to transfer any nucleic acid to control gene expression. For example, the DNA to be transferred could encode antisense RNA.

[0041] The present invention also encompasses the production by the disclosed method of transformed maize plants and progeny containing a gene or genes which encode for and express insecticidal proteins. Such genes may be derived from the genus Bacillus, for example Bacillus thuringiensis. In a particular embodiment of the present invention is the use of the claimed method to produce transformed maize plants containing a gene or genes whose nucleic acid sequence has been altered so as to be optimized for expression in maize. A complete description of the creation of said gene or genes may be found in U.S. Ser. No. 951,715 which is herein incorporated by reference. A summary of that disclosure is given below.

[0042] A nucleic acid sequence of interest in the present invention includes one which encodes the production of an insecticidal toxin, preferably a polypeptide sharing substantially the amino acid sequence of an insecticidal crystal protein toxin normally produced by Bacillus thuringiensis (BT). The synthetic gene may encode a truncated or full-length insecticidal protein (IP) Especially preferred are synthetic nucleic sequences which encode a polypeptide effective against insects of the order Lepidoptera and Coleoptera, and synthetic nucleic acid sequences which encode a polypeptide having an amino acid sequence essentially the same as one of the crystal protein toxins of Bacillus thuringiensis variety kurstaki, HD-1.

[0043] The present invention provides the use of synthetic nucleic acid sequences to yield high level expression of active insecticidal proteins in maize plants. The synthetic,nucleic acid sequences of the present invention have been modified to resemble a maize gene in terms of codon usage and G+C content. As a result of these modifications, the synthetic nucleic acid sequences of the present invention do not contain the potential processing sites which are present in the native gene. The resulting synthetic nucleic acid sequences (synthetic BT IP coding sequences) and plant transformation vectors containing this synthetic nucleic acid sequence (synthetic BT IP genes) result in surprisingly increased expression of the synthetic BT IP gene, compared to the native BT IP gene, in terms of insecticidal protein production in plants, particularly maize. The high level of expression results in maize cells and plants that exhibit resistance to lepidopteran insects, preferably European Corn Borer and Diatrea saccharalis, the Sugarcane Borer.

[0044] For example, the maize codon usage table described in Murray et al., Nucleic Acids Research, 17:477 1989, the disclosure of which is incorporated herein by reference, was used to reverse translate the amino acid sequence of the toxin produced by the Bacillus thuringiensis subsp. kurstaki HD-1 cryIA(b) gene, using only the most preferred maize codons. This sequence was subsequently modified to eliminate unwanted restriction endonuclease sites, and to create desired restriction endonuclease sites. These modifications were designed to facilitate cloning of the gene without appreciably altering the codon usage or the maize optimized sequence. During the cloning procedure, in order to facilitate cloning of the gene, other modifications were made in a region that appears especially susceptible to errors induced during cloning by the polymerase chain reaction (PCR).

[0045] In a preferred embodiment of the present invention, the protein produced by the synthetic nucleic acid sequence of interest is effective against insects of the order Lepidoptera or Coleoptera. In a more preferred embodiment, the polypeptide encoded by the synthetic nucleic acid sequence of interest consists essentially of the full-length or a truncated amino acid sequence of an insecticidal protein normally produced by Bacillus thuringiensis var. kurstaki HD-1. In a particular embodiment, the synthetic DNA sequence encodes a polypeptide consisting essentially of a truncated amino acid sequence of the BT CryIA(b) protein.

[0046] The present invention also encompasses the use of maize optimized coding sequences encoding other polypeptides, including those of other Bacillus thuringiensis insecticidal polypeptides or insecticidal proteins from other sources. For example, cryIB genes from Bacillus thuringiensis can be maize optimized, and then stably introduced into maize plants. It is also within the scope of this invention that the nucleic acid sequences of interest which encode insecticidal proteins may be either in the native or synthetic forms, optimized for expression in maize, and derived from any species of the genus Bacillus.

[0047] The insecticidal proteins produced by the nucleic acid sequences of interest in the present invention are expressed in a maize plant in an amount sufficient to control insect pests, i.e. insect controlling amounts. It is recognized that the amount of expression of insecticidal protein in a plant necessary to control insects may vary depending upon species of plant, type of insect, environmental factors and the like. Generally, the insect population will be kept below the economic threshold which varies from plant to plant. For example, to control European corn borer in maize, the economic threshold is 0.5 eggmass/plant which translates to about 10 larvae/plant.

