Proc. Natl. Acad. Sci. USAVol. 93, pp. 3487-3491, April 1996Applied Biological Sciences

Transgenic barley expressing a protein-engineered, thermostable(1,3-1,4)-(3-glucanase during germination

(Hordeum vulgare/codon usage/malt production/animal feed)

LISBETH GATH JENSEN*, OLE OLSEN*, OLIVER KOPSt, NORBERT WOLFt, KARL KRISTIAN THOMSEN*,AND DITER VON WETTSTEIN*t§*Carlsberg Laboratory, Department of Physiology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark; tWeissheimer Research Laboratory,Department of Biotechnology, Schaarstrasse 1, D-56626 Andernach, Germany; and *Departments of Crop and Soil Sciences and Genetics and Cell Biology,Washington State University, Pullmann, WA 99164

Contributed by Diter von Wettstein, December 14, 1995

ABSTRACT The codon usage of a hybrid bacterial geneencoding a thermostable (1,3-1,4)-13-glucanase was modifiedto match that of the barley (1,3-1,4)-p-glucanase isoenzymeEl gene. Both the modified and unmodified bacterial geneswere fused to a DNA segment encoding the barley high-pIa-amylase signal peptide downstream of the barley (1,3-1,4)-,3-glucanase isoenzyme EII gene promoter. When introducedinto barley aleurone protoplasts, the bacterial gene withadapted codon usage directed synthesis of heat stable (1,3-1,4)-p8-glucanase, whereas activity of the heterologous enzymewas not detectable when protoplasts were transfected with theunmodified gene. In a different expression plasmid, the codonmodified bacterial gene was cloned downstream of the barleyhigh-pI a-amylase gene promoter and signal peptide codingregion. This expression cassette was introduced into imma-ture barley embryos together with plasmids carrying the barand the uidA genes. Green, fertile plants were regenerated and-75% of grains harvested from primary transformants syn-thesized thermostable (1,3-1,4)-p3-glucanase during germina-tion. All three trans genes were detected in 17 progenies froma homozygous T1 plant.

The (1,3-1,4)-13-glucans from barley (Hordeum vulgare L.) arelinear polysaccharides consisting of glucose units joined by(1,3)-43 and (1,4)-,B glycosidic linkages (1-3). These polymersare the major constituents of barley endosperm cell walls (4)and their degradation is a prerequisite for the enzymaticmobilization of endosperm storage components, which serveas nutrients for the growing embryo. Efficient degradation ofendosperm cell walls is also important for utilization of barleyas a monogastric animal feed (5, 6) and in industrial processessuch as malting and brewing (7). Furthermore, extraction ofnon-food products deposited in the endosperm of transgenicbarley would be facilitated by the action of highly efficient,heat stable cell wall-degrading enzymes. The (1,3-1,4)-3-glucanases (EC 3.2.1.73) synthesized by the aleurone andscutellum tissues during barley grain germination (8) aresusceptible to irreversible thermoinactivation at temperaturesabove 55°C (9, 10), which may result in incomplete degradationof cell wall 13-glucans and limit the utility of barley forindustrial processes unless a thermostable (1,3-1,4)-43-glucanase is present during extraction at elevated temperatures.

Bacillus species synthesize and secrete (1,3-1,4)-,3-glucanases with the same specificity as the barley enzymes-i.e., hydrolysis of (1-4)-j3-glycosidic linkages joining 3-0 sub-stituted glucose units (1, 11, 12) but the bacterial enzymes aremore thermotolerant than their barley counterparts (13).Hybrid (1,3-1,4)-3-glucanases with improved thermostabilityat pH 5.0 have been obtained by intragenic recombination in

vitro (14) between the genes from Bacillus amyloliquefaciens(15) and Bacillus macerans (16)-e.g., H(A12-M)AY13 (H,hybrid; A, amino acid from B. amyloliquefaciens; M, amino acidfrom B. macerans), which exhibits a half-life of >4 h at 70°C(pH 5.0) (17). Computer modeling, using the coordinates ofH(A16-M) (1,3-1,4)-p3-glucanase (18) (Fig. 1 Left), suggestedthat hydrogen bond formation between the spatially close N-and C-terminal B-sheets forms the structural basis for theincreased enzymic stability.The promoter of the barley (1,3-1,4)-f3-glucanase isoenzyme

