Classifications

• • 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
• • 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
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
  • A01N65/08—Magnoliopsida [dicotyledons]
• • 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
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
  • A01N65/40—Liliopsida [monocotyledons]
  • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
• • 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
  • C12N15/8282—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 fungal resistance
• • 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
  • C12N15/8285—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 nematode resistance
• • 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
• • 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

• the present invention is directed to chimeric genes that express in cotton cells, plants and seeds, and encode insecticides and fungicides having substantially the insect toxicity and fungal toxicity of barley, nettle, and hevein lectins.
• Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. They are believed to be defense-related, and many exhibit insecticidal and/or anti-fungal activities (Murdock et. al, 1990; Lerner, D.R. and Raikhel, N.V., 1992). Lectins exhibit specific carbohydrate-binding properties. Lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine in susceptible pest species (Schroeder, M.R. and Raikhel, N. V. 1992).
• a gene responsible for the production of a useful polypeptide can be transferred from a donor cell, in which the gene naturally occurs, to a host cell, in which the gene does not naturally occur; Cohen and Boyer, U.S. Patent Nos. 4,237,224 and 4,468,464. There are, in fact, few inherent limits to such transfers. Genes can be transferred between viruses, bacteria, plants, and animals. In some cases, the transferred gene is functional, or can be made to be functional, in the host cell. When the host cell is a plant cell, whole plants can sometimes be regenerated from the cell. Genes typically contain regions of DNA sequences including a promoter and a transcribed region. The transcribed region normally contains a 5' untranslated region, a coding sequence, and a 3' untranslated region.
• the promoter contains the DNA sequence necessary for the initiation of transcription, during which the transcribed region is converted into mRNA.
• the promoter is believed to include a region recognized by RNA polymerase and a region which positions the RNA polymerase on the DNA for the initiation of transcription. This latter region, which is referred to as the TATA box, usually occurs about 30 nucleotides upstream from the site of transcription initiation.
• RNA sequence Following the promoter region is a sequence that is transcribed into mRNA but is not translated into polypeptide. This sequence constitutes the so-called 5 ' untranslated region and is believed to contain sequences that are responsible for the initiation of translation, such as a ribosome binding site.
• the coding region is the sequence that is just downstream from the 5 ' untranslated region in the DNA or the corresponding RNA. It is the coding region that is translated into polypeptides in accordance with the genetic code.
• Bacillus thuringiensis for example, has a gene with a coding sequence that translates into the amino acid sequence of an insecticidal crystal protein.
• the coding region is followed by a sequence that is transcribed into mRNA, but is not translated into polypeptide. This sequence is called the 3' untranslated region and is believed to contain a signal that leads to the termination of transcription and, in eukaryotic mRNA, a signal that causes polyadenylation of the transcribed mRNA strand. Polyadenylation of the mRNA is believed to have processing and transportation functions.
• Natural genes can be transferred in their entirety from a donor cell to a host cell. It is often preferable, however, to construct a gene containing the desired coding region with a promoter and, optionally, 5' and 3' untranslated regions that do not, in nature, exist in the same gene as the coding region. Such constructs are known as chimeric genes.
• Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole (Bednarek, S.Y., and Raikhel, N.V., 1991).
• the glycosylated carboxyl- terminal propeptide (CTPP) is removed before or concomitant with the deposition of the mature, active protein in vacuoles (Bednarek, et al., 1990).
• Mature barley lectin is a dimeric protein composed of two identical 18-kilodalton polypeptides (Wilkins, T.A., Bednarek, S.Y.
• barley lectin cDNA clone BLc3 The nucleotide sequence and deduced amino acid sequence of the barley lectin coding region (barley lectin cDNA clone BLc3) has been reported (see Lerner and Raikhel, 1989; and U.S. Patent No. 5,276,269, incorporated herein by reference).
• a chimeric gene construct was created by fusing the BLc3 coding region to the CaMV 35 S promoter, and transferring the chimeric gene construct into tobacco plants via Agrobacterium tumefaciens mediated transformation (U.S. Patent No. 5,276,269). Plants were reported to exhibit insecticidal and fungicidal properties.
