CA2198708A1

CA2198708A1 - Transgenic plants expressing acc oxidase genes

Info

Publication number: CA2198708A1
Application number: CA002198708A
Authority: CA
Inventors: Maury L. Boeshore; Rosaline Z. Deng; Kim J. Carney; Glen E. Ruttencutter; John F. Reynolds
Original assignee: Individual
Current assignee: Seminis Vegetable Seeds Inc
Priority date: 1994-09-02
Filing date: 1995-06-07
Publication date: 1996-03-14
Also published as: IL115116A0; AU715924B2; WO1996007742A1; AU2700095A; EP0779926A1

Abstract

The cDNA and genomic DNA encoding the ACC oxidase of broccoli are provided along with recombinant materials containing antisense constructs of these DNA
sequences to permit control of the level of ACC oxidase in and, thus, the maturation and aging of Brassica oleracea plants which allows one to influence, e.g., lengthen, the shelflife of these plants.

Description

= --~ W096/07742 2 1 9 8 7 0 8 PCT~S95/07233 TITLE

TRANSGENIC PLANTS EXPRESSING ACC OXIDASE GENES

Field o~ the Invention This invention relates to the plant enzyme ~CC oxidase which is essential for the production of ethylene in higher plants. More particularly, the invention relates to the DNA sequence of a Brassica oleracea ACC
oxidase, DNA constructs containing this sequence, plant cells cont~lnlng the constructs and plants derived therefrom.

Background of the Invention The enzyme ACC oxidase (also known as ethylene forming enzyme) is essential to the production o~ ethylene in higher plants. It is well known that ethylene is related to various events in plant growth and development including fruit ripening, seed g~rm~n~tion, abscission, and leaf and flower senescence. Ethylene production is strictly regulated by the plant and is induced by a variety of external factors, including the application of auxins, wounding, anaerobic conditionsl viral infection, elicitor treatment, chilling, drought SUBSTITUTE SHEET (RULE 26) W096/07742 PCT~S95/07233 ~

and ions such as cadmium and lithium ions, known as ethylene-inducible events. In addition, it recently has been shown that ethylene production begins after harvest (Tian et al. (1994) "A Role for Ethylene in the Yellowing of Broccoli After Harvest", J. Amer. Soc.
Hort. Sci. Vol. 119: 276-281).

The pathway for ethylene synthesis in plants was first described by Adams and Yang, PNAS USA 76:170-174 (1979) who identified 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. The physiology and biochemistry of ethylene synthesis was extensively reviewed by Yang and Hoffman in Ann. Rev. Plant Physiol. 35:155-189 (1984).
In the ethylene biosynthetic pathway, methionine is catalyzed by the enzyme S-adenosylmethionine synthetase to form S-adenosylmethionine (SAM). SAM is then catalyzed to form the three-membered-ring amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase. This three-membered-ring am no acid is then catalyzed by the enzyme ACC oxidase to form ethylene.

The ethylene forming enzyme genes in tomato plants were the first to be isolated. Smith et al. (1986) Planta 168:94-100 reported the rapid appearance of an mRNA
correlated with ethylene synthesis encoding a protein of molecular weight 35000.
A number of molecular strategies have been used to inhibit ethylene formation in transgenic plants.
Theologis et al., Cell, 70:181-184 (1992), report using updated antisense RNA and ACC d~m~ n~ ~e approaches.
Gray et al, Plant Mol. Biol. 19:69-87 (1992), report the manipulation of fruit ripening with antisense genes. Both ACC oxidase (Ham~ilton et al., Nature SUBSTITUTE SllEET (RULE 26) ~ WO 96/07742 2 1 9 8 7 0 8 PCT/US9S/07233 (1990) 346:284-296) and ACC synthase (Oeller et al, ~cience (1991) 254:437-439) antisense constructs have been used success~ully to inhibit ethylene production in transgenic tomato plants. Klee et al. ((1991) The Pla~t Cell 3:1187-1193) overexpressed a Pseu~om~n~,~ ACC
d~Am;n~e gene in transgenic tomato plants. ACC
d~m' n~ ~e converts ACC to a-ketobutyrate. This approach led to 90~-97~ inhibition of ethylene production during ~ruit ripening in transgenic plants.
As is well known, a cell manu~actures protein by transcribing the DNA of the gene for that protein,to produce messenger RNA (mRNA), which is then processed (e.g., by the removal o~ introns) and ~inally translated by ribosomes into protein. This process may be inhibited by the presence in the cell of "anti~ense RNA". By this term is meant an RNA sequence which is complementary to a sequence o~ bases in the mRNA in question: complementary in the sense ~hat each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA
sequence read in the 5' to 3~ sense. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, preventing the ~ormation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these ef~ects. Such antisense RNA may be produced in the cell by transformation with an appropriate DNA
=~ construct arranged to transcribe backwards part o~ the coding strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).

SUBSTITUTE SHEET ~RULE 26J

W096/07742 2 ~ 9 8 PCT~S9S/07233 The use of this technology to downregulate the expression of specific plant genes is well known.
Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference, e,y., lack of anthocyanin production in flower petals of petunia leading to colorless instead of colored petals (van der Krol et al., Nature, 333, 866-869, 1988); or at a more subtle biochemical level, e.g., change in the amount of polygalacturonase and reduction in depolymerization of pectin during tomato fruit ripening (Smith et al., Nature, 334, 724-726). Thus, antisense RNA has been proven to be useful in achieving downregulation of gene expression in plants.
Information Disclosure WO 92/04456 reports the isolation of a gene encoding the ACC synthase gene derived from zucchini and transgenic plants in which ethylene production is modified to control changes associated with fruit ripening.

WO 92/11371 reports a gene encoding an ethylene forming enzyme gene derived from melon and transgenic plants in which ethylene production is modified to control changes associated with fruit ripening, improved fruit quality, improved flavor and texture, and the possibility of production over a longer harvest period.
W0 92/11372 reports a peach gene encoding ethylene forming enzyme and plants transformed with the peach ethylene forming enzyme gene construct. These constructs modify ethylene-associated ripening changes, reduced rate of deterioration after harvest, and allowed storage for longer periods.

SUBSTITUTE SHEET (RULE 26) ~ W096/07742 2 ~ ~ 8 7 0 ~ PCT~95/07233 WC 94/0844g reports the isolation of a gene encoding the ACC synthase polypeptide derived from Crucifier and transgenic plants in which ethylene production is modified to control changes associated with fruit ripening.

Balague et al., (1993) ~ur. J. Biochem. 212:27-34 reported the isolation and sequencing o~ an ethylene ~orming gene ~rom melon (Cucumis melo h.) where the predicted amino acid sequence of the melon ACC oxidase gene appears to be closely related to the sequences reported for 3 tomato ACC oxidase genes (81~, 81~ and 77~ identity), an avocado ACC oxidase gene (73 identity), and a carnation ACC oxidase gene (75~
identity). The authors speculate that transforming melon with pM$~1 antisense transgene should allow them to determine whether ethylene biosynthesis can be inhibited in ripening melon and whether this inhibition will delay ripening processes. However, the engineering of constructs for plant trans ormation or expression was not reported.

Gray et al., Plant Mol. Biol. 19:69-87 (1992) report the molecular biology of fruit ripening and its manipulation with antisense genes.

Hamilton et al. (1990) Nature 346:284-286 report the transformation of ~h;mPric pTOM13 antisense gene construct into the tomato variety Ailsa Craig. All transformants showed reduced ethylene biosynthesis.
Ethylene production in wounded leaves of primary transformants was inhibited by 68~ and by 87~ in ripening fruit.

Holdsworth et al. (1987) Nucl. Acids Res. 15:731-739 report the structure and expression of an ethylene-related mRNA from tomato.
SUBSTITUTE SHEET ~RULE 26,) W096/07742 PCT~S95/07233 - 6 - 21 ~8 ~08 Holdsworth et al. (1987) Nuc, Acids Res. 15:10600 report the isolation and sequencing of a genomic clone (GTOMA) of tomato ethylene forming enzyme. Transgenic tomato plants expressing antisense RNA to tomato ethylene forming enzyme sequences displayed reduced ethylene synthesis.

Kende (1993) Ann. Rev. Plant Physiol. Plant Mol. Biol.
44:283-307 reports a history of the study of the ethylene biosynthetic pathway.

Kim, W.T. and Yang, S.F. (1993) Plant Physiol. Suppl.
102:26 reported the isolation and characterization of cDNAs encoding 1-aminocyclopropane-1-carboxylate oxidase homologs from mung bean hypocotyls.

Klee et al. ((1991) The Plant Cell 3:1187-1193) reports the overexpression of a Pseudomonas ACC d~Am;n~se gene in transgenic tomato plants to inhibit ethylene production during ~ruit ripening.

McGarvey et al. (1990) Plant Mol. Biol. 15:165-167 report the nucleotide sequence o~ a ripening-related cDNA ~rom avocado fruit.
Oeller et al. (1991) Science 254:437-439 report the reversible inhibition of tomato fruit senescence by antisense ACC synthase RNA.

Pua et al. (1992) Plant Mol. Biology 19:541-544, report the isolation and sequence analysis of a cDNA clone encoding ethylene-forming enzyme in Brassica juncea but did not report any genomic clone or genetic sequence and reported no engineering for plant expression or plant transformation.

SUBSTiTUTE SHEET (RULE 26,) =

W096l07742 2 1 9 8 7 08 PCT~S95107233 Smith et al. (1986) Planta 168:94-100 reported the rapid appearance of an mRNA correlated with ethylene synthe8is encoding a protein of molecular weight 35000.

Theologis, Cell 70:181-184 (1992) report using updated antisense RNA and ACC dP~m;n~e approaches to control fruit ripening.

Theologis et al. (1993) Dev. Genet. 14:282-295 report the reversible inhibition o~ tomato ~ruit senescence by antisense ACC synthase RNA.

Theologis et al. (1992) Plant Physiol. 100:549-551 report the modification of fruit ripening by suppressing gene expression.

Tian et al. (1994) J. Amer. Soc. Hort. Sci. Vol.
119:276-281 reports ethylene production and the yellowing of broccoli begins after harvest.
Wang et al. (1991) Plant Physiol. 96:1000-1001 isolated the ACC oxidase cDNA sequenced of a carnation (Dianthus caryophyllus) by screening a cDNA library with the tomato efe gene pTOM13 and an avocado efe gene pAVOe3.
Wang e~ al. (1992) Plant Physiol. 100:535-536 isolated the ACC oxidase cDNA sequence of Petunia corollas.
.