EII gene-directed transient expression of the chloramphenicolacetyltransferase gene upon transfection of barley aleuroneprotoplasts and showed a gibberellin A3 response (20), but thispromoter did not direct detectable expression of a hybridbacterial (1,3-1,4)-,3-glucanase gene in barley aleurone pro-toplasts. However, transient expression and secretion was

obtained with the same 13-glucanase gene controlled by thebarley low-pI a-amylase gene promoter and the low-pI a-amy-lase signal peptide (21). The codon usage for barley (1,3-1,4)-,3-glucanase isoenzyme EII exhibits strong preference for G orC in the third position (22, 23), resulting in a G+C content of65.9% in the coding region, while such codon bias is notobserved in Bacillus (1,3-1,4)-/3-glucanase genes (15, 16).Modification of a bacterial gene, crylA(b), toward plant genecodon usage increased its expression in planta (24, 25). Toobtain value-added barley lines synthesizing thermostable(1,3-1,4)-p3-glucanase during germination, the codons for hy-brid H(A12-M)AY13 were modified to match those of the geneencoding barley (1,3-1,4)-p1-glucanase isoenzyme EII, and themodified gene was tested for expression with the barley(1,3-1,4)-B1-glucanase isoenzyme EII gene promoter in aleu-rone protoplasts. The codon adapted hybrid gene, clonedbehind the barley high-pI a-amylase promoter and signalpeptide encoding sequences, was introduced into immatureembryo cells and germinating grains from regenerated plantswere analyzed for thermostable (1,3-1,4)-13-glucanase.

EXPERIMENTAL PROCEDURES

Organisms and Materials. Grains ofHordeum vulgare L., cv.

Himalaya (1985 harvest at Washington State University, Pull-man, WA) were used for preparation of protoplasts, andimmature embryos were isolated from cv. Golden Promise.Genomic DNA from cultivars Golden Promise and CarlsbergII was purified according to the procedure by Edwards et al.(26). Escherichia coli cells of strain DH5a (27) (Life Technol-ogies, Grand Island, NY) were used for propagation of plas-mids, which were purified using the Wizard System (Promega).Nucleotide sequence analysis was on an Applied Biosystems

Abbreviations: A, amino acid from Bacillus amyloliquefaciens; M,amino acid from Bacillus macerans; H, hybrid.§To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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* S i 0 6 6 .0i.6st