• HEV1 A full length cDNA clone (HEV1) encoding Hevea brasiliensis lectin was isolated from a H. brasiliensis latex cDNA library, sequenced, and characterized (see Broekaert et al. , 1990; Lee et al, 1991; and U.S. Patent No. 5,187,262, incorporated herein by reference).
• HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide.
• the amino-terminal region of 43 amino acids is identical to hevein and shows homology to several chitin-binding proteins and to the amino- termini of wound-induced genes in potato and poplar.
• Northern blots using HEV1 cDNA as a probe, showed that the gene is induced by wounding and the plant hormones abscisic acid and ethylene. Accumulation of these transcripts was seen in leaves, stems, and latex, but not in roots. Chimeric gene constructs fusing the hevein coding region with heterologous promoters were not reported. However, tests with hevein protein showed antifungal activity against Trichoderma, Phycomyces, Botrytis, Septoria, Pyricularia, and Fusarium.
• hevein anti- fungal activity was found to be stable even after heating to 90° C, a condition under which certain chitinase activities are completely destroyed.
• a full length cDNA encoding the nettle lectin (Urtica dioica agglutinin) has been cloned, sequenced, and characterized (Lerner and Raikhel, 1992).
• the protein is made up of 374 amino acids. 21 are a putative signal sequence and 86 amino acids encode the two chitin-binding domains of nettle lectin. These are fused to a 19 amino acid "spacer" domain and a 244 amino acid carboxyl extension with partial identity to a chitinase catalytic domain.
• This gene represents another lectin heretofore unavailable as a source for resistance to important cotton insect and fungal pathogens.
• lectins which might have activity against a given pest in a feeding assay following topical application to plant tissue (see, for example, Cavalieri et. al, U.S. Patent No. 5,407,454), may not exhibit that same activity when expressed in vivo.
• Cavalieri et al. provides somewhat suggestive evidence that a broad range of plant lectins may provide a level of control against certain corn pests. Unfortunately, those studies were carried out using isolated lectin preparations for which essentially no biochemical characterization was provided. Some may even have been from commercial providers, where composition can vary from preparation to preparation. Hence, commercial providers include lot numbers with their products so that problems can be traced back on a lot by lot basis. Purity of the preparations was not discussed by Cavalieri, nor did they provide information on how they obtained their lectins or discuss the actual number of different lectins which may have been present in a given preparation.
• any plant species may produce several different lectins, and protein preparations are readily contaminated with multiple protein species which may be present in trace amounts, but have a significant effect, positive or negative, on observed activity.
• the preparations tried may have actually been mixtures of lectins and even other proteins derived from the plants in question. No data were provided on the source of the lectin preparations used, on their purity, or hence on which of the lectin genes in a given plant the actual activity observed was based.
• Such preparations could have distinctly different insecticidal and fungicidal activities than a lectin provided in purified form from the expression in planta of a single lectin gene.
• proteins which do not have activity in a feeding assay following topical application to plant tissues may have activity when expressed in vivo. This could particularly be true in cotton, where plants normally express a compound called gossypol which is known to suppress feeding of certain insect pests. Thus, there could be synergistic effects between gossypol and lectins in such a way so as to enhance the insecticidal activity of a given lectin against important cotton pests. Alternatively, gossypol expression could suppress feeding just enough so that the target insect might never consume a potentially lethal amount of lectin. Hence, one could not know the insecticidal or fungicidal effect of a lectin gene transferred into cotton until such cotton cells, plants, and seeds were created.
• Raikhel U.S. Patent No. 5,276,269
• a chimeric barley lectin gene under control of the CaMV 35S promoter could be transferred into tobacco plants to produce a single species lectin protein which was transported properly and thereby create a plant with new insecticidal and fungicidal properties.
• pesticidal for example, insecticidal and fungicidal
• chimeric genes capable of expressing in cotton cells, plants, and seeds a polypeptide having substantially the pesticidal toxicity (for example, the insect toxicity)
• Figure 1 illustrates the gene map and 35S promoter region of the binary plant expression vector pGA643 (described in An et al, 1988), which is useful for expression in plants of lectin genes (Wilkins et al, 1990; Raikhel U.S. Patent No. 5,276,269, incorporated herein by reference).