[0048] In the present invention, the coding sequence of the nucleic acids of interest is a synthetic maize-optimized gene under the control of regulatory elements such as promoters which direct expression of the coding sequence. Such regulatory elements, for example, include monocot or maize and other monocot functional promoters to provide expression of the gene in various parts of the maize plant.

[0049] The regulatory element may be constitutive. That is, it may promote continuous and stable expression of the gene. Such promoters include but are not limited to the CaMV 35S promoter; the CaMV 19S promoter; A. tumefaciens promoters such as octopine synthase promoters, mannopine synthase promoters, nopaline synthase promoters, or other opine synthase promoters; ubiquitin promoters, actin promoters, histone promoters and tubulin promoters.

[0050] The regulatory element may also be a tissue-preferential or tissue-specific promoter. The term “tissue-preferred promoter” is used to indicate that a given regulatory DNA sequence will promote a higher level of transcription of an associated structural gene or DNA coding sequence, or of expression of the product of the associated gene as indicated by any conventional RNA or protein assay, or that a given DNA sequence will demonstrate some differential effect; i.e., that the transcription of the associated DNA sequences or the expression of a gene product is greater in some tissue than in all other tissues of the plant. Preferably, the tissue-preferential promoter may direct higher expression of the synthetic gene in leaves, stems, roots and/or pollen than in seed. “Tissue-specific promoter” is used to indicate that a given regulatory DNA sequence will promote transcription of an associated coding DNA sequence essentially entirely in one or more tissues of a plant, or in one type of tissue, e.g. green tissue, while essentially no transcription of that associated coding DNA sequence will occur in all other tissues or types of tissues of the plant. Numerous promoters whose expression are known to vary in a tissue specific manner are known in the art. One such example is the maize phosphoenol pyruvate carboxylase (PEPC), which is green tissue-specific. See, for example, Hudspeth, R. L. and Grula, J. W., Plant Molecular Biology 12:579-589, 1989. Other green tissue-specific promoters include chlorophyll a/b binding protein promoters and RubisCO small subunit promoters.

[0051] The regulatory element may also be inducible, such as by heat stress, water stress, insect feeding or chemical induction, or may be developmentally regulated.

[0052] In one preferred nucleic acid of interest, the regulatory element is a pith-preferred promoter isolated from a maize TrpA gene. That is, the promoter in its native state is operatively associated with a maize tryptophan synthase-alpha subunit gene (hereinafter “TrpA”). The encoded protein has a molecular mass of about 38 kD. Together with another alpha subunit and two beta subunits, TrpA forms a multimeric enzyme, tryptophan synthase. Each subunit can operate separately, but they function more efficiently together.

[0053] The nucleic acids of interest in the present invention also include purified pollen-specific promoters obtainable from a plant calcium-dependent phosphate kinase (CDPK) gene. That is, in its native state, the promoter is operably linked to a plant CDPK gene. In a preferred embodiment, the promoter is isolated from a maize CDPK gene. By “pollen-specific,” it is meant that the expression of an operatively associated structural gene of interest is substantially exclusively (i.e. essentially entirely) in the pollen of a plant, and is negligible in all other plant parts. By “CDPK,” it is meant a plant protein kinase which has a high affinity for calcium, but not calmodulin, and requires calcium, but not calmodulin, for its catalytic activity.

[0054] In another nucleic acid of interest, the regulatory element is a root-preferential promoter. A complete description of such a root promoter and the methods for finding one may be found in U.S. Ser. No. 508,207 filed Apr. 12, 1990, the relevant parts of which are herein incorporated by reference. Briefly, a root-preferential promoter was isolated from a gene whose cDNA was found by differential screening of a cDNA library from maize. A cDNA clone so obtained was used to isolate a homologous genomic clone from maize. The protein encoded by the isolated clone was identified as a metallothionein-like protein.