* c * ,, '6c, , : i ;

~~~~~~~~~~~~~ l;_ I .. . ..& I .. .. S . . 6

_~~~~~~~~~~~~~~~~~~a .5_ _ _. .e

N N d ; i I I i fi i i I i I i I i i I6 i6

___~~~r.U.0.. .o

1 1 _~~~~~ ~ ~ ., . , e - , C a 1 X;

. S ; . L ..3 S , a , .5 e.t6

.0

FIG. 1. Hybrid (1,3-1,4)-p3-glucanases. (Left) Three-dimensional structure of hybrid enzyme H(A16-M) derived from the structure amplitudesand coordinates deposited in the Protein Data Bank (reference 1AYH) by Keitel et al. (18). The peptide backbone of residues originating fromthe B. amyloliquefaciens and B. macerans parental enzymes are shown in blue and red, respectively. Side chains of active site residues Glu-105,Asp-107, and Glu-109 (Glu-129, Asp-131, and Glu-133 in the preenzyme listed on the right) are illustrated with white sticks. Since the parentalB. macerans enzyme adopts a similar conformation (19), only minor differences are expected for H(A12-M)AY13. (Right) Synthetic DNA sequenceused to direct synthesis and secretion of H(A12-M)AY13 in barley. Amino acid sequences of the barley a-amylase signal sequence, B.amyloliquefaciens (1,3-1,4)-j3-glucanase and B. macerans (1,3-1,4)-p3-glucanase are shown on green, blue, and red backgrounds, respectively.Nucleotides that have been changed to those preferred for barley (1,3-1,4)-13-glucanase isoenzyme EII are shown in capital letters. Of 215 codonsspecifying the reading frame for the mature enzyme, 141 (corresponding to 66%) have been changed.

model 373A nucleotide sequencer. Oligonucleotides weresynthesized on a model 380B synthesizer (Applied Biosys-tems). For PCRs Taq enzyme (Perkin-Elmer/Cetus) was usedaccording to the supplier's recommendations. Genomic DNAwas digested with BamHI when used as template in PCR.Other recombinant DNA techniques were as described (28).Polyclonal antibodies were raised in rabbits against purifiedH(A12-M)AY13 (1,3-1,4)-f3-glucanase produced in E. coli.Plasmid Constructions. The plasmid pEmuGN contains the

uidA (29) gene encoding the ,3-glucuronidase and has beendescribed (30). The plasmid pUBARN carries the bar gene(31). The plasmids pEII-aH(A12-M)AY13-N and pAMY-aH(A12-M)AY13-N carry the unmodified bacterial (1,3-1,4)-f3-glucanase gene fused to the high-pI a-amylase signal peptidecoding sequence under control of the promoters from barley(1,3-1,4)-j3-glucanase isoenzyme EII and high-pI a-amylasegenes, respectively. These and the plasmids carrying the modi-fied gene [pEII-aH(A12-M)AY13-GC-N and pAMY-aH(A12-M)AY13-GC-N] will be described elsewhere.

Transfection ofAleurone Protoplasts. Aleurone protoplastswere prepared (32) and transfected with 50 ,ug of plasmidDNA by PEG-mediated DNA uptake (33). Gibberellin A3 ata final concentration of 1 ,tM was included in all experimentsand incubation was for 65 or 110 h at room temperature.Protoplasts were removed by centrifugation at 1000 x g for 5min and the supernatant was assayed for (1,3-1,4)-,3-glucanaseactivity.

Plant Transformation, Selection, and Regeneration. Mediaused for tissue culture and plants were callus induction me-dium (CIM); plant growth medium (PGM), which is CIMwithout hormones added as described (34); as well as thehormone-free FHG medium (35). Plantlet induction medium(PIM) is CIM in which the auxin 3,6-dichloro-o-anisic acid(Dicamba) is replaced by the cytokinin 6-benzylaminopurine

(1 mg/ml). The selective agent bialaphos (Meiji Seika KaishaLtd., Tokyo) was used in the concentrations suggested by Wanand Lemaux (34).A 1:1:1 mixture of linearized pEmuGN, pUBARN, and

pAMY-aH(A12-M)AY13-GC-N was introduced into imma-ture embryos by particle bombardment using a DuPont 1000He device. After passage through the selection procedure (34)embryogenic clusters from uniformly growing callus lines weretransferred to PIM, and after 10-14 d in darkness pieces ofcallus were transferred to hormone-free FHG medium andexposed to light. When plantlets reached a size of 1-1.5 cm,they were transferred to PGM in culture cylinders for furtherdevelopment and finally transferred to soil and grown to maturity.For rapid propagation, immature embryos from mother

