Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
  • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers

Definitions

• TRANSGENIC CEREAL PLANTS BACKGROUND OF THE INVENTION The present invention relates to obtaining plants by a methodology that entails the biolistic bombardment of meristem tissue, at a very early stage of development, and the selective enhancement of transgenic sectors, toward genetic homogeneity, in cell layers that contribute to germline transmission.
• transgenic cereal plants e.g., maize, sorghum, wheat, barley, oat or rice plants, that will transmit introduced DNA to progeny, comprising the steps of
• the foreign DNA can be introduced into a plurality of meristems, at least some of which differentiate in step (C) to form a plurality of plantlets.
• the foreign DNA is introduced into a meristem that is not enclosed by sheathing leaves including meristems from early proembryo, mid proembryo. late proembryo, transitional and early coleoptilar stage embryos.
• reorganization is effected through at least one manipulation selected from the group consisting of (i) imposition of a nonlethal selective pressure on the meristems, (ii) mechanically- induced meristem reorganization, and (iii) hormonally- induced shoot multiplication.
• the conditions in step (C) are such that the meristems undergo maturation and plant differentiation to form shoot apices, and the method further comprises effecting reorganization of meristem tissue in the shoot apices to enlarge transformed sectors or to produce periclinal L2 chimeras.
• the reorganization in this regard can be effected, for example, by exposing the shoot apices to nonlethal selection pressure such that transformed cells have a competitive growth advantage over nontransformed cells in the shoot apices, and the proportion of transformed cells in the shoot apices is increased.
• the method further comprises a step before step (B) , e.g., before step (A) , of wounding the apical dome selectively.
• a method of the present invention also can comprise the further steps of (i) dissecting out an axillary bud from above the base of a leaf of a plantlet when a chimeric sector is observed in a substantial portion of the leaf, and then (ii) germinating the axillary bud into a whole plant or subjecting the axillary bud to shoot multiplication.
• the transgenic sector of a plantlet is stabilized by inducing tillers. The apex of a transgenic plantlet is removed, the wounded plantlet is grown to induce formation of a plurality of tillers, and transgenic tillers then are selected from that plurality.
• a transgenic cereal plant that (A) is the product of a method as described above, (B) transmits introduced DNA to progeny and (C) belongs to a cereal line that is recalcitrant to callus-based transformation.
• the transgenic cereal is a maize plant that is not produced by transformation of a genotype selected from the group consisting of A188, A188 x B73, H99, Pa91, FR16 and a genotype obtained from a cross involving any of the foregoing.
• a maize plant that transmits introduced DNA to progeny and that has a pedigree including a line selected from the group consisting of PHT47, PHP02, PHV78, PHK05, PHW20, PHR62, PHN37, PHM10, PHV37, PHJ65, PHBW8, PHK29, PHJ33, PHP60, PHN73 and PHHV4.
• FIGURE 1 is a series of line drawings depicting the structure of a typical cereal embryo (a) at the coleoptilar stage, which in maize occurs approximately 8 to 14 days after pollination, and (b) at the later third stage (about 22 to 28 days after pollination in maize) , respectively; (c) the model shoot tip of an angiosperm, including cereals, shown in longitudinal section; and (d) the shoot and root structures which pertain in cereals generally, with a unit phytomer of the shoot highlighted.
• FIGURE 2 is a schematic representation of transformation methodology according to the present invention.
• the nonlethal selection also can promote the development of a short-lived, mericlinal L2 event into a stable, periclinal event in which most or all of the cells contributing to the germ line (i.e., the L2 layer) are transformed.
• selective pressure can promote LI- to-L2 conversion events, thus increasing the probability of germ line transmission.
• the shoot apical meristem in cereals is highly variable between species. In most species, however, a stratified meristem exists that is composed of two or three visible layers which generate the entire shoot: a superficial LI, a subsurface L2 and, in some cases, a deeper L3 layer.
• the outer layer(s) comprise the tunica, which are characterized by anticlinal cell divisions. In contrast, divisions in the innermost layer, the corpus, occur randomly, both anticlinally and periclinally.
• the meristem is believed to be composed of only two layers, LI and L2, and possibly a third L3 layer. See Poethig, CONTEMPORARY PROBLEMS IN PLANT ANATOMY 235-39 (1984) . Cell differentiation to delimit the major tissues of the shoot is position-dependent rather than lineage-dependent.
• the epidermis is generated almost exclusively by the LI layer, with the L2 layer contributing to the germ line.
