CA2243269A1 - Transgenic maize with increased mannitol content - Google Patents

Abstract

The present invention provides a method for conferring tolerance or resistance to water or salt stress in a monocot plant, and/or altering the osmoprotectant content of a monocot plant, by introducing a preselected DNA segment into the plant. This invention also relates to the transformed cells and seeds, and to the fertile plants grown from the transformed cells and to their pollen.

Description

W O 97/26365 1 PCTrUS97/00978 SG~,NIC M~T7.~, WITH INCI~F,~SED M~NNITOL CONT~,NT

Bacl~roul,~td of the Invention Unpredictable rainfall, inclGases in soil salinity, and low tt;lllp~ LLu.~ at the begl~ g or end of the growing season often result in decreased plant growth and crop productivity. These three ellvi~ nt~l factors share at least one element of stress and that is water deficit or dehydration.
Drought is a significant problem in agriculture today. Over the last 40 years, for example, drought accounted for 74% of the total U.S. crop losses of corn (Agriculture, U. S. Dt;Pa~LLL1~1-L of, 1990. Agricultural St~ti~tics- US
Go~,~ ."",ent Printing Office, Washington, D.C.). To sustain productivity under adverse envhot""~ l conditions, it is illl~olL~ll to provide crops with a genetic 20 basis for coping with water deficit, for example by breeding water retention and tolerance m~ch~ni~m~ into crops so that they can grow and yield under these adverse conditions.
When the rate of transpiration exceeds that of water uptake or supply, water deficit occurs and wilting ~ylll~Lcllls appear. The responses of plants to25 water deficits include leaf rolling and ~heclcling, stomata closure, leaf temperature ~ ,reases, and wilting. Metabolism is also pl<:r(Julldly affected. General protein synt'nesis is inhibited and significant increases in certain amino acid pools, such as proline, become ~ lL (Barnett et al., P'lant Pllysiol. 41, 1222 (1966)).
During these water deficit periods, the photosynthetic rate decr~ases with t'ne 30 l~ltim~te result of loss in yield (Boyer, J. S., In: Water deficit~ and plant ~rowth, T. T. Kozlowski (ed.)., Academic Press, New Yorlc., pp. 154-190 (1976)). If carried to an extreme, severe water deficits result in death of the plant.
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WO 97/26365 PCT~US97/00978 Several mecl-A~ appear to enable water deficit-tolerant plants to survive and produce. For exarnple, a co"~ ison of drought-,e,i~L~ and drought-sensitive lines of Zea mays inclir~tes that higher levels of abscisic acid (ABA), which is known to regulate stomata opening and p~ ~s other signal S responses are correlated with re~i~t~nre (Milborrow, B.V., In: Th~ physiology ~n~l bioch(~m;~try of drou~ht resict~nt pl~nt~ Paleg and Aspinall (eds.), ~r~ mic Press, NY, pp.348-388 (1981)). In addition, ABA-in~ iv~; mnt~nt~ and ABA-deficient ~ ; o f Arabidopsis are prone to wilting (Koorneef et al., Th~oret Appl Gen~:t., 61, 385 (198Z); Finkt-lctein et al., pl~nt Pllysiol. 94, 1172 10 (1990)).
Of the merh~ni~m~ employed by water deficit-tolerant plants to grow and yield, those with major impact on plant productivity are osmotic adjlTctment through the ~ll~;l~sed ~yll~e:,is of osmoploLt;~;liv~; metabolites, control over ion uptake and partitioning within the plant, ability to increase water intake, and 15 acceleration of ontogeny. Examples of osmo~-~te-;Li~, metabolites include sugars, such as sugar alcohols, proline, and ~,ly-;ille-betaine (Bohnert et al.,Pl~nt Cell, ~Z, 1099 (1995); McCue et al., Tibtt~rl~ ~, 358 (1990)). Sugar alcohols, or polyols, such as m~nnit-ll and sorbitol, are major photosynthetic products of, and are known to ~rcllmlll~te to high levels in, various higher plant 20 species. While ~ ...;lol is the most abundant sugar alcohol in at least 70 plant families, it is not produced at ~l~t~oct~hle levels in any illl~olL~-l agricultural field or vegetable crop, other than celery (Apiaceae), coffee (Rubiaceae), and olive (Oleacea). Other sugar alcohols, such as ononitol and pinitol, sre known to be produced in some plants under con-liti-~n~ of stress from drought, salt, or low~25 tt;~ w ~.
To produce a plant with a genetic basis for coping with water deficit, T~;~yl~i et al. (proc. N~tl Acad. S~i US~ 89, 2600 (1992); WO 92/19731, published November 12, 1992; Sci~nr~ 259, 508 (1993)) introduced the b~ct~
ul-l-pllos~h~l~ dehydrogenase gene, m~lD, into tobacco cells via~0 ~grob~cterium-meAi~te(l L.dl~-rol.llation. Root and leaf tissues from Ll~lsgelfic W O 97/26365 PCTrUS97/00978 plants regenerated from these ~ ro~ ed tobacco cells contained up to 100 mM
ol. Control plants cont~in~cl no c~etect~hle m~nnitol To clct~ ....;.~t?
whether the Ll~lsgellic tobacco plants exhibited increased tolerance to water deficit, Tarczynski et al. col~ cd the growth of transgenic plants to that of S ....~ rclllled control plants in the ~ lce of 25(~ rnM NaCL After 30 days of exposure to 250 mM NaCI, I.~ ,PI~;C plants had decreased weight loss and increased height relative to their ~ ru....e~7 coullL~ . The authors con~ le-l that the ~resence of ~ ;lol in these ~ n~fiorm~d tobacco plants contributed to water deficit tolerance at the cellular level.
While Ta~ yllski et al. (WO 92/19731, p1lblished November 12, 19923) disclose that the same methodo}ogy might be applied to other higher plants, suchas field crops, the introduction of exogenous DNA into monocotyledonous species and subsequent legc.l.,,dlion of LldLkrollllcd plants ~A~l~ssing useful phenot,vpic ~lup~ ies has proven much more difficult than l~ rollllation and lege.l~dLion of dicotyledonous plants.
Thus, there is a need for L~s~cllic monocot plants that are resistant or tolerant to a reduction in water availability. Also, a method to produce transgenic monocot plants with ill~;lGascd levels of osmc~l~ Le~;~lL~ is needed.
S--mm~lry of the T~
The present invention provides a method to illcrea3e water stress resistance or tolerance in a monocot plant cell or monncot plant, cnmrri~ing introducing an ~A~I~,ssion cassette into the cells of a monocot plant to yield Ll~rc,lllled monocot plant cells. Monocot plant cells include cells of monocotyledenous plants such as cereals, including corn (Zea mays), wheat, oats,rice, barley, millet and the like. The ~,AI,lei,:~ion cassette cCJ~ e~ a preselected DNA segm~-nt encoding an enzyme which catalyzes the synthesis of an osmoprotectant, op~.~ly linked to a promoter functional in the monocot plant cell. The enzyme encoded by the DNA segment is ~A~ ed in the Ll~kir~lllled 3û monocot plant cells to increase the level of the osmc,~lote.;L~lL so as to render the WO 97/26365 PCTrUS97/0O978 L~ rv....r-7 cells !;Uh~ y tolerant or resistant to a rennr~ticm in water availability that inhibits the growth of l..",~ f l.l.ed cells of the plant.
As used herein, an "osmoplo~c.;~~ is an osmotically active molecule which, when that molecule is present in an c~e~;Livc arnount in a cell or plant,5 confers water stress tolerance or r~oeiets nre or salt stress tolerance or rl?cietslnre7 to the cell or plant. Osmoprotectants include sugars such as ,..t~nnsAr~ l.A. ;-les, slrçh~. ;n.os, oligos~crh~r~ s~ polysaccharides, sugar alcohols, and sugar d~,~;v~lives, as well as proline and gly~;hlc-betaine. A ~ f~ d embodiment of the invention is an osmoprotectant that ;s a sugar. A more p~cr~ d embodiment 10 of the invention is an osmo~.~ Le~ l that is a sugar alcohol. Thus, useful osmoprotectants include fructose, e-~Llllilol, sorbitol, dulcitol, glucoglycerol, sucrose, stachyose, rSlffinnse, ononitol, ms nnitol, inositol, methyl-inositol, galactol, hepitol, ribitol, xylitol, arabitol, trehalose, and pinitol. A ~,f~l~cd o ,..lo~.ote.iL~ll of the invention is ...~ .;lol.
Genes which encode an enzyme that catalyzes the synthesis of an osmc,~l~Jt~e~ include genes ~nc~.orling ~ 1 dehydrogenase (Lee and Saier, ,LRST~'.tl'.T'iOI., ~. (1982)) and trehalose-6-rh~lsph~te ,y~Lhase (Kaasen et al., L
~Slrt~orio~ , 889 (1992)). Through the :iubse lucllL action of native s~h~/~ces in the cell or by the introduction and co~ es~ion of a specific 20 phnsrhsTtslee7 these introduced genes result in the aCcTmnTllsltinn of either ...~....;lvl or trehSTlos~ lc;,l,e.,~ivt;ly, both of which have been well do~ .le~ as protective c~ oul.ds able to mitigat~ the effects of stress. Mannitol sT~TTmnTAtinn in ege~ic tobacco has been verified and pl~Ti...;.~s,. y results jn-lir~te that plants c ~ lg high levels of this metabolite are able to tolerate an applied osmotic 25 stress (~a..;~y~Li et al., cited supra (1992), (1993)).
Also provided is an isolated ~ fvl..~ecT monocot plant cell and an isolated IL~Çulllled monocot plant comrTieing said l.,...~ir~ e(i cells, which cell and plant are ~ b~ ;ATly tolerant or 1~;,;;,~l~ to a reduction in water availability.
The cells of the LLd~l~rt~ lcd monocot plant cornrri~e a rec-)mhinSTnt DNA
30 segm-ont con.~.isillg a preselected DNA segment encoding an enzyme which -W O 97/26365 PCT~US97/00978 catalyzes the synthesis of an osmoploL~-;l~,l. The pres~ircte~l DNA segm.-ntis present in the cells of the lLcul~fo~llled plant and the enzyme encoded by the preselrcte~l DNA segment is ~ es~ed in thos'e cells to yield an amount of oX,l,op~ e~ errc;~;live to confer tolerance or rçei~et~nr-e to said cells to a S recl~ction in water availability that inhibits the growth of the coLl~,~unding cells of the ~ ld~xr~l-..ed plant. A preferred embodiment of the invention inrh~des a olllled monocot plant that has an improved osmotic potential when the total water potential of the ll~srollllcd plant a~roa~hes ~ro relative to the osmotic potential of a c~ onding ~ .ef~ cl plant.
Another pL~rc~ ,d embodi~ .ll of the invention is an isolated ~.,.nx~ ic Zea mays cell or plant, cornrrixing a ~eC~ t DNA segm~nt cc,...l..;x;..~ a promoter operably linked to a first DNA ~ encoding an amino t~ rmin~l chloroplast transit peptide operably linked to a second DNA sc~ nco~in~ an enzyme which catalyzes the synthesis of an oxl.~opl~lecl~.l. The ~,l~ylllc 15 encoded by the DNA sequence is c;~ ;s~ed in the l~ -xg~-ic Zea mays plant or cell so that the level of the osmo~çut~-;l~ll in the cells of the 1....egenic Zea mays plant is ~ lly ~ sed above the level in the cells of a Zea mays plant which only differ from the cells of the t.,.l.x~,....iC Zea mays plant in that the DNA se~;...r-.l is absent. The DNA segment is ~ ..x...;l~~~1 through a complete 20 nolmal sexual cycle of the transgenic plant to its progeny and to further ~,~n~,.dlions.
A further embodiment of the invention is a method for altering the sugar content in a monocot plant, such as a Zea mays plant, or rnn~-~cot cell. The method co~ es introducing an ~,e~xion cassette into the cells of a monocot 25 plant so as to yield transformed monocot plant cells. The e~ ,i,_ion ç~e~e~
cnmprixe~ a ~l. srle~ DNA segm~nt encoding an enzy~ne which catalyzes the Xyl~ sis of a sugar, operably linked to a promoter filnr,tion~l in the plant cells.
A ~liLr~ cl plant is lege~e~ d from the ll~ x~lmed plant cells. The enzyme encoded by the preselec~ DNA ser.~ nl is t;A~exxed in the cells of the 30 differenti~t~cl plant in an amount effective to hlc~;ase the sugar content in the CA 02i43269 1998-07-16 W O 97/26365 PCTnUS97/00978 cells of the diLL.,.~ tecl plant relative to the sugar eontent in the cells of the ullLldllsr~ lcd .lirr~ d plant.
Yet another embodiment of the invention is an isolated L-~rolllled monoeot plant eell or L~ru~ ed monoeot plant, having an altered sugar 5 cellular cont~nt The Ll~,:jr~.ll,led monocot c~ a recombinant DNA
seg...~-.l COlllpl;~illg a y~scle~Lt;d DNA segm~nt eneoding an enzyme whieh eatalyzes the synthesis of a sugar. The enzyme eneoded by the DNA segm~nt is cAyicssed in an arnount crrt;~;Livc to alter the sugar eontent of the eells of the plant.
The present invention also provides an isolated hallsgcL~c Zea m~rys cell or plant, comprising a reeombinant DNA seg... ~.~ eo...~ g a promoter operably linked to a pr~selectec7 DNA se~ eneoding an enzyme which catalyzes the synthesis of a sugar. The enzyme ~neo-Jerl by the recombinant DNA se~ ~1 is cAyl'cssed so that the level of sugar in the eells of the ll~ulSg~.lic 15 Zea mays plant is ~u~ y increased above the level in the cells of a Zea mays plant whieh only differ from the eells of the 1.~ . -.ie Zea mays plant in which the reeG...l.;i~ DNA se~ll~ ~,l is absent. The 1~ eolllbill~L DNA se~... ..f is ~ iLLed ~vu~L a complete normal sexual eyele of the hdllSgel~iC plant to its progeny and further generations.
A ylcr~ d embodiment of the invention is a method for altering the .;(ol content in a monocot plant eell or plant, sueh as a Zea mays plant. The method e-,...p. ;CF.~ introdueing an c~ .ion eassette into the eells of the monoeot plant so as to yield lld,~l~ll,led plant eells. The cAI,le;,~ion eassette CU~ liSCS a pl~ ~elc~le(l DNA segtnrnt ~nroflin~ an C~L~Y11~C whieh catalyzes the synthesis of ~- ,.. ;~ol, operably linked to a ~lUIllOt~ filnr.tit~n~l in the plant eell.
A dirrcl~ plant is ,~ l from the l.,~ r(.. ed plant eells. The c ~ylllc encoded by the DNA se~ is ~A~i~ssed in the eells of the t~od plant in an amount effeetive to i"~ ase the .. ;1~1 content in the cells of tne difr~ fl plant relative to the .. ~.;lol content in the cells of anllntr~n~formed dirr~ ecl monocot plant. O

W O 97/26365 PCTrUS97100978 Also provided is an isolated ll~l~;ro~ ed monocot plant comprising an altered mannitol cellular content. The plant comprises a recombinant DNA
segment CO---p"~;t-g a preselected DNA se~ment encoding an enzyme which catalyzes the synthesis of m~tnnit~l. The enzyme eneoded by the DNA is S eAylessed in an amount crre.;live to alter the m~nnitol eontent of the cells of the plant.
Another embodiment of the invention is a method to inerease salt stress recictRnre or tolerance in a monocot plant. Ihe method comrriceC introducing an CA~l~ s~ion e~ccette into the eells of a monocot plant. The cA~ sion cassette 10 comprises a preselected DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in a monocot plant cell, to yield ~ r~,lllled monocot plant eells. These transformed eells are regenerated to form a dirrGl~ ;Rte~ monoeot plant. The enzyme eneoded by the DNA segment is Gx~ ssed so as to render the ll~ r."."P-l 15 monocot plant subst~ntiS3~ly lc~ to an amount of salt that inhibits the growth of an ulllld.~rolllled monoeot plant. Also provided is a LLcLL~lllled monocot plant which is salt stress tolerant or ~ The cells of the plant c~-mprice arecombinant DNA ScgTllpnt colll~ illg a preselected I~NA segrnPnt encoding an enzyme whieh eatalyzes the synthesis of an osmol loLe~iL~l. The enzyme is 20 e~le~:,ed in the eells of the plant in an amount t Lrc-; ive to confer tolerance or recictRn- e to the Lldl~rulllled plant to an amount of sRlt that inhibits the growth of the corresponding ullLlal~r~ led plant.
As used herein, the term "salt" inrludes~ but is not limited to, salts of R~ric ~ lral fertilizers and salts associated with RlkRIine or aeid soil conditions. A
25 p~ef~ d salt of the invention is sodium chloride ~aCI).
The present invention also provides an eA~ i.,;,ion cassette c--mpri~in~ a preselected DNA seE~mPnt encoding an enzyme whieh eatR~yzes the synthesis of an osmopl~te~ lL, operably linked to a promoter funetional in a host cell. The promoter in the GA~lGs:jion eassette is seleeted from, but not limited to, the group 30 consisting of the Glb promoter, the AdhI promoter, and the ActI promoter.

