AU616444B2 - Insecticidal cotton plant cells - Google Patents

Description

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S F Ref: 75975 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION 616444

(ORIGINAL)

FOR OFFICE USE: Class Int Class

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EVA,

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4 14 Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Ciba-Geigy AG Klybeckstrasse 141 4002 Basle

SWITZERLAND

A p Address for Service: c Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Insecticidal Cotton Plant Cells The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/11

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5-16768/CGC 1313/CIP Insecticidal cotton plant cells Abstract The present invention is directed to a chimeric gene that expresses in cotton cells insecticides having substantially the insect toxicity properties of the crystal protein produced by Bacillus thuringiensis.

The transformed cells are regenerated into plants that are toxic to the larvae of Leptidoptera, nj,, and Diptera.

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9o094 I0 090 0 41 t 9 44 44 __0 5-16768/CGC 1313/CIP Insecticidal cotton plant cells oS 4 00 0 04 9 0 0 00 .a 4.

*s 4 0 The present invention is directed to a chimeric gene that expresses in cotton cells insecticides having substantially the insect toxicity properties of the crystal protein produced by Bac.ilus thuringiensis.

B. thuringiensis (hereinafter Bt) is a species of bacteria that produces a crystal protein, also referred to as 6-endotoxin. This crystal protein is, technically, n protoxin that is converted into a toxin upon being ingested by larvae of lepidopteran, coleopteran and dipteran insects.

The crystal protein from Bt is a potentially important insecticide having no known harmful effects on humans, other mammals, birds, fish or on insects other than the larvae of lepidopteran, coleopteran and dipteran insects. The activity of the Bt toxin is extremely high, so that only nanogram amounts are required to kill susceptible insect larvae. Other advantages of the use of the crystal protein from Bt as an insecticide include its broad spectrum of activity against lepidopteran, coleopteran and dipteran insect larvae, and the apparent difficulty of such larvae to develop resistance against the crystal protein, even where the crystal protein is used on a large scale. Said larvae are a major problem in agriculture and forestry, and especially in cotton cultivation.

The crystal protein is effective as an insecticide when it is applied to plants subject to lepidopteran, coleopteran or dipteran larvae infestation. Such plants include broccoli, lettuce and cotton. Ledidopteran larvae infestation is especially serious in cotton plants.

000*0* S0 *0 *4 040444 2- So far, the Bt crystal protein (protoxin) was isolated from the Bacillus and applied to the plants by standard methods such as by dusting or spraying. Preparations containing the Bt crystal protein are used commercially as biological insecticides.

For example: Bactospeine, distributed by Biochem Products Ltd., Dipel, distributed by Abbot Laboratories; and Thurcide, distributed by Sandoz AG.

The fact that Bt produces the crystal protein only during sporulation represents a significant disadvantage in connection with the manufacture and use of this biological insecticide. Such growth phase limitation, particularly in an industrial process, can result in inconvenience and excessive time requirements during manufacture. In addition, costs Soo 9a associated with said manufacture made it difficult for such a biological o00 insecticide to compete effectively with other commercially available 9 99 products based on chemicals, such as, for example, pyrethroid a i derivatives.

oa A further disadvantage with respect to the use of the Bt toxin is, for example, the fact that the protein usually remains on the surface of the plants being treated, where it is effective only against surface-feeding o larvae, and where it is inactivated by prolonged exposure to ultraviolet radiation. This inactivation may be at least one cause of the general «oO lack of persistance of the crystal protein in the environment. According- :Ly, frequent and expensive application of the crystal protein is necessary.

SThese and other disadvantages can be overcome by incorporating and expressing a gene coding for a Bt crystal protein or for a protein having substantially the insect toxicity properties of a Bt crystal protein in plants. The present invention describes how these disadvantages can be overcome by incorporating and expressing a gene coding for a Bt crystal protein or for a protein having substantially the insect toxicity 3 3properties of a Bt crystal protein in corn protoplasts and regenerating fertile transgenic corn plants from the transformed protoplasts and culturing these insect resistant corn plants.

By taking advantage of genetic engineering, 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 patents 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 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 o transcription, during which the transcribed region is converted into SmRNA. In eukaryotic cells, the promoter is believed to include a region recognized by RNA polymerase and a region which positions the RNA 0 polymerase on the DNA for the initiation of transcription. This latter Sregion, which is referred to as the TATA box, usually occurs about nucleotides upstream from the site of transcription initiation.

S Following the promoter region is a sequence that is transcribed into mRNA Sbut is not translated into polypeptide. This sequence constitutes the S, so-called 5' untranslated region and is believed to contain sequences 0 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 untranslated region in the DNA or the corresponding RNA. It is the coding region that is translated into polypeptides in accordance with the

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4 -4genetic code. tB for example, has a gene with a coding sequence that translates into the amino acid sequence of the insecticidal protoxin crystal protein.

The coding region is followed downstream 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 Sthe coding region. Such constructs are known as chimeric genes.

L Genetic engineering methods have been described for improved ways of S* producing the crystal protein. For example, U.S. patents 4,448,885 4# and 4,467,036, describe plasmids for producing crystal protein in bacterial strains other than Bt. These methods permit production of the crystal protein, but do not overcome the disadvantages of using the crystal protein as a commercial insecticide.

4 Suggestions have been made to clone Bt toxin genes directly into plants in order to permit the plants to protect themselves (Klausner, 1984).

The European Patent Application EP-0,142,924 (Agrigenetics) alleges a i t method for cloning toxin genes from Bt in tobacco (page 59) and suggests protecting cotton the same way (page 77).

Such a suggestion constitutes mere speculation, however, until methods for transforming cotton cells and regenerating plants from the cells are available. Such methods are described in U.S. patent application serial no. 122,200 entitled "Regeneration and Transformation of Cotton", assigned to Phytogen, and in U.S. patent application Serial No. 122,162 entitled "An Efficient Method for Regenerating Cotton from Cultured Cells", assigned to Ciba-Geigy. U.S. patent applications serial no.

122,200 and 122,162 were filed the same day as the present application.

The methods for transforming cotton cells in the Phytogen patent application serial no. 122,200 anc the methods for regenerating cotton plants in the Phytogen and Ciba-Geigy patent applications serial no. 122,200 and 122,162, respectively, are incorporated herein by reference.

A need exists for developing new methods for producing the crystal protein of Bt or a similar polypeptide having substantially the insect toxicity properties (insecticidal activity) of a Bt crystal protein in cells of cotton plants and for new methods of controlling insect larvae which feed on said cotton plants. "Controlling" should be understood as referring either to killing the larvae, or at least reducing their feeding. Further need exists for a method for protecting cotton plants against damage caused by pests or pathogens comprising the production of 1 an amount of a pesticidal or a;itipathogenic protein in the plant cell and plant respectively, which amount is sufficiently effective for killing or controlling the pest or the pathogen. Further need exists preferably for o a method for protecting cotton plants against insect damage comprising o the production of an insecticidal effective amount of a Bt crystal protein or a protein having substantially the insect toxicity properties of a Bt crystal protein in the plant cell, which amount is sufficiently effective for killing or controlling the insects which feed on said o plants. Further need exists for a method for protecting cotton plants from damage by chemical agents, such as, for example by increasing the tolerability against certain herbicides, so that such chemicals can be •o safely applied to cotton plants which have minimal side effects on the 4 ecosystem.

S These and other objects of the present invention have been achieved by providing chimeric genes capable of expressing in cotton cells a polypeptide having substantially the insect toxicity properties of Bt crystal protein (hereinafter, chimeric Bt toxin gene), as can be seen from the following detailed description.

6 The present invention primarily relates to transgenic cotton plant cells having stably incorporated into the plant genome a chimeric gene that expresses in cotton cells a polypeptide having substantially the insect toxicity properties of the Bt crystal protein.

Also comprised by the present invention are transgenic cotton plants that can be regenerated from transgenic cotton plant cells and have stably incorporated into their genome said chimeric gene that upon expression renders the cotton plant unattractive and/or toxic to insects so that they stop feeding on the plant.

This invention also relates to the propagules and progeny of said transgenic cotton plants.

Propagules of said transgenic cotton plants include any material that can be sexually or asexually propagated or propagated in-vivo or in-vitro. Among this propagating material protoplasts, cells, calli, K Itissues, organs, seeds, embryos, pollen, ovules, zygotes or any other propagating material that can be obtained from said transgenic cotton plants are preferred.

t 1i A further object of the present invention includes the progeny of said transgenic cotton plant cells or plants.

The progeny of transgenic cotton plants includes mutants and variants thereof. Mutants and variants include, for example, those plants obtained from cell fusion or mutant selection that still have the characteristic properties of the starting material, caused by the previous transformation of exogenous DNA.

It is, therefore, an object of the present invention to provide a method for producing in cotton cells a toxin that has substantially the insect toxicity properties of Bt crystal protein. Especially preferred is a method of transforming cotton cells undergoing suspension culture on a callus growth medium which comprises, after a suspension subculture growth cycle,

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I t II I 7 a) recovering cells and any embryogenic callus from the callus growth medium; b) resuspending the cells and the embryogenic callus in a callus growth medium containing Agrobacterium vector having a gene that confers resistance to the untibiotic hygromycin on cotton cells while maintaining suspension growth conditions for a period of time sufficient to transform the suspended cells; c) recovering the suspended cells from the callus growth mediur containing the Agrobacterium; d) treating the transformed cells and the embryogenic callus with an antibiotic in sufficient concentration to kill the Agrobacterium; e) contacting the cells arid the embryogenic callus with the antibiotic hygromycin in order to select the transformed cells and embryogenic callus; f) filtering the suspension to remove embryogenic callus greater than about 600 microns (pm).

It is a further object of the present invention to provide a method for killing insect larvae by feeding them cotton plant cells containing chimeric genes that express an insecticidal amount of a toxin having substantially the insect toxicity properties of Bt crystal protein. The insecticidal cotton plant cells include those from whole plants and parts of plants as well as individual cotton cells in culture.

It is a further object of the present invention to provide a method for protecting cotton plants against insect damage comprising the expression of a Bt crystal protein or a protein having substantially the insect toxicity properties of a Bt crystal protein in the plant cells constituting the plant, in an amount sufficient to kill or at least to control the insect larvae.

44 41c I 4 8 Especially preferred is a method for protecting cotton plants against damage caused by lepidopteran larvae.

It is a further object of the present invention to provide a method wherein the crystal protein or a protein having substantially the insect toxicity properties of a Bt crystal protein is expressed in the plant cell in an amount sufficient to render the plant unattractive and/or toxic to insects, so that they stop feeding on the plants.

It is an additional object of the present invention to provide the genes and other DNA segments as well as the cells and plants associated with the above methods.

According to a first embodiment of this invention, there is "provided a cotton cell comprising a chimeric gene that expresses a polypeptide having substantially the insect toxicity properties of ;Bacillus thuringiensis crystal protein, exhibiting toxicity toward S, 15 Dipteran and Lepidopteran insects.

S' According to a second embodiment of this invention, there is provided a culture of cotton cells according to the first embodiment.

According to a third embodiment of this invention, there is provided a cotton plant comprising a gene that expresses a polypeptide S 20 having substantially the insect toxicity properties of Bacillus i thuringiensis crystal protein, exhibiting toxicity toward Dipteran and Lepidopteran insects.

According to a fourth embodiment of this invention, there is Sprovided propagules of a transgenic cotton plant according to the third embodiment.

According to a fifth embodiment of this invention, there is provided progeny of a transgenic cotton plant according to the third embodimert, or mutants and variants thereof, that still have the characteristic properties of the starting material, caused by the previous transformation of exogenous DNA.

According to a sixth embodiment of this invention, there is provided a method of producing transformed, embryogenic cotton callus which comprises: a) contacting a cotton explant with an Agrobacterium vector containing a chimeric gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein and a gene that confers resistance to an antibiotic on Si cotton cells, the period of the contacting being sufficient to CeiL S /LMM/TCW/1160v 8a transfer the genes to the explant; b) incubating the transformed explant in a callus growth medium for a period of from about 15 to about 200 hours at a temperature of from 250 to about 35 0 C under a cycle of about 16 hours light and 8 hours dark to develop callus from the explants; c) contacting the incubated explants with a callus growth medium containing an antibiotic toxic to Agrobacterium for a time sufficient to kill the Agrobacterium; d) culturing the callus free of Agrobacterium on a callus growth medium; e) contacting the resulting embryogenic callus with the antibiotic in a concentration sufficient to permit selection of callus resistant to i the antibiotic; and f) selecting transformed embryogenic callus.

15 According to a seventh embodiment of this invention, there is S, provided a method of transforming cotton cells undergoing suspension culture on a callus growth medium which comprises, after a suspension subculture growth cycle: a) recovering cells and any embryogenic callus from the callus growth medium; b) resuspending the cells and embryogenic callus in a callus growth medium containing an Agrobacterium vector containing a chimeric gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein and a gene that confers resistance to an antibiotic on cotton cells while maintaining suspension growth conditions for a period of time sufficient to transform the suspended cells; c) recovering the suspended cells from the callus growth medium containing the Agrobacterium; d) treating the transformed cells and the embryogenic callus with an antibiotic toxic to Agrobacterium in sufficient concentration and for a time sufficient to kill the Agrobacterium; e) contacting the cells and embryogenic callus with the antibiotic in order to select the transformed cells and embryogenic callus; f) filtering the suspension to remove embryogenic callus greater than about 600AUm.

According to an eighth embodiment of this invention, there is V. A A/LMM/1160v

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Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu 740 Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin 750 /3 8b provided cotton plants transformed to express a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein and have resistance to the antibiotic hygromycin.

According to a ninth embodiment of this invention, there is provided a method for protecting cotton plants against Dipteran and Lepidopteran insect damage comprising the expression of a Bt crystal protein or a protein having substantially the insect toxicity properties of a Bt crystal protein in the plant cells constituting the plant, in an amount sufficient to kill or to control the insect larvae.

According to a tenth embodiment of this invention, there is provided a method for killing or controlling Dipteran and Lepidopteran 4 t insect larvae by feeding them cotton plant cells containing chimeric genes that express an insecticidal amount of a toxin having substantially the insect toxicity properties of Bt crystal protein.

S 15 Additional embodiments of the present invention include the S: chimeric Bt toxin gene in vectors, bacteria, plant cells in culture, and plant cells in living plants, as well as methods for producing a S toxin having substantially the insect toxicity properties of Bt crystal protein in cotton cells and methods for protecting cotton plants against 20 insect damage, comprising the production of a controlling or insecticidal effective amount of a Bt crystal protein or a protein having substantially the insect toxicity properties of a Bt crystal protein in the plant cell.

S'Figures: Figure 1: Construction of mp 19/bt, a plasmid containing the 5' end of the Bt protoxin gene.

Figure 2: Construction of mp 19/bt, ca/del, a plasmid containing the CaMV gene VI promoter fused to the 5' end of Bt protoxin coding sequence.

Figure 3: Construction of p702/bt, a plasmid having the 3' coding region of the protoxin fused to the CaMV transcription termination signals.

Figure 4: Construction of pBR322/bt 14, containing the complete protoxin coding sequence flanked by CaMV promoter and terminator sequences.

Figure 5: Construction of pRK252/Tn903/BglII.

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4 41 44 4r 4 44i 9- Figure 6: Construction of Figure 7/8: Construction of pCIB4.

Figure 9: Construction of pCIB2.

Figure 10: Construction of pCIB10, a broad host range plasmid containing T-DNA borders and gene for plant selection.

Figure 11: Construction of pCIB10/19Sbt.

Figure 12: Construction of pCIB710.

Figure 13: Construction of pCIB10/710.

Figure 14: Construction of pCIB10/35Sbt.

Figure 15: Construction of pCIB1O//35Sbt (KpnI).

Figure 16: Construction of pCIB10/35Sbt (Bcll).

Figure 17: Construction of pCIB10/35Sbt (607).

Figure 18: not existing Figure 19: Construction of pCIB1300, a plasmid having a chimeric gene containing the CaMV 35S promoter/AMV leader/Bt(Bcl) terminator.

Figures 20, 21, and 22: Construction of pCIB 1301, having a chimeric gene containing the cotton rbs-gX promoter/Bt(607 deletion) coding sequence.

Figure 23: Construction of pCIB1302, having a chimeric gene containing the cotton rbs-gY promoter/Bt(607 deletion) coding sequence.

10 Figure 24: Restriction map of the cotton genomic clones carrying rbc-gX and rbc-gY.

Figure 25: Nucleotide and amino acid sequences of rbc-gY. The first ATG and methionine of the transit peptide are boxed in the figure.

Figure 26: Nucleotide and amino acid sequences of rbc-gX. The first ATG and methionine of the transit peptide are boxed in the figure.

Deposits: In connection with the present invention the following listed plasmids and/or microorganisms were deposited with the International Depository Authority "American Type Culture Collection, Rockville, Maryland" in accordance with the requirements of the Budapest Treaty.

1) Escherichia coli MC1061, pCIB10/35SBT....ATCC 67329 (date of deposit: February 27, 1987) t 2) Escherichia coli HB101, pCIB10/19SBT..... ATCC 67330 (date of deposit: February 27, 1987) 3) Plasmid pLV 11 ATCC 40235 (date of deposit: May 14, 1986) i i 4) Phage 40486 (date of deposit, August 25, 1988) i r 5) Phage X/rbc-gX ATCC 40487 (date of deposit, August 25, 1988) 4 1 The present invention is directed to the production of a chimeric Bt toxin gene. 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.

The above exemplification is included herein for purpose of illustration only, and is not intended to be limiting. Gossypium hirsutum is 11 preferred, and may be of the stripper or picker types. Stripper and picker cotton differ in their method of harvest, the stripper cotton bols being very firmly attached to the plant so that they are not released during late-season storms. Harvesting stripper cotton virtually destroys the plant. Picker cotton is less firmly attached and is harvested by less disruptive means. Some commercially available varieties of G. hirsutumn capable of being regenerated by the method of the present invention include: Acala 1515-75, Acala SJ-2, Acala SJ-4, Acala SJ-5, Acala SJC-1, Acala SJC-22, Acala SJC-28, Acala SJC-30, Acala B-1644, Acala B-1810, Acala B-2724, Acala GC-510.

Coker 304, Coker 315, Coker 201, Coker 310, Coker 312, DP 41, DP 4 e0 DPL 50, DPL 20, DPL 120, DPL 775, S* Lankart 611, Lankart 57, Paymaster 145, Paymaster HS 26, Stoneville 506, Stoneville 825, Funk 519-2, Funk FC 3008, Funk FC 3024, Funk C 1568R, Funk FC 2005, Funk C 0947B, Funk FC 2028, Funk FC 2017, Funk C 1379, McNair 235, Tomcot SP 21-S, Siokra, Tx-CAB-CS.

S The preferred varieties are Acala SJ-2, Acala SJC-1, Acala GC 510, Acala SJC-28, Acala SJC-30, Acala B-1644 and Siokra.

Acala SJ-2, Acala GC 510, Acala B-1644, and Siokra are especially preferred.

12 The term "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 crystal protein from Bt or for a polypeptide having substantially the insecticidal properties of the crystal protein from Bt. 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 0 the promoter nor the coding region. Preferably, either the 5' or the 3' )untranslated region is associated with the promoter in natural genes, and 0o. most preferably both the 5' and 3' regions are associated with the promoter in natural genes.

00 oe "no One could on': predict, based on the state of the art at the time this invention was made, that a chimeric gene could be stably and functionally o introduced into cotton cells. It was even less predictable that such S cells would express an insecticidal polypeptide at any level, and 0 9, especially at sufficient levels to impart insecticidal properties to the cells. In particular, a polypeptide as large and as insoluble as the Spolypeptide having the insect toxicity properties of Bt crystal protein So01 was expected to be especially difficult to express in plant cells.

@0 0 900 In order to be considered insecticidal, the plant cells must contain an 0:0" insecticidal amount of toxin having substantially the insecticidal activity of the crystal protein from Bt. An insecticidal amount is an amount which, when present in plant cells, kills insect larvae or at least reduces their feeding substantially.

13 Accordingly, the plant cells of the present invention are able to withstand attacks by insect larvae without, or with less, application of crystal protein or other insecticides when compared with plant cells that do not contain a gene producing an insecticidal polypeptide.

The chimeric gene of this invention contains transcription control sequences comprising promoter and 5' and 3' untranslated sequences that are functional in cotton plants. These sequences may, independently, be derived from any source, such as, virus, plant or bacterial genes.

The virus promoters and 5' and 3' untranslated sequences suitable for use are functional in cotton plants and are obtained, for example, from plant viruses such as Cauliflower mosaic virus (CaMV). CaMV has been characterized and described by Hohn et al. (1982) 194-220 and appendices A to G.

This description is incorporated herein by reference.

CaMV is an atypical plant virus in that it contains double-stranded DNA.

S.o At least two CaMV promoters are functional in plants, namely the 19S *e 0 promoter, which results in transcription of gene VI of CaMV, and the promoter of the 35S transcript. The 19S promoter and the 35S promoter are *o the preferred plant virus promoters for use in the present invention.

S CaMV 19S promoters and 5' untranslated regions may be obtained by means S of a restriction map such as the map described in Figure 4 on page 199 of P o .o the Hohn et al. article mentioned above, or from the sequence that S appears in Appendix C of the Hohn et al. article.

o e 0"0 In order to isolate the CaMV 19S promoter and, optionally, the adjacent 5' untranslated region, a restriction fragment of the CaMV genome containing the desired sequences is selected. A suitable restriction fragment that contains the 19S promoter and the 5' untranslated region is the fragment between the PstI site starting at position 5386 and the HindIII site starting at position 5850 of Figure 4 and appendix C of the Hohn et al. article.

By analogous methods, the 35S promoter from CaMV may be obtained, as is described below.

14 Undesired nucleotides in the restriction fragment may optionally be removed by standard methods. Some suitable methods for deleting undesired nucleotides include the use of exonucleases (Maniatis et al., 1982) and oligonucleotide-directed mutagenesis (Zoller and Smith, 1983).

A similar procedure may be used to obtain a desirable 3' untranslated region. For example, a suitable CaMV 19S gene 3' untranslated sequence may be obtained by isolating the region between the EcoRV site at position 7342 and the BglII site at position 7643 of the CaMV genome as described in Figure 4 and appendix C of the Hohn et al. article.

Examples of plant gene promoters and 5' and 3' untranslated regions suitable for use in the present invention also include those of the gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase and aoo, chlorophyll a/b-binding protein. These plant gene regions may be isolated o a o* from plant cells in ways comparable to those described above for isolate, ing the corresponding regions from CaMV (see Morelli et al., 1985).