[0055] Maize is easily hybridized because of the physical distance between the tassel (male part) and the ear (female part). The method of hybridization first involves the development of inbred lines. Inbred lines are maize plants that are essentially the same genetically from generation to generation. Inbreds are produced by taking the pollen from one maize plant and transferring the pollen to the silk of a receptive maize ear of that same plant. Selections for uniformity and agronomic performance are made and the process is repeated until the seeds from the ears of the plants produce genetically the same plants and the line is pure. A hybrid maize plant is produced by crossing one elite inbred maize plant with one or more other, genetically different and diverse, inbred maize plant. The crossing consists of taking the pollen from one inbred elite maize plant and transferring the pollen to the silk of a receptive ear of the other elite inbred maize plant. The seed from crossing of two inbreds is a first generation hybrid and is called a F1. The F1 of commercially valuable inbreds have better yields, standability, and improvement in other important characteristics than either of the parents. This phenomenon is called hybrid vigor.

[0056] In the present invention, commercially-valuable inbred lines of maize are directly transformed through the disclosed methods of delivering nucleic acid sequences of interest to either immature zygotic embryos obtained from such lines or Type I embryogenic callus derived from immature zygotic embryos of such lines. The ability to directly transform maize lines of commercial value is a distinct advantage of the claimed invention in that the generations of backcrossing required when the starting material is not commercially valuable can be avoided, thereby reducing the time and cost of commercialization. Alternatively, the present invention also discloses the direct transformation of hybrids of inbred lines using the claimed methods.

[0057] Many hybrid crosses have been successfully made using the transformed, commercially-valuable plants of the claimed invention. For example, the transformed genotype CG00526 of Example 2, below, has been crossed to genotypes CG00689, CG00716, CG00661, CG00642, and LH82 thereby creating hybrids possessing insecticidal activity.

[0058] Using the methods of the present invention any hybrid expressing a gene of interest can be created by transforming an inbred line with the gene of interest and using such transformed line to create the hybrid. A transformed hybrid may also be obtained according to the present invention by directly transforming either immature zygotic embryos obtained from said hybrid plant or by transforming Type I embryogenic callus derived from immature zygotic embryos obtained from said-hybrid plant.

[0059] In another embodiment of the claimed invention, it is also possible to produce maize plants that have an altered phenotype of anthocyanin pigmentation. This can be accomplished through the use of the disclosed chimeric genes for the constitutive promotion of the genes known as C1 and B-Peru. That activation of the biosynthetic pathway for anthocyanin can be achieved in this way in embryogenic callus was reported by Goff et al., EMBO Journal, 9: 2517-2522, 1990. In the present invention, the above said genes were used to produce plants and progeny according to the claimed method whose color phenotype was altered.

[0060] Commercially-valuable maize genotypes having altered color phenotype may have benefit to the process of plant breeding. For example, the use of the monoploid inducing gene known as id (Kermicle, Science 166:1422-1424, 1969) can be used to create a haploid having a paternal nuclear constitution. Monoploid inducers creating a haploid having a maternal nuclear constitution are also known (for example, Coe, The American Naturalist, XCIII: 381-382, 1959). Because of the low frequency of such an event, it would be advantageous to have an easily screened color phenotype which would allow the identification of the haploids. By using the claimed method and genes of interest, it is possible to obtain a transformed maize line having pigmented seeds, which can be used with a monoploid inducing line. Haploid seed can then be identified by either the presence or absence of seed pigmentation, depending upon the genotypes and crossing methods used.

[0061] Since a variety of altered color phenotypes can be produced by the present invention, examples of which are described below, it is further envisioned that other uses in plant breeding may be found for the claimed plants. As another example of such utility, it is possible to link operably, molecularly, biochemically or genetically, or any combination thereof, the expression of the altered color phenotype with the expression of the insecticidal activity produced by transformation of maize according to the claimed methods. Such a link would allow rapid, visual identification of plants within a segregating population of plants and possesing the gene or gene products. Linkages of the altered color phenotype and genotype to other traits of agronomic interest are also envisioned. The ability to perform such identification would translate into reduced costs and time for the plant breeder.

EXAMPLES

[0062] The following examples further describe the materials and methods used in carrying out the invention and the subsequent results. They are offered by way of illustration, and their recitation should not be considered as a limitation of the claimed invention.

Example 1

Bioassay of Transformed Maize for Insecticidal Activity and Quantitation of an Insecticidal Protein

[0063] Transformed plants were assayed for insecticidal activity and the presence of a BT protein resulting from the expression of the maize-optimized coding sequence of a synthetic BT gene. The procedure is similar for any maize plant transformed with a BT gene but is described here using as an example a cryIA(b) gene, its expressed product, and resistance to European corn borer.