Yang (1984) Ann. Rev. Plant Physiol. 35:155-189 report generally on ethylene biosynthesis and its regulation in higher plants.

SUMM~RY OF THE I~v~NllON

The present invention provides recombinant materials which permit control of the level of ACC oxidase in plants, specifically, Brassica oleracea and Cucumis SUBSTITUTE SHEET (RULE 26) W096/0774~ PCT~S95/07233 melo. The invention is also directed to DNA in purified and isolated form comprising a DNA sequence encoding the enzyme ACC oxidase of Brassica oleracea and Cucumis melo. The invention is also directed to expression systems effective in expressing the DNA
encoding said ACC oxidase and to recombinant hosts transformed with this expression system. The invention is further directed to methods to control ACC oxidase production and, thus, the growth and development of Brassica oleracea and Cucumis melo plants, using the coding sequences for ACC oxidase in an antisense construct or by replacing the ACC oxidase gene by a mutated ~orm thereof. The invention thus provides a method for controlling the maturation and aging of Brassica oleracea and Cucumis melo plants which allows one to influence, e.g., lengthen, the shelflife of these plants.

~RIEF DESCRIPTION OF THE FIGURES
Fig. 1 illustrates the amino acid sequence of B.
oleracea ACC oxidase [SEQ ID NO:1], the cDNA sequence of B. oleracea ACC oxidase [SEQ ID NO:2] and the restriction enzyme cloning sites for PCR oligomer reaction primers;

Fig. 2 illustrates the cDNA and amino acid sequences of B. oleracea ACC oxidase [SEQ ID NOS:1 and 2] compared to the cDNA and amino acid sequences of B. juncea ACC
oxidase [SEQ ID NOS:9 and 10];

Fig. 3 illustrates the PCR oligomer reaction primers and the novel restriction enzyme cloning sites ~or each of the primers used for the amplification of the DNA
nucleotide sequence of the B. oleracea ACC oxidase gene [SEQ ID NO:8] from the portion of the B. oleracea genome cont~;n;ng the DNA sequence of the B. oleracea SVBSTITUTE SHEET ~RUEE 26) ~ W0~6/07742 2 1 9 8 7 0 8 PCT~S95/07233 ACC oxidase and compares to the genomic DNA sequence with the cDNA sequence of B. oleracea ACC oxidase;

Fig. 4 illustrates a flow chart showing the engineering steps used to install the ACC oxidase cDNA coding sequence, both in the sense and the antisense orientation, into plant expression vectors and the subsequent insertion into binary plasmids; and Fig. 5 illustrates a ~low chart showing the engineering steps used to install the B. oleracea ACC oxidase genomic DNA coding sequence, both in the sense and the antisense orientation, into plant expression vectors and the subsequent insertion into binary plasmids.
Fig. 6 illustrates an RNA blot of total RNA extracted from Ro transgenic melon plants (leaves) hybridized with B. oleracea ACC oxidase sense RNA probe.

Fig. 7 illustrates an RNA blot of total RNA extracted from Rl transgenic melon progeny of line 4168-10 hybridized with B. oleracea ACC oxidase sense RNA
probe.

Fig. 8 illustrates an RNA blot of total RNP. extracted ~orm Rl transgenic melon progeny of lines 4168-19 and 4168-20 hybridized with B. oleracea ACC oxidase sense RNA probe.

Fig. 9 illustrates a comparison o~ melon ACC oxidase nucleotide sequence with B. oleracea nucleotide sequence. Sequences were aligned with the use of the Pileup Program in the UWGCG program package.

SUBSTiTUTE SHEET (RULE 26) W096l07742 PCT~S95/07233 ~

- 10 - ~ 7 a s PETAI~ED DESCRIPTION OF THE PREFERRED EMBODIMENT

Most of the recombinant DNA methods employed in practicing the present invention are st~n~rd procedures, well known to those skilled in the art.
Enzymes are obtained from commercial sources and are used according to the vendor's recomm~n~tions or other variations known to the art. Reagents, buffers, and culture conditions are also known to those in the art.
General references cont~;n;ng such standard techniques include the following: R. Wu, ed. (1979) Methods in Enzymoloqy, Vol. 68; J.H. Miller (1972) Experiments in Molecular Genetics; D.M. Glover, ed. (1985) DNA
Cloning, Vol. II; S.B. Gelvin and R.A. Schilperoort, eds. Introduction. Expression, and Analysis of Gene Products in Plants; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor ~aboratory Press, Cold Spring Harbor all of which are incorporated by reference.
As used herein, "recombinant" refers to a nucleic acid sequence which has been obtained by manipulation of genetic material using restriction enzymes, ligases, and similar recombinant.techniques as described by, for example, Sambrook et al., Molecular Cloning: A
~aboratQry M~nll~l, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
"Recombinant", as used herein, does not refer to naturally-occurring genetic recombinations.
As defined herein, "ACC oxidase" includes enzymes which are capable of catalyzing the conversion of ACC to ethylene. The amino acid sequence of the oxidase may or may not be identical with the amino acid sequence which occurs natively in higher plants. An example of such a native se~uence is shown in Fig. 1 [SEQ ID NO:1]
which occurs in broccoli. Naturally occurring allelic SUBSTITUTE SHEET (RULE 26) ~ WO 96/07742 2 1 9 8 7 3 8 PCT~S95/07233 variants undoubtedly occur as well. In addition, arti~icially induced mutations are also included so long as they do not destroy acti~ity. In general, conservative amino acid substitutions can be made for most o~ the amino acids in the primary structure as shown without effecting destruction of activity. Thus, the definition of ACC oxidase used herein includes those variants which are derived by direct or indirect manipulation of the disclosed sequence.
It is alRo understood that the primary structure may be altered by post-translational processing or by subsequent chemical manipulation to result in a derivatized protein which contains, for example, glycosylation substituents, oxidized forms of, for example, cysteine or proline, conjugation to additional moieties, such as carriers, solid supports, and the like. These alterations do not remove the protein from the de~inition of ACC oxidase so long as its capacity to convert ACC to ethylene is maintained.

Thus, the identity of an enzyme as "ACC oxidase" can be confirmed by its ability to effect the production of ethylene in an assay performed as follows: 5 ng to 0.5 mg of enzyme protein in a 500-uL volume is added to 2.5 mL of assay buffer [50mM Tris-HCl (pH 7.2), 10~ (v/v) glycerol, 0.1 mM FeSO4, 10 mM ascorbate, 1 mM ACC, and 1 mM 2-oxoglutarate] in 25-mL Erlenmeyer flasks. The vials are sealed with serum caps and incubated for 1 hr at 23C shaking gently. Air in the headspace is analyzed by gas chromatography on a Varian 3400 gas chromatograph equipped with a flame ionization detector and an 80~ Porapak N/20~ Porapak Q column. Ethylene production is quantitated by comparison with a 97.7 ppm ethylene gas mixture in helium (Alltech Associates). A
unit is defined as 1 nL/hr. Pirrung et al. (1993) Biochemistry 32:7445-7450, teach the purification and SUBSTITUTE SHEET (RULE 26) W096/07742 PCT~S95/07233 - 12 - 2l 98 7~8 properties of the apple fruit ethylene-forming enzyme.
While alternative forms of assessment of ACC oxidase can be devised, and variations on the above protocol are certainly permissible, the foregoing provides a definite criterion for the presence of ACC oxidase activity and classification of a test protein as ACC
oxidase.

The amino acid sequence for ACC oxidase in broccoli is shown in Fig. 1 [SEQ ID NO:1]. Preferred ~orms of the ACC oxidase of the invention include that illustrated herein, and those derivable therefrom by systematic mutation of the genes. Such systematic mutation may be desirable to enhance the ACC oxidase properties of the enzyme, to enhance the characteristics of the enzyme which are ancillary to its activity, such as stability, or shelf life, or may be desirable to provide inactive forms useful in the control of ACC oxidase activity in vivo.
As described above, "ACC oxidase" refers to a protein having the activity assessed by the assay set forth above; a "mutated ACC oxidase" refers to a protein which does not necessarily have this activity, but which is derived by mutation of a DNA encoding in ACC
oxidase. By "derived from mutation" is meant both direct physical derivation from a DNA encoding the starting material ACC oxidase using, for example, site specific mutagenesis or indirect derivation by synthesis of DNA having a sequence related to, but deliberately different from, that of the ACC oxidase.
As means for constructing oligonucleotides of the required length are available, such DNAs can be constructed wholly or partially from their individual constituent nucleotides.

SUBSTITUTE SHEET (RUEE 26) -wos6/07742 2 I q 8 7 0~ PCT~S95/07Z33 Initial Isolation of the ACC Oxidase cDNA
-In view of the recent studies which have shown that ethylene production begins after harvest (Tian et al.
(1994) ~. Amer. Soc. Hort. Sci. Vol. 119:276-281), one does not have to wait until a plant illustrates visible signs of senescence to ensure one harvests the mRNA
needed ~or ethylene production. After isolating total mRNA from plants such as Brassica oleracea var. Italica or Cucumis melo by methods well known in the art, such as single step liquid-phase separation, the mRNA is purified. The mRNA is then treated with reverse transcriptase to produce total ~irst strand cD~A.

Polymerase chain reaction (PCR) primers can then be used to amplify the ACC oxidase gene ~rom the cDNA
template. In the case of Brassica oleracea and Cucumis melo, because it was suspected that its ACC oxidase DNA
sequence would be similar-to the ACC oxidase cDNA
sequence o~ other species, oligonucleotides used to prime the PCR were modeled after sequences of a cDNA
clone o~ the ACC oxidase gene found in Brassica juncea (Pua et al. (1992) Plant Mol. Biology 19:541-544).

With the ACC oxidase gene available because o~ PCR
amplification, ACC oxidase can be produced in a variety of recombinant systems. Specifically, the ACC oxidase can be expressed in tran~genic plants both in enhanced amounts and in an antisense mode to control the aspects of plant development which are ethylene sensitive, and ~ in particular, to delay plant senescence.