P, Suitable promoters and 5' and 3' untranslated regions from bacterial S genes include those present in the T-DNA region of Agrobacterium plasmids. Some examples of suitable Agrobacterium plasmids include the Ti plasmid of A. ttmefaciens and the Ri plasmid of A. rhizogenes. The a Agrobacterum promoters and 5' and 3' untranslated regions useful in the a oo present invention are, in particular, those present in the genes coding for octopine synthase and nopaline synthase. These sequences may be 0 obtained by methods similar to those described above for isolating CaMV 0 *00~ and plant promoters and untranslated sequences (see Bevan et al., 1983).

o0 V' The coding region of the chimeric gene contains a nucleotide sequence Q" that codes for a polypeptide having substantially the toxicity properties of a Bt 6-endotoxin crystal protein. A polypeptide, for the purpose of the present invention, has substantially the toxicity properties of Bt 6-endotoxin crystal protein if it is insecticidal to a similar range of insect larvae as is the crystal protein from a subspecies of Bt. Some suitable subspecies include, for example, Bt var. kurstaki, Bt var.

berliner, Bt var. alesti, Bt var. tolworthi, Bit var. sotto, Bt var.

i; i i dendrolimus; Bt var. tenebrionis; Bt var. san diego; and Bt var. sizawal.

The preferred subspecies is Bt var. kurstaki, and especially Bt var.

kurstaki HD1.

The coding region may exist naturally in Bt. Alternatively, the coding region may contain a sequence that is different from the sequence that exists in Bt, but is equivalent because of the degeneracy of the genetic code.

The coding sequence of the chimeric gene may also code for a polypeptide that differs from a naturally occuring crystal protein 6-endotoxin but that still has substantially the insect toxicity properties of the crystal protein. Such a coding sequence will usually be a variant of a natural coding region. A "variant" of a natural DNA sequence is a modified form of the natural sequence that performs the same function.

cti,. The variant may be a mutation, or may be a synthetic DNA sequence, and is Sc substantially homologous to the corresponding natural sequence.

t "Substantial sequence homology" should be understood as referring to is e either: a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment to produce a protein having similar properties; or a polypeptide having an amino acid sequence sufficiently similar to S another polypeptide to exhibit similar properties.

S Normally, a DNA sequence is substantially homologous to a second DNA sequence if at least 70 preferably at least 80 and most preferably S at least 90 of the active portions of the DNA sequence are homologous.

q, Two different nucleotides are considered to be homologous in a DNA sequence of a coding region for the purpose of determining substantial 4L homology if the substitution of one for the other constitutes a silent mutation.

The invention thus includes cotton cells and plants containing any chimeric gene coding for a sequence of amino acids having the insecticidal properties satisfying the requirements disclosed and 16 claimed. It is preferred that the nucleotide sequence is substantially homologous at least to that portion or to those portions of the natural sequence responsible for insecticidal activity.

The polypeptide expressed by the chimeric gene of this invention will generally also share at least some immunological properties with a natural Bt crystal protein, since it has at least some of the same antigenic determinants.

Accordingly, the polypeptide coded for by the chimeric gene of the present invention is preferably structurally related to the crystal 6-endotoxin protein produced by Bt. Bt produces a crystal protein with a subunit which is a protoxin having an Mr of about 130,000 to 140,000.

This protoxin can be cleaved by proteases or by alkali to form insecticidal fragments having an Mr as low as 80,000, preferably about 70 000, more preferably about 60,000 and possibly even lower. The fragments Spreferably have a maximum Mr of about 120,000, more preferably about S 110,000 and most preferably about 100,000. Chimeric genes that code for

I

such fragments of the protoxin or for even smaller portions thereof according to the present invention can be constructed as long as the fragments or portions of fragments have the requisite insecticidal activity. The protoxin, insecticidal fragments of the protoxin and S insecticidal portions of these fragments can be fused to other molecules t st, such as polypeptides and proteins.

i Coding regions suitable for use in the present invention may be obtained t from crystal protein toxin genes isolated from Bt (for example, see PCT V application WO 86/01536 and U.S. patents 4,448,885 and 4,467,036). A o* preferred sequence of nucleotides that codes for a crystal protein is that shown as nucleotides 156 to 3623 in the sequence of the formula I or a shorter sequence that codes for an insecticidal fragment of such a crystal protein. The disclosure of this sequence in Geiser et al. (1986) is incorporated herein by reference.

A 1 17 000 400 0 0 04 o '3 00 o 0~j 0000 0'~ 0 0 000 0 00 0 0 0 00 0 00 00 '3 00 0 0 00 0 000000 000000 0 0 00 0 0 O 000 000000 0 Formula I

GTTAACACCC

TCATAAGATG

130

AATTGGTATC

190

AATGCATTCC

250

TAGAAACTGG

310

AATTTGTTCC

370 CT CCCT CICA 430

AAGAATTCGC

490

TTTACGCAGA

550

AGATGCCTAT

610

CAGTTCAAAA

670 TAT CAGTTTT 730

TCAATAGTCG

790

GCTGGTACAA

850

ATAATCAATT

910

ACTAIGATAG

970

CAAACCCAGT

1030

GAAGTATTAG

20

TGGGTCAAAA

80

AGTCATATGT

140

TTAATAAAAG

200

TTATAATTGT

260 TTACACC CCA 320

CGGTGCTGGA

380

ATGGGACGCA

440

TAGGAACCAA

500 ATOTT TTAGA 560

TCAATTCAAT

620 TI \TAAGT 680 GAGAGAT OTT 740

TTATAATGAT

800

TACGGGATTA

860

TAGAAGAGAA

920

TAGAACGTAT

980

ATTAGAAAAT

1040

GAGTCCACAT

30

ATTGATATTT

90

TTTAA.ATTGT

150

AGATGGAGGT

210

TTAAGTAACC

270

ATCGATATTT

330

TTTGTGTTAG

390

TTTCTTGTAC

450

GCCATTTCTA

510

GAGTGGGAAG

570

GACATGAACA

630 C CT CTTT TAT 690

TCAGTGTTTG

750

TTAACTAGGC

810

GAGCGTGTAT

870

TTAACACTAA

930

CCAATTCGAA

990

TTTGATGGTA

1050

TTGATGGATA

40

AGTAAAATTA

100

AGTAATGAAA

160

AACTTATGGA

220

CTGAAGTAGA

280 C CTTGT COCT 340

GACTAGTTGA

400

AAATTGAACA

460

GATTAGAAGG

520

CAGATCCTAC

580

GTGCCCTTAC

640

CAGTATATGT

700

GACAAAGGTG

760 TTATT GGCAA 820

GGGGACCGGA

880

CTGTATTAGA

940

CAGTTTCCCA

1000

GTTTTCGAGG

1060

TACTTAACAG

50

GTTGCACTTT

110

AACAGTATTA

170

TAACAATCCG

230

AGTATTAGGT

290

AACGCAATTT

350

TATAATATGG

410

GTTAATTAAC

470

ACTAAGCAAT

530

TAATCCAGCA

590

AACCGCTATT

650

TCAAGCTGCA

710

GGGATTTGAT

770

CTATACAGAT

830 TT CTAGAGAT 890 TAT CGTTT CT 950

ATTAACAAGA

1010 CT CGCT CAG 1070

TATAACCATC

GTGCATTTTT

120 TAT CATAATG 180

AACATCAATG

240

GGAGAAAGAA

300

CTTTTGAGTG

360

GGAATTTTTG

420

CAAAGAATAG

480

CTTTATCAAA

540

TTAAGAGAAO

600 C CT CTTTTTG 660

AATTTACATT

720

GCCGCGACTA

780

CATGCTGTAC

840

TGGATA.AGAT

900 C TAT TT C CGA 960

OAAATTTATA

1020

GGCATAGAAG

1080

TATACGGATG

t.1 4

U

U

I

'1

I

U

4

I

18 1090 1100 CTCATAGAGG AGAATATTAT 1150 1160 CGGGGCCAGA ATTCACTTTT 1210 1220 GTATTGTTGO TCAACTA3GT 1270 1280 GACCTTTTAA TATAGGGATA 1330 1340 CTTATGGAAC CTCCTCAA.AT 1390 1400 CGCTGGATGA AATACCGCCA 1450 1460 GATTAAGCCA TGTTTCAATG 1510 1520 GAGCTCCTAT GTTCTCTTGO 1570 1580 CACAAATTAC ACAAATACCT 1630 1640 TTAAAGGACC AGGATTTAOA 1690 1700 OAACCTTAAG AGTAAATATT 1750 1760 ACGCTTCTAC CACAAATTTA 1810 1820 GGAATTTTTC AGCAACTATG 1870 1880 TAGGTTTTAC TACTCCGTTT 1930 1940 ATGTCTTCAA TTCAGGCAAT 1990 2000 TAACCTTTGA GGCAGAATAT 2050 2060 CTTCTTCCAA TCAAATCGGG 1110 TGGT GAOGO 1170

CCGCTATATG

1230

CAGGGCGTGT

1290

AATAATCAAC

1350 TTG COAT CC C 1410

CAGAATAACA

1470 T TT C TT GAG 1530

ATACATCGTA

1590

TTAACAAAAT

1650

GGAGCAOATA

1710 ACTGCAC CAT 1770

CAATTCCATA

1830

AOTAGTOGGA

1890

AACTTTTCAA

1950

GAAGTTTATA

2010

GATTTAGAAA

2070

TTAAAAACAG

1120

ATCAAATAAT

1180

GAACTATCGGG

1240

ATAGAACATT

1300

AACTATCTCT

1360

CTGTATAOAO

1420 ACCT GC CAC C 1480

GCTTTAGTAA

1540

GTGCTGAATT

1600

CTACTAATCT

1660

TTCTTCOAAG

1720

TATCACAAAO

1780

CATCAATTGA

1840

CTAATTTACA

1900

ATGGATCAAG

1960

TAOATCGAAT

2020

GAGCACAAAA

2080

ATGTGACGGA

1130 GOOTTO TOOT 1190

AAATGCAOOT

1250

ATOGTOCAOT

1310

TOTTGAOGGG

1370 AAAAAG COCA 1430

TAGOAAGGA

1490

TAGTAGTGTA

1550

TAATAATATA

1610

TGGCTCTCOA

1670

AAOTTOACOT

1730

ATATOCOGTA

1790

CGGAAGAOOT

1850

GTCCGGAAGO

1910

TGTATTTAOO

1970

TCAATTTGTT

2030

OOOGGTOAAT

2090

TTATCATATT

1140

GTAGGGTTTT

1200

CCACAACAAO

1260

TTATATAGAA

1320

AOAOAATTTG

1380

ACOGTAGATT

1440 TTTAOT CAT C 1500

AGTATAATAA

1560 ATTOOTT OAT 1620

ACTTOTGTOO

1680

GOOAGATTT

1740

AGAATTOOOT

1800

ATTAATOAGG

1860

TTTAOCACTG

1920

TTAAGTGCTO

1980

OCGGOAGAAG

2040

GAGCTGTTTA

2100

GATCAAGTAT

2110 2120 2130 2140 2150 2160 OCAATTTAGT TGAGTGTTTA TOTGATGAAT TTTGTOTOCA TGAAAAAAAA GAATTGTOCCG 19 444 ,,4 o 4 44 4 0 4 4 44 4 9 .44.

4 44 4 4 444 4 94 4 4* 4 4 4 49 4 9 4 44 *4 44 4 4 *.4440 44 44 4 444 44.44, 4 4 2170

AGAAAGTCAA

2230

TTAGAGGGAT

2290

AAGGAGGCGA

2350 GO TAT CCAAC 2410

ACCAATTAAG

2470

ATGCCAAACA

2530 CAAGT CCAAT 2590

GATGTACAGA

2650

ATGGCCATGC

2710

CACTAGCTCG

2770

GGCAAACAAA

2830

CTCAATATGA

2890

GCGTTCATAG

2950

CGGCTATTTT

3010

GAAATGTCAT

307j

ATGTAGATGT

3130

CAGAAGTGTC

3190

CGTACAAGGA

3250

ACGAACTGAA

2180

ACATGCGAAG

2240

CAATAGACAA

2300

TGACGTATTC

2360

GTATTTATAT

2420 AGCGT.ATAT C 2480

CGAAACAGTA

2540

CGGAAAATCT

2600

CTTAAATGAG

2660

AAGACTAGGA

2720

TGTGAAAAGA

2780

TATTGTTTAT

2840

TAGATTACAA

2900

CATTCGAGAA

2960

TGAAGAATTA

3020

TAAAAATGGT

3080

AGAAGAACAA

3140

ACAAGAAGTT

3200

GGGATATGGA

3260

GTTTAGCAAC

2190

CGACTTAGTG

2250

CTAGACCGTG

2310

AAAGAGAATT

2370

CAAAAAATAG

2430

GAAGATAGTC

2490 AATGTGC GAG 2550

GCCCATCATT

2610

GACTTACGTG

2670

AATCTAGAAT

2730

GCGGAGAAAA

2790

AAAGAGGCAA

2850

GCGGATACCA

2910

GCTTATCTGC

2970

GAAGGGCGTA

3030

GATTTTAATA

3090 AACAAC CACC 3150

CGTGTCTGTC

3210

GAAGGTTGCG

3270

TGTGTAGAAG

2200

ATGAGCGGAA

2260

GCTGGAGAGG

2320

ACGTTACGCT

2380

ATGAGTCGAA

2440

AAGACTTAGA

2500

GTACGGGTTC

2560

CCCATCATTT

2620

TATGGOTGAT

2680

TTCTCGAAGA

2740

AATGGAGAGA

2800

AAGAATCTGT

2860

ACATOGCGAT

2920 CT GAGCTGT C 2980

TTTTCACTGC

3040

ATGGOTTATC

3100 OTT CGGT CCT 3160

CGGGTCGTGG

3220

TAACCATTCA

3280

AGGAAGTATA

2210

TTTACTTCAA

2270

AAGTACGGAT

2330

ATTGGGTACC

2390

ATTAAAAOCC

2450

AATCTATTTA

2510 CTTATOG C C 2570 CT CCTTOGAC 2630

ATTCAAGATT

2690 GAAAC CAT TA 2750

CAAACGTGAA

2810

AGATGCTTTA

2870

GATTCATOCG

2930 TOT GATT C C 2990 AT TC TC C TA 3050

CTGCTGGAAC

3110

TOTTGTTOCC

3170 OTATATO OTT 3230

TGAGATCGAG

3290

TCCAAACAAC

2220

GATCCAAACT

2280 ATTAC CATC C 2340

TTTGATOAGT

2400

TATACCCGTT

2460

ATTCGCTACA

2520 CTTT CAG CC C 2580

ATTGATGTTG

2640

AAGACGCAAG

2700

GTAOGAGAAG

2760

AAATTGGAAT

2820

TTTOTAAACT

2880 GCAGATA,4AC 2940

GGTGTCAATO

3000

TATGATGCOA

3060

GTGAAAGGGC

3120

GAATGGGAAG

3180

CGTGTCAOAG

3240

AACAATACAG

3300

ACGGTAACGT

20 t 3310

GTAATGATTA

3370

GATATGACGG

3430

AAGAAAAAGC

3490

GGGATTACAC

3550

CCGATAAGGT

3610

AATTACTTCT

3670

GATTACTOAC

3730

TCACTCTTAA

3790 T TTTTTGC CGA 3850

CACTACCCCO

3910 AT TT TTTAT C 3970

TCATTTAACC

4030

ACGAAAGTTT

4090

GAGTGATTCT

4250 CCAGAAG GAC 3320

TACTGCGACT

3380

AGCCTATGAA

3440

ATATACAGAT

3500 AC CACTAC CA 3560

ATGGATTGAG

3620

TATGGAGGAA

3680

TTGTATTGAC

3740

AAGAATGATG

3800

AGGCTTTACT

3860

AAGTGTCAAA

3920

AATCTTTCAA

3980

CCTTCTCTTT

4040

TCAGGAAATG

4100 CT CGTTC GAG 4160

TCAATAAACG

CAAGAA

AGCAAI

GGACCA

CCTGG C

ATCGGA

TAATAT

AGATM

TCCGTI

TAACGG

AAACGI

TTCAAC

TGGAAC

AATTA(

TATGCI

CTTTG)

3330 3340 ~GAAT ATGAGGGTAC 3390 3400 TCTT CTGTACCAGC 3450 3460 AGAG ACAATCCTTG 3510 3520 TATG TGACAAAAGA 3570 3580 ~GAAA CGGAAGGAAC 3630 3640 'ATGC TTTATAATGT 3690 3700 ATAA GGAAATTTTT 3750 3760 ~TTTT GTATGATTTA 3810 3820 GGTA CCGCCACATG 3870 3880 'TATT CTTTCTAAAA 3930 3940 ATGA ATTACAACTA 3990 4000 ;AACT CGCTAAAGAA 4050 4060 ;CTAC CATATGTATC 4110 4120 ~GTCA ATTACACGCC 4170 4180 ~TAAA AAAGCGGTTG 4230 4240 'TAAA ACATCAGCCA 4290 4300 MTCG ACGATTTTCC 4350 4360 'TGCA CAAACTGCAG 3350

GTACACTTCT

3410

TGATTATGCA

3470

TGAATCTAAC

3530

ATTAGAGTAC

3590

ATTCATCGTG

3650

AAGGTGTGCA

3710

ATATGAATAA

3770

ACGAGTGATA

3830

CCCATCAACT

3890

AGCTAGCTAG

3950

TTTTCTGAAG

4010

TTAGGTTTTG

4070

TGGGGCAGTC

4130

GCCACAGCAC

4190

AATTTTTGAA

4250

TTTCAAGTGC

4310

AAGTACCGAA

3360 CGTAATC GAG 3420 TCAGCC TAT C 3480

ACAGGATATG

3540

TTCCCAGAAA

3600

GACAGCCTGG

3660

AATAAAGAAT

3720

AAAACCGGCA

3780

TTTAAATGTT

3840

TAAC',AATTTG

3900

AAAGGATGAC

3960

AGCTGTATCC

4020

TAAAAAGAAA

4080

AACGTACAGC

4140

TCTTATGAGT

4200

ATATATTTTT

4260

AGCACTCACG

4320

ACATTTAGCA

1.4 4210 4220 TnTGCATTAT 4270

TATTTTCAAC

4330

CATGTATATC

GGAAAAGTAA ACTTT( 4280 GAATCCGTAT TTTAG 4340 CTGGGTCAGG TGGTTC 21 The coding region defined by nucleotides 156 to 3623 of the sequence (I) encodes the polypeptide of the sequence of the formula (II).

SEQUENCE OF THE FORMULA (II) Met Asp Asn Asr Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gin Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Gin Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu 100 Glu Gly Leu Ser Asn Leu Tyr Gin Ile Tyr 110 Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp 120 Pro Thr Asn Pro Ala Leu Arg Glu Glu Met 130 Arg Ile Gin Phe Asn Asp Met Asn Ser Ala 140 Leu Thr Thr Ala Ile Pro Leu Phe Ala Val 150 Gin Asn Tyr Gin Val Pro Leu Leu Ser Val 160 Tyr Val Gin Ala Ala Asn Leu His Leu Ser 170 Val Leu Arg Asp Val Ser Val Phe Gly Gin 180 Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn 190 Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile 200 S Gly Asn Tyr Thr Asp His Ala Val Arg Trp 210 Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly 220 Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn 230 Gin Phe Arg Arg Glu Leu Thr Leu Thr Val 240 Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr 250 Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val 260 Ser Gin Leu Thr Arg Glu Ile Tyr Thr Asn 270 S Pro Val Leu Glu Asn Phe Asp Gly Ser Phe 280 Arg Gly Ser Ala Gin Gly Ile Glu Gly Ser 290 Ile Arg Ser Pro His Leu Met Asp lie Leu 300 Asn Ser Ile Thr Ile Tyr Thr Asp Ala His 310 Arg Gly Glu Tyr Tyr Trp Ser Gly His Gin 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly 330 Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr 340 SMet Gly Asn Ala Ala Pro Gin Gln Arg Ile 350 Val Ala Gln Leu Gly Gin Gly Val Tyr Arg 360 Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro 370 Phe Asn Ile Gly Ile Asn Asn Gin Gin Leu 380 Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr 390 Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu 410 Asp Glu Ile Pro Pro Gin Asn Asn Asn Val 420 Pro Pro Arg Gin Gly Phe Ser His Arg Leu 430 Ser His Val Ser Met Phe Arg Ser Gly Phe 440 Ser Asn Ser Ser Val Ser Ile Ile Arg Ala 450 Pro Met Phe Ser Trp Ile His Arg Ser Ala 460 Glu Phe Asn Asn lie Ile Pro Ser Ser Gin 470 Ile Thr Gin Ile Pro Leu Thr Lys Ser Thr 480 Asn Leu Gly Ser Gly Thr Ser Val Val Lys 490

I

-22- Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu 500 Arg Arg Thr Ser Pro Giy Gin Ile Ser Thr 510 Leu Arg Val Asn Ile Thr Ala Pro Leu Ser 520 Gin Arg Tyr Arg Val Arg Ile Arg Tyr Ala 530 Ser Thr Thr Asn Leu Gin Phe His Thr Ser 540 Ile Asp Gly Arg Pro Ile Asn Gin Gly Asn 550 Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 560 Leu Gin Ser Gly Ser Phe Arg Thr Val Gly 570 Phe Thr Thr Pro Phe Asn Phe Set Asn Giy 580 Set Ser Val Phe Thr Leu Ser Ala His Val 590 Pbe Asn Ser Gly Asn Glu Val Tyr Ile Asp 600 Arg Ile Giu Phe Val Pro Aia Giu Val Thr 610 Phe Giu Ala Giu Tyr Asp Leu Giu Arg Ala 620 Gin Lys Ala Val Asn Glu Leu Phe Thr Ser 630 Ser Asn Gin Ie Gly Leu Lys Thr Asp Val 640 Thr Asp Tyr His Ile Asp Gin Val Ser Asn 650 Leu Val Glu Cys Leu Ser Asp Giu Phe Cys 660 Leu Asp Giu Lys Lys Giu Leu Ser Glii Lys 670 Val Lys His Ala Lys Arg Leti Ser Asp Ohu 680 Arg Asn Leu Leu Gin Asp Pro A~n Phe Arg 690 Gly Ile Asn Arg Gin Leu Asp Arg Gly Trp 700 t Arg Gly Ser Thr Asp Ile Thr Ile Gin Giy 710 r r Gly Asp Asp Val Phe Lys Giu Asn Tyr Val 720 16*t Thr Leu Leu Gly Thr Phe Asp Ohu Gys Tyr 730 Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glii 740 Set Lys Leu Lys Ala Tyr Thr Arg Tyr Gin 750 Leu Arg Gly Tyr Ile Giu Asp Set Gin Asp 760 Leu Giu Ile Tyr Leu Ile Arg Tyr Asn Ala 770 Lys His Glu Thr Val Asn Val Pro Gly Thr 780 c Gly Ser Leu Trp Pro Leu Ser Ala Pro Ser 790 Pro Ile Giy Lys Gys Ala His His Ser His 800 His Phe Ser Leu Asp Ile Asp Val Gly Cys 810 Thr Asp Leu Asn Glu Asp Leu Gly Val fTp 820 *~Val Ile Phe Lys Ile Lys Thr Gin Asp Gly 830 His Ala Arg Leu Giy Asn Leu Giu Phe Leu 840 Glu Ohu Lys Pro Leu Val Guy Glu Ala Leu 850 Ala Arg Val Lys Arg Aia Ohu Lys Lys Trp 860 S Arg Asp Lys Arg Ohu Lys Leu Giu Trp Ciii 870 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu 880 Ser Val Asp Ala Leu Phe Vai Asn Set Gin 890 Tyr Asp Arg Leu Gin Ala Asp Thr Asn Ile 900 Ala Met Ile His Ala Ala Asp Lys Arg Vcd 910 0 His Ser Ile Arg Glu Ala Tyr Leu Pro Clu 920 Leu Ser Val Ile Pro Gly Val Asn Ala Ala 930 S le Phe Glu Giu Leu Glu Gly Arg Ile Phe 940 Thr Ala Phe Ser Leti Tyr Asp Ala Arg Asn 950 Val Ile Lys Asn Gly Asp Phe Asn Asn Guy 960 Leu Set Cys Trp Asn Val Lys Gly His Val 970 Asp Val Glu Giu Gin Asn Asn His Arg Ser 980 Vai Leu Val Val Pro Glu Trp Glu Ala Glu 990 Val Ser Gin Glu Val Arg Val Cys Pro Gly 1000 Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr 1010 Lys Glu Gly Tyr Gly Glu Giy Cys Val Thr 1020 Ie His Ci Ile Glu Asn Asn Thr Asp Glu 1030 Leu Lys Phe Ser Asn Cys Val Giu Giu Giu 1040 Val Tyr Pro Asn Asn Thr Vai Thr Cys Asn 1050 23 Asp Tyr Thr Ala Thr Gin Glu Glu Tyr Glu 1060 Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr 1070 Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val 1080 Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu 1090 Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn 1100 Pro Cys GIu Ser Asn Arg Gly Tyr Gly Asp 1110 Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr 1120 Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1130 Lys Val Trp Ile Glu lie Gly Glu Thr Glu 1140 Gly Thr Phe Ile Val Asp Ser Val Glu Leu 1150 Leu Leu Met Glu Glu End 1156 Hence, the present invention is further directed to the polypeptide having the sequence of the formula (II) or to insecticidal parts of this polypeptide.

