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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
  • A01N37/46—N-acyl derivatives
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
  • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
  • C07K14/8114—Kunitz type inhibitors
  • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• This invention relates to materials and methods for killing insect larvae which are harmful to plants, and materials and methods for imparting insect resistance to plants, material harvested from the plants, and products derived from the harvested material.
• insects are serious pests of common agricul ⁇ tural crops.
• One method of controlling insects has been to apply insecticidal organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems.
• a more recent method of control of insect pests has been the use of biological control organisms which are typically natural predators of the troublesome insects. These include other insects, fungi (milky-spore) and bacteria (Bacillus thuringiensis cv., commonly referred to as "Bt").
• Bact Bacillus thuringiensis cv.
• this invention provides a method for killing susceptible insect larvae, including larvae of European corn borer and corn rootworm comprising administering enterally to the larvae a larvicidal amount of aprotinin or a serine proteinase inhibitor which is at least 90% homologous to aprotinin by amino acid sequence.
• insect and larva although not equivalent when used specifically, should be understood to include both adult and larval forms of a species when used generically.
• insect resistance should be understood to include resistance to larval forms as well as adults, and “larvicidal” materials should be considered insecticidal, particularly since killing larvae produces a corresponding absence of adults.
• the proteinase inhibitor can be effectively applied to plants, harvested materials or products consumed by the insects by spray, dust or other formulation common to the insecticidal arts.
• harvested plant material herein is meant any material harvested from an agricultural or horticultural crop, including without limitation grain, fruit, leaves, fibers, seeds, or other plant parts.
• Products derived or obtained from such harvested material include flour, meal, and flakes derived from grain; and products in which such materials are admixed, such as, for example, cake, cookie, muffin, pancake and biscuit mixes.
• the larvicidal proteinase inhibitor can be incorporated into the tissues of a susceptible plant so that in the course of infesting and consuming the plant, its harvested material, or a product derived from harvested plant material, the larvae consume larvicidal amounts of the proteinase inhibitor.
• One method of doing this is to incorporate the proteinase inhibitor in a non-phytotoxic vehicle which is adapted for systemic administration to the susceptible plants.
• This method is commonly employed with insecticidal materials which are designed to attack chewing insects and is well within the purview of one of ordinary skill in the art of insecticide and larvicide formulation, but is a method which may not be as suitable for active enzyme blockers such as proteinase inhibitors.
• a dietary bait containing one or more of the selected proteinase inhibitors can be employed, with, optionally, an added pheromonal or other larval attractant material.
• the genes which code for these peptides can be isolated and cloned. Alternatively, they can be synthesized directly using a DNA sequence obtained by working backwards from the known amino acid sequence for aprotinin or a related proteinase inhibitor, preferably using plant-preferred codons.
• the resulting sequence can be inserted into an appropriate expression cassette and introduced into cells of a susceptible plant species, so that an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA sequence coding for one or more insecticidal proteinase inhibitors selected from aprotinin and serine proteinase inhibitors having at least 90% homology to aprotinin by amino acid sequence, in proper reading frame relative to transcription initiator and promoter sequences active in the plant. Transcription and translation of the DNA sequence under control of the plant-active regulatory sequences causes expression of the larvicidal gene product at levels which provide an insecticidal amount of the proteinase inhibitor in the tissues of the plant which are normally infested by the larvae.
• Insecticide-resistant insects become a problem as a result of application of strong selection pressure which highly favors naturally resistant individuals and any resistant mutants which occur. As a result, over the course of a few generations the resistant insects become the predominant type. Heavy application of insecticidal materials generally to a field or a geographical area by dust or spray or by soil incorporation tends to impose strong selection pressures of the kind described, since insects have no "safe havens" where non-resistant individuals can survive. However, many insect pests of crop plants also attack non- crop species.
• This method also offers advantages from the standpoint of soil and groundwater contamination, since no application vehicle is required.
• the insecticidal components themselves are of natural origin and break down naturally when the plant is digested or decomposes.
• the method offers further advantages from the standpoint of cost, since no application expense is involved and the cost of the insecticidal materials is factored into the price of the seed or other reproductive material which the grower purchases.
• the plant should be a plant which is susceptible to infestation and damage, or whose harvested material or products are susceptible to infestation and damage by the larvae of European corn borer and corn rootworm.