Accordingly, a variety of expression systems and hosts can be used for the production of this enzyme. A
variety of prokaryotic hosts and appropriate vectors is known in the art; most commonly used are E. coli or other bacterial hosts such as B. subtilis or SUBST~TUTE SHEET (RULE 26) W096/07742 PCT~S95107233 - 14 - 21 98 7a8 Pseudomonas and typical bacterial promoters include the trp, lac, tac, and beta-lactamase promoters. A readily controllable, inducible promoter, the lambda-phage promoter can also be used. A large number of control systems suitable for prokaryote expression is known in the art.

Similarly, a large number of recombinant systems have been developed for expression in eukaryotic hosts, including yeasts, insect cells, m~mm~l ian cells, and plant cells. These systems are well characterized and require the ligation of the coding se~uence under the control of a suitable transcription initiating system (promoter) and, if desired, termination se~uences and enhancers. Especially useful in connection with the ACC oxidase gene of the present invention are expression systems which are operable in plants. These include systems which are under control of a tissue-specific promoter, as well as those which involve promoters that are operable in all plant tissues.

Transcription initiation regions, for example, include the various opine initiation regions, such as ocotopine, mannopine, nopaline and the like. Plant viral promoters can also be used, such as the cauliflower mosaic virus 35S promoter. In addition, plant promoters such as ribulose-l,3-diphosphate carboxylase, flower organ-specific promoters, heat shock promoters, seed-specific promoters, promoters that are transcriptionally active in associated vegetable tis~ue, etc. can also be used.

The cauliflower mosaic virus (CaMV) 35S promoter has been shown to be highly active in many plant organs and during many stages of development when integrated into the genome of transgenic plants including tobacco and SUBSTITUTE SHEET (RULE 26) W096/07742 2 ~ ~ ~ 7 a 8 pcT~ss5lo7233 petunia, and has been shown to confer expression in protoplasts of both dicots and monocots.

The CaMV 35S promoter has been demonstrated to be active and may be used in at least the following monocot and dicot plants with edible parts:
blackberry, RUbUS; blackberry/raspberry hybrid, Rubus, and red raspberry; carrot, Daucus carota; maize;
potato, Solanum tuberosum; rice, Oryza sa~iva;
strawberry, Fragaria x ,inAn~.~sa; and ~omato, Lycopersi con escul en tum .

The nopaline synthase (Nos) promoter has been shown to be active and may be used in at least the ~ollowing monocot and dicot plants with edible parts: apple, Malus pumila; cauliflower, Brassica oleracea; celery, Apium graveolens; cucumber, Cucumis s~tivus; eggplant, Solanum melongena; lettuce, Lactuca sativa; potato, Solanum tuberosum; rye, Secale cereale; strawberry, Fragaria x An~n~sa; tomato, Lycopersicon esculentum;
and walnut, ~uglans regia.

Organ-specific promoters are also well known. For example, the E8 promoter is only transcriptionally activated during tomato fruit ripening, and can be used to target gene expression in ripening tomato fruit (Deikman and Fischer, EMBO ~ (1988) 7:3315). The activity of the E8 promoter is not limited to tomato fruit, but is thought to be compatible with any system wherein ethylene activates biological processes. Other organ-specific promoters appropriate for a desired target organ can be isolated using known procedures.
These control sequences are generally associated with yenes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, Trans., R. Soc. ~ondon (1986) B314:343).
SUBSTITUTE SHEET (RULE 26~

W096/07742 PCT~S95/07233 ~

- 16 - 21~8708 To create an expression system, the gene coding ~or ACC
oxidase in hand i8 ligated to a promoter using st~n~rd - -techniques now common in the art. The expression system may be ~urther optimized by employing ~ _ supplemental elements such as transcription terminators and/or enhancer elements.

Thus, for expression in plants, the recombinant expression cassette will contain in addition to the ACC
oxidase-encoding sequence, a plant promoter region, a transcription initiation site (if the coding sequence to be transcribed lacks one), and a transcription tPrm;n~tion sequence. Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.

Sequences controlling eukaryotic gene expression have been extensively studied. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20-30 base pairs (bp) upstream cf the transcription start site. In most instances, the TATA
box is required for accurate transcription initiation.
By convention, the start site is called +1. Sequences extending in the 5' (upstream) direction are given negative numbers and sequences extending in the 3' (downstream) direction are given positive numbers.

In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T)NG (Messing, J. et al., in Genetic Enqineerinq in Plan~s, Kosage, Meredith and Hollaender, eds. (1983) pp. 221-227). Other sequences conferring tissue specificity, response to environmental signals, or m~;mllm efficiency of transcription may also be found in the promoter region.
SUBST~TUTE SHEET (RULE 26) ~ W096/07742 2 1 98 7~8 PCT/US95/07233 Such sequences are often found within 400 bp of transcription initiation site, but may extend as ~ar as 2000 bp or more.

In the construction of heterologous promoter/structural gene combinations, the promoter is preferably positioned about the same distance from the heterologous transcription start site as it i~ ~rom the transcription start site in this natural setting. As 10 i9 known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

AS stated above, any o~ a number o~ promoters which direct transcription in plant cells is suitable. The promoter can be either constitutive or inducible.
Promoters of bacterial origin include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids (Herrera-Estrella et al., Nature (1983) 303:209-213).
Viral promoters include the 35S and l9S RNA promoters of cauliflower mosaic virus (O'Dell et al., Nature (1985) 313:81Q-812. Plant promoters include the ribulose-1,3-diphosphate carboxylase small subunit promoter and the phaseolin promoter.

In addition to a promoter sequence, the expression cassette should also contain a transcription t~rm; n~ tion region downstream of the structural gene to provide for efficient t~rm;n~tion. The term;n~tion region may be obtained from the same gene as the promoter sequence or may be obtained from different genes .

If the mRNA encoded by the structural gene is to be efficiently processed, DNA sequences which direct polyadenylation of the RNA are also commonly added to SUBSTITUTE SHEET (RULE 26) W096/07742 - PCT~S95/07233 ~

the vector construct (Albert and Kawaski, Mol. and Appl. Genet. (1982) 1:419-434). Polyadenylation is of importance for expression of the ACC oxidase-encoding RNA in plant cells. Polyadenylation sequences include, ; -but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., BMBO J (1984) 3:835-846) or the nopaline synthase signal (Depicker et al., Mol. and Appl. Genet. (1982) 1:561-573).

The resulting expression system or cassette is ligated into or otherwise constructed to be included in a recombinant vector which is appropriate for higher plant transformation. The vector will also typically contain a selectable marker gene by which transformed plant cells can be selected for and identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. After transforming the plant cells, those cells having the vector will be identified by their ability to grow on a medium cont~;n;ng the particular antibiotic. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bac~erial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct.
Suitable prokaryotic selectable markers also include 30 resistance to antibiotics such as kanamycin or --tetracycline.

Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
For instance, in the case of Agrobacterium transformation, T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
SUBST~TUTE SHEET (RULE 26) --~ wosG/07742 2 ~ 9 8 7 0 8 PCT~S95/072~3 In addition, vectors can also be constructed that contain in-~rame ligations between the se~uence encoding the ACC oxidase protein and sequences encoding other molecules of interest resulting in ~usion proteins, by techni~ues well known in the art.

When an appropriate vector is obtained, transgenic plants are prepared which contain the desired expression system. A number of techniques are available for transformation; in general, only dicots can be transformed using Agrobacterium-mediated infection.

In one ~orm o~ direct transformation, the ~-ector is microinjected directly into plant cells by use of micropippettes to mechanically transfer the recombinant DNA (Crossway, Mol. Gen. Genetics (1985) 202:179-185).
In another form, the genetic material is transferred into the plant cell using polyethylene glycol (Krens, et al. Nature (1982) 296:72-74), or high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, is used (Klein, et al., ~ature (1987) 327:70-73). In still another method protoplasts are fused with other entities which contain the DNA whose introduction is desired. These entities are minicells, cells, lysosomes or other ~usible lipid-surfaced bodies (Fraley, et al., Proc. Natl. Acad. Sci.
USA (1982) 79:1859-1863.
DNA may also be introduced into the plant cell~ by electroporation (Fromm et al., Proc. Natl. Acad. Sci.
USA (1985) 82:5824). In this technique, plant protoplasts are electroporated in the presence o~
plasmids cont~; n; ng the expression cassette.
Electrical impulses of high field strength reversibly perme~hilize biomembranes allowing the introduction of SUBSTITUTE SHEET (RULE 26) W096/07742 PCT~S95/07233 the plasmids. Electroporated plant protoplasts reform the cell wall, divide and regenerate.

For transformation mediated by bacterial infection, a _ plant cell is infected with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the DNA to be introduced. Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefaciens) and hair root disease (A.
rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria. The bacterial genes responsible for expression of opines are a convenient source of control elements for ch'm~ric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
-Heterologous genetic sequences can be introduced intoappropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on --infection by Agrobacterium and is stably integrated into the plant genome (Schell, J., Science (1987) 237:1176-1183). Ti and Ri plasmids contain two regions essential for the production of transformed cells. One of these, named transferred DNA (T-DNA), is transferred to plant nuclei and induces tumor or root formation.
The other, termed the virulence (vir) region, is essential for the transfer of the T-DNA but is not itself transferred. The T-DNA will be transferred into a plant cell even if the vir region is on a different plasmid (Hoekema, et al., Nature (1983) 303:179-189).
The transferred DNA region can be increased in size by the insertion of heterologous DNA without its ability to be transferred being affected. Thus a modified Ti SUBSTITUTE SHEET (RULE 26) W096/07742 ~ t ~ ~ 7 ~ ~ PCT~S95/07233 or Ri plasmid, in which the disease-causing genes have been deleted, can be used as a vector for the transfer of the gene constructs of this invention into an appropriate plant cell.

Construction of recombinant Ti and Ri plasmids in general ~ollows a method typically used with the more common bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes ~ound with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to "shuttle vectors~, (Ru~kum and Ausubel, Nature (1981) 298:85-88), promoters (Lawton et al., Plant Mol. Biol. (1987) ~:315-324) and structural genes for antibiotic resistance as a ~election ~actor (Fraley et al., Proc. Natl. Acad. Sci. (1983) 80:4803-4807).