Furthermore, it has been shown that the toxins of some Bt strains are toxic to other than lepidopteran insects. Specifically the toxin of Bt var. tenebrionis is, for example, toxic to coleopteran insects. The toxicity of Bt strain san diego toward coleopteran insects and the Ssequence of the associated toxin gene is disclosed in EP-0,202,739 and EP-0,213,818.

2 In order to introduce the chimeric gene of the present invention into plant cells, the gene is first inserted into a vector. If the gene is not I o, available in an amount sufficient for transformation, the vector may be amplified by replication in a host cell. The most convenient host cells for amplification are bacterial or yeast cells. When a sufficient amount S of the chimeric gene is available, it is introduced into cotton cells or tissue. The introduction of the gene into cotton plant cells or tissue may be by means of the same vector used for replication, or by means of a different vector.

S Some examples of bacterial host cells suitable for replicating the chimeric gene include those selected from the group consisting of the genera Escherichia such as E. coll and Agrobacterium such as A. tumefaciens or A. rhizogenes. Methods for cloning heterologous genes in bacteria are described in U.S. patents 4,237,224 and 4,468,464.

The replication of genes coding for the crystal protein of Bt in E. coli is described in Wong et al. (1983).

i 24 The preferred bacterium host cell for amplifying the chimeric Bt genes of this invention is Agrobacter-um. The advantage of amplifying the gene in Agrobacterium is that the Agrobacterium may then be used to insert the amplified gene into plant cells without further genetic manipulation.

Some examples of yeast host cells suitable for replicating the genes of this invention include those of the genus Saccharomyces.

Any vector into which the chimeric gene can be inserted and which replicates in a suitable host cell, such as in bacteria or yeast, may be used to amplify the genes of this invention. The vector may, for example, be derived from a phage or a plasmid. Some examples of vectors derived from phages useful in the invention include those derived from M13 and from X. Some suitable vectors derived from M13 include M13mpl8 and M13mpl9. Some suitable vectors derived from X include Xgtll, Xgt7 and XCharon4.

Ae 9 r Some vectors derived from plasmids especially suitable for replication in bacteria include pBR322 (Bolivar et al., 1977); pUC18 and pUC19 (Norrander et al., 1983); and Ti plasmids (Bevan et al., 1983). The preferred vectors for amplifying the genes in bacteria are pBR322, pUC18 and pUC19.

In order to construct a chimeric gene suitable for replication in bacteria, a promoter sequence, a 5' untranslated sequence, a coding c sequence and a 3' untranslated sequence are inserted into or are assembled in the proper order in a suitable vector, such as a vector described above. In order to be suitable, the vector must be able to replicate in the host cell.

4 4 The promoter, 5' untranslated region, coding region and 3' untranslated region, which comprise the chimeric gene of the invention, may first be combined in one unit outside the vector, and then inserted into the vector. Alternatively, portions of the chimeric gene may be inserted into the vector separately. The vector preferably also contains a gene that confers a trait on the host cell permitting the selection of cells containing the vector. A preferred trait is antibiotic resistance. Some examples of useful antibiotics include ampicillin, tetracycline, hygromycin, G418, chloramphenicol, kanamycin and neomycin.

Insertion or assembly of the gene in the vector is accomplished by standard methods such as the use of recombinant DNA (Maniatis et al., 1982) and homologous recombination (Hinnen et al., 1978).

Using known recombinant DNA methods, the vector is cut, the desired DNA sequence is inserted between the cut pieces of the vector, and the ends of the desired DNA sequence are ligated to the corresponding ends of the vector.

The vector is most conveniently cut by means of suitable restriction endonucleases. Some suitable restriction endonucleases include those which form blunt ends, such as SmaI, Hpal and EcoRV, and those which form cohesive ends, such as EcoRI, SacI and BamHI.

e The desired DNA sequence normally exists as part of a larger DNA molecule S such as a chromosome, plasmid, transposon or phage. The desired DNA sequence is excised from its source, and optionally modified so that the ends can be joined to the ends of the cut vector. If the ends of the S desired DNA sequence and of the cut vector are blunt ends, they are joined by blunt end ligases such as T4 DNA ligase.

The ends of the desired DNA sequence may also be joined to the ends of S the cut vector in the form of cohesive ends, in which case a cohesive end ligase, which may also be T4 DNA ligase is used. Other suitable cohesive S end ligases include, for example, E. coli DNA ligase.

Cohesive ends are most conveniently formed by cutting the desired DNA sequence and the vector with the same restriction endonuclease. In such a case, the desired DNA sequence and the cut vector have cohesive ends that are complementary to each other.

i 26 The cohesive ends may also be constructed by adding complementary homopolymer tails to the ends of the desired DNA sequence and to the cut vector using terminal deoxynucleotidyl transferase, or by adding a synthetic oligonucleotide sequence recognized by a particular restriction endonuclease, known as a linker, and cleaving the sequence with the endonuclease (see, for example, Maniatis et al., 1982).

The Bt toxin genes of the present invention may be introduced directly into plant cells by taking advantage of certain plasmids present in Agrobacterium. These plasmids contain regions that are naturally inserted into the genome of plant cells infected by Agrobacterium. The inserted region is called T-DNA (transferred-DNA). These plasmids, examples of which include the Ti (tumor inducing) plasmid of A. tumefaciens and the Ri (root inducing) plasmid of A. rhizogenes, contain T-DNA border tt sequences, at least one of which is believed to be necessary for the Stransfer of the T-DNA region from the plasmid to the genome of the S infected plant cell. Natural Ti and Ri plasmids also contain virulence S regions, the location of which is believed to be outside of the T-DNA region. The virulence regions are needed for the transfer of T-DNA to plant cells.

S In modified systems the virulence regions may exist on plasmids different from the plasmids that contain the T-DNA. Such virulence regioncontaining plasmids are called helper plasmids.

The T-DNA regions that occur naturally are oncogenic and cause plant tumors. The oncogenic portions of these T-DNA regions may be partially or fully removed before, or simultaneously with, the insertion of the desired DNA sequence. The plasmids containing such modified T-DNA regions are said to be disarmed.

i The genes suitable for use in the present invention are assembled in or are inserted into a T-DNA vector system by methods known in the art (Barton and Chilton, 1983; Chilton, 1985). The T-DNA vector may be j oncogenic (Hernalsteens et al., 1980), partially disarmed (Barton and Chilton, 1983), fully disarmed (Zambryski et al., 1983),or may be based on artificial T-DNA vectors having synthetic T-DNA border-like sequences, i:i 1 111-- 27 (Wang et al., 1984). Some suitable disarmed vectors containing T-DNA border regions include pGA436, pGA437 and pGA438, as are described in An et al. (1985); pMON120 (see Fraley et al., 1983) and pCIBIO (Rothstein et al., 1987). The transfer of T-DNA is usually accomplished by incubating Agrobacterium with plant cell protoplasts or wounded plant tissue (see Caplan et al., 1983).

In addition to the chimeric gene coding for a Bt or a Bt-like toxin, the vectors preferably further comprise a DNA sequence that permits the selection or screening of cotton plant cells containing the vector in the presence of cells that do not contain the vector. Such selectable or screenable markers may naturally be present in the vector into which the chimeric gene of this invention is introduced, or may be introduced into the vector either before or after the chimeric gene is introduced.

SAlternatively, the selectable or screenable marker gene or a portion thereof may first be joined to the desired chimeric gene or any portion thereof and the recombined genes or gene segments may be introduced as a unit into the vector. The selectable or screenable marker may itself be rt t chimeric.

The preferred selectable marker is a gene coding for antibiotic resistance. The gene must be capable of expression in the cells to be trans- S formed. The cells can be cultured in a medium containing the antibiotic, and those cells containing the vector, which have an enhanced ability to S survive in the medium, are selected. Genes that confer resistance to S C chloramphenicol, kanamycin, hygromycin, G418 or, in principle, any other antibiotic may be useful as a selectable marker.

Some examples of genes that confer antibiotic resistance include, for example, those coding for neomycin phosphotransferase [kanamycin and G418 resistance, Velten et al., 1984]; hygromycin phosphotransferase [hygromycin resistance, van den Elzen et al., 1985]; and chloramphenicol acetyltransferase.

An example of a gene useful primarily as a screenable marker in tissue culture for identification of plant cells containing genetically engineered vectors is a gene that encodes an enzyme having a chromogenic P i I l I I- i i i I- -1 -28 substrate. For example, if the gene encodes the enzyme 8-galactosidase, the plant cells are plated on a tissue culture medium containing the chromogenic substrate Xgal (5-chloro-4-bromo-3-indolyl-8-D-galactoside), and under appropriate conditions, plant cells containing copies of this gene are stained blue by the dye indigo which is released when 8-galactosidase cleaves Xgal.

The introduction of chimeric genes into plants in accordance with the present invention may be carried out with any T-DNA-derived vector system capable of introducing genes into cotton plant cells from Agrobacteria.

The vector system may, for example, be a co-integrate system (Comai et al., 1983; Zambryski et al., 1983) for example the split-end vector system (Fraley et al., 1985), as described by Chilton (1985). The vector system may, on the other hand, be a binary system (de Framond et al., 1983; Hoekema et al., 1983), or a Ti plasmid engineered by homogenotization of the gene into the T-DNA (Matzke and Chilton, S1981). A further possibility is a system wherein the T-DNA is on a plasmid and the virulence genes are on the chromosonal DNA.

t The preferred T-DNA vector system is a binary vector system, and especially a system utilizing pCIB10 (Rothstein et al., 1987) (see figure The introduction of heterologous genes by recombinant DNA manipulation (into a binary vector system is described by Klee et al., 1985. The insertion of genes into a T-DNA vector may be by homologous recombination using a double recombination strategy (Matzke and Chilton, 1981); single recombination strategy (Comai et al., 1983); Zambryski et al., 1983); or S a single recombination strategy with no repeats in the T-DNA (Fraley et al., 1985) as described by Chilton (1985).

If the vectors containing the chimeric gene are not assembled in Agrobacterium, they may be introduced into Agrobacterium by methods known in the art. These methods include transformation and conjugation.

A

T

29 Transformation involves adding naked DNA to bacteria. Agrobacterium may be made susceptible to the introduction of naked DNA by freezing and thawing. The transformation of Agrobaceterium is described by Holsters et al. (1978).

Conjugation involves the mating of a cell containing the desired vector, usually E. coli, with Agrobacterium. This method is described by Comai et al. (1983) and Chilton et al. (1976).

The Agrobacterium spp. may be any strain of Agrobacterium capable of introducing genes into cotton plant cells. Some suitable examples include A. tumefaciens, A. rhizogenes, and A. radiobacter.

Transformed cotton plant cells containing the chimeric gene may be maintained in culture or may be regenerated into living plants.

S Expesssion is preferably of sufficient efficiency to render the plant Scells insecticidal.

It, t The medium capable of sustaining a particular plant cell in culture depends on the particular variety of cotton plant cell. For example, some suitable media include approximately 10 mg/liter of 2,4-dichlorophenoxyacetic acid and either Murashige and Skoog inorganic salts (Murashige and SSkoog, 1962) or Gamborg B-5 inorganic salts (Gamborg et al., 1968).

St* Cotton (Gossypium spp.) embryos capable of germination and regeneration can be efficiently produced through somatic embryogenesis by developing S pro-embryonic cell masses and, from them, embryos in a cell suspension culture system.

The present method permits the production, for example, in a standard 250 ml DeLong flask of about 10,000 globular embryos, from which about 1000 mature embryos and about 50 plants may be obtained.

The cotton plants produced in accordance with this method may be cultivated or wild. Cultivated cotton plants are preferred.

Step a: Embryogenic Cotton Callus The first step is to induce cotton callus formation from cotton explant tissue. Some examples of suitable cotton explant tissue include somatic embryos, mature and immature zygotic embryos, cotyledons or hypocotyls from a seedling, and young tissue from a mature plant. Somatic embryos and seedling cotyledons or hypocotyls are preferred.

Zygotic embryos, for example, may be obtained by excision from ovules.

The ovules are preferably excised about 7 to 30 days after pollination, preferably about 10 to 21 days after pollination, and most preferably about 12 to 16 days after pollination.

Cotyledons and hypocotyls may be obtained from young seedlings. The seedlings are preferably between about 3 and 21 days old, more preferably between about 4 and 9 days old, and most preferably about 7 days old.

Hypocotyls are sliced longitudinally and cut into convenient sections for example between 1 and 20 mm, preferably about 2 mm. Cotyledon tissue is ki |cut into sections between I and 400 mm 2 preferably between 5 and 100 mm 2 and most preferably about 10 mm 2 Somatic embryos derived from this procedure are the most preferred source for obtaining embryogenic callus according to the present method.

t Somatic embryos may, for example, be obtained by using the method described above for hypocotyl and cotyledonary tissue as the explant -source. Any somatic embryo taken before primary leaf expansion is suitable. The size of the somatic embryo is not critical. Preferably, the somatic embryo is less than about 5 mm in length.

l Young tissue from a mature cotton plant may conveniently be obtained by excising the apical 10 cm, preferably about 5 cm, of a shoot tip. Stem V and petiole tissue are sliced longitudinally and cut into the same size sections as are hypocotyls (see above). Leaf tissue is cut into the same size section as cotyledon tissue (see above).

31- The cotton plant tissue is placed on a suitable callus induction medium at about 200 to 40 0 C, preferably 230 to 35 0 C, more preferably about 31 0

C.

Any medium capable of inducing callus from the tissue may be used in this regeneration method. The medium may be liquid or solid, although a solid medium is preferred since it is more convenient.

One medium capable of inducing callus under the conditions of the invention contains inorganic salts, vitamins, a carbon source, an auxin, and a cytokinin. The medium is adjusted to a pH between 3.5 and preferably between 4.5 and 6.5, and most preferably about 5.7.

Any inorganic salts and vitamins capable of contributing to callus induction are suitable. Some examples of suitable inorganic salts and vitamins include those described by Murashige and Skoog (1962) (MS) and Gamborg et al. (1968) Another example is a modification of MS or Gamborg's B-5 media described by Cheng et al. (1980). The preferred inorganic salts are MS inorganic salts. The preferred vitamins are 0 940 Gamborg's B-5 vitamins.

49 0 *o The carbon source may be any carbon source on which callus can be grown.

The preferred carbon sources include sugars and derivatives of sugar. The o preferred sugars are glucose and sucrose. It is especially desirable to 4 a initiate callus in a callus induction medium containing glucose in order 0 to reduce browning of the tissue, and then to transfer the callus to a S callus induction medium containing sucrose.

9 The concentration of the carbon source is 5 to 60 g/liter, preferably about 30 g/liter.

The auxin present in the callus induction medium may be any auxin capable of inducing callus. Some suitable auxins include a-naphthaleneacetic acid, picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lactic acid, indole-3pyruvic acid, adole-3-acetic acid, and p-chlorophenoxyacetic acid. A preferred auxin is a-naphthaleneacetic acid.

32 Any concentration of auxins capable of inducing callus formation may be used in the method of the invention. A suitable concentration is 0.1 to mg/liter. A preferred concentration is about 2 mg/liter, especially when the auxin is a-naphthaleneacetic acid.

The cytokinin present in the callus induction medium may be any cytokinin capable of inducing callus. Some suitable cytokinins include kinetin, 6-benzyladenine, 2-isopentenyladenine, and zeatin. A preferred cytokinin is kinetin.

Any concentration of cytokinin capable of inducing callus formation may be used in the method of the invention. Suitable concentrations are 0.1 to 10 mg/liter. A preferred concentration is 1 mg/liter, especially when the cytokinin is kinetin.

a If the medium is solid, it contains a component that causes solidification, for example about 0.8 agar such as Agar Noble (Difco) or about 0.8 agarose. (All percents in this specification are by weight).

0 a The tissue is cultured on the callus induction medium for a period of time sufficient for the callus to form. For example, tissue may be S. cultured on a callus induction medium containing glucose as the carbon source. A five week induction period is typical. Subcultures are performed as necessary to prevent browning. Weekly subcultures are preferred.

I The callus that forms may be unorganized, or may contain pro-embryonic «o cell masses, embryogenic callus and/or embryos. Normally, when hypocotyls or cotyledons are used as the explant source, the callus appears to be S° unorganized. When somatic embryos are used as the explant source, at least part of the callus appears to comprise embryogenic callus, which is characterized by a light yellow color and nodulation.

The resulting callus may then advantageously be transferred to a callus subculture medium, which is similar to a callus induction medium except that it contains sucrose as the carbon source, for a period of time up to i: i i i i ~rr 33 months. One month, or two months with a subculture into fresh medium after one month, on a sucrose-containing callus induction medium is preferred.

The callus may be induced in the dark, but is preferably induced in the light. The light may have an intensity of, for example, 0.5 to -2 -1 150 PEm2 s 41.75 to 12525 lx).

Step b: Clumpy Aggregates of Pro-embryonic Cell Masses The callus from step is suspended in a liquid medium promoting the development of pro-embryonic or proliferating embryonic cell masses. It is important for the cell density to be low. Therefore, not more than mg of callus/ml of culture medium, preferably not more than 15 mg of callus/ml of culture medium and more preferably not more than 5 mg of o. So callus/ml of culture medium is suspended.

e e a oa SThe medium useful in step may be any medium capable of inducing .oo pro-embryonic cell masses. The medium comprises inorganic salts, a vitamins, a carbon source, and an auxin. The medium may also include organic nitrogen sources, cytokinins, amino acids and other addenda such as casein hydrolysate or coconut water.

9 D The inorganic salts and vitamins may be the same as in step (supra).

*4oa MS inorganic salts and B-5 vitamins are preferred.

a The carbon source may be the same as in step (supra). Sucrose is preferred. The concentration of the carbon source is 0.1 to 100 g/liter.

About 20 g/liter is preferred, especially when the carbon source is 0.

o a *o sucrose.

o The auxin may be selected from the auxins used in step The preferred auxins are 2,4-dichlorophenccyacetic acid and picloram. Picinram is most preferred.

The concentration of the auxin in step is relatively low. The exact concentration depends on the specific auxin used. The relatively low auxin concentration is generally similar to that usually used in sus- ~_1Lii_ iiiiiliilli~il-l 34 pension culture media and is significantly lower than the corresponding auxin concentration used in step When picloram is the auxin used in step the concentration is 0.01 to 5 mg/liter, preferably 0.1 to 1 mg/liter, and most preferably about 0.5 mg/liter. When 2,4-dichlorophenoxyacetic acid is the auxin used in step the concentration is 0.01 to 0.5 mg/liter, preferably 0.05 to 0.25 mg/liter, and most preferably about 0.1 mg/liter.

The induction of pro-embryonic cell masses is preferably carried out in an aerated medium at a temperature between 200 and 35 0 C, preferably between 220 and 33°C and most preferably between 25° and 31°C. One may aerate the medium by any method known in the art, for example by shaking.

Step may be carried out in the dark or in light up to about -2 -2 -1 Em-2s 1 6262,5 lx), preferably between 5 and 10 lEm- s 417,5 and 835 lx).

6 00 s The callus is maintained in the medium preferably without subculture until clumpy aggregates of pro-embryonic cell masses form and begin to s* 0 0 proliferate rapidly. The onset of rapid proliferation usually takes between 3 and 8 weeks, more typically between 5 and 7 weeks. During the induction period, the medium may be replaced by fresh medium, although it is preferable not to disturb the medium during this period.

The change from callus to clumpy aggregates of pro-embryonic cell masses will be readily apparent to those of ordinary skill in the art of plant S tissue culture. It is distinguishable by the light yellow color and 3| clumpy nature of the pro-embryonic cell masses.

io Once the clumpy aggregates of pro-embryogenic cell masses begin to proliferate rapidly, they may be introduced directly into the medium described in step or they may be subcultured in order to prevent browning. Subculturing every 3 to 7 days, preferably every 5 to 7 days, is convenient. The cell masses survive without subculture for about fourteen days.

35 Step c: Finely Dispersed Pro-embryonic Cell Masses The clumpy aggregates of pro-embryonic cell masses from step are transferred to a liquid medium that is capable of causing the clmpy aggregates of pro-embryonic cell masses to become finely dispersed. The medium may be similar to that described in step except that the medium of step comprises a relatively high concentration of an auxin.

The auxin may be any auxin used in step The preferred auxins are 2,4,5-trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid. The most preferred auxin is 2,4-dichlorophenoxyacetic acid.

