AU2007202314A1 - Regeneration and transformation of cotton - Google Patents

Description

S 4*

AUSTRALIA

E

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Mycogen Corporation Actual Inventor(s): Thirumale Srinivasa Rangan, David Maurice Anderson, Kanniah Rajasekaran, John William Grula, Richard Lorne Hudspeth, Richard Lee Yenofsky Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: REGENERATION AND TRANSFORMATION OF COTTON Our Ref: 802545 POF Code: 112152/460044 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6006q 1A REGENERATION AND TRANSFORMATION OF COTTON This is a divisional application divided out of Application No. 2002300516, which is a divisional of AU 56002/99, which is a divisional of AU 64247/96, which is a divisional of AU 35284/93, which is a divisional of AU 29266/89, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION This invention is directed to plant regeneration and transformation of cotton, particularly cotton of the species Gossvpium hirsutum L.

In recent years many tissues of diverse origin from plants belonging to different taxonomic groups have been established as in vitro tissue culture. Some of the factors controlling growth and differentiation of such cultures have also been determined. The establishment of subtle interactions among the different groups of plant hormones, and plant growth regulators operating either directly or indirectly, alone or in synergistic combination, have given to some degree an insight into certain interrelationships that may exist among cells, tissues and organs. The information is however by no means complete.

For some time it has been known that plant cell culture can be maintained in a nondifferentiating proliferative state indefinitely. It has, however, only been recently found that redifferentiation of tissues, organs or whole plant organisms can be experimentally induced. Since the demonstrations by Skoog et al "Chemical regulation of growth and organ formation in plant tissues culture in vitro," Symp. Soc. Exp. Biol.

11:18-130, U WEGANIMNB\NODELETESeofcatnOrs\fl of 20023W518 doc -2- 1 1958, incorporated herein by reference, that the relative ratio of a cytokinin to an auxin determines the nature of organogenesis in tobacco pith tissue. Reorganization or regeneration from callus cultures includes the formation of shoot primordia or embryos, both of which ultimately lead to plantlet development in vitro.

The tendency for organogenesis vs. embryogenesis still depends upon the species involved and the presence of certain triggering factors which are chemical and/or, physical in nature.

In 1902, Haberlandt "Kulturversuche mit isolierten pflanzenzellen," MHft. I. Kis, Aad, WissJ. jie 111:62, incorporated herein by reference, postulated that plant cells possessed the ability to produce entire plants and predicted that this would someday be demonstrable in cell cultures. In 1965, Reinert "Untersuchungen uber die morphogenese an Gewebekulturen," er. dt. Bot. Ges.

71:15, and Steward et al, "Growth and organized development of cultured cells/II. Organization in cultures grown from freely suspended cells," Am. I. Bot. 45:705-708, working independently, confirmed the occurrence of in vitro somatic embryogenesis. Both references are incorporated herein by reference. In experimentally manipulating somatic embryogenesis it is believed that two components of the culture media, an auxin and the nitrogen source, play crucial roles.

It has also been shown that the process of somatic embryogenesis takes place in two stages: first, the induction of cells with .embryogenic competence in the presence of a high concentration of auxin; and second, the development of embryonic cell masses into embryos in the absence of or at a low concentration of auxin.

The induction of organogenesis or embryogenesis leads to distinct structural patterns in the callus.

Detailed study of several plant species has enabled -3- 1 certain generalizations to be made about the developmental pathways leading to shoot, bud or embryo development.

The application of tissue culture techniques to'the regeneration of plants via organogenesis or embryogenesis remains perhaps the most important contribution of basic studies in morphogenesis to commercial application.

Beasley reported the formation of callus in ovule cultures of cotton in 1971, "In vitro culture of fertilized cotton ovules," Bicience 21:906:907, 1971, incorporated herein by reference. Later, Hsu et al "Callus induction by (2-chlorethyl) phosphonic (CPA) acid in, cultured cotton ovules," Phvsiol. Plant 36:150-153, 1976, incorporated herein by reference, observed a stimulation of growth of calli obtained from ovules due to the addition of CPA and gibberellic acid to the medium.

Callus cultures from other explants such as leaf Davis et al., "In vitro culture of callus tissues and cell suspensions from okra (Hibiscus esculentus) and cotton (GossVDni hirsutum)," In vitro 9:395-398, 1974, both incorporated herein by reference; hypocotyl Schenk et al. "Medium and technique for induction and growth of monocotyledonous and dicotyledonous plant cell cultures," Can. 3. Bot. 50:199-204, 1972, incorporated herein by reference; and cotyledons Rani et al.

"Establishment of Tissue Cultures of Cotton," Plant Soi.

Lett. 7:163-169, 1976, incorporated herein by reference) have been established for Gossvpium hirsutun and f.

arboreum.

Katterman et al, "The influence of a strong reducing agent upon initiation of callus from the germinating seedlings of GossVpiun barbadense," Physiol. Pla 40:98- 101, 1977, incorporated herein by reference, observed that the compact callus from cotyledons of Q. barbadense formed roots, and in one instance regeneration of a complete plant was also obtained. Smith et al "Defined -4conditions for the initiation and growth of cotton callus in vitro, Coyrij arborein," In vitro 13:329-334, 1977, Incorporated herein by reference, determined conditions for initiation and subculture of hypocotylderived callus of G. arboreu Subsequently, Price et al "Callus cultures of six species of cotton (§&asvpi= L) on defined media," L1 So. 5- &tt. 8:115-119, 1977, and "Tissue culture of GOSSyrium species and its potential in cotton genetics and crop improvement," Beltwide Cotton tin Resarch Conference Pro. pp. 51-55, 1977, of the National Cotton Council, Memphis, each incorporated herein by reference, defined conditions for the initiation and subculture of callus from five species of Gosypun.

One of the common problems in establishing cultures of many plant species is the "browning" of the explant.

in the culture medium. In cotton, this leaching of polyphenols was overcome by replacing sucrose with glucose, and by transferring the cultures to a fresh medium every days. After 3 or 4 passages on glucose supplemented medium, the browning completely disappeared and the cultures could be transferred back to sucrose-supplemented media. Although difficulties with the induction, browning and maintenance of calli during subcultures have been overcome with certain ossvyium species, all attempts to regenerate plants from callus cultures have been either unsuccessful or have involved several timeconsuming steps. Davidonis and Hamilton "Plant Regeneration from Callus Tissue of Gossypium hirsutum." L. lan 5. f.&tI. 32:89-93, 1983, incorporated herein by reference, reported the eventual formation of embryos two years after the initiation of culture.

Although many growth substances, such as natural phytohormones and synthetic growth regulating compounds have been utilized in tissue culture media to bring about plant regeneration in yitr, no generalization, much less specifics, of the effects of different substances on plant regeneration has been arrived at. Indeed, the same substances, when applied to different plant species, may either inhibit growth, enhance growth, or have no effect whatsoever. Therefore, aside from certain standard procedures, it remains necessarily a difficult task to arrive at a working protocol for plant regeneration for any new species and by many orders of magnitude a more difficult task to achieve plant transformation.

The present invention provides a method for the rapid regeneration of cotton plants from segments exercised from seedlings. The method described offers a high degree of repeatability and reliability and it enables genetic transformation of cotton plants.

Summary of the Invention The present invention also provides a method for transforming cotton which includes the steps of a) providing a cotton explant; b) culturing the cotton explant in a first solid callus growth medium containing glucose as a carbon source from a period of time sufficient for phenolic secretion from the explant forming the callus to end and for undifferentiated callus to develop from the explant; c) transferring the callus at the end of phenolic secretions to a second solid callus growth medium containing sucrose as the carbon source; d) culturing the callus in the second solid callus growth medium from a period of item sufficient to allow development of embryogenetic callus providing at least one embryo; e) transferring the embryo to a plant germination medium; wherein the transformation involves exposing the explant during regeneration to an Agrobacterium vector containing an expressible gene sequence foreign to cotton for a time sufficient for the gene to transfer to the plant cells.

The present invention also provides a vector for conferring antibiotic resistance to a cotton plant which comprises two T-DNA right border sequences from A.

Tunmefaciens capable of integration with the plant genome flanking a chimeric gene capable of expression in cotton and of conferring resistance to the antibiotics Kanamycin and G418.

The present invention also provides a vector for transforming cotton comprising: integration sequences for integrating into the genome of cotton plants; a cotton plant expressible promoter for promoting transcription in cotton plants; a DNA border sequence encoding a selectable marker wherein the selectable marker is a herbicide resistance gene; and a cotton plant expressible termination signal for terminating transcription in cotton plants.

Seed is sterilised and grown in the dark to a seedling. The seedling is one source of an explant, usually the hypocotyl and the cotyledon. Another source are immature zygotic embryos of developing fruit. The explant is subdivided and cultured in a first callus growth medium (containing glucose) for a period of time to allow a callus to develop from the explant on a culture medium which copes with phenolic secretions and stimulates cells of the explant to divide and proliferate. The callus, after passing through the phenolic secretion stage, is transferred to a fresh callus growth medium (containing sucrose) which develops the callus to an embryogenetic callus. The embryo may then be subcultured to produce more embryogenetic callus or transferred to another growth medium (plant germination medium) and cultured for a period of time sufficient to develop a plantlet wbich after another period of growth is transferred to a greenhouse, then into the field and grown to a mature plant from which seeds can be harvested.

The embryos may also be cultured in suspension.

I

this procedure, after the period of growth, the embryo containing embryogenic clumps greater than about 600 microns, preferably greater than about 800 microns in size are isolated and utilized for plant production.

Smaller callus are recycled to the callus growth medium for growth to plant forming callus or maintained as an embryos source.

Transformation may occur at the explant, callus or suspension development stage. Transformation involves exposing the explant, callus and/or embryogenic callus to the action of an Agrobacterium vector containing an expressible gene. sequence foreign to cotton for a time sufficient for the gquene to transfer into the cells. The residual Agrobacterium is then killed off with an antibiotic which is toxic to the Agrobacterium. This is followed by selection of the transformed cells and/or embryogenic callus for development into transformed plantlets. In suspension culture, transformation and/or selection can occur prior to or following separation of embryogenic callus from cells and callus too immature to be embryogenic.

Plants of unique phenotypic traits are obtainable, and there is provided new cotton plants which having resistance to antibotics normally inhibitory to plant cell growth; cotton plants which have increased resistance or tolerance to herbicides, fungal pathogens and cotton plants which exhibit better yield and improved fiber quality.

-7- 1 Brief Description of the Drawinos FIG. 1 presents diagramatically preferred procedures for development of cotton plants from seed by tissue culture techniques with a showing of establishing zones of transformation.

FIG. 2 is a photo illustration of embryogenic callus of cotton with somatic embryos (12) at various stages of development including leaf (14) and root (16).

FIG. 3 is a photo illustration of a somatic cotton' embryo at a late globular stage isolated to form the embryogenic callus culture as depicted in FIG. 2.

FIG. 4, as with reference to FIG. 2, is a photo illustration of embryos and young plantlets (18) of cotton developing on an embryo germination medium.

FIG. 5 is a photo illustration of small clumps of embryogenic cells from suspension cultures of cotton.

FIG. 6 is a photo illustration of a globular stage embryo from a suspension culture.

FIG. 7 illustrates germinating embryos obtained from suspension cultures showing emerging leaves (14) and roots (16).

FIG. 8 illustrates the development of plantlets of cotton growing on the embryo germination medium.

FIGS. 9 to 15 depict the genetic transformation of cotton, with FIG. 9 showing the development of cell colonies (20) from transformed cotton cells containing a gene for kanamycin resistance.

FIG. 10 shows somatic embryos developing from the selected antibiotic resistance cells of FIG. 9 on an l antibiotic-supplemented medium.