There are two classes of recombinant Ti and Ri plasmid vector system now in use. In one class, called ~cointegrate," the shuttle vector cont~;n;ng the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJ1 shuttle vector of DeBlock et al., EMBO J (1984) 3:1681-1689 and the non-oncogenic Ti plasmid pGV2850 described by Zambryski et al., EMBOJ (1983) 2:2143-2150. In the second class or "binary" system, the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector described by Bevan, Nucleic Acids Research (1984) 12:8711-8721 and the non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al., Nature SUBSTITVTE SHEET (RULE 26) W096/07742 PCT~S95/07233 ~

._ (1983) 303:179-180. Some of these vectors are commercially available.
-There are two common ways to transform plant cells with - _ Agrobacterium: co-cultivation of Agrobacterium with cultured isolated protoplasts, or transformation of intact cells or tissues with Agrobacterium. The first requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts. The second method requires (a) that the intact plant tissues, such as cotyledons, can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
Most dicot species can be transformed by Agrobacterium as well as species which are a natural plant host for Agrobacterium are transformable in vitro.
Monocotyledonous plants, and in particular, cereals, are not natural hosts to Agrobacterium. Attempts to transform them using Agrobacterium have been unsuccessful until recently (Hooykas-Van Slogteren et al., Nature (1984) 311:763-764). However, there is growing evidence now that certain monocots can be transformed by Agrobacterium. Using novel experimental approaches cereal species such as rye (de la Pena et al., Nature (1987) 325:274-276), maize (Rhodes et al., Science (1988) 24Q:204-207), and rice (Sh;m~moto et al., Nature (1989) ~:274-276) may now be transformed. --Identification of transformed cells or plants is generally accomplished by including a selectable marker in the transforming vector, or by obt~;n;ng evidence of successful bacterial infection.
Plant cells which have been transformed can also be regenerated using known techniques.
SUBSTITUTE SHEET (RULE 26) _ ~ WO 96/07742 2 l q 3 ~ 3 ~ PCT~S95/07233 Plant regeneration from cultured protoplasts is described in Evans et al., Handbook o~ Plant Cell Cultures, Vol. 1: (MacMilan Publishing Co~ New York, 1983); and Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II, 1986). It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugar-cane, sugar beet, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, but generally a suspension of trans~ormed protoplasts or a petri plate cont~; n~ ng transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently root. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos g~rm;n~te as natural embryos to form plarts. The culture m~ will generally contain various amino acids and plant hormones, such as auxin and cytok; n; n.q, It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

A large number of plants have been shown capable o~
regeneration from trans~ormed individual cells to obtain transgenic whole plants. For example, regeneration has been shown for dicots a~ ~ollows:
apple, Malus pumila; blackberry, Rubus;
Blackberry/raspberry hybrid, Rubus; red raspberry, Rubus; carrot, Daucus carota, cauliflower, Brassica oleracea; celery, Apium graveolens; cucumber, Cucumis SUBSTITUTE SHE~T (RULE 26) W096/07742 PCT~S95/07233 ~

sativus; eggplant, solAnum melongena; lettuce, ~actuca sativa; potato, Solanum tuberosum; rape, Brassica napus; soybean (wild), Glycine Canescens; strawberry, Fragaria x AnAnAlqsa; tomato, Lycopersicon esculentum;
walnut, Juglans regia; melon, Cucumis melo; grape, Vitis vinifera; mango, Mangifera indica; and for the following monocots; rice, Oryza sativa; rye, Secale cereale; and maize.

In addition, regeneration of whole plants ~rom cells (not necessarily transformed) has been observed in: -apricot, Prunus armeniaca; asparagus, Asparagus officinalis; banana, hydrib Musa; bean, Phaseolus vulgaris; cherry, hybrid Prunus; grape, Vitis vinifera;
mango, Mangifera indica; melon, Cucumis melo; ochra, Abelmoschus esculentus; onion, hybrid Allium; orange, Citrus sinensis; papaya, Carrica papaya; peach, Prunus persica and plum, Prunus domestica; pear, Pyrus c~mm7~nis; pineapple, ~nAnA.~ comosus; watermelon, Citrullus vulgaris; and wheat, Triticum aesti V~m~

The regenerated plants selected from those listed are transferred to standard soil conditions and cultivated in a conventional m~nner.
After the expression cassette is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number o~ st~n~rd breeding techniques can be used, depending upon the species to be crossed.

The plants are grown and harvested using conventional procedures.

SUBSTITIJTE SHEET (RULE 26,) ~ W096/07742 2 1 ~ ~ 7 Q 8 ~CT~S95/07233 AC~ OXIDASE GENE OBTAINED FROM B. OLERACEA cDNA CLONES

EXAMP~E 1 Isolation of total RNA from broccoli beads (florets) Total RNA was isolated from broccoli florets (beads) by use of TRI-REAGENT RNA/DNA/protein isolation reagent (a single step liquid-phase separation) (Molecular Research Center, Inc., Cincinnati, Ohio). The instructio~s provided with the reagent were followed to accomplish the isolation.

EXAMP~E 2 Enrichmen~ for ~olyA+ RNA

Oligo dT-cellulose chromatography was then used to enrich for polyA+ RNA. The procedure involved mixing total broccoli floret RNA (this includes messenger RNA
or polyA+ RNA) with oligo dT-cellulose in 20mM NaCl and Tris buffer. The oligo-dT cellulose was washed to eliminate non-polyadenylated RNAs from the cellulose.
Subsequently, polyA+ RNA was eluted from the cellulose by elution in Tris buffer that includes no NaC1.
Sambrook et al. (1989) "Selection of poly(A)+ RNA~
Molecular Cloning: A LaboratorY Manual, Second Edition, pp. 7.26-7.29.

Synthesis of sinqle-stranded cDNA

Single-stranded cDNA was synthesized using the polyA+
RNA template from Example 2. A 50uL reaction included 1 X First Strand cDNA Synthesis Buffer (GIBCO BRL, Gaithersburg, Maryland), 1 ug polyA+ RNA, 1 mM dNTP~s SU8STITUTE SHEET (RULE 26,) WO 96/07742 PCT/US95/07233~

(USB, Cleveland Ohio), 1 ug oligo dT, 1 uL RNasin (Promega, Madison, Wisconsin), 3.3 uM dithiothreitol, 5 uL 32PdCTP (3000 Ci/mmol, NENDuPont NEG013~l Wilmington, Delaware), and luL RTase Superscript (GIBCO BRL, Gaithersburg, Maryland). Single-stranded B. oleracea cDNA was purified by the use of columns (Qiaquick-spin PCR column) obtained from Qiagen (Chatsworth, Cali~ornia). First strand cDNA was characterized by hydroxide agarose gel electrophoresis; based on electrophoretic mobility, the size distribution of ~irst strand cDNA was estimated to center near 1 kilobase.

PCR amplification of tar~et cDNA ACC oxidase sequences An ACC oxidase cDNA sequence was PCR amplified from total Brassica oleracea ~irst strand cDNA with the use -_ of the cDNA template obtained as above. The polymerase chain reaction (PCR) was carried out using reagents supplied with the Perkin Elmer Cetus Gene Amp PCR Kit under the following conditions: ~0.1 ug/mL total cDNA
of Brassica oleracea, 1.5mM MgCl2, 24ug/mL of each oligomer primer, 200uM each dNPT, kit reaction buffer, and AmpliTaq DNA ploymerase supplied with the kit.
Reaction tubes were subjected to 93C for 1 min, 55C
for 1 min, the 72C for 3 min for 30 cycles in a Perkin Elmer Thermocycler. Oligonucleotides used to prime the PCR were modeled after sequences of a cDNA clone of the ACC oxidase gene found in brassica juncea (Pua et al.
(1992) Plant Mol. Biology 19:541-544). Oligomer primers RMM389 (5' GAGAGAGCCATGGAGAAGAACATTAAGTTTCCAG
3', complementary to the 5' end of the cDNA clone of brassica juncea ACC oxidase gene) (SEQ ID NO:3) and RMM391 (5' CGGCATCTCTGAAAGA~ ~'l'~GATCCTCAAACTCGC 3', complementary to the 3' end of the cDNA clone of SUBST~TUTE SHEET (RULE 26) -219870g B~assica juncea ACC oxidase gene) (SEQ ID NO:4) were used to prime this reaction. Fig. 2 illustrates the cDNA and amino acid sequences of B. oleracea ACC
oxidase [SEQ ID NOS:1 and 2] compared to the cDNA and amino acid sequences of B. juncea ACC oxidase [SEQ ID
NOS:9 and 10].

Cloninq an ACC oxidase PCR fraqment into the pCRII
vector The 1 kb ACC oxidase PCR fragment was cloned into ~he pCRIITM vector, included in the TA Cloning Kit available ~rom Invitrogen Corporation (San Diego, California) to obtain a clone known as EFEG3 (Fig. 4). The sequence of the inserted gene in EFEG3 was verified by nucleotide DNA sequencing using a U.S. Biochemical (Cleveland, Ohio) dideoxy se~uencing kit (Fig. 1 ) (SEQ
ID NO:2).

Insertion of the ACC oxidase codinq sequence into an ex~ression cassette (cp express) in antisense orientation EcoRI digestion of clone EFEG3 produced an EFEG3 fragment cont~; n; ng the Brassica aleracea ACC oxidase gene. An NcoI restriction site was fitted onto the 3' end of the EFEG3 fragment during a second PCR
amplification by the use of the primer RMM480 (5' CGGCATCTCTGAAAGA~ GGTACCTCA~A 3', complementary to the 3' end of the ACC oxidase gene) (Figs 2 and 4) (SEQ
ID NO:5). Its sequence is located at the 3' end of the gene and includes a novel NcoI site (Figs. 2 and 4).
During this second PCR amplification one Of two SUBSTITUTE S~IEET (RULE 26) W096/07742 2 1 9 8 7 0 8 PCT~S9Sl07233 ~
!

internal NcoI sites was also eliminated by the use of oligomer primer RMM470 (5' GAGAGCCATGGAGAAGAACATTAAGTTTCCAGTTGTAGACT
TGTCCAAGCTCATTGGTGAAGAGAGAGACCAAACAATGGCTTTGATCAACGATGC _ 3', complementary to the 5' end of the ACC oxidase gene) (Figs. 2 and 4) (SEQ ID NO:6); RMM470 does not include the first internal NcoI site located in EFEG3 (Fig. 2). The resulting PCR ~ragment was cloned into --the pCRII cloning vector included in the TA cloning kit available from Invitrogen Corporation to obtain a clone known as EFEG3'.
._ To begin transfer of the Brassica oleracea cDNA ACC
oxidase gene into a plant expression cassette, EFEG3' was digested with NcoI to produce an NcoI cDNA fragment encoding B. oleracea ACC oxidase. Using standard methods (see J.L.-Slightom, 1991, Gene, Vol. 100, pp.
251-255, "Custom PCR Bngineering of a Plant Expression Vectorl'), this fragment was inserted into the expression cassette pUC18cp express in an antisense orientation to obtain EFEG3cel and in the sense orientation to obtain EFEG3ce7 (Fig. 4). pUC18cp express includes about 330 base pairs of the CaMV 35S
transcript promoter and 70 bp of the cucumber mosaic virus 5'-untranslated region. The region flanking the 3' end of the inserted gene includes 200 bp of the CaMV35S transcript poly(A) addition signal. The Nco I
site maintains the ATG translation initiation site found in the ACC oxidase gene. Sense orientation constructs are designed to give sense mRNA that can be translated into ACC oxidase in the plant. The antisense orientation of the NcoI fragment in EFEG3cel is designed to transcribe mRNA in the plant that is complementary to the sense mRNA; no B. oleracea ACC
oxidase protein can be translated in the plant from this construct.