The concentration of auxin depends on the particular auxin used. The auxin concentration in the medium of step is generally higher, or at least at the high end of the range of, than concentrations that are usually used in suspension culture media, and in any event is significantly higher than the corresponding auxin concentration used in S' step 4 i For example, when 2,4-dichlorophenoxyacetic acid is the auxin in the ec' medium of step the concentration may be about 0.5 to 100 mg/liter, S preferably 1 to 10 mg/liter, and most preferably about 2.5 to mg/liter.

t S Except for the concentration and possibly the identity of the auxin, the S medium, temperature, and amount of light in step may be the same as that described in step The conditions of step are maintained until the clumpy aggregates of pro-embryonic cell masses become smaller, more finely dispersed proembryonic cell masses. The appearance of the smaller, more finely 1 dispersed pro-embryonic cell masses will be readily apparent to those skilled in the art. These cell masses are characterized by their yellow color, smooth surface, intermediate density and small size. The change to the smaller, more finely dispersed cell masses usually occurs within 6 weeks, more typically 2 weeks.

L

36 The culture of the small finely dispersed pro-embryonic cell masses may be maintained indefinitely, and may be subcultured so as to maintain active growth. It is convenient to subculture, for example, every 3 to 28 days, preferably every 5 to 10 days.

Step d: Mature Embryos The smaller, more finely dispersed pro-embryonic cell masses are added to a medium that induces the development of mature embryos. The medium is preferably a liquid.

Embryos pass through a number of developmental stages before they mature and are able to germinate. These stages include globular, heart, torpedo and mature stages. The names of the stages are based on the approximate shapes of the embryos.

t The medium useful in step may be any medium that induces the develop- *o ment of mature embryos. One useful medium comprises inorganic salts, too vitamins, a carbon source and an organic compound containing reduced nitrogen.

t The salts and vitamins and concentrations thereof may be the same as those described in step The carbon source may also be one of the 4 4 S carbon sources described in step The concentration of the carbon source is about 1 to 10 g/liter preferably about 2 to 6 g/liter. A preferred carbon source is sucrose.

The organic nitrogen source may be any such compound which, when added to the medium of step induces the development of mature embryos. The preferred compounds are amino acids. A preP i amino acid is glutamine.

The concentration of the organic nitrogen source depends on the particular compound used. An effective concentration of glutamine as the organic nitrogen source is 2 to 260 mM, preferably 5 to 100 mM, and most preferably 10 to 50 mM.

i i 1 i 37 The medium of step may contain an auxin. Auxins are desirable during the early stages of embryo development, but not during the later stages.

Therefore, if auxins are present at all, they are preferably present only until the heart stage of development. Then, the embryos are transferred to a medium that contains no auxin.

If present, the auxin concentration may be 0.01 to 0.1 mg/liter.

The auxin may be one of the auxins useful in step The preferred auxins are picloram and 2,4-dichlorophenoxyacetic acid.

The embryos may be cultured in the medium of step at temperatures of 200 to 35°C in the dark or in light. The intensity of the light may be, -2 -1 for example, 5 to 75 iEm s 6262.5 lx).

0 oooro a a The embryos are maintained in the medium of step until the embryos o" have matured into torpedo or mature states. Those skilled in the art of 0 00 o 60o plant tissue culture will be able to recognize the globular, heart, 0 0 00 torpedo and mature embryos as they form. The embryos mature, typically, o 0 in 2 to 5 weeks, usually in 3 to 4 weeks. It is usually unnecessary to 0 0o subculture the embryos or to transfer the embryos to fresh medium, except possibly to change from an auxin-containing medium to a medium not containing an auxin at the heart stage.

oo0 0 00 Step e: Germination The mature embryos are placed on a solid medium capable of inducing 0ofo germination. The medium comprises inorganic salts, vitamins, and a carbon o 8 source. The iiedium is solidified with a suitable solidifying agent such I as Gelrite (Kelko, San Diego, California), agarose or agar.

0 1& The inorganic salts may be those described in step modified so that nitrate is present at high concentration while ammonium is either absent or is present at very low concentration. The concentration of nitrate may be 20 to 60 mM, preferably 30 to 60 mM, more preferably 35 to 45 mM. The concentration of ammonium ion should be no greater than 5 mM.

38 The source of carbon is preferably a sugar. The preferred sugar is sucrose. The concentration of the carbon source depends on the particular carbon source used. For example, when sucrose is the carbon source, the concentration is 0.1 to 6 by weight, preferably 0.5 to 4 more preferably 1 to 3 An organic nitrogen source is optionally present in the medium of step The organic compound is preferably an amino acid or a mixture of amino acids capable of supporting germination. Preferred amino acids or mixtures thereof are glutamine or casein hydrolysate.

The concentration of the organic nitrogen source depends on the specific compound used. For example, when the compound is glutamine, the concentration may be 2 to 50 mM, preferably 5 to 30 mM, more preferably 10 to mM. When the compound is casein hydrolysate or modified casein hydrolysate, the concentration is 100 to 3000 mg/liter, preferably 1' 000 to 2800 mg/liter, more preferably 1500 to 2500 mg/liter.

Preferably, germination occurs on a medium containing an organic nitrogen source until shoots develop. The embryos are then transferred to a medium containing no organic nitrogen source for elongation of roots.

SThe density of embryos in the medium is limited to a density less than that which causes development to be self-inhibitory. Suitable densities include 1 to 100 embryos in a 9 cm petri disk containing about 10 to ml of medium, preferably 25 to 50 ml of medium, and most preferably about 35 ml of medium.

The medium or media of step are maintained at 200 to 30 0 C. Preferably, the temperature is about 250C.

Some light is necessary in step An intensity of light between 5 and -2 -1 -2 -1 150 pEm2 s 417.5 to 12525 lx), preferably between 10 and 75 pEm- s 835 to 6262.5 lx), is suitable.

39 The embryos are maintained on the medium or media of step until the embryos have germinated, typically 1 to 20 days, usually 2 to 4 days.

Those skilled in the art will know when the embryos have germinated.

Step f: Plants Following germination, the plantlets are transferred to soil for growth into plants. The transferred plants are initially covered with a glass to maintain high humidity. After one week under glass, no special treatment of the plantlets or of the plants is necessary.

Utility Propagation Mature embryos may be used for mass propagation and cloning. This entails either germinating the embryos and transplanting the plantlets to soil, to other growth substrates, or other plant growth environments. Mature embryos may also be enclosed in an artificial seed coat and planted as i t r "somatic seeds". Mass propagation and cloning is beneficial if hybrid parents or a hybrid itself needs to be mass produced.

SCells, pro-embryos, embryos, plantlets and plants may be analyzed at any 4 .time during the stages described above in order to determine whether any new trait is present as a result of genetic alteration. The trait may be a useful in vitro or in planta trait. Some examples of useful traits include phytotoxin tolerance, drought tolerance, cold tolerance, disease tolerance, etc.

The cells of steps and may also be subjected to tissue culture methods capable of produicing cells or plants having desirable properties, such as herbicide tolerance. Some examples of such methods include, for example, Chaleff and Ray (1984).

The invention therefore also includes living cotton plants, the cells of which contain the chimeric gene that encodes and expresses the polypeptide having substantially the insect toxicity properties of Brt crystal protein.

40 The plant cells of this invention contain the chimeric gene and may be used to produce the polypeptide having substantially the insect toxicity of a Bt crystal protein. The plant cells per se may constitute the insecticide. Plant cells used directly as insecticides may be cultured plant cells, or may be components of a living plant.

The toxin may also be isolated from the plant cells by known methods such as, for example, by extraction or chromatography. The extract may be the total plant cell extract, a partially purified extract, or a pure preparation of the polypeptide. Any such extract or chromatographic isolate may be used in the same way as crystal protein from Bt (see, for example, Deacon, 1983, Miller et al., 1983).

The insecticidal cells of the present invention are toxic to insects o that attack cotton cells and plants.

.4 Hence, the present invention provides a method for producing in cotton S a polypeptide having substantially the insect toxicity properties of a Bt crystal protein, which method comprises: introducing into cotton cells a gene coding for a polypeptide having substantially the insect toxicity properties of a Bt crystal protein 0 wherein the promoter, 5' untranslated region, and optionally, the 3' untranslated region of the gene are derived from a plant or plant virus gene, and expressing the polypeptide.

kPPlic^+LI' o iescoibes The present ir- nir-t- a method of controlling insect larvae S comprising feeding the larvae an insecticidal amount of transgenic cotton cells containing a gene coding for a Bt crystal toxin or a polypeptide having substantially the insect toxicity properties of a Bt crystal protein.

The present 4n,,to e .a a method for killing or controlling insect larvae comprising feeding the larvae an insecticidal amount of transgenic cotton plant cells that contain the chimeric gene of the invention. Furthermore, the present invention also includes a method for 41 killing coleopteran larvae by feeding them an insecticidal amount of cells containing the chimeric gene having the coding sequence of the Bt var. tenebrionis crystal toxin or insecticidal parts thereof.

The plant cells may be cultured plant cells, or may be components of living plants.

The present invention further includes corn seeds of plants genetically engineered in accordance with this invention as long as the seeds contain the inserted chimeric gene and the desirable trait resulting therefrom.

Progeny of plants produced by the method of this invention, including sexual and vegetative progeny, are further embodiments. Sexual progeny may result from selfing or cross pollination.

Non limiting Examples General Recombinant DNA Techniques Since many of the recombinant DNA techniques used in this invention are routine for those skilled in the art, a brief description of these commonly used techniques is included here rather than at each instance where they appear below. Except where noted, all of these routine procedures are described in the reference by Maniatis et al. (1982).

A. Restriction endonuclease digestions Typically, DNA is present in the reaction mixture at approximately 50-500 pg/ml in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA. 2 to 5 units of restriction endonucleases are added for each jig of DNA, and the reaction mixture incubated at the temperature recommended by the manufacturer for one to three hours. The reaction is terminated by heating to 65°C for ten minutes or by extraction with phenol, followed by precipitation of the DNA with ethanol. This technique is also described on pages 104-106 of the Maniatis et al.

reference.

-42- B. Treatment of DNA with polymerase to create flush ends DNA fragments are added to a reaction mixture at 50-500 ig/ml in the buffer recommended by the manufacturer, New England Biolabs. The reaction mixture contains all four deoxynucleotide triphosphates at a concentration of 0.2 mM. The reaction is incubated at 15 C for 30 minutes, and then terminated by heating to 65°C for ten minutes. For fragments produced by digestion with restriction endonucleases that create truding ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of DNA polymerase is used. For fragments produced by endonucleases that produce 3'-protruding ends, such as PstI and SacI, T4 DNA polymerase is used. Use of these two enzymes is described on pages 113-121 of the Maniatis et al. reference.

I C. Agarose gel electrophoresis and purification of DNA fragments from gels Agarose gel electrophoresis is performed in a horizontal apparatus as described on pages 150-163 of Maniatis et al. reference. The buffer used Sis the Tris-borate buffer described therein. DNA fragments are visualized by staining with 0.5 pg/ml ethidium bromide, which is either present in the gel and tank buffer during electrophoresis or added following S electrophoresis. DNA is visualized by illumination with short-wavelength or long-wavelength ultraviolet light. When the fragments are to be t, ,isolated from the gel, the agarose used is the low gelling-temperature i variety, obtained from Sigma Chemical, St. Louis, Missouri. After electrophoresis, the desired fragment is excised, placed in a plastic tube, heated to 65°C for approximately 15 minutes, then extracted with S t• phenol three times and precipitated with ethanol twice. This procedure is slightly modified from that described in the Maniatis et al. reference at page 170.

D. Addition of synthetic linker fragments to DNA ends When it is desired to add a new restriction endonuclease site to the end of a DNA molecule, that molecule is first treated with DNA polymerase to create flush ends, if necessary, as described in the section above.

43 Approximately 0.1 to 1.0 ig of this fragment is added to approximately 100 ng of phosphorylated linker DNA, obtained from New England Biolabs, in a volume of 20 to 30 p1 containing 2 pl of T4 DNA ligase, from New England Biolabs, and 1 mM ATP in the buffer recommended by the manufacturer. After incubation overnight at 15 0 C, the reaction is terminated by heating the mixture at 65CC for ten minutes. The reaction mixture is then diluted to approximately 100 pl in a buffer suitable for the restriction endonuclease that cleaves at the synthetic linker sequence, and approximately 50 to 200 units of this endonuclease are added. The mixture is incubated at the appropriate temperature for 2 to 6 hours, then the fragment is subjected to agarose gel electrophoresis and the fragment purified as described above. The resulting fragment will now have ends with termini produced by digestion with the restriction endonuclease.

SThese termini are usually cohesive, so that the resulting fragment is now easily ligated to other fragments having the same cohesive termini.

E. Removal of 5'-terminal phosphates from DNA fragments r During plasmid cloning steps, treatment of the vector plasmid with phosphatase reduces recircularization of the vector (discussed on page 13 of Maniatis et al. reference). After digestion of the DNA with the appropriate restriction endonuclease, one unit of calf intestine alkaline i phosphatase, obtained from Boehringer-Mannheim, Indianapolis, IN, is added. The DNA is incubated at 37 0 C for one hour, then extracted twice with phenol and precipitated with ethanol.

F. Ligation of DNA fragments i When fragments having complementary cohesive termi.ni are to be joined, approximately 100 ng of each fragment are incubated in a reaction mixture of 20 to 40 p1 containing approximately 0.2 units of T4 DNA ligase from New England Biolabs in the buffer recommended by the manufacturer. The incubation is conducted for 1 to 20 hours at 150C. When DNA fragments having flush ends are to be joined, they are incubated as above, except the amount of T4 DNA ligase is increased to 2 to 4 units.

i 7- 44 G. Transformation of DNA into E. coll E. coli strain HB101 is used for most experiments. DNA is introduced into E. coli using the calcium chloride procedure described by Maniatis et al.

on pages 250-251. Transformed bacteria are selectively able to grow on medium containing appropriate antibiotics. This selective ability allows the desired bacteria to be distinguished from host bacteria not receiving transforming DNA. Determining what antibiotic is appropriate is routine, given knowledge of the drug resistance genes present on incoming transforming DNA and the drug sensitivity of the host bacteria. For example, where the host bacterium is known to be sensitive to ampicillin and there is a functional drug resistance gene for ampicillin on the incoming transforming DNA, ampicillin is an appropriate antibiotic for selection of transformants.

H. Screening E. col for plasmids Following transformation, the resulting colonies of E. coly are screened S fr the presence of the desired plasmid by a quick plasmid isolation procedure. Two conventient procedures are described on pages 366-369 of Maniatis et al. reference.

i I. Large scale isolation of plasmid DNA Procedures for isolating large amounts of plasmids in E. coll are found on pages 88-94 of the Maniatis et al. reference.

J. Cloning into M13 phage vectors.

SIn the following description, it is understood that the double-stranded r replicative form of the phage M13 derivatives is used for routine procedures such as restriction endonuclease digestions, ligations, etc.

Example 1: Construction of chimeric gene plasmid pBR322 In order to fuse the CaMV gene VI promoter and protoxin coding sequences, a derivative of phage vector mpl9 (Yanish-Perron et al., 1985) is constructed.

45 First, a DNA fragment containing approximately 155 nucleotides 5' to the protoxin coding region and the adjacent approximately 1346 nucleotides of the coding sequence are inserted into mpl9. Phage mpl9 ds rf (doublestranded replicative form) DNA is digested with restriction endonucleases SacI and Smal and the approximately 7.2 kbp (kilo base pairs) vector fragment is purified after electrophoresis through low-temperature gelling agarose by standard procedures. Plasmid pKU25/4, containing approximately 10 kbp of Bt DNA, including the protoxin gene, is obtained from Dr. J. NUesch, CIBA-GEIGY Ltd., Basle, Switzerland. The nucleotide sequence of the protoxin gene present in plasmid pKU25/4 is shown in formula I. Plasmid pKU25/4 DNA is digested with endonucleases Hpal and SacI, and a 1503 bp fragment (containing nucleotides 2 to 1505 in formula is purified as above. (This fragment contains approximately S 155 bp of bacterial promoter sequences and approximately 1346 bp of the start of the protoxin coding sequence). Approximately 100 ng of each fragment is then mixed, T4 DNA ligase added, and incubated at 15 0

C

S, overnight. The resulting mixture is transformed into E. coli strain tIS S HB101, mixed with indicator bacteria E. coll JM 101 and plated as described (Messing, 1983). One phage called mpl9/bt is used for further construction below (Figure 1).

I Next, a fragment of DNA containing the CaMV gene VI promoter, and some of the coding sequences for gene VI, is inserted into mpl9/bt. Phage mpl9/bt #tit ds rf DNA is digested with BamHI, treated with the large fragment of DNA 4t-, polymerase to create flush ends and recleaved with endonuclease PstI. The larger vector fragment is purified by electrophoresis as described above.

i Sr Plasmid pABDI is described in Paszkowski et al., 1984. Plasmid pABD1 DNA :iit s digested with Pstl and HindIII. The fragment approximately 465 bp long containing the CaMV gene VI promoter and approximately 75 bp of the gene VI coding sequence is purified. The two fragments are ligated and plated as described above. One of the resulting recombinant phages, called mpl9/btca is used in the following experiment.

Phage mpl9/btca contains CaMV gene VI promoter sequences, a portion of the gene VI coding sequence, approximately 155 bp of Bt DNA upstream of the protoxin coding sequence, and approximately 1346 bp of the protoxin 46 coding sequence. To fuse the CaMV promoter sequences precisely to the protoxin coding sequences, the intervening DNA is deleted using oligonucleotide-directed mutagenesis of mpl9/btca DNA. A DNA oligonucleotide with the sequence TTCGGATTGTTATCCATGGTTGGAGGTCTGA is synthesized by routine procedures using an Applied Biosystems DNA Synthesizer. This oligonucleotide is complementary to those sequences in phage mpl9/btca DNA at the 3' end of the CaMV promoter (nucleotides 5762 to 5778 in Hohn et al., 1982) and the beginning of the protoxin coding sequence (nucleotides 156 to 172 in formula I above). The general procedure for the mutagenesis is that described in Zoller and Smith (1983). Approximately 5 }ig of single-stranded phage mpl9/btca DNA is mixed with 0.3 pg phosphorylated oligonucleotide in a volume of 40 Pl.

The mixture is heated to 65°C for 5 minutes, cooled to 50 0 C, and slowly cooled to 4 0 C. Next, buffer, nucleotide triphosphates, ATP, T4 DNA ligase S and the large fragment of DNA polymerase are added and incubated overnight at 15 0 C as described (Zoller and Smith, 1983). After agarose gel eleLcrophoresis, circular double-stranded DNA is purified and transfected into E. coll strain JM101. The resulting plaques are screened for S sequences that hybridize with 32 P-labelled oligonucleotide, and phage are analyzed by DNA restriction endonuclease analysis. Among the resulting phage clones will be ones which have correctly deleted the unwanted S. sequences between the CaMV gene VI promoter and the protoxin coding S sequence. This phage is called mpl9/btca/del (see Figure 2).

Next, a plasmid is constructed in which the 3' coding region of the protoxin gene is fused to CaMV transcription termination signals. First, plasmid pABD1 DNA is digested with endonucleases BamHI and BglII and a 0.5 kbp fragment containing the CaMV transcription terminator sequences is isolated. Next plasmid pUC19 (Yanish-Perron et al., 1985) is digested with BamHI, mixed with the 0.5 kbp fragment and incubated with T4 DNA ligase. After transformation of the DNA into E. coll strain HB101, one of the resulting clones, called plasmid p702, is obtained, which has the structure shown in Figure 3.

Next, plasmid p702 DNA is cleaved with endonucleases SacI and SmaI, and the larger, approximately 3.2 kbp fragment is isolated by gel electrophoresis. Plasmid pKU25/4 DNA is digested with endonucleases AhaIII and 47 SacI, and the 2.3 kbp fragment (nucleotides 1502 to 3773 in formula I above) containing the 3' portion of the protoxin coding sequence (nucleotides 1504 to 3773 of the sequence shown in formula I) is isolated after gel electrophoresis. These two DNA fragments are mixed, incubated with T4 DNA ligase and transformed into E. coll strain HB101. The resulting plasmid is p702/bt (see Figure 3).

Finally, portions of phage mpl9/btca/del ds rf DNA and plasmid p702/bt are joined to create a plasmid containing the complete protoxin coding sequence flanked by CaMV promoter and terminator sequences. Phage mpl9/bt ca/del DNA is digested with endonucleases SacI and Sphl, and a fragment of approximately 1.75 kbp is purified following agarose gel electrophoresis. Similarly, plasmid p702/bt DNA is digested with endonucleases SacI and SalI and a fragment of approximately 2.5 kbp is isolated.

Finally, plasmid pBR322 DNA (Bolivar et al., 1977) is digested with Sall and SphI and the larger 4.2 kbp fragment isolated. All three DNA fragi ments are mixed and incubated with T4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid, pBR322/btl4 is a derivative of pBR322 containing the CaMV gene VI promoter and translation start signals fused to the Bt crystal protein coding sequence, followed by CaMV transcription termination signals (shown in Figure 4).

Example 2: Construction of a Ti plasmid-derived vector.

The vector pCIB10 (Rothstein et al., 1987) is a Ti-plasmid-derived vector useful for transfer of the chimeric gene to plants via Agrobacterium 4 tumefaciens. The vector is derived from the broad host range plasmid pRK252, which may be obtained from Dr. W. Barnes, Washington University, St. Louis, Mo. The vector also contains a gene for kanamycin resistance in Agrobacterium, from Tn903, (Oka et al., 1981) and left and right T-DNA border sequences from the Ti plasmid pTiT37. Between the border sequence are the polylinker region from the plasmid pUC1l and a chimeric gene that confers kanamycin resistance in plants.

First, plasmid pRK252 is modified to replace the gene conferring tetracycline-resistance with one conferring resistance to kanamycin from the transposon Tn903, and is also modified by replacing the unique EcoRI site in pRK252 with a BglII site (see Figure 5 for a summary of these modifi- 48 cations). Plasmid pRK252 is first digested with endonucleases SalI and Smal, then treated with the large fragment of DNA poJymerase I to create flush ends, a.-d the large vector fragment purified by agarose gel electrophoresis. Next, plasmid p368, which contains Tn903 on an approximately 1050 bp BamHI fragment, is digested with endonuclease BamHI, treated with the large fragment of DNA polymerase, and an approximately 1050 bp fragment is isolated after agarose gel electrophoresis; this fragment contains the gene from transposon Tn903 which confers resistance to the antibiotic kanamycin (Oka et al., 1981). Both fragments are then treated with the large fragment of DNA polymerase to create flush ends.

Both fragments are mixed and incubated with T4 DNA ligase overnight at 1500. After transformation into E. coli strain HB101 and selection for kanamycin resist--t colonies, plasmid pRK252/Tn903 is obtained (see .o 0. Figure So o 0 0 SPlasmid pRK252/Tn903 is digested at its unique EcoRI site, followed by treatment with the large fragment of f. cold DNA polymerase to create flush ends. This fragment is added to nynthetic BglII restriction site linkers, and incubated overnight with T4 DNA ligase. The resulting DNA is S,O digested with an excess of BglII restriction endonuclease and the larger vector fragment purified by agarose gel electrophoresis. The resulting fragment is again incubated with T4 DNA ligaae to recircularize the fragment via its newly added BglII cohesive ends. Following trans- Sformation into E. cold strain HB101, plasmid pRK252/Tn903/BglII is e obtained (see Figure S A derivative of plasmid pBR322 is constructed which contains the Ti Splasmid T-DNA borders, the polylinker region of plasmid pUC19, and the selectable gene for kanamycin resistance in plants (see Figure 6).

