CA2181418A1 - Transgenes with floury2 gene signal peptide - Google Patents

Abstract

A transgene is disclosed that contains a polynucleotide segment coding for a portion of a signal sequence from a floury2 gene of maize linked to an agronomically high-value protein. Cereal plants that contain such a transgene also are disclosed, as are millet or sorghum plants that contain a maize floury2 gene.

Description

~ 2181418 Irlventor (s): ~rian A. 1arkin~
L~rry R. ~
Craig ~. Colemzm ll~ANSGENES W~I~ FLOURY~ ~EN13 SIGNAL ~
P~C~~ OF S~ TNV~n~
The present invention ~-slateb to a L~ th~t contains a polynucleotide fiegm~nt encoding at least a portion of a sign~l se~uenc~ fro~ a floury2 g-ne. For 5 eYample, a transgene within the inv~ntlon al&o cah.
contain a ~ecand segmQnt ~:oding rOr ~n al,~L~ iCIlly high-value protein, such th,~t the l~_ , - - e ~ ~,ies a fusion protein cosprised of' the si~nal s~u~:...,e moiety and a~ino acid sequenc~ of ths high-value protein. ~Ae O present inventlon also rel,~tes to cereal plant3 that contain a transgene, such as millet or 60r~hum plants c~ ihin~ ~ naize or other heterologou~ flou~y2 gene.
~ etween 50~ and 60% of the ~rotein Ln ~aize kernels consi6ts of a D~iYt-~re Or pr~la~ain storago proteins known 15 as "zeins," which are D~ nt;Ally devoid Or lysine. This makes the seed nutritionally inferior for v.,~,~J~ ic Anis~ls The lysiné d~ficiency of saize spurred extensive efforts to id~ntify rutants with higher levels of thi6 e~sential amino acid. The mai2e floL~ry2 ~flZ~
20 mutant ~as first described by Emerson et al., and was reported to re6ult ~rom zl 5D~;~ ihant mutation that causes ~ ~ioft, starchy emiosper;3. CO~N_~ U~ S
AGXICULTORAL EXPRR~MENTAI STA~ON ~PORT 180 (l935~.
~The content~i of this docu~lent and all other6 ~ntioned 25 herein ~re incorpor~ted by ~eferencQ. ~ fl2 allele occurs on the short arm o~ 4, ~nd for ~any years it served as ~ u~eful genetic sarker for the ~hort arm o~ LI ~~ ~ 4.
In lg64, 1'12 ~nd anothl~r ~ut~tion in ~ize, Op~qUGZ
30 ~o2~, took on special in~erest when it wa6 reporte~ that both of theso ~utations leall to a ~ubstantial increase in lysine content of naize geed8. Nelgo- et al., Sci~c~

2'i81418 150; i46~3-7~ 11965), and ?~ert~ e~ ~1., soience l- S:
27g-80 (1964~ . Kernels in normal maize ~Qn~yy 3 average around 0.20 to 0.25~ ly~ine, whlle kernels rrO~ o2 and fl2 mutations hav~ lysine contents of 0.3 to 0.35%. I~ut S the soft starchy endu~ a~sociated ~ith thQ fl2 and o2 ~L~ Ly~ causes ths lc~n~ to be susceptible to r- --nir~ll das~age, whicn cr~ates a higher s~F~eptihi1ity to in~cct and ~ungal da~age. co~s2ciusn~1y, neither mutant gain~d Yide~pread co~ercial applic~tion.
For many years, o2 ancl rl2 were considered to be de~ects of genes reclulating zein synt~e~i~. This conc~usion was based on the significant and fairly specific efiect these r~utations have on storage protein synthesis. Both o2 2nd fl2 reduce zein synthesis by 15 about 50S of the wild type ~evel, with thQ o2 ~utation specifically a2'~ecting the 22-kDd 4-zeins, and th~ ~12 mutation ec~lly affecting synthesis of all classe21 of zelns .
Other distinctiv~ hi~sh~m~r~l di~erences have be2n 20 reported for o~ and fl2 mutant~. Proteln bodies in both o2 and fl2 are smallar than normal, but fl2-encodad protein bodi~s are asy~ettical and m~hAr~r~ co~parad to the spherical protein boc~i~s o~ nor~al and o2 ~I~A~perm.
Lending ~ Larkins, Pli~nt C811 1: 123-133 (1989). ~he o2 25 mutation is recessi~e, ~hile th~ ~12 _utation is semiao~inant, with thc~ seYerit~ of the p~_..o~y~"e correlated to dosage of t~-e ~tant allele.
qh~ hy~othesis that o;! is a zein regulatory qen2 was conf irmed eventually, fc~llo~ing its tagging with a transposable el21nent. Ihis led to the ~ r cloning of 02 and the A ' ~tion that it encc~des a leucine z~ppQr-type tr~n~cription factor that binds th~ yL. ~e.
o~ certain ~--2Qin genes and controls e~cpression o~ tbe 2Z-~cDa fa~ily of c~-zein genes. Schmidt et ~1., Science 23~: 96~-63 (1987). N~erous atte~pt~ to tag rl2 by a ~imilar strat~cly were ~ . ~ce,~rul, and the ba~i~ of the ~12 defect r2mained unknc~-n.

~ 2181418 The failure to discern the nature of t3a ~12 defsct c(~nt~ despite ~airly ext~!nsive study of this ~hutant.
Several studie~ not~d the appearanc~ of an unusual ~-zein protein in rl2 with a nolecu3.ar w~ight of 24 kDa, higher s than normal zein. Lee et A7., Biocham. Genet. ~: 641-50 (1976); Soave et ~ ydic~l 23: 145-5~ (l97a); GalantQ
~t al., ~ol. Gen. G n~t. ~92: 316-21 ~1983). ThQ level o~ this protein w2s found to be dependent on the dosage o~ the f l Z allele . A high ~ lon of b-70, the 10 maize ho~D.ologus of the BiP ch.~peronin, al~o Wd5 reported to be associated with protein botie~ in fl2. Zhanq ~
Boston, Protoplasma 171: 142--52 ~1~93~. 8iP is a ~e~ber of the hsp-70 protein faslily that bindG malfolded polypeptide chdins. The levQl of b-70 is a~ected by the 15 dosage of flZ double m~tants, as is the degree to whic the protein bodieS become ~i~5h~r~". It al2~0 was reported th~t in o2/~12 douo:le 7~utants, thc unu~ual 24-kDa ~-z~in W~5 not synthesize~ and the Dorphology of the protein bodios was ~imilar to that ln o2. ~hus, the o2 20 gene was reported to be epist.atic to rl2. Lopes et ~1., Mol. ~;en. Genst. 2~5: 537-47 (lY94~.
Lopes et al. report~d three ~-~eins protein~ in ~ddition to the abnormal Z4-kDa proteln in thQ storage proteln fraction of fl2, Witll molecular ~.reights ranqing 2~ ~rom ~3~out 25 to 27 kDa~ ~hey also detected 2 restrictioh ~ragment length polymorphism (RPIP~ linked to th~ ~12 locus with a 22-kDa ~-zein 3?robe. They hypothe~ized th~t the characteristiCs ;o~ fl2 might be a response to the Al lAt;on of the defective 24-kDa 30 protein, but were unable to 3?rove that the acc~ ulation of this protein wa6 regpon~ or the rlz ph_.~oty~ ~.
~IMAR~ OF ~
It is sn object o~ the p~esent invention to provide 35 ~ fusion protein Or ~ 21 amino acid Glgnal seguenc- from fl2 ~ith a desired protein.