W O 97/26365 PCT~US97/00978 Also provided is an c~.cs~ion cassette comprising a presçlected first DNA segmPn~ encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter filnrtion~l in a host cell, wherein a second DNA segm~nt sep~aLes the first preselected DNA se~ nt encoding the 5 enzyme from the promoter. A ~.ef~ ,d second DNA segm~nt is the AdhI
intron 1.
Further provided is an G~ ,,.,ion cassette comprising a ~.csele~;led first DNA segm~-nf encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in a host cell, wh~"c 11 a 10 second DNA segment enCo~iing a maize chlùnl~pla~,l transit peptide is op~bly linked to the preselected first DNA segment encoding the ~,lLCylllc.
As used herein, a "preselected" DNA sequence or segment is an exogenous or recombinant DNA sequence or se~ .I that encodes an enzyme which catalyzes the synthesis of an osmo~.o~e~ L, such as a sugar. The enzyme 15 plG~Iably utilizes a substrate that is aLulld~lL in the plant cell. More preferably, the ~ub~LldlG is present in either, or both, the cytosol and chloroplasts of the plant cell. It is also plGr~,.lcd that the pres~i~cted DNA se~m~nt or se.lu~ ce encodean enzyme that is active ~,vithout a co-factor, or with a readily available co-factor.
For exarnple, the mtlD gene of E. coli encodes a lll~ ul~ 1-phnsph~t~
20 dehydrogenase (MIPD). The only co-factor l~c~ for the enzymatic activity of MIPD in plants is NADH and the ~.b~ for MlPD in plants is fructose-6-ph~sph~te Both NADH and fructose-6-phosphate are plentiful in higher plant cells As used herein, "~ub~ lly ill~ ,ased" or "elevated" levels of an 25 osmopl~,LG~ in a Ll~r~,ll.led plant cell, plant tissue, plant part, or plant, are greater than the levels in an ulll~ rullllcd plant cell, plant part, plant tissue, or plant, i.e., one where the genome has not been altered by the p.~,.,ence of a preselected DNA sequence. In the ~ ;vG, "~b~ ly in~ ased" or "elevated" levels of an osmoprotectant in a water-stressed Ll~rol.lled plant cell, 30 plant tissue, plant part, or plant, are levels that are at least about 1.1 to S0 times, W O 9712636S PCTrUS97/00978 .

preferably at least about 2 to 30 times, and more ~ref~lably about 5-20 times, greater than the levels in a non-water-stressed L~ fo.lllcd plant cell, plant tissue, plant part or plant.
For example, the levels of m~nnitol in a monocot plant L,~r(i.,lled with a preselected DNA sequence encoding an er~yme which catalyzes the synthesis of ms~nnitol, are co,l~d~d to the levels in an lmtr~n~formed plant. In the ;vc~ the levels of m~nnitol in a homozygous backcross converted inbred plant ll~r(,.ll-ed with a preselected DNA sc~lucl~ce enrof~in~ an enzyme which catalyzes the synthesis of ~l~n~ l, are co"l~ d to the levels in a ~ecu~
inbred plant. A homo~;ygous backcross cu~ lcd inbred transformed plant is a tran~rul,lled plant which has been l~edLcdly crossed to the lc~ ll inbred parent until the ll~rc,lll.ed plant is ~ ";~lly isogenic with the lc,;ullc,ll inbred parent except for the presence of the pr~selocted DNA sequence, and is then self-pollinated (selfed) at lea~st once, and preferably 5 or more times.
As used herein, '~sllbst~nti~lly isogenic'l means that the genomic DNA
content of a homo~ygoL~ backcross coll~ Gd inbred ll~r~ll"cd plant is at le~t about 92%, ~lcÇ~.dl)ly at least about 98%, and most ~ Ç~,.dl~ly at least about 99%, iclrntir.~l to the genomic DNA content of a ~ lC.ll inbred parent of the Ll~sr~ l..led plant.
As used herein, a plant cell, plant part, plant tissue or plant that is "~-b~ lly lesi:,~L or tolerant" to a re~ rtion in water availability is a plant cell, plant part, plant tissue, or plant that grows under water-stress con-litions~
e.g., high salt, low ttlll~.~.d~ S, or decreased water availability, that nnrm~lly inhibit the growth of the ~mtr~n~formed plant cell, plant tissue, plant part, orplant, as ~ by methodologies known to the art. Methodologies to ...;..C plant growth or response to stress inr,l~ e~ but are not limited to, height me~.,.G.llc.,L~, weight mea~ ."~ , leaf area, plant water rel~tion ability to flower, ability to gel.~"dLe progeny, and yield. For example, a homo~y~,ous backcross converted inbred Lldl~irollllGd plant of the invention has a W O 97/26365 PCT~US97/00978 .

superior osmotic potential during a water deficit relative to the cu~lc:~lJonding, i.e., ~ub~ ily isogenic, recurrent inbred plant.
As used herein, an "exogenous" gene or "recombinant" DNA is a DNA
sequence or segment that has been iso}ated from a cell, purified, and arnplified.
As used herein, the term "isolated" means either physically isolated from the cell or syntheci~d ~n vitro in the basis of the sequence of an isolated DNA
segm~nt As used herein, a "native" gene means a DNA sequence or se~ that has not been manipulated in vitro, i.e., has not been isolated, purified, and arnplified.
As used herein~ "altered" levels of an osmoprotectant in a lld~rc,..lled plant, plant tissue, plant part, or plant cell are levels which are di~.,lc.lL, preferably greater, than the levels found in the collc:,~onding ~ rol.lled plant, plant tissue, plant part, or plant cells. In the ~11 ...~livc, altered levels of 15 the osmoprotectant in a backcross coll~ cd inbred Ll~rulllled plant are L, preferably greater, than the levels found in the cc,.lGi,~ondillg lG~ lcllL
inbred plant.

Rrief n~s "l.lion of ~he Fig~res 20 Figure 1. A srh~m~tic diagram of rl~cmi~l pDPG451.
Figure 2. A s~ h~",i1l;c diagram of pl~cmi~ pDPG165.
Figure 3. A sf~.h~m~tic diagram of plasmid pDPG480.
Figure 4. A s~h~ tic ~ m of pl~mid pDPG493.
Figure 5. A sçh- ...~l ;c diagram of rl~mifl pDPG586.
Figu}e 6. A s~ hP~ic diagram of plasmid pDPG587.
Figure 7. A time course of leaf osmotic l.u~.l~ial values collected from a population of llculsg~;llic maize platlts. All plants were derived from AT824 cells bombarded with pDPG165 and pDPG480 which were subsequently selected on bialaphos-co.,~ medium. (A) S80HO-5201, (B) S80HO-5205, and ((:~) S80HO-5208.

W O 97/26365 PCT~US97/00978 .

Figure 8. Leaf tell~dlul~ data from Glufosinate(~) sensitive (mt~D negativej andresistant (mtlD positive) plants grown under water stress conditions in the field.

Detailed D~ ion of the ~ L~,,.
S The i~lPntific~tion and ch~clf .;,~I;on of plants that are resistant or tolerant to water deprivation has long been a goal of a~lunollly. However, it has not been possible to accomplish the identification and isolation of genes that can provide re~i~t~nce or tolerance to water stress. The insertion of such genes into monocots has the potential for long term ilnpl~ov~ ent in, and expansion of, l 0 agriculture world-wide.
The ability of a plant to adapt to changes in water and salt cnncçn~tions is clepenAent on the ability of the plant to osmotically adjust its intrz~re11~ r ~,.lviloll Llt;nL by ~1tPrinp the cu~lc~ lnl;on of osmop,~ ;L~L~ within the cells of the plant. These osmc,~luLt;~ LlL~ include, but are not limited to, various sugar molecules, such as monnc~rr1~ e, f1i~ct~h~r~ e~ oli~os~cf~ es~
poly~crh~ritlPs, sugar alcohols, and sugar d liv~liv~s. Thus, to provide a plantthat is tolerant or ~ L~.L to a re~ rtinn in water availability, a prl~eP1ected DNA
se~n.ont or "gene" or 1'LI1.-~g~ ..P" Pnro~1ing an enzyme which catalyzes the sy"Ll~e~is of a particular osrn ,~uL~ ~L~ can be introduced into the ~enomP of the 20 plant. The osmop.u~.,L~.L may be one that is not normally synthpei7~cl by theplant, but one which can be synth~o-ei~d from a sllhstr~1~ that is abundant in the cells of the plant after the introduction of the p.~ e1~ d DNA se~mPnt In the ;v~, the osmoprotectant may be one that is n~h1r~11y sy..l~ 1 by the plant but the levels of the osmo~LoLe~L~.L in the plant are inellfficient to render 25 the plant tolerant to a red~letion in water availability.
The ~c~ tion of a non-naturally oc-;--..;--~ osmc,~lote~L~,L in a plant, plant cell, plant part, or plant tissue, could result in a ~l~imPnt~l effect be~use the ~ua~sLL~Le employed to synth~ei7e the osmc~-ot~-;L~LlL is being depleted and a non-naturally oc~;~...;..g product is produced, which most likely would not be 30 degraded. Moreover, a single introduced preselected DNA se~ ~.1 in the W O 97/26365 PCT~US97/00978 lla,lsgellic maize plant resulted in a beneficial effect to the Ll~sg~ ic maize plant when it is placed under water stress, i.e., the plant becarne more water stress-tolerant than its untransformed coul~ ~L. Furth~nnore~ the expression of the preselecte~1 DNA segmt?nt in the ~ ~gGlliC plant did not ,ul~L~lli~lly affect the S reproduction or growth of the plant, relative to its llntr~,n~formed COU~lt~ ~L.
Thus, the present invention provides a method of g~nt~tic~l1y en~h~e~ g monocot plants so as to produce altered agronomic or physio}ogic ch~n~es in the plants by the ~lt~r~ti~n in the levels of an osm~Jplol~ e~ such as a sugar, or more preferably a sugar alcohol, within the tissues of the plant. Alterations inthese levels result in more n~Li~G osmotic water potentials in transformed planttissues under either, or both, water stress or non-water stress conditions relative to the osmotic potentials in ~ulLl~l,ro,l"ed plant tissues.
Yet another embodiment of the invention is a method to confer tolerance or resistance to a redllrtion in water availability to a monocot plant, plant tissue, plant part or plant cell. Methods and compositions are provided for producing callus cultures, plant tissues, plants and seeds that are tolerant and/or resistant to a reduction in water availability under conditions that normally inhibit the function or growth of these cultures, tissues, plants or seeds. Such plants and seeds sexually can ~ this trait to their progeny.
The methnr~ provided in the present invention may be used to produce ill~Gased levels of osmoprol~ x, such as a sugar in monocots and other cereal crops in-~.ln-lin~, but not limited to, maize, rice, rye, millet, wheat, barley,sorghum, and oats.
In accord with the present invention, a preS~l~CteC1 DNA segm~?nt is icl~ntifietl, isolated, and cnmhin~ll with at least a promoter functional in a plant cell to provide a recombinant eA~.ession c~sette Once forrned, an e~ ssion cassette co,..l" ;~i~.g a preselected DNA segm~nt can be subcloned into a known ~A~ ion vector. Suitable known eA~s~ion vectors include plasmids that autonomously replicate in prokaryotic and/or eukaryotic cells. Specific e~mples 30 include plasmids such as pUC, pSK, pGEM, pBS and pSP-derived vectors, the W O 97/26365 PCTrUS97/00978 pBI121 or pBI221 plasmid constructed as described by JGrr~ u~l (Pl. Mol. ~iol.
~pL, 5, 387 (1987)), or a binary Ti plasmid vector such as pG582 as described by An (Plant Cell. 1, 115 (1989)), and the like.
An G~ ion cassette of the invention can be subcloned into an S G,.~.es~ion vector by standard methods. The G~lession vector can then be introduced into prokaryotic or eukaryotic cells by ~ lclllly available methods inelllriing, but not limited to, protoplast tr~n~ on~ ,4grobacterium-merli~t~d tran~rul~ ion~ ele~kupuldlion, microprojectile bombaldlllc~ll, tllng.~ten whiskers (Coffee et al., U.S. Patent No. 5,302,523, issued April 12, 1994) and liposr)me.s The vector can be introduced into prokarvotic cells such as E. coli or Agrobacterium. Tlans~llned cells can be selected typically using a selectable orscreenable marker encoded on the G~res~ion vector.
The G~lG:i~ion cassette or vector can be introduced into monocot plant cells. Plant cells useful for transformation include callus, illllll~l/lllG embryos, 15 m~ri~tP n~tic tissue, gametic tissue, or cultured suspension cells. Optionally, other preselect~ DNA se,~.,.~,.l~ encoding enzymes which catalyze the synthesis of osmc,~lole-;l~ll~ can be introduced into the plant cell. The k~l~l..led plantcell can then be lG~ d into a plant and the plant tested for its ability to grow or thrive under stress cont1ition~, such as high salinity or reduced water 20 availability. DGpC~ g on the type of plant, the level of gene G~ iion, and the activity of the enzyme encoded by the preselected DNA se~ l, introduction of the preselected DNA into the plant can confer the phenotype of tolerance or nce to water deficit to the plant.
The introduced preselected DNA SG~ G11l:; can be G~yl. ssed in the 25 L~rol.lled monocot plant cells and sta~ly ll,...~ d (s~m~tir~lly and sexually) to the next generation of cells produced. The vector should be capable of introducing, .,.~ ,;.,g, and G~ e~ g a preselected DNA segrnent in plant cells, whG~eill the preselected DNA can be obtained from a variety of sources, including but not limited to plants and ~nim~l~, bacteria, fungi, yeast or virus.
30 Additionally, it should be possible to introduce the vector into a wide variety of W O 97/2636~ PCTnUS97/00978 cells of monocot plants. The preselected DNA segm~n~ is passed on to progeny by normal sexual tran~mi~ion.
Introduction and ~ c;s~ion of foreign genes in dicotyledonous (broad-leafed) plants such as tobacco, potato and alfalfa has been shown to be possibleS using the T-DNA of the turnor-in~lllcing (Ti) pl~mic~ of Agrobacterium tumefaciens. Using recombinant DNA techniques and bacteri~l genetics, a wide variety of foreign DNAs can be inserted into T-DNA in ~grobacterium.
Following infection by the ba~ih,.;ulll c~ the reco~ llL Ti plasmid, the foreign DNA is inserted into the host plant chr~ mr~somto~, thus producing a 10 genetically ~ng;,,~el~1 cell and eventually a g~qnetic~lly et y~ plant. Asecond approach is to introduce root-in~lcing (Ri) plasmids as the gene vectors.While Agrobacterium appear to attack only dicots, many hll~olL~t crop plants including maize, wheat, rice, barley, oats, so~llulll, millet, and rye are monocots and are not known to be ~usc~lible to !.,~ r.,lll7~1;nn by 15 ,4grobacterium. The Ti plasmid, huwt;~ , may be lll~i~ulated in the future toact as a vector for monocot plants. Additionally, using the Ti pl~cmi~l as a model system, it may be possible to artificially Co~ u~ ~rlJllll;~l;on vectors for monocot plants. Ti-plasmids rnight also be ir troduced into monocots by artificial methods such as microinjection, or fusion between monocot protoplasts and 20 bacterial spheroplasts coll~ g the T-region, which can then be integrated into the plant nuclear DNA.
Tl~ro~ Lion of plant cells witn a pres~l~cte(l DNA segm~ont may also be accG.ll~lished by introducing a l)leselecl~ DNA into other nueleic acid molecules that can transfer the hlselLed DNA into a plant gc.~..lll~., e.g., plant 25 pathogens such as DNA viruses like CaM~ or geminiviruses, RNA viruses, and viroids; DNA moleeules derived from unstable plant genome eollll)o~ like extrachromosomal DNA elem~n~ in organelles ~e.g., ehloroplasts or mitochondria), or nuclearly encoded controlling elements, DNA molecules from stable plant genome co~ ,unc~ (e.g., origins of replication and other DNA
30 sequences which allow introduced DNA to illLt;~ le into the organellar or nuclear W O 97/26365 PCTrUS97/00978 genomes and to replicate norrnally, to a--tonomously replicate, to segregate normally during cell division and sexual reproduction of the plant and to be inherited in s-lccee-1inF~ generations of plants) or transposons.
A preselected DNA may be delivered into plant cells or tissues directly by 5 microorg~ni~m~ with infectious plasmids, infectious viruses, the use of liposomes, microinjection by m~r~h~nic~l or laser beam methods, by whole chromosomes or chromosome fragments, electroporation, and microprojectile bombardment.