Plasmid pBR325/Eco29 contains the 1,5 kbp EcoRI fragment from the nopaline Ti plasmid pTiT37. This fragment contains the T-DNA left border sequence (Yadav et al., 1982). To replace the EcoRI ends of this fragment with HindIII ends, plasmid pBR325/Eco29 DNA is digested with EcoRI, then incubated with nuclease S1, followed by incubation with the large fragment of DNA polymerase to create flush ends, then mixed with synthetic HindIII linkers and incubated with T4 DNA ligase. The resulting DNA is digested with endonucleases ClaI and an excess of HindIII, and the -49 resulting 1.1 kbp fragment containing the T-DNA left border is purified by gel electrophoresis. Next, the polylinker region of plasmid pUC19 is isolated by digestion of the plasmid DNA with endonucleases EcoRI and HindIII and the smaller fragment (approx. 53 bp) is isolated by agarose gel electrophoresis. Next, plasmid pBR322 is digested with endonucleases EcoRI and ClaI, mixed with the other two isolated fragments, incubated with T4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid, pCIB5, contains the polylinker and T-DNA left border in a derivative of plasmid pBR322 (see Figure 6).

A plasmid containing the gene for expression of kanamycin resistance in plants is constructed (see Figures 7 and Plasmid pBIN6 is obtained from Dr. M. Bevan, Plant Breeding Institute, Cambridge, UK. This plasmid o° 0, is described in the reference by Bevan, 1984. Plasmid pBIN6 DNA is o ,o digested with EcoRI and HindIII and the fragment approximately 1.5 kbp in size containing the chimeric neomycin phosphotransferase (NPT) gene is S, isolated and purified following agarose gel electrophoresis. This t t fragment is then mixed with plasmid pUC18 DNA which has been cleaved with endonucleases EcoRI and HindIII. Following incubation with T4 DNA ligase, S, the resulting DNA is transformed into E. coli strain HB101. The resulting S plasmid is called pUC18/neo. This plasmid DNA contains an unwanted BamHI recognition sequence between the neomycin phosphotransferase gene and the terminator sequence of nopaline synthase gene (see Bevan, 1984). To remove this recognition sequence, plasmid pUC18/neo is digested with 1 endonuclease BamHI, followed by treatment with the large fragment of DNA polymerase to create flush ends. The fragment is then inubated with T4 DNA ligase to recirC.uarize the fragment, and is transformed into E. colii strain HB101. The resulting plasmid, pUC18/neo (Bam) has lost the BamHI recognition sequence.

The T-DNA right border sequence is then added next to the chimeric NPT gene (see Figure Plasmid pBR325/Hind23 contains the 3.4 kbp HindIII fragment of plasmid pTiT37. This fragment contains the right T-DNA border sequence (Bevan et al., 1983). Plasmid pBR325/Hind23 DNA is cleaved with endonucleases SacII and HindIII, and a 1.0 kbp fragment containing the right border is isolated and purified following agarose gel electrophoresis. Plasmid pUC18/neo(Bam) DNA is digested with endonucleases SacII I ,I 50 and HindIII and the 4.0 kbp vector fragment is isolated by agarose gel electrophoresis. The two fragments are mixed, incubated with T4 DNA ligase and transformed into E. coll strain HB101. The resulting plasmid, pCIB4 (shown in Figure contains the T-DNA right border and the plant-selectable marker for kanamycin resistance in a derivative of plasmid pUC18.

Next, a plasmid is constructed which contains both the T-DNA left and right borders, with the plant selectable kanamycin-resistance gene and the polylinker of pUC18 between the borders (shown in Figure Plasmid pCIB4 DNA is digested with endonuclease HindIII, followed by treatment with the large fragment of DNA polymerase to create flush ends, followed by digestion with endonuclease EcoRI. The 2.6 kbp fragment containing the chimeric kanamycin-resistance gene and the right border of T-DNA is S, isolated by agarose gel electrophoresis.

0.4 Plasmid pCIB5 DNA is digested with endonuclese AatII, treated with T4 DNA I polymerase to create flush ends, then cleaved with endonuclease EcoRI.

The larger vector fragment is purified by agarose gel electrophoresis, S mixed with the pCIB4 fragment, incubated with T4 DNA ligase, and trans- L formed into E. coli strain HB101.

04 The resulting plasmid, pCIB2 (shown in Figure 9) is a derivative of plasmid pBR322 containing the desired sequences between the two T-DNA borders.

The following steps complete the construction of the vector pCIB1O, and Sare shown in Figure 10. Plasmid pCIB2 DNA is digested with endonuclease SEcoRV, and synthetic linkers containinig BglII recognition sites are added as described above. After digestion with an excess or BglII endonuclease, the approximately 2.6 kbp fragment is isolated after agarose gel electrophoresis. Plasmid pRK252/Tn903/BglII, described above (see Figure is digested with endonuclease BglII and then treated with phosphatase to prevent recircularization. These two DNA fragments are mixed, incubated with T4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid is the completed vector, r 51 Example 3: Insertion of the chimeric protoxin gene into vector pCIB1O.

The following steps are shown in Figure 11. Plasmid pBR322/btl4 DNA is digested with endonucleases Pvul and Sail, and then partially digested with endonuclease BamHI. A BamHI-SalI fragment approx. 4.2 kbp in size, containing the chimeTic gene, is isolated following agarose gel electrophoresis, and mixed with plasmid pCIB10 DNA which has been digested with endonucleases BamHI and Sail. After incubation with T4 DNA ligase and transformation into E. coli strain HB101, plasmid pCIB10/19Sbt is obtained (see Figure 11). This plasmid contains the chimeric protoxin gene in the plasmid vector pCIBlO.

In order to transfer plasmid pCIB10/19Sbt from E. coli HB101 to Agrobacterium, an intermediate E. coll host strain S17-1 (Simon et al., 1983) o o is used. This strain, obtainable from Agrigenetics Research Corp., S Boulder, Co., contains mobilization functions that can transfer plasmid pCIB10/19Sbt directly to Agrobacterium via conjugation, thus avoiding the Snecessity to transform naked plasmid DNA directly into Agrobacterium.

S" First, plasmid pCIB10/19Sbt DNA is introduced into calcium chloridetreated S17-1 cells. Next, cultures of transformed S17-1 cells and Agrobacterium tumefaciens strain LBA 4404 (Ooms et al.,1982) are mixed S and mated on an N agar (Difco) plate overnight at room temperture. A loopful of the resulting bacteria are streaked onto AB minimal media, (Chilton et al., 1974) plated with 50 g/ml kanamycin and incubated at 28°C. Colonies are restreaked onto the same media, then restreaked onto N agar plates. Slow-growing colonies are picked, restreaked onto AB minimal media with kanamycin and single colonies isolated. This procedure selects for Agrobacteria containing the pCIB10/19Sbt plasmid.

Example 4: Transfer of the chimeric gene to tobacco plant cells.

Protoplasts of Nicotiana tabacum cv. "Coker 176" are prepared as follows: Four to five week old shoot cultures are grown aseptically in MS medium (Murashige and Skoog, 1962) without hormones at 26 0 C with a 16 hour .ight/8 hour dark photoperiod. Approximately 1.5 g lea tissue are removed from the plant and distributed equally among 8 to 10 Petri dishes (100 X 25 mm, Lab-Tek), each containing 10 ml of enzyme solution. Enzyme solution contains 1 cellulase R-10 (Yakult Pharmaceutical 0.25 macerase (Calbiochem 1 pectolyase Y-23 (Seishin Pharma-

I

ret we l I C till 4 C Ce.: U Ci ic 52 ceutical 0.45 M mannitol and 0.1 x K3 salts (Nagy and Maliga, 1976). Tobacco leaves are cut into thin strips with a scalpel, the dishes are sealed, placed on a gyrotory shaker at 35 rpm and incubated with the enzymes for 4 to 5 hours at room temperature.

Next, contents of the dishes are filtered through a cheesecloth-lined funnel and collected in a flask. The filtrate is pipetted into babcook flasks containing 35 ml each of rinse solution. [Rinse solution contains 0.45 M sucrose, MES (2-[N-morpholino]ethanesulfonic acid), and 0.1 x K3 salts.] The bottles are centrifuged at 80 x g for ten minutes, after which the protoplasts will have floated to the top of the bottle. The protoplasts are removed with a 1 ml pipet, combined into one bottle, and rinsed twice more. The resulting protoplasts are suspended in K3 medium in a 15 ml disposable centrifuge tube.

Concentration of protoplasts is determined by counting in a Fuchs- Rosenthal hemocytometer. Protoplasts are then plated at a density of 100,000/ml in 6 ml of liquid K3 medium per 100 x 20 mm Petri dish (Corning). The dishes containing the protoplasts are incubated at 26 0

C

in the dark for two days, during which time cell wall regeneration will occur.

After the two-day incubation, 5 ;il of a stationary culture of A. tumefaciens containing pCIB10/19Sbt are added to the dish of protoplasts. (The Agrobacteria are grown in YEP medium plus 50 ig/ml kanamycin at 28 0 C until stationary phase is reached.) After incubation for three more days at 26 0 C, cefotaxime (Calbiochem Co.) is added (500 Pg/ml) to kill the Agrobacteria. The following day, cells are diluted with 3 ml fresh K3 medium per dish, and cefotaxime added again (500 ig/ml). Cells are then grown at 26 0 C for 2 to 3 weeks and then screened on selective medium as described by DeBlock et al. (1984).

Example 5: Construction of a Bt protoxin chimeric gene with the CaMV promoter.

5.1. Construction of a CaMV 35S Promoter Cassette Plasmid pCIB710 is constructed as shown in Figure 12. This plasmid contains CaMV promoter and transcription termination sequences for the

U:

1 53 RNA transcript (Covey et al., 1981). A 1149 bp BglII restriction fragment of CaMV DNA [bp 6494-7643 in Hohn et al., 1982] is isolated from plasmid pLV111 (obtained from Dr. S. Howell Univ. California-San Diego).

Alternatively, the fragment can be isolated directly from CaMV DNA by preparative agarose gel electrophoresis as described earlier and mixed with BamHI-cleaved plasmid pUC19 DNA, treated with T4 DNA ligase, and transformed into E. coli. (Note the BamHI restriction site in the resulting plasmid has been destroyed by ligation of the BglII cohesive ends to the BamHI cohesive ends.) The resulting plasmid, called pUC19/35S, is then used in oligonucleotide- directed in-vitro mutagenesis to insert the BamHI recognition sequence GGATCC immediately following CaMV nucleotide 7483 in the Hohn reference. The resulting plasmid, pCIB710, contains the CaMV 35S promoter region and transcription termination region separated by a BamHI restriction site. DNA sequences inserted into this BamHI site will be expressed in plants by these CaMV transcription regulation sequences. (Also note that pCIB710 does not 't contain any ATG translation initiation codons between the start of transcription and the BamHI site.) 5.2. Insertion of the CaMV 35S promoter/Terminator Cassette into pCIB1O.

I The following steps are outlined in Figure 13. Plasmids pCIB10 and S pCIB710 DNAs are digested with EcoRI and SalI, mixed and ligated. The resulting plasmid. pCIB10/710 has the CaMV 35S promoter/terminator cassette inserted into the plant transformation vector pCIBIO. The CaMV 35S sequences are between the T-DNA borders in pCIB1O, and thus will be inserted into the plant genome in plant transformation experiments.

S5.3. Insertion of the Bt protoxin gene into pCIB10/710 The following steps are outlined in Figure 14. As a source of the protoxin gene, plasmid pCIB10/19Sbt is digested with BamHI and NcoI, and the 3.6 kbp fragment containing the protoxin gene is isolated by preparative gel electrophoresis. The fragment is then mixed with synthetic NcoI-BamHI adapter with the sequence 5'-CATGGCCGGATCCGGC-3', then digested with BamHI. This step creates BamHI cohesive ends at both ends of the protoxin fragment. This fragment is then inserted into BamHI- 54 cleaved pCIB10/710. The resulting plasmid, pCIB10/35Sbt, shown in Figure 14, contains the protoxin gene between the CaMV 35S promoter and transcription termination sequences.

5.4. Transfer of the plasmid pCIB10/35Sbt into Agrobacterium tumefaciens for plant transformation.

The plasmid pCIB10/35Sbt is transferred into A. tumefaciens strain LBA4404 as described in example 4, above.

Example 6: Construction of pTOX, containing a chimeric gene encoding the insecticidal toxin gene of Bt var. tenebrionis A gene encoding the insecticidal crystal protein gene of Bt var. tenebrionis has been characterized and sequenced (Sekar et al., 1987). This S coding sequence is isolated on a convenient restriction fragment, such as S' a HindIII fragment of approximately 3 kbp in size, and inserted into an Sappropriate plant expression vector, such as the plasmid pCIB770 (Rothstein et al., 1987). The plasmid pCIB770 contains a chimeric Skanamycin gene for expression in plants, as well as the promoter and terminator of the 35S RNA transcript of CaMV separated by a unique BamHI site. The restriction fragment bearing the toxin coding sequence is made compatible to the unique BamHI site of pCIB770 by use of the appropriate molecular adapter and ligated together.

Example 7: Construction of pSAN, containing a chimeric gene encoding the I insecticidal toxin gene of Bt strain san diego A 4A gene encoding the insecticidal protein of Bt strain sa, diego has been characterized and sequenced by Herrnstadt et al., EP-0-202-739 and 'EP-0-213-818. This coding sequence is isolated on a convenient restriction fragment and inserted into the appropriate plant expression vector, such as pCIB770. The plasmid pCIB770 contains a chimeric kanamycin gene for expression in plants, as well as the promoter and terminator of the RNA transcript of CaMV separated by a unique BamH site. The restriction fragment bearing the toxin coding sequence is made compatible to the unique BamHI site of pCIB770 by use of the appropriate molecular adapter and ligated together.

-r Example 8: Construction of a deleted Bt protoxin gene encoding a polypeptide of approximately 725 amino acids, and construction of a chimeric gene containing this deleted gene with the CaMV promoter A deleted protoxin gene encoding a polypeptide of approximately 725 amino acids is made by removing the COOH-terminal portion of the gene by cleaving at the KpnI restriction endonuclease site at position 2325 in the sequence shown in formula I.

Plasmid pCIBlO/35Sbt (Figure 14) is digested with BamHI and KpnI, and the approximately 2.2 kbp BamHI/KpnI fragment containing the deleted protoxin gene is isolated by preparative agarose gel electrophoresis. To convert the KpnI site at the 3' nd to a BamHI site, the fragment is mixed with a S KpnI/BamHI adapter oligonucleotide and ligated. This fragment is then mixed with BamHI-cleaved pCIB10/710 (Figure 13). The resulting transformants, designed pCIB10/35Sbt (KpnI) and shown in Figure 15, contain the deleted protoxin gene encoding a polypeptide of approximately 725 amino acids. These transformants are selected on kanamycin.

Example 9: Construction of a deleted Bt protoxin gene encoding a polypeptide of approximately 645 amino acids, and construction of a chimeric gene containing this deleted gene with the CaMV promoter.

A deleted protoxin gene encoding a polypeptide of approximately 645 amino acids is made by removing the COOH-terminal portion of the gene by cleaving at the Bell restriction endonuclease site at position 2090 in the sequence shown in Formula I.

Plasmid pCIB10/35Sbt (Figure 14) is digested with BamHI and BclI, and the approximately 1.9 kbp BamHI/BclI fragment containing the deleted protoxin gene is isolated by preparative agarose gel electrophoresis. Since BclI creates a cohesive end compatible with BamHI, no further manipulation is required prior to ligating this fragment into BamHI-cleaved pCIB10/710 (Figure 13). The resulting plasmid pCIB10/35Sbt(BclI) which has the structure shown in Figure 16, is selected on kanamycin.

~IIUCLI~LIW~IIIII

56 Example 10: Construction of i; deleted Bt protoxin gene encoding a polypeptide of approximately 607 amino acids, and construction of a chimeric gene containing this deleted gene with the CaMV 35S promoter.

A deleted protoxin gene is made by introducing a BamHI cleavage site (GGATCC) following nucleotide 1976 in the sequence shown in Formula I.

This is done by cloning the BamHI fragment containing the protoxin sequence from pCIB10/35Sbt into mpl8, and using standard oligonucleotide mutagenesis procedures described above. After mutagenesis, doublestranded replicative form DNA is prepared from the M13 clone, which is then digested with BamHI. The approximately 1.9 kbp fragment containing the deleted protoxin gene is inserted into BamHI-cleaved pCIBI0/710. The 11 resulting plasmid pCIBl0/.5Sbt(607) which has the structure shown in Figure 17, is selected for on kanamycin.

The remaining Examples describe specific protocols for transforming cotton cells and regenerating cotton plants from cotton cells and callus.

It should be understood that those with ordinary skill in the art may Svary the details of the protocols while still remaining within the limits Sof the present invention. For example, numerous plant tissue culture media are known, some of which are described in detail below. The ordinarily skilled tissue culture scientist would know how to vary these solutions in order to achieve the same or similar results. Thus, Example 12 discloses a modified White's stock solution as a seed germination and callus development medium; Example 13 describes a Murashige and Skoog stock solution as a callus growth/maintenance medium; Exampl- 14 describes a Beasley and Ting stock solution as a plant germination medium. The ordinarily skilled tissue culture scientist knows how to vary these solutions in order to achieve results similar to those described in the Examples. Thus, the sugar in the callus growth medium may be glucose, which minimizes phenolic secretions, or sucrose, which promotes the formation of embryogenic callus.

The explants used in the transformation procedure may be from any suitable source, such as from seedlings, especially a hypocotyl or cotyledon, or from immature embryos of developing fruit.

-57 Any antibiotic toxic to Agrobacteriwn may be used to kill residual Agzrobact-rum after the transformation step. Cefotaxime is preferred.

Example 11: Regeneration of cotton plants 11.1. Media All media in this example contain Murashige and Skoog inorganic salts and Gamborg's B-5 vitamins, are adjusted to pH 5.7, and have the following composition (mg/liter): lMacronutrients MgSil 71120 370 ~4o: KH 2 PO11 170 47~ KNO 3 1900 NHi;N0 3 1650 GaCl 2 *21120 440 Micronutrients 131303 6.2 0 00 MnS04 1712 15.6 -0 ZnS04~ 7H2~0 8.6 *NaMoOi# 2H 2 0 0.25 CuS0i 51120 0.025 CaCl 2 6H2~0 0.025 1( 0.83 FeS04 7H 2 0 27.8 0 Na 2 EDTA 37.3 0 Vitamines Thiamine HCl Pyridoxine HU0 I Nicotinic acid 1 Myo-Inositol 100 In addition, the various media have the following components: 58 Medium #I Additional Components g/liter sucrose, 0.6 noble agar (Difco) g/liter glucose, 2 mg/liter a-naphthaleneacetic acid 1 mg/liter kinetin, 0.8 noble agar g/liter sucrose, 2 mg/liter a-naphthaleneacetic acid 1 mg/liter kinetin, 0.8 noble agar g/liter sucrose, 0.5 mg/liter picloram g/liter sucrose, 5 mg/liter 2,4-dichlorphenoxyacetic acid g/liter sucrose, 15 mM glutamine 9 9t 9,99 *9I 9 49 99 Media at 250, 280 and 31 0 C refer, in addition to the temperature, to a photoperiod of 16 hours light: 8 hours dark at a light intensity of -2 -1 20 IE m s 1 1670 lx).

11.2. Seed Sterilization and Planting Seeds of cotton (Gossypium hirsutum var. Coker 310) are delinted by placing seed in concentrated H 2 SOt for 2 min. Seeds are then washed 4 times with sterile, distilled water, dipped in 95 ethanol, flamed and planted on Medium #1 at 31 0

C.

11.3. Callus induction Seven days following planting, seedling hypocotyls are excised, sliced longitudinally, cut into 2 mm sections and placed on Medium #2 at 31 0

C.

Hypocotyl sections (2 mm) are transferred weekly to fresh Medium #2 and these cultures are also maintained at 31°C. Following 4 weekly transfers to Medium callus tissue proliferating on the hypocotyl sections is removed from the original explant and placed on Medium #3 at 310C. The callus is transferred to fresh Medium #3 after one month and maintained for an additional 1 to 2 months.

S59 11.4. Suspension Culture Initiation For initiation of suspension cultures, 100 mg of callus tissue is placed into 35 ml of Medium #4 in a 125 ml DeLong flask. Suspensions are rotated for 6 weeks at 140 rpm, and 2800, at which time they begin rapidly to proliferate.

11.5. Embryo Development and Plant Regeneration The embryos that form in Medium #4 proliferate even faster following replacement of Medium #4 by Medium This embryogenic suspension is divided and subcultured every 3 to 7 days into fresh Medium For i development of embryos proliferating in Medium the embryos are washed with, and then placed into, Medium Three to four weeks following transfer to Medium the mature embryos are placed on a solid medium at t* 25C. The solid medium consists of a modified MS medium containing MS .1 salts with 40 mM KNO 3 in place of KN0 3 and NHi(N0 3 B-5 vitamins, 2 sucrose, 15 mM glutamine, and solidified with 0.2 Gelrite (pH 5.7).

Embryos are placed in petri dishes at 25°C. Shoot development is sporadic on this medium and root elongation is enhanced with the transfer of the embryos to the above modified MS medium without glutamine. Germinating embryos are then planted in vermiculite in pots and covered with a beaker 0 After plantlets are established in vermiculite, the beaker is removed. Following one week at 280C, the plantlets are placed in the greenhouse for further development into plants.

60 Example 12: Seed germination and callus development media [Composition of modified White (1961)'s stock solution (incorporated herein by reference)]

I,

~oooq 00 ~0 0 05 0 4 0 4 0050 0 44 00 4 004 4 00 4 0 0 00 4 04 44 44 a 0 S 45 0040.54 *0404 04 Concentration Component per 1000 ml. Comments MgS0i4.7 H 2 0 3.6 g Dissolve and make up Na 2 S0 4 2.0 g the l.inal volume to NaH 2 POi 0

-H

2 0 1.65 g 1000 ml. Label White's A Stock. Use 100 ml/liter of final medium Ca(N0 3 )2.4 H20 2.6 g Dissolve and make up KN0 3 800 iuig the final volume to KCl 650 mg 1000 ml. Label White's B Stock. Use 100 ml/ liter of final medium.

Na 2 M00 4 -2H 2 O 2.5 mg Dissolve and make up CoC1 2 *6H 2 0 2.5 mg the final volume to 100 MnSOi 0

*H

2 0 300 mg ml. Label White's C ZnS04*7 H20 50 mg Stock. Use 1.0 ml/liter CUS04-5 H 2 0 2.5 mg final medium.