• the process of introducing a foreign gene into a subset of apical meristem cells one creates a plant that necessarily is "chimeric," i.e., a plant in which portions have been altered in genetic composition.
• chimeric plants There are three major categories of chimeric plants, based on the characteristic pattern of genetic differences: (1) sectoral chimeras, in which a portion of the plant is "genetically distinct" through all cell layers by virtue, for example, of displaying a mutant somatic phenotype, a change in chromosome number, or the presence of transformed cells; (2) periclinal chimeras, in which an entire cell layer (LI alone or L2 alone, for instance) is different from the rest of the plant; and (3) mericlinal chimeras, which represent an intermediate between the other two types, i.e., a genetic difference characterizes only a portion of one layer.
• sectoral chimeras in which a portion of the plant is "genetically distinct" through all cell layers by virtue, for example, of displaying a mutant somatic phenotype, a change in chromosome number, or the presence of transformed cells
• periclinal chimeras in which an entire cell layer (LI alone or L2 alone, for instance
• biolistically denote an approach to genetic transformation described, for example, in U.S. patents No. 4,945,050 and No. 5,141,131, the respective contents of which are hereby incorporated by reference.
• force is transmitted to small particles that carry DNA, for example, coated on particulate surfaces or absorbed into the particles, in such a way that the exerted pressure forces particles into a targeted cell or tissue
• microprojectiles or “microcarriers.”
• the microprojectiles are propelled at the biological sample, accelerating to such speed that, upon impact, they penetrate cellular surfaces and are incorporated into the interior of a cell or cells in the sample.
• the microprojectiles should have an average diameter sufficiently small to permit penetration of and retention by cells of the biological sample without killing the cells.
• Particles of gold or tungsten in the size range of about 0.1 to 4 microns, are illustrative of microprojectiles that are suitable for delivering exogenous nucleic acid into a host.
• Other types of biolistic delivery vehicles are disclosed, for example, by U.S. patents No. 5,120,657 (electrical discharge propels a carrier sheet toward target) and No. 5,240,842
• the present invention contemplates the biolistic targeting of apical meristem cells at an early developmental stage.
• meristem cells are bombarded at a developmental phase that is no later than the coleoptile-ring stage, when the apical dome is fully exposed, lacking protection from leaf primordia, and is composed of fewer cells in the meristem than are present at later stages.
• the stages of maize embryogenesis are described in detail by Poethig et al . , Developmental Biology 111 : 392-404 (1986) , the contents of which are incorporated by reference.
• the transformation method of the present invention focuses on coleoptilar and earlier stage embryos, namely, early proembryo, mid proembryo, late proembryo, and the transitional stages of embryo development.
• the meristem is not defined; instead, a group of cytoplasmically dense cells undergo more rapid division and, ultimately, form the apical meristem.
• DNA is introduced (i) into cells that make up the meristem proper (i.e., at the coleoptilar stage) or, (ii) in the earlier stages of development, into cells that are destined, by position or fate, to contribute to the meristem.
• Biolistic bombardment according to the present invention is effected by orienting the embryo so that cells that are within a meristem or that are destined to contribute to the meristem are exposed directly to the biolistic projectiles. In late proembryos, the axis side of the embryo is slightly flattened, allowing this side of the embryo to be placed face up (away from the agar) for bombardment.
• Transition stage and coleoptilar stage embryos are similarly oriented. There is no such orientation, however, for mid and early proembryos on agar after isolation (i.e., before shooting). Rather, when proembryos are placed in a random orientation on the agar medium, the meristem apparently develops on the upper side of the embryo (away from the medium) . Thus, placement on the medium may be stimulating the embryo to re-orient its growth axis, for example, by virtue of the in vitro conditions which are provided (i.e., the new hormonal gradient that is being established within the embryo) .
• a convenient and, hence, preferred source of meristems for use in the present invention are coleoptilar stage embryos.
• the coleoptilar stage of cereal embryo development the coleoptile is visible as a ring of leaf primordium surrounding an exposed meristem.
• the early coleoptilar stage can generally be obtained 10 to 12 days after pollination.
• the days-after-pollination criterion, or "DAP, " is affected by embryonic environment and genotype, and therefore is an adjunct to developmental staging based on morphology, which is an important criterion for timing of transformation in the present invention.
• DAP days-after-pollination criterion
• the boundary of the meristem is distinct, with a visible tunica and corpus (LI and L2 layers, respectively) .
• a particularly preferred source of target cells for use in the present invention are present in the early proembryo, mid proembryo, late proembryo and the transitional stages in embryo development.