SUBSTITUTE SHEET (RULE 26) Insertion of ACC oxidase DNA cassettes into a binary vector The antisense cassette EFEG3FL AS (Fig. 4) was inserted into the unique HindIII site of binary vector pGA482G
to produce plasmid pEPG604 (Fig. 4). pGA482G is available from Gynehung An, Institute of Biological Chemistry, Washington State University in the form of pGA482 followed by the insertion of a gentamicin resistance gene. The sense cassette EFEG3FL (Fig. 4) was inserted into the unique HindIII site of binary vector pGA482G to produce plasmid pEPG606 (Fig. 4).
The structures shown in Fig. 4 were verified by restriction analysis.

Transformation of the binary vectors into Brassica oleracea plants by Agrobacteria-mediated transformation The binary plasmids pEPG604 and pEPG606 are transformed into strains of Agrobacterium tumefaciens, e.g., strain C58Z707 and Agrobacterium rhizogenes, e.g., strain A4.Strain C58Z707 is available from August Hebpurn at Indiana University, Bloomington, Indiana (Hepburn et al., (1985) J. Gen. Micro. 131:2961-2969). Strain A4 is available from Jerry Slightom, The Upjohn Company, Kalamazoo, Michigan. Evidence of the origin of the strain A4 is presented by Slightom et al. J. Biol. Chem.
(1986) Vol. 261, No. 1 pp. 108-121. The resulting Agrobacterium strain is used to perform B. oleracea plant transformation procedures.

Agrobacterium-mediated transfer of the plant expressible Brassica oleracea ACC oxidase is done using procedures known to those skilled in the art. For W096/07742 2 t q 8 7 0 8 PCT~S95/07233 ~

example, David and Tempe (1988) Plant Cell Reports 7:88-91) and Damgaard and R~Rmll~sen (1991) Plant ~ _ Molecular Biology 17:1-8, transformed cauliflower and rapeseed hypocotyl cells and regenerated transformed plants. Specifically, aseptically grown hypocotyls with or without an intact root system are inoculated with engineered A. tumefaciens or A. rhizogene.
Hypocotyls are then transferred to Murashiges and Shogg -(1962) Physiol Plantarum 15:473-497) medium (MS) 10 containing 200 micromolar acetsyringone. Two to three_ days later, hypocotyls are transferred to MS medium cont~ln;ng 50 mg/1 kanamycin sulfate, 500 mg/1 car~enicillin and 200 mg/1 cefotaxime (MS-O).
Hypocotyls are continuously subcultured every 21 days on MS-0 medium until shoots form. Shoots are then removed from agar and potted in soil. Transgenic plants (~) are grown to sexual maturity in a green house and Rl transgeneic seed is produced. Transfer of this gene into plant cells can also be accomplished using other methods, such as direct DNA uptake (Paszkowski, et al, EMB0 J., 1084, 3:2717), microinjection (Crossway, et al., Mol Gen. Genet.
202:179), electroporation (Fromm et al., Proc. Natl.
Acad. Sci. U.S.A. 82:5824), or high velocity microprojectiles (Klein, et al., Nature 327:70).
-EXAMP~E ~ -Evaluation of transqenic plants for inhibition of ethylene biosynthesis Transgenic status of Ro plants and their segregating progeny is verified by routine methods. These include ELISA assays for NPTII protein detection; DNA assays such as PCR amplification (detection) of transgenes and Southern blot hybridization for detection of transgenes.
SUESTITUTE SHEET (RULE 26) ~ w096/07742 2T~8708 PCT~S95/07233 For example, protein in leaf tissue samples taken from - R1 transgenic lettuce seedlings is extracted and analyzed for NPTII protein by enzyme-linked immunosorbant assay (ELISA). The procedure and kit supplied by 5 Prime ---~ 3 Prime, Inc., Boulder, Colorado, is used to assay NPTII expression in R1 transgenic lettuce seedlings. In an initial screen of R1 transgenic seedlings for NPTII protein by ELISA, it is expected that ll independent transgenic proprietary B. ol erace lines express NPTII. The date indicate that these initial lines are segregating for the NPTII
marker gene.

Evaluation of transgenic plants for inhib_~ion of ethylene biosynthesis can be accomplished by assaying transgenic B. ol eacea materials ~or expression of ACC
oxidase an~isense RNA using a Northern analysis or a RNase protection assay. In a Northern analysis of transgenic materials, RNA extracted ~rom transgenic B.
2 0 ol eracea is subjected to agarose electrophoresis and blotted onto a Nylon membrane. A radioactive (32p_ labelled) RNA probe (sense RNA) synthesized in vi tro is used ~o hybridize the blot. Only antisense RNA of the ACC oxidase trangene in the plant will bind to the 32p_ labelled RNA probe; thus antisense ACC oxidase RNA will be detected by autoradiography. Parallel hybridization of replicate blots with antisense ACC oxidase RNA probe serves as a check on the hybridization with the sense RNA probe.
The RNase protection assay involves hybridizing a labelled RNA molecule (pure sequence synthesized in vi tro) with total tissue RNA in solution in a tube.
Only complementary RNA will hybridize with the pure RNA
labelled and sythesized in vitro. The total pool o~
RNA is subjected to RNase A and RNase Tl digestion;
protected ~RNAs are resistant to RNase digestion.
SU~ST~TI.JTE SHEET (RllLE 26~

W096/07742 2 ~ 9 8 7 0 8 PCT~S95/07233 ~

Protected mRNAa are evaluated quantitatively and qualitatively on an acrylamide gel. - _ Following the determination of whether B. oleracea ACC
oxidase antisense RNA is expressed, the transgenic materials or tissues are assayed for ACC oxidase activity. This can be accomplished by the assay methods outlined above for measuring ACC oxidase activity. In addition, it is possible to employ immunological methods (for example, ELISA or Western blots) to assay transgenic materials for levels of ACC
oxidase protein. It is expected that transgenic would exhibit reduced levels of ACC oxidase protein compared with non-transgenic materials. Tian et al. (1994) J.
Amer. Soc. Hort. Sci. Vol. ~ 276-281 outline in some detail their procedures for evaluating "degreening" in response to ethylene in harvested broccoli. They measured chlorophyll content in the flore~s after harvest.
A~ QXIDASE GENE OBTAINED FROM B. OLERACEA GENOMIC
CLQNES
-EXAMP~E 10 Extraction of tQ~al cellular DNA from broccoli by a CTAB extrac~ion method Three or 4 newly expanding leaves (0.5 - 1 gm fresh weight) were placed into the bottom corner of a Ziplock bag. One mL of preheated CTAB extraction buffer was added to the leaf sample. CTAB extraction buffer (1~
(w/v) CTAB Sigma H-5882; 1.4 M NaCl; 100 mM Tris HCl pH
8.0; 30 mM EDTA pH 8.0) was prepared and preheated to 65C 5-10 minutes prior to use. The following was added to each mL of CTAB extraction buffer just before using: 10uL of 2-mercaptoethanol, 6 ~L of Ribonuclease SUBSTITUTE SHEET (RULE 26) -_ wos6/07742 PCT~S95107233 ~ 2~ 9~7~8 T1 (5,000 U/ml) Sigma R-8251, and 25 ~L of Ribonuclease A(10 mg/ml~ Sigma R-4875.

The Ziplock bag was placed flat on a hard surface. A
one-liter Corning media-bottle was ~irmly rolled across the surface of the bag repeatedly until the leaf tissue was disrupted and had the consi~tency of applesauce.
The macerated sample was moved to a botto~l corner of the Ziplock bag and the corner was cut with a scissors.
The entire sample was squeezed into a sterile 15-mL
Falcon tube and incubated at 70C ~or 30 minutes. The sample was cooled for 5 minutes at room temperature.
One mL of cholororm-octanol (24:1, v:V) was added, and the sample was vortexed 1 second to mix thoroughly.
The samples were then centrifuged in a Beckman GH 3.7 rotor (Beckman GPR centri~uge) at 2500 rpm, 25OC for 5 minutes to separate phases. The aqueous phase (~1000 ~1) was then transferred to a sterile 1.5-mL Eppendorf tube. 1.5 ~L of RNAse Tl (10mg/m~) was added. An equal volume of 1~ CTAB precipitation buf~er was added to each sample. The tube was inverted a few times and incubated at room temperature for 30 minutes.

The sample was centrifuged in a Eppendorf microfuge for 60 seconds to pellet the precipitate. The supernatant was discarded, and the tube was inverted on a paper towel to drain. 500 ~l of high salt solution (10 mM
Tris pH 8.0, 1 M NaCl, 1 mM EDTA pH 8.Q) was added, and the sample was incubated at 65C for 15 minutes to dissolve the DNA. One ml of 100~ ethanol was added and the sample was placed at -20C for one hour or overnight to precipitate DNA. DNA was hooked or spooled with a 1.5 ml capillary pipet and placed into a sterile 1.5 ml ~ppendorf tube. The DNA pellet was washed by adding 1 ml of wash solution (80~ ethanol, 15 mM ~mmon; um acetate) and incubated at room temperature SUBSTITUTE Sl IEET (RULE 26?