• Figure 2 shows the nucleotide sequence of barley lectin cDNA clone BLc3 (Lerner and Raikhel, 1989; Raikhel U.S. Patent No. 5,276,269).
• Figure 3 shows the nucleotide sequence of the hevein cDNA clone "HEVl” (Broekaert et al, 1990; Raikhel U.S. Patent No. 5,187,262, incorporated herein by reference).
• Figure 4 shows the nucleotide sequence of the nettle lectin cDNA clone MK209 (Urtica dioica agglutinin; Lerner, D. R. and Raikhel, N.V., 1992, incorporated herein by reference).
• the present invention is directed to a chimeric gene that expresses in cotton cells, plants, and seeds, and encodes pesticides such as insecticides and fungicides having substantially the insect toxicity and fungal toxicity of barley, nettle, and hevein lectins.
• the cotton plant cells contemplated include cells from any and all cotton plants into which foreign DNA can be introduced, replicated, and expressed.
• Some suitable examples of cotton plant species include Gossypium hirsutum. Gossypium arboreum. and Gossypium barbadense.
• plant cell refers to any cell derived from a cotton plant.
• Some examples of cells encompassed by the present invention include differentiated cells that are part of a living plant; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors; seeds; embryos; propagules; and pollen.
• the chimeric gene of this invention contains a promoter region that functions efficiently in cotton plants and a coding region that codes for the barley lectin encoded in pBLc3, the hevein lectin encoded in the cDNA clone HEV1, and/or the nettle lectin encoded in the cDNA clone
• the coding sequence of the chimeric gene is not known to be associated with the promoter in natural genes.
• the 5 ' and/or 3 ' untranslated regions may, independently, be associated in nature with either the promoter or the coding region, or with neither the promoter or the coding region.
• either the 5' or the 3' untranslated region is associated with the promoter in natural genes, and most preferably both the 5' and 3' regions are associated with the promoter in natural genes.
• a chimeric barley, hevein, or lectin gene could be functionally introduced into cotton cells. It was even less predictable that such cells would express such lectins at sufficient levels to impart pesticidal (for example, insecticidal or fungicidal) properties to the cells.
• pesticidal for example, insecticidal or fungicidal
• the plant cells In order to be considered pesticidal (for example, insecticidal or fungicidal), the plant cells must contain an insecticidal or fungicidal amount of lectin having substantially the insecticidal and fungicidal activity of purified lectin from barley, rubber, or nettle.
• substantially the insecticidal and fungicidal activity of purified lectin means exhibiting activity against substantially the same range of insects or fungi as does the corresponding lectin purified from its native host.
• An insecticidal or fungicidal amount is an amount which, when present in plant cells, kills insects or fungi or at least significantly inhibits a function necessary for growth, such as feeding. Such inhibition is that which can be measured as statistically significant when compared with a control.
• the plant cells, plants, or seeds of the present invention are able to withstand attacks by cotton pests such as insects, nematodes, or fungi without, or with less, application of purified barley lectin, hevein, nettle lectin, or other insecticides or fungicides when compared with plant cells, plants, or seeds that do not contain a gene producing barley lectin, hevein, or nettle lectin.
• chimeric plant lectin genes barley, hevein, and nettle
• Each comprised a cDNA for a given specific lectin driven by a promoter active in cotton.
• the CaMV 35S promoter was used, but any promoter proven to be active in cotton, such as the A. tumefaciens T-DNA promoters, A. rhizogenes T-DNA promoters, or the cotton chlorophyll A/B binding protein gene promoter (Anderson, et al, 1993) would be useful.
• This list is exemplary, but not intended to be all inclusive.
• One skilled in the art will recognize other useful promoters which can be used to express barley, hevein, and nettle lectins in appropriate cotton cells, plants, and seeds to control problematic cotton pests such as insects and fungi.