FIG. 11 shows germinating embryos of transformed somatic embryos containing a gene confering resistance to the herbicide glyphosate.

FIG. 12 shows cotton plantlets developed from the embryos of FIG. 11.

-8- 1 FIG. 13 shows germinating somatic embryos transformed to confer resistance to Lepidopterous insects with leaf 14 and root 16 development.

FIG. 14 shows plantlets developed from the embrycs of FIG. 13.

FIG. 15 shows a plantlet of the variety Siokra developed from transformed embryos exhibiting a resistance to kanamycin.

FIG. 16 shows the construction of mp 19/bt, a plasmid containing the 5' end of the Bt protoxin gene.

FIG. 17 shows the construction of np 19/bt ca/del, a plasmid containing the CaMV gene VI promotor fused to the 5' end of Bt protoxin coding sequence.

FIG. 18 shows the construction of p702/bt, a plasmid having the 3' coding region of the protoxin fused to the CaMV transcription termination signals.

FIG. 19 shows the construction of pBR322/bt 14, containing the complete protoxin coding sequence flanked by CaMV promotor and terminator sequences.

FIG. 20 shows the construction of pRK252/Tn903/BglII.

FIG. 21 shows FIGS. 22 23 FIG. 24 shows FIG. 25 shows host range plasmid plant selection.

FIG. 26 shows FIG. 27 shows FIG. 28 shows FIG. 29 shows the construction of PCIB shows the construction of pCIB 4.

the construction of pCIB 2.

the construction of pCIB 10, a broad containing T-DNA borders and gene for the construction of the construction of the construction of the construction of pCIB10/19Sbt.

pCIB 710.

pCIB10/710.

pCIB10/35Sbt.

FIG.

FIG.

FIG.

30 shows the construction of pCIB10//35Sbt(XpnI).

31 shows the construction of pCIB10/35Sbt(BclI).

32 shows the construction of pCIB10/35Sbt(607) FIG. 33 depicts the plasmid prom 3 containing a T-DNA right border sequence (rb) and a nopaline synthase gene (nos) from the wild-type A. tumefaciens strain C58, was digested with Hind III and Sal I The fragment containing the right border sequence was cloned into pBR322 to generate pProm 1 and this construction was digested with Cla Plasmid pCon 1, containing a marker gene (nos/npt II) that allows selection of plant transformants on medium containing kanamycin, was digested with Hind III (4) and the restriction fragments generated from pCon 1 and pProm 1 were treated with DNA polymerase I (Klenow fragment) and "blunt-end" ligated to obtain promcon 1 The last cloning steps (6 and 7) involved the Eco RI digestion and ligation of promcon 1 and pRK290. The plasmid pRK290 contains a wide host replicon required for replication in A. tumefaciens. The final binary vector construction (DEI) contains a unique Hind III cloning suit and the selectable marker gene between two T-DNA right border sequences. Restriction sites: H, Hind III; S, Sal I; E, Eco RI; C, Cla I; Co, Cla I (Klenow filled).

FIG. 34 is a photo showing a field trial made up of cotton regenerants planted in a Verticillium infested field.

FIG. 35 is a photo showing progeny of a regenerated SJ4 plant in the field trial show in FIG. 33. A somaclonal variant with improved tolerance to Verticillium fungus is indicated by the arrow.

1 Detailed Description The present-invention is directed to the regeneration by tissue culture of cotton plants particularly plants of the genus GossvYPn hrirsutun from somatic cells for propagation in the field. Optionally, the cells may be transformed to include foreign genetic information.

The various growth medium useful in accordance with this invention are as follows: .0 SEED GERMINATION GROWTH MEDIUM COMPOSITION OF MODIFIED WHITE'S STOCK SOLUTION (Phytomorphology 11:109-127, 1961 incorporated herein by reference) Concentration per 1000 ml.

Component Comments MgSO 4 .7 H20 Na2SO 4 NaH 2

PO

4 .H20 Ca(N0 3 2 .4 H 2 0 KN03 KC1 3.6 2.0 1.65 2.6 800 650 Dissolve and make up the final volume to 1000 ml. Label White's A Stock. Use 100 ml/i of final medium.

g Dissolve and make up mg the final volume to mg 1000 ml. Label White's B Stock. Use 100 ml/1 of final medium.

Na2MoO 4 2H 2 0 CoC12.6H 2 0 MnSO4.H 2 0 ZnSO 4 .7 H 2 0 CuSO 4 .5 H20

H

3 B0 3 Fe EDTA Organic 2.5 2.5 300 50 2.5 Dissolve and make up the final volume to 100 ml. Label White's C Stock. Use 1.0 ml/1 of final medium.

Use 10 ml/i of MSFe

EDTA.

Use 10 ml/i of MS organic.

-11- CALLUS GROWTH/MAINTENANCE MEDIUM COMPOSITION OF MURASHIGE SKOOG (MS) STOCK SOLUTIONS (Physiol. Plant 15:473-497, 1962 in6orporated herein by reference) Concentration per 1000 ml.

of Stock

NH

4

NO

3

KNO

3 CaC1 2 .2 H20 MgSO 4 .7 H 2 0

XH

2

PO

4

XKI

H

3 B0 3 MnSO 4 H20 ZnSo 4 .7 H20 Na 2 MoO 4 .2 H 2 0 CuSO 4 .5 H20 CoC1 2 .6 H 2 0 Nicotinic acid Pyridoxin HC1 Thiamine HC1 41.26 47.50 11.00 9.25 4.25 83 620 1690 860 25 2.5 2.5 g g g g g mg mg mg mg mg mg Big Comments Dissolve and make up the final volume to 1000 ml. Use 40 ml/l of final medium.

Dissolve and make up the final volume to 1000 ml. Label MS Minor. Use 10 ml/1 of final medium.

Dissolve and make up the final volume to 1000 ml. Label MS Organic. Freeze in ml aliquots. Use ml/i of final medium.

-12- Fe EDTA Fe S0 4 .7" 2 0 Na 2 EDTA.2 H 2 0 2.78 g 3.73 g Dissolve 2.78 g of FeSO, 4 .7 H 2 0 In abou.t 200 ml of deionized water. Dissolve 3.73 g of N8 2 EDTA.2 H 2 0 (disodium salt of ethylenediaininotetraacetic acid dihydrate) in 200 ml of deionized water in another beaker.

Heat the Na 2

EDTA

solution on a hot plate for about minutes. Wile constantly Btirring, add FeS0 4 solution to Na 2 EDTA solution.

Cool the solution to room temperature and make up the volume to 1000 nl. Label M EDT. Cover bottle with foil and store in refrigerator. Use 7ml/l of final medium.

Thiamine RCd 50 rag Dissolve and make up the volume to 500 ml.

Label MS Thiomine.

Use 4.0 mI/l of final medium.

As if required.

Inositol Glycine 10 g 0.2 g Dissolve and make up the final volume to 1000 ml. Label MS glvcine/inositol. Use ml/l of final medium.

-13- Cc) PLANT GERMINATION

MEDIUM

COMPOSITION OF BEASLEY AND TING'S STOCK SOLUTIOGS.

(Am. J. Bot. 60:130-139, 1973 incorporated herein by reference) Concentration Cper 1000 comnonent mBl Coments

KH

2P

O

4 0 H 3 B0 3 1 Na2Mo04.2 H20 CaCl 2 .2 H20

KI

CoC1 2 .6 H20 MgS0 4 7

H

2 0 MnSO 4 .HO2 ZnSO 4 .7 H20 CuS0 4 .5 H 2 0 2.72 61.83 2.42 4.41 8.3 0.24 4.93 169.02 86.27 0.25

KNO

3 25.275 g Dissolve and make up the volume to 100 ml.

Label B6T A Stock.

Use 10 ml/i of final medium.

Dissolve and make up the volume to 100 ml.

Label B&T B Stock.

Use 10 ml/1 of final medium.

Dissolve and make up the volume to 100 ml.

Label B&T C Stock.

Use 10 ml/l of final medium.

Dissolve and make up the volume to 200 ml.

Label B&T D Stock.

Use 40 ml/1 of final medium.

Dissolve and make up the final volume to 100 ml. Label B&T prsanics. Use 10 ml/l of final medium.

Use 10 ml/l of MS Fe

EDTA.

100 mg/1 of final medium.

1200.6 mg/l of final medium.

Nicotinic acid Pyridoxin HCL Thiamine HCL Fe EDTA 4.92 8.22 13.49 Inositol

NH

4

NO

3 (15 uM) -14- 1 With any of the above solutions, the following, procedure is used to prepare one liter of the medium.

There is provided as a base, 200 ml of deionized water and the various stock solutions are added in the amounts stated for 1 liter. For example, if there is to be employed 10 ml of a stock in the final medium, then ml of the stock are added to the 200 ml of the distilled water. To ensure the salts stay in solution, stock solutions are normally added in the order shown in the formulations above. After thoroughly mixing additional deionized water is added to the mixture to bring it to, as required 500 l, and the mixture adjusted in pH to a value. of from about 5.8 to 6.0. The final volume is brought to 1,000 ml and there is normally added tissue culture Agar, or its equivalent to a level of about 0.8% by weight. This is to provide some solidity to solution to reduce flow. The mixture is then autoclaved for about to 20 minutes at a pressure 15-21 lbs/in 2 to kill any contaminating organism, and suitably labeled and stored as a sterile medium.

'Briefly, cotton seeds are sterilized and germinated on a suitable seed germination medium such as a basal agar medium in the dark for a time sufficient to produce seedlings. The normal period of growth is up to about 4 weeks, typically 7 to 14 days.

Segments of explants are excised from the seedling.

It is preferred that the explant come from the hypocotyl or cotyledon. In the alternative, one can equally use immature embryos obtained from the developing fruits of greenhouse or field grown cotton plants as the explant.

The explant segments are cultured on a suitable first callus growth medium, preferably a or full Murashige and Skoog (MS) nutrient medium containing glucose. Growth occurs by culturing at a temperature of from about 25 to about 35'C in a light/dark cycle of about 16 hours of 1 light and above 8 hours of dark. Culturing is the procedure whereby the medium is replaced at periodic intervals as the nutrients are consumed and continued for approximately about 3 to about 4 weeks, or until undifferentiated callus are formed. The callus are transferred to a second callus growth medium, preferably an MS medium supplemented with naphthaleneacetic acid (NAA) and sucrose as the carbon source and cultured for three to four months to produce embryos.

The embryos may then be maintained in the second callus growth medium to maintain an embryo supply or transferred to a plant germination medium such as Beasley and Ting's medium preferably containing casein hydrolysate and source of ammonuim cultured for 2 to 3 weeks to produce plantlets.

The plantlets are transferred to soil under high humidity conditions, then transplanted to larger pots in a greenhouse and finally transferred to the field for growth to maturity.

The methods briefly described herein have been successfully employed to induce somatic embryo formation in cotton of the species Gossviu hirsutum by tissue and suspension cultures and, ultimately, to obtain mature plants from hypocotyl and cotyledon derived callus cultures of Acala varieties of Gossypium hirsutum including SJ2, SJ4, SJ5, B1644, B1810, B2724, GC510 and Cl and non Acala "picker" Siokra and "stripper" variety FC 2017.

Cultures have been transformed to normal plants with novel traits or properties.

More particularly, the procedure involves first the sterilizing of the cotton seeds. Suitable sterilization may be achieved by immersing the seeds in 95% ethanol for 2 to 3 minutes, rinsing in sterile water one or more -16- 1 times, then soaking the seeds in a 15% solution of sodium hypochlorite for 15 to 20 minutes, and rinsing several times with sterile water.

The sterilized seeds are then transferred to a first medium, termed a seed germination medium. A seed germination medium is one of normal salt content. A suitable germination medium is a basal agar medium, including White's medium or half-strength MS medium.