plants (To) and their offspring (Ti and T2) were allowed togerminate in darkness with the scutella facing downward onFHG medium without the selective agent. Green seedlingswith well developed roots were transferred to soil and grownto maturity in the greenhouse. Mature grains from putativetransgenic plants were surface sterilized and left to germinatein darkness on sterile, humidified filter paper at 10°C. Whencoleoptiles were '10 mm, the germinating grains were trans-ferred to room temperature (low light conditions) for furtherdevelopment for 1-2 d before separation of the seedlings fromthe grains. Residual grain material was ground in liquidnitrogen and extracted with 50 mM 2-(N-morpholino)ethane-sulfonic acid/5 mM CaCl2 using 500 ,ll per grain. Extracts werekept on ice for 30 min with occasional mixing followed bycentrifugation at 15,000 x g for 10 min. Supernatants werestored at -20°C.Enzyme Assays. (1,3-1,4)-/-Glucanase activity was deter-

mined by the method of McCleary (36) using 200 ,ul ofazobarley f3-glucan substrate. Analysis of protoplast superna-tants was in 50 mM sodium acetate, pH 6.0/5 mM CaCl2 and

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incubation was at 56°C. Determination of bacterial (1,3-1,4)-P3-glucanase activity in extracts from germinating grains was in50 mM BisTris (pH 7.4) at 65°C, and homologous barley(1,3-1,4)-43-glucanase activity was determined in 50 mM so-dium acetate (pH 4.5) at 30°C. Samples to be analyzed wereincubated for 10 min in 200 ,il of buffer before addition ofsubstrate and then further incubated for 30 min. Reactionswere stopped by addition of 1 ml of precipitation solution (36).Samples were centrifuged at 5000 x g for 3 min and theabsorbance at 590 nm was measured. In this report, 1 unit of(1,3-1,4)-/3-glucanase is defined as the amount of enzyme thatin the described assay results in an A590 of 1.0. One unit ofH(A12-M)AY13 corresponds to 8 ng of protein.

Analysis of Proteins from Transfected Protoplasts andGerminating Grains. Proteins were separated by SDS/PAGE,transferred to nitrocellulose membranes, and probed withantibodies (37-39). Grain extracts were analyzed by isoelectricfocusing using IsoGel agarose plates (FMC) with a separationrange from pH 3 to 10. Focused proteins were transferred tonitrocellulose filters and probed with the antibodies. Alterna-tively the focusing gel was overlaid with a 1% agarose gelcontaining 0.5% lichenin and incubated at 55°C for 1 h.Undigested lichenin was stained with Congo Red (40) andclear zones of ,3-glucanase activity were revealed.

RESULTS AND DISCUSSIONEffect of Codon Modification. Three different protoplast

preparations were transfected with the (1,3-1,4)-j3-glucanaseencoding plasmids pEII-aH(A12-M)AY13-N and pEII-aH(A12-M)AY13-GC-N. In experiment B (Fig. 2), seven wellscontaining protoplasts transfected with the codon adapted,G+C-rich construct gave an average production of 40 ng(1,3-1,4)-p3-glucanase per 2 x 105 protoplasts (s = ± 18 ng)after incubation for 110 h, while no (1,3-1,4)-13-glucanaseactivity was detectable in any of the six wells containingprotoplasts transfected with pEII-aH(A12-M)AY13-N. Twodifferent protoplast preparations were incubated for 65 h upontransfection with (1,3-1,4)-j3-glucanase encoding plasmids. In12 wells, protoplasts were transfected with the high G+Cconstruct, giving an average production of 30 ng per 2 x 105protoplasts (s = ± 12.8 ng), whereas no (1,3-1,4)-p3-glucanaseactivity was detected when the nonmodified gene was used fortransfection (Fig. 2B). Furthermore, in all experiments per-

A 1kDa36.8 -

27.7-

formed, essentially all (1,3-1,4)-j3-glucanase activity was in thesupernatant and only negligible amounts were associated withthe pelleted protoplast fraction.

Proteins from aleurone protoplasts, transfected with pEII-aH(A12-M)AY13-GC-N were separated by SDS/PAGE,transferred to nitrocellulose membranes, and probed withantibodies. Fig. 2A shows the reaction with proteins fromtransfected protoplasts (lanes 4 and 5). Three bands of differ-ent Mr react with the antibodies, indicating that the (1,3-1,4)-,3-glucanase has been exported via the secretory system re-sulting in an array of glycoforms as encountered in yeastexpression studies (41, 42) (lane 2). The protein band with thelowest Mr (24,000) comigrates with (1,3-1,4)-f3-glucanase fromE. coli (lane 1).

Plant Transformation and Regeneration. The plasmidpAMY-aH(A12-M)AY13-GC-N encoding heat stable (1,3-1,4)-,3-glucanase was introduced into immature barley em-bryos together with plasmids carrying the bar and uidA genes.In 10 experiments, 293 bisected and 45 whole embryos werebombarded. Twenty-two lines survived the selection proce-dure and the callus obtained grew well on bialaphos-containing medium. Long-term resistance was never observedwith wild-type tissue. Two of the transformation experimentsgave a total of 14 green plants. Four were from experiment 6(37 half embryos) and 10 were from experiment 8 (17 wholeembryos). These plants were morphologically indistinguish-able from wild-type Golden Promise plants grown underidentical conditions in the green house (Fig. 3).