• An expression cassette comprising the coding region for barley lectin operably linked to the CaMV 35S promoter was created by ligating the pBLc3 cDNA sequence ( Figure 2) into the plant cloning vector pGA643 ( Figure 1 ; An et al. , 1988) as described in Raikhel, U.S. Patent No. 5,276,269 and incorporated herein by reference, taking advantage of the Xbal restriction endonuclease sites in pBLc3 and pGA643. Transformation was into the E. coli strain DH5 ⁇ . Proper orientation of the coding region of the insert relative to the promoter region was confirmed by restriction endonuclease mapping and DNA sequence analysis.
• the clone comprising the coding region barley lectin cDNA pBLc3 in pGA643 can be obtained from Dr. N. Raikhel, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824.
• An expression cassette comprising the coding region for hevein (Hevea brasiliensis aglutinin) operably linked to the CaMV 35S promoter was created by ligating the hevein cDNA sequence HEVl ( Figure 3; Broekaert et al, 1990; Raikhel, U.S. Patent No. 5,187,262) into the plant cloning vector pGA643 ( Figure 1; An et al, 1988) taking advantage of the Xbal and Bglll restriction endonucleases which release the insert from HEV 1 and cleave within the polylinker region of pGA643. Transformation was into the E. coli strain DH5cc.
• the clone comprising the HEVl cDNA inserted into pGA643 can be obtained from Dr. N. Raikhel, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824.
• An expression cassette comprising the coding region for nettle lectin operably linked to the CaMV 35S promoter was created by ligating the nettle cDNA sequence (Figure 4) into the plant cloning vector pGA643 ( Figure 1 ; An et al, 1988). This was accomplished by releasing the insert from the nettle cDNA clone MK209 with Xbal and ligating this fragment into the Xbal restriction endonuclease site within the polylinker region of pGA643. Transformation was into the E. coli strain DH5 . Proper orientation of the coding region of the insert relative to the promoter region was confirmed by restriction endonuclease mapping and DNA sequence analysis.
• the nettle cDNA clone MK209 and the clone comprising the nettle coding region inserted into pGA643 can be obtained from Dr. N. Raikhel, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824.
• HB101 harboring the wide-host range mobilizing plasmid pRK2013 (Clonetech, Palo Alto,
• Transconjugates were selected on minimal nutrient plates (An et al, 1988) containing kanamycin (5 ⁇ g/ml) and tetracycline (12.5 ⁇ g/ml).
• Seeds were surface sterilized by first treating with 70% ethanol for 3 min, followed by a 20 min treatment with a 20% CLOROX solution (1% available chlorine) containing 0.01% of the surfactant TWEEN-20. Seedlings were grown under 16 h light (40-60 ⁇ E m "2 s "1 ) and 8 h dark at 26 ⁇ 2° C on agar-solidified (TC Agar, Hazleton Biologies, Lenexa, KS) White's medium (Singh and Krikorian, 1981) containing 1 mg/1 kinetin. Embryogenic callus cultures were first established from seedling explants according to the procedures of Rangan (U.S. Patent No. 5,244,802).
• cotyledon and hypocotyl explants from 7- to 10-day old seedlings were placed on a callus induction medium (MS, Murashige and Skoog, 1962) supplemented with 0.4 mg/1 thiamine HC1, 30 g/1 glucose, 2.0 mg/1 ⁇ -naphthaleneacetic acid (NAA), 1.0 mg/1 kinetin, 100 mg/1 myo-inositol and 0.8% (w/v) agar.
• the cultures were incubated at 27 ⁇ 2 °C under conditions of 16h light and 8h dark, light intensity at 60 ⁇ E m 2 s "1 , in an environmentally controlled incubator (Percival, Boone, IA). Callus formed on these explants within three to four weeks.
• Callus pieces were selectively subcultured to enrich for friable, yellowish-green callus every three to four weeks on the same medium, except the carbon source was sucrose (20 g/1) instead of glucose.
• the carbon source was sucrose (20 g/1) instead of glucose.
• embryogenic callus capable of forming small globular somatic embryos appeared one to four subcultures after initiation.
• Embryogenic callus was maintained and multiplied by routine subculture every three to four weeks on MS medium containing 100 mg/1 myo-inositol, 20 g/1 sucrose, 2.0 mg/1 NAA and 0.8% (w/v) agar (maintenance medium).