H

3 B0 3 50 mg Fe-EDTA Use 10 ml/liter of NSFe EDTA. (See below) Organic Use 10 ml/liter of MS organic. (See below) -61- LI Example 13: Callus growth/maintenance media 000021 0 0 0 00 0 0 0 00 00 0 4 0000 0 00 O I I 000 0 00 0 0 0 I 0 40 to 00 0 0 0 00 0 00 *4 0 0 00 00000 I 000000 04 0 4

I

[Composition of blurashige Skoog (MS) (1962) stock solutions (incorporated herein by reference)] Concentration per Component 1000 ml. of stock Comments NR4NO 3 41.26 g Dissolve and make up KN0 3 47.50 g the final volume to CaCla.2 H120 11.00 g 1000 ml. Label MS-Major.

MgS0ij.7 1q20 9.25 g Use 40 ml/liter of final KiJ 2 P0~ 4 4.25 g medium 1(1 83 mg Dissolve and make up 131303 620 mg the final volume to MnS0O*1120 1690 mg 1000 ml. Label MS-Minor. 'Use ZnS~i 1 .7 H230 860 mg 100 ml/liter of final medium.

Na 2 M 4-2 H120 25 mg hi 2 0 2.5 mg CoCl 2 .6 1120 2.5 mg Nicotinic acid 50 mg Dissolve and make up Pyridoxine HCl 50 mg the final volume to Thiamine HCI 10 mg 1000 ml. Label MS-Organic.

Freeze in 10 ml aliquots. Use 10 ml/liter of final medium.

62 aaa9aa a 09 00 ,9 *9 a *ap 0 £0 a 4*0 a 94 a 09 a a Ca.S 4 4a 4* Concentration per Component 1000 ml. of stock Comments Fe EDTA 2.78 g Dissolve 2.78 g of FeSOi47 HO Fe SOi.7H 2 0 3.73 g in about 200 ml of deionized Na 2 EDTA*2 H 2 0 water. Dissolve 3.73 g of Naz- EDTA*2 HO0 (disodium salt of ethylenediaminotetraacetic acid dihydrate) in 200 ml of deionized water in another beaker.-Heat the Na 2

-EDTA

solution on a hot plate for about 10 minutes. While constantly stirring, add FeS04 solution to Na 2 -EDTA solution.

Cool the solution to room temperature and make up the volume to 1000 ml. Label MSFe-EDTA. Cover bottle with foil and store in refrigerator. Use 10 ml/liter of final medium.

Thiamine HC1 50 mg Dissolve and make up the volume to 500 ml. Label MS Thiamine. Use ml/liter of final medium.

m-Inositol 10 g Dissolve and make up the Glycine 0.2 g final volume to 1000 ml.

Label MS glycine/inositol.

Use 10 ml/liter of final medium.

I

63 Example 14: Plant germination media [Composition of Beasley and Ting (1973)'s stock solutions] *a~c

I

9 ,t

I

1 t I t Concentration Component per 1000 ml. Comments

KH

2 P04 2.72 g Dissolve and make up the

H

3 B0 3 61.83 mg volume to 100 ml.

NazMo04-2 H 2 0 2.42 mg Label B T A Stock.

Use 10 ml/1 of final medium.

CaC12"2 H20 2.6 g Dissolve and make up the KI 8.3 mg volume to 100 ml. Label CoC1 2 *6 H 2 0 0.24 mg B T B Stock. Use 10 ml/l of final medium.

MgSOq*7 H20 4.93 g Dissolve and make up the MnSOi 4

H

2 0 169.02 mg volume to 100 ml. Label ZnSO 4 7 H20 86.27 mg B T C Stock. Use CuS04-5 H 2 0 0.25 mg 10 ml/l of final medium.

KN03 25.275 g Dissolve and make up the volume to 200 ml. Label B T D Stock. Use ml/l of final medium.

Nicotinic acid 4.92 mg Dissolve and make up the Pyridoxinrf HC1 8.22 mg final volume to 100 ml.

Thiamine HC1 13.49 mg Label B T Organics.

Use 10 ml/l of final medium.

Fe-EDTA Use 10 ml/l of MS-Fe-EDTA.

Inositol 100 mg/1 of final medium.

NH

4 N0 3 (15 iM) 1200.6 mg/l of final medium.

I t' I C I I *1*1 -64 Example 15: Regeneration of plants starting from cotyledon explants Seeds of Acala cotton variety SJ2 of Gossyplum hirsutum are sterilized by contact with 95 alcohol for three minutes, then twice rinsed with sterile water and immersed with a 15 solution of sodium hypochlorite for 15 minutes, then rinsed in sterile water. Sterilized seeds are germinated on a basal agar medium in the dark for approximately 14 days to produce a seedling. The cotyledons of the seedlings are cut into segments of 2 to 4 mm 2 which are transferred aseptically to a callus inducing medium [see above] consisting of Murashige and Skoog (MS) major and minor salts supplemented with 0.4 mg/liter thiamine-HCl, 30 g/liter glucose, 2.0 mg/liter naphthaleneacetic acid (NAA), 1 mg/liter kinetin, 100 mg/liter m-inositol, and agar (0.8 The cultures are incubated at S about 30 0 C under conditions of 16 hours light and 8 hours darkness in a 0e Percival incubator with fluorescent lights (cool daylight) providing a S' light intensity of about 2000 to 4000 lx.

t Calli are formed on the cultured tissue segments within 3 to 4 weeks and are white to gray-greenish in color. The calli formed are subcultured every three to four weeks onto a callus growth medium comprising MS medium containing 100 mg/liter m-inositol, 20 g/liter sucrose, 2 mg/liter (a naphthaleneacetic acid (NAA) and agar. Somatic embryos form four to six months after first placing the tissue explants on the callus inducing medium. The callus and embryos are maintained on callus growth medium by subculturing onto fresh callus growth medium every three to four weeks.

Somatic embryos which formed on tissue pieces are explanted either to fresh callus growth medium, or to Beasley Ting's medium (embryo S germination medium).

The somatic plantlets which are formed from somatic embryos are transferred onto Beasley and Ting's medium which contains 1200 mg/liter ammonium nitrate and 500 mg/liter casein hydrolysate as an organic nitrogen source. the medium is solidified by a solidifying agent (Gelrite) and plantlets are placed in Magenta boxes.

The somatic embryos develop into plantlets within about three months. The plantlets are rooted at the six to eight leaf stage [about 7.5 and 10 cm tall], and transferred to soil and maintained in an incubator under high humidity for three to four weeks, after which they are transferred to the greenhouse. After hardening, plants are transferred to open tilled soil.

Example 16: Regeneration of plants starting from cotyledon explants Variation 1 The procedure of Example 15 is repeated using instead half-strength MS medium in which all medium components have been reduced to one-half the specified concentration. Essentially the same results are obtained.

Example 17: Regeneration of different cotton varieties from cotyledon a c explants.

The procedure of Examples 15 and 16 is repeated with Acala cotton S, varieties SJ4, SJ2C-1, GC510, B1644, B2724, B1810, the picker variety ar Siokra and the stripper variety FC2017. All are successfully regenerated.

S" Example 18: Regeneration of cotton plants from cotyledon explants with suspension cell culture as intermediate step.

t The procedure of Example 15 is repeated to the extent of obtaining callus S capable of forming somatic embryos.

a 4 Pieces of about 750 to 1000 mg of actively growing embryogenic callus are suspended in 8 ml units of liquid suspension culture medium comprised of SMS major and minor salts, supplemented with 0.4 mg/liter thiamine HC1, 1 20 g/liter sucrose, 100 mg/liter m-inositol and naphthaleneacetic acid S (2 mg/liter) in T-tubes and placed on a roller drum rotating at 1.5 rpm Sunder 16:8 light:dark regime. Light intensity of about 2000 to 4500 lx is again provided by fluorescent lights (cool daylight).

After four weeks, the suspension is filtered through an 840 micron size nylon mesh to remove larger cell clumps. The fraction smaller than 840 microns is allowed to settle, washed once with about 20 to 25 ml of fresh suspension culture medium. This cell suspension is transferred to T-tubes (2 ml per tube) and each tube diluted with 6 ml of fresh suspension culture medium. The cultures are maintained by repeating the i, i PO I*--rC r- -66 t. above at 10 to 12 day intervals. At each subculture, the suspension is filtered and only the fraction containing cell aggregates smaller than 840 microns is transferred to fresh suspension culture medium. In all instance, the fraction containing cell clumps larger than 840 microns are placed onto the callus growth medium to obtain mature somatic embryos.

The somatic embryos that are formed on callus growth medium are removed and transferred to embryo germination medium. Using the protocol of Example 15, these are germinated, developed into plantlets and then field grown plants.

Example 19: Regeneration of cotton plants from cotyledon explants with suspension cell culture as an intermediate step-Variant 1.

s! The procedure of Example 18 is repeated except that suspension cultures I are formed by transferring 750 to 1000 mg embryogenic calli to a DeLong flask containing 15 to 20 ml of the MS liquid medium containing 2 mg/liter NAA. The culture containing flask is placed on gyrotory shaker and shaken at 100 to 110 strokes/minute. After three weeks the suspension 1 8 is filtered through an 840 micron nylon mesh to remove the large cell clumps for plant growth, as in Example 18. The less than 840 micron suspension is allowed to settle, washed once in the MS liquid medium and resuspended in 2 to 5 ml of the MS liquid medium. The suspension is A "subcultured by transfer to fresh medium in a DeLong flask containing 1 to S2 ml of suspension and 15 ml of fresh MS liquid medium. The cultures are maintained by repeating this procedure at seven to ten day intervals. At each subculture only the less than 840 micron suspensions are subcultured and the large clumps (840 microns or greater) used for plant growth.

Example 20: Production of plants from large clumps of suspension cultured cells After three or four subcultures using the suspension growth procedure of Examples 18 and 19, 1.5 ml to 2.0 ml of cell suspension from the T-tube and DeLong flask are in each instance plated onto agar-solidified MS medium containing 2 mg/liter NAA and Beasley Ting medium containing 500 mg/liter casein hydrolysate. Within three to four weeks embryogenic calli with developing embryos become visible. Again, the 840 micron or 67 greater cell clumps are plated on the callus growth medium, give rise to embryogenic clumps with developing embryos, which ultimately grow into plants.

Example 21: Transformation of cotton suspension culture cells to tumorous-phenotype by Agrobacteria LBA 4434.

21.1. Growth of the plant suspension culture.

An Acala cotton suspension culture [as described in Example 18, above] is subcultured into tubes with the medium (MS medium containing 2 mg/liter NAA) being changed every seven to ten days. After a medium change, the tube is rotated 90° and the cells allowed to settle out.

The supernatant is removed by pipeting prior to transformation and the resulting cells treated as described below.

S 21.2. Description of Agrobacterium vector.

r The Agrobacterium strain LBA 4434 (Hoekema et al., 1983) contains a Ti Splasmid-derived binary plant transformation system. In such binary systems, one plasmid contains the T-DNA of a Ti-plasmid, the second plasmid contains the vir-region of a Ti-plasmid, and together the two plasmids function to effect plant transformation. In the Agrobacterium A strain LBA 4434, the T-DNA plasmid pAL1050 contains T of pTiAch5, an

SL

I 4 octopine Ti-plasmid. The vir plasmid in strain LBA 4434, pAL4404, contains the intact virulence regions of pTiAch5 (Ooms et al., 1982).

See Strain LBA 4434 is available from Dr. Robert Schilperoort of the Department of Biochemistry, University of Leiden, the Netherlands.

21.3. Growth of Agrobacteria.

The transforming Agrobacterium strain is taken from a glycerol stock, inoculated in a small overnight culture, from which a 50 ml culture is inoculated the following day. Agrobacteria are grown on YEB medium [YEB is per liter in water: 5 g beef extract, 1 g yeast extract, 5 g peptone, g sucrose, adjusted to pH 7.2 with NaOH. After autoclaving, 1 ml of 2 M MgC12 is added] to which antibiotics as appropriate have been added. The absorbance at 600 nm (OD 60 0 of the 50 ml overnight culture is read, the culture is centrifuged and the pellet resuspended in the plant cell

-P_

68 growth medium (MS medium plus NAA at 2 mg/ml) to a final absorbance at 600 nm of 0.5. 8 ml of this bacterial suspension is added to each "T" tube containing the plant cells from part 21.1 above.

21.4. Infection.

The "T"tube containing the plant and bacteria cells is agitated to resuspend all cells and returned to a roller drum for three hours to allow the Agrobacteria to attach to the plant cells. The cells are then allowed to settle and the residual supernatant removed. A fresh aliquot of growth medium is added to the tube and this allowed to incubate on a roller drum for a period of 18 to 20 hours in the presence of any residual Agrobacteria which remained. After this time, the cells are again allowed to settle, the supernatant is removed and the cells are washed twice with a solution of growth medium containing cefotaxime (200 g/ml). After washing, the cells from each T-tube are resuspended in ml growth medium containing cefotaxime (200 .ig/ml in all cases) and 1 ml aliquots of this plated on petri dishes.

21.5. Growth of transformed tissue.

The cells infected with Agrobacteria grow on the growth medium which had S no added phytohormones, indicating the tissue has received the wild-type phytohormone genes in T-DNA. These cells develop into tumors, further indicating transformation of the cultures.

i Example 22: Transformation of cotton suspension culture cells to a kanamycin-resistant non-tumorous phenotype.

The same procedure as in Example 21 is followed except that different transforming Agrobacteria are used and that the plant selection medium contains an antibiotic for the selection of transformed plant tissue.

22.1. Growth of plant tissue.

As in Example 21, part 21.1.

22.2. Description of Agrobacterium vector.

The transforming Agrobacteria contain the T-DNA containing binary vector pCIBIO (Rothstein et al., 1987) as well as the pAL4404 vir plasmid.

The T-DNA of pCIBlO contains a chimeric gene composed of the promoter 69 from nopaline synthase, the coding region from Tn5 [encoding the enzyme neomycin phosphotransferase], and the terminator from nopaline synthase.

The Agrobacterium strain LBA4404, containing the vir helper plasmid pAL4404 [described above], is similarly available from Dr. Schilperoort.

22.3. Growth of Agrobacteria.

Agrobacteria containing pCIB10 are grown on YEB containing kanamycin pg/ml). Otherwise, conditions are as in Example 21, part 21.3.

22.4. Infection.

Transformation is accomplished as detailed in Example 21 with the change that the 1 ml aliquots resulting in part 21.3 are plated immediately on medium containing selective antibiotics. Selection medium contained either kanamycin (50 4g/ml) or G418 (25 ig/ml). Expression of the 0 9 nos/neo/nos chimeric gene in transformed pl'nt tissue allows the selec-

S

t tion of this tissue on either of these antib:,tics.

22.5. Growth of transformed tissue.

"S Plant growth media in this and all following examples contain phytohormones as indicated in Example In 2 to 4 weeks, transformed tissue becomes apparent on the selection plates. Uninfected tissue or control tissue shows no signs of growth, turns brown and dies. Transformed tissue grows very well in the presence of kanamycin or G418. At this time, tissue pieces which are growing well are subcultured to fresh selection medium.

22.6. Growth of Somatic Embryos.

SSomatic embryos form on these tissue pieces. Somatic embryos are explanted to fresh medium (non selective).

22.7. Germination.

When the embryos have begun to differentiate and germinate, i.e. the point where they are beginning to form roots and had two or three leaves, they are transferred to Magenta boxes containing growth medium. Growth is allowed to proceed until the plantlet has 6 to 8 leaves, at which time it is removed from the agar medium.

22.8. Growth of plantlet.

The plantlet is now placed in potting soil, covered with a beaker to maintain humidity and placed in a Percival incubator for 4 to 8 weeks. At this time, the beaker is removed and the plant transferred to the greenhouse.

22.9. Growth of plant in greenhouse.

The plants grow in the greenhouse, flower and set seed.

Example 23: Transformation of cotton suspension culture cells to a glyphosate-tolerant phenotype The same procedure as in Example 22 is followed except where changes are noted below. Different transforming Agrobacteria are used. Also, after plant tissue is selected on an antibiotic for the selection of trans- S. formed material, it is further selected for herbicide tolerance.

23.1. Growth of plant tissue SAs in Example 21, part 21.1.

23.2. Description of Agrobacerium vector.

Transforming Agrobacteria contain the T-DNA vector pPMG85/587 (Fillatti et al., 1987) as well as the pAL4404 vir plasmid. The plasmid pPMG85/587 carries three chimeric genes capable of expression in plants. Two of these genes code for neomycin phsphotransferase (NPT) which confers resistance to antibiotic kanamycin or G418. The third chimeric gene, containing the coding sequence from a mutant aroA gene of Salmonella typhimurium, confers tolerance to the herbicide glyphosate (Comai et al., i 1983).

23.3. Growth of Agrobacteria.

Agrobacteria containing pPMG85/587 are grown on medium containing kanamycin (100 gg/ml).

71 23.4. Infection.

Transformation is accomplished as detailed in Example 21 with the change that the 1 ml aliquots resulting in part 21.3 are plated immediately on medium containing selective antibiotics. This selceton medium contains either kanamycin (50 ig/ml) or G418 (25 ig/ml). Expression of the NT chimeric gene in transformed plant tissue allows the selection of this tissue on either of these antibiotics.

23.5. Growth of transformed tissue.

In 2 to 4 weeks, transformed tissue becomes apparent on the selection plates. Plant material is originally selected on kanamycin.

S Plant tissue [either individual embryos or callus] is then placed on medium containing the herbicide glyphosate. Transformed tissue continues o .0 to grow well.

S Example 24: Transformation of cotton suspension culture cells to a *o hygromycin-resistant non-tumorous phenotype.

The same procedure as in Example 22 is followed except where noted.

Different transforming Agrobacteria are used and the plant selection medium contains an antibiotic appropriate for the selection of trans- 0 ,o0 formed plant tissue.

24.1. Growth of plant tissue.

0 As in Example 21, part 21.1.

24.2. Description of Agrobacterium.

The transforming Agrobacteria contain the T-DNA containing binary vector 4 pCIB2115 (Rothstein et al., 1987) as well as the vir plasmid. The T-DNA of pCIB2115 contains a chimeric gene composed of the promoter and terminator from the CaMV 35S transcript [Odell et al., 1985] and the coding sequence for hygromycin B phosphotransferase [Gritz and Davies, 1983].

24.3. Growth of Agrobacteria.

Agrobacteria containing pCIB2115 are grown on YEB containing kanamycin ug/ml).

I

72 24.4. Infection.

Transformation is accomplished as detailed in Example 21 with the change that the 1 ml aliquots resulting in part 21.3 are plated immediately on medium containing selective antibiotics. Selection medium contains ig/ml hygromycin. Expression of the chimeric hygromycin gene in transformed plant tissue allows the selection of this tissue on medium containing hygromycin.

24.5. Growth of transformed tissue.

As in Example 22, part 22.5 except that the antibiotic hygromycin is used in the plant selection growth medium.

0 'a Example 25: Plant extraction procedure o Plant tissue is homogenized in extraction buffer [ca 100 mg in 0.1 ml ao Extraction Buffer].

a o 0* 0 Leaf extraction buffer Na 2

CO

3 (pH 9.5) 50 mM EDTA 10 mM o Triton X-100 0.05 S" T 'en 0.05 4 04 NaCl 1000mM PMSF (add just prior to use) 1 mM leupeptine (add just prior to use). 1 mM 0. After extraction, 2 M Tris pH 7.0 is added to adjust the pH of the 4,,0 extract to a pH of 8.0 to 8.5. The extract is then centrifuged 10 minutes 0 in a Beckman microfuge and the supernatant used for ELISA analysis.

Example 26: ELISA analysis of plant tissue ELISAs [enzyme-linked immunosorbent assay] are very sensitive, specific assays for antigenic material. ELISAs are very useful for studying the expression of polypeptide gene products. The development of ELISA techniques as a general tool is described by Clark et al. (1986); this is herein incorporated by reference.

1_ i 73 An ELISA for the Bt toxin was developed using standard procedures and is used to analyze transgenic plant material for expression of Bt sequences.

The steps used in this procedure are as given below: Media and Buffers EPBS (ELISA Phosphate Buffered Saline) mM Na Phosphate: NazHPO 4.68 g/4 liter.

NaH 2 POi-H 2 O 0.976 g/4 liter 140 mM NaCl NaCI 32.7 g/4 liter pH should be approximately 7.4 t It Borate Buffered Saline 100 mM Boric acid t 25 mM Na Borate mM NaCl Adjust pH to 8.4 to 8.5 with HC1 or NaOH as needed.

ELISA Blocking Buffer In EPBS, 1 BSA 0.02 Na Azide ELISA Wash Buffer mM Tris-HC1 pH 0.05 Tween 0,02 Na A-.de I i i r 74 M TRIS ELISA Diluent In EPBS: 0.05 Tween 1 BSA 0.02 Na Azide ELISA Substrate Buffer In 500 ml, 48 ml Diethanolamine, 24.5 mg MgCla; adjust to pH 9.8 with HC1.

ELISA Substrate 15 mg p-nitrophenyl phosphate 00*000 0 0 00, *r 0 0 0 000 o 00 0 00 0 1 0 0 40000.40

SI

4 1 in 25 ml substrate buffer.

Procedure: 1. ELISA plate is pretreated with ethanol.

2. Affinity-purified rabbit anti-Bt toxin antiserum (50 ul) at a concentration of 3 ig/ml in Borate Buffered Saline is added to the plate and this allowed to incubate overnight at 4°C. Antiserum is produced in response to immunizing rabbits with gradient-purified Bt toxin crystals (Ang and Nickerson, 1978) solubilized with sodium dodecyl sulfate.

3. Wash with ELISA Wash Buffer.

4. Treat 1 hour at room temperature with Blocking Buffer.

5. Wash with ELISA Wash Buffer.

6. Add plant extract in an amount to give 50 ig of protein (this is typScally ca. 5 pl of extract). Leaf extraction buffer is described in example 25; protein is determined by the Bradford method (Bradford, 1976) using a commercially available kit [Bio-Rad, Richmond, California]. If dilution of the leaf extract is necessary, ELISA Diluent is used. Allow this to incubate overnight at 7. Wash with ELISA Wash Buffer.

75 8. Add 50 pl affinity-purified goat anti-Bt toxin antiserum at a concentration of 3 pg protein/ml in ELISA Diluent. Allow this to incubate for one hour at 37 0

C.

9. Wash with ELISA Wash Buffer.

Add 50 pl rabbit anti-goat antibody bound to alkaline phosphatase [commercially available from Sigma Chemicals, St. Lous, This is diluted 1:500 in Diluent. Allow to incubate for one hour at 37 0

C.

11. Wash with ELISA Wash Buffer.

12. Add 50 pl substrate [0.6 mg/ml p-nitrophenyl phosphate in ELISA Substrate Buffer. Incubate for 30 minutes at room temperature.