• the early proembryo, mid proembryo, late proembryo and the transitional stages can generally be isolated 2, 4, 7-8 and 8-10 DAP, respectively (see Poethig et al . (1986), cited above) .
• the developmental stage is the important criterion. Rate of development and, hence, DAPs at which embryos are isolated vary with growth environment and genotype.
• immature staminate inflorescences tassels
• pistillate inflorescences ears
• transformation can serve as sources of meristems for transformation in accordance with the present invention.
• "Immature” here denotes a developmental state when floral meristems still are developmentally plastic, i.e., are capable of shoot differentiation. This developmental plasticity should be exploitable, pursuant to the present invention, for transformation of many Graminaceous species, given the recognized similarities in inflorescence development among the grasses.
• a trained technician can isolate 200 to 600 mid proembryo, late proembryo, transitional or coleoptilar- stage embryos per day, the ease of isolation and number of isolated embryos increasing with embryo size.
• Another important advantage associated with using floral explants as meristem sources is that many genotypes exhibit better meristem growth and shoot multiplication when the floral explants are the starting material. This advantage is pronounced, for example, with respect to an inbred maize line with a lineage that includes line PHV78.
• immature embryos are the preferred explant for some genotypes, such as maize inbreds having a lineage including line PHBW8. Access to both options significantly extends the genotype range for meristem transformation pursuant to the invention.
• the bombarded cells are subjected to a first, nonlethal selection pressure in the course of generating plantlets which are grown out directly (course I in FIGURE 2) or, alternatively, are subjected to meristem reorganization, induced mechanically or hormonally, in advance of a second nonlethal selection (course II in FIGURE 2) .
• a first, nonlethal selection pressure in the course of generating plantlets which are grown out directly
• meristem reorganization induced mechanically or hormonally, in advance of a second nonlethal selection
• course II in FIGURE 2 the aspects of nonlethal selection and induced meristem reorganization are addressed in greater detail below.
• the developmental fate of cells within the meristem normally is rigidly determined. Thus, transformation of a particular cell within the meristem typically will result in a small transgenic sector made up only of the descendants of that cell.
• a selective growth advantage is imparted to transformed cells in the form of NPTII-encoded resistance to tobramycin, kanamycin or a related compound. It is acceptable, however, to confer resistance to another "bleaching" antibiotic (by means of a streptomycin-resistance gene, for instance) or herbicide, for example, by transformation with the crtl gene, which imparts resistance to norflurazon.
• the present invention contemplates similar non-lethal strategies which entail the use of other selective agents, such as bialaphos and hygromycin, with a corresponding, resistance-imparting gene, so long as the resulting selective pressure retards the growth of non-transformed cells relative to cells in the transgenic sector.
• a model experiment in this regard would involve exposing samples of isolated meristem tissue to a graded series of dilutions of the selection agent in the medium of choice, and then determining a concentration threshold below which the selective pressure favoring transformed cells is not so stringent as to be detrimental to general meristem development. While this approach typically would result in continued meristem growth during selection, the present invention also envisages establishing conditions of little or no meristem growth ("static conditions") which are punctuated by brief exposure(s) to a higher concentration of selection agent (“pulsed selection”) which otherwise would adversely affect overall meristem development.
• cereal transformation according to the present invention optionally involves a reorganization of the meristem, for example, by wounding of the apical dome. While other methods of wounding also result in reorganization, a preferred method is to pierce the apical dome using a micromanipulation needle. The reorganization thus effected alters growth in the apical dome and, it has been discovered, prompts a proliferation of multiple meristems which, in turn, enhances transformation frequency and sector size. For example, mechanically-induced meristem proliferation in conjunction with selective pressure results in an increase in frequency and size of the transgenic sectors observed in subsequent leaves.
• Meristem reorganization may precede biolistic treatment, followed by germination and selection leading to the production of chimerically transformed plants (course I) .
• mechanical wounding can be performed after bombardment of the meristems in order to effect a proliferation of meristems.
• the sectors can enlarge because the reorganized meristems are derived from a smaller number of cells and, hence, the percentage of transformed cells in the meristems is increased.
• a reorganization is brought about by hormonally-induced shoot multiplication with respect to the developing shoot meristem of a plantlet selected for the presence of a transformed sector.
• the hormonally induced reorganization need not be exclusive of the optional, mechanically induced reorganization mentioned above, and brings about meristem proliferation via shoot multiplication.
• the developing shoot meristem first is localized, typically in a swelling that occurs in the germinated plantlet at the junction between the mesocotyl and the epicotyl (see FIGURE 2) .