~ 2 1 8 1 4 1 8 It i8 a ~urther obiect of th~ inventlon to pro~ride plants that contain an r~V~ g DNA sec,~lenc~ comprising this fusion protein, in wh/~:h expre~ision o~ the desired protein i~ increased ln seecls Or the plant.
It also i~ an object Or the present inventian to provide a ~Dethcd Of increasing the content ot Ps~n~
amino acids in an ani.al fe~d vithout ~ppl~ ~nll.
In accompllshing these and other objectives, there has been prcvided~ in ~ccortlance vith on~ aspect of the present inventlon, a cereal plant thzt contains a C.Ipnr compri5ed of ~ first polynucleoeid~ ~eg~ent co~prisinq a nucLeotide seq-lence that encodes the amino acid secuence MATKTTArr1~rTATrvsATNv j~md tii) a second polynucleotide segment coding for a protein. In on~
preferred '~ , polyn~cleotide seg_ent (il) ha~
high content o~ an amino at:id selected from th~ group consisting of _ethionine, ly~ine, LLy~L, ' 'n and threonine, snch th~t the amount o~ said amino aoid in seeds Or said cereal plant i5 increased a~ compared to zo seeds fro_ other~ise idenl:ical plant~ that are not trans~ormed. In another preLerred ~mh~ the fir~t and seccnd pclynuclectide ~egments are cperably linked to a promoter, such as the fl2 prcmoter, SO that s~id cer~l plant ~AiJ~ ._.; both segmQnt:s under the contrcl of said prcmo~er. Pursuant to other . ' ~1 L~, the cereal plant can be a maize plant, wht~re segment (ii) is not native to naize, or can be rice, :rheat, barley, millet cr sorghum, for examplQ.
In acocrdance with cther aspects c~ the pres~nt inventicn, there alsc is provided seed produced by a plant ~s descrlced abcve and a feed product cc_prising ~eal obtained fro~ s~ch seed.
Pur3uant tc ancther aspe~:t of the present in~ention, a transgene is provided that co~prise~ (i) a first pol~nuclectidQ segment ccmpri.sing the nuoleotide seguenc~
Coding ror the amino acid ~eSluence MA~rTr~Tr~rT~rTVSATNV
and ~ii) a second polynuclectidQ seg~ent ccding for a protein. In a prefQrred ~mho~lir , th~ L.e.,,s~n~

~ 2181418 ~dditLonally cocpriDes ~ ~e~ence of fl2 ~!lected fro~
one or bcth of nucleotides 761-3824 and e61~-8335 o~
Figure 1, described in g~-eater d,tail belo,r. In addition, a transgene i5 prolrided that co211pri~-s thn fl2 5 pro~ote~ op-r~bl~ lin~A~ed to a polynucleot$t~ 6egrAent.
other obiects~ features and advantages of the preScnt invention will ~ecome apparent frcm 'ch~ follo~ing detailed de~cription. It shc~uld be understood, however, that the detailed descriptioll and the ~pecific ex~pl~, 10 while ind~catLng preferred ~--'i 1,5 0~ the inYention, re given by way of illustration only, 3ince various changes and modifications wil:hin the spirit and scope of the invention will beco~le aE)parent to t:hosa skilled in the art frosl this detailed dencription.
~3RI~F ~8CRIP~I0~l OF ~ DaAN~lG
E~igure 1 is the nucleotide fiequence of a clone of fl2. Positions 1-760 and 8,336-~0,539 are vector sequences, and positions 761-~3,33 are the coDIplete nucleotide seoyence o~ ~12.
Figure 2 sho~ the ~u~le~n~ sequence and deduced a3nino acid seiuence of th,e coding region of fl2, i nr~ the gignal secuence. Nu~bers on thc left correspond to position of the f irst ~II;ino acid o~ eaoh llne beginning witb -21 to re~lect the signal peptide, so th~t the -1 position is ol:cupied by the C-terminal residue of the signal peptide and the +1 position is occupied oy the first a~ino acid of the predicted ~ature polypeptide .
It has been discovered that a gene encoding a ZZ-kDa ~-ze$n protein, an,d not a regulatory gene, i~ r~vpnr~qihl~
for the fl2 ~utant ph~2not~p~ diAr~lr~n~d above. In ~ -6- 2181418 particular, it has be~n fo~md th/~t the Z4-kDa protein identified in fl2 mutants cn2priass th- amino acid ~:e~uence o~ a 22-k3~ a-zein plus ar; unclQaved, 2~-amino acid signal peptide. I
The ccmpl~tQ nucleotide sequencs of the rlz i9 sb.cwn in Figurc 1, while Figure 2 depictR the nucleotide seguence and deduced amino acid 6equence o~ th~ codinq region of the 24-kDa prote:Ln. Th~ deduc~d amino acid sequence of the sign~l pept~de begins a'c poRltion -21 in Figure 2, so that the -1 })osition iL occupied by the C-terminal residue of the sign~ pQptide and the +1 position is occupied by the first anino aoid o~ the predicted ~ature polypeptide ~he lieq;uence of the signlal peptide is MhrKTTJ~T-A~TAr.rV~AT23V. A co~parison of thi~
deduced U-terminal amino acid sequence of th~ 24-kD~
a-zein protein with other a-~eins haR revealed an alanine to valine substitution at the C-termin~l position of the Signal peptide, a histidine insertion within the sevsnth a-helical repeat, and ~n ~lanine to threonine substitution Yith the same a-helical repeat of the protein. Iihen an alanin~ codon is substituted for ths valine codon Or the mutan~ zein gene, the in vivo translated protein product is procesoed corrsctly in the presence o~ maize microsome~.
The ~ional peptide targets th~ a-zein protein to thq lu~en of the rough ~n~lorlR~mic reticulum (R~. The signal peptide is retainled on the 24-kDa ~-zein p~L~ r; that is, the Z4-kDa a-zein is not pl cessed in l'lZ endosperm. Tho Z4-kDa ~-zein is 'b~lieved to remain anchored to the RE~ me~brane, disrupting the normal h~ n~ of protein bodies. In normal protein body developr~ent, zein protein ~r~ retained within the ER
where they coalesce into spherical bodies in which a-zeins ~re lor:~l;2ed to the interior Or a shell Or cross-linked ~- zmd ~-zein. At~r I o~ an a-~ein to the RER membrane i~hibits its nlovement into the interlor of th~ protein body. Xore particularly, inter~ction o~' h- h~--ttA~hed ~ 1n vitll the ~hu11 of ,~ y-~