I. Recipient Cells Practicing the present invention inchlr~ee the gG.~.~lion and use of recipient cells. As used herein, the term "recipient cells" refers to monocot cells that are receptive to L~ Çol.l.ation and subsequent regeneration into stably LL~l~..lled, fertile monocot plants.

A. Sources of Cells Recipient cell targets include, but are not lirnited to, mPri~t~tn cells, Type I, Type II, and Type III callus, ;~ llG embryos and g~m~tic cells such as microspores pollen, sperm and egg cells. Type I, Type II, and Type III callus may be initi~t~l from tissue sources in~ ling, but not limited to, ;Il~lO~IlllG
20 embryos, see~11ing apical ".~ , miclus~ules and the such. Those cells which are capable of proliferating as callus are also recipient cells for genetic rol~ lion. The present invention provides techniques for LL~l,r~,.l. i~lg i"....~ , embryos followed by initiation of callus and ~ubsc~uGlll le~ G...,.dlion of fertile L.~ g~.~ic plants. Direct ~ "~iru",.,,l;cn ûf ;~ h~ c c.ll13~yc)s ûbviates the 25 need for long term development of recipient cell cultures. Pollen, as well as its precursor cells, microspores, may be capable of fimctioning as recipient cells for genetic l~ r~ll..ation, or as vectors to carr~ foreign DNA for incul~,ol~lion during fertilization. Direct pollen tr~n!iru.."nl;on would obviate the need for cell culture. Meristematic cells (i.e., plant cells capable of co"~ l cell division and 30 characterized by an undirrG ~ iated cytological appe~cu~ce~ normally found at W O 97/26365 PCTfUS97/00978 , growing points or tissues in plants such as root tips, stem apices, lateral buds, etc.) may r~lc;sel,l another type of recipient plant cell. Because of their undifferenti~tPd growth and capacity for organ dirr~ I;on and totipotency, a single transformed meri~t.om~tic cell could be l~,coveled as a whole L~l.r,~ ed S plant. In fact, it is proposed that embryogenic ~ ..xion cultures may be an invi~ro meri~tem~tic cell system, ref~ining an ability for co~tin-le~ cell division in an undirr~ te~l state, controlled by the media cllvilol~llc~
In certain emboriiment~, cultured plant cells that can serve as recipient cells for ~l,...~r(...";.,g with desired DNA se~".~ include maize cells, and more 10 specific~lly, cells from Zea ma,vs L. Somatic cells are of various types.
Embryogenic cells are one exarnple of somatic cells which may be in~uce~ to legel,~ L~; a plant through embryo formation. Non-embryogenic cells are those which will typically not respond in such a fashion. An exarnple of non-embryogenic cells are certain Black Mexican Sweet (BMS) maize cells. These 15 cells have been l,~ul.ru.",ed by microprojectile bombardment using the neo gene followed by selection with the aminoglycoside, k~ly-;ill (Klein et al., Plant P~ysiol, ~, 440 (1989)). ~Iowever, this BMS culture was not found to be regenerable.
The development of embryogenic rnaize calli and .~ el~iQn cultures 20 useful in the context of the present invention, e.g., as reripient cells for Ll~rol~ ion, has been described in Gordon et al. (U.S. Patent No. 5,134,074, issued July 28, 1992, inco.~olaled herein by ,~,r~.~llce).
The present invention also provides certain techniques that may enrich recipient cells within a cell population. For ~ lc, Type II callus development, 25 followed by manual selection and culture of friable, embryogenic tissue, generally results in an enrirhm~ont of recipient cells for use in, e.g., microproJectile r~ )n Suspension culturing, particularly using the media disclosed herein, may also improve the ratio of recipient to non-recipient cells in any given population. Manual selection techniques which employed to select recipient cells may include, e.g., ~e~es~inp cell morphology and ~ ion, or may use CA 02243269 l998-07-l6 W O 97/26365 PCT~US97/00978 various physical or biological means. Cr~ol)lesc~lation is also colllc~ lated as a possible method of selecting for recipient cells.
Manual selection of recipient cells, e.g., by selecting embryogenic cells from the surface of a Type II eallus, is one means employed in an attempt to S enrich for reçipient cells prior to cllltllring (whcll~ eultured on solid media or in s-T~ren~inn). The p~cr~ ,d cells may be those loeated at the surface of a eell eluster, and may further be i(lentifi~ble by their laek of dirr.,.~ liation, their size and dense cytoplasm. The plGr~ d cells will gPn~r~lly be those eells which are less dirrt;r~ or not yet c~ ..l.;lled to diLr~ inn Thus, one may wish 10 to identify and seleet those cells which are ~;y~opl~llliç~ily dense, relatively unvacuolated with a high nucleus to cytoplasm ratio (e.g., ~t~nninrd by eytological observations), small in size (e.g., 10-20 ,um), and capable of s-let~in~d divisions and somatic proembryo forrn~tio~
It is proposed that other means for id~nLiryillg such eells may also be 15 employed. For example, through the use of dyes, sueh as Evan's blue, which are eYrl-lcleA by cells with relatively non~ .P~hle m~rnhr~n~s, such as embryogenic cells, and taken up by relatively di~e~ 1~1 cells such as root-like cells and snake cells (so-called due to their snake-like ~pc.~
Other possible means of identifying reçipirnt cells include the use of 20 isozyme m~rk~rs of emb~ug~-ic cells, such as ~ dehydrogenase, which can be ~etected by cytorh-onnir.~l stains (Fransz et al., Pl~nt Cell Rep., 8, 67(1989)). However, it is e~lltione~l that the use of iso;~yll~c m~rk~.r~ such as dehydrogenase may lead to some degree of false ~o~ ,3 from non-embryogenic cells such as rooty cells which ..~ have a relatively high 25 metabolic activity.

B. Media In certain emboll;.. l~i, recipient cells are selected following growth in culture. Where employed, cultured cells will preferably be grown either on solid30 ~u~,~o~ or in the form of liquid su~el~;on~ In either in~t~nre, mltrient~ may CA 02i43269 1998-07-16 WO 97/26365 PCTrUS97/00978 be provided to the cells in the form of media, and envirf~nm~nt~l conditions controlled. There are many types of tissue culture media comprised of amino acids, salts, sugars, growth regulators and vit~mine Most of the media employed in the practice of the invention will have some similar co,l~one~ (see, e.g., S Table 1 hereinbelow), the media differ in the composition and ~lu~ollions of their ingredients depending on the particular application envisioned. For example, various cell types usually grow in more than one type of media, but will exhibit di~ growth rates and ~li~el.,llL morphologies, dep.-.n~ing on the growth media. In some media, cells survive but do not divide.
Various types of media suitable for culture of plant cells have been previously described. Examples of these media inchl(le but are not limited to, the N6 medium described by Chu et al. (Sci~nti~ Sinica, ~, 659 (1975)) and MS
media described by Murashige & Skoog (Pl~nt Pllysiol., 15, 473 (1962)). Media such as MS which have a high ~mmoni~/nitrate ratio are coullt~ ductive to the 15 generation of recipient cells in that they promote loss of morphogenic capacity.
N6 media, on the other hand, has a somewhat lower ~..,...~ ..;~/nitrate ratio, and is co~ lated to promote the generation of l~ ll cells by .--~;..Ji.;.,;..g cells ina proembryonic state capable of sllet~in~rl divisions.

C. Cell Cultures 1. Tniti~ n In the practice of the invention it is SOl, ~I;".~e~ but not always, .,~c~es,~. y to develop cultures which contain recirilont cells. Suitable cultures can be initi~t.od from a number of whole plant tissue exr1~nte inr~ ing, but not limited 25 to, imm~tllre embryos, leaf bases, ;1(~ , tassels, ~nth~re, microspores, and other tissues co~ l.;..g cells capable of in vi~ro prolif~-~tinn and ,~,~e~ J;on of fertile plants. In one exemplary embodiment, recipient cell cultures are initi51t~
from imm~ re embryos of Zea mays ~. by growing excised ;.. ~ c embryos on a solid culture me~ m co- J~;.-;--~ growth regulators inclu~ing~ but not limited 30 to, dicamba, 2,4-D, NAA, and IAA. In some in~t~n~es it will be L~lGr~ d to add silver nitrate to culture medium fior callus initiation as this compound has been reported to çnhs7nce culture initiation (Vain et al., Plant Celi. tissue and Or~an Clllture., 1~ 143 (1989)). Embryos will produce callus that varies greatly in morphology including from highly u"~ li;Ged culLu,~3 cc ~ very early S embryogenic ~l~u~iLules (such as, but not limited to, type II cultures in maize), to highly o,~ cl cultures contz7ining large late embryogenic ~llu~ s (such as, but not limited to, type I cultures in maize). This variation in culture morphology may be related to genotype, culture l~f.lil,.,, composition, size of the initial embryos and other factors. Each of these types of culture morphologies is 10 a source of recipient cells.
The development of s..~ )n cultures capable of plant regeneration may be used in the context of the present invention. S--epçnc;on cultures may be initis7tçcl by trs7nefio7Ting caUus tissue to liquid culture m~7illm co..~ g growth regulators. ~7.~iition of coconut water or other s-lhstz7n~es to su~l,G"~ion culture 15 lllediulll may enhance growth and culture morphology, but the utility of ~u ,~.,.~ion cultures is not limited to tllose c~ ;..;..g these cu",pou"ds. In some embo~iim~nt~ of this invention, the use of ~u~ ~ion cultures will be pler~"~,d as these cultures grow more rapidly and are more easily manipulated than callus cells growing on solid culture m~ 7 When imms7t-7re embryos or other tissues directly removed from a whole plant are used as the target tissue for DNA delivery, it will only be ..~cç~ to initiate cultures of cells insofar as is I~Pce~ for i~7.~ntificz7tion and isolation of h al~r(J~ a7lts. In an illu~LI~liv~ embor7im~nt, DNA is inhroduced by par~ticle bomb~ nL into imms7h-re embryos following their excision from the plant.
25 Embryos are L d.~r~ d to a culhure .~ ll that will support proliferation of tissues and a.low for selection of L.~r~l."ed sectors, at about 0-14 days following DNA delivery. In this embodiment of the invention it is not nPce~
to establish stable callus cultures capable of long term .~ S~ e and plant ,egenc.dLion.

W O 97/26365 PCT~US97/00978 ~ 20 2. ~intPn~nce The method of ~ e~)~nce of cell cultures may contribute to their utility as sources of recipient cells for transformation. Manual selection of cells for LL~irc~ to fresh culture mediurn, r~ ucll~;y of LL~l~rcl to fresh culture medium, S composition of culture m~ m, and e.lvilolLLllent factors incll~rlin~, but not lirnited to, light quality and ~u~llily and lclllpeldLulc are all i.llpolL~IL factors in m~ .g callus and/or su~cl~ion cultures that are useful as sources of recipient cells. It is cQ~ te~ that ~ callus beLwccll dirr~ l culture conditions may be bçn~fici~l in enriching for reCipient cells within a culture. ~or 10 example, it is proposed that cells may be cultured in ~u~ .~ion culture, but ll~r~Llcd to solid 1"~ ."~ at regular interv-als. After a period of growth on solid m~ m cells can be m~nll~lly selected for return to liquid culture mer~ m It is proposed that by l~eaLulg this sequence of h~lL~r~,~ to fresh culture me~ m it is possible to enrich for recipient cells. It is also cont~mpl~t~d that passing cell 15 cultures through a sieve, e.g., a 1.9 mm sieve, is useful in m5.;1~;i~.g the friability of a callus or ~ ,c.l~;on culture and may be b~n~fici~l is t?nric~hin~ for Ll~rollllable cells.

3. Clyo~ . vz.lion Additionally, clyu~l_sci.v~LLion may effect the development of, or perhaps select for, recipient cells. Clyu~!les~,v~LLion selection may operate due to a selection again3t highly vacuolated, non-embryogenic cells, which may be sclccli~,~,ly killed during clyu~l~sel~ration. There is a ~l~,ol~Ll window in which cultured cells retain their l~ ;Ve ability, thus, it is believed that they must be IJL~,s_. v-ed at or before that t~;lll~olal period if they are to be used for future tranaçu~ l;Qn and 1~ ;f)n For use in ~a~LarullllaLion~ suspension or callus culture cells may be ~;lyo~ s~l ~/ed and stored for periods of time, thawed, then used as recipient cells for l d,lsru.lll~ion. An illustrative embodiment of ~,lyupl~,sel~/ation methods 30 comprises the steps of slowly adding ~;lyo~!loLe~ to ~u~ell~ion cultures to W O 97/2636~ PCTrUS97/00978 give a final concentration of 10% dimethyl sulfoxide, 10% polyethylene glycol (6000MW), 0.23 M proline and 0.23 M glucose. The Illixlul~ is then cooled to -35~C at 0.5~C per minute. After an isotherrnal period of 45 mimltes, sarnples are placed in liquid N2 (modification of methods of Withers et al., Pl~nt Pllysiol., 5 64, 675 (1979), and Finkle et al., Pl~nt Sci.. 42, 133 (1985)~. To reinitiate ~u~ ion cultures from ~;l~p~,sc~.,d material, cells may be thawed rapidly and pipetted onto feeder plates similar to those described by Vaeck et al. (N~hlre, ~, 33 (1987~).

10 II. DNA Sequences Virtually any DNA composition may be used for delivery to l~cipie.lL
monocotyledonous cells to llltim~tely produce fertile ~.,...e~ ic plants in accordance with the present invention. For ~ox~mpl~, a preselected DNA segment encoding a gene product whose c~ ion confers an in "~ase in intracellular 15 mannitol levels, or drought re~ict~nc~e, in the forrn of vectors and pl~mi~ie, or linear DNA fir~gmPnte, in some ;~ e co~ g only the DNA element to be c~ scd in the plant, and the like, may be employed.
In certain emboriimente, it is co~ pi~t~d that one may wish to employ replication-cc..ll.cLc..L viral vectors in monocot l.;.l-~r~ n. Such vectors 20 include, for exarnple, wheat dwarf virus (WDV) "shuttle" vectors, such as pWI-11 and PW1-GUS (Ugaki et al., Nucl. Acid Res., 1~, 391 (1991)). These vectors are capable of autonomous replication in maize cells as well as E coli, and as such may provide inc,Gased sel~ilivily-for detecting DNA dcli~ ,1 to ~lSgGl,ic cells. A replicating vector may also be useful for delivery of genes fl~nk~t1 by25 DNA sequences from transposable elements such as Ac, Ds, or Mu. It has been proposed ~Laufs et al., Proc. N~tl Acad. Sci. USA. ~, 7752 (1990)) that transposition of these elements within the maize genome lG~IUilGS DNA
replication. It is also co~lGll~lated that transposable el~ would be useful for introducing DNA fr~E~n~nt~ lacking elements ~ ces~.y for selection and m~ .,ce of the p}asmid vector in bacteria, e.g., antibiotic reci~t~n~e genes and origins of DNA replication. It is also ~lu~osed that use of a transposable element such as Ac, Ds, or Mu would actively promote hllcgldLion of the desired DNA
and hence increase the frequency of stably l~ arlJLllled cells.
Vectors, pl~cmi ic, coxmi~ie~ YACs (yeast artificial chromosomes) and 5 DNA se~ for use in ~ r...."i,.g such cells will, of course, generally compriee the pr~eP!lected cDNA(s), preselected DNA(s) or genes which one desires to introduce into the cells. These DNA constructs can further include structures such as promoters, ~nhzlnc~rs, polylinkers, or even regulatory genes as desired. The DNA segrnt~nt or gene chosen for cellular introd~ction will often 10 encode a protein which will be ~ rejsed in the ~ recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotvpe to the l~gcl~eLdlcd plant. EIowever, this may not always be the case, and the present invention also enco...l.~Xx. ~ neg~ . plants inco.~ol~Lli.lg non c~cs:~ed LL~sg~"les related to drought-r~ciet~nre or ..~ ol 15 e~yl~s~ion.
DNA useful for introduction into maize cells includes that which has been derived or isolated from any source, that may be ~ubs~uently ch~a~ ~cd as to u~ c, size and/or function, chemically altered, and later ullloduced into maize. An example of DNA "derived" from a source, would be a DNA sequence 20 or segm~nt that is i~ntified as a useful fragment within a given o~ and which is then ~h~mic~ily synth~ox-;7~ in ece~nt1~lly pure form. An example of such DNA "isolated" from a source would be a useful DNA seyLl. ,~e that is excised or removed from said source by chemical means, e.g., by the use of restriction ~n~lonlTcle~e~s~ so that it can be further manipulated, e.g., amplified, 25 for use in the invention, by the methûdology of genetic çn~;,.Pe~ Such DNA
is commonly lcr~ d to as "recombinant DNA."
Therefore useful DNA inr~n~T~s completely synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from RNA. It is within the scope of the invention to isolate a preselected DNA se~ t from a 30 given maize genotype, and to subsequently introduce multiple copies of the =

preselected DNA segment into the same genotype, e.g., to çnh~nfe production of a given gene product such as a protein that confers tolerance or resistance to water deficit.
The introduced DNA includes, but is not limited to, DNA from plant S genes, and non-plant genes such as those from b~Ct---ri~ yeasts, ~nim~l~ or viruses. The introduced DNA can include modified genes, portions of genes, or chimeric genes, including genes from the sarne or dirr~le,.t maize genotype. I'he term "fhimf-ric gene" or "çhimf-ric DNA" is defined as a gene or DNA sequence or segtn~nt comprising at least two DNA sequences or se~ from species 10 which do not combine DNA under natural conditions, or which DNA sequences or segment~ are positioned or linked in a manner which does not normally occur in the native genome of ~-n~n~formed maize, or other monocot.
The introduced DNA used for ll~rul.llation herein may be circular or linear, double-stranded or single-stranded. Generally, the DNA is in the form of15 chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by regulatory sequences which promote the c~ e;~:iion of the recombinantDNA present in the resultant maize plant. For e~mplf-, the DNA may itself comrri~e or consist of a promoter that is active in maize which is derived from a non-maize source, or may utilize a promoter already present in the maize 20 genotype that is the ~ r"",~liQ~ target.
Generally, the introduced DNA will be relatively small, i.e., less than about 30 kb to Illill;llli~.~ any susceptibility to physical, ch~mif~l~ or el~yll~Llic ~1~gr~ ;on which is known to increasè as the size of the DNA inc.~,2ses. As noted above, the number of ploleil~ RNA Ll.u s~i.ip~s or llli~ s thereof which 25 is introduced into the maize genome is ~lereldbly p~s~rlr~l~d and defined, e.g., from one to about 5-10 such products of the introduced DNA may be formed.