• the suspension cultures were enriched for small, isodiametric, densely cytoplasmic and highly embryogenic cells by periodically discarding free floating cells and large aggregates ⁇ > 840 ⁇ m) every week. Two days before use, these cultures were subcultured in 250 ml Erlenmeyer flasks containing 40 ml of maintenance medium.
• the cell suspension cultures used in our experiments were rapid growing embryogenic cells that exhibited a doubling of fresh weight in four to six days (the logarithmic phase of growth begins two days after subculture). All cell suspension cultures used for biolistic transformation experiments had a cumulative age of three to four months.
• the three plasmids (barley lectin coding region in pGA643; hevein coding region in pGA643; and nettle lectin coding region in pGA643) were used to coat 1.0 uM gold particles, and then projected into embryogenic cotton suspension cell cultures using an improved helium- driven biolistic device (PDS 1000/He; BioRad). Briefly, 50 ⁇ l of a gold micro-carrier suspension (l ⁇ gold particles) in water was used.
• the bombardments were performed using a membrane rupture pressure of 1550 psi and other device settings as described by Hamilton et al. (8).
• One ml of suspension cells (1 X 10 6 cells) was transferred to each dish.
• a 400 mesh nylon screen was placed over the surface of the suspensions to serve as a baffle.
• the optimal bombardment conditions included the use of 10 MPa rupture disks, a distance between the stopping screen and the cell suspensions of 7.5 cm and a macro-carrier travel distance of 10 mm.
• the vacuum in the sample chamber was 95 kPa. Bombardment of the cells was repeated three to five times at two-day intervals to maximize the transformation frequency. Following particle bombardment, the cell suspension cultures were grown for a week without any selection in maintenance medium.
• the pGA643 binary vector carries a neomycin phosphotransferase II gene for selection of transformed cells ( Figure 1). Accordingly, cell suspensions were selected with the antibiotic G418 (lO ⁇ g/ml). Selection with the antibiotic G418 was applied by gradually increasing the concentration each week.
• G418 was initiated at 10 ⁇ g/ml and increased by 10 ⁇ g/ml increments at five to seven day intervals to achieve a final concentration of 50 ⁇ g/ml after three to four weeks.
• cells were directly exposed to only one high level of antibiotic (G418 at 50 ⁇ g/ml) at the beginning of the selection process.
• Independent transformation events arose as separated growing colonies in the presence of the selective agent. Each colony so arising was maintained separately and verified as a true transformant via NPTII ELISA (Firoozabady et al, 1987).
• Cotton plants are regenerated from embryogenic suspension cultures as described in Rangan et al, U. S. Patent No. 5,593,036 (incorporated herein by reference).
• Example 4 Agrobacterium transformation with lectin genes
• the three binary vector plasmids (barley lectin coding region in pGA643; hevein coding region in pGA643; and nettle lectin coding region in pGA643) were mobilized into the binary A. tumefaciens host strain LBA4404 by triparental mating as previously described. Transformation of cotton primary explants can be accomplished by a number of approaches (Firoozabady et al, 1987; Umbeck et al., 1987; Rangan et al., U. S. Patent No. 5,244,802). For convenience, the method of Rangan et al, U. S. Patent No. 5,244,802, as modified by Rajasekaran et al, 1996, is briefly described.
• Agrobacterium cultures for transformation experiments were initiated in 50ml of YEB liquid medium using frozen glycerol stocks (500 ⁇ l) as inoculum. These cultures were grown overnight for about 18 h at 26 ⁇ 2° C on a gyratory shaker. The optical density (A600) values were adjusted to 0.6-0.8 in liquid MS medium prior to use.
• Cotyledon (1 cm 2 ) explants for Agrobacterium transformations are prepared from 5- to 7-day old seedlings. The explants are treated with an Agrobacterium suspension as prepared above for 15 to 30 min, blotted dry, and then plated on 12 cm diameter filter paper (Whatman No. 1) placed on freshly made, agar-solidified callus induction medium (Rangan U. S. Patent No. 5,244,802) in 15 cm diameter Petri dishes containing 60 ml of medium. Cocultivation is carried out for 48 h in a Percival incubator maintained at 26 ⁇ 2 ° C, 16 h light, 60-90 ⁇ E m" 2 s "* .