(One-half ingredient strength). Germination normally occurs in the dark over an about 12 to about 14 day period.

Hypocotyl and/or cotyledons are preferably excised from the germinated seed, subdivided or cut into segments and cultured on a first callus growth medium such as an MS medium supplemented with growth substances. The presently preferred medium is the MS medium supplemented with about 0.4 mg/1 thiamine hydrochloride, about 30 g/l glucose, about 2 mg/l naphthaleneacetic acid, about 1 mg/1 kinetin, a common growth regulator, and about 100 mg/l inositol and agar. Thiamine hydrochloride can generally range in concentration from 0.1 to about mg/l, glucose about 20 to about 30 g/l, about 1 to about mg/l naphthaleneacetic acid, about 1 to about 2 mg/1 kinetin and about 50 to about 100 mg/l inositol.

The cultures are maintained at a temperature of about 25 to about 35'C, preferably about 30'C and with a light/dark cycle of about 16 hours of light and about 8 hours of dark. It is preferred to have a light intensity of about 2000 to 4000 lux, more preferably about 3000 to 4000 lux.

The calli formed are periodically subcultured at 3 to 4 week intervals and transferred to a fresh first callus growth medium. In the culturing of the explants, secretions of phenolic compounds from the explants can occur as evidenced by darkening of the cultured medium. In (N -17- 1 this instance, the medium is changed more regularly.

SDarkening has been avoided by changing the culture medium every 10 days. Normally, after three to five medium changes, phenolic secretions will disappear. When this occurs, the first callus growth medium can be replaced by fresh callus growth medium containing sucrose or supplemented with sucrose as a carbon source.

Cr After 3 to 4 weeks of culture, active calli develop on the cut surfaces of the explants. The calli are then transferred to a fresh second callus growth maintenance medium which is preferably an MS medium combined with about 1 to about 10 mg/l, preferably about 1 to about mg/l NAA. Cytokinin is employed at a concentration of from 0 to about 1 g/l. A callus growth medium is characterized as a high salt content medium containing as much as 10 times more salt than the seed germination medium. The essential difference between first and second callus growth medium is the carbon source. Glucose is used during period of phenolic secretions. Sucrose is used when secretion have stopped. The balance of the callus growth medium can remain the same or changed.

The calli are transferred in regular intervals to a fresh callus growth medium and, after generally about to 7 passages or until an anthocyanin pigmentation becomes evident in a portion of the calli, which is followed by development of a yellowish-white embryogenic callus.

The embryogenic callus are then selectively subcultured and maintained by regular subculturing. The embryogenic callus contain somatic embryos at various stages of development. Some may have reached the point of development that enables growth into small plantlets.

Most, however, require further development. Some may be advanced to germination. Other may be maintained as a source of embryos for future use.

-18- 1 With reference to FIG. 2, there is illustrated this stage of development showing calli of AclaI cotton with somatic embryos 12 of differing size with some r having emerging leaves 14 and roots 16. FIG. 3 illustrates 0 5 a somatic embryo isolated at a late globular stage.

With reference to FIG. 4, further development may O be achieved by transferring the somatic embryos to a third growth medium termed herein an embryo germination medium, a medium rich in nitrogen usually in the form of ammonia or its equivalent. Suitable media include Beasley and Ting's medium, preferably supplemented with up to about 500 mg/l casein hydrolysate.

Germination occurs from somatic embryos and, within 2 to 3 weeks, a well developed plantlet 18 of up to 6 leaves and good root system is generally formed.

At this stage, the plantlets are transferred to soil in small clumps and grown in a standard incubator under conditions of high humidity. Temperature is normally maintained at about 25 to 30'C (See Fig. 7).

After a period of growth, the small plants are transferred to larger pots in a greenhouse and thereafter transferred to field and grown to maturity. All the regenerated plants are preferably self-pollinated either while growing in the green house or in field conditions and the seeds collected. Seeds are then germinated and 4 to 5 week old seedlings transferred to the field for progeny row trials and other standard plant breeding procedures. Practicing the above procedure produces viable cotton plants from about 35% of the explants in the period of time from about 6 to about 8 months.

Proliferation of Embryogenic Cotton Cells In Suspension Cultures As an alternative to allowing the growing embryogenic calli to be developed into a plant, the callus may be cut -19- 1 into smaller pieces and further developed using suspension culture techniques.

In this procedure, suspension concentration is normally from about 750 to 1000 mg of callus parts to 8 ml. callus growth medium such as the second callus growth medium (MS medium supplemented with NAA), and allowed to grow in suspension. In a preferred embodiment, the suspension of -the callus is inserted in T-tubes and placed on a roller drum rotating at about 1.5 rpm under a light regime of about 16 hours of light and about 8 hours of dark. Growth is for about 3 to 4 weeks.

After about every 3 to 4 weeks, the suspension is filtered to remove large cell clumps of embryogenic callus depicted in groups in FIG. 5 and as isolated at late globular stages as shown in FIG. 6. The filtrate is returned to a nutrient medium for a 3 to 4 week period of growth. This procedure is repeated over and over with harvesting of large clumps at about 3 to 4 week intervals, at which time the medium is supplanted in whole or in part with fresh callus growth medium. Preferably, about 4 volumes or more of the fresh medium are added to about one volume of residual suspension. It is presently preferred that the filter employed have a mesh size greater than about 600 microns, preferably greater than 800 microns, as it has been observed the cell masses of a particle size less than 600 microns will not develop into plants, whereas cell masses greater than 600 microns and preferably greater than 800 microns have undergone sufficient differentiation so as to become embryogenic and capable of developing into viable plants.

Suspension cultures can also be initiated by transferring of embryogenic calli to a flask, such as a DeLong or Erlenmeyer flask, containing the liquid embryo growth medium in an amount of about 20 ml of MS and NAA at a concentration of 2.0 mg/3. The flask is placed on a 1 gyrotory shaker and is shaken at about 100-110 strokes per minute. After 3 to 4 weeks the suspension is suitable for filtration as described above to remove the large cell clumps for plant development.

More typically, after the third or fourth subculture, the cell suspension from the tube or De Long or Erlenneyer flask is plated onto agar-solidified MS medium containing NAA (2.0 mg/1) or Beasley Ting's medium containing casein hydrolysate (500 mg/1) media and a source of nitrogen. Within 3-4 weeks embryogenic calli with developing embryos become visible. Likewise, the larger cell clumps when plated on the above media give rise to embryogenic clumps with developing embryos.

In both suspension growth methods, the MS media is used to promote and/or sustain embryos whereas the germination medium is employed for rapid plant development.

The seedling explants, if desired, can be transformed.

In this procedure, cotyledon and/or hypocotyl segments of the sterilized seed can be used. Cotyledons are preferred.

The segments are placed in a medium containing an Agrobacterium vector containing a code (genetic marker) such as resistance to an antibiotic, such as for instance kanamycin for a time sufficient for the vector to transfer the gene to the cells of the explant. Generally, contact times ranging from 1 minute to 24 hours may be used and may be accompanied with intermittent or gentle agitation.

The explants are then removed and placed on agar-solidified callus growth medium such as a MS medium supplemented with NAA (2 mg/1) and incubated about 15 to 200 hours at 25 to 35'C, preferably 30"C, on a 16:8 hour light: dark regime.

After incubation, the explants are transferred to the same medium supplemented with the antibiotic cefotaxime preferably in a concentration of 200 mg/l. Cefotaxime is included to prevent any remaining Agrobacterium from -21- 1 proliferating and overgrowing the plant tissues.

Alternatively, the explants can be rinsed with MS medium supplemented with NAA (2mg/l) and incubated an additional 4 to 28 days before rinsing, then incubating the same medium containing cefotaxime. At the end of 4-5 weeks of culture on fresh medium, the developing callus, i.e., primary callus, is separated from the remainder of the primary explant tissue and transferred to MS medium containing NAA (2 mg/1), cefotaxime (200 mg/1) and an antibiotic such as kanamycin sulfate (50 mg/1).

Transformed primary callus, identified by virtue of its ability to grow in the presence of the antibiotic (kanamycin), is selected and embryos developed, germinated and plants obtained following the procedure set forth above.

It is also feasible to achieve transformation of a cell suspension. Following a normal subculture growth cycle of 7 to 14 days, usually 7 to 10 days, cells are allowed to settle leaving a supernatant which is removed: The remaining concentrated suspended cells may be centrifuged at 4000Xg for 5 minutes and the excess medium is discarded. The concentrated suspension cultures are resuspended in the 8 ml of the same medium which contains the Agrobacterium. The suspension is transferred to "T" tubes and suitably agitated for incubation.

Following about 2 to 24 hours, preferably 3 to hours, of incubation to allow for bacterial attachment and DNA transfer, the suspension is removed and allowed to settle. The supernatant containing the bacteria is discarded and the cells are washed with fresh medium.

The suspension may, if desired, be centrifuged for about minutes and the supernatant removed. In either event, the cells are resuspended in the same medium and transferred to a tube or flask and suspension subculture resumed. The object is to minimize the amount of unattached Agrobacterium vector left in the cell suspension.

S-22- After about 15 to about 200 hours, typically 15 to CK1 1 about 72 hours, preferably 18 to 20 hours, the suspension is filtered to remove large clumps and washed with fresh liquid medium and allowed to settle. The suspension is 5 n resuspended in the fresh liquid medium containing 0 cefotaxime (200 mg/1) plated on a solidified medium in Petri dishes.

0 Alternatively, the suspension may be resuspended in r- fresh medium containing cefotaxime and allowed to grow an additional 4 to 28 days prior plating on solidified medium in Petri dishes. Cell concentration is 1 vol. of suspension cells plus 3 vol. of medium with cefotaxime.

Kanamycin at 10 to 300 mg/l preferably about 20 to 200 mg/l more preferably about 40 to 80 mg/l is included in the medium for selection of transformed cells expressing the neomycin phosphotransferase (NPT) gene. Cells and embryos proliferating in the selective concentration of kanamycin are further grown as set forth above to mature somatic embryos capable of germinating and regenerating into whole plants according to the procedures described herein.

Using the above procedure and with reference 06 FIG. 9, there is shown variable cell colonies which is consequence of transformation. There exists cotton cells 20 exhibiting resistance to the antibiotic kanamycin.

With reference to FIG. 10, transformed calli are shown developing into somatic embryos on an antibiotic MS medium. FIG. 11 shows transformed somatic embryos established to have kanamycin resistance and transformed to have resistance to the herbicide glyphosate. FIG. 12 shows plants from the embryos of FIG. 11. FIG. 13 shows cells transformed to have resistance to lepidopterous insects growing on an MS medium and in FIG. 14 transferred to a Beasley and Ting's medium whereas FIG. shows further development of the plantlets of FIG. 14 to more mature plantlets.

-23- 1 COTTON REGENERATION Examnle 1 Regeneration of plants starting from cotyledon explants Seeds of Acala cotton variety SJ2 of Gossvpium hirsutum were 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 minutes, then rinsed in sterile water. Sterilized seeds were germinated on a basal agar medium in the dark for approximately 14 days to produce a seedling. The cotyledons of the seedlings were cut into segments of 2-4mm 2 which were transferred aseptically to a callus inducing medium consisting of Murashige and Skoog (MS) major and minor salts supplemented with 0.4 mg/l thiamine- HC1, 30 g/l glucose, 2.0 mg/l naphtaleneacetic acid (NAA), 1 mg/l kinetin, 100 mg/l of m-inositol, and agar The cultures were incubated at about 30'C under conditions of 16 hours light and 8 hours darkness in a Percival incubator with fluorescent lights (cool daylight) providing a light intensity of about 2000-4000 lux.

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

-24- 1 Somatic embryos which formed on tissue pieces were explanted either to fresh callus growth medium, or to Beasley Ting's medium (embryo germination medium).