• a section of 2 to 3 mm in size which contains the meristem then can be excised at the swelling point and cultured on a shoot proliferation medium of the sort described, for example, by Lowe et al., Plant Science 41: 125 (1985), and by Zhong et al . , Planta 187: 483 (1992), respectively.
• meristems typically are cultured on MS medium with 2 mg/1 BAP (6-benzyl- aminopurine) , 3% sucrose and 9 mg/1 agar.
• BAP 6-benzyl- aminopurine
• a shoot multiplication medium will utilize a cytokinin, such as Kinetin, BAP, Thidiazuron or Zeatin, at a concentration between 0.5 and 10 mg/1.
• auxin also may be required in some genotypes.
• Murashige and Skooge (MS) salts are adequate but probably not optimal, in that preliminary experiments using media with ammonium levels higher than those in MS resulted in an improved culture response.
• Additional additives such as the auxin transport inhibitor, TIBA, and ethylene inhibitors like silver nitrate and cefotaxime also appear to be beneficial.
• the shoot proliferation medium forces the generation of a few to hundreds of shoots from each excised shoot meristem, thereby increasing ' the likelihood of obtaining a subpopulation of shoots, some of which may arise from a transformed sector.
• a significant and reproducible percentage of the resulting shoots are periclinal chimeras and, hence, are "stabilized” in the sense that genetic homogeneity is promoted within a cell layer, such as the L2 layer, that ultimately contributes to germline transmission.
• the large population of induced shoots is screened to identify non-sectored, periclinal chimeras. This is accomplished via a nonlethal assay which brings about an enrichment of transformed cells through the use of selective agents (i) that bleach normally green tissue at levels that do not inhibit growth or (ii) that inhibit growth of non-transformed meristem sectors without significantly reducing viability of the meristems.
• one aspect of the present invention relates to forcing meristem reorganization, before bombardment, after bombardment or both, by suppressing cell growth through selective wounding of the apical dome, prompting generation of multiple meristems, or by exposing excised meristems to hormonal stimuli likewise leading to multiple meristems, albeit in the form of proliferated shoots.
• the axillary bud of a transformed plantlet can be dissected out, from just above a leaf base, when a chimeric sector is observed in a substantial portion of that leaf.
• the isolated axillary bud represents an additional meristem that can be grown into a whole plant, or taken through a brief cycle of shoot multiplication as described, thereby to obtain a more homogeneously transformed plant.
• Another method of stabilizing transgenic sectors is to induce tillers in the transformed plant. In those cases in which transgenic sectors are limited to the lowermost leaves or domains of maize plants, tillering is induced to stabilize these transgenic sectors.
• a wide range of cereal varieties can be transformed stably, in a genotype-independent manner, for the first time.
• the genes implicated in this regard include but are not limited to those categorized below. Genes That Confer Resistance To Pests or Disease And That Encode;
• a Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon See, for example, Geiser et al., Gene
• DNA molecules encoding ⁇ -endotoxin genes can be purchased from American Type Culture Collection (Rockville, MD) , under ATCC accession Nos. 40098, 67136, 31995 and 31998.
• C A vitamin-binding protein such as avidin. See U.S. patent application serial No. 07/911,864, the contents of which are hereby incorporated by reference. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.
• E An insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al . , Nature 344: 458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.
• F An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol . Chem. 269: 9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor) , and Pratt et al . , Biochem. Biophys . Res . Comm. 163:
• an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of
• (M) A viral-invasive protein or a complex toxin derived therefrom.
• the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses.
• Coat protein- mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus.
• N An insect-specific antibody or an immunotoxin derived therefrom.
• an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect.
• O A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.
• (Q) A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10: 305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease. Genes That Confer Resistance To A Herbicide.
• (A) A herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea.
• Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee et al., EMBO J. 7: 1241 (1988), and Miki et al. , Theor. Appl .
• a DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession No. 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. patent No. 4,769,061 to Comai. European patent application No. 0 333
• nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L- phosphinothricin.
• the nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European application
• C A herbicide that inhibits photosynthesis, such as a triazine ⁇ psbA and gs+ genes) and a benzonitrile (nitrilase gene) .
• Przibilla et al., Plant Cell 3: 169 (1991) describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in
• Higher lysine content A cereal such as maize could be transformed with a gene that increases lysine content, making the cereal nutritionally more complete and thereby eliminating need for added lysine, for example, in poultry and swine feeds.