,~ 2~814~8 disrupts the spatial or~anl~ation of developing protein oodie~ ~y forlDing multiple foci ~or ~-z~in aggregation n~ar the sur~ace Or thQ ~ ~eDbrane, whic~ th~ irr~gular budding that occurs in fl.2 protein bodiGs. The retention o~ the signal peptide on the Z2-kD4 ~-z~in also provide~
an explanation for the ~ y~ ion o~ 8iP in fl2, since thi~ would afrect the notmal folding Or t~h-prot~in.
The conversion of valine to 21anine in the sign~
peptide of this 3-zein l~rovides an explan~tion for it~
retention on the protein, and rOr 1;2ny o~ the phenotypic e~ects of the fl2 mutation. According to von Neijne'~
"-3- rule" for signal peptides, the -1 positi3n is critical for recognitic~n ~y signal peptidase ~nd i~
gen~rally occupied by ~n uncharged amino ~cid with a s~all side chain. 5~e von Heijne, ~ur. J. ~ioc~e~. 133:
17 -27 ( 198 3 ) .
A point mutation in ~:he signal peptide is consistcnt with the fact that onl~r one fl2 allole has been identiried. A point mutation algo helps to explain the difficulty in conventional ~Ipproaches to tag fl2 by transposon mu~es~cic.
The ~o~plete ~12 sequence contains 7575 b~se pairs, nucleotides 761-8,335 of Figure l. The flZ coding region (open reading fra~e), inrl~ in~ t~e stop codon, comprlse3 nucleotides 3,825-~,613. This sequence has be~n trans~ormed into maize. Transgenic seed that contained t4e gene expressed the fl2, 24-kDa zein, and seed segregAting r~hich did not have the rl2 protein did not have the gene Since the coding region matches the sequence o~ fl2, 24-kDa protein, it is understood th2t the sequence shown in Figure 1 include~ the pro~oter for fl2, Nucleotide~:
oetween nucleotides 761 and al~out 3,8z4 in the sequence Or ErLgure 1 e4code the rlz pro~oter. 6everal motifs comrlon t~ 22-kr)a 2ein promoters are found in this region of the fl2 sequence of Figure 1. For example, locatQd up~tream of the start o~ initiatiot~ is a ~equence 218t418 -a-5 ' -~;~CA~CCAC-3 ' . ~he rirSt nucleotide is at -300 with respect to the start of inltiatl~n. ~is uur.., '- to part of the sequence rsco~Jnized by the 02 gQnR pro~Uct, also locate~ 300 bp upstrea3L of th~ start of initiation, ar ~1 i Crl oq~d in Figura S of Sch~idt et al ., Plant CQ~1 ~: 68 9 ~ 199 2 ~ .
Similarly, Morton et al, rerer to a prolamin-speciric 5 '-TGTAAAG-3 ' motif co3u~on to a~l zein genes of ~aiz~, comn~only rQferred to as the U_300 boY" by virtue o~ its location 300 bp fro~ the start o~ tr~nslation.
"Regula~ion of Seed Stor~ge Protein Gene ~xpr~ssion," in sEEl~ D~vEr3~ENT hND ~ N ~Kigel and Ga~ili, eds. J, Ncw ~ork: ~farcel Dek3cer, Inc. ~1995). A ~.L'~7~J ~in~
sequence ig found at nucleotide 3500 in the fl2 segu~nce o~ Figure 1. Mort~n Qt al. also disclo~a a S~-CATGCATG-3 ' elenent co~Don to r.any ~La ~,. ci~i¢
gqmes. Thi5 sequencQ i91 sirilar to thQ sequence 5'-CATGCG~G-3' of fl2, vhich beqins at nucleotida 3517 in ~igure l The retention o~ the Z4-kl~a prot-in on the RER and its ~ lation in the endosper~ leads to a concollitant decrease in the levels o~ other zein proteins and, hQn~a, to a decreased lcvel of total 6torag~ proteins. The reduction in Sotal storage protein lead~ to the soft, st rchy ph~",~r~e of fl2 and the reductio;n of the zein ~ractiOr, as a percf~ntage of total 3torag~ protein lead~
to an apparent increase in lysine content, sinc~ the otller storage proteins in ~aize, suo~ as globulins and albu~Ding, have higher lys,ine contents.
~he discovery or t~le nature of the fl2 defect provides the basis rOr seed progcny , i ri-l ly ongineered to Qxpress various phL~ y~.. o~ a~, ; r interest. That is, a fusion ~orotein of the 21 a~ino acid ~lqnal GequenC~ from fl2 with a desired polypeptide can 35 ~e u~ed to provide increased ~xpression of the desired polypeptide in seedG of a host plant. ~be r~gion fro~

3, 888-4, 613 ls replaced ~ith the ooding region ~open read~ng rralle) of tha desired polypeptid~.

g Alternatively, rl2 ~equonces upstrea~ ~nd d~ ,~ o~
the coding region a~ ~la can be used to drive expre~sion of a desired polypQptide. In tbis case, the region ~rom 3,825-4,613 in ~12 18 r2placed by the coding rQgion o~
5 the desir~d polypeptide.
A wide variety of polypeptide6 csn be fu6ed to th-rl2 signal seguenc~ and/or to t~e r~gions upstrealD or do~rnstrea~ of th~ flZ coding region, in or~er to achleve higher levels o~ expre~aion andJor accumula~ion o~ the 10 polypeptide ~han might otherwise be attained. The palypeptides may or ~Ry not be native to the plant in which they are being ~i.ee~e.l. IlluYtrative of suc4 polypeptides are short polypeptides o~ less th~n about 40 ~ino acids, polvpeptides which are ~ither unuGually 15 hydrophilic or ~Iy~Jpl~obic or amphip~thic, polypeptides which have unioue solubility characteristlcs, polypeptides which have a unique thre~-dir~ension~l structure, polypeptides ~hich have ~Otir~ which lnclude binding domains, polypepl:ld~ which have either vsry few 20 or many disulfide bonds, polypeptides which have a high content of charged amino ~cidY, polyp~ptide6 ~hich have a particularly high cont~nt of specific amino acids, ~
~stnionine, ly~ine, tL-yptophan or thrsonine, and polypeptides which have been altered rrO:G their native 25 structure such that th~y ~ight otherwis~ not be stable.
DNA coding for the polypeptide may be modified to reflect preferred codon usag~ in the partlcular crop that is the target of the transfor~ation.
A fusion protQin of tlle slgn~l p~aptid~3 with a protein 30 other than 8 zein protein should not afrect the total amount of zein storaqe proteins that are accumulated by the plant. Inas~uch a~ it i~ the decrease in storag~
protein~ in fl2 that is reported to give rise to the starchy ~ rr~m characteriYtic Or the ~Z mutant, zl 35 phenotype like that o~ the fl2 ~utant is not expected when plants are tran~or~Ded with i fusion prot~-in o~ thQ
fl2 signal peptide and a protein other than a zcin protQin .