21 ~8708 --W096/07742 PCT~S95/07233 15 minutes. The washed DNA was dissolved in 300 ~ of --sterile water. ~ _ EXAMP~E 11 -PCR amplification of target genomic ACC oxidase Polymerase chain reactions (PCRs) were carried out 3 using reagents supplied with the Perkin Elmer Cetus 10 Gene Amp PCR Kit under the following condltions: ~0.1 ug/mL total cellular DNA of Brassica oleracea 1. 5 mM
MgCl2, 24 ug/mL of each oligomer primer, 200 uM each dNPT, kit reaction buffer, and AmpliTaq D~ polymerase supplied with the kit. Reaction tubes were subject to 93C for 1 min, 55 C for 1 min., the 72C for 3 min.
for 30 cycles in a Perkin Lemer Thermocycler.
Oligonucleotides used to prime the PCR were modeled after sequences of a cDNA close of the ACC oxidase gene found in Brassica juncea (Pua et al. (1992) Plant Mol.
~iology 19:541-544). Oligomer primers RMM389 (5' GAGAGAGCCATGGAGAAGAACATTAAGTTTCCAG 3', complementary to the 5' end of the cDNA clone of Brassica juncea ACC
oxidase gene) (SEQ ID N0:3) AND rmm390 (5' CCGCCAATTAACAACCAGGTACCACA~ATTTCACACCC 3', complementary to the 3' end of the cDNA clone o~
Brassica juncea ACC oxidase gene) (SEQ ID N0:7) were used to prime this reaction. (Fig. 3).

E~AMP~E 12 Cloninq genomic ACC oxidase PCR fragment into the pCRII
vec~or The genomic ACC oxidase PCR fragment was cloned into the pCRII vector (Invitrogen Corporation, San Diego, California) to obtain a clone known as EFE3-1 (Fig. 5).
The sequence of the insert gene in EFE3-1 was verified SUBST~TUTE SHEET (RIJLE 26) _ _ _ -_ wos6lo7742 PCT~S95/07233 ~ 2 ~ 987~8 by nucleotide DNA sequencing using a U.F. Biochemical (Cleveland, Ohio) dideoxy sequencing kit (Fig. 3) (SEQ
ID NO:8). Comparison of B oleracea genom c clone BFE3-1 with cDNA clone EFEG-3 revealed 4 exons and 3 introns in B. oleracea ACC oxidase genomic clone 3-1 (Fig. 3). The coding regions of genomic clone 3-1 are identical to the sequence for the cDNA clone EFEG-3 (Fig. 3). The structure of Brassica oleracea ACC
oxidase iS highly related to the intron/exon arrangement in the tomato genomic ACC oxidase clone GTOMA (Holdsworth et al. (1987) Nuc. Acids Res.
15:10600).

Insertion of the ACC oxidase codinq sequence into an expression cassette (cp express) To begin trans~er of the genomic Brassica oleracea ACC
oxidase gene into a plant expression cassette, EFE3-1 was digested with NcoI to produce a 1528 bp NcoI
~ragment encoding genomic B. oleracea ACC oxidase; two internal NcoI sites near the 5' end of the gene resulted in the elimination of about 220 bp of the gene by NcoI digestion (Figs. 3 and 5). Using stAn~rd methods (see J.L. Slightom, 1991, Gene. Vol. 100, pp.
251-255), this fragment was inserted into the expression cassette pUC18cp express in an antisense orientation to obtain EFE2.7 and in the sense orientation to obtain EFE3.3 (Fig. 5).

SU~ST~TVTE SltEET (RULE 26) W096/07742 2 1 9 8 7 a 8 PCT~S95/07233 -Insertion o~ genomic ACC oxidase DNA cassettes into a binarY vector HindIII fragments harboring full-length cDNA clone antisense and sense cassettes were isolated. The antisense cassette EFE3.7 AS (Fig. 5) was inserted into the unique HindIII site of binary vector pGA482G to produce plasmid pEPG600 (Fig. 5). The sense cassette EFE3.3 SENSE (Fig. 5) was inserted into the unique HindIII site of binary vector pGA482G to produce plasmid pEPG602 (Fig. 5). The structures shown in Fig.
5 were verified by restriction analysis.

-Transformation of the binary vectors into Brassica oleracea plants by Aqrobacteria-mediated transformation procedures.
-The binary plasmids are trans~ormed into Agrobacteriumstrains ~ and C58Z707 as in Example 8. The resulting Agrobacterium strain is used to perform B. oleracea plant transformation procedures.

Evaluation Qf transgenic plants for inhibition of ethylene biosynthesis Evaluation of transgenic plants for inhibition of ethylene biosynthesis is accomplished as described in Example 9.

SUBSTITUTE SHEET (RULE 26) ~p~ W096/07742 PCT~S95/07233 EXAMP~E 17 Brassica oleracea ACC oxidase antisense constructs were transferred to melon ( Cucumis melo) plants via Agrobacteria-mediated ~ransformation using procedures published by Fang and Grumet (1990 and 1993). The pEPG600 and pEPG604 constructs were trans~ormed into melon (see Figures 4 and 5 ~or restriction maps o~
thesa binary plasmids).
A~ter shoots were regenerated on kanamycin-containing solid tissue culture media, they were rooted and tested for transformation status. We veri~ied t.~,ans~ormation status either by testing regenerated organized shoots ~or ability to form callus on kanamycin-cont~;n;ng solid media (only transformed materials expressing NPTII can grow on these media) or by NPTIII expression detected by ELISA. The results are summarized in Table I.

TABLE I -- SUMM~RY OF CANTALOUPE LINES TRANSFORMED WITH
B. OLERACEA ACC OXIDASE CONSTRUCT
Plant Inbred Exp.line Construct Ploidy Status Rl Seed 4140.3 604 AB discarded 4168.10 604 harvested 0162 4168.11B 604 potted 4168.14 604 died 4168.15 604 died 4168.15B 604 harvested 0010 4168.17D 604 potted 4168.18 604 harvested 0016 4168.19 604 harvested 0084 4168.20 604 harvested 0146 4168.21B 604 AB discarded 4168.22B 604 harveRted 0173 4168.25B 604 harvested 0047 4168.29 604 died SUBSTITU r~ ET (RULE 26) W O 96/07742 2 1 9 8 7 0 8 PCTrUS95/07233 ~

;

Plant Inbred Exp.line Construct Ploidy Statuæ R1 Seed _ 4168.33 604 potted 4168.33B 604 potted CA95 4132.6 600 potted CA95 4132.9 600 harve~ted 019 Accordingly, stable transgenic lines have been produced cont~n;ng the ACC antisense constructs.
Further, seed has been harvested from these plants.
-EX~MPLE 18 -ACC oxidase antisense transgene expression was evaluated in a number of Ro and Rl melon plants by Northern blot hybridization. This assay measures -levels of accumulated B. oleracea ACC oxidase antisense RNA. RNA was extracted from transgenic Cucumis melo leaves with the use of an RNA extraction kit (Trireagent) supplied by Molecular Research Center, Inc. (Cincinnati, OH). Total melon leaf RNA was subjected to glyoxalation before separation by agarose gel electrophoresis. After electrophoresis, RNA was pressure blotted onto a Nylon membrane (Hybond N, Amersham) with the use a Stratagene pressure blotter (La Jolla, CA).
Radioactive (32p _ labelled) RNA probe (sense RNA) was synthesized in vitro with t-he use of RNA transcription vectors, for example pGEM-3 (Promega, Madison, WI).
First the coding sequence for B. oleracea oxidase was inserted into the RNA transcription vector pGMM, a modification of pBluescript II SK (+). The pGMM
plasmid harboring the ACC oxidase coding sequence was linearized with BamHI and used as template for sense RNA synthesis in vi tro . Radioactive 32p_ labelled probe was synthesized under the following reaction conditions: 2 ~g linearized template DNA, T3/T7 buffer S~8ST~lES~ R~263 W 096/07742 2 1 9 8 7 ~ 8 PCTrUS95/07233 (lX) (BRL), 10 ~ ~2P-UTP, 10 mM dithiothreitol, 2 ul RNAsin (Promega, Madison, WI), 2 mM ATP, CTP, and GPT
and 1 mM UTP, and 1 ~1 T7 RNA polymerase (BRL) in a 50-~1 total reaction volume. Blots were hybridized at 65C with the use of Megablock (Cel Associates, Houston, TX) and instructions provided with the Megablock reagent. Following hybridization blots were washed according to instructions provided with Megablock reagent. Hybridization signals were detected by autoradiography. The results are summarized in Table II and Table III.

Table II -- SUMMARY OF Ro PLANT RNA BLOT RESULTS

Species Binary Gene Construct ~Plant Tran-script?
Melon CA10 pEPG 604 EFE cCNA fl AS 4168-33 (as-) 4168-llB (as+) 4168-18 (as+) 4168-19 (as-) 4168-25B (a8-) 4168-35 (as-) 4168-19 ~a~-) 4168-10 (as+) 4168-20 (a8+) 4168-15B (a8+) SUBSTITUTE SHEET (RULE 26) W O 96/07742 2 1 9 8 7 08 PCTIUS95/07233 ~ _ -TABLE III - SUMMARY OF Rl PLANT RNA BLOT ANALYSIS
Species Binary Gene ~Plant Tran- NPTII
Construct ~cript?
Melon CA10 pEPG 604 EFE cCNA fl 4168-10-1 (a8-) AS
4168-10-2 (a~-) +
4168-10-3 (a~-) -4168-10-4 (as-) -4168-10-5 (as+) +
4168-10-6 +
4168-10-7 (a~+) +
4168-10-8 (as+) +
4168-10-9 +
4168-10- (as+) 4168-19- +

4168-20-1 (as+) +
4168-20-2 (as+) +
4168-19- +

4168-19- +

4168-20-3 +
4168-20-4 +
4168-20-5 (as+) +
4168-20-6 (as+) +
4168-20-7 (as+) +
4168-20-8 (as+) +
4168-20-9 ~a~+) +
4168-20- (a~+) +

4168-20- (a~+) +
4168-20- (a8+) +

4168-20- (as+) +

4168-20- (a8+) +
4168-20- (as+) +

SUBSTITUTE SHEET (RULE 26,) ~ W096/07742 2 1 9 ~ 7 u 8 PCT~S95/07233 RNA blot analysis o~ melon plants transgenic ~or the B.
oleracea ACC oxidase antisense construct in pEPG604 shows accu~ulation of ACC oxidase antisen~e RNA
(Figures 6, 7, and 8). For example, transgenic ~ melon plants 4168-18, 4168-10, 4168-20, and 4168-21 accumulate substantial levels o~ ACC oxidase antisense ~ranscript (Figure 6 and Table II).