• the explants are thoroughly washed in MS liquid medium containing 200 mg/1 cefotaxime (Cal-Biochem) and 200 mg/1 carbenicillin (Sigma), blotted dry, and placed on freshly prepared callus induction medium containing the antibiotic G418 (10 mg/1; Gibco BRL, Life Technologies, Gaithersburg, MD) as the selection agent and the same concentrations of cefotaxime and carbenicillin as above to control bacterial growth.
• Cotyledon segments are plated at seven per Petri dish (9 cm diameter) containing 25 ml callus induction medium. After the first subculture the explants are transferred to freshly made callus induction medium to encourage more callus production in the presence of selection pressure.
• Transformed (antibiotic resistant) callus develops 3-8 weeks after transformation. Individual callus colonies are subcultured separately to maintain identity of separate integration events. NPT II ELISAs are carried out according to the procedures of Firoozabady et al, 1987 to confirm that antibiotic resistant callus colonies are transformed. Transformed colonies are regenerated to plants as described (Rangan, et al. , U. S. Patent No. 5,244,802)
• Cotton cell lines (embryogenic colonies) transformed with barley, nettle, or hevein lectin genes in pGA643 were maintained as independent colonies in culture and confirmed to be transformed by NPTII ELISA as described above.
• NPTII ELISA positive colonies transformed with BLc3 were assayed using double-bind ELISA in methods similar in principle to those of Raikhel et al, 1984, but modified to be more suitable for transformed cotton cells.
• Wheat germ agglutinin antibody which is available commercially, will cross react with barley lectin (Wilkins et al. , 1990) and hence can be used in detecting expression of BLc3 protein in transformed cotton cells using WGA ELISA. It was observed in initial studies with transformed cotton cells that cotton extracts give a high background reading when in these WGA ELISA tests for transformation. The following protocol was developed which overcomes this background problem and enabled the confirmation of co-transfer of lectin genes along with the antibiotic marker gene using the methods in the present invention.
• Rabbit anti-wheat germ aglutinin (6mg/ml) and biotinylated rabbit anti WGA (3.5mg/ml) were purchased from E.Y. Laboratories.
• Primary antibody solution (1 ⁇ g/ml) was prepared by diluting 1.8 ⁇ l of rabbit anti-WGA stock with 1 1 ml carbonate binding buffer (Na 2 C0 3 1.59g, NaHC0 3 2.93g, H 2 0 to IL, pH 9.6) and kept on ice. lOO ⁇ l were applied to each well of a 96 well ELISA plate (Corning #25805-96), sealed and kept overnight at 4° C. Pre- adsorbed antibody was then prepared as follows.
• control (non-transformed) callus was homogenized in 6ml of PBS Tween prepared from 50X concentrate (Agdia, Elckhart, Indiana) containing 1% PVP 40,000 and centrifuged at 8,000 rpm for 10' to pellet cell debris. 5.5ml of the supernatant was mixed with 5.5ml of PBS TWEEN containing 0.1% BSA and 4% PEG 8,000. To this was added 9.4 ⁇ l biotinylated rabbit anti- WGA (E.Y. Laboratories, 3.5mg/ml) for a final antibody concentration of 3 ⁇ g/ml. This was then incubated on ice for 3 hours to preadsorb the antibody.
• PBS Tween prepared from 50X concentrate (Agdia, Elckhart, Indiana) containing 1% PVP 40,000 and centrifuged at 8,000 rpm for 10' to pellet cell debris. 5.5ml of the supernatant was mixed with 5.5ml of PBS TWEEN containing 0.1%
• ELISA plates were removed from the overnight incubation and washed thoroughly (4X) with PBS TWEEN.
• a blocking step was performed by filling each well of the plate with 1% BSA in PBS without TWEEN.