Analysis of Primary Transformants and Their Offspring.Analysis of the 14 primary transformants by PCR showed thatall plants carried all three genes. An example is shown in Fig.4A. Eight T, plants were obtained by germination of immatureembryos from transgenic mother plant 6.2.2. Six of the off-spring had inherited all three heterologous genes, while theother two did not carry any of them (examples shown in Fig.4B), indicating Mendelian segregation and linkage of theintroduced genes. Immature embryos from one spike of eachof four TI plants were germinated to produce T2 plants. FromTI plant 6.2.2.2, 17 offspring plants were obtained and ana-

2 3 4 5

B1 -.. --

2 ', .,

3

0 10 20 30 40 50 60ng (1,3-1,4)-rI-glucanase

FIG. 2. Heat stable (1,3-1,4)-/3-glucanase from aleurone proto-plasts. (A) Proteins secreted by two independent sets of transfectedaleurone protoplasts were subjected to SDS/PAGE and Western blotanalysis (lanes 4 and 5). H(A12-M)AY13 (1,3-1,4)-p3-glucanase fromE. coli (lane 1), an N-glycosylated preparation (lane 2), and adeglycosylated preparation from yeast (lane 3) were used as standards.(B) Quantitation of heat stable (1,3-1,4)-,3-glucanase synthesized bytransfected aleurone protoplasts. 1, Protoplasts transfected with pEII-aH(A12-M)AY13-GC-N incubated for 110 h; 2, as in 1, but incubationwas for 65 h; 3, protoplasts transfected with pEII-aH(A12-M)AY13-N.

FIG. 3. Spikes of regenerated plants. A spike from transformant8.2.1 (red tag) is compared with a spike from a regenerated wild-typeplant (white tag). Spikes are fully fertile and grains are morphologicallyindistinguishable.

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29 4 t

bp-845- 705~,539509

A

B

C

bp

_972869

bp

-972.539-429

1 2 3 4 5 6 7 8 9 10

FIG. 4. PCR analysis of transgenic barley plants. Genomic DNA ofTo plant 6.2.2 (A), four T1 plants (B), and two T2 plants (C) were usedas templates for amplification of homologous barley sequences [a509-bp fragment spanning bases 3036-3545 of the barley (1,3-1,4)-3-glucanase isoenzyme EII gene (20) (A, lane 1) and bases -679 to + 190of the gene encoding high-pI a-amylase (43) (B, lanes 7-10). DNAsequence analysis has shown that the minor band is due to a deletioncorresponding to -610 to -531]. A heterologous 845-bp fragmentspanning the entire bar and nos sequences (31) was amplified from To(A, lane 4), while a 429-bp fragment, covering the coding region wasobtained from T2 plants (C, lanes 9 and 10). A 539-bp fragment of theuidA gene (29) was identified in To (A, lane 5) and in T2 (C, lanes 6and 7). The gene encoding H(A12-M)AY13 was detected with primersspanning the signal peptide code to the stop codon in To (A, lane 3)and with primers spanning the signal peptide code to the terminatorregion in T1 (B, lanes 3-6) and T2 (C, lanes 3 and 4) giving rise tofragments of 705 and 972 bp, respectively. The H(A12-M)AY13 genecould not be detected in a sister plant segregating as wild type (B, lane1). Lane 2 in B and lanes 2, 5, and 8 in C are plasmid control reactionsfor H(A12-M)AY13 uidA and bar, respectively.

lyzed by PCR, which demonstrated the presence of all threeheterologous genes in all 17 T2 plants (examples shown in Fig.4C), indicating that T, plant 6.2.2.2 was homozygous. Aqueousextracts of germinating grains from primary transformants6.2.1, 6.2.2, and 8.2.1 were assayed for heat stable (1,3-1,4)-f3-glucanase activity. Plants originating from grains producingheat stable (1,3-1,4)-j3-glucanase were positive for the pAMY-aH(A12-M)AY13-GC-N gene. The numbers obtained are inagreement with a Mendelian 3:1 segregation for all threemother plants (Table 1). Protein extracted from leaf, root, andstem tissues of transgenic plants showed no heat stable (1,3-1,4)-f3-glucanase activity. Plants obtained from grains withoutheat stable (1,3-1,4)-,3-glucanase were subsequently analyzedby PCR and the absence of the corresponding genes wasconfirmed in all cases. PCR analysis of T2 seedlings generatedby germination of immature embryos from a single spike of thehomozygous T, plant 6.2.2.2 confirmed the presence of thegene encoding heat stable (1,3-1,4)-j3-glucanase. Aqueousextracts were prepared from 10 germinating grains of this plantand all 10 extracts contained heat stable (1,3-1,4)-,3-glucanase,demonstrating activity of the introduced gene in the secondgeneration after transformation. Extracts of randomly chosentransformed and untransformed grains were analyzed forendogenous barley (1,3-1,4)-/3-glucanase. This enzyme was