--~.o--Alternatlv~ly, the entlr- ~12 gene say b-7 tran~rorr~ed into a plant that produc~s s~eds that are - .nic:~7 ly stronger than mai2e. Fo~- exa31pl4, sorg~um arld r~ t produc~,7 smaller and rounder seeds which are less a~ected 5 by shear force~ and, bence, to oy any .7~ernel ~ ~k~nir~
as~ociate~ ~ith flZ expr~ssion. ~xpression o~ rl2 in these crops can ~rovide ~e~.ds ~Irlth improved digestibility ~ithout unacceptable leve~.s of kernel ~ -keni~q. It is expected that rice, wheat and barley liJcewisQ could lC ~- - te rl2 expres~ion in accordance with the pres~nt invehtion, In accordanCe with the present invention, a DNA
raolecule comprising a transformation/expresSion vector i~
enqineered to inolude th4 21 amino acid signal sequence from ~12 and/or regions up~,tteam or down3trea~ Or thc ~12 coding region, and eithqr the rer~ainder of the fl2 gene or a DNA segment ~ncoding a high-value protein a8 described abova. A copy of the sequence of fl2 or o~ the 21 ar~ino aoid signal segue:~c~ fro~ rl2 coupl~d to a 20 desired high-value protein is plaoed into nn exp~ession vector by st~ndard ~ethods. The r~~t~ ~r1 of an appropriate expression vect:or will depend upon the blethOd of in~roducing the eXprefisiOn vector into host cl311s.
A typical expres~ion vectcr contains: prokaryotlc DNA
ZS elehlents coding for a b~cterial rep7ication origin and an ~ntibiotic resistanc~ gene to provide for the gro~th and tiorl Of the expres~ion vector in the baoterial ho!~t;
a cloning site ~or insertion of the ~ J-?nv.l~ DNA
sequence; eukaryotic DNA el,~ments that control initiation 30 of transcription o~ the t,~g- cvu, DNA seqUenGe, such as a promoter and an optional enhancer; and DNA elements that control th~ processir~g Or tran~cripts, such as a transcription termination-polyadenylation ~equence. ~h-vector also could contaln .~dditional 5_, ~' that arQ
35 necessary to allow ror the eventual lntegration o~ th~
vect or into a chror~osoms. ~or a gen~ral de~oriptlon o~
p7ant expression vector~ ~e Gruoer t7t al., "Vectors ror Plant ~ransformation, " in ~T~ODS IN P~N~ MOLE~ULAR
sIO~GY AND BIOTECE~OLOGY 89-119 (CRC Prefis, 1993).
~xprQsslon of the g~lls sequence i~ under the ccntrol of n promoter . EYampl-t~, o~ suitabl- p. tc~ J are the S promotor ~or the sr~ll 6ubunit Or ribulose-1,5-bi~-phosphate carboxylas~ pro20ters fro~D t~or-inducing pl~smids o~ Agrobacteriun ~e~aci~ns, ~uch ~8 the nopaline synt~ase and ocrtopin~ synthase pro~ot~rs, and viral pro~otQrs such as th~ oauliflower ~osaic virus (CaXV~ l9S and 35S pL~ or the flg~ror'c r,losaic viru~
35S promoter. The promoter can b~ constitutive or ;n~lr~ihl~, I
Fc:p~ri:~lly preferred is a "seed tissue-preferredn or ~'seed tissue-specific" prouoters, that is, pror~ot~rs that drive hiqh eYpression o~ the hQterclogous DNA sagslent in E eed tlssue where control of genes that ar~- involved in seed metabolism is desir~cd, and little ar no expression in other pa~s o~ the plant. I~anufacture of the protein encoded by the heterolog~us D:~J~ segment $n other parts of the plant n~ A~.c~:1y expends the plant's energy.
Ex~mples of known seed tis6ue-prererred or seed tissue-specific pro~oters include thQ seed-directed ~/L~
from the zein qenes o~ maize ~-Y~ rh,. Pederfien et al., Cell 2g: lolS ~1982). The flZ promoter is particularly preferred.
In addition to a suitable promoter, one or more Qnhancers ar~ useful Ln the invention to increa~e transcription of the intr~duced DNA segment. ~he enhancer or enhancer-like elemen~ can~ be i~serted into the promoter to provide 'higher levels of transcription.
Exalsples of such c-nhancers include, inter alia, viral enhancerfi like those wit,h,in the 35S promoter, as sho~rn by od--ll et al., Pl~n~ Mol. ~lol. 10: Z63-72 (1988), and an enhz~ncer ~rorD an opine gene as desoribed by Tror~ et ~7., Pla~t Cell 1: 977 ~1989~.
selectable ~arker ~erles, in phy~ic~l proximity to th-int~u, ~ NA seglsent, are used to allow transforlse~l c~ 9 to be Lcco~clcd by either positiv~ gen~tlc ~ -~2- 2181418 sel~ction or screQnin~. The ~electable marker genes alsO
allow for ~aintainihg sel~ction pressur- on ~ , ic plant population, to 6~n6ure that the i-d,L~' ' D~A
~eg:;ent, and its controlling promotars and enhancQ~s, are retain~d by the transgel~ic plant.
Many o~ the comronl~ used positive ~olectAbl~ nlarker g~nQs itor plant transformation have 'oeen isolated ~ro bacteri~- and code itor enz~es that ~etabolically d~toxii~y a selectiv~ chemical ay~nt which 2ay be an antLbiotlc or a herblcide. Other p~sitive ~ ;nn ~arker genes encodQ an a ltered 'carget which i8 insensitive to the ihhibitor~
A preferre~ sele~tion marker gene for plant tranGforration is the BAR or PAT gene, which i8 used with ths selecting aqent h~ ~1 arhrs Spencer ~t al ., ~E~R.
APpL. GENEr., Berlin: Sp~.ingcr Int~rnational, vol. 79, pp 62S-631, 1990. Another usefUl s~lecticn marker gQne is the neomycin phosphotranafer2se II (~IptII~ gen~, isolated fro~ ~n5, which conferl~ resl~tance to Xanan~ycin ;rhen placed under th(3 control Or plant regulatory signals.
Fraley et ~l., Pr~c. NAt'l Acad. sci. 17SA ao: 4803 119~33~. 'rhe ~Iy~r~ n ph~Jayl~ U.~r~LCISe qene, ~hich con~ers resistance to the antibiotic hygro~ycin, ~s a further example of a us~ful s~l~ct~hl~ ~;arker. Vanden El2en Qt al., Plan Mol. Riol 5: 299 (1985~. Additional po~itive select2ble ~ar3cers qenes Or bacterial origln that confer resistahce to antibiotics include ~ nt~rnir;n acstyl transfera8e, str~pto:llycin phosphotransrer~Q, a~inoglycoside-3 ~-adenyl tran6feras~ and thc ~lecmycin resistance determinant. Hayford et ~ lant Physiol.
~C: 1216 (1988~; Jone~ et al., Mol. Gen. Genet. 21~: 86 (1987~; Svab et al., Piant ~ol. 13iol. 1~: 197 (l990);
Hill- et 21., loc. cit, 7: 171 (1986~.
other positLve selectable ~arker gones i~or pl~nt transformation are not oi' ~acterial oriqin. I'hes~ genes include 130U8e dlhyc~roi'olate reduatase, plant 5-enc~lpyruvylshikimate-~ pl,o~ c.te ~y~thase and pl~nt acetolactate synthase. Elchh~ltz et ~l., SocatiC C~ll , ~ 2181418 ~ol. Genet. ~3: 67 (lge~); Shah et al., Scier~ce 233: 478 ~19~6); Charest et al., Plant C311 R~p. 8: 643 (1990).
Another clas~ o~ useful ~a.rker gen~s ~or plant tn~nsrOr~ation witll the DNA ~equ~ncc re~lrea screehing of presu~Dptively tranbrori~ed pl~nt cells rather than dir~ct genetic 8 ~~~t'iOIl 01~ ~L~I:irU ~ cells for resistance to a toxic ,ui,,t~n-.~ such as ~n antibLotic.
~hese genes i~re particularly userul tc quantitat~ or visua1izQ the spatial ~attern o~ sxpres610n Or th~ DNA
sequence in speclric tissues and are ~requently rererred to as reporter genes because they can ~e rused to ~ gene or qene regulatory sequence ~or the investigation of qene expression. Com~only used genes ~or screening pres~ ptiv~ly transfor~ed cells includ~ glucuronidase (G~7S), ,B-gdlactosid~s~, luciferase, ~nd ehl~L ,' ;CO1 acetyltransrerase Jef~erson, ~lant Hol. ~iol. R~p. 5:
3B7 (igs7); Teeri et ~ S30J. 8: 343 ~1989~; Konc~ e~
al., Proc. Nat~ Acad. ,sci. U~;A 8~: 131 (1987~; De Block et ~1., E~30 J. 3: 1681 ~1984~. Another approach to the identi~ication of relatively rare transrormatlon events has been use c~ a gene that encodes a dc~inant con6titutive regulator of the z~a mays anthocyanin pigi~cntation pathway. 1udwig et al., Sclence 2~7: 449 tl990) .
~n order to create an e~cpres5ion vector cnn~aln;ng the gene or DNA segment Or interest, an expre~sion cassette first i6 i~ade ~>y inserting a cloned fl2 gene, or a D~IA segl;ent comprising the fl2 signal sequence ru~ed to a de6ired high-valu~ protein d5 described above, into ~
plas~id under the control Or a regulatory sesIuence. l~e resulting expression cassette can be ligated back to itself to produce an e~pression cassettc with a tandem repeat Or the cloned g~n~. A rurther ligation can be psr~orr~ed to generate a ~ L.LU''t that contalns four tdnde~ copies of the gene.
one or more COpl~L of the ~xpression cassetto rr~n~ n; nq the introduced DNA segment CO~L - AL V~ ; n7 to thc rl2 g-nQ or to thq ~NA seg~ent compri~ing th~ fl~