The somatic plantlets which were formed from somatic embryos were transferred onto Beasley and Ting's medium which contained 1200 ag/l ammonium nitrate and 500 ag/ casein hydrolysate as an organic nitrogen source. The medium was solidified by a solidifying agent (Gelrite) and plantlets were placed in Magenta boxes.

The somatic embryos developed into plantlets within about three months. The plantlets were rooted with six to eight leaves and about three to four inches tall and were transferred to soil and maintained in an incubator under high humidity for three to four weeks and then transferred to a greenhouse. After hardening, plants were also transferred to open tilled soil.

Example 2 The procedure of Example 1 was 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 were obtained.

Example 3 The procedures of Examples 1 and 2 were repeated except that the explant was the hypocotyl segments. The same results were obtained.

Example 4 The procedure of Examples 1 and 2 were repeated except that the explant was the immature zygotic embryo.

Essentially the same results were obtained.

1 Exampie The procedure of Examples 1 and 2 was repeated with Acalt cotton varieties SJ4, SJ5, SJ2C-1, GC510, B1644, B 2724, B1l10, the picker variety Siokra and the stripper variety FC2017. All were successfully regenerated.

Example 6 The procedure of Example 1 was repeated to the extent of obtaining callus capable of forming somatic embryos. Pieces of about 750-1000 mg of actively growing embryogenic callus was suspended in 8 ml units of liquid suspension culture medium comprised of MS major and minor salts, supplemented with 0.4 mg/l thiamine HC1, g/l sucrose, 100 mg/l of inositol and naphthaleneacetic acid (2 mg/l) in T-tubes and placed on a roller drum rotating at 1.5 rpm under 16:8 light:dark regime. Light intensity of about 2000-4500 lux was again provided by fluorescent lights (cool daylight).

After four weeks, the suspension was filtered through an 840 micron size nylon mesh to remove larger cell clumps. The fraction smaller than 840 microns were allowed to settle, washed once with about 20-25 ml of fresh suspension culture medium. This suspension was transferred to T-tubes (2 ml per tube) and each tube diluted with 6 ml of fresh suspension culture medium.

The cultures were maintained by repeating the above procedure at 10-12 day intervals. Namely, the suspension was filtered and only the fraction containing cell aggregates smaller than 840 microns was transferred to fresh suspension culture medium. -In all instances, the fraction containing cell clumps larger than 840 microns was placed onto the callus growth medium to obtain mature somatic embryos.

-26- 1 The somatic embryos that were formed on callus growth medium were removed and transferred to embryo germination medium and using the protocol of Example 1 were germinated, developed into plantlets and then field grown plants.

Example 7 The procedure of Example 6 was repeated except that suspension cultures were formed by transferring 750-1000 mg of embryogenic calli to a DeLong flask containing ml of the MS liquid medium containing 2 mg/1 NAA.

The culture containing flask was placed on a gyrotory shaker and shaken at 100-110 strokes/minute. After three weeks the suspension was filtered through an 840 micron nylon mesh to remove the large cell clumps for plant growth, as in Example 4. The less than 840 micron suspension was allowed to settle, washed once in the MS liquid medium and resuspended in 2 to 5 ml of the MS liquid medium. The suspension was subcultured by transfer to fresh medium in a DeLong flask containing 1-2 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' suspension was subcultured and the large clumps (840 microns or greater) were used for plant growth.

Example 8 After three or four subcultures using the suspension growth procedure of Examples 6 and 7, 1.5 to 2.0 ml of cell suspension from the T-tube and DeLong flask were in each instance plated onto agar-solidified MS medium containing 2 mg/l NAA and Beasley Ting medium containing 500 mg/1 casein hydrolysate. Within three to four weeks

I

-27- 1 embryogenic calli with developing embryos became visible.

Again, the 840 micron or greater cell clumps were plated on the callus growth medium giving rise to embryogenic clumps with developing embryos which ultimately grew into plants.

COTTON TRANSFORMATION Example 9 Transformation To Form Tumorous-PhenotvDe Vith Acrobacteria LBA 4434 An Acala cotton suspension culture was subcultured for three to four months in T-tubes with the medium (MS medium containing 2 mg/l NAA) being changed every seven to ten days. After any medium change thereafter the cells can be allowed to settle and harvested for transformation. The supernatant was removed by pipeting and cells transformed with the Agrobacterium strain LBA 4434. The Agrobacterium strain LBA 4434 is described in (Hoekema, A. et al. Nature 303: 179-180, 1983, incorporated herein by reference) contains a Ti plasmid-derived binary plant transformation system. In such binary systems, one plasmid contains the T-DNA of a Ti-plasmid, the second plasmid contains the yir-region of a Ti-plasmid.

The two plasmids cooperate to effect plant transformation.

In the strain -LBA 4434, the T-DNA plasmid, pAL 1050, contains TL of pTiAchS, an octopine Ti-plasmid and the yir-plasmid in' strain LBA 4434, pAL4404, contains the intact virulence regions of pTiAch 5 (Ooms, G. et al.

Plasmid 7:15-29, 1982, incorporated herein by reference).

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

-28- 1 The transforming Agrobacterium strain was taken from a glycerol stock, inoculated in a small overnight culture, from which a 50-ml culture was inoculated the following day. Agrobacteria was grown on YEB medium containing per liter in water adjusted to pH 7.2 with NaOH, 5 g beef extract, 1 g yeast extract, 5 g peptone, g sucrose. After autoclaving, 1 ml of 2 M MgC12 is C< added after which antibiotics, as required to kill other strains. The absorbance at 600 nm of the 50 ml overnight culture is read, the culture centrifuged and the formed pellet resuspended in the plant cell growth medium (MS medium plus NAA at 2 mg/l) to a final absorbance at 600 nm of Eight ml of this bacterial suspension of Agrobacterium LBA 4434 was added to each T-tube containing the suspension plant cells after removal of the supernatant liquid.

The T-tube containing the plant and bacteria cells was agitated to resuspend the cells and returned to a roller drum for three hours to allow the Agrobacteria to attach to the plant cells. The cells were then allowed to settle and the residual supernatant removed. A fresh aliquot of growth medium was added to the T-tube and the suspension 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 were again allowed to settle, the supernatant removed and the cells washed twice with a solution of growth medium containing cefotaxime (200 ug/ml). After washing, the cells from each T-tube were resuspended in 10 ml growth medium containing cefotaxime (200 ug/ml in all cases) and 1 ml aliquots of the suspension plated on petri dishes.

Infected cells grew on the growth medium to which no phytohormones were added establishing the tissue had received the wild-type phytohormone genes in T-DNA. The -29- 1 cells developed tumors, further indicating transformation of the cultures.

Example Trensfornation. of Cotton To Form a Kanamcin-Resistant Non-Tumorous Phenot-pe The suspension culture as obtained in Example 9 was transformed using an Agrobacteria which contained the T- DNA containing binary vector pCIB 10. (Rothstein, S.J.

et al. Gene 53: 153-161, 1987, incorporated herein by reference) as well as the pAL 4404 yir-plasmid. The T- DNA of pCIB 10 contains a chimeric gene composed of the promoter from nopaline synthase, the coding region from encoding the enzyme neomycin phosphotransferase, and the terminator from nopaline synthase. The Agrobacteria containing pCIB 10 were grown on YEB medium containing kanamycin (50 ug/ml). Transformation was accomplished in the same manner as in Example 10 except that the 1 ml aliquots resulting in cells and Agrobacteria were immediately plated on selective media containing either kanamycin (50 ug/ml) or G418 (25 ug/ml). Expression of the nos/neo/nos chimeric gene in transformed plant tissue allows the selection of this tissue in the presence of both antibiotics. The existence in two to four weeks of transformed tissue became apparent on the selection plates. Uninfected tissue as well as added control tissue showed no signs of growth, turned brown and died.

Transformed tissue grew very well in the presence of both kanamycin and G418.

At this time, tissue pieces which were growing well were subcultured to fresh selection medium. Somatic embryos formed on these tissue pieces and were explanted to fresh non-selective growth media. When the embryos began to differentiate and germinate, at the point where they were beginning to form roots and had two or 1 three leaves, they were transferred to Magenta boxes containing growth medium described in Example 1. Growth was allowed to proceed until a plantlet had six to eight leaves, at which time it was removed from the agar medium.

The plantlets were now placed in potting soil, covered with a beaker to maintain humidity and placed in a Percival incubator for four to eight weeks. At this time, the plant was removed from the beaker and transferred to a greenhouse. The plants grew in the greenhouse, flowered and set seed.

Example 11 The procedure of Example 10 was followed, except that the transforming Agrobacteria used contained the T- DNA vector DEI PEP10 as well as the pAL4404 yir plasmid.

DEI PEP10, shown in Figure 33, utilizes two T-DNA PstI cleaved right border sequences from h. Tuefaciens (strain C-58) which had been further subdivided with BamHI for integration in the plant genome, a passenger maize phosphoenolpyruvate carboxylase gene (Pepcase gene), and a chimeric gene (NOS/NPT/TK) capable of expression in plants and conferring resistance to the antibiotics kanamycin and G418. This chimeric gene utilizes a nopaline synthetase promoter, the neomycin phosphotransferase

II

coding region from Tn5, and the terminator from the herpes simplex virus thimidine kinase gene. Following transformation, embryogenic callus and embryos were obtained by selection on kanamycin (50 mg/l). No resistant callus was obtained from the control (non-transformed callus) plated on kanamycin at this level (50 mg/l).

Example 12 Transformation of Cotton Suspension Culture Cells To A GCwvhosate-Tolernnt PhenotvDe The procedure of Example 10 was followed, except that the transforming Agrobacteria used contained the T- -31- 1 DNA vector pPMG85/587 (Fillatti, J. et al., Mol Gen.

Genet. 206: 192-199, 1987, incorporated herein by reference) as well as the pAL4404 yit plasmid. The c plasmid pPMG85/587 carries three chimeric genes capable of expression in plants. Two genes code for neomycin phosphotransferase (NPT) which confers resistance to the antibiotics kanamycin and G418. The third chimeric gene, containing the coding sequence from a mutant aroA gene of S. typhimurium, confers tolerance to the herbicide glyphosate (Comai, et al., Science 221: 370-371, 1983, incorporated herein by reference). The Agrobacteria containing pPMG85/587 were grown on medium containing kanamycin (100 ug/ml). Transformation is accomplished as detailed in Example 10 except that the suspension is allowed to grow for 28 days at which time 1 ml aliquots were plated on medium containing selective antibiotics.

Expression of the NPT chimeric gene in transformed plant tissue allowed selection of this tissue on both antibiotics. In this instance the selective antibiotic was kanamycin (50 ug/ml).

In two to four weeks, transformed tissue became apparent on the selection plates. Plant tissue, individual embryos and callus were then placed on growth medium containing the herbicide glyphosate ImM and transformed tissue continued to grow well. Extraction and analysis of the proteins of both callus and embryos confirmed the presence of the product of the glyphosate tolerance gene.

Examrle 13 Transformation of Cotton Suspension Culture Cells To a Hvaromvcin-Resistant Non-Tumorous PhenotvDe The transformation procedure of Example 10 was followed except there was used as the transforming Agrobacteria one containing the T-DNA binary vector pCIB -32- 1 715 (Rothstein, S. J. et al. Gene 53: 153-161, 1987) as well as the yir plasmid. The T-DNA of pCIB 715 contains a chimeric gene composed of the promoter and terminator from the cauliflower mosaic virus (CaHV) 35S transcript (Odell et al, Nature 111: 810-812, 1985, incorporated herein by reference) and the coding sequence for hygromycin B phosphotransferase (Gritz, L. and J. Davies, Gene 179-188, incorporated herein by reference). Agrobacteria containing pCIB 715 was grown on YEB containing kanamycin (50 ug/ml).