• Higher methionine content A gene would be added to increase methionine levels in a cereal crop to offset an overall low methionine content, for example, in a poultry feed which combines lower- and higher-methionine components such as soybean and maize, respectively.
• B Decreased phytate content
• C Modified carbohydrate composition effected, for example, by transforming maize with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al . , J. Bacteriol . 170: 810 (1988) (nucleotide sequence of Streptococcus mutans fructosyltransferase gene), Steinmetz et al . , Mol . Gen . Genet . 200:
• Maize lines that can be transformed via the present invention include, among others, inbreds that are employed in producing commercial hybrids. These inbreds, both proprietary and publicly-available, span many heterotic families. The preferred representatives within the heterotic groupings, and the relative use of entire heterotic patterns, vary with the market in question. For example, different germplasm is favored when breeding for the continental United States, including different geographic areas of adaptation (for example, the South, the East, the West, the North and the Central Corn Belt) , for Europe and for South America, as well as for other international markets.
• Callus-mediated methodology is unsuitable for many inbreds which do not produce the required callus response or which provide callus that grows in a manner rendering the methodology unusably inefficient ("recalcitrant" inbreds) . Accordingly, such methodology has been limited to a large extent to transformation of a few genotypes, such as, in maize, A188, A188 x B73, H99, Pa91, FR16 and genotypes obtained via a cross involving one of these genotypes.
• meristem transformation pursuant to the present invention is applicable to any line, regardless of how that line responds to callus-mediated transformation. Thus, even cereal lines heretofore deemed recalcitrant to transformation can be transformed stably via the present invention.
• the present invention should be applicable to newly-developed inbreds and to new heterotic groups which are created through the combination of existing germplasm, including "exotic" material brought into breeding programs from sources in the tropics and elsewhere. According to a preferred embodiment, therefore, the present invention contemplates a transgenic plant that belongs to a cereal line that is recalcitrant to callus- based method transformation.
• transgenic maize plants that are not produced by transformation of A188, A188 x B73, H99, Pa91 or FR16.
• the phrase "cereal line” denotes a group of gramineous plants of the sub-family Poaoideae which display relatively little variation between individuals with respect to more than one distinctive trait, generally although not exclusively by virtue of several generations of self-pollination.
• line here is used sufficiently broadly to include a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques.
• a plant is said to "belong” to a particular line if it (A) is a primary transformant (T 0 ) plant regenerated from material of that line or (B) has a pedigree comprised of a T 0 plant of that line.
• T 0 primary transformant
• B has a pedigree comprised of a T 0 plant of that line.
• the term “pedigree” denotes the lineage of a plant, e.g., in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
• 50 ⁇ l of the sonicated tungsten microprojectile suspension were pipetted into a 1.5 ml tube, to which was added 1 to 10 ⁇ g of the foreign DNA. After mixing, 50 ⁇ l of 2.5 M CaCl 2 solution were added and, with further mixing,20 ⁇ l of 0.1 M spermidine also were introduced.
• Ears of a proprietary maize genotype were harvested seven days after pollination at the early coleoptilar stage of development. Harvested ears were surface- sterilized in 50% Chlorox with Tween 20 for 20 minutes, and then rinsed three times with sterile deionized water. Kernel tops were removed with a scalpel and embryos were dissected from endosperm.
• DP6212 contains the 2xhistone-143 promoter, the first intron of the maize ADH1 gene, the ⁇ ptll gene encoding neomycin phosphotransferase (NPTII) , and a 3' transcript processing region from the Proteinase Inhibitor II ⁇ Pinll) gene of potato.
• DP3953 contains the ubiquitin promoter, the first intron of the ubi gene, the gene encoding 3-glucuronidase (GUS) , and a 3' transcript processing region from the Pinll gene.
• Embryos were bombarded with DP6212 and DP3953, mixed at a 1:1 ratio, at a concentration of one 1 ⁇ g of total DNA per tube of acid-washed tungsten particles. This concentration, at ten times less than the standard, was optimal for yielding transformants with more uniform GUS staining patterns and yet having no detrimental effect on the function of the selectable marker or the frequency of transformation.
• the particles were delivered as five 5- ⁇ l shots per tungsten tube, using a PDS-1000 Helium gun with 1100 p.s.i. rupture disks. All embryos received one bombardment per plate.
• embryos were maintained at 28°C in the dark for seven days on Maturation Medium. Embryos then were transferred to 272K shoot elongation medium (MS salts, 0.1 g/L myoinositol, MS vitamins, 30g/L sucrose, and 4g/L gelrite) which contained 150 mg/L tobramycin sulfate as the selection agent. Embryos were incubated in the light at 28°C. At the time of transfer, embryos had elongated cotyledons.