~ 2~8~418 !
Gignal s-quence fused tc a deslred protein i~ transferred to an e~pression vector. In a preferred ' ~; ', thQ
vector also contains a gene ~ncodirlg a sel~ction ~arlcer which is functlonally llnked to pro3l0tor~ that control S tr~nscription initiatio~l.
To creats a transg~!~nic plant, an ex.oression vector cnntAinln~ the fl2 g-ne or the DNA seg~ent co3lp~ising th-rl2 signal ~iequenc~ fu~,ed to a desired protein can be in~ into protopla8ts; into intact ti~r~ue~, such a~
i~nature embryos and me~-LstelD~; into calluri c~lture~ or into isolated cells. Preferably, expression vectors aro inserted into intact tissues, such as explants derived from hypocotyl or cotyledonary nodes of a yer3cinat~d seed. (In this regard, an explant is a pieco of tissu-that is taken from a dono~ plant and i5 capable of producing callu6 in cultu~r~. ~ypocotyl tis~e. i8 thAt portiOn of th~ ste~ of a plant enbryo or seedling below tho cotyledonL and above the root. A cotyledon is an ~mbryonic loaf, and a cotyledoni~ry nodo is that part of ~0 the seedllnq between the embryonic axis and the cotyledons which ~otanically de~inos the divisian of the h~pocotyl and the epicotyl, or elrbryonic shoot. ) General ~ethods of culturing plant tissuos are provid~d, for exa3~ple, by ~5iki ~t ~ Plv.~duY~s ~or Introducing Foreign DNA into Plants," in ~ETHODS ~N Pl,ANT Mor~-rrr.
~IOLOGY AND ~Io~ECE~OLOGY 67-aa ~CRC Pre3s 1993) .
Prefera~ly, the ~12 gene or DNA aegment co~prisir~g tbe flZ siqnal sequence ~u.sed to a desired high-value protoin is transforned into ~ v~ ic naize callus by p~rticle ~ - ~ t. Tran~genic ~aize pl~nts are produced by bonbarc~ent of c ' y~enically responsive ir~mature e~ryos T;ith tullgsten p~rticles associ2ted ~ith DNA plasmids. Th~ plaslnids consist of a ~e~ t~hle and an unselectable ~rker gene.
The present invention is described fUrther by refer~nc~ to the ~ollot~irl~, illustrative exaD~ples.