Transformation was accomplished as detailed in Example 10 again with the change that the 1 ml aliquots were plated immediately on medium containing as the selective antibiotic 50 ug/ml hygromycin. Expression of the chimeric hygromycin gene in transformed plant tissue allows the selection of this tissue on the medium containing hygromycin. Transformed tissue was grown in the manner described in Example 8 on the selection growth medium establishing transformation had occurred.

Example 14 Transformation of Cotton Suspension Culture Cells To Confer Resistance To Lepidopteran Insects The procedure of Example 10 was followed except where changes are noted below. Different transforming Agrobacteria were used. Also, after plant tissue was selected on an antibiotic for the selection of transformed material, it was further selected for expression of the BT gene as defined herein.

The Agrobacteria used contained the T-DNA vector (Rothstein et al, Gene 53:153-161 (198) incorporated herein by reference into which had been inserted the following chimeric Bacillus thurinaiensis andotoxin genes ("BT Genes"): Ct -33- 1 To prepare the Agrobacterium vector there was fused the CaMV gene VI promotor and protoxin coding sequences.

_A derivative of phage vector mpl9 (Yanish-Perron et al., c 1985) was first constructed. The steps are shown 'in FIGS. 16 and 17. First, a DNA fragment containing approximately 155 nucelotides 5' to the protoxin coding Sregion and the adjacent approximately 1346 nucleotides Ci of coding sequence are inserted into mpl9. Phage mpl9 ds rf (double-stranded replicative form) DNA was digested with restriction endonucleases SacI and SmaI and the approximately 7.2-kbp vector fragment was purified after electrophoresis through low-gelling temperature agarose by standard procedures. Plasmid pKU25/4, containing approximately 10 kbP (kilobase pairs) of Bacillus thuringiensis DNA, including the protoxin gene, was obtained from Dr. J. Nueesch, CIBA-Geigy Ltd., Basle, Switzerland. The nucleotide sequence of the protoxin gene present in plasmid pkU25/4 is shown in Formula 1 below. Plasmid pKU25/4 DNA was digested with endonucleases Hpal and SacI, and a 1503 bp fragment containing nucleotides 2 to 1505 of Formula 1 and purified. This fragment contains approximately 155 bp of bacteria promotor 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'C overnight. The resulting mixture was transformed into E. coli strain HB 101, mixed with indicator bacteria E. coli JM 101 and plated. One phage (mpl9/bt) was used for further construction below.

Next, a fragment of DNA containing the CaMV gene VI promotor, and some of the coding sequences for gene VI, was inserted into mpl9/bt. Phage mpl9/bt ds rf DNA is digested with BamHI, treated with the large fragment of DNA polymerase to create flush ends and recleaved with endocuclease Pstl. The larger vector fragment was purified -34- 1 by electrophoresis as described above. Plasmid pABDI is described in Paszkowski et al., EMBO J. 3, 2717-2722, (1984) incporated herein by reference. Plasmid pABDl DNA is digested with PstI and HindIII. The fragment approximately 465 bp long containing the CaMV gene VI promotor and approximately 75 bp of gene VI coding sequende was purified. The two fragments were ligated and plated as described above. One of the resulting recombinant phages, mpl9/btca contained the CaMV gene VI promotor sequences, a portion of the gene VI coding sequence, approximately 155 bp of Bacillus thuringiensis DNA upstream of the protoxin coding sequence, and approximately 1346 bp of the protoxin coding sequence. To fuse the CaMV promotor sequences precisely to the protoxin coding sequenes, the intervening DNA was deleted using oligionicleotide-directed mutagensis of npl9/btca DNA.

A DNA oligonucleotide with the sequence TTCGGATTGTTATCCATGGTTGGAGGTCTGA was synthesized by routine procedures using an Applied Biosystems DNA Synthesizer. This oligonucleotide is complimentary to those sequences in phage mpl9/btca DNA at the 3' end of the CaMV promotor (nucleotides 5762 to 5778 in Hohn, Current Topics, in Microbiology and Immunology, S, 193- 235 (1982) incorporate herein by reference and the beginning of the protoxin coding sequence (nucleotides 156 to 172 in formula I above). The general procedure for the mutagensis is that described in Zoller and Smith, Meth, Enzym., J19 468-500 (1983) incorporated herein by reference. Approximately five micrograms of single-standed phage mpl9/btca DNA was mixed with 0.3 mg of phosphorylated oligonucleotide in a volume of 40 ul. The mixture was heated to 65'C for 5 min, cooled to 50'C, and slowly cooled to 4*C. Next, buffer, nucleotide triphosphates, ATP, T 4 DNA ligase and large fragment of DNA polymerase were added and incubated overnight at 15*C as described 1 [Zoller and Smith Meth. Enzym., 2_Q, 468-500 (1983)] incorporated herein by reference. After agarose gel electrophoresis, circular double-stranded DNA was purified and transfected into E. coli strain JM101. The resulting plaques are Acreened for sequences that hybridize with 32P-labeled oligonucleotide, and phage are analyzed by DNA restriction endonuclease analysis. Among the resulting phage clones were ones which have correctly deleted the unwanted sequences between the CaMV gene VI promotor ahd the protoxin coding sequence. This phage is called mpl9/btca/del (see FIG. 17).

Next, a plasmid was constructed in which the 3' coding region of the protoxin gene was fused to CaMV transcription termination signals. The steps are shown in FIG. 18.

First, plasmid pABDI DNA was digested with endonucleases BamHI and BglII and a 0.5 kbp fragment containing the CaMV transcription terminator sequences isolated. Next plasmid pUC19, Yanisch-Perron et al., Gene, 103-119 (1985) incorporated herein by reference was digested with BamHI, mixed with the 0.5 kbp fragment and incubated with T 4 DNA ligase. After transformation of the DNA into E. coli strain HB101, one of the resulting clones, called plasmid p 7 02, was obtained which has the structure shown in FIG. 18. Next, plasmid p702 DNA was cleaved with endonucleases Sad and Smal, and the larger, approximately 3.2 kbp fragment isolated by gel electrophoresis. Plasmid pKU25/4 DNA was digested with endonucleases AhaIII and SacI, and the 2.3-kbp fragment (nucleotides 1502 to 3773 of Formula 1) containing the 3' portion of the protoxin coding sequence (nt 1504 to 3773) was isolated after gel electrophoresis. These two DNA fragments are mixed, incubated with T 4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid was p702/bt (FIG. 18).

-36- 1 Finally, portions of phage mpl9/btca/del ds rf DNA and plasmid p702/bt were joined to create a plasmid containing the complete protoxin coding sequence flanked by CaMV promoter and terminator sequences (Eee FIG. 18).

Phage mpl9/btca/del DNA was digested with endonucleases SacI and SphI, and a fragment of approx. 1.75 kbp is purified following agarose gel electrophoresis. Similarly, plasmid p702/bt DNA is digested with endonucleases SacI and Sail and a fragment of approximately 2.5 kbp is isolated. Finally, plasmid pBR 322 DNA (Bolivar et al., Gene, 2, 95-113 (1977) incorporated herein by reference was digested with SalI and SphI and the larger 4.2-kbp fragment isolated. All three DNA fragments were 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 Bacillus thuringiensis crystal protein coding sequence, followed by CaMV transcription termination signals (shown in FIG. 19).

The vector pCIB0 is a Ti-plasmid-derived vector useful for transfer of the chimeric gene to plants via Agrobacterium tumefaciens. The vector is derived from the broad host range plasmid pRK 252, 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, and left and right T-DNA border sequences from the Ti plasmid pTiT37.

Between the border sequences are the polylinker region from the plasmid pUC18 and a chimeric gene that confers kanamycin resistance in plants.

First, plasmid pRK252 was modified to replace the gene conferring tetracycline-resistance with one conferring resistance to kanamycin from the transposon Tn903 [Oka, et al., J. Mol. Biol., 147, 217-226 (1981) incorporated 1 herein by reference], and was also modified by replacing the unique EcoRI site in pRX252 with a BglII site (see FIG. 20 for a summary of these modifications). Plasmid pRK252 was first digested with endonucleases Sail and SmaI, then treated with the large fragment of DNA polymerase I to create flush ends, and the large vector fragment purified by agarose gel electrophoresis. Next, plasmid p368 was digested with endonuclease BamHI, treated with the large fragment of DNA polymerase, and an approximately 1050-bp fragment isolated after agarose gel electrophoresis; this fragment containing the gene from. transposon Tn903 which confers resistance to the antibiotic kanamycin [Oka et al., J. Mol. Biol., 11, 217-226 (1981) incorporated herein by reference]. Both fragments were 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 After transformation into E. coli strain HB101 and selection for kanamycin resistant colonies, plasmid pRK252/Tn903 is obtained (see FIG. 19).

Plasmid pRK252/Tn90 3 was digested at its EcoRI site, followed by treatment with the large fragment of E. coli DNA polymeraSe to create flush ends. This fragment was added to synthetic BglII restriction site linkers, and incubated overnight with T 4 DNA ligase. The resulting DNA was digested with an excess of BglII restriction endonuclease and the larger vector fragment purified by agarose gel electrophoresis. The resulting fragment was again incubated with T4 DNA ligase to recircularize the fragment via its newly-added BglII cohesive ends.

Following transformation into E. coli strain HB101, plasmid pRK252/Tn903/BglII is obtained (see FIG. A derivative of plasmid pBR322 was constructed which contains the Ti plasmid T-DNA borders, the polylinker region of plasmid pUC19, and the selectable gene for -38- 1 kanamycin resistance in plants (see FIG. 21). 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., Proc.

Natl. Acad. Sci. USA, 79, 6322-6326 (1982) incorporated herein by reference. To replace the EcoRI ends of this fragment with HindIII ends, plasmid pBR325/Eco29 DNA was digested with EcoRI, then incubated with nuclease S1, followed by incubation with the large fragment of DNA polymerase to treate flush ends, then mixed with synthetic HindIII linkers and incubated with T4 DNA ligase., The resulting DNA was digested with endonucleases Clal and an excess of HindIII, and the resulting l.l-kbp fragment containing the T-DNA left border purified by gel electrophoresis. Next, the polylinker region of plasmid pUC19 was isolated by digestion of the plasmid DNA with endonucleases EcoRI and HindIII and the smaller fragment (approx. 53 bp) isolated by agarose gel electrophoresis.

Next, plasmid pBR322 was digested with endonucleases EcoRI and Clal, mixed with the other two isolated fragments, incubated with T4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid, contains the polylinker and T-DNA left border in a derivative of plasmid pBR322 (see FIG. 21).

A plasmid containing the gene for expression of kanamycin resistance in plants was constructed (see FIGS. 22 and 23). Plasmid Bin6 obtained from Dr. M.

Bevan, Plant Breeding Institute, Cambridge, UK. This plasmid is described in the reference by Bevan, Nucl.

Acids Res., 12, 8711-8721 (1984) incorporate herein by reference. Plasmid Bin6 DNA was digested with EcoRI and HindIII and the fragment approximately 1.5 kbp in size containing the chimeric neomycin phosphotransferase

(NPT)

gene is isolated and purified following agarose gel electrophoresis. This fragment was then mixed with plasmid

I

-39- 1 pUC18 DNA which had been cleaved with endonucleases EcoRI and HindIII. Following incubation with T4 DNA ligase, the resulting DNA was transformed into E. coli strain HB101. The resulting plasmid is called pUCl8/neo.

This plasmid DNA containing an unwanted BamHI recognition sequence between the neomycin phosphotransferase gene and the terminator sequence for nopaline synthase; see Bevan, Nucl. Acids Res., 12, 8711-8721 (1984) incorporated herein by reference. To remove this recognition sequence, plasmid pUC18/neo was digested with endonuclease BamHI, followed by treatment with the large fragment of DNA polymerase to create flush ends. The fragment was then incubated with T4 DNA ligase to recircularize the fragment, and transformed into E. coli strain HB101. The resulting plasmid, pUC18/neo(Bam) has lost the BamHI recognition sequence.