[0064] Insecticidal activity was determined by insect bioassay. One to four 4 cm sections are cut from an extended leaf of a transformed maize plant. Each leaf piece is placed on a moistened filter disc in a 50×9 mm petri dish. Five neonate European corn borer larvae are placed on each leaf piece. Since each plant is sampled multiple times this makes a total of 5-20 larvae per plant. The petri dishes are incubated at 29.5° C. and leaf feeding damage and mortality data are scored at 24, 48, and 72 hours.

[0065] Quantitative determination of a cryIA(b) IP in the leaves of transgenic plants is performed using enzyme-linked immunosorbant assays (ELISA) as disclosed in Clark M F, Lister R M, Bar-Joseph M: ELISA Techniques. In: Weissbach A, Weissbach H (eds) Methods in Enzymology 118:742-766, Academic Press, Florida (1986). Immunoaffinity purified polyclonal rabbit and goat antibodies specific for the B. thuringiensis subsp. kurstaki IP were used to determine ng IP per mg soluble protein from crude extracts of leaf samples. The sensitivity of the double sandwich ELISA is 1-5 ng IP per mg soluble protein using 50 ug of total protein per ELISA microtiter dish well. Corn extracts were made by grinding leaf tissue in gauze lined plastic bags using a hand held ball-bearing homogenizer (AGDIA, Elkart Ind.) in the presence of extraction buffer (50 mM Na2CO3 pH 9.51 100 mM NaCl, 0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 μM leupeptin). Protein determination was performed using the Bio-Rad (Richmond, Calif.) protein assay.

Example 2

Transformation of the CG00526 Genotype of Maize by Direct Bombarding of Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0066] Immature embryos for experiment KC-65 were obtained approximately 14 days after self-pollination. The immature zygotic embryos were divided among different target plates containing medium capable of inducing and supporting embryogenic callus formation at 36 immature embryos per plate. The immature zygotic embryos were bombarded with a mixture of the plasmids pCIB3064 and pCIB4431 using the PDS-1000/He device from DuPont. The plasmids were precipitated onto 1 um gold particles essentially according to the published procedure from DuPont, as described above. Each target plate was shot one time with the plasmid and gold preparation. Since the plasmid pCIB3064 contained a chimeric gene coding for resistance to phosphinothricin this substance was used to select transformed cells in vitro. This selection was applied at 3 mg/L one day after bombardment and maintained for a total of 12 weeks. The embryogenic callus so obtained was regenerated in the absence of the selection agent phosphinothricin. Plants were obtained from one isolated line of embryogenic callus and given the Event Number 176. All plants were tested by the chlorophenol red (CR) test for resistance to PPT as described in U.S. patent application Ser. No. 759,243, filed Sep. 13, 1991, the relevant portions of which are hereby incorporated herein by reference. This assay utilizes a pH sensitive indicator dye to show which cells are growing in the presence of PPT. Cells which grow produce a pH change in the media and turn the indicator yellow (from red). Plants expressing the resistance gene to PPT are easily seen in this test. Of the 38 plants regenerated for Event Number 176, eight were positive in this test and 30 were negative. Plants positive by the CR test were assayed by PCR for the presence of the synthetic BT gene. Of the eight positive plants from the CR test, 5 were positive for the presence of the synthetic BT gene, 2 were negative and 1 died during propagation. These five remaining plants were bioassayed and found to be resistant to European Corn Borer. DNA was isolated from plant #11 using standard techniques and analysed by Southern blot analysis. It was found to contain sequences which hybridize with probes generated from the synthetic cryIA(b) gene and with a probe generated from the PAT gene. These results showed integration of these genes into the genome of maize. Plant #11 was shown by ELISA to contain 2,195 ng BT protein per mg soluble protein in the leaf tissue, consistent with the use of the leaf-specific promoter from PEPC operably linked to a synthetic BT gene. Plants resistant to European Corn Borer and expressing the introduced BT gene are transformed.