W O 97/2636~ PCTAUS97/00978 .

A. R~ ory F~
The construction of vectors which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure.
S Ultimately, the most desirable DNA sç~.. - ~.l~ for introduction into amonocot genome may be homologous genes or gene families which encode a desired trait (e.g., increased yield per acre) and which are introduced under the control of novel promoters or enh~nrers, etc., or perhaps even homologous or tissue-specific (e.g., root-, collar/sheath-, whorl-, stalk-, e~r~h~nk-, kernel- or leaf-10 specific) promoters or control cle.~ Indeed, it is envisioned that a particular use of the present invention will be the ~L~ ;Lill~, of a l~.e3ele~ik~f1 DNA segm~rlt in a t;ssue- or organelle- or turgor- specific manner.
Vectors for use in tissue-specific targeting of a preselected DNA segment in transgenic plants will typically include tissue-specific promoters and may also 15 include other tissue-specific control elenn~nt~ such as enh~ncer sequences.
Promoters which direct specific or ~nh~nred c..~ ion in certain plant tissues will be known to those of skill in the art in light of the present ~ clc snre. These include, for example, the rbc~ promoter, $pecific for green tissue; the ocs, nosand mas promoters which have higher activity in roots or wounded leaf tissue; a 20 Llullc~Led (-90 to +8) 35S promoter which directs ~nh~n~ec~ ~;A~ie3~ion in roots, an a-tubulin gene that directs ~ s:,ion in roots and l)roll-oL.,l~ derived from zein storage protein genes which direct ~A~l~s:,ion in en-1ospçrrn It is particularly contemplated that one may adv~nt~geo~ y use the 16 bp ocs Al~ element from the o~;~opille ~ hasc (ocs) gerle ~Ellis et al., supra 25 (1987); Bouchez et al., supra (1989)), especially when present in multiple copies, to achieve ~nh~n~ed ~A~ ion in roots.

W O 97t26365 PCTAUS97/00978 B. Marker Genes In order to improve the ability to identify l-~ro~ ants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the s~ible preselected DNA segm~t "Marker genes" are genes that impart a 5 distinct phenotype to cells t~ t~ g the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker.
Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can 'select' for by chemical means,i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), 10 or whether it is simply a trait that one can identify through observation or testing, i.e., by 'screening' (e.g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the mvenhon.
Included within the terms select~hle or screenable marker genes are also 15 genes which encode a "secretable marker" whose secretion can be ~1~ectec~ as a rneans of identifying or se!~cting for L,d~ro..l~ed cells. F~mples include r~:i which encode a secretable antigen that can be id~ntifi~A by antibody interaction, or even secretable enzymes which can be de~ct~-l by their catalyticactivity. Secretable pr~,Leills fall into a nurnber of classes, inclu~linp small, 20 diffusible ~ ins cletect~hle, e.g., by ELISA; and p~ illlS that are inserted or trapped in the cell wall (e.g., pl~ ills that include a leader sequence such as that found in the t;~ ion unit of ~x~ or tobacco PR-S).
With regard to selectable secretable m~rk~rq, the use of a gene that encodes a protein that becom~c sequestered in the cell wall, and which protein 25 incl~l-les a unique epitope is considered to be particularly advantageous. Such a secreted antigen marker would ideally employ an epitope sequence that would provide low ba~ oul,d in plant tissue, a promoter-leader sequence that would impart effici~nt ~ .,ssion and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet ~ccesqihle to antibodies. A

W 097/26365 PCT~US97/00978 normally secreted wall protein modified to include a unique epitope would satisfy all such ~4uh~ ents~
One example of a protein suitable for modification in this manner is exten~Tn, or hy~h~xylJloline rich ~lycoplotein (HPRG). The use of the maize S HPRG (Steifel et al., The PlRnt Cell, ~, 785 (1990)) is ~lc~r~,Ll~,d as this molecule is well chh. n~ ;~;d in terms of molecular biology, ~ le~iion, and protein structure. However, any one of a variety of ~ and/or glycine-rich wall proteins (Keller et al., F.l\~R~O J.. ~, 1309 (1989)) could be modified by the addition of an antigenic site to create a screenable marlcer.
F.lemf~nt~ of the present disclosure are exemplified in detail through the use of particular marlcer genes, however in light of this disclosure, nulll~,lous other possible selectable and/or screenable marker genes will be a~ lll to thoseof skill in the art in addition to the one set forth hereinbelow. Therefore, it will be understood that the following ~ cl~cion is e~rPmplRry rather than ~xhdu~ e.
In light of the technic~ues disclosed herein and the general recombinant techniques which are known in the art, the present invention renders possible the introduction of any gene, including marker genes, into a reçipient cell to gcl~Ldle a transformed monocot.

1. Sr~cL ble Markers Possible selectable i~h.l~...'i for use in c~"",e.~;on with the present invention include, but are not limited to, a neo gene (Potrykus et al., Mol. GenGenet., 1~2, 183 (1985)) which codes~for kanarnycin rçsi~tRn~e and can be selected for using ka~alny~ l, G418, and the lilce; a bar gene which codes for 25 bialaphos re~i~tRn~e; a gene which encodes an altered EPSP ~ylll~lase protein(Hinchee et al., Riotech ~, 915 (1988)) thus confernnP gly~hosdl~ resi~tRnce a nitrilR~e gene such as ~xn from Klebsiella ozaenae which confers re~i~tRnre to bromoxynil (Stalker et al., Scicnce, ~, 419 (1988)); a mutant acetolactate synthase gene (ALS) which confers resistance to imi<~7t~1inone, sulfollylulea or30 other ALS-inhibiting chPnn;rRI~ (Eulv~eal1 Patent Application 154,204, 1985); a W O 97/26365 PCTrUS97/00978 methotrexate-resistant DHFR gene (Thillet et al., J. Riol. Chem, 263, 12500 (1988)); a dalapon dehalogenase gene that confers resict:~nce to the herbicide dalapon; or a mllt~ted ~ dnilate synthase gene that confers r~sict~nre to 5-methyl ll~lo~ an. Where a mutant EPSP synthase gene is employed, additional 5 benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0,218,571, 1987).
An illustrative embodiment of a selectable marker gene capable of being used in systems to select tran~roLlll,ulls is the genes that encode the enzyme phosphinothricin ac~:lyll~ sferase~ such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes ~U.S. patent application Serial No. 07/565,844, which is incol~o~ d by reference herein).
The enzyme phosphinothricin acetyl ~ Ç~Iase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits pl~ .."ii~e synth~t~cP, (Mllr~k~mi et al., Mol. Gen. G~net., ~, 42 (1986); Twell15 et al., Pl~nt Pbysiol.. 21, 1270 (1989)) causing rapid accumulation of ~rnmnrliz and cell death. The success in using this selective system in conjunction with monocots was particularly surprising because of the major difficulties which have been reported in tran:jrol.--~ion of cereals (Potrykus, Tren~lc P~iot~rh 1, 269 (1989))-2. S~ able Markers Screenable mz~rk~rc that may be employed inrlu-lP, but are not limited to, a ,B-glu ;ulu~dase or uidA gene (GUS~ which encodes an el~y-.lc for which various chromogenic ~ c are knûwn; an R-locus gene, which encodes a 25 product that regulates the production of anthocyanin pigmrnt.c (red color) in plant tissues (Dellaporta et al., in Chro!T osonl~ Stnlr.hlre ~nd Fl-nction, pp. 263-282 (1988)); a ~ rt~m~ce gene (Sutcliffe, PNA~ US~ 75, 3737 (1978)), which encodes an enzyrne for which various chromogenic ~u~):ilrdles are known (e.g., PADAC, a chromogenic cephalosporin); a ~ylE gene (Zukowsky et al., PNAS
30 .USA, 80, 1101 (1983)) which encodes a catechol dioxygenase that can convert W O 97/26365 PCT~US97~00978 chromogenic catechols; an a-amylase gene (Ikuta et al., P~iotech ~, 241 (1990));a tyrosinase gene (Katz et al., J. Gen. Microbiol., 1~, 2703 (1983)) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopa~uinone which in turn con~1PncPs to form the easily tletect~hle compound mel~nin a ~-S galactosidase gene, which encodes an enzyme for which there are chromogenic~ h~ lP~, a ln~ir~ e (1~) gene (Ow et al., Scipnr~ ~, 856 (1986)), which allows for biolnm;..Psce...-e detection, or even an aequorin gene (Prasher et al., Riorh~m. Riopllys Res. Cornm ~, 1259 (1985)), which may be employed in c~lril-m-sensitive biolnmin~sc~nr-G detection, or a green fluo.cscGll~ protein gene 10 (Niedz et al., Pl~nt Cell Reportc ~4, 403 (1995)).
Genes from the maize R gene complex are cu..~ tecl to be particularly useful as screenable markers. The R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pi~m~nte in most seed and plant tissue. Maize strains can have one, or as many as four, R alleles which 15 combine to regulate pigmPnt~tion in a develop..~ l and tissue specific manner.
A gene from the R gene cnmrle~ was applied to maize l~ alioll~ because the ~ iion of this gene in /~,1..cr~ d cells does not harm the cells. Thus, an R gene introduced into such cells will cause the ~ ion of a red pi~m~nt and, if stably inco~porated, can be visually scored as a red sector. If a maize line is 20 carries do~ alleles for genes encoding the enzymatic intPrme~i~tes in theanthocyanin biosynthetic ~lhw~y (C2, Al, A2, Bzl and Bz2), but carries a recessive allele at the R locus, ~ "..~tion of any cell from that line with R
will result in red pi mPnt fonn~tion F.~emrl~ry lines include Wieconein 22 which col~klins the rg-Stadler allele and TR112, a KSS d~,.;v~liv~ which is r-g, b, 25 Pl. ~ltPm~tively any genotype of maize can be utilized if the C1 and R alleles are introduced together.
It is further proposed that R gene regulatory regions may be employed in ~himPric constructs in order to provide merh~nicme for controlling the ~ ,;,sionof chirneric genes. More div~ y of phenotypic c;~ession is known at the R
30 locus than at any other locus (Coe et al., In: Corn ~nrl Corn Irr~rovernent W O 97/26365 PCT~US97/00978 Sprague et al. (eds.) pp. 81-258 (1988)). It is co,.~ t~cl that reF~ tc)ry regions obtained from regions S' to the ~ u~ dl R gene would be valuable in directing the c;A~ ion of genes, e.g., insect l~ ;X~ re drought reeiet~nl~e, herbicide tolerance or other protein coding regions. For the purposes of the 5 present invention, it is believed that any of the various R gene family members may be sllccçeefully employed (e.g., P, S, Lc, etc.). However, the most ~ r~,L~dwill generally be Sn (particularly Sn:bol3?. Sn is a dc,...;~ member of the R
gene complex and is functionally similar to the R and B loci in that Sn controlsthe tissue specific deposition of anthocyanin pi m~onte in certain see~lling and10 plant cells, therefore, its phenotype is similar to R
A further screenable marker c.,llt~ lated for use in the present invention is firefly luciferase, encoded by the l20C gene. The presence of the l2~x gene in transformed cells may be cletpc~tecl using, for e~r~mple, X MY filrn, scint~ tic n counting, fluol~ ecL,opll(~Lo.ll~ , low-light video r~mer~e, photon 15 counting c~mf r~e or multiwell lurninometry. It is also envisioned that this system may be developed for poplll~ti-n~l s~ ,lfillg for biol...... ;.. ~ ~cf-.re, such as on tissue culture plates, or even for whole plant s-,~f~ lg.

C. Tr,~,~s~ or Maize Moflifi-Improvement of the ability of maize to tolerate various environmf nt~l stresses in~ ling, but not limited to, drought, excess moisture, chilling, freezing, high lelllp~la~ salt, and oxidative stress, can be effected through ~ ;saion of heterologous, or ov~ ion of homologous, genes.
Expression of novel p.~ rPlç~;~e(l DNA se~ that favorably effect plant water content, total water potential, osmotic pot~nti~l, and turgor can enh~nl e the ability of the plant to tolerate dLv~hl. As used herein, the terrns "drought e" and "drought tolerance" are used to refer to a plants h1cl~ ascd r~si~t~n~ e or tolerance to stress inrlrl~ed by a red~ .tion in water availability, as con~aLed to norrnal circllm~t~n~ , and the ability of the plant to function and 30 survive m lower-water ~llvir~ - ~nt~, and pl~llll in a relatively superior llla~ CL.

W 097/26365 PCTfUS97/00978 In this aspect of the invention it is proposed, for e~ mple, that the e~ ,ssion of a preselected DNA segment encoding the biosynthesis of osmotically-active solutes can irnpart protection against drought. Within this class of preselectedDNA segm~ntc are DNAs encoding m~nnitcl dehydrogenase (Lee and Saier, L
5 Racteriol., 153 (1982)) and trehalose-6-phosphate synthase (Kaasen et al., 1 Racteriol. 1~, 889 ~19923). Through the subseguent action of native phosph~t~ees in the cell or by the introduction and co~ ession of a specific phosphatase, these introduced presf lected DNAs will result in the acc--mnl~tiQnof either m~nnitol or trehalose, respectively, both of which have been well 10 docnrnentçd as protective compounds able to mitig~te the effects of stress.
Mannitol ~cc Im--l~ti~ n in transgenic tobacco has been verified and prelimin~ryresults inrlir~tç that plants ~ es~ g high levels of this metabolite are able totolerate an applied osmotic stress (Tarczynski et al., cited supra (1992), ~1993)).
Sirnilarly, the efficacy of other metabolites in ~lote.;lillg either ~l~y~llc 15 function (e.g. alanopine or propionic acid7 or m~mhr~ne integrity (e.g., alanopine) has been doc~-mentrd (Loomis et al., J. Fxpt. 7 -ol.. ~, 9 (1989)), and therefore expression of a pres~olected DNA segrn~nt encoding the biosynthesis of these compounds can confer drought rç~i~t~nre in a manner similar to or compli".e..~y to ...~ . Other examples of n~tllrally oCc~lrring metabolites 20 that are osmotically active and/or provide some direct protective effect during drought and/or desiccation include sugars and sugar derivatives such as fructose, c ~yllllik~l (Coxson et al., Bio1ropica. ~, 121 (1992)), sorbitol, dulcitol (Karsten et al., Rot~nica M~rin~ 35, 11 (1992)), glucosylglycerol ~Reed et al., l~çn~
Microbiol., ~Q, 1 (1984); Erdmann et al., J. G~n Microbiol~, 138, 363 (1992)), 25 sucrose, stachyose (Koster and Leopold, Pl~nt Plursiol~, ~, 829 (1988); Bl~rl~m~n et al., Pl~nt Ph,ysiol ~, 225 (1992)), ononitol and pinitol (Vernon and Bohnert,F~RO J.. ~, 2077 (1992)), and raffinose (Bernal-Lugo and Leopold, ~n~
P~ysiol.. 98, 1207 (1992)). Other osmotically active solutes which are not sugars include, but are not limited to, proline (Ren~ et al., 1993) and glycine-betaine30 (Wyn-Jones and Storey, ln: Ph~ysiolo~y ~nd Rioch~mi~try of Drol~ht R~ t~nce W O 97/26365 PCTrUS97/00978 .