The T-DNA right border sequence was then added next to the chimeric NPT gene (see FIG. 24). 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., Nucl, Acids Res., I, 369-385 incorporated herein by reference. Plasmid pBR325/Hind23 DNA was cleaved with endonucleases SacII and HindIII, and a 1.0 kbp fragment containing the right border isolated and purified following agarose gel electrophoresis. Plasmid pUC18/neo(Bam) DNA was digested with endonucleases SacII and HindIII and the 4.0 kbp vector fragment isolated by agarose gel electrophoresis.

The two fragments were mixed, incubated with T4 DNA ligase and transformed into E. coli strain HB101. The resulting plasmid, pCIB4 (shown in FIG. 23), contains the T-DNA right border and the plant-selectable marker for kanamycin resistance in a derivative of plasmid pUC18.

Next, a plasmid was constructed which contains both the T-DNA left and right borders, with the plant selectable 1 kanamycin-resistance gene and the polylinker of pUC18 gy between the borders (see FIG. 28). Plasmid pCIB4 DNA was digested with endonuclease HindIII, followed by S treatment with the large fragment of DNA polymerase to create flush ends, followed by digestion with endonuclease l^ EcoRI. The 2.6-kbp fragment containing the chimeric kanamycin-resistance gene and the right border ot T-DNA <C was isolated by agarose gel electrophoresis. Plasmid DNA was digested with endonuclease AatII, treated with T4 DNA polymerase to create flush ends, then cleaved with endonuclease EcoRI. The larger vector fragment was purified by agarose gel electrophoresis, mixed with the pCIB4 fragment, incubated with T4 DNA ligase, and transformed into E. coli strain HB101. The resulting plasmid, pCIB2 (shown in FIG. 24) is a derivative of plasmic pBR322 containing the desired sequences between the two T-DNA borders.

The following steps complete construction of the vector pCIBO0, and are shown in FIG. 25. Plasmid pCIB2 DNA was digested with endonuclease EcoRV, and synthetic linkers containing BglII recognition sites are added as described above. After digestion with an excess of BglII endonuclease, the approximately 2.6-kbp fragment was isolated after agarose gel electrophoresis. Plasmid pRX252/Tn903/BglII, described above (see FIG. 20) was 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, pCIB0O.

Insertion of the chimeric protoxin gene into vector pCIBlO is by the steps shown in FIG. 26. Plasmid pBR322/btl4 DNA was digested with endonucleasas Pvul and SalI, and then partially digested with endonuclease BamHI. A BamHI-SalI fragment approx. 4.2 kbp in size, -41- 1 containing the chimeric gene, was isolated following agarose gel electrophoresis, and mixed with plasmid pCIBl0 DNA which had been digested with endonucleases BamHI and Sail. After incubation with T4 DNA ligase and transformation into E. Coli strain HB101, plasmid shown in FIG. 26 and contained 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. coli host strain S17-1 was used. This strain, obtainable from Agrigenetics Research Corp., Boulder, Co. contains mobilization functions that transfer plasmid directly to Agrobacterium via conjugation, thus avoiding the necessity to transform naked plasmid DNA directly into Agrobacterium (reference for strain 517-1 is Simon et al., "Molecular Genetics of the Bacteria-Plant Interaction", A Puhler, ed, Springer Verlag, Berlin, pages 98-106, 1983, incorporated herein by reference).

First, plasmid pCIB10/19Sbt DNA is introduced into calcium chloride-treated S17-1 cells. Next, cultures of transformed S17-1 cells and Agrobacteriuam tumefaciens strain LBA4404 (Ooms et al., Gene, 1A, 33-50 (1981) incorporated herein by reference] were mixed and mated on an N agar (Difco) plate overnight at room temperature.

A loopful of the resulting bacteria are streaked onto AB minimal media; Chilton et al., Proc. Natl. Acad. Sci.

USA, 27, 7347-7351 (1974), incorporated herein by reference, plated with 50ug/ml kanamycin and incubated at 28'C. Colonies were restreaked onto the same media, then restreaked onto NB agar plates. Slow-growing colonies were picked, restreaked onto AB minimal media with kanamycin and single colonies isolated. This procedure selects for 'Agrobacteria containing the pCIB10/19SBt plasmid.

ct S-42- 1 Construction of a Bacillus thuringiensis protoxin 1 chimeric gene with the CaMV 35S promoter was achieved by construction of a CaMV 35S Promoter Cassette Plasmid n pCIB710 was constructed as shown in FIG. 27. This plasmid 0 contained CaMV promoter and transcription termination Ssequences for the 35S RNA transcript [Covey,

S.N.,

SLomonossoff, G.P. and Hull, Nucleic Acids Research Svol. 9, 6735-6747 (1981) incorporated herein by reference].

A 1149-bp BglII restriction fragment of CaMV DNA in Hohn et al., Current Topics in Microbiology and Immunology, 1 0 194-220 and Appendices A to C (1982) incorporated herein by reference] was 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. 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, was then used in oligonucleotide-directed in-vitro mutagensis to insert the BamHI recognition sequence GGATCC immediately followingCaMV nucleotide 7483 in the Hohn reference.

The resulting plasmid, pCIB710, contains the CaMV promotor region and transcription termination region separated by a BamHI restriction site. DNA sequences inserted into this BamHI site will be expressed in plants by the CaMV transcription regulation sequences. pCIB710 does not contain any ATG translation initiation codons between the start of transcription and the BamHI site.

Insertion of the CaMV 35S promoter/Terminator Cassette into pCIBIO occurred by the steps outlined in FIG. 28.

Plasmids pCIBO0 and pCIB 7 10 DNAs were digested with EcoRI and Sall, mixed and ligated. The resulting plasmid, 1 pCIB10/710 has the CaMV 35S promoter/terminator cassette inserted into the plant transformation vector pCIBlO.

The CaMV 355 sequences are between the T-DNA borders in and thus will be inserted into the plant genome in plant transformation.

Insertion of the Bacillus thuringiensis protoxin gene into pCIBO0/710 occurred by the steps outlined in FIG.

29. As a source of the protoxin gene, plasmid pCIB10/19Sbt was digested with BamHI and NcoI, and the 3.6-kb fragment containing the protoxin gene was isolated by preparative gel electrophoresis. The fragment was then mixed with synthetic NcoI-BamHI adapter with the sequence CATGGCCGGATCCGGC-3', then digested with BamHI. This step creates BamHI cohesive ends at both ends of the protoxin fragment. This fragment was then inserted into BamHI-cleaved pCIBO0/710. The resulting plasmid, pCIB10/35Sbt, shown in FIG. 29, contains the protoxin gene between the CaMV 35S promoter and transcription termination sequences.

Transfer of the plasmid pCIB10/35Sbt into Agrobacterium tumefaciens strain LBA4404 was as described above.

Construction of a deleted Bacillus thuringiensis protoxin gene containing approximately 725 amino acids, and construction of a chimeric gene containing this deleted gene with the CaMV .35S promoter was 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 1. Plasmid pCIB10/35Sbt (FIG. 29) was digested with BamHI and KpnI, and the approximately 2.2-kbp BamHI/KpnI fragment containing the deleted protoxin gene isolated by preparative agarose gel electrophoresis. To convert the KpnI site at the 3' end to a BamHI site, the fragment was mixed with a KpnI/BamHI adapter oligonucleotide and -44- 1 ligated. This fragment is then mixed with BamHI-cleaved pCIB10/710 (FIG. 28).

A deleted protoxin gene containing approximately 645 amino acids was made by removing the COOH-terminal portion of the gene by cleaving at the BclI restriction endonuclese site at position 2090 in the sequence shown in Formula 1. Plasmid pCIB10/35Sbt (FIG. 29) was digested with BamHI and BclI, and the approximately 1.9-kbp BamHI/BcII fragment containing the deleted protoxin gene isolated by reprarative 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 pCIB0O/710 (FIG. 28).

The resulting plasmid, which has the structure pCIB10/35Sbt(BclI) shown in FIG. 31 was selected on kanamycin.

The resulting transformants, designated pCIB10/35Sbt(Kpnl) and shown in FIG. 30, contain the deleted protoxin gene of approximately 725 amino acids.

These transformants are selected on kanamycin.

A deleted protoxin gene was made by introducing a BamHI cleavage site (GGATCC). This is done by cloning the BamHI fragment containing the protoxin sequence from pCIB10/35Sbt into mpl8, and using standard oligonucleotide mutagensis procedures described above. After mutagensis, double-stranded 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 pCIBO/710.

The resulting plasmid, which the structure pCIB10/35Sbt(607) shown in FIG. 32 is selected for on kanamycin.

The pCIBlO/Sbt 607 was used. Transformation was accomplished as detailed in Example 7 with the change that the 1 ml aliquots were plated immediately on medium 1 containing selective antibiotics. This selection medium contained kanamycin (50 ug/ml) or G418 (25 ug/ml).

Expression of the NPT chimeric gene in both transformed plant'tissue allows the selection of this tissue on either antibiotic.

In 2-4 weeks, transformed tissue became apparent on the selection plates. Plant material was selected on kanamycin or G418. Plant tissue (either individual embryos or callus) was then extracted with buffer and assayed for expression of the BT gene product by ELISA assay. The conditions of extraction are as follows: per 100mg of tissue, homogenize in 0.1 ml of extraction buffer containing 50 mM NaC03 (pH9.5), 0.05% Triton, 0.05% Tween, 100nMNaCl, 10mM EDTA, ImM leupeptine, and 1mM PMSF. The leupeptine and PMSF are added immediately prior to use from 100x stock solutions. The tissue was ground with a motor driven pestle. After extraction, 2M Tris pH7 was added to adjust pH to 8.0-8.5 then centrifuged at 12,000 RPM in a Beckman microfuge 12 (10 minutes at and the supernatant saved for enzyme linked immunosorbent assay ("ELISA").

ELISA techniques as a general tool is described by M. F. Clark et al in Methods in Enzvmoloqv 118:742-766 (1986), incorporated by reference.

An ELISA for the Bt toxin was developed using standard procedures and used to analyze transgenic plant material for expression of Bt sequences. For this procedure, an ELISA plate is pretreated with ethanol and affinitypurified rabbit anti-Bt antiserum (50 ul) at a concentraction of 3 ug/ml in borate-buffered saline (see below) is added to the plate. This was allowed to incubate overnight at 4'C. Antiserum was produced in response to immunizing rabbits with gradient-purified Bt crystals (Ang, B.J. Nickerson, Appl. Environ. Microbiol.

3: 625-626 (1978)], incorporated herein by reference, -46- 1 solubilized with sodium dodecyl sulfate and washed with ELISA Wash Buffer (see below). It was then treated for 1 hour at room temperature with Blocking Buffer (see below) washed with ELISA Wash Buffer. Plant extract was added in an amount to give 50 ug of protein (this is typically ca. 5 microliters of extract). Leaf extraction buffer as protein is determined by the Bradford method [Bradford, Anal. Biochem. 72:248 (1976) incorporated herein by reference] using a commercially available kit obtained from Bio-Rad, Richmond, California. If dilution of the leaf extract is necessary, ELISA Diluent (see below)] is used. Allow this to incubate overnight at 4'C. After a wash with ELISA Wash Buffer, 50 ul affinitypurified goat anti-Bt antiserum is added at a concentration of 3 ug/ml protein in ELISA Diluent. This is allowed to incubate for 1 hour at 37"C, then washed with ELISA Wash Buffer. 50 ul r6bbit anti-goat antibody bound to alkaline phosphatase [commercially available from Sigma Chemicals, St. Louis, Mo.] is diluted 1:500 in ELISA Diluent and allowed to incubate for 1 hour at 37'C, then washed with ELISA Wash Buffer. 50 microliters substrate [0.6 mg/ml p-nitrophenyl phosphate in ELISA Substrate Buffer (see below) are added and incubated for 30 minutes at room temperature. Reaction is terminated by adding microliters of 3 M NaOH. Absorbance is read at 405 nm in modified ELISA reader [Hewlett Packard, Stanford, Ca.].