• the methods of this invention are readily applicable via conventional techniques to numerous plant species, if they are found to be susceptible to the plant pests listed hereinabove, including, without limitation, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Triticum, and Datura.
• Preferred plants that are to be transformed according to the methods of this invention are cereal crops, including maize, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean, fiber crops, such as cotton, fruit crops, such as melons, and vegetable crops, including onion, pepper, tomato, cucumber, squash, carrot, crucifer (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce.
• cereal crops including maize, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean
• fiber crops such as cotton, fruit crops, such as melons
• vegetable crops including onion, pepper, tomato, cucumber, squash, carrot, crucifer (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce.
• the DNA sequence which when expressed imparts insecti ⁇ cidal activity is a structural gene which codes for aprotinin, or a proteinase inhibitor having at least 90% homology to aprotinin. It has been found that these proteinase inhibitors have sufficient insecticidal (larvicidal) activity to be operative in a plant cell expression system. That is, while certain other proteinase inhibitors such as cowpea trypsin inhibitors have some larvicidal activity at high concentrations in pure form, plant cell expression at such high concentrations is either not possible in a living plant cell system, or is not feasible if the commercially useful characteristics of the plant are to be preserved in terms of production of oils, starches, fibers, or other materials.
• a tissue-specific promoter can be used in any instance where it may be desirable to localize production of the proteinase inhibitor to an infested tissue or to a tissue which is efficient in production of the proteinase inhibitor.
• expression cassette is meant a complete set of control sequences including initiation, promoter and termination sequences which function in a plant cell when they flank a structural gene in the proper reading frame.
• Expression cassettes frequently and preferably contain an assortment of restric ⁇ tion sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence.
• the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA.
• vector herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell.
• the vector has one or more endo- nuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme.
• Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as selectable markers to identify and separate transformed cells.
• Preferred selection agents include kanamycin, chlorosulfuron, phosphonothricin, glyphosate, hygromycin and methotrexate, and preferred markers are genes conferring resistance to these agents.
• a cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant" .
• a particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule that is not a part of the chromosomes of the cell.
• genomic, synthetic and cDNA encoding the gene of interest may be used in this invention.
• the vector of interest may also be constructed partially from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone.
• genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene in the host cell.
• the genetic construct will contain (a) a first genetic sequence coding for the proteinase inhibitor of interest and (b) one or more regulatory sequences operably linked on either side of the structural gene of interest.
• the regulatory sequences will be selected from the group comprising of promoters and terminators.
• the regulatory sequences may be from autologous or heterologou ⁇ sources. Promoters that may be used in the genetic sequence include nos, ocs, phaseolin, CaMV, FMV and other promoters isolated from plants or plant pests.
• An efficient plant promoter that may be used is an overproducing plant promoter.
• Overproducing plant promoters that may be used in this invention include the promoter of the small sub-unit (ss) of the ribulose-1,5-biphosphate carboxylase from soybean (Berry-Lowe et al, J. Molecular and App. Gen., _l:483-498 (1982)), and the promoter of the cholorophyll a-b binding protein. These two promoters are known to be light-induced, in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, An Agricultural Perspective, A. Cashmore, Pelham, New York, 1983, pp. 29-38, G. Coruzzi et al. , J. Biol. Che . , 258;1399 (1983), and P. Dunsmuir, et al. , J. Molecular and App. Gen., _2:285 (1983)).
• the expression cassette comprising the structural gene for the proteinase inhibitor of interest operably linked to the desired control sequences can be ligated into a suitable cloning vector.
• plasmid or viral (bacterio- phage) vectors containing replication and control sequences derived from species compatible with the host cell are used.
• the cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells. Typically, genes conferring resistance to anti ⁇ biotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers.
• an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector.
• the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells.
• Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S . typhimurium, and ⁇ J. marcescens.
• Eukaryotic hosts such as yeast or filamentou ⁇ fungi may al ⁇ o be used in thi ⁇ invention.
• the isolated cloning vector will then be introduced into the plant cell using any convenient technique, includ- ing electroporation (in protoplasts), retroviruse ⁇ , microparticle bombardment, and microinjection, into cells from monocotyledonous or dicotyledonous plants, in cell or tissue culture, to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette.