Example 3

Transformation of the Hi II Genotype of Maize by Direct Bombarding of Immature Zygotic Embryos and Isolation of Transformed Callus Without the Use of a Selection Agent

[0067] Ear number ED42 was self-pollinated and immature zygotic embryos were obtained approximately 10 days later. Two hundred and eighty eight immature zygotic embryos were divided among 7 different target plates containing a medium capable of inducing and supporting the formation of embryogenic callus. After two days, the immature zygotic embryos were transferred to the same medium but containing 12% sucrose. After 5 hours, the immature zygotic embryos were bombarded with a mixture of the plasmids pCIB3089, pCIB4430, pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont. The plasmids were precipitated onto 1 um gold particles essentially according to the published procedure from DuPont, as described above. The particles were delivered using burst pressures of 450, 650 and 900 psi of helium. Each target plate was shot twice with the plasmid and gold particle preparation. After overnight incubation, the immature embryos were transferred to fresh maintenance medium containing 2% sucrose. Since the plasmids pCIB3089 and pCIB4436 contain the C1 and B-Peru genes which regulate anthocyanin production, the appearance of red, multicellular sectors on the developing embryogenic callus was used to select and isolate transformed cells, eventually obtaining a homogeneous callus line. Embryogenic callus was regenerated in both the absence and presence of the selection agent phosphinothricin, resistance to which was conferred by a chimeric gene present in plasmid pCIB4433. Plants were obtained from a total of nine isolated embryogenic callus lines and were given the Event Numbers 197, 198, 208, 211, 219, 255, 261, 281 and 284. Leaf tissue from plants from each event were assayed for resistance to European Corn Borer. Plants from Event Numbers 208 and 211 were susceptible to European Corn Borer whereas plants from Event Numbers 197, 198, 219, 255, and 261 were resistant. All the plants that were resistant to European Corn Borer also expressed the introduced, leaf-specific PEPC-promoted synthetic BT gene as evidenced by the detection of BT protein using an ELISA assay. Plants resistant to European Corn Borer and expressing the introduced BT gene are transformed.

Example 4

Transformation of the Hi II Genotype of Maize by Direct Bombarding of Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0068] Ear number ED47 was self-pollinated and immature zygotic embryos were obtained approximately 10 days later. Approximately two hundred and sixty immature zygotic embryos were divided among 8 different target plates containing a medium capable of inducing and supporting the formation of embryogenic callus. After two days, the immature zygotic embryos were transferred to the same medium but containing 12% sucrose. After 5 hours, the immature zygotic embryos were bombarded with a mixture of the plasmids pCIB3089, pCIB4430, pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont. The plasmids were precipitated onto 1 um gold particles essentially according to the published procedure from DuPont, as described above. The particles were delivered using a burst pressure of 900 psi of helium. Each target plate was shot twice with the plasmid and gold particle preparation. After overnight incubation, the immature embryos were transferred to fresh maintenance medium containing 2% sucrose. Since the plasmid pCIB4433 contained a chimeric gene coding for resistance to phosphinothricin this substance was used to select transformed cells in vitro. The selection agent was applied at 10 mg/L 14 days after gene delivery and increased to 20-40 mg/L after approximately one month. The embryogenic callus so obtained was regenerated in the presence of the selection agent phosphinothricin. Plants were obtained from a total of eleven isolated embryogenic callus lines and were given the Event Numbers 220, 221, 222, 223, 225, 230, 231, 232, 233, 269, 274. Plants from each event were assayed for resistance to European Corn Borer. Leaf tissue of plants from Event Numbers 220, 221, 222, 223, 225, 231 and 233 were resistant. All the plants that were resistant to European Corn Borer also expressed the introduced, leaf-specific PEPC-promoted synthetic BT gene as evidenced by the detection of BT protein using an ELISA assay. Plants resistant to European Corn Borer and expressing the introduced BT gene are transformed. Plants from Event Numbers 230, 232, 269 and 274 were not completely tested.

Example 5

Transformation of the CG00526 Genotype of Maize by Bombarding of Type I Callus Derived From Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0069] Type I callus was obtained from immature zygotic embryos using standard culture techniques. For gene delivery, approximately 300 mg of the Type I callus was prepared by chopping with a scalpel blade, rinsing 3 times with standard culture media containing 18% sucrose and immediately placed onto semi-solid culture medium again containing 18% sucrose. After approximately 4 hours, the tissue was bombarded using the PDS-1000/He Biolistic device from DuPont. The plasmids pCIB4430 and pCIB4433 were precipitated onto 1 um gold particles using the standard protocol from DuPont. Approximately 16 hours after gene delivery the callus was transferred to standard culture medium containing 2% sucrose and 10 mg/L phosphinothricin as Basta. The callus was subcultured on selection for 8 weeks, after which surviving and growing callus was transferred to standard regeneration medium for the production of plants.