13. Terminate reaction by adding 50 il of 3 M NaOH.

14. Read absorbance at 405 nm in modified ELISA reader [Hewlett Packard, Stanford, Ca.].

Plant tissue transformed with the pCIB10/35Sbt(BclI) [see Figure 16] construction, when assayed using this ELISA procedure shows a positive S reaction, indicating expression of the Bt gene.

r f t Example 27: Bioassay of transformed cotton *i t L SHeliothis virescens eggs laid on sheets of cheesecloth are obtained from the Tobacco Insect Control Laboratory at North Carolina State University, Raleigh, North Carolina. The cheesecloth sheets are transferred to a large covered glass beaker and incubated at 29 0 C with wet paper towels to maintain humidity. The eggs hatch within three days. As soon as possible after hatching, the larvae (one larva per cup) are transferred to covered small plastic cups. Each cup contains cotton leaf discs. Larvae are transferred using a fine bristle paint brush.

Leaf discs one cm in diameter are punched from leaves of cotton plants and placed on a circle of wet filter paper in the cup with the larva. At least 6 to 10 leaf discs, representing both young and old leaves, are tested from each plant. Leaf discs are replaced at two day intervals, or as necessary to feed the larvae. Growth rates [size or combined weight of all replica worms] and mortality of larvae feeding on leaves of transformed plants are compared with those of larvae feeding on untransformed cotton leaves.

76 Larvae feeding on discs of cotton transformed with pCIB 10/35Sbt (BclI) show a decrease in growth rate and an increase in mortality compared with controls.

Example 28: Construction of pCIB1300, for high level expression in plants.

pCIB1300 is engineered for high level expression of the Bt toxin gene and contains an untranslated leader sequence 5' to the Bt toxin gene to enhance Bt toxin gene expression in plants. The untranslated leader is a bp sequence 5' to the initiation codon of the Bt toxin gene and 3' to the CaMV 35S untranslated leader. The final pCIB1300 construct is engineered by the insertion of the 40 bp leader and deleted Bt toxin gene into the BamHl site of pCIB10/710 as shown in Figure 19. A 1.9 kbp NcoI-BamHI fragment from pCIB10/35Sbt(Bcl) deletion is purified in low-temperature gelling agarose. The 40 bp leader is chemically synthesized as a double-stranded oligonucleotide with a 5' overhanging BamHI site and a 3' overhanging Ncol site using an Applied Biosystems DNA SSynthesizer. The sequence of the untranslated leader as shown in the center of Figure 19 is derived from the alfalfa mosaic virus (AMV) coat S protein untranslated leader described by Koper-Zwarthoff et al. (1977).

The 40 bp leader, 1.9 kbp Bt fragment and BamHI linearized pCIB710 vector Sre joined in a three-part ligation using T4 DNA ligase to construct pCIB1300.

Example 29: Isolation of cDNA clones coding for the small subunit of RuBPCase in Cotton Sossypium hirsutum (Funk line RF522) plants are grown from seeds in the greenhouse with 14 hour daily light periods. Total RNA is isolated from young green leaves following the procedure of Newbury and Possingham S(1977). PolyA RNA is purified as described in Maniatis et al. (1982), p. 197. Double-stranded cDNA (complementary DNA) is synthesized according to the procedure of Okayama and Berg (1982) with the following modifications: -I i II_.

77 A. First strand cDNA is primed with oligo-dT; B. After tailing the double-stranded cDNA with oligo-dG using polynucleotidyl-transferase, it is cloned into oligo-dC tailed pUC9 (Pst I site from Pharmacia), and annealed; and C. the DNA is transformed into E. coll strain HB101.

Since, with the chlorophyll a/b binding protein (Cab), RuBPCase is the most abundant protein in green leaves, the cDNA library is then screened for cDNA clones of the most abundant mRNAs. Nitrocellulose (Schleichez and Schuell) filter replicas of the cDNA clones are screened with the first cDNA strand, radioactively labeled with a-dCT 32 P and reversetranscriptase, the template being the same polyA RNA as that used to construct the cDNA library. Six cDNA clones out of 275, are selected and analyzed further.

~Northern analysis (done as described in Maniatis et al., 1982, p. 202) S shows that two of these cDNA clones hybridize to a class of mRNA about 1100 nt long. They cross-hybridize with a Cab gene probe from S .tobacco. The other four hybridize to a class of mRNA 900 to 1000 nt long, S a size consistent with that of the rbcS (small subunit of Rubisco).

SCotton leaf mRNA, after hybrid selection using one of these four cDNA clones, is released and translated in vitro (as described in Maniatis et al. 1982, p. 329) using rabbit reticulocytes in vitro i translation kit (Promega Biotec). Electrophoresis on polyacrylamide gel of the translation products showed one major polypeptide of about 20 kD, a molecular weight consistent with that of the precursor of the RuBPCase The other 3 cDNA clones cross-hybridize with the clone used for the hybrid-release experiment.

Large portions of these cDNA clones are sequenced, using the dideoxy chain-termination technique (Sanger et al., 1977) after subcloning into M13. Comparison of their sequences with formerly published rbcS sequences from other species shows that they are indeed rbcS cDNA clones.

j 78 Example 30: Isolation of genomic clones of small subunit RuBPcase of cotton 30.1. Cotton Genomic Southern analysis.

Genomic Southern blots are prepared by standard procedures using nitrocellulose filters. Prehybridization, hybridization and washing conditions are as described in Klessig et al. (1983). Genomic Southern analysis, using our rbcS cDNA clone as a probe, reveals 4 to 5 genomic fragments depending on the restriction enzyme used to digest the DNA. The RuBPCase is encoded by a small gene family in cotton, as in other species previously studied by others. The cotton rbcS multigene family is estimated to contain at least 5 members.

30.2. Isolation of rbcS genomic clones In order to construct a cotton genomic library, partial Sau3a digests of cotton genomic DNA are size-fractionated on a 10 to 40 sucroset gradient, and ligated into X EMBL3 arms (Stratagene) digested with BamHI.

Packaging of X recombinants, done using Packagene kit (Stratagene), is followed by transfection into E. coli strain K802. Nitrocellulose filter i duplicate replicas are screened as described in Maniatis et al. (1982) p. 320, using the rbcS cDNA clone from above as a probe. Twelve positive clones out of 450,000 plaques are purified. DNA is isolated form plate lysates of these recombinants phages, as described in Maniatis et al.

S(1982) p. After comparing these genomic clones by their restriction digest pattern i with various enzymes, five different rbcS genes are identified. Each one is subcloned into the plasmid vector pBSM13 (Stratagene). These subclones are then mapped and partially sequenced in order to localize S the 5' end of the gene and the first ATG (translational start site). A map of two of these genomic subclones, rbc-gX and rbc-gY is shown on Figure 24. The X EMBL3 phages containing the genomic DNA of subclones rbc-gX and rbc-gY have been deposited with the International Depository American Type Culture Collection, Rockville, Maryland.

~ii~-i-i 79 30.3. Study of the level of expression of the rbcS gene fragments in cotton leaves Forty-one additional rbcS cDNA clones are isolated from the cotton leaf cDNA library. Restriction mapping analysis, sequencing and hybridization of these cDNA clones to gene specific probes allows to conclude that the gene carried by the genomic clone rbc-gX is responsible for about 17 of the cotton leaf rbcS transcripts.

Example 31: Construction of chimeric genes using cotton rbcS promoter.

31.1. Insertion of an Nco I site at the first ATG of the rbcS genes encoding transit peptides.

The sequences of the transit peptides of rbc-gX and rbc-gY are shown on Figures 26 and 25 respectively. An Nco I cleavage site (CCATGG) is introduced at the first ATG of these two genes of the encoding transit peptide. This is done by cloning the PstI-EcoRI fragment of gene rbc-gX Sand the XbaI-SphI fragment of gene rbc-gY (hatched fragments on Figures 22 and 23 respectively) into mpl8 and wpl9 respectively, and Susing standard oligonucleotide site-directed mutagenesis procedures described above to introduce the NcoI site.

31.2. Construction of pCIB 1301, a plasmid bearing a chimeric gene *0 containing the deleted Bt protoxin gene (607 deletion) with the rbc-gX gene promoter.

S After the site-directed mutagenesis, double-stranded replicative form (ds rf) DNA is isolated from the M13 clone, which is then digested with Hind III and Eco RI. The Hind III-Eco RI fragment containing the rbc-gX promoter is ligated together with Hind III and Eco RI digested plasmid S pUCl9 and the ligation mix then transformed into E. coll strain HB101.

Plasmid DNA is isolated from ampicillin-selected transformants and digested with HindIII. The ends of the resulting molecule are made blunt-ended by treatment with the Klenow subunit of DNA polymerase I and Sal I linkers are ligated to these ends. The resulting linear molecule is digested with Sal I and Nco I and gel-purified. In a three-part ligation the gel-purified Sal I-Nco I fragment is joined to a gel-purified Bam HI-Sal I fragment from pCIB770, a broad-host range replicon used as

A

i 80 an Agrobacterium Ti plasmid cloning vector (Rothstein et al., 1987) and a gel-purified Nco I-Bam HI fragment containing the truncated 607 amino acid Bt gene. The ligation mix is transformed into E. col- strain HB101.

The resulting plasmid, pCIB1301, which is depicted graphically in Figures 20, 21 and 22, is selected on kanamycin.

31.3. Construction of pCIB1302, a plasmid bearing a chimeric gene containing the deleted Bt protoxin gene (607 deletion) with the rbc-gY gene promoter.

After the mutagenesis, double-stranded replicative form (ds rf) DNA is isolated from the M13 clone, which is then digested with Xba I-Nco I. The approximately 1.97 kbp NcoI-BamHI fragment, containing the deleted protoxin gene, is then ligated, together with the XbaI-NcoI rbc-gY promoter fragment, in a three way ligation, into XbaI-BamHI cleaved S pCIB10/710. The resulting plasmid, pCIB1302, the structure of which is S shown in Figure 23, is selected on kanamycin.

I i t I i -81 Lif"erature An, Watson, Stachel, Gordon, Nester EMBO J. 4, 277, 1985 Ang, Nickerson, Appi. Environ. Microbial. 36, 625, 1978 Barton, hloM-. n Wu, Grossmann, Moldave, K., Methods in Enzymnology 10J., 527, 1983 Beasley, Ting, Am. J. Bot. 60, 130, 1973 Bevan, Barnes, Chilton, Nuci. Acids Res. 11, 369, 1983 Bevan, Flavell, Chilton, Nature 304, 184, 1983 Beva: Nuci. Acids Res. 12, 8711, 1984 Bolivar, Rodriguez, R.L. Greene, Betlach, Heyneker, H.L., Boyer, Crosa, Falkow, Gene 2, 95, 1977 Bradford, Anal. Biochem. 72, 248, 1976 Caplan, Herrera-Estrella, Tnz6, van Haute, van Montagu, Schell, Zambryski, Science 222, 815, 1983 Chaleff, Ray, Science 223, 1148, 1984 Cheng et al., Plant Sdi. Lett. 19, 91, 1980 Chilton, et al., Proc. Natl. Acad. Sci., USA 77, 7347, 1974 Chilton, Farrand, Levin, Nester, Genetics 83, 609, 1976 Chilton, Bevan, Yadav, Matzke, Byrne, M., Crula, Barton, Vanderleyden, de Framond, Barnes, W.M., Stadler Genetics Symposia Series 13, 39, 1981 Chilton, in: the Role of Plant Biotechnology in Plant Breeding, Report of 1984 Plant Breeding Research Forum, 21.-23. August 1984, 177, 1985 Clark M.F. et al., Methods in Enzymology 118, 742, 1986 Comai, Schilling-Cordaro, Mergia, Houck, Plasmid 21, 1983 Covey, Lomonossoff, Hull, Nucl. Acids Res. 9, 6735, 1981 Deacon, Aspects of Microbiology 7, ed. Cole et al., American Society of Microbiology, 1983 DeBlock et al., EMBO Journal 3, 1681, 1984 i i _I 82 van den Elzen, Townsend, Lee, Bedbrook, Plant.

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IC

C C C ae' C CC C I C Miller, Lingg, lBulla, Science 219, 715, 1983 Morelli, Nagy, Fraley, Rogers, Chua, N.H., Nature 315, 200, 1985 Murashige, Skoog, Physiol. Plant. 15, 473, 1962 Nagy, I.J, Maliga, Z. Pflanzenphysiol. 78, 453, 1976 Newbury, Possingham, Plant Physiol. 60, 543, 1977 Norrander, Kempe, Messing, Gene 26, 101, 1983 Odell, et al., 1985 Oka, Sugisaki, Takanami, J. Mol. Biol. 147, 217, 1981 Okayama, Berg, Hal. Cell, Biol. 2, 161, 1982 Ooms, Regensburg-Tuink, Hofker, Hoekema, Hooykaas, Schilperoort, Plant Moleculax Biology 1, 265, 1982 Paszkowski, Shillito, Saul, Mandak, Hohn, Hohn, B., Potrykus, EMBO J. 3, 2717, 1984 Rothstein, Labmers, Lotstein, Carozzi, N.B., Jayne, Rice, Gene 53, 153, 1987 Sanger et al., 1977 Sekar, et al., Proc. Natl. Acad. Sci, USA 84, 7036, 1987 Simon, Priefer, PUhler, in: PUhler, A. Molecular Genetics of the Bacteria-Plant Interaction, Springer Verlag, Berlin, 98, 1983 Velten, Velten, Hain, Schell, EMBO J. 3, 2723, 1984 Wang, Herrera-Estrella, van Montagu, Zambryski, Cell 38, 455, 1984 White, Phytomorphology 11, 19, 1961 Wong, Schnepf, Whiteley, J. Biol. Chem. 258, 1960, 1983 Yadav, Vanderleyden, Bennett, Barnes, W.M., Chilton, Proc. Natl. Acad. Sci. USA 79, 6322, 1982 Yanish-Perron, Vieira, Messing, Gene 33, 103, 1985 Zambryski, Joos, Hi. Genetello, Leemans, van Montagu, N., Sc',ell, EMBO J. 2, 2143, 1983 Zoller, Smith, in: Wu, Grossmann, Moldave, Methods in Enzymology 100, 468, 1983

Claims (21)

1. A cotton cell comprising a chimeric gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein, exhibiting toxicity toward Dipteran and Lepidopteran insects.

2. The cell according to claim 1 wherein the plant cells are cells of Gossypium hirsutum, Gossypium arboreum, or Gossypium barbadense.

3. The cell according to claim 2 wherein the plant cells are cells of Gossypium hirsutum.

4. The cell according to claim 1 wherein the plant cells are of the variety Acala SJ-2, Acala GC 510, Acala B-1644 or Siokra.

5. The cell according to any one of claims 1 to 4 wherein the promoter, 5' untranslated region, and, optionally the 3' untranslated 15 region of the chimeric gene are derived from plant or plant virus genes.

6. The cell according to claim 5 wherein the promoter, untranslated region and/or optionally 3' untranslated region of the chimeric gene are derived from a plant gene that codes for the small subunit of ribulose-1,5-bisphosphate carboxylase or chlorophyll 20 a/b-binding protein.

7. The cell according to claim 5 or 6 wherein the promoter, untranslated region and/or optionally 3' untranslated region are derived from a gene of a plant DNA virus.

8. The cell of claim 7 wherein the plant virus is cauliflower mosaic virus.

9. The cell of claim 8 wherein the cauliflower mosaic virus promoter is the 35S promoter of gene VI. 1160v 85 I f The cell of claim 1 wherein the promoter, 5' untranslated region and optionally the 3' untranslated region of the chimeric gene are derived from DNA sequences that are present in Agrobacterium plasmids, and that cause expression in plants.

11. The cell of claim 10 wherein the promoter is derived from the Ti plasmid of Agrobacterium tumefaciens.

12. The cell of claim 10 wherein said DNA sequences gene that codes for octopine synthase.

13. The cell of claim 10 wherein said DNA sequences gene that codes for nopaline synthase.

14. The cell of claim 1 wherein the polypeptide has 130,000 to about 140,000, or insecticidal fragments

15. The cell of claim 14 wherein the polypeptide is molecule. are derived from a are derived from a an Mr of about thereof. fused to another 4b 4 o et 9 it 4*4 4t I I *r

16. The cell of claim 1 wherein the chimeric gene is substantially complementary to the nucleotide sequence that codes for the crystal protein 6-endotoxin in Bacillus thuringiensis.

17. The cell of claim 1 wherein the chimeric gene is capable of hybridizing to the coding region of the gene that codes for the crystal protein 6-endotoxin in Bacillus thuringiensis.

18. The cell of claim 14 wherein the polypeptide has substantially the same immunological properties as the crystal protein from Bacillus thuringiensis.

19. The cell of claim 16, 17 or 18 wherein said Bacillus thuringiensis is a subspecies selected from the group consisting of Bt var. kurstaki, Bt var. berliner, Bt var. alesti, Bt var. tolworthi, Bt var. sotto, Bt var. dendrolimus, Bt var. tenebrionis, Bt var. san diego and Bt var. aizawai. 86 The cell of claim 19 wherein the BacillZus thuringiZensis is the variety kur-stak-i HDl.

21. The cell of claim 20 wherein the gene expresses a polypeptide having the amino acid sequence: Sequence of the formula (II) 40*444 4 o 04 4 4 4 44 4 *4 4 0 44 44 0 4 444 4 *4 4 4 4* 4* o *0 p p p 44 44 *4 4 4 *0 44*044 4411:4 Met Ile Val Thr Ser Val Val Set G lu Phe Glu Ala Pro Ar g Leu Gln Tyr Val Ar g Ser Oly Ty r Pro Gln Leu Asp Ser Pro Ar g I le Asn Arg I le Pro Met Val Thr Phe Set Gly Ty r Asp Pro Ser Ser Asp Pro Glu Gly Leu Pro Asp Gln Gln Ala Gly G lu Thr I le Thr Asn Val Le u Trp Ar g Asn Asn Asp Phe Asp Ser Gln Val Gly Ar g Ser Gly Met Glu Gly Ala Leu Asn Val Thr Arg Glu Pro His Asn Asn Tyr Val1 Tyr Thr Gly Ile Trp Le u Ar g Leu Ser Asn G ln Tb r Tyr Gln Arg Gly Tyr Tyr Tb r Ser Ar g I le Ar g Leu Leu Ser Ser I le Glu Ala Phe Asn Gln Ser I le Leu Ser Ly s Ile Arg Val Set Asn Asn Leu Thr Gln Ala Ile Asp I le Asn Ser Phe Pro Phe Ala O ln Ala Asp Ph e Asn Thr Gly Ar g Ar g Val Thr Thr O lu Ala Pro Thr Tyr Ser Thr Ala Leu Set Gly Asp Ser Ser Pro Gln Ser Ser Pro Cy s Gly Pro Phe Gly Trp Ala Asn Gin As n Ar g Ala Asn I le Val1 Ala Val Asp Asp Asp Leu Asp 0 lu Ser Tyr Arg Asn G ln His Ile Tyr Pro Phe Ala Gly Thr I le G ly Asn Gly Pro Gly Met Val Asn Lett Gly Ile Leu Phe Gly Phe O ln Ala Leu Glu Le u Asp Pro Pro Asn Ser Ala Leu His Glu T rp Leu Leu Pro Glu Ph e Gly Leu Tyr T rp ValI Pro Pro Gln Le u Asn Thr Leu Thr G ln Phe Phe Ser Ile Set Glu Asp Leu Val I le Leu Ar g I le Tyr T rp Arg Met Le u Leu Le u Val1 Ala Tb r Ala Ar g Ile Thr Phe I le Ile Asp I le Met Thr Set Gly Leu Gln Gly Tyr Asn Glu Pro Val Asn Set Arg Ile Asn Asn Arg I le Set Leu Ph e Val I le Set Gln Glu Giu Asn Phe Leu His Phe Thr Arg Val Val Arg Le* u Pro Ar g Tyr Gly Giu Asp Asp Gly Phe Tyr Gln Val Ar g Gln Phe Set Asp Asn His Set I le Glu Pro I le Ser Glu Gly Gly O ln Glu Arg I le Al a Glu Set Ala Ser Leu Gly I le Leu Ar g Trp Tyt Asn Thr Thr Set Gly I le Ala His Set Gly Ar g Tyr At g Gln Ala Al a Set Asn Atg Gly Ar g Cy s Glu Glu Leu Phe Leu Pro I le Glu Leu Ty r Asp Met Ala Val Val1 Set O ln Asn Ile Trp Gly Asn Val Tyr Val Asn Phe Set Leu His Gln G ly Thr I le Arg Pro Leu Tyt Val Lett Val Leu Phe Ala 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 0L 4 1 4 0 t14~. -87 Pro Met Phe Set Trp Ile His Arg Set Ala 460 Giu Phe Asn Asn Ile Ile Pro Set Set Gin 470 Ile Thr Gin Ile Pro Leu Thr Lys Ser Thr 480 Asn Leu Gly Set Gly Thr Ser Val Val Lys 490 Gly Pro Gly Phe Thr Giy Gly Asp Ile Leu 500 Arg. Arg Thr Ser Pro Gly Gin Ile Set Thr 510 Leu Arg Val Asn Ile Thr Ala Pro Leu Set 520 Gin Arg Tyr Arg Val kqy Tle Arg Tyr Ala 530 Set Tht Thr Asn Let. Gln P'lie His Thr Set 540 Ile Asp Gly Arg Pro Ilez Asn Gin Gly Asn 550 Phe Set Aia Tht Met Set Set Gly Set Asn 560 Leu Gin Set Giy Sef Phe Atg Tht Vai Gly 570 Phe Thr Thr Pro Phe Asn Phe Set Asn Gly 580 Set Set Val Phe Thr Leu Set Ala His Val 590 Phe Asn Set Giy Asn Glu Val Tyr Ile Asp 600 Ar Ile Giu Phe Vai Pro Ala Giu Vai Thr 610 Phe Giu Ala Giu Tyr Asp Leu Giu Arg Ala 620 Gin Lys Ala Val Asn Giu Leu Phe Thr Set 630 Set Asn Gin Ile Gly Leu Lys Thr Asp Val 640 0 Thr Asp Tyr His Ile Asp Gin Val Set Asn 650 00 Leu Val Giu Cys Leu Set Asp Giu Phe Gys 660 000 Leu Asp Glu Lys Lys Giu Leu Set Giu Lys 670 0 Val Lys His Ala Lys Arg Leu Set Asp Giu 680 Ara Asn Leu Leu Gin Asp Pro Asn Phe Arg 690 0 Gly Ile Asn Arg Gin Leu Asp Arg Gly Trp 700 0Arg Giy Set Thr Asp Ile Thr Ile Gin Giy 710 Cily Asp Asp Vai Phe Lys Giu Asn Tyr Val 720 Thr Leu Leu Giy Thr Phe Asp Giu Cys Tyr 0Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Giu 740 Set Lys Leu Lys Ala Tyr Tht Arg Tyr Gin 750 00Leu Arg Giy Tyt Ile Giu Asp Set Gin Asp 760 Leu Giu Ile Tyt Leu Ile Atg Tyr Asn Ala 770 Lys His Glu Tht Val Asn Val Pro Gly Thr 780 S Giy Set Leu Trp Pro Leu Set Ala Pro Set 790 Pro Ile Gly Lys Gys Ala His His Set His 800 His Phe Set Leu Asp Ile Asp Vai Gly Cys 810 Thr Asp Leu Asn Glu Asp Leu Gly Val Trp 820 Val Ile Phe Lys Ile Lys Thr Gin Asp Gly 830 His Ala Arg Leu Giy Asn Leu Giu Phe Leu 840 Giu Giu Lys Pro Leu Val Gly Giu Ala Leu 850 Ala Arg Val Lys Arg Ala Glu Lys Lys Trp 860 Atg Asp Lys Arg Giu Lys Leu Glu Trp Giu 870 Tht Asn Ile Val Tyr Lys Giu Ala Lys Giu 880 Set Val Asp Ala Leu Phe Val Asn Set Gin 890 Tyr Asp Arg Leu Gin Ala Asp Thr Asn Ile 900 Ala Met Ile His Ala Ala Asp Lys Arg Val 910 His Set le Arg Giu Ala Tyr Leu Pro Giu 920 Leu Set Val Ile Pto Gly Val Asn Ala Ala 930 Ile Phe Giu Giu Leu Giu Gly Ar Ile Phe 940 Thr Ala Phe Set Leu Tyt Asp Ala Arg Asn 950 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly 960 Leu Set Cys Ttp Asn Val Lys Gly His Val 970 Asp Val Giu Glu Gin Asn Asn His Arg Set 980 Val Leu Val Val Pro Glu Ttp Glu Ala Giu 990 Val Set Gin Glu Val Arg Val Cys Pro Gly 1000 Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr 1010 1 88- ,Ly s Ile Leu Val Asp G ly Asp Pro Ly s Pro Tyr by s Ly s Gly Leu Gly Glii Asn Asn Thr Set 0 lu Ala Asp As n Pro Tyr G lu Val G lu 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1156 000900 6 6 0 0*