Plant tissue transformed with the pCIB10/35SBt(Bcll) when assayed using this ELISA procedure showed a positive reaction, indicating expression of the Bt gene.

EPBS (ELISA Phosphate Buffered Saline) mM NaPhosphate: Na 2 HP0 4 4.68 grams/4 liters NaH 2

PO

4 .H20 0.976 grams/4 liters 140 mM NaCl NaCl 32.7 grams/4 liters -47pH should be approximately 7.4 Borate Bu.ffered Saline 100 rM Boric a&cid 2 5 Ml' 14a BorAte mM' NaCi Adjust pH to 8.4-8.5 with HC1 or NaoH as needed.

EL18A B 1o ck in ct Buffer In EPBSI It BSA 0.02% Na azide ELISA Wash Bu)ffer Tris-HCl pH 0.05% Tween 0.02% Va Azide -48- 1 ELISA Diluent In EPBS: 0.05% Tween 1% BSA 0.02% Na Azide ELISA Substrate Buffer In 500 mis, 48 ml Diethanolamine, 24.5 mg MgC1 2 adjust to pH 9.8 with HC1.

ELISA Substrate mg p-nitrophenyl phosphate in 25 ml Substrate Buffer.

For bioassays, cell suspensions from antibioticresistant cell cultures obtained from transformations with these Agrobacteria were initiated. Suspensions were grown in medium supplemented with G418 and subcultured into fresh antibiotic-containing medium on ,7-10 day intervals. Samples of these cultures were then used in bioassays to test for toxicity to lepidopterous insects. Twenty ml aliquots of these cultures were allowed to settle (cell volume 3-4ml), and resuspended in medium lacking antibiotics. Suspensions were then allowed to grow for an additional two days in this medium to deplete the cells of any residual antibiotic. Two circles of wet Whatman 2.3 cm filter paper were placed in the .bottom of a 3/4 oz portion cup.

A layer of transformed suspension culture cells 0.2 cm deep was placed onto the filter paper disk. A newlyhatched Manduca serta or Heliothis virescens larva was placed into each portion cup. Controls were made up of larvae fed on non-transformed suspension culture cells.

Discs were replenished on 2-day intervals or as needed.

-49- 1 Manduca larvae generally require more plant material.

The growth rate and mortality of the larvae feeding on transformed cells compared with the growth rate of larvae feeding on untransformed cells was scored after 5 days, and clearly affirmed the toxicity of the BT gene product in transformed cotton cells.

EXAMPLE Heliothis 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 degrees,C with vet paper towels to maintain humidity. The eggs hatched within three days. As soon as possible after hatching, the larvae (one larva per cup) are transferred to covered 3/4 oz. plastic cups. Each cup contains cotton leaf discs. Larvae are transferred using a fine bristle paint brush.

Leaf discs one centimeter 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 6leaf 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 larva feeding on untransformed cotton leaves.

Larvae feeding on discs of cotton transformed with pCIB10/35SB5(Bcll) show a decrease in growth rate and increase in mortality compared with controls.

C(

(N

1 It was observed that a certain number of our regenerated plants appeared to have acquired genetically heritable phenotypic variations as a r r consequence of the process of regeneration. This variation is known as somaclonal variation. The following examples r illustrate how somaclonal variation as a consequence of Sour regeneration procedure has been used to introduce Cq commercially useful new traits into cotton varieties.

EXAMPLE 16 Cotton Recenerants Tolerant to Fungal Pathogens The procedure of Example 1 was followed, and regenerated cotton plants obtained of the variety and SJ4 were hardened and placed in the soil. These plants were self-pollinated and the seed, representing the Fl generation, collected.

To obtain regenerants (somaclonal variants) more tolerant to Verticillium, the Fl generation was planted in a Verticillium infested field for progeny row analysis.

Seed, of the varieties SJ4 and SJ5 were planted in the field as controls. Somaclonal variants more tolerant than the parental varieties to the Verticillium fungus were identified in a few of the progeny rows by assessing overall plant vigor, yield, and the absence of foliar symtoms 'associated with the disease. FIG. 33 shows the progeny rows of regenerants planted in a Verticilium infested field. FIG. 34 shows a Verticillium tolerant somaclonal variant of variety SJ4. This improvement in tolerance to the fungal pathogen was found to be genetically stable and passed on to subsequent generations.

-51- 1 EXAMPLE 17 Cotton Reaenerents with altered growth habits The procedure of Example 13 was followed except that, rather than planting in disease-infested soil, the Fl generation was planted in a cotton breeding nursery.

The overall growth habit of the Fl regenerated progeny was compared to that of the control varieties. Somaclonal variants were identified which were more uniform in growth habit and shorter in stature than the parental variety. One SJ5 regenerant, identified in our trials as Phy 6, was 20% shorter in stature than the parental variety. This kind of growth habit is desireable in cotton grown under narrow row (30" row spacing) cultural conditions. These traits were found to be genetically stable and passed on to subsequent generations.

EXAMPLE 18 Cotton regenerants with imnroved fiber traits The procedure of Example 13 was followed except that the F1 progeny of regenerants were planted in a cotton breeding nursery and allowed to set fruit. When the bolls were mature, the cotton was harvested and subjected to an analysis of several fiber quality traits including length, uniformity, tensile strength, elasticity, and micronaire. Somaclonal variants were identified which were iiproved significantly over the parental variety in one or more of these traits. Representative data from F2 progeny (cell pollination of the Fl) are included in the following Table 1. Values marked with an asterisk represent improvements in SJ5 regenerants which are statistically significant and have been found to breed true in subsequent generations.

-52- Cc, Fiber Properties Variety Length Uniformity Tensile Elasticity Micronaire or strain _Index tn- 5 DS 1.13 48.7 51.2 24.7 24.6 3SP16 3SP20 1.27* 1.28* 1.11 1.18 53.1* 23.1 53.2* 25.7* 6.8 8.0* 7.6* 6.2 7.1 4.27 4.10* 4.13* 4.55 4.43 5SP10 5SP17 51.7 26.7* Cotton recenerants with improved yield The procedure of Example 13 was followed except that the F1 progeny of regenerants of the variety SJ4 were planted in replicated yield trials along with nonregenerated controls. One variant, which exhibited a more uniform growth habit and more vigorous growth habit, yielded 4% more cotton than the parental variety in the same trial. The data are given in Table 2 below.

Table 2 Variety or Strain SJ4 Control X Yield per clot (Ib) X Yield Increase Ibs/Acre 28.0 29.1 3049 3169 Phy 4 *This differec-a was significant at the 95% confidence level.

A 4% increase in yield would represent a return of almost $20 per acre to the average cotton grower in C1 C 5 3- 1 California, where over one million acres of cotton are grown annually.

SEXAMPLE 2 Cotton Regenerants tolerant to a herbicide. (kanamvcinL 9 Suspension cultures of the cotton variety B1644 Swere developed according to the method of Example SSuspension cultures were then plated onto an agar medium as described in Example 6, but supplemented with the herbicide (antibiotic) kanamycin (25mg/l). Most of the cells in the population died, but a few (1 to were tolerant and survived. These were selectively subcultured onto agar-solidified media supplemented with increasing concentrations of kanamycin, until the final concentration reached 50mg/l. Embryos were then developed from this callus, and those resistant embryos were germinated into kanamycin resistant plants.

5 4dr-TAACA=t 70 AATTOOTAT C Ito A.ATD CArT cC TASAAACT0O 330 AATTTOTtCC 270

OTCC-.TCTCA

430

AAGAATTCOC

490 iTTTA.X C A- A rZo AOATCCG TAT 0

FADTTCAAAA

TATCAOTTTT

7.1o 7TCAAT140TCO 790

CTOTACAA

1550

ATAAICAATT

910 AC 7ATO AT AG

CAAACCCAOT

SAAG07 ATT7AG 1 C-90 CT CAT ADAO 20 30 40 ba0A T0WOTCAAAA ATTCDATATTT ADIAJAAT-TA OT7CA-TTT DICCATYTTT to Ito 100 ti 13070 ACTC-ATAT." MM..T A' .AAT.t U C'AAAaz--..A'ACA- 140 ISO TTAATAAAAO ACATOOADOT 200 230 7TATAATTOT TTAADTALAC No0 270 TTACACzccrA ATCOATATT-T CCOTOLCTGGA TTTOTGTTAG to0 390 AIOACDC-A TTTCCTOTAC 440 4".0 YAOOAACCAA OCCATTTCTA goo 510 ATCTTITADA DACTGOOALAO E40 570 TCAATTCAAT OACA1BAACA 620 &;0 TIATCAAGTT CCTCIYTTAT 630 0 CAADATOTT TCAOTOTTTG 740 7:o0 TTATAATOAT TTAACTADOC TAZGGGATTA OACCOTOTAT 96D. eD70 S40 170 AACrTATUDA TAACAAICCO 2:0 230 CTCD.AOTA&A A0TAT1A0aT 230 210 CCTTOTCGCT AADCA.ATT to0

AACATCAATO

240 0.OAOAAAOA6A 300 LCT 71 TOTO 340 250 360 CACTAOTTOA IATA6ATATOO OOAAYTTTTG AAAT IDA

CATTAGA

CA DATICC 07DC CC7 CAD TA TA

DACA.AAD

TTATTOC

CGODDAC

400 410 CA OTTAATTAAC 460 470 AGO ACAACAAT TAC IAATC-ADCA to0 1590 TAC AACCOCTATT Al ~TGT ICAA"CTOCA 700 710 1070 OCCATITOAT 740 770 C.AA CTATACAZAT sz :OCA ITCTADAZGA7 l80 390 TADA TATCCTTICT 940 IS0 :C~r ATTAACAAOA S000 1020 GAGO rTCODCTCAD S0L0 3070 ACAD TA.7AACCA7C 1320 11,3o TAAT OCC77CICCT 420 r.AAAGDA AT AD C7TTAICAkA 540 TrTAACAGAAD CCTCTTT7TO AA7TTACATT

OCCO:GACTA

7110 CAT OCIT 7Ar 0 0AT AA AT qQ0

CYATTICCOA

OAAA777ATA 0rC AT$.A 0 I risc

YATACCZAYG

1140

O;TAOOOII

YA-DAAGAGAA TTAACACTAA. C107AT 920 1930 TAGAACOTAT CCAATTCGAA C.ACTTT3 930 %g0 ATIAOAAAAT TTTOATOOTA GTTTTCI 1040 lOS 0 GADTCCACAT TTGATGGATA TACTTA 3300 3110 AGAATA77AT TOGICADOC AT CASA 11-10 3360 1 370 1380 CDOOCCAGA A77CACY71T CCOCTA7ATO OAAC7A7O 3390 AAATOCADCC CCACAACAAC