Example 6

Transformation of the LH51 Genotype of Maize by Bombarding of Type I Callus Derived From Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0070] Type I callus was obtained from immature zygotic embryos using standard culture techniques. For gene delivery, approximately 300 mg of the Type I callus was prepared by chopping with a scalpel blade, rinsing 3 times with standard culture media containing 12% sucrose and immediately placed onto semi-solid culture medium again containing 12% sucrose. After approximately 4 hours, the tissue was bombarded using the PDS-1000/He Biolistic device from DuPont. The plasmids pCIB4430 and pCIB4433 were precipitated onto 1 um gold particles using essentially the standard protocol from DuPont as described above. Approximately 16 hours after gene delivery the callus was transferred to standard culture medium containing 2% sucrose and 1 mg/L phosphinothricin as Basta. The callus was subcultured on selection for 8 weeks, after which surviving and growing callus was transferred to standard regeneration medium for the production of plants.

Example 7

Transformation of the CG00526 Genotype of Maize by Direct Bombarding of Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0071] Ear numbers JS21, JS22, JS23, JS24 and JS25 were self-pollinated and immature zygotic embryos were obtained approximately 10 days later. Approximately eight hundred and forty immature zygotic embryos were divided among 14 different target plates containing a medium capable of inducing and supporting the formation of embryogenic callus. The immature zygotic embryos were transferred immediately to the same medium but containing 12% sucrose. After 5 hours, the immature zygotic embryos were bombarded with a mixture of the plasmids pCIB3089, pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont. The plasmids were precipitated onto 1 um gold particles essentially according to the published procedure from DuPont, as described above. The particles were delivered using a burst pressure of 1550 psi of helium. Each target plate was shot twice with the plasmid and gold particle preparation. Since the plasmid pCIB4433 contained a chimeric gene coding for resistance to phosphinothricin this substance was used to select transformed cells in vitro. The selection agent was applied at 10 mg/L on the day of gene delivery and increased to 40 mg/L after approximately one month. The embryogenic callus so obtained was regenerated in the presence of the selection agent phosphinothricin. Plants were obtained from a total of eight isolated embryogenic callus lines and were given the Event Numbers 187, 188, 191, 192, 193, 196, 228 and 229. Plants from each event were assayed for resistance to European Corn Borer. Plants from Event Numbers 191 and 193 exhibited insect resistance in the pith in accordance with the use of the pith-preferred synthetic BT construct. All the plants that were resistant to European Corn Borer also expressed the introduced chimeric BT gene as evidenced by the detection of BT protein in the pith using an ELISA assay. Plants resistant to European Corn Borer and expressing the introduced BT gene are transformed.

Example 8

Transformation of the (H99xFR16)xPa91 Genotype of Maize by Direct Bombarding of Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0072] Ear numbers GP5 and JS26 were self-pollinated and immature zygotic embryos were obtained approximately 10 days later. Approximately three hundred and thirty immature zygotic embryos were divided among 5 different target plates containing a medium capable of inducing and supporting the formation of embryogenic callus. After two days the immature zygotic embryos were transferred to the same medium but containing 12% sucrose. After approximately 5 hours, the immature zygotic embryos were bombarded with a mixture of the plasmids pCIB3089, pCIB4430, pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont. The plasmids were precipitated onto 1 um gold particles essentially according to the published procedure from DuPont, as described above. The particles were delivered using a burst pressure of 1300 psi of helium. Each target plate was shot twice with the plasmid and gold particle preparation. Since the plasmid pCIB4433 contained a chimeric gene coding for resistance to phosphinothricin this'substance was used to select transformed cells in vitro. The selection agent was applied at 10 mg/L 3 weeks after the day of gene delivery. The embryogenic callus so obtained was regenerated. Plants were obtained from three isolated embryogenic callus lines and were given the Event Numbers 242, 247 and 260. Plants from each event were assayed for resistance to European Corn Borer. Plants from Event Numbers 247 and 260 exhibited insect resistance indicating that they were transformed.