;n Pl~nts, Paleg et al. (eds.), pp. 171-204 (1981)~. Continllr~l canopy growth and increased reproductive fitness during times of stress can be ~ mentPd by introduction and c2~le:i~ion of preselected DNA segmente such as those controlling the osmotically active compounds ~1ieclleeerl above and other such 5 compounds, as l~lcscllLed in one e~pmr!slry embodiment by the enzyme myoinositol 0-methylLldllsrcldse.
It is corltPmplated that the c~l~ssion of specific proteins may also increase drought tolerance. Three classes of Late Embryogenic Proteins have been ~eei~nP~l based on structural eimil~rities (see Dure et al., Pl~nt Mol P~iol., 10 ~, 475 (1989)). All three classes of these proteins have been demonstrated inm~tllring (i.e., desiccating) seeds. Within these 3 types of proteins, the Type-II
(dehydrin-type) have generally been implic~tPIl in drought and/or desiccation tolerance in vegetative plant parts (i.e. Mundy and Chua, Fl\~RO J-,l, 2279 (1988); Piatkowski et al., Pl~nt Physiol.. ~L 1682 (1990), Yamaguchi-ShinnYzlki 15 et al., pl~nt Cell P~ysiol.. 33, 217 (1992)). Recently, c~,cs~ion of a Type-lII
LEA (HVA-1) in tobacco was found to inflllPnre plant height, maturity and drought tolerance (Fit~tricl~. Gen. F,r~i~.r..;~ News, ~, 7 (1993)). Expression of structural genes from all three groups may therefore confer drought tolerance.
Other types of proteins in~nre-l during water stress include thiol ~.~uleases, aldolases and tr~nememhrane ~ olL~l~ (Guerrero et al., Pl~nt Mol. ~iol., lS, 11 (l9gO)), which may confer various ~,lotc~ e and/or repair-type functions during drought stress. The c~-es~ion of a presp-lectrc~ DNA se~nent that effectslipid biosynthesis and hence l~lc.llbLaile composition can also be useful in c~ r~ g drought reeist~nre on the plant.
Many genes that improve drought r~siet~nce have complrm~nt~ry modes of action. Thus, combinations of these genes might have additive and/or synergistic effects in improving drought r~sief~nce in maize. Many of these genes also improve freezing tolerance (or reeiet~nce); the physical stresses incurred during freezing and drought are similar in nature and may be mitig~ted in similar fashion. Benefit may be conferred via col~liLuLi~e expression of these W O 97/2636~ PCT~US97/00978 3, genes, but the ~-c~..~,d means of cA~.esiil~g these novel genes may be through the use of a turgor-ind~lce(l promoter (such as the promoters for the turgor-in~ red genes described in Guerrero et al. (p'l~nt Mo~ r Riolo~y, 15, 11 (1990)) and Shagan et al., Pl~nt Pllysiol., 101, 1397 ~1993), which are 5 incc~ u~dlcd herein by .er~ ce). Spatiai and h.~ u~a} e.~.c~:~ion p~tt~rn~ of these genes may enable maize to better withct~n~l stress.
It is ~-uposed that G~l..c~ion of genes that are involved with speçifi~
morphological traits that allow for in,.~sed water ~Yt~ctinn~ from drying soil would be of benefit. For example, introduction and GA~ sion of genes that alter 10 root cl.~c~ ;ctics may ~nh~nre water uptake. It is also co~ p~ i that t:A~ ion of DNAs that enh~nre reproductive fitness during times of stress would be of ~ignific~nt value. For exarnple, eA~ ion of DNAs that improve the syl.~ u..y of pollen shed and receptiveness of the fema}e flower parts, i.e., siL~cs, would be of benefit. In ~.litio~ it is proposed that cA~le~ion of genes that 15 ~ lf~ kernel abortion during times of stress would increase the amount of g~ain to be l~ Lcd and hence be of value. It is further cU~lp~ t~ that regulation of cytokinin }evels in monnçots~ such as m~ize, by intro~ ctinn and .c~ion of an isû~enLc--yl L~ dse gene with a~ru~ Lc regul~tnry se~ can ~mprove .~n.~ncùt stress r~ t~nre and yield (Gan et al., Sci~ nce.
20 ~:ZQ, 1986 (~ 995)).
Given the overall role of water in d~'~--...;..~.~g yield, it is cO!.I. ..l.l~te~l that en~hling maize to utilize water more ~ffici~ntly, through the introduction and cA~ ion of novel genes, will ull~ur~ overall ~- . ru....~ e even when soil water av~ hility is not limitin~ By introducing genes that i y~ur~ tne ability 25 of maize to mnX;...;,S~ water usage across a full range of stresses relating to water availability, yield stability or co~ y of yield ~. r,....~ c may be realized.

D. Preparation of an E~ ;.;o~
An eA~.c~:,ion cassette of the invention can comrri~e a recombinant DNA
30 mo}ecule c~ n;..;..g a pres~7ecte.7 DNA segment operably linked to a promoter W O 97/26365 PCTrUS97/00978 functional in a host cell. A preselected DNA segmPnt can be identified and isolated by standard methods, as described by Sambrook et al., Molecular Clonin~: A J ~horatory Manual, Cold Spring Harbor, NY (1989). The preselected DNA segmerlt can also be obtained from water stress-tolerant cell lines. The S preselected DNA segmPnt can be identified by SC~ lng of a DNA or cDNA
library genl L~tecl from nucleic acid derived from a particular cell type, cell line, primary cells, or tissue. Screening for DNA fr~gm.ont~ that encode all or a portion of the pr~sel.octe~ DNA segment can be ~ccomrli~h~o<i by ~ G~lLing plaques from a genomic or cDNA library for hybri~i7~tion to a probe of the 10 DNA from other O~ or by s.,l.,c,ling plaques from a cDNA e~ ion library for binding to antibodies that specifically recognize the protein encoded by the preselected DNA segment. DNA fragments that hybridize to a preselected DNA segment probe from other O.~ c and/or plaques carrying DNA
fi~gment~ that are imm-lnoreactive with antibodies to the protein encoded by the15 preselected DNA segm~nt can be subcloned into a vector and se~ Gllced and/or used as probes to identify other cDNA or genomic seqL.~llc~s Pn-~o~ling all or aportion of the preselected DNA segmPnt Portions of the genomic copy or copies of the presel~ctecl DNA segrnPnt can be sequenced and the 5' end of the DNA identified by standard methods 20 including either DNA se~u~LIce homology to other homologous genes or by RNAase protection analysis, as described by Sambrook et al., Molec~ r Clo~in~:
A T~hol~lul~ Manual. Cold ~pring Harbor Press, Cold Spring Harbor, New Yorlc (1989). Once portions of the 5' end of the pre~electe(l DNA segment are ntified, complete copies of the preselected DNA se~ .I can be obtained by 25 standard methods, inrl~ltling cloning or polymerase chain reacbon (PCR) synthesis using oligonucleotide primers cnmplem~nt~ry to the pres~lecterl DNA
segrnent at the 5' end of the DNA. The y~Gscllce of an isolated full-length copyof the preselected DNA can be verified by hybn-li7~ti~ n, partial sequence analysis, or by C~l. .sion of the preselected DNA se~...~..l -W O 97/26365 PCT~US97/00978 The construction of such c~p~ssion c~ettes which may be employed in conjunction with the present inverition will be known to those of skill in the art in light of the present disclosure (see, e.g., Sambrook et al., Molec~ r Clo~
A T.~hol~loly M~ml~l Cold Spring Harbor, New York (1989~, Gelvin et al., ~a~
5 Molecu~ar Riolo~v Mam-~, (1990)~.

1. Prolnoters Once a preselected DNA segment is obtained and amplified, it is operably combined with a promoter to form an CA~ie~7 ,ion c~e~te Most genes have regions of DNA sequence that are known as promoters and which regulate gene eAy~c"ion. Promoter regions are typically found in the fl~nking DNA sequence uy~lL~dlll from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the dow.LslL~l gene se~ ce and typically in~ des from about 15 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as ~nh~nf~er sequences that can infhlen-e the level ofgene ~Xylc~ ,ion. Some isolated plVlllOt~. sequences can provide for gene c~yles~ion of heterologous DNAs, that is a DNA difrclcl-l from the native or homologous ~NA. Promoter se~ucnces are also known to be strong or weak or 20 inducible. A strong promoter provides for a high level of gene c~yfe.,:iion, whereas a weak yr~llol~ provides for a very low }evel of gene c~yL~ion. An inducible promoter is a promoter that provides for turning on and off of gene c;~yl~s~ion in .e.,~olL~c to an exogenously added agent or to an en~ ....L ..1~l or develo~m.ont~l stimulus. Promoters can also provide for tissue specific or 25 develolJ...~ l regulation. An isolated promoter se~u~.lce that is a strong promoter for heterologo~s DNAs is advantageous l~ec~Luse it provides for a sufficient level of gene c~yl~ ,ion to allow for easy ~l~-tecti~ n and selection of transformed cells and provides for a high level of gene e~yl~s~ion when desired.The promoter in an c~yLc;~ion cassette of the invention can provide for~0 ~-cs~ion of the y.e3clecled DNA segmpnt Preferably, the presçll-ctlo~l DNA

W O 97/26365 PCT~US97/00978 segment is cx~lc;.sed so as to result in an increase in tolerance of tne plant cells to water deficit, or to increase tne content of an osmoploLe~ t in the plant cells.
The promoter can also be inducible so t'nat gene ~ Jle~.ion can be turned on or offby an exogenously added agent. For ex~mple7 a b~teri~l promoter such as 5 the P",~ promoter can be in~ ed to varying levels of gene c;~,ul~s~.ion depending on tne level of isot'niopropylgalactoside added to the lla~7ru~ ed bacterial cells.
It may also be preferable to col..l,i.lc the preselected DNA segment with a promoter that provides tissue specific c~rc~ion or develol~ment~lly regulated gene c~ 7sion in plants.
Pl~,f~ ;d constructs will generally incluc~e, but are not limited to, a plant promoter such as the CaMV 35S promoter (Odell et al., ~, ~ , 810 (1985)), or others such as CaMV l9S (Lawton et al., p]~nt l~ol. Riol,, 2, 31F (1987)), nos (Ebert et al., PNAS USA, 84, 5745 (1987)), A& (Walker et al., PNAS USA, 84, 6624 (1987)), sucrose ~.y..Lhas~ (Yang et al., PNAS USA, 87, 4144 (1990)), a-15 tubulin, ubiquitin, actin (Wang et al., Mol. Cell Riol" 12, 3399 (1992)), ca~(Sullivan et al., Mol. Gen. Genet ~,, 431 (1989)), PEPCase (I~ lcpeth et al., Plant Mol. Riol~, ~, 579 (1989)) or tnose associated witn the R gene complex (Chand}er et al., ~he pl~nt Cell 1, 1175 (1989)). Otner promoters usefill in tnepractice of the invention are known to those of skill in the art, including, but not 20 limited to, water-stress, ABA and turgor-inducible promoters.
A pr~s~lect~<l DNA seO~ can be combined with t'ne promoter by standard methods as described in Sambrook et al., cited supra. Briefly, a plasmid co~ a promoter sucn as tne 35S CaMV promoter can be constructed as described in J~ on, pl~lt Moleclll~r Riolo~y Reporter. 5, 387 (1987) or 25 obtained from Clontech Bab in Palo Alto, California (e.g., pBI121 or pBI221).Typically, these plasrnids ~re co~sl.ucLcd to provide for mllltiple cloning sites having specificity for diLr~ restriction C.~ C3 d~wl~L~ from the promoter. The prese}ected DNA segrnent can be subcloned dowl~ l from the promoter using restriction enzymes to ensure that the DNA is inserted in proper 30 orientation with respect to the promoter so that the DNA can be expressed. In a .

p~cr~lcd version, a bacterial ~IPD gene is operably linked to a 35S CaMV
promoter in a plasmid. Once the preselected DNA segment is operably linked to a promoter, the e~ ,ssion cassette so formed can be subcloned into a plasmid or other vectors.
2. Optior~l Sequerlces in ~he ~,x~ressioll C~ssettç
The e~ s~ion cassette can also optionally contain other DNA sequences.
Transcription ~nh~nr~rs or duplications of lorlh~nr~r~ can be used to increase c,~ ion from a particular ~lÇUIllU~ . Fx~mrles of such ~nh~nr~r~ include, but 10 are not limited to, elemPnt~ from the CaMV 35S promoter and octopine synthasegenes {Last et al., U.S. Patent No. 5,290,924, issued March 1, 1994). ~or example, it is colll~lllplated that vectors for use in accordance with the present invention may be constructed to include the ocs enh~nrer el~m~nt This elernPnt was first icl~ontified as a 16 bp palindromic enh~ncP-r from the octopine synthase 15 (ocs) gene of f~grobacterium (Ellis et al., F~O J., Ç, 3203 (1987)), and is present in at least 10 other promoters (Bouchez et al., Fl\~O J., 8, 4197 (1989)).
It is proposed that the use of an e nh~n~r el~m~ont~ such as the ocs element andparticularly multiple copies of the element, will act to increase the level of ,Lion from ~dj~ce.nt ~ llluL~ when applied in the context of monocot 20 tran~rO~ Lion. Tissue-specific promt~ , inc~ ng but not limited to, root-cellpromoters (Conkling et al., Pl~nt Phy~ l.. ~, 12û3 (1990)), and tissue-specific enh~nre-~ (Fromm et al., Thlo Pl~nt Cell, L 977 (1989)) are also cc ..~ te-l to be particularly useful, as are inducible promoters such as water-stress-, ABA- and turgor-inducible promoters (Guerrero et al., Plant Molecular Biology 15: 11-26),25 and the like.
Tissue specific t;~yle~:~ion may be filnction~lly ~ecomrli~h~ by introducing a c~ ;t~.l;vely ~re;,sed gene (all tissues) in combination with an e,-~e gene that is c;~ s~ed only in those tissues where the gene product is not desired. For example, a preselect~d DNA seg."L ~1 ellcodh.g an e..cymc 30 which catalyzes the synthesis of an osmo~ e.;l~ll, may be introduced so that it W O 97t26365 PCT~US97/00978 is expressed in all tissues using the 35S promoter from Cauliflower Mosaic Virus.
Expression of an ~nti~n~e l~ s~ t of this preselected DNA segment in a maize kernel, using, for example, a zein promoter, would prevent ~cl-mlll~tio~ of the gene product in seed. Hence the protein encoded by the preselected DNA would S be present in all tissues except the kernel.
Alternatively, one may wish to obtain novel tissue-speeific promoter sequences for use in accordance with the present invention. To achieve this, onemay first isolate cDNA elones from the tissue e~ ..Pd and identify those clones which are c~ ;,sed spec-ifie~lly in that tissue, for example, using 10 Northern blotting. Ideally, one would like to identify a gene that is not present in a high copy number, but which gene product is relatively abundant in specifie tissues. The promoter and eontrol elements of cu.lc.,~onding genomic clones may then be loealized using the teehniques of molecular biology known to those of skill in the art.
Expression of some genes in tr~n~genie plants will oceur only under speeified eonditions. For example, it is an objeet of the present invention thateA~resaion of presçleete~l DNA segment that confer rçs~ nl~e to e.~vi~
stress factors such as drought will occur only under actual stress eonditions.
~A~ ion of such genes throughout a plants development may have del,;...
20 effects. It is lcnown that a large nurnber of genes exist that respond to theel.vh~ ll. For eY~mrle c~yl~ ion of some genes such as r~cS, f~nrUfling the small subunit of ribulose bisphosphate earboxylase, is regulated by light as mP~ te~ u~h phytocl~r~lllc. C)ther genes are infhlcecl by secu~ stimuli.
For example, :jyl~lc~ of abscisic acid (ABA) is i..~ by certain ~5 ellvhu.. ~ l factors, including, but not limited to, water stress. A number of genes have been shown to be intlll-erl by ABA (Skriver et al., Pl~nt Celt ~, 503~1990)). Therefore, inducible G~le~ion of a pr~leet~r1 DNA se~;...~ -.1 in transger~ic plants can occur.
In some embo-lim~nts of the present invention c~l,lession of a preselected 30 DNA segment in a llallsgenic plant will occur only in a certain time period _ W O 97/26365 PCTrUS97/00978 during the development of the plant. Develorment~l timing is frequently correlated ~,vith tissue specific gene eA~rcsaion~ For example, cA~lei~aion of zein storage yroLcills is in;ti~t~l in the endosperm about 15 days after pollination.As the DNA Sc4ucllCC inserted belw~ the l.~sc~ lion initiation site and 5 the start of the coding sequence, i.e., the ....~ d leader sequence, can infl~lf~nre gene c~ aion~ one can also employ a particular leader sequence.
P~cfc~l~ d leader sequence include those which comprise seq~l~nr~s selected to direct o~lh"uln e~ aion of the :ltt~rhed gene, i.e., to include a ~l~fc.l~d COI.~f~ leader seql-enre which can i,l~lease or ...S.;..l~;., mRNA stability and10 prevent i~a~ ulJlidlc initiation of tr~n.cl~tion (Joshi, Nucl Acid Res.. ~, 6643 (1987)~. Such sequences are known to those of skill in the art. Sequences that are derived from genes that are highly eAy~ ssed in plants, and in maize in particular, will be most p~cr~lcd.
R~ tnry elements such as Adh intron 1 (Callis et al., Genes neve 15 L 1183 (1987)), sucrose :iyllLhase intron (Vasil et al., Pl~nf Physiol., 91, 517~
(1989)) or TMV omega clc,.l~ Gallie et al., The Pl~nt Celt 1, 301 (1989)) can also be incllldetl where desired. Other such regulatory elements useful in the practice of the invention are known to those of skill in the art.
~1tlitit)n~11y";~ on c~ett~s can be cu~Llu~;led and employed to 20 target the gene product of the preselected DNA 5~ to an intr~ r c~)lll~aLLl.~..l within plant cells or to direct a protein to the extracellular ~"lVil..llll....ll This can gen~lly be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding se.lu.,llce of the pro~electerl DNA seg-.~ The lc~,ulL~l transit, or signal, peptide will hd~polL
25 the protein to a particular intr~celllll~r, or ~Yfr~el~ r ~e~t;n~tion~ ~;liveand can then be post-translationally removed. Transit or signal peptides act by f~f ilit~tinE the L~ u~L of proteins through intr~qr~ r membranes, e.g., vacuole, vesicle, plastid and mitoch~n~lri~l memhr~nf~c, wl..,leas signal peptides direct proteins through the e~-.ellul~r membrane. By f~cilit~t;nE transport of .