Table 1. Segregation analysisTo No. tested Positive Negative

6.2.1 30 20 106.2.2 74 58 168.2.1 25 18 7

present in all grain extracts, irrespective of expression of theheterologous (1,3-1,4)-3-glucanase gene.

Characterization of Heterologous (1,3-1,4)-f3-Glucanase.Aliquots of single grain extracts were analysed by isoelectricfocusing. Fig. 5A shows that heat stable (1,3-1,4)-p3-glucanasefrom a germinating grain of To plant 6.2.1 (lane 2) has the samepI value as a control sample of H(A12-M)AY13 (1,3-1,4)-/3-glucanase produced in E. coli (lane 1). Extract from a wild-typegrain gives no activity zone. Fig. SB shows the reaction offocused proteins with antibodies. Soluble proteins from ger-minating grains of transgenic barley were separated by SDS/PAGE, transferred to nitrocellulose filters, and probed withantibodies. Fig. 5C shows four samples reacting with differentintensities. This difference is in agreement with the activitiesmeasured using azobarley glucan as substrate. The mobility ofthe transgenic (1,3-1,4)-,3-glucanase is slightly decreased incomparison with the enzyme from E. coli. This is most likelydue to N-glycosylation, since there are three sites for N-glycosylation in H(A12-M)AY13. The enzyme from germinat-ing grains appeared as a single band, whereas three majorforms are released from aleurone protoplasts. WhenH(A16-M) is secreted from Saccharomyces cerevisiae twomajor molecular weight classes appear, each representing anarray of different glycoforms (Fig. 2A) (41, 42). Analysis ofbarley (1,3-1,4)-,3-glucanase isoenzyme EII showed that a

seemingly uniform preparation contained four major glyco-forms (44). It is therefore to be-expected that the bacterialenzyme with three sites for N-glycosylation will appear indifferent glycoforms when synthesized in barley.

Conclusion. In this plant breeding project, we endeavouredto produce barley plants that during steeping and germinationexpress a (1,3-1,4)-f3-glucanase that survives the high temper-atures used for kiln drying of green malt. This would allow theenzyme to act in the mash tun as do the thermostablea-amylases from barley, wheat, and rice. Such a heat stable(1,3-1,4)-f3-glucanase synthesized during germination mighteliminate the requirement of complete endosperm wall depo-lymerization in the malting schedule and thereby provide newopportunities for the application of malting and mashing inproduction of conventional and novel biotechnological com-modities. The design of the enzyme with dramatically im-proved thermostability rested on the discovery that hybrid(1,3-1,4)-,3-glucanases obtained by intragenic recombinationcan exhibit molecular heterosis or hybrid vigor (45). The heatstability of the bacterial enzyme was further optimized withreference to the three-dimensional structure of H(A16-M)(18). The improved enzyme has been produced in transgenicE. coli and successfully tested in pilot mashing (14) and feedpellet production (K.K.T., unpublished data). In the present

1 2 3

B

C -

1 2 3 4 5 6

FIG. 5. Analysis of heat stable (1,3-1,4)-p-glucanase from trans-genic barley. (A) Zones of activity after isoelectric focusing andstaining with Congo Red. Lanes: 1, (1,3-1,4)-p3-glucanase from E. coli;2 and 3, extracts from germinating sister grains. (B) Same threesamples were blotted onto nitrocellulose and probed with antibodies.(C) SDS/PAGE and Western blot analysis of extracts from germinat-ing grains. Lanes: 1, (1,3-1,4)-/3-glucanase from E. coli; 2-5, extractswith different amounts of heat stable (1,3-1,4)-/3-glucanase; 6, extractfrom a germinating wild-type grain.

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communication, we report the synthesis of the engineeredthermotolerant (1,3-1,4)-p3-glucanase during germination andthe fidelity of its inheritance in fully fertile transgenic barley.

Dr. S0ren Knudsen is thanked for valuable advice regarding planttransformation and regeneration. This work was supported by a grantto D.v.W. from the Danish Programme for Food Technology, Projekt6028 and by a Ph.D. fellowship to L.G.J. from the Danish AgriculturalScience Research Council.

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