Example 9

Transformation of the CG00526 Genotype of Maize by Direct Bombarding of Immature Zygotic Embryos and Isolation of Transformed Callus With the Use of Phosphinothricin as a Selection Agent

[0073] Immature zygotic embryos for the experiment KM-124 were obtained approximately 14 days after self-pollination. Approximately one hundred and five immature zygotic embryos were divided among 4 different target plates containing a medium capable of inducing and supporting the formation of embryogenic callus. The immature zygotic embryos were bombarded with a mixture of the plasmids pCIB4421 and pCIB4433 using the PDS-1000/He device from DuPont.

[0074] The plasmids were precipitated onto 1 um gold particles essentially according to the published procedure from DuPont, as described above. The particles were delivered using a burst pressure of 1550 psi of helium. Each target plate was shot once with the plasmid and gold particle preparation. Since the plasmid pCIB4433 contained a chimeric gene coding for resistance to phosphinothricin this substance was used, as Basta, to select transformed cells in vitro. The selection agent was applied at 5 mg/L one day after gene delivery and maintained for a total of 12 weeks. The embryogenic callus so obtained was regenerated in the absence of the selection agent phosphinothricin. Plants were obtained from one isolated embryogenic callus line and was given the Event Number 268. Plants were assayed for resistance to European Corn Borer. One of the 5 plants obtained is resistant to European Corn Borer and is transformed.

Example 10

Color Phenotypes Exhibited by Plants Transformed With the C1 and B-Peru Coding Sequences According to the Claimed Methods

[0075] Both genotypes Hi II and CG00526 were transformed with the C1 and B-Peru chimeric genes recited above in FIGS. 2 and 7. A variety of stably expressed altered color phenotypes were obtained, a partial listing of which appears in Table IV, below.

TABLE IV
BT Event Number Color Phenotype
197             Red roots, red anthers
208             Red roots
211             Red roots
213             Red stripe shoot
204             Red anthers, pink silks
210             Red stripe shoot, red root, red anther, red
                silk, red embryo
239             Red shoot, red root, red silk, normal
                embryo
207             Red anthers, red silks

[0076] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0077] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A method for producing stably transformed fertile maize plants, said method comprising: obtaining an immature embryo from a maize plant; delivering a nucleic acid sequence of interest by microprojectile bombardment within 14 days to said immature embryo; selecting for transformed cells; and, regenerating fertile transformed slants.

2. The method of claim 1 wherein said maize plant is selected from the genotypes CG00526, LH51, CG00708, LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 and CG00689.

3. The method of claim 1 wherein an immature embryo is pretreated with an osmotically-active substance while in the presence of a nutrient medium.

4. The method of claim 3 wherein an osmotically-active substance is selected from sucrose, sorbitol, polyethylene glycol, glucose and mannitol.

5. The method of claim 1 wherein said nucleic acid sequence codes for an insecticidal protein.

6. The method of claim 5 wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.

7. The method of claim 6 wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.

8. The method of claim 7 wherein said nucleic acid sequence is a maize optimized coding sequence.

9. The method of claim 5 wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.

10. The method of claim 5 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

11. The method of claim 6 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

12. The method of claim 7 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

13. The method of claim 8 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

14. The method of claim 9 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase-promoter and a root-preferred promoter.

15. The method of claim 1 wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.

16. The method of claim 15 whereby the transformed cells are selected visually using the expression of anthocyanin controlled by the introduced genes.

17. A stably transformed maize plant produced by: obtaining an immature embryo from a maize plant; delivering a nucleic acid sequence of interest by microprojectile bombardment within 14 days to said immature embryo; selecting for transformed cells; and, regenerating fertile transformed plants.

18. The stably transformed maize plant of claim 17 having essentially one of the genotypes known as CG00526, LH51, CG00708, LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 or CG00689.