W O 97/26365 PCTrUS97100978 .

the protein into co~ aLLIllellLi7 inside or outside the cell, these sequences can increase the ~cc~mlliRti~-n of gene product.
The preselected DNA seg~nG~ll can be directed to a particular organelle, such as the chloroplast rather than to the cytoplasm. Thus, the G~lci..ion S cassette can further be comprised of a ch~ loroplast transit peptide ~nrotling DNA
sequence operably linked bGLv~eell a promoter and the preselected DNA segm~nt (for a review of plastid targeting peptides, see Heijne et al., F.ur. J. ~ioch~m .
180, 535 (1989); Keegstra et al., .Ann Rev. Pl~nt physiol Pl~nt Mol. P.iol.~ 40,471 (1989)). This is exemplified by the use of the rbcS (RuBISCO) transit 10 peptide which targets proteins specifically to plastids.
An exogenous chloroplast transit peptide can be used. A chlo~opla.L
transit peptide is typically 40 to 7û amino acids in length and functions post-translationally to direct a protein to the chloroplast. The transit peptide is cleaved either during or just after import into the chloroplast to yield the mature protein.
15 The complete copy of the IJL'. ~ e]e~ C~ DNA se~ " may contain a chloroplast transit peptide sequence. In that case, it may not be .,~c. ~!~;.l y to combine an exogenously obtained chloroplast transit peptide se.lu~ ce into the ~ ,sion c~ette Fxogenous chloroplast transit peptide encoding sequences can be obtained 20 from a var~ety of plant nuclear genes, so long as the products of the genes are ssed as ~ lulei~ls co"~p,;~ an amino terrl7in~1 transit peptide and transported into chloroplast. E~..plcs of plant gene products known to include such transit peptide sequences inrl~l~lr, but are not limited to, the small subunit of ribulose biph- sph~te carboxylase, ferredoxin, chlorophyll a/b binding protein, 25 chloroplast ribosomal proteins encoded by nuclear genes, certain ht~t~h~ r~
olt;il-s, arnino acid biosyl.~ ;Lic ~l~y~ ,s such as acetolactate acid ~yllL~as~, 3-enoll,y~lvylpllospho~hikim~t~ :iyllLhase, dihydrodipicolinate synthase, and the like.
;v~ly, the DNA ~nPnt coding for the transit peptide may be chemically synthr~i7~1 either wholly or in part from the known sequences of transit peptides 30 such as those listed above.

W O 97/26365 PCT~US97/00978 Regardless of the source of the DNA ~gtnçnt coding for the transit peptide, it should include a translation initiation codon and an amino acid sequence that is recogni~d by and will function ~lupelly in chloroplasts of the host plant. Attention should also be given to the arnino acid sequence at the 5 junction between the transit peptide and the protein encoded by the prçs~lected DNA ~e~m~nt where it is cleaved to yield the mature enzyme. Certain conserved amino acid sequences have been idçntifie~1 and may serve as a gll;r1~1in~. Precise fusion of the transit peptide coding sequence with the plcs~lee,led DNA se~m~nt coding sequence may require manipulation of one or both DNA sequences to 10 introduce, for c~..plc, a convenient restriction site. This may be accomplished by methods including site-directed mutagenesis, insertion of rht~rnir~lly synth~i7~-1 oligonucleoffde linkers, and the like.
Once obtained, the chloroplast transit pepffde ~-c~ucllce can be a~lvpl;ately linked to the promoter and the preselected DNA segm.ont in an 15 e~ iOn c~ettç using standard methods. Briefly, a plasmid co..l~irl;..g a promoter fim~ tion~l in plant cells and having multiple cloning sites downstreamcan be constructed as described in Jelr~.~o.., cited supra. The chloroplast transit peptide sequence can be inserted dow.l,lll,all. from t_e promoter using restriction enzymes. The ,u.~ s~le~;~~d DNA sçg~.. l can then be inserted imm~;~tely 20 duw~ ~ll from and in frame with the 3' l~ -..-;----~ of the chloroplast transit peptide sequence so that the chloroplast transit peptide is lir~ed to the arninol~-...;....~: of the protein encoded by the pre~elrctçd DNA s~..~ Once forrned, the c,~3lc~:~ion cassette can be subcloned into other plasmids or vectors.
Targeting of the gene product to an intr~çll~ r cu~ llt within plant 25 cells may also be achieved by direct delivery of a preq~olect~d DNA segnrnt to the intr~r~llular cu ~p~LIl~ t. For e~mple, an ~ e~,ion cassette encoding a protein, the prese.lce of which is desired in the chlorûplast, may be directly introduced into the chlûroplast ~e.lolnc using the method described in Maliga etal., U.S. Patent No. 5,451,513, issued September 19, 1995, incol~o~aled herein by 30 l~ rei~.lce.

W O 97/26365 PCT~US97/00978 It may be useful to target DNA itself within a cell. For example, it may be useful to target an introduced preselected DNA to the nucleus as this may increase the frequency of tr~n~ru~ n Within the nucleus itself, it would be useful to target a gene in order to achieve site-specific i~ dLion. For ~Y~mp~e,5 it would be useful to have a gene introduced through tr~n~rol~ on replace an existing gene in the cell.
When the cA~ ion cassette is to be introduced into a plant cell, the eAylc~sion cassette can also optionally include 3' nontr~n~l~t~f d plant regulatory DNA sequences that act as a signal to ~f~ e l~ I;on and allow for the 10 polyadenylation of the le~u~ l mRNA. The 3' l~v~ tt?d regulatory DNA
sequence preferably includes from about 300 to 1,000 nucleotide base pairs and cullL~ills plant Ll~ls-,fiylional and translational t~....i..~l;f~n sequences.
Pl~f~ d 3' elennent~ are derived from those from the nopaline synthase gene of Agrobacterium fumefaciens (Bevan et al., Nucl. Acid Res., 11, 369 (1983)), the 15 ~ lor for the T7 ~ s~iliy~ from the octopine ~yl~lilase gene of ~grobacterium tumefaciens, and the 3' end of the ylul~se jnhibitor I or II genesfrom potato or tomato, although other 3' eJ~m~nt~ known to those of skill in theart can also be employed. These 3' nontr~n~l~t~ regulatory sequences can be obtained as ~les~ rihe~ in An, ~thods in Fn7~rn~1O~y. 1~, 292 (1987) or are 20 already present in pl~micl~ available from collllllel~ial sources such as Clontech, Palo Alto, California. The 3' nontr~ncl~t~l re~ll~tnry s~luellces can be operably linked to the 3' t~-...;l...~ of the preselecte~l DNA segm~nt by standard methods.
An eA~res~ion cassette of the iu~.llioll can also be further c~-mpri~e pl~mi(l DNA. Plasmid vectors include ~ ticm~l DNA sequences that provide 25 for easy selection, a-m--plification~ and tran~ ;on of the c;A~ression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUCll9, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. The additional DNA sequences include origins of replication to provide for ~ltn~omcus 30 replication of the vector, selectable marker genes, pl~r. l~ly encoding antibiotic W O 97/26365 PCTrUS97/00978 or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the ~A~ ion ~ set~e, and sequences that enhance transforrnation of prokaryotic and eukaryotic cells.
Another vector that is useful for e~y.c~ion in both plant and prokaryotic 5 cells is the binary Ti plasmid (as disclosed in Schilperoort et al., U.~. Patent No.

4,940,838, issued July 10, 1990) as exemplified by vector pGA582. This binary Ti plasmid vector has been previously characterized by An, cited supra, and is available from Dr. An. This binary Ti vector can be re~lic~t~A in prokaryotic bacteria such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors 10 can be used to Ll~r~l the cA~i~s:iion cassette to plant cells. The binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell L~ r.. l~ion, a selectable marker gene, unique multiple cloning sites in the T border regions, the colE1 replication of origin and a wide host range replicon. The binary Ti vectors carrying an ~y~ssion cassette of the 15 invention can be used to tld.lsîulL~l both prokaryotic and c.~ lic cells, but is .~,f~iably used to ~ ~r~Jml plant cells.

~II. DNA Dcliv_. ~
Following the generation of recipient cells, the present invention generally 20 next inclll~es steps directed to introducing a preselected DNA se~rnt?nt or segment, such as a preselected cDNA, into a recipient cell to create a ~alLsfull~ed cell. The rlc~lu~ ;y of oc~;...l~nce of cells receiving DNA is believed to be low.
Moreover, it is most likely that not all ~ )ic-~l cells ~e~ g DNA se~n~ont~ or sequences will result in a transformed cell Wl~ ,ll the DNA is stably ;,l~e~ t4~1 25 into the plant ~ ...c and/or t;;ip,c~ed. Some may show only initial and transient gene c~r~s:jion. However, certain cells from virtually any monncot species may be stably L,d~ Lùlllled, and these cells developed into l~al~g~lliC
plants, through the application of the techni~lues ~lic~losed herein.
An ~A~le;,:.ion cassette of the invention can be introduced by methods of 3û transformation especi~lly effective for monocots, including, but not lirnited to, W O 97/26365 PCT~US97/00978 .

microprojectile bombardment of imm~hlre embryos (U.S. patent application 08/249,458, filed May 26, 1994, incorporated by .~Çt;.~lce herein; U.S. Patent Application Serial No. 08/112,245, filed Augùst 25, 1993, inco-~o-dl~d by reference herein) or Type Il embryogenic callus cells as described by W.J.

5 Gordon-Kamm et al. (Pl5~nt Cell 2, 603 (1990)), M.E. Fromm et al.
(;P~io/Te hnt logy. 8, 833 (1990)) and D.A. Walters et al. (Pl~nt Molecl~ r Biolo~, 18, 189 ~1992)), or by ele iL opo.dlion of type I embryogenic calluses described by DHalluin et al. (The Plant Cel~ _, 1495 (1992)~, or by Krzyzek et al. (U.S. Patent No. 5,384,253, issued January 24, 1995).
A. Elc~L, 1 o~..lion Where one wishes to introduce DNA by means of electroporation, it is co..L~lllplated that the method of Krzyzek et al. (U.S. Patent No. 5,384,253, issued Jan. 24, l9gS, inc ~l~OlaLed herein by .~rere~lce~ will be particularly 15 advantageous. In this method, certain cell wal~ gr~ ng el~y,.-es, such as pectin-de~-l;n~ cl~yll~CS, are employed to render the target reciI~ient cells more :~u~y~ible to L~ru~ on by elecLlupo.dLion than ~ Lc;d cells.
~ll~.. ,.l;v~ly, recipient cells are made more :iusct;~ible to L.,.. ! r~.. ~'ion, by l W ,ull li.lg.
To effect Llal~rollllaLion by ele-;Llu~u.dLion one may employ either ~iable tissues such as a ~ c.-~ion culture of cells, or embryogenic callus, or iv~ly~ one may l,dl.~Ç,..n imm~tllre embryos or other o.~li;~d tissues directly. One would partially clegrf de the cell walls of the chosen cells by e~osing them to pectin-degrading enzymes (pectolyases) or ...~ ..ic~lly 25 wounding in a controlled marmer. Such cells would then be recipient to DNA
l.~r.,. by ele.;L ù~c,-alion, which may be carried out at this stage, and L~d~ cd cells then ici~ntified by a suitable selection or s irt,.,~lillg protocol dependent on the nature of the newly incol~Joldl~d DNA.

CA 02i43269 1998-07-16 W O 97/26365 PCTrUS97/00978 B. Microprojectile Bombardment A further advantageous method for delivering transforming DNA segme.ntc to plant cells is micropro3ectile bomb~ll~nt. In this m~thn~l particles may be coated with nucleic acids and delivered into cells by a propelling force.
5 Fxempl~ry particles include those co~nrrice~ of tl-n~.ct~n, gold, plRtinllm, and the like.
It is contemplated that in some inctRnres DNA ~le.,ipiL~lion onto metal particles would not be n~ce~c~ly for DNA delivery to a reripient cell using microprojectile bombardment. In an illustrative embodiment, non-embryogenic 10 BMS cells were bombarded with intact cells of the bR~t~riR E. coli or Agrobacterium lumefaciens co~ plasmids with either the ,13-glucoronidase or bar gene en~ ,ed for ~ ion in maize. l~rt~qri~ were inactivated by ethanol dehy~hdlion prior to bomb~d~.,.l~. A low level of trRnci~nt ~x~lGssion of the ~-glucoronid~e gene was observed 2448 hours following DNA delivery. In 15 R~ ;tiQn, stable ~ r.,..,.n..l~ ct...~;.;..;,.~ the bar gene were .~icu~ d following bomb~nc.lL with either E coli or Agrobacterium tumefaciens cells. It is c.~ plated that particles may contain DNA rather than be coated with DNA.
Hence it is proposed that DNA-coated particles may increase the level of DNA
delivery via particle bomb~dll.c..l but are not, in and of L~ms~ s, nece~
An advantage of microprojecti}e bomb~nel-L, in R~l~lition to it being an e~l;ve mearl.e of reproducibly stably L. ~ r~ monocots, is that neither the isolation of protoplasts (Cristou et al., PlRnt P'hy~siol 87, 671 (1988)) nor the :iusc~ibility to Agro~,acterium infection is required. An ill~ ;vt; embodiment of a method for delivering DNA into maize cells by R-~cl~ ;on is a Biolistics Particle Delivery System, which can be used to propel partic}es coated with DNA
or cells through a screen, such as a stRinl~oce steel or Nytex screen" onto a filter surface covered with maize cells c~ultured in su~c.~ion (Gordon-Kamm et al., The PlRnt Cell, ;~, 603 (1990)). The screen disperses the particles so that they are not delivered to the recipient cells in large aggLe~Les. It is believed that a screen 30 illL~L~ lg b~L\~"l the projectile a~pd.a~-ls and the cells to be bombarded W O 97/2636S PCT~US97/00978 reduces the size of projectiles aggregate and may eontribute to a higher frequency of l,~lxrollllation by reducing damage inflieted on the recipient cells by projeetiles that are too large.
For the bombardment, eells in _us~ ~lxion are preferably concc~li,cLLed on S filters or solid culture medium. ~ltrrn~tively~ i,l.",~ "c embryos or other target cells may be arranged on solid culture met~ m The cells to be bolllbarded are positioned at an a~ -iate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned b~L~ the aceeleration deviee and the cells to be bombarded. Through the use of techniques set forth herein 10 one may obtain up to 1000 or more foci of cells ll~xit~ ly ~.ci,xi-~g a marker gene. The number of cells in a foeus whieh express the exogenous gene produet 48 hours post-bolnbal.llllent often range from about 1 to 10 and average about 1to 3.
In bombardment l~ xrolll~l;on, one may O~Jtill i;~C the prebomba~
15 eT-Ihlring conditions and the bombardment p~u..cters to yield the m~cimnm numbers of stable ~ rO,."~."x Both the physieal and biological parameters for bombaLd.l~ are important in this teehnology. Physical factors are those that involve manipulating the DNA/mieroprojeetile ~,.eci~ik.Le or those that affeet the flight and velocity of either the maero- or mieroprojeetiles. Biological factors20 include all steps involved in manipulation of cells before and immPrli~t~ly after bombal~,.cl.l, the osmotie adj~ ..1 of target eells to help alleviate the trauma~Ccosi~trci with boml)dlL--~.-L, and also the nature of the ~ - cr,r.,.;.~ DNA, sueh as 1;"~ d DNA or intaet xu~ oiled pl~cmi~lx It is believed that pre-bo"~ba.,ll.,e"L ,..~ i~ul~ions are e~speei~lly .I.-~.~ulL~ for ~ (cce~xr 25 ~,n..~r~,~."~tion of ;".,.,~1"." embryos.
Aeco..liugly, it is co~l~r..~plated that one may wish to adjust various of the boml~a.&..cn~ p..,..,.. t~,,x in small seale studies to fully û~ lli~ the eonditions.
One may partieularly wish to adJust physieal p~ .el~ .i sueh as gap dict~nre7 flight ~lict~nr~ tissue ~lict~nr,e, and helium ~l~-x-xule. One may also minimi7~ the 30 trauma reduction factors (TRFs) by modifying eonditions which inflllence the W O 97/26365 PCTrUS97/00978 physiological state of the recipient cells and which may there~ore influence transfor~nation and int~gr~t;on efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for op~ um transform~tic)n- Results from such small scale optimi7~tion S studies are disclosed herein and the execution of other routine adjll~tnnent~ will be known to those of skill in the art in light of the present disclosure.