• the monocotyledonous species will be selected from maize, sorghum, wheat and rice
• the dicotyledonous specie ⁇ will be selected from soybean, sunflower, cotton, rape ⁇ eed (either edible or industrial), alfalfa, tobacco, and Solanaceae such as potato and tomato.
• a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression ca ⁇ ette of this invention.
• Thi ⁇ invention al ⁇ o provide ⁇ method ⁇ of imparting resistance to European corn borer and corn rootworm to plants of a susceptible taxon, comprising the step ⁇ of: a) culturing cell ⁇ or ti ⁇ ue ⁇ from at lea ⁇ t one plant from the taxon, b) introducing into the cell ⁇ of the cell or tissue culture at lea ⁇ t one copy of an expre ⁇ ion ca ⁇ ette compris ⁇ ing a ⁇ tructural gene coding for a proteina ⁇ e inhibitor selected from aprotinin and serine proteina ⁇ e inhibitors having at least 90% homology thereto by amino acid sequence, or a combination of such proteinase inhibitors, operably linked to plant regulatory sequence ⁇ which cau ⁇ e the expression of the protein structural gene in the cell ⁇ , and c) regenerating insect-resistant whole plants from the cell or tissue culture.
• Such intermediate method ⁇ will compri ⁇ e the further steps of a) sexually crossing the insect-resistant plant with a plant from the insect-susceptible taxon; b) recovering reproductive material from insect- re ⁇ i ⁇ tant progeny of the cross; and c) growing insect-re ⁇ i ⁇ tant plant ⁇ from the reproductive material.
• the agronomic characteristics of the susceptible taxon can be sub ⁇ tantially preserved by expanding thi ⁇ method to include the further steps of repetitively: a) backcrossing the insect-resistant progeny with insect-susceptible plant ⁇ from the susceptible taxon; and b) selecting for expression of insect resi ⁇ tance (or an associated marker gene) among the progeny of the back- cro ⁇ , until the desired percentage of the characteristic ⁇ of the susceptible taxon are present in the progeny along with the gene imparting insect resistance.
• Thi ⁇ will be important, for example, where the taxon i ⁇ a ⁇ ubstantially homozygous plant variety, such as an inbred line of maize or a variety of a self-pollinated crop such as ⁇ oybeans.
• substantially homozygous is meant homozygous within the limit ⁇ commonly accepted in the commercial production of certified seed of the species.
• an inbred line of maize used in commercial seed production is typically 95% to 100% homozygous, and preferably 98% to 100% homozygous, as measured by RFLP analysis using 50 to 200 probes well distributed across the genome. If necessary, an RFLP-guided process of self-pollination and selection can be used to achieve thi ⁇ degree of genetic uniformity.
• taxon herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varietie ⁇ , variant ⁇ , and other minor taxonomic group ⁇ which lack a con ⁇ istent nomenclature. It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens or Agrobacterium rhizogenes, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous specie ⁇ .
• thi ⁇ invention provide ⁇ a method for imparting insect resistance in Agrobacterium-su ⁇ ceptible dicotyledonous plants in which the expression cas ⁇ ette is introduced into the cells by infecting the cell ⁇ with an Agrobacterium species, a plasmid of which ha ⁇ been modified to include a plant expre ⁇ ion cassette of this invention.
• the probability of such a double mutation is potentially (if there is no as ⁇ ociation between the mutations) as low as the product of the probabilities of the individual mutation ⁇ , which would be quite low indeed — perhap ⁇ 1.0 x 10 "10 , or less than fifty potentially survivable individuals per year in the entire United States. Also, the low or reduced selection pressure for each individual mutation further reduce ⁇ it ⁇ ⁇ pread within the population.
• this invention also provides a method for killing European corn borer and corn rootworm, comprising administering enterally to the larvae of those species a larvicidal combination of (a) aprotinin, a serine proteinase inhibitor having at lea ⁇ t 90% homology to aprotinin by amino acid sequence, or a combination thereof; and (b) an insecticidal lectin.
• in ⁇ ect ⁇ screened has resulted in several different bioas ⁇ ay ⁇ being u ⁇ ed to determine the effect of aprotinin and combinations of aprotinin plu ⁇ lectin on larval growth and ⁇ urvivor ⁇ hip.