-- 21~418 F 1 ~ 1. Chnracteri~r~tion of 24-~a p~ote.Ln of fl2 Wild-type maize, W64A+, W15 crossed with W6~A~12 to gl~Q an F2 population, according to Lopes ~t ~1., 19~4, thQ contents of whil:h are inco.~ tad hereln 3~y S referenc~. The proqeny were sorted into floury, semi-floury And norm~l kernl21s, and DNA was isolated fro~ 30 individuals in each pbenotypic Cl~fiS. To ldentify DNA
polyrorphisms li~ked ~ith the fl2 mutation, a bulked seqreqant analysls w~ uaed. ~i~h~~ e et ~-1., P~oc.
Nat'l Acat. USA 8a: 9~28-32 ~19gl~. A~t~r ext~nsiv-~creening with different DNA prcbe~restricticn enzym~
combinations, an RF3.P was detectcd iD SstI-di~ested DNA
vith a 22-kDa ~-zein c~NA. A 7.7 3cb DNA frsgment was pres~nt in t31e ~ ~U.Il~ fl2 bulked sample and the 15 heteLvzy~,a~ semi-floury bulX, but not in the norm21 buLk .
The ~. 7 kb Sstl fragment wa~ i~olatea from genomic DN~ of ~64~12, And t:he resulting clone, pCC515, was mappet by restrictlon enzyme digestion, as described in Z~ Cole~an et al., Proc. Nat'l Acsd. sci. USA 92: 6828-31 ~19~5~, the contents ol. ~hich are inc-JL~-oL~ed herein by rererence. Clone pCC515 was found to contain a single 22-kDa ~-zein coding sequence, vhich was obtained ag ~
1. 6 kb EcoR1 fragment. Upon nucleotide se~Iuence 25 analysis, the deduced ami~o acid sequence vas found to coL~ .o,ld to a 22-kDa ~I-zein. The protein contains Z62 amino acids, including a Zl a~ino ~oid fiignal peptide.
Comparison of the ~educed a~ino acid sequence of the slgnal peptide with th~ signal peptides o~ other ~-z-ins 3û reveals an alanine to valine substitution at the C-terminal (-13 residue of the signal peptide, insertion of a hifitidine following the seventh re3idue in the seventh ~r-helical repeat and an alanine to thoureonin-sub~titutlon Ln the sa~e ~-helical repeat. N-terninAl 35 sequence analysis of th~ purified 24-kDa protein ~ron ~12 ~.dos3J~ Ghowed an Ldentical match ~or the f ir~t ~.
amino acid residue~ oet~een pCC~515 and the 24-kDa pol~peptide~. ~he fiignz~l peptide that targets the protein 218~418 into the lu~en of the rough ~ndoplas~ic r~ticulum ~ras found to be attzch~d to thc protein.
r le 2. ~rransfor~tion of ~i~:e vltb the rl2 ~7e~e To prove that pCC515 contains th_ fl2 gen-, the gen~
~as transformed into ~v~ ic maize callu~ by p~rticl~
b,ombardment. ~ran~genic llaize plants wer~ produced by bombardment Or ~ Lr~ ir~lly re6ponsive i~ature embryos with tungsten partlcles a~Lociatsd wit~ DNA
plasmid¢. The plasmids consi6t of a ~electaole and an unselPct~hle ~arker qene.
Prop~ra~cion Or ti~ue Immature eDroryos of maize variety High ~ype II were the target for particlo ~ ~ ~ ,t-mediated transformation. ~his yenotype is the F~ of two purebred genetic lines, parents P. and B, derived rrol3 the cross of two know maize inbreds, A188 and B73. Both parents were ~elected ror high competenae of so~atic . y~gd~ 3iU, according to Armstrong et al., ~ais~e Genetlc~ Coop. News 65: 92 ~1991). The High q'~'pQ II genotype does not pos~ess the native mutant r12 gene.
Ears from ~, plasts were selfed or sibbed, and er~oryos were aseptically ~l~cc~c~d ~rom developing caryOpseS when the scul ellum ~irst became opaque. ~his stage occurred about 9-13 days post-pollination, and ~ost ~ener~lly about lO days post-pollinatlon, clPr~n~lin7 on growth conditlons . ~he e~bryo~ were about 0 . 75 to 1. 5 milli~eters long. Ears were surface sterilized with 20-50% Clorox for 30 Dlinutes, ~ollo~red by three rinses with 6terilo distilled water.
~ature embryo-~ were cultured with the sc~t~l ~
oriented upward, on ~ vy~l,ic induction mediu~n comprised Or N~ ba~-al salts, EriXsson VitaD~ins, 0.5 mg/l thlamin~ }}CI., 30 gm/l ~ucrose, 2.88 gm/l L-proline, 1 mg/l 2,4-dichlorophenoxyacetic ACid, Z q~/l Gelrite, ana 8.S mgll Ag~103. Chu et al., Scl. Sin~ 18: 659 (1975); ~rlksson, Physiol. Plant 1~: 976 ~1965~. The medium was sterilized by autoclavinq at 121-C for 15 ~ 2 1 ~