• 272K shoot elongation medium MS salts, 0.1 g/L myoinositol, MS vitamins, 30g/L sucrose, and 4g/L gelrite
• Transgenic plants maturing in the greenhouse were sampled for GUS activity or NPTII protein in each new leaf and in tassel and ear tissues to characterize the expression pattern in each plant. Pollinations were completed as selfs or as sibs. Eight to ten days after pollination, embryos were rescued by harvesting and surface sterilizing the ears, excising the embryos and placing the embryos on shoot elongation medium for germination. (This procedure was not required but was preformed to accelerate the analysis * process.) Tl leaf tissue was sampled for GUS histochemical assays and painted with 2% kanamycin sulfate in 0.2% SDS buffer to verify transmission of the transgenes. Samples of mature leaves were harvested from the TO transformant for Southern analysis to further characterize the transformation.
• Genotype NIOOOO and several other proprietary genotypes designated “P10000,” “W20000,” “E10000,” “PHP02” and “R20000,” respectively, for purposes of this description, were transformed with plasmids DP6212 and DP3953 as described above.
• Table 2 below enumerates data showing that the non-lethal selection method is applicable to various maize lines, based on co- transformation experiments where the expression of the second nonselected gene was used to assess stable sector frequency and size.
• Ears of genotypes E10000 and W20000 were harvested at the early coleoptilar stage of development and at 11 and 9 days, respectively, after pollination.
• One hundred and sixty embryos were isolated and incubated on maturation medium, as described above.
• the apical dome of several embryos was disrupted prior to bombardment to force the meristem to reorganize and form new meristematic areas.
• Mechanical disruption was performed by means of micromanipulation needles, ranging from 0.5 ⁇ m to 5 ⁇ m in diameter, which were attached to a World Precision
• Instruments M3301 micromanipulator Needle penetration of each embryo was effected in the center of the apical dome to a depth ranging from a few microns to a few hundred microns, depending on the morphology of the embryo (embryos with a larger scutellem will tolerate deeper penetration) .
• the preferred targeted depth of penetration was between 50 ⁇ m and 150 ⁇ m.
• Embryos then were bombarded with NPTII and GUS constructs, as described above. Embryos were maintained in the dark at 28°C for seven days on Maturation Medium and then transferred to 272K medium containing 150 mg/L Tobramycin sulfate. At time of transfer, embryos had multiple meristem formation with elongated cotyledons. Embryos were incubated in the light at 28°C.
• Embryos at the coleoptilar stage were isolated and cultured scutellum side down on an embryo maturation medium (10-20 embryos/plate) . Since there can be considerable seasonal and genotypic variation affecting embryo ontogeny, embryo stage rather than size or days after pollination, was monitored carefully. Embryos typically were matured on MS-based medium containing 0.5 mg/L zeatin, 1 mg/L indoleacetic acid, and elevated sugar levels which serve as an osmoticum. The embryos were cultured for a period ranging from 0-48 hours post-isolation, with 12-24 hours being optimal. Meristems then were bombarded with genes conferring kanamycin or streptomycin resistance, along with other nonselected genes, such as agronomic or visual marker genes.
• the embryos were cultured in the dark to promote germination. After one to two weeks, the embryos were moved to a germination medium, such as hormone-free or low-hormone MS medium.
• a germination medium such as hormone-free or low-hormone MS medium.
• the germinated plantlets generally had a swelling at the junction between the mesocotyl and epicotyl. This swelling occurred in the region containing the developing shoot meristem.
• Two to three millimeter sections including the meristem were excised and cultured on a shoot proliferation medium which contained the appropriate hormones and a selection agent.
• the sections were regularly trimmed of elongated leaves and transferred to fresh medium every 10 to 14 days.
• Cultured meristems were incubated at 28 ⁇ C in the dark. After three to nine weeks, the proliferating meristems were transferred to an illuminated culture room.
• Transformed sectors were identified one to two weeks after culture in the light, based on their green phenotype, i.e., nontransformed tissue remained bleached upon selection.
• plants were regenerated by lowering the hormone concentration, although in some genotypes cytokinin concentrations were increased to promote plant regeneration. Since regenerated plants sometimes have difficulty rooting, rooting was promoted by culture on SH medium with 1 mg/1 NAA, or by nicking the base of the stem and dipping the shoots in a 1 mg/ml NAA solution.