19. A hybrid obtained by crossing the stably transformed plant of claim 18 with another maize plant.

20. The maize plant of claim 18 wherein said nucleic acid sequence encodes an insecticidal protein.

21. The maize plant of claim 20 wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.

22. The maize plant of claim 21 wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.

23. The maize plant of claim 18 wherein said nucleic acid sequence is a maize optimized coding sequence.

24. The maize plant of claim 20 wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.

25. The maize plant of claim 20 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

26. The maize plant of claim 21 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

27. The maize plant of claim 22 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

28. The maize plant of claim 23 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

29. The maize plant of claim 24 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

30. The maize plant of claim 18 wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.

31. The hybrid of claim 19 wherein said nucleic acid sequence encodes an insecticidal protein.

32. The hybrid of claim 31 wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.

33. The hybrid of claim 32 wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.

34. The hybrid of claim 31 wherein said nucleic acid sequence is a maize optimized coding sequence.

35. The hybrid of claim 31 wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.

36. The hybrid of claim 31 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

37. The hybrid of claim 32 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

38. The hybrid of claim 33 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

39. The hybrid of claim 34 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

40. The hybrid of claim 35 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

41. The hybrid of claim 19 wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.

42. A method for producing stably transformed fertile maize plants, said method comprising: obtaining Type I embryogenic callus derived from a viable portion of a maize plant; delivering a nucleic acid sequence of interest by microprojectile bombardment to said Type I embryogenic callus; selecting for transformed cells; and, regenerating fertile transformed plants.

43. The method of claim 42 wherein said maize plant is selected from the genotypes CG00526, LH51, CG00708, LH82, G00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 and CG00689.

44. The method of claim 42 wherein a Type I embryogenic callus is pretreated with an osmotically-active substance while in the presence of a nutrient medium.

45. The method of claim 44 wherein an osmotically-active substance is selected from sucrose, sorbitol, polyethylene glycol, glucose, mannitol.

46. The method of claim 42 wherein said nucleic acid sequence codes for an insecticidal protein.

47. The method of claim 46 wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.

48. The method of claim 47 wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.

49. The method of claim 46 wherein said nucleic acid sequence is a maize optimized coding sequence.

50. The method of claim 46 wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.

51. The method of claim 46 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

52. The method of claim 47 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

53. The method of claim 48 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

54. The method of claim 49 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

55. The method of claim 50 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

56. The method of claim 42 wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.

57. A stably transformed maize plant produced by: obtaining Type I embryogenic callus derived from a viable portion of a maize plant; delivering a nucleic acid sequence of interest by microprojectile bombardment to said Type I embryogenic callus; selecting for transformed cells; and, regenerating fertile transformed plants.

58. The stably transformed maize plant of claim 57 having essentially one of the genotypes known as CG00526, LH51, CG00708, LH82, CG00716, CG00717, LH213, LH216, CG00721, CG00637, CG00642, CG00623, CG00675, CG00678, CG00653, CG00683, CG00685, CG00686, CG00656, CG00657, CG00661, CG00632, CG00662, CG00712, CG00684 or CG00689.

59. A hybrid obtained by crossing the stably transformed plant of claim 58 with another maize plant.

60. The maize plant of claim 58 wherein said nucleic acid sequence encodes an insecticidal protein.

61. The maize plant of claim 60 wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.

62. The maize plant of claim 61 wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.

63. The maize plant of claim 58 wherein said nucleic acid sequence is a maize optimized coding sequence.

64. The maize plant of claim 60 wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.

65. The maize plant of claim 60 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

66. The maize plant of claim 61 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

67. The maize plant of claim 62 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

68. The maize plant of claim 63 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

69. The maize plant of claim 64 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

70. The maize plant of claim 58 wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.

71. The hybrid of claim 59 wherein said nucleic acid sequence encodes an insecticidal protein.

72. The hybrid of claim 71 wherein said nucleic acid sequence codes for an insecticidal protein from the genus Bacillus.

73. The hybrid of claim 72 wherein said nucleic acid sequence codes for the protein endotoxin of Bacillus thurigiensis.

74. The hybrid of claim 71 wherein said nucleic acid sequence is a maize optimized coding sequence.

75. The hybrid of claim 71 wherein said nucleic acid sequence is one of the sequences recited in Tables 5 to 11 of the above specification.

76. The hybrid of claim 71 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

77. The hybrid of claim 72 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

78. The hybrid of claim 73 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

79. The hybrid of claim 74 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

80. The hybrid of claim 75 wherein said nucleic acid sequence comprises a promoter from a pith-preferred promoter, a pollen-specific promoter, a PEP Carboxlase promoter and a root-preferred promoter.

81. The hybrid of claim 59 wherein said nucleic acid sequence codes for the regulatory sequences known as C1 and B-Peru which control anthocyanin expression.