mlnuSes and ~iFr~n~q~ cl into lC0 X 25 mm PQtri dishes .
Ag~03 is rilter-sterilized and added to the mediun aftQr autoclavlng. ~ha tisLiues wOEe cultured in cosplete darkneJs At 28~C. Ar'cer about 3 to 7 ~lay3, most usually 5 n~out 4 days, the scuto~lu~ of ehe embry-o had fiw-lled to about double it~ origin,~l size and th- protu~er~nces at t~e colPnrhi~ surface of the ~ at~llu~ indlc~ted the inception of _~.y;~nic ti~sua. 1~ to 100~ o~ the embryo~ di5played this respons-, but nost co3;~only, the 10 e2lb y ,_ ~ responsc rL~ y~ was about 80t.
Wnen the ~_JLyc~ ic response was observlad, the ellbryos ~ere trans~erred to a nedium co~prised o~
induction ;~lediU~ modi~ied to contain 120 g~l suoro~e.
The enoryos wsrs orient~d with thc coleorhizal pole, th~
15 allLbryo~n1t~11y responsive ti~sue, upwards from the culture medium. $en e~ry-os per Petri dir~ were located in the center of a Petri Aisn in an area about 2 cn in diametsr. The emb~yos wlare naintaineq on thls ~edium rOr 3-1~ hour, prererably 4 hours, in complete darXness at 20 28-C just prior to bombard2ent with pArticles zssoclated with plasmid DN~s ~ontaining the selectAble and unselectable marker gane~.
To effect particle! bombardr~ent of e~bryos, the particle-DNA ~gglomerates were accelerate~ using a DuPont 25 prlS_1000 particle acceleration devicd. The partlcle-~NA
agqlomeration was briefly sonicated and 10 ~1 were depositad on ~acrocarriers and the ethanol was allowed to Qvaporat~. The macrocarrier was accalerat~d onto a stainless-steel Ltopping screen by the rupture of a 30 p~lymer diaphrag~ (rupt~rQ disk). Rupture was e~fectad by pressurize~ helium ~ha veloclty o~ parti~le-~NA
~ccelaration was c~t~r~inD~I ba~ed on the rupture dLslc ~reaking pressure Rupture dis~c lJL._ ~Du~Ls of 200 to 1800 psi ~ere used, with ~50 to 110~ psi being preferred, 35 and ~bout 900 psi bcing IDost highly preferred. Nultiple diDks were used to ~ffeot a r~nge of rupture ~ r~lL~_L~
~ rhe shelf cnnt ~1 n j nq th~ plate with e~bryos ~as placed 5.1 c~ belo~ the botto~ o~ the ~L~ L.ier -lB -platrorm ~shelS' t3~. 'ro effect particl~ bo~b2rd~ t Of cultured i~ature ~bryos, a rupture difik 1nd a ~cacrocarrlsr with driecl particle-DNA agglo~erates wer-installcd in tne device. The E~e pressure delivsred to 5 the device wa~ adju~lted to 200 psi above tl~ rupturQ disX
breaking pre~sure. A Pe:tri di~h with the t~rget embryo~
was placed into the va~:uu~n chamber and loc~ted in the pro~cted path o~ ~ccel~rated ~nrticles. A vacuuD was created in tl~e chan~er, preferably about 2a in Hg. I~fter 10 operatioh Qf the devlce, the vacuu~o wa~ relea3ed ~nd the Pet~i dish was removed.
~ ' .l.-d embryos re~lained on the osmotically-ad~usted mediu~ durinq bo~bardlDent, and 1 to 4 day~
subsequently. The e31bryos were transferred to selectlon 15 Dediu~ conpriscd of t~6 ~asal s~lts, Erik~on vita~ins, 0.5 ~g/l thia~ine HCL, 30 g~3/l sucros~ /l 2,4-dichl~,L~ .4~acetic acid, 2 gu/l Gelrit~, 0.85 mg/l Aq NO3 and 3 mg/1 bialaphos tElerbiace, ll~ rhr~
was ;~dded f ilter-steriliz~d. ~he enbryos were 20 subc~ltured to fresh se~ection medium at 10 ~o li d~y int~rvals. A~ter aoaut 7 weeks,: ~., tc tiss~e, putatively trans~orr~ed for both selectable and unselected ~arXer genes, proliferaced fro:~ about 7~ of the bo~ba~ded embryos. Putative transgenic tissue ~ras rescued, and 25 that tissue derived ~ro~ individual ellLbryos was CQne 1 ' ~t d to be an eve~t and Wl18 propagated lnA~-r~nd..r~tly Oh so~ t~ ~edi~. rwo cycles o~ clonal propagation were acnieved by visual s~lection ror the slDa~llest contiquous ~ragrents of organized: ~-,g~"ic 3r~ ti55Ue.
A sa~ple Or tl~sue ~rQm each even~ ~a~ y~Occ~ d to recover DNA. ~he DNA was restricted with a re~triction endonuclease and probed witb pri~er 5~LaCllC._3 desisned to anpli~y ~lrA ~ n~ s overlapping thc rl2 and non-flZ
35 portion of the plasnid. r yot, I) ~ tissue with anpllfiable ~equence wag advanced to pl~nt rc4~...aL~tion.
~ or Lt~,. rl~iOn of transgenic pl~nts, y~...ic tissue was ~ubculturcd to ~ ~edi~ conprising ~l-e salt~