• One hundred eighty coleoptilar-stage maize embryos of the Honey and Pearl variety were harvested nine days after pollination.
• the scutella of the isolated embryos averaged 0.48 mm in length. These embryos were placed on embryo Maturation Medium (10 embryos per plate) and cultured overnight in the dark at 28°C.
• Plasmid DP551 contains ADH intron 1, GUS gene, and nos terminator, as well as ADH intron 1, NPTII gene, Pinll terminator. Both GUS and NPTII genes are regulated by 35S CaMV sequences. Plates containing these embryos were cultured and matured in the dark at 28°C. Eight days later, a few of the embryos were placed in X-Gluc histochemical stain. All embryos contained intense blue staining, indicating GUS activity.
• Plates 1 to 4 were bombarded with plasmid DP4790, which contains a 35S CaMV promoter, omega' , aadA and ocs terminator (provided by Dr. Jonathan Jones, John Innes Institute), and with plasmid DP460, which contains a 35S CaMV promoter, ADH intron, GUS gene, and nos terminator. Plates 5 to 8 were bombarded with plasmids DP4790 and DP3536. The latter plasmid contains a cab promoter, ADH intron 6, GUS gene, and ocs terminator. All embryos were grown and germinated as described in part (B) of this example, supra.
• the regions containing the meristems were cultured on agar- solidified MS medium containing 2 mg/L BAP and 100 mg/L streptomycin sulfate. After cultured meristems were moved to an" illuminated culture room, a green sector was observed on a proliferating meristem on plate 6. All other cultured meristems were white due to streptomycin bleaching. GUS staining at this time revealed a mix of sectored and non-sectored blue staining leaves.
• coleoptilar stage embryos of an elite inbred designated "B30000" for purposes of this description, were isolated and cultured on 288L medium in fifteen plates containing twenty embryos per plate. Twelve plates were bombarded, using standard protocols. Briefly, particle bombardment was performed with six shots using 650 psi rupture disks and 1 ⁇ m tungsten particles, which were coated with plasmids DP5397 (proprietary agronomic gene) and DP5606 (Ubi promoter/Ubi-intron/ NPTII/pin II terminator linked to cab promoter/ADH intron 6/GUS/ocs terminator) at a concentration of 5 ⁇ g DNA/particle preparation tube for each plasmid.
• DP5397 proprietary agronomic gene
• DP5606 Ubi promoter/Ubi-intron/ NPTII/pin II terminator linked to cab promoter/ADH intron 6/GUS/ocs terminator
• Plasmid DP5397 is a proprietary agronomic plasmid which contains a Bt gene, while plasmid DP5606 contains the Ubi promoter, Ubi intron, NPTII gene, and Pinll terminator, which is linked to a cab promoter, ADH intron 6, GUS gene, and ocs terminator.
• agar solidified MS medium containing 2 mg/L BAP, 0.25 mg/L, 2,4-dichlorophenoxy acetic acid and 3% sucrose.
• Five weeks after bombardment meristems were placed on kanamycin selection (100 mg/L) . To avoid irreversible bleaching of the meristems, this tissue was cycled between selective and non-selective media.
• a preferred medium used in the initial stage of floral meristem culture (used for various genotypes) consisted of Murashige and Skoog salts, MS vitamins, 0.1 mg/1 2,4-D, 0.5 gm/1 6- BAP, 1-proline at 12.2 ⁇ M, 8% sucrose, and silver nitrate at 30 mg/1.
• a preferred gelling agent is GELRITE (product of Merck and Co, Inc./Kelco division, Rahway, NJ) at 3.5 g/1. (D) Bombardment
• Immature ear explants were bombarded using 650 psi rupture disks and a stainless steel screen (100 um mesh size) suspended approximately 0.5 to 1.0 cm above the tissue. DNA precipitation and other bombardment parameters were as described in Example 1.
• Putatively transformed shoots clumps were transferred to medium lacking plant growth regulators. Varying degrees of leaf development occurred on 1 mg/1 BAP, and shoots soon formed and elongated in the absence-of hormones.
• Rooting at high frequency was effected via several days of exposure to MS- or SH-based media with 1-5 mg/1 NAA.
• PROEMBRYO, LATE PROEMBRYO, TRANSITIONAL AND EARLY COLEOPTILAR-STAGE EMBRYOS Immature embryos at the mid proembryo, late proembryo, transitional and early coleoptilar stage were harvested and cultured on culture medium 610A, containing high concentrations of cytokinin and osmoticum.