IV. Production alld Chs~racl~.~,.iion of Stable Transgenic Maize After effecting delivery of a pr~qelectç(l DNA se~m~nt to recipient cells 10 by any of the methods .li~ f (l above, tne next steps of the invention generally concern identifying the ~ srulllled cells for further c~ -ring and plant regeneration. As mentioned above, in order to improve the ability to identify tran~rolma.l~, one may desire to employ a selectable or screenable marker gene as, or in addition to, the c~ ,ssible preselected DNA segrn~nt In this case, one15 would then generally assay the potentially ~ r~"c~ cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait.

A. S~ *~
An exenlpla. y embodiment of methods for identifying ll~rollllcd cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like. Cells which have beentransformed and have stably ;..~ f (1 a marker gene co lrcll~g resistance to thes~leclive agent used, wvill grow and divide in culture. Sensitive cells will not be 25 amenable to further C~ . .. ;..g To use the bar-bi~l~rhl~s or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselec~iv~i m~ m and subsequently transferred to medium co..~ from about 1-3 mgA bi~l~phc-s or about 1-3 mM glyphos~t~, as ~L~ id~C. While ranges of about 1-3 mgA
30 bialaphos or about 1-3 mM glyphosate will typically be ~lereL.cd, it is proposed W O 97126365 PCTrUS97100978 that ranges of at least about 0.1-50 mg/l bialaphos or at least about 0.1-50 mM
glyphosate will find utility in the practice of the invention. Tissue can be placed on any porous, inert, solid or semi-solid support for bomb~.l~ l, including but not limited to filters and solid culture m.oAi--m Bi~l~rhos and glyphosate are 5 provided as examples of agents suitable for selection of tr~nxr~ but the technique of this invention is not limited to them.
An example of a screenable marker trait is the red pigrnent produced under the control of the R-locus in maize. This rigm~nt may be ~l~t~ct~od by clllt lring cells on a solid support COI.l;.;.~l..g llull;enl media capable of supporting 10 growth at this stage and sç~ecting cells from colonies (visible ag~Les of cells) that are pi~nPn~l ~rhese cells may be cultured further, either in _u_~cl~iiOn oron solid media. The R-locus is useful for selecfion of tr~n~;ro~ from bombarded i"..~ e embryos. In a similar fashion, the intro~ ction of the C1 and B genes will result in pigrnentecl cells and/or tissues.
The enzyme h~rifrr~e is also useful as a sclccllable marker in the context of the present invention. In the plGsence of the X~ ;r. ;.., cells CA~lG:ixillg ll.. ;L.,.~e emit light which can be ~let~cte~l on photogr~rhic or x-ray film, in a hlminnmetçr (or liquid srintill~tion counter), by devices that ~onh~nre night vision, or by a highly light sensitive video r~mpr~ such as a photon 20 counting r~m~r~ All of these assays are nnnrle~ c and l ,-- Xr~ c~l cells may be cultured further following idP-nfific~ti~n. The photon counting camera isespecially valuable as it allows one to identify specific cells or groups of cells which are ~A~ lg l~I--;r~ ~ee and manipulate those in real time.
It is further col~ lnl~cl that combin~tiorle of screenable and select~ble 25 mzlrk~rs will be useful for idçntifir~tion of L,clL~rc,lll~ed cells. In some cell or tissue types a selection agent, such as bi~l~rhns or glyrh- s~te~ may either notprovide enough killing activity to clearly recognize l,n.l~rol."~cl cells or maycause substantial nonselective inhibition of L-~.r~ X and nn~ ,...xr~llllantS
alike, thus causing the sçlçctinn technique to not be t;Lre.;live. It is proposed that 30 selection witn a growth inhibiting compound, such as bi~l~rhns or glyphosate at W O 97/26365 PCT~US97/~0978 concentrations below those that cause 100% inhibition followed by screening of growing tissue for e~ ,;,sion of a screenable marker gene such as luciferase would allow one to recover ~ .s~o.,.,ants from cell or tissue types that are notamenable to selection alone. In an illu:iL,dlive embodiment embryogenic type II
S callus of Zea mc~ys L. was selected with sub-lethal levels of bialaphos. Slowly growing tissue was subsequently screened for G~ ion of the luciferase gene and tran~r~"manl~ were i~l~ntifie~i In this ex_mple, neither selection nor S~ Cning contlitil n~ employed were sufficient in and of themselves to identifyru~ . Therefore it is ,u-oposed that co.l.l,i..;.lion~ of selection and 10 s~,c.,nillg will enable one to identify LL~ r~,..,.~nt~ in a wider variety of cell and tissue types.

B. R~g~ ion and Seed Production Cells that survive the c,~o:j~e to the selective agent, or cells that have 15 been scored positive in a SC~ lillg assay, may be cultured in media that ~iu~lJull~
,cg. ..~ n of plants. In an eY~mrlAry embonim~nt MS and N6 media have been modified (see Table 1) by including further s17hstAnres such as growth regulators. A l~lc~cd growth regulator for such ~u~oses is dicamba or 2,4-D.
However, other growth regulators may be employed, inrlll~ing NAA, NAA + 2,4-20 D or Y~L1~a~S even picloram. Media in~,u~,."cnt in these and like ways wasfound to f~r-ilit~te the growth of cells at specific develo~ 1 stages. Tissue ispreferably .,.~ on a basic media with growth ~ ldl~ until sufficient tissue is available to begin plant ,~ c,dLion efforts, or following repeated rounds of manual S~lectinn~ until the morphology of the tissue is suitable for 25 ,cg~ ;on, at least two weeks, then l.di~f~ d to media coi~-1..civc to lu~dlion of embryoids. Cultures are ~ r~ .~d every two weeks on this ...~-li,..., Shoot development will signal the time to L1 ~ ~ to m~flil-m lacking growth regulators.
The L,~ls~,."ed cells, identified by selection or scle~ g and cultured in 30 an a~,u~liate medium that ~U~O1L~regCn~dliOn~ WiII then be allowed to mature W O 97/26365 PCT~US97/00978 into p}ants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environment~l1y controlled chamber at about 85% relative humidity, about 600 ppm CO2, and at about 25-250 microeinsteins m~2 -s~' of light. Plants are preferably matured either in a growth chamber or greenhouse.
S Plants are regenerated from about 6 weeks to lO months after a transformant isid~n1ifie-1, depending on the initial tissue. During reg~n~r~tinn, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Con~s. Regenerating plants are preferably grown at about l9 to 28~C. After the regene~ting plants have reached the stage of shoot 10 and root development, they may be ~ r~ d to a greenhouse for further growth and testing.
Mature plants are then obtained from cell lines that are known to express the trait. If possible, the regenerated plants are self po11in~terl In addition,pollen obtained from the l~ gell~.ated plants is crossed to seed grown plants of15 agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate l~,gel~laLGd plants. The trait is g~nPti~ y ~h~ractt~rT7~ by ev~ ting the segregation of the trait in first and later generation progeny. The heritability and G~ ssion in plants of traits select~-l in tissue culture are of particular h.l~u~ ce if the traits are to be c~ lLl~ef~ially 20 useful.
Re~e~ aled plants can be repeate~Ty crossed to inbred maize plants in order to introgress the preselect~(l DNA se~.. 1 into the genome of the inbred maize plants. This process is referred to as backcross col~ ion. When a sufficient number of crosses to the 1G~ 1t inbred parent have been completed 25 in order to produce a product of the backcross coll~ ion process that is substantially isogenic with tne recurrent inbred parent except for the presence of the introduced preselected DNA se~.. l, the plant is self-pollinated at least once in order to produce a homu~ygu-~s backcross con~c.led inbred co~ ;..;..g the preselected DNA seg.~. ..1 Progeny of these plants are true breeding and the 30 level of an o~.llo~-ul~ctant, or the degree of resistance or tolerance to a re~ tion W 097/2636S PCTAUS97/0097$

in water availability, in these progeny are cOlllp~ d to the level of the osmoprotectant, or the degree o~ rçe;et~nre or tolerance to a reduction in wateravailability, in the l~ l parent inbred, in the field under a range of e~lvil~ nt~l conditions ~see below3. The cl~ ion of the level of 5 tolerance or reeiet~nre to a reduction in water availability are well known in the art.
~ ltf-rn~tively, seed from ll~ n~ d monocot plants re~n~l~lGd from ll~irc,lllled tissue cultures is grown in the field and self-pollinated to ~Gl.~.dlc true breeding plants. Progenies from these plants become true breeding lines 10 which are evaluated for r~iet~nre or tol~r~nre to reduced water availability, or production of an osmo~,.ote.i~ll, in the field under a range of environmenf~i conditions.
Progeny and subsequent generations are grown in the field and assayed for their p- Lro,l.la..ce under a range of water availability c~m~lifion~ Both qualitative 15 and ~ e lllG~iUlGS of the plant's ability to w~ 1 water stress are made. Seeds are g~..lll;~A~r.d in the greenhouses, growth r.h~mh~rs and field conditions under ample water supply. At one or more tirnes during the plant's life cycle, water availability is reduced in order to identify plants that exhibit tolerance or le~ .re to a re~ rtiorl in water availability. In ~drlitinn to the 20 visual signs of wilting, which may only be observed under more pronounced drought stress, measures of plant water status are made. These ...e~ul~ s inrl~lde, but are not limited to, total water pot~ ial, osmotic potential and turgor pol~ ial are q~ l;vely measured and ~etect~c-n of differences in turgor or the ability ofthe plants not to wilt. These m~.u~ .,.-l. can be made even when no signs of 25 plant stress are visible to the eye. Plants ~l,.essi~g the most favorable water status result in superior growth under water stress. Dirr~ 7UlCS of growth are used to doc~ nt tbis superior ~.. r.. A.. ~e inrhl~in~, but not lirnited to, measures of cell and leaf area ~p~n~ion.
The physiological and biochemical activity of the Ll~lsL;J--lled plant tissue 30 is indicative of its improved stress tolerance. Such scle~ ~ing of plants with the W O 97/26365 PCT~US97/00978 mea~u.~n~ent of photosynthetic activity or llalls~i~dLional activity are only two examples of the types of me~ulGlllent that can be done to identify the superiority of the transgenic plants conl~.d to non~ ~rolllled plants. Measurements of reproductive capacity inrhl-l;ng, but not limited to, the synchrony of pollen shed 5 and silk emergence are infli~tors of improved stress tolerance when the pr~celect~P(l DNA se~nP-nt is eAl~L~ ed. It is collLclllL)lated that barrenness will not be a problem.
Once the ir~itial breeding lines are selected by criteria, which may include the criteria described above, test crosses are made and hybrid seed is produced.10 The testcross hybrids and brGe1il.g pop-llRtiorl~ are planted in several dirrcL~
arrays in the field. One scheme of ev~ tion is to grow populations of hybrid plants contRining the preselected DNA segm~nt in many different locations and measure the ~clrJl~ ce of the plants at these dirr~,rclll locations. Given the variability of rainfall distribution, the dirrclGlrL locations receive diLr~
15 quantities of rainfall and in some locations, the plants will receive stress. Yield illr~lln~lion as well as measures which ~u~Liry plant rc;,~onse to stress as described earlier, are made. The inf )nn~tion ,eg~dillg the lJGlro~ LLlce of these hybrids along with that of the p~lr ~ ~ of non-trRncf~ nnP-l hybrids is co~ d. It is anticipated that the hybrids CA~lG~illg the pre~elect~Pci DNA
20 segn~Pnt will be higher in yield ~elr.,.... ~re and stability at a given level of water availability than the controls.
Where irrigRtic~ is available, more controlled c-""l.nl;~on~ are made through the establi~hmPnt of dirrG~GllLial irrigation Ll~ ; The same entries of hybrids or lines are grown under co..~ g irrigation treRtment~ Such an a~ uach limits the nulnbe~ of variables at work in the evaluation. Aside from the same types of mea~ulGlllelll:, as defined above, dirrGlGllLial responses arecRir~ Rte~l bec~usG of the contrast in the data. It is anticipated that preselected DNA segment GA~es:~ing hybrids will have less yield reduction when grown f under itngRtf~l versus non-inigRteci conditions when compared to hybrids without the gene.

W O 97/26365 PCTnUS97/00978 Upon the identification of the superior p~,L~ n~-e of ~ cgP~-ic plants, the parent selections are advanced and inbred lines are produced through conventional breeding techni~ues. Hybrid plants having one or more parents C~ .i..g the ~.~,scle.~led DNA segment are tested in cor..~n~ ial testing and 5 evaluation programs and pc~ llr~Ce f1n~ A This testing includes perform~n-e trials over a wide geographical area as well as ~e-~ir~tecl trials where water availability is varied to reveal p~,lr~ ance advantage and hence value.
An ~ itif~n~l advantage of the G~lllesaion of the presçlectecl DNA
segment is the superior ~P. r~ nce of the parental inbred lines in production of10 hybrids. Less stress related parent yield loss is associated with higher green seed yield and thereby higher economic margins.
It is anticipated that the perfollll~u~ce advantage will not only be present under stress con~itionc Given the overall role of water in d~ ;.lg yield, it is co..~ .lated that maize plants GA~Jr~saillg the pr~selP~cted DNA segrn~nt may 15 utilize water more effiçi~ntly. This will h,~ vG overall pclruLlll~lcc even when soil water availability is not limitinp Through the introduction of the preselected DNA se~ -l(s3 and the hll~ ,d ability of maize to ll~x ;lll;i.~? water usage across a fi}ll range of con~itionr~ relating to water availabilitv (i.e., incl~l~ing normal and stressed c~n-lition~)~ yield stability or coo~ PI-~y of yield 20 ~,.rollllallce will be achieved. These studies are cnn~ tPcl in mai~ and other monocots.

C. Char..~ lion To confirm the pl~,3ellce of the presel~ctPd DNA se~ .ll(s) or 25 "transgene(s)" in the Leg~ aLil~g plants, a variety of assays may be performed.
Such assays inc~ e~ for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting and PCR;
"bioçhP.mic ~1" assays, such as rl~tPcting the pl~,~Gllce of a protein product, e.g., by immnnnlogical means (ELISAs and Western blots) or by enzymatic function; r W O 97/26365 PCTrUS97/00978 plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

1. DNA ILIt~,l;r~lion~ RNA Expressio~l and Inheritance Genomic DNA may be isolated from callus cell lines or any plant parts to det~rmine the presence of the preselected DNA se~ through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rP,~rrAn~emPnt or deletion of sequencesin the cell.
The ~ ,se.rce of DNA cl~ ; introduced through the methods of this invention may be detPrmined by polymerase chain reaction (PCR). Using this technique discreet ~gm~nt~ of DNA are amplified and ~iPteGteA by gel electrophoresis. This type of analysis permits one to detPrmin~ whether a preselected DNA se~ is present in a stable ,.<.. ,~r.. ~ , but does not prove 15 integration of the introduced preselected DNA se ment into the host cell genome.
In ~AAition~ it is not possible using PCR techniques to ~ f~ f whether trar~r.. ~ have exogenous genes introduced into diLr~ l sites in the gellon~e, i.e., whether ~ rl.. ,.. l~i are of inA~pPnrl~nt origin. It is cn.. ~ .,,pl~te~i that using PCR techniques it would be possible to clone frA~m~nt~ of the host 20 genomic DNA adjacent to an introduced preselecte~ DNA segmPnt Posilive proof of DNA integration into the host ~Pnnme and the independent icientitipc of ,.~.. xr~.. ~.. ,~ may be cl~ using the technique of Southern hybriAi7~ticn. Using this tec~hnique sperific DNA se.lucllccs that wereintroduced into the host gPnnm~ and fl~nking host DNA sequences can be 25 iti~ntifietl Hence the ~outhern hybriAi7~tion pattern of a given lldnsL~llll~ll serves as an identifying r~Ar~ctPri~tic of that L,t~ r~.. A~.L In addition it is possible through Southern hybri(1i7~tinn to clemn~ the presence of introduced preselected DNA segmf-ntC in high mnlec~ r weight DNA, i.e., confirrn that the introduced pr~elect~A DNA segm~n~ has been integrated into 30 the host cell genome. The technique of Southern hybri-1i7~tion provides .