Figure 7 shows an autoradiogram o~ RNA blot o~ total RNA extracted ~rom Ro transgenic melon plants (leaves) hybridized with B. oleracea ACC oxidase sense RNA probe (approximately 50 x lo6 cpm 32P-labelled RNA probe). RNA
extracted from melon plants transformed with virus coat protein cassettes and RNA extracted ~rom red cabbage plants trans~ormed with pEPG604 are also included Approximately 10 ug total plant RNA was loaded in each well. Lane 1, RNA MW Markers; lane 2, melon line CA10 trans~ormed with pEPG328 (virus coated prctein cassettes); lane 3, melon line CA40 transformed with pEPG328; lane 4, line 4168-llB; lane 5, line 4168-18;
lane 6, 4168-19; lane 7, melon line 626 transformed with pEPG212 (~irus coat protein cassettes); lane 8, CA10 melon nontransgenic control; lane 9, 4168-10; lane 10, 4168-20; lane 11, 4168-21; lane 12, 4168-15B; lane 13, red cabbage transgenic line 604-30 transformed with PEPG604; lane 14,, nontransgenic red cabbage; lane 15, B. oleracea ACC oxidase antisense RNA synthesized in vitro; and lane 16, B.oleacea ACC oxidase sense RNA
synthesized in vitro. Number 4168 re~ers to melon line CA10 trans~ormed with PEPG604 (see Table II for details).

. This result strongly indicates that B. ol~racea ACC
oxidase antisense constructs are actively transcribed a~ter being trans~erred into melon.

SVBSTITVTE SI~EET (RlJLE 26) W096/07742 2 1 9 8 7 0 8 PCT~S95/07233 ~

RNA blot analysis of R~ progeny of 4168-lO, 4168-19, and 4168-20 shows that some progeny accumulate ACC oxidase antisense RNA to high levels, and others accumulate lower levels o~ antisense RNA (Figures 7 and 8 and Table III).
-Figure 7 shows an RNA blot of total RNA extracted fromRl transgenic melon progeny o~ line 4168-10 hybridized with B. oleracea ACC oxidase sense RNA prG~e (about 50 x 106 cpm 32P-labelled RNA probe). Approximately 10 ug total RNA was electrophoresed in each lane. Seed taken from a ~ruit produced on ~ plant 4168-10 was germinated and RNA samples were extracted ~rom seedlings for analysis. Lane 1, RNA MW markers; land 2, melon line CA10 transformed with pEPG328; lane 3, _ 4168-10-1; lane 4, 4168-10-2; lane 5, 4168-10-3; lane 6, 4168-10-4; lane 64168-10-4; lane 7, 4168-10-5; lane 8, CA10 transformed with pEPG196; lane 9, 4168-10-6;
lane 10, 4168-10-7; lane 11, 4168-10-8; lane 12, 4168-10-9; lane 13, 4168-10-11; lane 14, 4i68-18 Ro; lane 15, B. oleracea ACC oxidase antisense RNA synthesized in vitro; and lane 16, B. oleracea ACC oxidase sense RNA
synthesized in vitro. Number 4168 refers to melon line CA10 trans~ormed with PEPG604 (see Table II ~or details).
-Figure 8 shows an RNA blot o~ total RNA extracted ~romRl transgenic melon progeny of lines 4168-19 and 4168-20 hybridized with B. oleracea ACC oxidase sense RNA
probe. Electrophoresis and hybridization conditions were similar to conditions used in Figures 3 and 4.
Seed taken from produced on ~ plants 4168-19 and 4168--20 was germ;n~ted and RNA samples were extracted from seedlings for analysis. Lane 1, RNA MW markers; ~ane 2, CA10 transformed with PEPG328; lane 3, 4168--19-12;
lane 4, 4168-20-1; lane 5, 4168-20-21ane 6, 4168-19-13;
lane 7, 4168-19-14; lane 8, 4168-20-3; lane 9, 4168-20-SUBSTlTUTE SHEET (RULE 26,~ -~ W096/07742 2 1 ~ 8 7 ~ 8 PCT~S95/07233 4; lane 10, 4168-20-5; lane 11, CA10 transformed with pEPG208; lane 12, 4168-20-6; lane 13, 4168-20-7; lane 14, 4168-20-8; lane 15, 4168-20-9; lane 16, 4168-20-10;
lane 17, 4168-20-11; lane 18, 4168-20-12; lane 19, 5 4168-20-13; lane 20, 4168-20-14; lane 21, 4168-20-15;
lane 22, 4168 - 18 Ro; lane 23, B. ol eracea ACC oxidase antisense RNA synthesized in vitro; and lane 24, B.
oleracea ACC oxidase sense RNA synthesized in vitro.
Numbers 4168-19 and 4168-20 refer to melon line CA10 transformed with PEPG604 (see Table II for details).

These results demonstrate clearly that the transgene is heritable and that it produces antisense RNA in R
progeny.
It is highly unlikely that the hybridization signals shown in Figures 6, 7, and 8 result ~rom non-speci~ic hybridization. Each RNA blot included an antisense and sense in vitro transcript of ACC oxidase (for example, lanes 15 and 16, respectively, in Figures 6 and 7).
ACC oxidase sense RNA in vi tro transcript probe hybridized specifically with antisense i~ vi tro transcript (for example, ~ee Figures 6 and 7, lanes 15 and 16). The sense RNA transcript probe did not hybridize with blotted antisense transcript (Figures 6, and 7, lane 16).

Hybridizations signals produced in RNA extracted from nontransgenic red cabbage, melons, and broccoli were compared with RNA extracted from pEPG604-transformed red cabbage melons, and broccoli. Only RNA samples extracted from transgenic plants produced an ACC
oxidase antisense signal (for example, Figure 6, lanes 13 and 14).
The mobility of ACC oxidase antisense transcripts produced from the cassette in pEPG604 (ACC oxidase full SUBSTITUTL SHEET (RULE 26~) 2 1 ~8 708 --W096/07742 PCT~S95/07233 length antisense) were also compared with transcripts produced from the cassette in pEPG608 (ACC oxidase truncated antisense) following transformation into red -cabbage. ACC oxidase transcripts detected in red _ cabbage plants transformed with the full iength construct are longer than the transcripts detected in red cabbage plants trans~ormed with the truncated ACC
oxidase construct. This result demonstrates conclusively that the sense RNA problem is detecting only ACC oxidase antisense RNA transcripts.

These results demonstrate that only antisense RNA
transcribed by the B. oleracea ACC oxidase transgene in the plant is being detected by the 32p_ labelled RNA
probe.

Lack of detectable ACC oxidase antisense accumulation does not indicate that the transgene will be ine~ective in inhibiting ethylene biosynthetic pathway gene expression. Published results indicate that the degree of endogenous sense RNA reduction is not related to levels of antisense RNA accumulation (for example, see Stockhaus et al., 1990). Endogenous melon ACC
oxidase mRNA is produced in transgenic lines.
Melon, red cabbage, and broccoli plants transformed with pEPG610 and pEPG612 are analyzed in the.same way.
These binary plasmids include ACC synthase antisense RNA constructs. The analysis includes Northern analysis to evaluate B. oleracea ACC synthase antisense RNA accumulation and reduction in levels of endogenous ACC synthase antisense RNA accumulation and reduction in levels of endogenous ACC synthase sense RNA levels.
The analysis shows expression of RNA in these plants.
While specific embodiments of the invention have been described, it should be apparent to those skilled in SUBSTITUTE SHEET (RULE 26?

W096/07742 2~ 8 7 3 ~ PCT~Sg~/07233 the art that various modifications thereof can be made without departing from the true spirit and scope of the invention. Accordingly, it is intended that the following claims cover all such modifications with the full inventive concept.

SUBSTITUTE SHEET (RULE 26) W096/07742 PCTIUS9~/07233 ~ ---A. One of our first goals was to determine whether our ACC oxidase constructs produce antisense RNA in a transgenic situation. To answer this question, we transformed ACC oxidase constructs into red cabbage.
5 Transgenic red cabbage lines were generated with the use of the following binary plasmids; pEPG600, 604, 606, and 608. We verified the transgenic status of ~
many of the plants by NPTII ELISA and PCR analysis of _ the ACC oxidase transgene. These are summarized in the Tables.

B. Next we isolated, electrophoresed, and blotted total RNA by methods described in the melon ACC oxidase disclosure. Antisense ACC oxidase RNA transcripts were detected in RNA extracted from plants transformed with pEPG604 and 608 (see Tables).

C. We next verified unambiguously that hybridization signals detected in t~tal RNA of red cabbage Ro transgenics correspond to Brassica oleracea ACC oxidase antisense messenger RNA. We analyzed cabbage Ro plants transformed with pEPG60~ (ACC oxidase full-length CDNA
AS cassette) and plants transformed with pEPG608 (ACC
oxidase truncated cDNA AS cassette). We electrophoresed both "604" and "608" transgenic RNAs on the same gel to compare mobilities of transgene messages produced by the full length and the truncated genes. The resulting blot clearly shows smaller messages in the "608" transgenic RNA's and longer messages in the "604" RNA's. This hybridization result can only be explained by expression of ACC oxidase antisense genes.

SUBSTITUTE SHEET (RULE 26 PCT~S95/07233 ~ W096/07742 2 1 9 g 7 3 ~

D. Brocoli plants transgenic for ACC oxidase constructs have also been obtained. These include the following lines:
Transgenic pEPG
Line Number Construct Status 173-10 604 potted 133-1 604 potted 173-50 604 potted 173-40 604 potted 173-20 604 potted 133-19 604 potted 224-55 604 potted 238-33 604 potted 294-77 600 potted 287-68 600 potted 294-99 600 potted 238-6 604 potted 266-7 604 potted 133-22 604 potted 294-45 600 potted 224-81 604 potted 290-9 600 potted 224-62 604 potted 133-14 604 potted 294-27 600 potted 287-67 600 potted 294-53 600 potted 294-84 600 potted 294-88 600 potted 238-77 604 potted 287-72 600 shoots 238-77 604 shoots 294-144 600 shoots 294-35 600 shoots 294-3 600 shoots 287-36 600 shoots 287-123 600 shoots 294-122 600 shoots 294-109 600 shoots 294-4 600 shoots 294-47 600 shoots SUBSTITUTE SHEET (RULE 26,) WO 96/07742 2 1 9 8 7 0 8 PC7.`1US95/07233 ~

TRANSGENIC RED-CABBAGE EVALUATION
Germ-line:~16)NC9317424 Gene construct: pEPG606 NPTII
5Transformant# PCR-Gene ELISA PCR presence RNA Transcript 606-1 0.329 blot 11:??
606-2 1.298 blot 12: degraded RNA
606-3 1.028 blot 12:??