• PBS without TWEEN is prepared by combining 5ml of a 10% stock w/v of Bovine Serum Albumin (Fraction V, ICN Pharmaceuticals #81-066 in water) with 5 ml of PBS(NaCl 8.0g, Na 2 HP0 4 .2H 2 0 1.44g, KH 2 P0 4 0.2g, KCl 0.2g, H 2 0 to IL, adjusted to pH 7.4).
• the plates were incubated at room temperature (22°C-24 ° C) for 1 hour and then washed 4X with PBS TWEEN.
• Extracts from embryogenic cell lines transformed with BLc3 were prepared as follows. About 0.5g of callus was homogenized in 130 ⁇ l PBS Tween containing 1% PVP 40,000 in a 1.5ml micro-centrifuge tube, centrifuged at 10,000 rpm to pellet cell debris, and held on ice. 1 OO ⁇ l of the supernatant was added to each well of the ELISA plates following the 1 hr blocking, washing step noted above. Plates were incubated for 3 hours at room temperature and washed 4X with PBS TWEEN.
• Results of assays with several transformed embryogenic lines are presented in the following Table. Table 1. Results of Immunoassays in WGA ELISA with cotton cells transformed with Blc3 in pGA643 Sample # Colony # Lectin DNA ELISA Result
• Example 6 Cotton cells transformed with BLc3 are insecticidal In order to confirm the insecticidal nature of cotton cells transformed with BLc3, feeding assays were preformed with larvae of the genus Heliothis. Heliothis species are economically important pests of cotton. Transformed embryogenic callus cultures which scored positive in both NPTII ELISA and WGA ELISA were selected for assay. Colonies were divided in two, with one half maintained in culture for regeneration to plants and one half used for the feeding assay. For the assay, >lg of tissue was frozen in liquid nitrogen, lyophilized, and stored at -75° C until used in the feeding assay.
• corn ear worm diet was used for this example.
• Insect diet was prepared as follows. 2.6g agar was dissolved in 157ml H 2 0, boiled for 1 minute, and 40.6g of corn ear worm diet (Bioserv Product #9394) was added and the mixture was stirred well. 1.5ml of this mixture was added to each well of a 16 well insect feeding tray. To each well was then added 25mg of lyophilized callus sample or control (non-transformed) sample as appropriate.
• BLC numbers correspond to sample numbers in Table 1. Eight Heliothis larvae were tested for each callus test sample prepared as described in the text. The % growth weight increases shown are the average for the eight larvae after 6 days of feeding on the indicated test sample mix.
• the negative control sample was prepared using callus transformed with the NPTII marker alone (no lectin gene).
• the positive control was from tissue transformed with pPHY3.
• Example 7 Cotton cells transformed with HEVl (hevein) and MK209 (nettle lectinl are insecticidal Corn ear worm diet supplemented with lyophilized callus was prepared as described in
• Example 6 except that the callus samples were derived from transformations carried out with HEVl and with MK209. Newly hatched larvae (1 per feeding test plate well) were placed on the test medium, incubated at room temperature, and then scored after 7 days. The data are summarized in the following Table 3.
• HEV 30 95% 35% of suppression achieved with Bt.
• insect diet formulations employed in the present study included a very small percentage by weight of the test callus. Accordingly, the extent of insecticidal activity observed is to be deemed significant when one considers the relative activity versus the positive control. Although all lectin genes tested showed significant activity against Heliothis. the nettle lectin MK209 demonstrated the highest level of activity relative the B.t. endotoxin in this study.
• insects and pests include cutworms (Agrotis spp., Paridroma spp., Euxoa spp., Feltia spp.), thrips ( " Franklinialla spp.), aphids (Aphis gossypii " ).
• bollworms Heliothis spp., Pectinophora spp., Helicoverpa spp.), budworms (Heliothis spp.), plant bugs (Lygus spp., Euschistus spp.), boll weevil (Anthonomus grandis), armyworms (Spodoptera spp.), loopers (Alabama spp.), caterpillars (Estigmene spp.), cotton leaf perforator (Bacculatrix spp.), spider mites (Tetranychus spp.), whiteflies (Bemisia spp., Trialeurodes spp.), nematodes (Meloidogyne spp., Rotylenchulus spp.

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