~ -19- 2181418 ~nd vit~inq (lSura6hi~- L Skooq, PhyJiol. Plant ~5: 473 1196ZJ), 100 mgtl myo-ino~itol, 60 gm~l sUcrO5~, 3 g~tl Gelrite, 0.5 3~gJl z~a~ in, 1 n~g/l lndole-3-acetic acid, Z6.4 ng~1 ~cLs-trans-~c~ jcqic acid, nd 3 21lgJ1 bial~phos s ln 100 X 2~ 3UII P~tr~ dishe~, and w~ lnc~ated ln darkness at 28-C unti l the de~elopD~nt of v~ll-for~ed, m:-tur-d so~atic e~ryos coult be se-n. ~his requir4d about 14 day~. ~iell-formed so~atic enb~yos Yere opaqu~
and cream-colored, and ~cre coDlprised o~ ~n identi~iable scutellum ~nd coleoptile. Ths embryos wer~ individually su'ocultured to a ~er~ination DediUDI co~prising HS sAlts and vitamins, 100 mgll myo-inositol, 40 g~/l sucrose and 1.5 g~/l G~lrite in 1~0 X 25 ~ Petri dishes and incuba~ed under a 16 hl~ur liqht;8 hour dark photoperlod and 40 ~einstein~ sec-~ ~roill cool-white i'lUv,.3~
tubes . Af ter about 'J days, thc 50natio e~bryos had germinated and produced a well-dcrined shoot and root.
T'Ae individual plants were subcultur~d to gP~;n~tior~
modlum in 125 X 25 ~ gl~ss tube~ to allo~r furt~-r plaAt dev~l:, n~ The plants were Daintained under a 16 hour light:8 hour dark E,h~lu~_~lod and 40 ,U~;nstP~n~--'7sec-fro~ cool-wh$te fluore~;cent tubes. After about 7 d~ys, the plants were well-esta~lished and were tran~planted to h~rticultural soil, hardened o~f, and potted lnto Z5 coDmercial greenhouse soil mixture and grOwn to ~exual maturity iA a greenhouse. An elite inbred lina wa5 used as a male to pollinate regenerated transgenic plants.
Prep~r~tio~ of p~r1:iclell Firtcen ;llg of tungsten particlos ~Gener~l Electrlc~, 0.5 to 1.8 Il, pr~ferably 1 to 1.~ IL, and 3~o_t pre~erably 1 ~, were added to 2 ~l of COnCG~lt.La~l nitric acid This s~lsponci~n was ~ohioated ~t 0~C ~o~ 20 ~inutes ~Bran~on Sonl~ier Mod~l ~,50, 40% output, constant duty cycle). Tungs~en particles wGro pelloted by centrifuSI~tion at lOoOO rp!l (Hio~uqe) ~or one ~inute, ~nd the supcrhatant wa3 re311oved. T~o milliliter~ Or stG~rilc dit;tilled wat~r werc added to the pellet, and brief ~onication wa-~ used to r~ ., ~ the particle~. The 2l8l4l8 suspen?,ion wa~ pslleted, one ~illiliter of Ab~olute ethanol va6 added to the pellet, ~nd bri~f F~ 'at ~ was used to resusp-nd the p,~rticles. Rlj~slng, pell~ting, and r~Cucp~n~ ~ n~J 0~ the particles was p~rformed two sar-S ti~es with sterile di.stilled water, ard rin~lly th-~particles w~re r~C~C~ in tYo nillilit~rs of sterile dittilled water. The particles w~re suhdivided into 250~ a~iquots ~md stored frozen., ic~ of particl~-pl~ia ~IA a-90ci~tior.
~hc ~tock ol tungst,~n particles ~Jas sonicat2d briefly in a water bath sonicator ~firanson Sonifier .~lod~l ~.50, 2-~% output, constant duty cycle) and 50 ~ as.
transferred to a microfuge t~loe. ~qui~nolar anounts o~
selectable and unselectable plas~id DNA ~ere atded to the particles for a final DNA anount o~ 0.1 to 10 llg in 10 yl total volune, a~ d brierly sonicated. Preferably, 1 ~Lg total DYA was used. Specifically, 3 . 5 ,ul o~ DP~810 (U~2~:ubiint~ :pinIr~, 5.6 kltp) plus 6.5 ~1 Or DP6645 (flZ?::fl2!:fl21, 10.2 kbp), both at 0.~ ,ug/~l in Tl:
buffcr, were added to the particle suspension. Firty ~icroliters of sterile a.queous 2 . 5 M CaCll were added, and ths ~ixture ~as l riefly sonicated and vortexed.
Twenty microliters o~ sterile aqueous ~.1 M sr~
wsre added and the ~ ture was ,~rief ly sonicated and Z5 vortexed. ~he nixture ~i/as inc.Loated at roolL te~nper~ture f:or 20 minutes with interlrittent brief 30nication. The particle su~pe--,sion wa8 centrifuged, ~nd the gl~r~2i2tAnt wa3 renoved. Two hundred fifty nicroliters of absolute ethanol wer~ added to the pellet, rollowed by brief 82r~2n;~?tir~n. ~le 8~ pen~1r~ was pelleted, the supcrnatant ~as re~oved, and ~t ILl of absolute ethanol were added.
The suspension was sonic~ted briefly before loading the particla-DNA agglo:~eration onto Dlacrocarriers.
F ~ tn 3 . ~ractic~n a~td ~ t;~n of p~otcLn 3 5 rrO~ tr~r~ en ~ c seed ED~ryos wcre hand-d1~ s~to~ fron dry, ~atUrQ kernel~
ss~pled fron Siully d~eloped ears snd ~ I w~re 2~814~8 pulveriz~d to a ~ine ~eal ~ith a b~ll nill. Alph~-zeina were extracted overnight in 70% ~v/~ ethanol with con6tant sh~kiDg at 37'C. Arter centrlfugation ~or 15 minute$ at 12, 000 rlp3~, the aupernatant waY ro~ t~, 5 vacuu~ dried, and stored at 4~C until use. ~ot~l ~ein~
and non-zein proteinl; were isolaSed ~cco~ding to Wallace et al, Pla~.7t Pl~ysio.~. 92: 191-96 (1950~.
SDS-polyacrylamide gels ~10 and 12.~9~, w/v) and qradient gels ~7.5~ %, ;r/v) wera prepa~ed according to I,ae~D~li, Naturo 22,1: 680-8~ 70~, but the TR~S
~,OI~ e"~._tlons used in the re~olving g~l and running ouffer ~ere doubled. P~otcin samples were diluted ln I,ae~li sa~ple bu~rer and boiled for 3 31inutes be~ore loadlng. Gradient and 12 . 5% gels were run at room t, at,uL~:: at a constant current until th~ dye front ~iqrat~d throught the stacking gel, ar~d then at 25G ~A
through the resolvillg gel. Gels were stained with C_ sCie overni~ht, and destained in 40% (v/V) ~ethanol antS lû% fv/v) oc~tic acid ~or at lea~t 8 hour~.
7 ~70ttlnq analy6es were Lsed specifically to deteat Q-~eins in protein extracts. ~rotein e~acts were separated by SDS-P~ as described above, transferred to nitrocellulose f llters, and treated with a rabbit anti-~-zein polyclonal antibody. Lending et al., Protopla~:3a ~3: 51-62 ~19~8) . Go~t anti-ra~bit alkaline ph~oph~ta8e con~ugate waa u3ed rOr indirect detection of ~-~ain, as de~cribed by l~necht et al., A~al. ~iocl~e~. ~36: 180-84 (1984). The 24 kDa ~--zein was detected in the seed fr~m the tran~igenic plants.

Claims (17)

1. A cereal plant that contains a transgene comprised of (i) a first polynucleotide segment comprising a nucleotide sequence that encodes the amino acid sequence MATKILALLALLALLVSATNV and (ii) a second polynucleotide segment coding for a protein.

2. A cereal plant as claimed in claim 1, wherein said first and second polynucleotide segments are operably linked to a promoter, such that said cereal plant expresses both segments under the control of said promoter.

3. A cereal plant as claimed in claim 2, wherein said promoter is the fl2 promoter.

4. A cereal plant as claimed in claim 1, wherein said plant is a maize plant and wherein said segment (ii) is not native to maize.

5. A cereal plant as claimed in claim 1, wherein said plant is rice, wheat, barley, millet or sorghum.

6. A cereal plant as claimed in claim 5, wherein said plant is rice or wheat.

7. A cereal plant as claimed in claim 1, in which said polynucleotide segment (ii) has a high content of an amino acid selected from the group consisting of methionine, lysine, tryptophan and threonine such that the amount of said amino acid in seeds of said cereal plant is increased as compared to seeds from otherwise identical plants that are not transformed.

8. A seed produced by a plant as claimed in claim 1.

9. A feed product comprising meal obtained from seed as claimed in claim 8.

10. A transgene comprised of (i) a first polynucleotide segment comprising the nucleotide sequence coding for the amino acid sequence MATKILALLALLALLVSATNV
and (ii) a second polynucleotide segment coding for a protein.

11. A transgene as claimed in claim 10, wherein said first and second polynucleotide segments are operably linked to a promoter.

12. A transgene as claimed in claim 11, wherein said promoter is the fl2 promoter

13. A transgene as claimed in claim 10, comprising the coding region of fl2.

14. A transgene as claimed in claim 10, additionally comprising a sequence of fl2 selected from nucleotides 761-3824 of Figure 1.

15. A transgene as claimed in claim 10, additionally comprising a sequence of fl2 selected from nucleotides 4613-8335 of Figure 1.

16. A transgene as claimed in claim 14, additionally comprising a sequence of fl2 selected from nucleotides 4613-8335 of Figure 1.

17. A transgene comprising the fl2 promoter operably linked to a polynucleotide segment.