• the 610A culture medium comprised MS salts, MS vitamins, 100 mg/L myo-inositol, 0.4 mg/L thiamine- HC1, 1 mg/L zeatin riboside, 0.1 mg/L BAP, 60 g/L sucrose, 400 mg/L asparagine, and 7 g/L Hazelton TC agar. After one day of recovery, the embryos were bombarded with DNA, by means of the particle gun as described above, and punctured in the center of the area of where the apical meristem will develop with a 0.5 ⁇ m micromanipulation needle.
• Embryos were allowed to mature for 7 days in the dark and then transferred to a hormone-free medium containing l mg/1 bialaphos. Following another 7 days of culture on hormone-free medium m the dark, the embryos were transferred to germination medium, and cultured in the light for continued germination. As leaves developed, plant phenotype was observed and samples were taken to check for sector formation by histochemical assay (GUS) as described above.
• GUS histochemical assay
• embryos were cultured at day 0, axis up, onto modified 610A medium, containing 150 g/1 sucrose, 1 mg/1 zatin, and 12 g/1 agar, and incubated at 28 ⁇ C overnight in the dark.
• modified 610A medium containing 150 g/1 sucrose, 1 mg/1 zatin, and 12 g/1 agar
• the apical meristems of all embryos were disrupted in the center of the apical dome using a 0.5 ⁇ m Femtotip micromanipulation needle. All embryos were returned for an overnight incubation at 28C in the dark.
• bombardment was effected with the PDS-1000 Helium particle gun, one shot per plate, using 650PSI rupture disks.
• DNA employed in this regard was DP3528+DP3953 [2x35S: :BAR+UBI: :GUS] at 1 ⁇ g/tube of 1- ⁇ m tungsten.
• day 2 all embryos were maintained in the dark at 28°C for 7 days on 610A medium to allow meristem maturation to occur.
• day 7 (after 7 days on 610A) , embryos were transferred to 612 medium containing MS salts and vitamins, 0.001 mg/1 kinetin, 0.1 mg/1 adenine sulfate, 20 g/1 sucrose, 6 g/1 agar and 0.5 mg/L bialaphos, for germination and selection.
• the total number of embryos cultured and bombarded was 48, of which 37 developed normally.
• the number of embryos that grew beyond leaf 1 was 17, with four plants showing GUS expression.
• SID 180741 and 180742 both showed GUS expression at the time of greenhouse transfer and had normal leaf and root development, whereas all other plants died. SID 180742 showed GUS expression in leaves 1-8 only.
• Transformant 180741 was bombarded by the particle gun at the late proembryo stage of development, before meristem layer organization occurs. It contained a saddle sector which, by definition, is a sector which traverses the apical dome and bisects the meristem, in a region of the meristem that will later develop into the tassel (see Poethig (1986) , supra) . It also was wounded by a micro-manipulation needle, to encourage meristem reorganization, and exposed to bialaphos as the selective agent. GUS histochemical data showed expression in both layers of the leaves, in the anther wall, and in about 50% of the pollen from the central stalk.
• tillering of transformed plants is an alternative to shoot multiplication for stabilization of transgenic sectors. Accordingly, elite lines may be induced to tiller, pursuant to the present invention, thereby stabilizing transgenic sectors.
• tillering was induced in control plants using the method described by De Wolff, Euphytica 2J2.: 524-26 (1971) .
• a triangular incision was made with a number 11 scalpel blade at the approximate height of, or slightly above, the shoot apex of two week-old seedlings. The incision was made perpendicular to the plane of the leaves in order to avoid damage to the midribs. The shoot apex was removed from P10000, PHP02, G30000 and E10000 seedlings. Each of these genotypes represents inbreds from significantly different heterotic families. Untreated plants of the same genotype were used as controls. If the incision was too far above the apex the procedure was repeated just below the initial incision.
• the wounded plants and untreated controls were maintained in 24 hour continuous light (greenhouse during the day, growth chamber by night) for two weeks. A replicate treatment was grown under light/dark conditions. Significant tillering was observed in the plants from which apices were removed. The influence of continuous light on tillering frequency, relative to normal light/dark conditions, was variable and may depend on genotype. The untreated controls did not tiller.
• the hole made by the incision could have been plugged with lanolin and phytohormones, such as TIBA (1 mg/L) or BAP (10 mg/L) , to increase tillering frequency.
• phytohormones such as TIBA (1 mg/L) or BAP (10 mg/L)
• selective agents such as kanamycin could have been added to the incision to identify and select transgenic sectors.

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