OAAYIGDTC

1220

ICA-ACTADOT

1==O 3:40 1-0 1:40 CADCCCOTOT ATAOAACAIT ATCGTCCACT 7TA7AI IOA 1:170 120 12'90 GA=MTTAA IAYAOCAIA AAIATrAP.C 1330 1:40 1550 CTAYTOAAC C7CTCAAAT fl0.CAY=O 3390 1400 1410 CODC701ATGA 1&ATAtCZA CAOAAIAACA 1450 14L0 1470 DATTAAOCCA TGTT~T.AATD MTTCAD CDACrTAT OTYCTCYIDD 6TACAICGTA 1510 130 1 Vto r-ACAA.ATTAC ACA.AATACCT TTAACAAAAT *36!10 1440 2450 TTAAAGOACC AGATTTACA 0OAC5ADA7A 19 11700 1710 CA4CCT7AAG ACTAAATATT ACTGCACCAT 17:0 1760 17)0' 'ACC0TTCrAC CACA4AY17A CAATtA7A 3830 36:0 18:0 CCAATTrrTC ACCAACTATO AG.7AU70UGA 2970 logo 1090J 7ADGTYTIAC IAC7CCSITT AACT ITICAA 1 IV0 1940 39SO, ATCTCTICAA TICAGOCART CAACTTIATA 2990 2,oo :010 ,7AAC:TTTGA OCAOAAAT GATTTAGAAA 2c-o 2060 Z070 C7TCTTCCAA ICAAYCCDG ITAAALAACAG AACTAT CTh T C7OTATAZ AG ACO T CCA= O CTTTA137A.A 1:40

IDTOCTOMATT

2400O CT ACIA ATC~T 3660

YTICTTCCAAG

131 13i Tt7ahCo~ AC4A.AT 0 t370 1360 44AADCrOOA A=TCAGATT 130 1440 rAZGCAA00A TTTACITCATC 1410 TAOTA07OTA ADTATAATA 1530 3560 TAATAATATA ATTCCITC-AT 1&30 24:0 TOOCICTOCA A1TICTOTCO 34670 1480 AATrArCT GCCCAD4TTT 17:0 1730 3740) CAC.AAAO ATATCCCTA AGAATTCCCT 7 AI .1700

CAICAATYCA

1940

GTAATTTACA

1900 AT 0 0ATCAA 0 3940 TAGATco4AT :20

SACCACAAAA

2000

AIGTGACGOA

:140 TITOICT03A =100

ATGAUCUGAA

3790 21-.

CDOAABACCT AT TAA1 CA G CITCC0OGAAC.C T T 7AD r,&TO IGTATTTACO .TTAAO!TCCTC 1970 1 qbk TOAATT70TT CCOOCACAAG DOZODGAAT 1DAGC70YT7A 2090 Z 1(10 TTATCATATT OATCA"0TAT TGAAAAAAAA IDAATI VCCD ::l0 ITTACTTCAA GAICCAAACT AA13TACOGAT ATIACCATqt :110 :1:0 CCAATTADYT OATO7T7A 21:0

TCYGATSAAT

217CI :I90 :I190 A0AAArG1CAA ACATOCCAAD COACTTAUID 0 :0z 2260 77AGA55GAT CAATA040AA C740ACCOTO OCTOGAGADG

AAGOAOCCA

OCIA7CCAAC 2410

ACCAATTAAG

".470 AT CC t.AACA ::oo 2=10 ICACCTATIC AAAGCOAATT 2560 z170 GIATTIAIAT CAAAAAATAG 24':0 240O AGGG7ATAIC CAAOAIADTC 2400 2490 CGAAACAUTA AAITOCCAO .:z:0 AC017ACCCT ::so

ATCAGICOAA

:440 AAGAC1 TACA :"Do

GTACGGGTTC

ATIOOTACC 7AIAGYO 2390 Z 0 AITAAAPrICC TATACCCGIT 24:0 -:460 AATCTAITIA ATICCCACA 1150 CTTATOCCCG CTIICAOCCCC I0 -56nu3. 2540 5 CAA C.AAC-MT CMA.%TOT aCA TCATT CATGTACAOA CTTAMATCAO CACTTA&OTG U650 2ib&0 2670 AT00CCATGC A.AOAZrTAOGA AATCIA.OAAT 271dr 270 2730 CACTAGCTC0 TOTOAAAAGA OCDDAGAAAA 2770 2790 2790 GOOAAACAAA TATTGTITAT MAAOADOCAA :850 :240 2950 CTCAAYATGA TAGATTACAA OCGGATACCA 2890 ".900 2910 C-COTICATAO CATTCOAGAA GCTTATC7OC CCDCTATITT TGAAGAAITA 0iAACCGCDTA' Z010 30o zozo CAAATCTCAT TAAAAATGCT OATTTTAAYA 3070 309e) ATOTAGA7OT ADAAGAACAA AACAACCI.CC 31 3140 Z15(1 CAGAACIOTC ACAACAAGTT CGTDTCTGTC 310 ::00 5 :10 CDTACAA0DA GODATAYGOA GAAGCTIOCG 3:50 3:60 3270 ACCAACIGAA GITTAGOAAC.YTTYADAAG 5:10 =0 330 OTAATGATTA IACTGCOACT CAAGAADAAT 370 3330 3390 CATATGACO AOCCIATCAA ACCAATTCTI 34.30 3440 341-0 AACAAAAADC*A7ATAC4CAT OCACCAAGAD 3490 2:00 31-10 DOCATTACAC ACCAC7ACCA 0CT00C7A70 rCCATAAGOT ATGGATTGAO ATCGOAOAAA 3L30 36:!o 36:"0 AATTACITCT TATCGAGOAA TAA7ATATOC 1670 3680 3890 CATTACIOAC TICTATICAC ACATAAATAA Z730 3740 37:;o ICAC7CTTAA AAGAAIGATO CCCTTT7I =.A7 TAT 004 AAT 00 AAC AA ACAT C Cr. C CC T77CC ASG0AA

ATBAG

ACAAT

TDACA

ITTAT

0GDA A 2560 Z570 :-G .ATTT CTCCTTODAC ATTUATOTTO 2620 2L30. 2.640 27OAT ATTCAAG.AT-T A~aACLCC.AAO 2&00 2&q.0 2700 ~APLA CAAACCATTA OTADOAOA.AG 2740 2750O 2740 AAA CAAAr a7oTAA AAATTGGAA7 2200 2630 2820 TCTOT ADATOCTTTA TTTOTA&AZT 2860 2370 2300 OCGAT OATTCATCCO 0OAAAAAC -720 :130 nj CTGTC 7GTCATTCCO CCTCTCAA~b 980B M990 ,"C ACIUC ATTCICCCIA Z(140 30-10 Z060 TTATC CTOCTCDAAC zl100 3130 330 GICCT TGTTCTTCM OA0ATCCCAAG zi6 IL =70 SO 38 COTOG CTA AICCYT COICCACA3 Z2: Z4 ATTCA 7OAGATCCAO AACAATACAG 3:80 =M)3qCo OTATA ICCA.ArAAC ACCTAACGT 3340 GOTAC OTACAC77CT CGIAAICZAG 3400 Z410 14! CAOC TGATTAICCA ICACCTIAIC 3460 3470 .19CD CCTTO T0AAICTAAC AZAOCATATC Z570 D 3t"( AAAGA ATTACAGTAC TTCCCAG.AAA 31-BO :590 :;660 ,CGAAC ATTCAAtG10 G;ACACCOTOG =840 Z&to 6 AATCT AADDTGTCCA OLATPAAAAT 3700 5710 7 :!0 1777T ATATCAATAA AAAACCGCCA Z760 3770 OTAICAI7A ACOACTOATA YTAAATGTY

'A

3710 00 U-10 3820 3930 i940 oaTTTaCGA AD.OCTT1YAC 7 T&OOTIA CrDCZACATO C~ATCAACT TfAAA7 T T G 380 3840 Z070 Z080 T3O 3900 C.AtTACC CVAA SYTCAAA AAACUTTATT tTICTAAA W vwi.AOCA' ,AA 3910 ZIN 3yc 3q40 zq:30 39460 ATTTTTATO MTCITTTC&A TYCAADATOA 'AITACAACTA TTT=O7AA" AOCYTOTATCO 3970 3930 31910 4000 4010 40:0 IMATTTAACC CETTCTCT71 TOOAAGAACT CDCTAAAGAA TTAZOTTTTG TAAAAAOAAA 4030 .4040 40:0 4040 4070 .8 ACOAAAGTTT TCAGGAAAY0 AAYTAOCTAC CATATGTAIC 70 COCAOIC AACOTACAGC *4090 4100 4110 41:0 4130 4340 GACTOATICT CTCGT7COAC TATOCAUTCA ATTACACOCC OCCACAOCAC TCTTAIGAOT 4 1.0 4160 4170 4180 IIVO A6 CCAGAAGGAC TCAATAAACO CTTTOATAAA AAAGCGGTTG P.ATITTTAA ATATATTIT 4210 4::0 4:i) A-40 4:za2c TC'TCCATTAT OCAAAAGIAA ACYTTOTAAA ACA7CACCCA Y7&CACTOZ AGACTCACO 4-70 4.-00 A:9 t 43C0 4:=4 IATTTYCAAC GAAItCCGTAT ITTTA0ATO ACOATTTTCC AA57AtCCAA ACATT1.ACCA 4 0 d'40 A:so A6 CAITATATC CTUG:TCACC TOGTTCYOCA CAAACTOCRO

Claims (13)

1. A transformed cotton plant comprising a heterologous DNA sequence encoding an altered form of 5-enolpyruvyl shikimate 3-phosphate synthase, wherein said DNA sequence is expressed, whereby said transformed cotton plant is rendered glyphosate resistant.

2. A transformed cotton plant according to claim 1 wherein said heterologous DNA sequence is a mutant aroA gene.

3. A transformed cotton plant comprising a heterologous DNA sequence that, when expressed in the transformed cotton plant, confers a phenotype not present in a non-transformed cotton plant, wherein said phenotype is resistance to glyphosate.

4. A method for producing a transformed cotton plant comprising the steps of: providing a cotton plant selected from the group consisting of hypocotyl, cotyledon, immature zygotic embryos and mixtures thereof; incubating the cotton plant in the presence of a vector comprising DNA that, when expressed in the transformed cotton plant, confers glyphosate resistance not present in a non-transformed cotton plant, to produce a treated explant; growing the treated explant to produce callus; and selecting transformed embryogenic callus.

5. A transformed cotton embryo comprising DNA that, when expressed in the transformed cotton embryo, confers glyphosate resistance not present in a non-transformed cotton embryo.

6. A transformed cotton embryo according to claim 5, wherein the DNA comprises a mutant aroA gene.

7. A transformed cotton embryo according to claim 5, wherein the DNA encodes an altered form of 5-enolpyruvyl shikimate 3-phosphate synthase. U XMEGANIMNNV"ODELETEX\Slfi~bomVO02318 dOC

8. A method for producing a transformed embryogenic cotton callus comprising the steps of: providing a cotton explant selected from the group consisting of hypocotyl, cotyledon, immature zygotic embryos and mixtures thereof; incubating the cotton explant in the presence of a vector comprising DNA that, when expressed in the transformed cotton plant, confers glyphosate resistance not present in a non-transformed cotton plant, to produce a treated explant; growing the treated explant to produce callus; and selecting transformed embryogenic cotton callus.

9. A method according to claim 8, wherein the DNA comprises a mutant aroA gene.

A method according to claim 8, wherein the DNA encodes an altered form of 5-enolpyruvyl shikimate 3-phosphate synthase.

11. A transformed embryogenic cotton callus comprising a heterologous DNA sequence encoding an altered form of 5-enolpyruvyl shikimate 3- phosphate synthase, wherein said DNA sequence is expressed, whereby said transformed embryogenic cotton callus is rendered glyphosate resistant.

12. A transformed embryogenic cotton callus according to claim 11, wherein said heterologous DNA sequence is a mutant aroA gene.

13. A transformed embryogenic cotton callus comprising a heterologous DNA sequence that, when expressed in the transformed embryogenic cotton callus, confers a phenotype not present in a non-transformed cotton callus, wherein said phenotype is resistance to glyphosate. U WEGANMNBNODELETE\Speafication\200230516 daisdOC