• all of the bioa ⁇ ay ⁇ allow the te ⁇ t material ⁇ to be enterally administered to the insect.
• In vitro bioa ⁇ say ⁇ for the European corn borer (Ostrinia nubali ⁇ ) , and ⁇ outhern corn rootworm (Diabrotica undecimpunctata howardii) were done by incorporating the te ⁇ t protein into the artificial diet.
• Thi ⁇ is referred to herein as an "Incorporated Bioassay”.
• Soybean PI (Bow an-Birk) 4.7 0
• Soybean PI (Bowman-Birk) 2.4 1.5 50 Cystatin 2.5 1.4 30
• Test ⁇ were performed employing Wheat Germ Agglutinin (WGA) , aprotinin, and combinations of the two in 7-day incorporated bioas ⁇ ay ⁇ . The results are shown in Table 3.
• WGA Wheat Germ Agglutinin
• aprotinin aprotinin
• the remainder of the gene can be cloned using restriction enzymes that flank the protein coding region or, more preferably, by cloning the precise protein coding region by oligonucleotide- directed amplification of DNA (polymerase chain reaction or PCR) .
• the gene ha ⁇ can be cloned into a bacterial expre ⁇ ion vector with linker ⁇ added to create all three reading frame ⁇ (u ⁇ ing 8mer, lOmer, and 12mer ⁇ each of which contain an ATG tran ⁇ lational ⁇ tart ⁇ ite).
• the re ⁇ ulting vector ⁇ containing the fragment ⁇ of intere ⁇ t, can be inserted into, for example, BRL's Maximum Efficiency DH5 F' IQ tran ⁇ formation competent E_ ⁇ coli cell ⁇ . All three tran ⁇ formation ⁇ , one for each linker, are then ⁇ creened via minipreps for the presence and orientation of insert. Appropriate clones are then chosen to test for expression of the protein gene.
• Clones containing the properly oriented inserts are grown in culture medium conducive to the induction of the gene (LB medium with added IPTG) .
• the cells are lysed and bacterial protein ⁇ are subjected to electrophoresi ⁇ in SDS polyacrylamide gels and then transferred to nitrocellulose.
• the resulting protein blots are easily screened for presence of protein using rabbit polyclonal and mouse monoclonal anti-protein antibody.
• a plant expression ca ⁇ ette employing the regulatory sequences developed by Beach, et al . , and containing the protein gene, is constructed.
• Thi ⁇ plasmid contains an enhanced 35S promoter spanning nucleotide ⁇ - 421 to +2 of Cauliflower Mo ⁇ aic Virus with the region from - 421 to - 90 duplicated in tandem, a 79 bp Hindlll Sail fragment from pJIIlOl spanning the 5' leader sequence of Tobacco Mosaic Viru ⁇ , a 579 bp fragment ⁇ panning the first intron from maize AdHl-S, and a 281 bp fragment spanning the polyadenylation site from the nopaline synthase gene in pTiT37.
• pPHl412 pla ⁇ mid shown in Figure 2. it differs from pPHl414 in that it lacks the AdH intron segment. However, like pPHl414, it is constructed to have numerous restriction sites between the 0' segment and the NOS segment, which sites can be conveniently used for splicing any desired protein structural gene into position.
• This vector can be cotransformed with a similar pla ⁇ mid containing a selectable marker for antibiotic resistance into Black Mexican Sweet corn protopla ⁇ t ⁇ by electroporation. The ⁇ e protopla ⁇ ts can then be induced to regenerate cell walls and develop into callus by conventional techniques.
• this callus can then be subjected to antibiotic selection to select for transformed colonies, and these colonies can be tested for expre ⁇ ion of protein with antisera for the appropriate protein using known methods.
• the efficiency of protection can be measured by infesting callus (or suspension cultures derived from callus) with the target insect and measuring survival percentages.
• the protein gene can be introduced into embryogenic maize callus by methods similar to those used for Black Mexican Sweet. Embryogenic callus can be regenerated to whole fertile plants.
• the insect resistance imparted by the endogenous production of the protein is a simply inherited, dominant trait and can, if desired, be introduced into other plant varieties of the ⁇ pecie ⁇ by ⁇ imple cro ⁇ ing or backcro ⁇ sing.
• aprotinin ha ⁇ been expressed in maize suspension cells as determined by transient assays.

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• 1994
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