SUBSTITUTE SHEET (RULE 26~ _ ~ W 0 96/07742 2 ~ ~8 708 PC~rrUS95/07233 TRANSGENIC RED-CABBAGE EVALUATION
Germ-line: (15)NC9317405 Gene construct: pEPG608 NPTII
5Transforman~# ELISA PCR presenceRNA Transcript 608-1 1.398 blots 7 & 12: AS+
608-2 0.334 no RNA
608-3 1.776 blot 7: AS+
608-4 1.649 blot 7: AS+
10 608-5 1.651 blot 6 608-6 1.681 not tested 608-7 1.924 blot 13: AS+
608-8 1.743 no RNA
608-9 1.909 no RNA
15 608-10 1.210 no RNA
608-11 1.555 blot 12: AS +
608-12 0.007 blot 13: AS +
608-13 0.828 blot 13: AS+
608-14 1.892 no RNA
2 0608-15 1.725 not tested 608-16 blot 12:AS +
608-17 blot 13:AS +

SUBSTITUTE SHEET (RULE 26) WO 96/07742 2 1 9 8 7 0 8 PCTIUS95/07233 ~ _ TRANSGENIC RED-CABBAGE EVALUATION
Germ-line: (4)PC929090 Gene construct: pEPG600 NPTII
5Transformant# ELISA PCR presenceRNA Transcript 600-1 1.545 blot 12: AS-600-2 1.472 blot 12: AS-600-3 1.792 no RNA
600-4 1.801 no RNA
10600-5 not not tested tested 600-6 not not tested tested -SUBSTITUTE SHEET (RULE 26) --2 1 ~8708 TRANSGENIC RED-CABBAGE EVALUATION
Germ-line: ~16)NC931724 Gene construct: pEPG604 NPTII
5Transformant# PCR-Gene ELISAPCRpresence RNA Trdnsc,i,u 604-1 1.300 - - blot 8: AS+
604-2 0.557 + + blot 9: AS?
604-3 0.573 + + blot 9: AS+
604-4 0.757 + +
10604-5 0.973 + + blot 8: AS+
604-6 0.670 + + blot 8: AS?
604-7 1.041 + + blots 8 & 13: AS?
604-8 1.632 + + blot 9: AS?
604-9 1.406 + + blot 9: AS+
15604-10 1.007 + + blot 8: AS +
604-11 1.131 + + .blot 9: AS+
604-12 0.552 - - blot 9: degraded RNA
604-13 A,B 1.125+ ++ + blot 9: AS+
604-14 1.004 + + blot 11: degraded 20604-15 1.153 - - blots 6 & 7: AS+
604-16 1.291 + + blot 11: degraded RNA
604-17 0 043 +
604-18 0.277 + + blot 8: AS+
604-19 1.329 - - blot 8: AS +
25604-20 0.911 + + blot 10: AS+
604.21 1.479 + + blot 10: AS+
604-22 1.535 + + blot 10: AS-604-23 1.486 + + blot 13: degraded RNA

604-24 1.037 + + blot 10: AS-SUBSTITUTE SHEET (RULE 26~

WO 96/07742 2 1 9 8 7 0 8 PCT/US95/07233 ~ _ TRANSGENIC RED-CABBAGE EVALUATION
Germ-line: (16)NC931724 Gene construct: pEPG604 Transformant# NPTII PCR-Gene ELISA PCRpresenceRNA Transcript 604-1 1.300 - - blot 8: AS +
604-26 1.556 + +blot 13: AS?
604-26 1.704 + +blots 10 & 11: AS+
604-27 1.537 + +blot 12: AS+
604-28 1.293 ~- +blots 6 & 7: AS+
604-29 1.702 - -blots 6 & 11: AS +
604-30 1.178 ~ tblots 6 & 7: AS+
604-31 1.810 + +blot 10: AS+
604-32 1.575 + + not tested 604-33 1.597 ~ + blot 10: AS-604-34 + +blot 11: degraded RNA
604-35 - -blots 6, 7, 11, 15: AS+
604-36 + +blot 10: AS+
604-37 - + blot 10: AS-604-38 + + not test 604-39 ~- + no RNA
604-40 not not tested tested 604-41 not not tested tested -SUBSTITUTE SHEET (RULE 26)

Claims

IT IS CLAIMED:

1. A DNA isolate comprising a DNA sequence encoding Brassica oleracea ACC oxidase polypeptide.

2. An isolated nucleic acid comprising a portion of the Brassica oleracea genome which encodes a Brassica oleracea protein, wherein said nucleic acid comprise a nucleotide sequence selected from the group consisting of:
the nucleotide sequence as shown in SEQ ID NO:2;
the nucleotide sequence as shown in SEQ ID NO:8;
and the nucleotide sequence which encodes the same sequence of amino acids as encoded by the nucleotide sequence shown in SEQ ID NO:2.

3. A plant transformation vector comprising a nucleotide sequence as recited in claim 2 a promoter, and a polyadenylation signal, wherein said promoter is upstream and operably linked to said nucleotide sequence, and said nucleotide sequence is upstream and operably linked to said polyadenylation signal.

4. A plant transformation vector according to claim 3 wherein said promoter is Cauliflower mosaic virus 35S
promoter.

5. A plant transformation vector according to claim 4 wherein said polyadenylation signal is the polyadenylation signal of the cauliflower mosaic CaMV
35S gene.

6. A bacterial cell comprising the plant transformation vector of claim 5.

7. A bacterial cell 6 in which said bacterial cell is selected from the group consisting of an agrobacterium tumefaciens cell and an agrobacterium rhizogenes cell.

8. A transformed plant cell comprising the plant transformation vector of claim 3.

9. A transformed plant cell of claim 8 further including the 35S promoter of cauliflower mosaic virus and the polyadenylation signal of the cauliflower mosaic 35S gene.

10. A transformed plant selected from the species Brassica oleracea L. comprising transformed cells of claim 9.

11. A transformed plant seed comprising the plant transformation vector of claim 3.

12. A transformed plant seed of claim 11 further including the 35S promoter of cauliflower mosaic virus and the polyadenylation signal of the cauliflower mosaic 35S gene.

13. A transgenic plant selected from the species Brassica oleracea L. comprising transformed cells of claim 12.

14. A transgenic plant containing a transgene comprising a mutant ACC oxidase DNA sequence.

15. A seed from a transgenic plant of claim 14.

16. A cell from a transgenic plant of claim 14.

17. A method of producing a recombinant Brassica oleracea ACC oxidase polypeptide comprising:

(a) providing a cell transformed with DNA encoding a Brassica oleracea ACC oxidase polypeptide positioned for expression is said cell;

(b) culturing said transformed cell under conditions for expressing said DNA, and (c) isolating said recombinant Brassica oleracea ACC
oxidase polypeptide.

18. A method of inhibiting an ethylene-inducible event in a plant comprising:
providing a transgenic plant with DNA encoding a Brassica oleracea ACC oxidase polypeptide positioned for expression in a cell of said plant; and culturing said transgenic plant under conditions for expressing said DNA.

19. The method of claim 18, wherein said ethylene-inducible events comprise maturation or senescence.

20. A substantially pure Brassica oleracea ACC oxidase polypeptide.

21. A DNA isolate comprising an antisense DNA sequence complementary to a DNA sequence encoding Brassica oleracea ACC oxidase polypeptide.

22. An isolated nucleic acid comprising a portion of the Brassica oleracea L. genome which encodes a Brassica oleracea L. protein, wherein said nucleic acid comprises an antisense nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of:

the nucleotide seguence as shown in SEQ ID NO:2;
the nucleotide sequence as shown in SEQ ID NO:8;
and a nucleotide sequence which encodes the same sequence of amino acids as encoded by the nucleotide sequence shown in SEQ ID NO:2.

23. A plant transformation vector comprising the DNA
of claim 22, a promoter, and a polyandenylation signal, wherein said promoter is upstream and operably linked to said antisense nucleotide sequence, and said antisense nucleotide sequence is upstream and operably linked to said polyandenylation signal.

24. A plant transformation vector according to claim 23 wherein said promotor is Cauliflower mosaic virus CaMV 35S promoter.

25. A plant transformation vector according to claim 24 wherein said polyandenylation signal is the polyandenylation signal of the cauliflower mosaic CaMV
35S gene.

26. A bacterial cell comprising the plant transformation vector of claim 25.

27. A bacterial cell of claim 26 in which said bacterial cell is selected from the group consisting of an Agrobacterium tumefaciens cell and an agrobacterium rhizogenes cell.

28. A transformed plant cell comprising the plant transformation vector of claim 23.

29. A transformed plant cell of claim 28 further including the 35S promoter of cauliflower mosaic virus and the polyandenylation signal of the cauliflower mosaic 35S gene.

30. A transgenic plant selected from the species Brassica oleracea L. comprising transformed cells of claim 29.

31. A transformed plant seed comprising the plant transformation vector of claim 23.

32. A transformed plant seed of claim 31 further including the 35S promoter of cauliflower mosaic virus and the polyandenylation signal of the cauliflower mosaic 35S gene.

33. A transgenic plant selected from the species Brassica oleracea L. comprising transformed cells of claim 32.

34. A method of inhibiting an ethylene-inducible event in a plant comprising:
providing a transgenic plant with DNA encoding a Brassica oleracea ACC oxidase polypeptide positioned for expression in a cell of said plant, where said DNA
comprises an antisense nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of:

the nucleotide sequence as shown in SEQ ID NO:2;
the nucleotide sequence as shown in SEQ ID NO:8;
and the nucleotide sequence which encodes the same sequence of amino acids as encoded by the nucleotide sequence shown in SEQ ID NO:2; and culturing said transgenic plant under conditions for expressing said DNA.

35. The method of claim 34, wherein said ethylene-inducible events comprise maturation or senescence.