22. The cell of claim 1 wherein the DNA. sequence of the coding region of o the gene comprises: Formula I 20 30 40 50 GTTAACACCC TGGGTCAAAA ATTGATATTT AGTAAAATTA GTTGCACTTT GTGCATTTTT O 070 80 90 100 110 120 TCATAAGATG AGTCATATGT TTTAAATTGT AGTAATGAAA AACAGTATTA TATCATAATG 130 140 150 160 170 180 AATTGGTATC TTAATAAAAG AGATGGAGGT AACTTATGGA TAACAATCCG AACATCAATG 0190 200 210 220 230 240 AATGCATTCC TTATAATTGT TTAAGTAACC CTGAAGTAGA AGTATTAGGT GGAGAAAGAA 250 260 270 280 290 300 TAGAAACTGG TTACAGCCCA ATCGATATTT CCTTGTCGCT AACGCAATTT CTTTTGAGTG 310 320 330 340 350 360 AATTTGTTCC CGGTGCTGGA TTTGTGTTAG GACTAGTTGA TATAATATGG GGAATTTTTG 370 380 390 400 410 420 GTCCGTCTCA ATGGGACGCA TTTCTTQTAC AAATTGAACA GTTAATTAAC CAAAGAATAG 430 440 450 460 470 480 AAGAATTCGC TAGGAACCAA GCCATTTCTA GATTAGAAGG ACTAAGCAAT CTTTATCAAA 510 520 530 540 GAGTGGGAAG CAGATCCTAC TAATCCAGCA TTAAGAGAAG TTTACGCAGA ATCTTTTAGA 550 560 570 580 590 600 AGATGCGTAT TCAATTCAAT GACATGAAGA GTGCCCTTAC AACCGCTATT CCTCTTTTTG

89- 610 620 630 640 650 660 440444 4 4 0 00 0 4 4 o 44 44 0 0 4 0444 o .4 4 0 4 404 4 44 4 0 0 4 44 4 40 4 4 44 0 40 4 0 0 4 94 444444 0 9 409444 4 4 04 0 4 *44 44*4; CAGTTCAAAA 670 TATCAGTTTT 730 TCAATAGTCO 790 CCTGGTACAA 850 ATAATCAATT 910 ACTATGATAG 970 CAAACCCAGT 1030 GAAGTAT TAG 1090 CTCATAGAGG 1150 CGGGCCCAGA 1210 G TAT TGT TGCC 1270 GACCTTTTAA 1330 CTTATGGAAC 1390 CGCTGGATGA 1450 GATTAAGCCA 1510 CAGCTCCTAT 1570 CACAAATTAC 1630 TTAAAGGACC TTATCAAOTT 680 GAGAGAT OTT 740 TTATrAAT OAT 800 TACGGGATTA 860 TAGAAGAOAA 920 TAGAACGTAT 980 ATTAGAAAAT 1040 GAGTCCACAT 1100 AGAATATTAT 1160 ATTCACTTTT 1220 TCAACTAGGT 1280 TATAGGGATA 1340 CTCCTCAAAT 1400 AATACCGCCA 1460 TGTTTCAATG 1520 OTT CTCTTGC 1580 ACAAATACCT 1640 ACCATTTACA C CT CTT TTAT 690 TCAOTGTTTC 750 TTAACTACGC 810 CAC CTCT AT 870 TTAACACTAA 930 CCAATTCGAA 990 TTGATGGTA 1050 TTCATGCATA 1110 TOOT CACO CC 1170 CCCCTATATC 1230 CACCOCOTOT 1290 AATAATCAAC 1350 TTCCAT C C 1410 CAGAATAACA 1470 TTTCCTTCAC 1530 ATACATCGTA 1590 TTAACAAAAT 1650 COAGCACATA CACTATATOT 700 GACAAAGGTC 760 TTATTGGCAA 820 COOCAC COCA 880 CTOTATTACA 940 CACTTTCCCA 1000 GTTTTC GAO C 1060 TACTTAACAG 1120 ATCAAATAAT 1180 GAACTATOGGG 1240 ATAGAACATT 1300 AACTATCTGT 1360 CTGTATACAG 1420 ACGTGCCACC 1480 CCTTTACTAA 1540 GTCCTCAATT 1600 CTACTAATCT 1660 TTCTTCGAAG TCAAOCTOCA 710 GGGATTT CAT 770 CTATACAGAT 830 TTCTAGAGAT 890 TATCGTTT CT 950 ATTAACAACA 1010 CTCGCCTCAG 1070 TATAACCATC 1130 GGCTTCTCCT 1190 AAATCCACCT 1250 ATCGTCCACT 1310 TCTTCACGGG 1370 AAAAACCA 1430 TACCCAACCA 1490 TACTACTGTA 1550 TAATAATATA 1610 TCGCTCTCGA 1670 AACTTCACCT AATTTACATT 720 OCCOCCACTA 780 CATOCTGTAC 840 TOOATAACAT 900 CTATTTCCGA 960 CAAATTTATA 1020 OCCATACAAC 1080 TATACCOATO 1140 GTAGGGTTTT 1200 CCACAACAAC 1260 TTATATACAA 1320 PLCACAATTTC 1380 ACGGTAGATT 1440 TT TACT CAT C 1500 ACTATAATAA 1560 ATTCCTTCAT 1620 ACTTCTCTCC 1680 GCC AGATTT 1690 1700 1710 1720 1730 1740 CAACCTTAAC AGTAAATATT ACTOCACCAT TATCACAAAG ATATCCGGTA ACAATTCGCT 90 000009 0 0 0 00 0 0 0 0 00 0 0~ 0 0 0 0000 0 00 0 000 9 0e 0 0 0 *0 4 00 09 0 0 00 0 00 0 0 0 00 000004 0 000000 0 i L *00 000400 C 1750 ACGCTTCTAC 1810 GGAATTTTTC 1870 TAGGTTTTAC 1930 ATGTCTTCAA 1990 TAACCTTTGA 2050 CTTCTTCCAA 2110 CCAATTTAGT 2170 AGAAAGTCAA 2230 TTAGAGGGAT 2290 AAGGAGGCGA 2350 GCTATCCAAC 2410 ACCAATTAAG 2470 ATGCCAAACA 2530 CAAGTCCAAT 2590 GATGTACAGA 2650 ATGGCCATGC 1760 CACAAATTTA 1820 AGCAACTATG 1880 TACT CC GTTT 1940 TTCAGGCAAT 2000 GGCAGAATAT 2060 T CAAAT C GOG 2120 TGAGTGTTTA 2180 ACATGCGAAG 2240 CAATAOACAA 2300 TGACOTATTC 2360 GTATTTATAT 2420 AGOTATATC 2480 CGAAACAGTA 2540 CGGAAAATGT 2600 CTTAAATGAG 2660 AAGACTAGGA 1770 CAATrTCCATA 1830 AGTAGTGGGA 1890 AACTTTTCAA 1950 GAAOTTTATA 2010 GATTTAGAAAk 2070 TTAAAAACAG 2130 TCTOATGAAT 2190 CGACTTAGTG 2250 CTAGACCOTG 2310 AAAOAOAATT 2370 1780 CATCAATTGA 1840 GTAATTTACA 1900 ATGGATCAAG 1960 TAGATCOAAT 2020 GAGCACAAAA 2080 ATGTGACGGA 2140 TTTOTCTOOA 2200 ATGAOCOGAA 2260 OCTGGAOAGG 2320 ACOTTACGCT 2380 1790 CO GAAOAC CT 1850 GT CC GAAGC 1910 TGTATTTAGG 1970 TGAATTTGTT 2030 0000 GTOAAT 2090 TTATCATATT 2150 TGAAAAAAAA 2210 TTTACTTCAA 2270 AAGTACGGAT 2330 ATTGGGTACC 1800 ATTAATCAGG 1860 TTTAGGACTG 1920 TTAAGTGCTC 1980 CCG00CAGAAG 2040 GAGCTGTTTA 2100 GATCAAGTAT 2160 GAATTGTCCG 2220 GATCCAAACT 2280 AT TAC CATC C 2340 TTTGATGAGT 2390 2-400 CAAAAA GAAGAT AATOTG GCC CAT GACTTA AATCTA AAG ATGAGTCGAA ATTAAAAGCC 2430 2440 2450 AGTC AAGACTTACA AATCTATTTA 2490 2500 2510 CCAG GTACCGGTTC CTTATGGCCG 2550 2560 2570 CATT CCCATCATTT CTCCTTOGAC 2610 2620 2630 .OGTG TATGGCTGAT ATTCAAGATT 2670 2680 2690 .OAAT TTCTCGAAGA GAAACCATTA 2730 2740 2750 *AAAA AATOGAGAGA CAAACGTGAA 2790 2800 2810 GCAA AAGAATCTGT AGATGGTTTA TATACCCGTT 2460 ATTCGCTACA 2520 C TTT CAC CCC 2580 ATT OAT OTT 2640 AAGACGCAAG 2700 GTAGGAGAAG 2760 AAATTGGAAT 2820 TTTGTAAACT 2710 2720 CACTAGCTCG 2770 GGGAAACAAA TGTGAAAAGA OCGGAG 2780 TATTGTTTAT AAAGAG I 91 04 o"44 4 v 4. 44 2830 CTCAATATCA 2890 CCGTTCATAG 2950 CGGCTATTTT 3010 GAAATCTCAT 3070 ATGTACATCT 3130 CAGAAGTGTC 3190 CGTACAAGCA 3250 ACGAACTGAA 3310 GTAATGATTA 3370 GATATGACG 3430 AACAAAAAC 3490 CGGATTACAC 3550 CCGATAAGCT 3610 AATTACTTCT 3670 CATTACT GAG 3730 TCACTCTTAA 3790 TTTTTTGCA 3850 CACTACC CCC 3910 ATTTTTTATG 2840 TACATTACAA 2900 CATTCGAGAA 2960 TCAAGAATTA 3020 TAAAAATGGT 3080 AGAACAACAA 3140 ACAAGAAGTT 3200 GCGATATGGA 3260 GTTTAGCAAC 3320 TACTGCGACT 3380 AGCCTATGAA 3440 ATATACAGAT 3500 AC CACTAC CA 3560 ATCCATTCAC 3620 TATGGACGAA 3680 TTGTATTGAC 3740 AAGAATGATG 3800 AGCCTTTACT 3860 AAGTGTCAAA 3920 AATCTTTCAA 2850 GCCCATACCA 2910 C CT TAT CTGCC 2970 GAACCCCGTA 3030 CATTTTAATA 3090 AACAACCACC 3150 CCT GT CTCT C 3210 GAACCTTCCG 3270 TCTGTACAAG 3330 CAAGAACAAT 3390 AGCAATTCTT 3450 CCACGAACAG 3510 CCTGCCTAT C 3570 ATCCGACAAA 3630 TAATATATC 3690 ACATAAATAA 3750 TCCGTTTTTT 3810 TAACCCCGTA 3870 AAACCTTATT 3930 TTCAAGATGA 2860 ACATCCAT 2920 CTCACCTCTC 2980 TTTTCACTC 3040 ATGCCCT TAT C 3100 CTTCGCTCCT 3160 CCCCTCCT CC 3220 TAACCATTCA 3280 ACCAAGTATA 3340 ATCACCCTAC 3400 CTGTACCAC 3460 ACAATCCTTC 3520 TGACAAAAGA 3580 CCGAACGAAC 3640 TTTATAATCT 3700 CCAAATTTTT 3760 CTATCATTTA 3820 CCCCCACATC 3880 CTTTCTAAAA 3940 ATTACAACTA 2870 CATTCATC 2930 TCT CAT TCC C 2990 ATT CTC CCTA 3050 CTCCTCCAAC 3110 T CTTCT TCC C 3170 CTATATCCTT 3230 TGACATCGAG 3290 TCCAAACAAC 3350 CTACACTTCT 3410 TGATTATCCA 3470 TCAATCTAAC 3530 ATTACACTAC 3590 ATTCATCCTG 3650 AAGGTGTGCA 3710 ATATGAATAA 3770 ACCACTCATA 3830 CCCATCAACT 3890 ACCTACCTAC 3950 TTTTCTGAAG 2880 GCACATAAAC 2940 CCT CT CAAT C 3000 TAT CATC CA 3060 GTGAAACGGC 3120 CAATGCAAC 3180 C CTCT CACAC 3240 AACAATACAC 3300 ACCCTAACCT 3360 CGTAATCCAC 3420 TCACCCTATC 3480 ACACCATATC 3540 TTCCCAGAAA 3600 CACACTCC 3660 AATAAACAAT 3720 AAAACCCCCA 3780 TTTAAATGTT 3840 TAACAATTTC 3900 AAACCATCAC 3960 AGCTGTATCC 92 3970 3980 3990 4000 TCATTTAACC CCTTCTCTTT TGGAACAACT CGCTAAAGAA 4030 4040 4050 4060 ACGAAAGTTT TCAGGAAATG AATTAGCTAC CATATGTATC 4090 4100 4110 4120 GACTGATTCT CTCCTTCGAC TATGCACTCA ATTACACGCC 4150 4160 4170 4180 CCACAAOCAC TCAATAAACG CTTTOATAAAk AAAGCCGTTG 4210 4220 4230 4240 TCTGCATTAT GCAAAAGTAA ACTTTCTAAA ACATCAGCCA 4270 4280 4290 4300 TATTTTCAAC GAATCCGTAT TTTAGATGCG ACGATTTTCC 4330 4340 4350 4360 CATGTATATC CTGGGTCAGG TGGTTCTGCA CAAACTGCAG 15 23. The cell of claim 20 wherein the insecticidal polypeptide having substantial amino acid sequence of claim 21. 4010 4020 TTAGGTTTTG 4070 TGGGGCAGTC 4130 SC CACAOGAG 4190 AATTTTTGAA 4250 TTTCAAGTGC 4310 AAGTACCGAA TAAAAAGAA.A 4080 AACCTACAGC 4140 TCTTATGAGT 4200 ATATATTTTT 4260 AGCACTCACG 4320 ACATTTAGCA a a a a a a a a a. aa4 a a. a a. a a chimeric gene expresses an sequence homology to the *aaO.a a a a a.. I A ta a a 4 a a a a a a, 2 24. The cell of claim 20 wherein the chimeric gene shows substantial sequence homology to the DNA sequence given in claim 22. 25. A culture of cotton cells according to any one of claims 1 to 24. 26. The culture of claim 25 wherein the cotton cells are cells of Gossypiumn hirsutum, Gossypium arboreumn and Gossypium barbadense.- 27. The culture according to claim 26 wherein the plant cells are cell1s of Gossypium hirsutumn. 28. The culture of any one of claims 25 to 27 wherein the cells are protoplasts. 29. A cotton plant comprising a gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein, exhibiting toxicity toward Dipteran and Lepidopteran insects. 0 -93- A cotton plant according to claim 29 comprising a gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein in sufficient amounts to render the plant unattractive and/or toxic to insect larvae. 31. A cotton plant according to claim 30 comprising a gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein in sufficient amounts to render the plant toxic to lepidopteran, dipteran and coleopteran larvae. 32. The plant of claim 29 selected from the group consisting of Gossypium hirsutum, Gossypium arboreum and Gossypium barbadense. 33. The plant according to claim 32 wherein the plant cells are cells of Gossypium hirsutum. 34. Propagules of a transgenic cotton plant according to any one of claim 29 to 33. 35. The propagules of claim 34 selected from the group consisting of protoplasts, cells, o, o calli, tissues, embryos, organs, seeds, pollen, ovules, zygotes or any other propagules that can be obtained from a transgenic cotton plant. 9 36. The propagules of claim 34 that can be sexually or asexually propagated or that can be propagated in-vitro or in-vivo. 4o, o 37. Progeny of a transgenic cotton plant according to any one of claims 29 to 33, or mutants and variants thereof, that still have the characteristic properties of the starting material, caused by the previous transformation of exogenous DNA. 38. A method of producing transformed, embryogenic cotton callus which comprises: a) contacting a cotton explant with an Agrobacterium vector containing a chimeric gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein and a gene that confers resistance to an antibiotic on cotton cells, the period of the contacting being sufficient to transfer the genes to the explant; -94- b) incubating the transformed explant in a callus growth medium for a period of from about 15 to about 200 hours at a temperature of from 250 to about 35°C under a cycle of about 16 hours light and 8 hours dark to develop callus from the explants; c) contacting the incubated explants with a callus growth medium containing an antibiotic toxic to Agrobacterium for a time sufficient to kill the Agrobacterium; d) culturing the callus free of Agrobacterium on a callus growth medium; S e) contacting the resulting embryogenic callus with the antibiotic in a concentration sufficient to permit selection of callus resistant to the antibiotic; and f) selecting transformed embryogenic callus. Ii 39. The method of claim 38 further comprising the step of germinating the transformed callus and developing plantlets therefrom. 40. The method of claim 38 in which the transformed callus prior to contact with the i. callus growth medium in step c is rinsed in callus growth medium free of the antibiotic toxic to Agrobacterium. 41. The method of claim 38 wherein the cotton seedling explant is selected from hypocotyl, cotyledon and mixtures thereof. 42. The method of claim 38 wherein the callus growth medium is a Murashige and Skoog medium supplemented with about 1 to about 10 mg/liter naphthaleneacetic acid. 43. The method of claim 38 wherein the antibiotic toxic to Agrobacterium is cefotaxime. 44. A method of transforming cotton cells undergoing suspension culture on a callus growth medium which comprises, after a suspension subculture growth cycle: a) recovering cells and any embryogenic callus from the callus growth medium; resuspending the cells and embryogenic callus in a callus growth medium containing an 4 i grobacterium vector containing a chimeric gene that expresses a polypeptide having v-s *W $n<!i substantially the insect toxicity properties of Bacillus thuringiensis crystal protein and a gene that confers resistance to an antibiotic on cotton cells while maintaining suspension growth conditions for a period of time sufficient to transform the suspended cells; c) recovering the suspended cells from the callus growth medium containing the Agrobacterium; d) treating the transformed cells and the embryogenic callus with an antibiotic toxic to S Agrobacterium in sufficient concentration and for a time sufficient to kill the Agrobacterium; 4 c e) contacting the cells and embryogenic callus with the antibiotic in order to select the transformed cells and embryogenic callus; I f) filtering the suspension to remove embryogenic callus greater than about 600 lm. The method of claim 44 wherein steps d and e occur before step f. S 46. The method of claim 44 wherein steps d and e occur after step f. 47. The method of claim 44 wherein step d occurs before step f and step e occurs after stepf. 48. The method of claim 44 wherein step e occurs before step f and step d occurs after step f. 49. The method of claim 44 wherein the antibiotic of step d is cefotaxime. The method of claim 44 wherein the suspension subculture growth cycle is from about 7 to about 14 days. 51. The method of claim 44 further comprising the step of developing the transformed cotton cells into plantlets. I 96 52. Cotton plants transformed to express a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein and have resistance to the antibiotic hygromycin. 53. A method for protecting cotton plants against Dipteran and Lepidopteran insect damage comprising the expression of a Bt crystal protein or a protein having substantially the insect toxicity properties of a Bt crystal protein in the plant cells constituting the plant, in an amount sufficient to kill or to control the insect larvae. 54. A method according to claim 53 wherein the insect larvae are lepidopteran or dipteran larvae. A method according to claim 54 wherein the insect larvae are lepidopteran larvae. S: 56. A method for killing or controlling Dipteran and Lepidopteran insect larvae by feeding them cotton plant cells containing chimeric 15 genes that express an insecticidal amount of a toxin having substantially o, the insect toxicity properties of Bt crystal protein. 57. A method according to any one of claims 53 or 56, wherein the crystal protein is of the Bt variety kurstaki HD1. 58. A cotton cell comprising a chimeric gene that expresses a 20 polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein, exhibiting toxicity toward Dipteran and Lepidopteran insects substantially as herein described with reference to any one of Examples 11 to 32. 59. A cotton plant comprising a gene that expresses a polypeptide having substantially the insect toxicity properties of Bacillus thuringiensis crystal protein, exhibiting toxicity toward Dipteran and Lepidopteran insects substantially as hereinbefore described with reference to Examples 11 to 32. A method of producing transformed, embryogenic cotton callus substantially as hereinbefore described with reference to Examples 11 to 32. 61. A method of transforming cotton cells undergoing suspension culture on a callus growth medium substantially as hereinbefore described with reference to Examples 11 to 32. I/TCW/1160v 97 62. A method for killing or controlling Dipteran and Lepidopteran insect larvae comprising feeding said larvae an insecticidally effective amount of a cotton plant cell as defined in claim 58. DATED this TNENTY-SECOND day of AUGUST 1991 Ciba-Geigy AG Patent Attorneys for the Applicant SPRUSON FERGUSON t t I C It S i GSA/LMM/TCW/I160v