W O 97/26365 PCT~US97/00978 information that is obtained using PCR, e.g., the presence of a preselected DNA
segmçnt, but also demonstrates i~ aliOn into the genome and characterizes each individual llal~llll~ull.
It is co~ lated that using the tech~ques of dot or slot blot hybridization which are modifications of Southern hybridization techniques one could obtain the same ;l~rol.llAI;on that is derived from PCR, e.g., the presence of a preselecte~i DNA se~
Both PCR and Southern hybridization techniques can be used to demonstrate l,~ ."i~ion of a presPlect~d DNA se~ ,I to progeny. In most in~t~nces the ch~a~ Lic Southern hybridization pattern for a given Llallsrollll~ulL will se~ ; in progeny as one or more ~Pn~iPii~n genes (Spencer et al., Pl~nt Mol. Riol ~, 201 (1992~; T ~llr~n et al., Pl~nt Mo. Riol 24, 51 (1994~) in~ic~ting stable inh~rit~nf e of the gene. For P~mplç, in one study, of28 progeny (R,) plants tested, 50% (N=14) con~ine l bar, confirrnin~ trans-mission through the germline ofthe marker gene. The llo,-.l,;",eric nature ofthecallus and the parental L~al~rJlll~ ~ (Ro) was ~,.gge~leA by germline tr~n~mi~ion and the i~enti~l Southern blot hyhri~ tion palt~,~l~ and i~ iPS of the LLal~ro.~",.g DNA in callus, R" plants and Rl progeny that segregated for the l,~f;,l,l,ed gene.
Whereas DNA analysis techniques may be cnn~ cte.l using DNA isolated from any part of a plant, RNA may only be ~yl~;sscd in particular cells or tissue types and hence it will be n~ceSs~.y to prepare RNA for analysis from these tissues. PCR techniques may also be used for ~i~-tectio~ and 4l~n~ nl;0n of RNA
produced from introduced preselected DNA seg~ In this application of PCR
it is first nPce~ to reverse tr~n~nhe RNA into DNA, using el~y"lcs such as reverse ~".n~ se, and then through the use of Co"~ n~l PCR techniques amplify the DNA. In most in~t~n~ PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give inforrnation about the W O 97/2636S PCT~US97/00978 .

integrity of that RNA. The presence or ~hsPnre of an RNA species can also be determinrcl using dot or slot blot Northern hybri~i7~t;f-n~ These techniques aremo-'ific~ti--n~ of Northern blotting and will only dP no.~ the presence or hsenre of an RNA species.

2. Gene E~
While Southern blotting and PCR may be used to detect the prese1ected DNA se~ --f in question, they do not provide i~ru~ ;on as to whether the preselected DNA segmrnt is being Gx~.~ssed. Expression may be evaluated by 10 specific~lly idellLiryin~ the protein products of the introduced prese1~ cte~l DNA
segments or ev~ ting the phenotypic changes blou~lt about by their c..~ ion.
Assays for the production and icl~ntific~tion of specific ~luLeuls may make use of physical-chrmir~ Llu~L~ ~,fnn~.ti~n~l or other ~lop~.lies of the ~luteiLs.
Unique physical-chemical or structural pLùpGlLies allow the proteins to be 15 separated and i~lrntifiefT by electrophoretic ~luCG.~ ,S, such as native or de~&lu~ g gel ele~;l-ophoresis or isoelectric foc1~c~inf~, or by cl~ùl~l~Lographic techniques such as ion e~ch~nge or gel rYc111~ion cll.~o..~ gT~rhy. The unique :iLIu;Lu~. s of individual ploLeills offer o~pu.lul-~lies for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combin~tion~ of 20 approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene ~ludu~;L~ that have been se~dlaLGd by electrophoretic techniques. ~lrlition~l techniques may be employed to absolutely confirm the identity of the product of interest such as ev~ tion by arnino acid seq~rnring following pnrific~tion Although these are 25 among the most cornmonly employed, other plucGlLues may be ~A~itinn~lly used.Assay proce.lu,~ s may also be used to identify the c,~ple;.~ion of l~luLGuls by their functionality, especially the ability of Gl~ylll~S to catalyze specificchemical reactions involving specific :~ut~LlaLes and products. These re~rtio~
may be followed by providing and qu~lliryiu~g the loss of substrates or the 30 generation of products of the reactions by physical or r.h~mir:~l procedures.
-W O 97/26365 PCTnUS97/00978 Examples are as varied as the enzyme to be analyzed and may inelude assays for PAT enzymatic activity by following production of radiolabelled aeetylated phosphinot_ricin from phosFhinothricin and t4C-acetyl CoA or for ~~ ~,ilate synthase activity by following loss of fluoreseenee of ,1..ll..~.-il~t~, to name two.
S Very fre~uent}y the e~cyl~ ion of a gene product is ~ cl by evaluating the phenoty-pic results of its c~ ,ion. These assays also may take many forms including but not limited to analyzing ch~nge,c in the eh~-mi~
composition, morphology, or physiologieal y.~llies of the plant. (~h~mi~
eomposition may be altered by c~ ssion of p.~ selev~ DNA seg n~nt~ eneoding enzymes or storage proteins which change amino aeid eol"posilion and may be tleteet~ot1 by amino aeid analysis, or by el.~yl"es whieh change stareh quantitywhich may be analyzed by near infrared reflectance ~ye~ llletry. Morphological changes may include greater stature or thicker staL~cs. Most often changes in pollse of plants or plant parts to imposed tre~tm~ntc are evaluated under carefully eontrolled con~litionc termed bioassays.

D. Establ;~hrr t of the I~.h.JC~ -e~ DNA in Other Maize Varieties Fertile, I.,...c~ ..ie plants may then be used in a collv~ n~l maize breeding program in order to iLICol~old~G the pr~seleete~l DNA segrnton~ into the 2~ desired lines or v~rieti~oc Methods and lef~ ",ces for eonvergent improvement of maize are given by Hallauer et al. (In: Com sm~1 Corn T~ v~ l Sprague et al. (eds.), pp. 463-564 (1988)), inc~lyo,aled herein by ,cf,lence. Among the a~roaclles that eon~,. ..lion~l b.. cdiL~ programs employ is a eonv~,~ion proeess (bacL~,os,illg). Briefly, co~ ion is p~.r~ cl by ~il'U~ iillg the initial LLa~sgt;l~ic 25 fertile plant to elite inbred lines. The progeny from this cross will sc~lc~lc such that some of the plants will carry the ~c~l~led DNA se~ wL.,.~as some will not. The plants that do carry the yl~ A DNA segment are then crossed again to the elite inbred lines re,sT-lting in progeny which se~ once more.
This backcrossing process is repeated until the onginal elite inbred has been 30 c<,,lvc,~ed to a line c~ ;..;..g the presçlect~l DNA segment yet po~eqqin~ all W O 97/26365 PCT~US97/00978 ill.poll~ll attributed originally found in the parent. Generally, this will require about 6-8 generations. A se~d,dLe backcrossing program will be generally used for every elite line that is to be converted to a p~nPtir~lly PnginPPred elite line.
Generally, the cu~...-.c.~;ial value of the ll~ sfo----ed maize produced herein 5 will be ~-~ale:iL if the preselect~Pd DNA segm-Pnt can be incul~uldl~d into many dirr~.e.l~ hybrid combinations. A farmer typically grows several hybrids based on dirr~ ces in llldlulily, standability, and other agronomic traits. Also, the farmer must select a hybrid based upon his or her geographic location since hybrids rt~PA to one region are generally not adapted to another because of dirr~.~,..ces 10 in such traits as maturity, disease, drought and insect ,~ c;~f;..l. e As such, it is nPcejs~.y to inco.~u.al~ the gene into a large nurnber of parental lines so thatmany hybrid combinations can be produced CoI~t~ininF the preselrct~rl DNA
segm~nt Maize breeding and the techniques and skills required to L d ~r~. genes 15 from one line or variety to another are well known to those skilled in the art.
Thus, introducing a p~ sele~ ,d DNA se~ preferably in the form of recombinant DNA, into any other line or variety can be ~ccomrlichPrf by these breeding procedures.

2û E. Uses of Tr,.~s,~,.. ic Plants The ~ ic plants produced herein are expected to be useful for a variety of commPrcial and resea~ purposes. Tr~n~grnic plants can be created for use in traditional agriculture to possess traits b~nPfici~l to the grower (e.g., a~.ono..,ic traits such as rpciet~nre to water deficit, pest resict~nce herbicide 25 r~cict~nre or inc~ased yield), beneficial to the co~.c -mPr of the grain h~ ve~Led from the plant (e.g., in~,uvc;d nutritive content in human food or animal feed), or bPnefici~l to the food ~,ucessor (e.g., hn~uvt;d proc~ccin~ traits). In such uses, the plants are generally grown for the use of their grain in human or animal foods. However, other parts of the plants, including stalks, husks, vege~livt;
30 parts, and the like, may also have utility, including use as part of animal silage or W O 97/2636S PCT~US97/00978 for ornslm~ntS l ~ul~oses. Often, chemical con~titllent~ (e.g., oils or starches) of maize and other crops are extracted for foods or inrill~trisll use and transgenic plants may be created which have t-nhsmrec~ or mo~lifie~l levels of such components.
T~ s~ ~nic plants may also find use in the c~.. --;ial msmnf~tTlre of .s or other molecules, where the molecule of interest is extracted or purified from plant parts, seeds, and the like. ~ells or tissue from the plants may also be cultured, grown in vitro, or r ....~ .i to m~mlf~r~lre such molecules.
The trsln~gçnic plants may also be used in co.~ ial breeding ~ ~dlllS, 10 or may be crossed or bred to plants of related crop ~re~iec. Improvements encoded by the preselected DNA se~,....~ may be l.,...~r~ d, e.g., from maize cells to cells of other species, e.g., by protoplast fusion.
The T~ .iC plants may have many uses lnl~S~ or l.-~e.li~g, inc~hlAinp~ creation of new mutant plants through insertional rntltslgen~ci~, in order 15 to identify beneficial ~ ; that might later be created by trsT~iitionsll mTltsltion and selection. An ~Yslmrle would be the intrc!~llrtiC n of a recomhinsTnt DNA
sequence encoding a L.~,s~o~al)le elem~nt that may be used for ge~f.~
genetic vSl i~tinn The methods of the invention may also be used to create plants having unique 'l~ignslttlre se.lu~ nce:," or other marker seqllrnr~s which can be 2û used to identify proprietary lines or varieties.
Success in producing fertile LL~ gr~lic monocot plants (maize3 has now been achieved where others have failed by methods ~lesc~ihe~7 herein. Aspects ofthe methods of the present invention for producing the fertile, l~ulsgenic maizeplants compri~e but are not lirnited to, isolation of ~ 1 cells using media 25 conducive to spec}fic growth p~ , choice of selective ~y~ s that permit efflcient ~etection of ~u~ru~ ;on; mnfiifirsltions of DNA delivery ml~th-uls to introduce genetic vectors with exogenous or recom~inS~nt DNA into cells;
invention of methods to ,eg~ plants from l.,,...iri,. ",r~ cells at a high frequency; Slnd the pro~llct~ of fertile transgenic plants capable of surviving and r 30 reproducing.

W O 97/2636S PCTrUS97/00978 F. P~er~ d Methods of D~ w ;~, DNA to Cells Pl~r~ DNA delivery ~y~L~ S do not require protoplast isolation or use of Agrobacterium DNA. There are several potential cellular targets for DNA
S delivery to produce fertile Llallsge~lic plants: pollen, microspores, rn~ ten~, ;...1-..~...~ embryos and cultured embryogenic cells are but a few exarnples.
One of the newly emerging techniques for the introduction of preselected DNA segrnPnt~ into plant cells involves the use of microprojectile bomb~.,L.
The details of this technique and its use to hlLIud~lce preselected DNA se~...~ .l 10 into various plant cells are ~1icc~ e~1 in Klein et al. (Pl~nt Plwsiol., ~1, 440 (1989)), Wang et al. (Pl~nt Mol. Riol.. 11, 433 (1988)) and Christou et al. (~
Physiol.. ~, 671 (1988)). One method of clot~ the efficiency of DNA
delivery into the cells via microprojectile bomb~-lcnL employs ~letection of tr~n~i-?nt e2~,le.,:jion of the enzyme ,B-glucuronidase (GUS) in bombarded cells.
15 For this method, plant cells are bombarded with a DNA construct which directs the .7yll11le~is of the GUS enzyme.
Apparati are available which p~.r~lll microproJectile bombardment. A
c~mmercially available source is an a~p~àlus made by Biolistics, Inc. (now DuPont), but other microproJectile or acceleration methods are within the scope 20 of this invention. Of course, other "gene guns" may be used to introduce DNA
into cells.
Several modifications of the microprojectile bombardment method were made. For ~ .le, st in1~ss steel mesh screens were introduced below the stop plate of the bombardment ~ LaLuS, i.e., bcL~en the gun and the cells.
25 Furthermore, mo-lific<~tjon~ to t~ ting techniques were developed for plc~ JiLdLi, g DNA onto the microprojectiles.
Another newly e111~L~,i11g technique for the introduction of preselected DNA segTn~nt into plant cells is ele.i~lupoldLion of intact cells. The details of this technique are described in Kr~zek et al. (U.S. Patent No. 5,324,253, issued30 Jan. 24, 1995). Similar to particle bombardment, the efficiency of DNA delivery W O 97/2636~ PCT~US97/00978 into cells by ele-;L~ol,oldLion can be Aet~ mined by using the ~3-glucuronidase gene. The method of electroporation of intact cells and by extension intact tissues, e.g., i,,,,,,;~ ,c; embryos, were developed by Krzyzek et al., and le~lt;sGllL
nlents over published procedures. Gene~tic-n of fertile plants using these S techniques were described by Spencer et al. (cited supra (1993)) and Laursen et al. (cited supra (1994)).
Other methods may also be used for introduction of DNA into plants cells, e.g., a~it~tion of cells with DNA and silicon carbide fibers.
Histological analysis of stressed and ull~Llcis~ed tissue from Ll~ sr(3.ll-cd 10 and lmtriqncforrned plants are p~Lrulllled (Sylvc~t~. et al., T i~ht Microscopy l:
nicc~ti~ n ~ntl M;crotec~lnique. In: The Maize Handbook, pp. 83-95, Springer-Verlag, NY (1994)). Cross sections through the a~ropliaLc tissues (e.g. leaves or roots) reveal any sL.Il.;L~dl aberrations. Tr~ncmic~iQn and sc~nnin~ cle~,L~ol1 microscopy are used to ~~h~r~- teri7p ~r~nc~f~ rmed and llntr~ncform~oA cell structure 15 and epidermal sn~f~-~ec Leaf sll~f~ ec are also e~ ".~-l for normal storn~tP
::jL~lclulc and density using epidermal peels (Ristic et al., Rot. G~7 ~2, 173 (1991)).

The invention has been described with lGr~ lce to various specific and 20 ~lGr~,~ embodiments and will be further described by ~ .~rce to the following ~et~iled P~mplec It is lm~l~nctr~od~ however, that there are many ct~.nci~n.c, Vs~ri~tinnc, and mo-lifi~tionc on the basic theme of the present invention beyond that shown in the examples and description, which are within the spirit and scope of the present invention.

W O 97/26365 PCT~US97/00978 F.Y~
Because a link was observed between (I) the ms~ P~ e of turgor level via shifts in osmotic potential and yield of hybrids under stress cnnflition~, and (2) more negative osmotic potentials and ill~Gased yield levels in hybrids under5 il'ri~tP~ Con~ monocot cells were ~ r~... Pcl with a preselected DNA
segment encoding an enzyme which catalyzes the synthesis of an osmoplole~;LaLlL
so as to result in a Ll~r~ ed monocot plant with improved cellular osmotic relations. The GA~ ion of the preCpl~ctecl DNA segnent in~ ies t-Ayl~;~sion in the cytosol or the chloroplast, or both. In addition to col~liluli~e gene 10 eA~ies:jion, ~lirr~e"Lial GA~ ion in shoots, roots and reproductive tissues, develop~ lL~l, temporal, as well as inducible ~A~ ion of a preselected DNA
segmPnt, is within the scope of the invention.
Monocot plant cells can be transformed with more than one prPsPlectP~l DNA segment, so as to result in a synergistic effect for plant ~c.~....~n-~, under 15 either, or both, water-stress and non water-stress 50n~1ition~. Thus, it is also col.tr.,lplated that .,A~.e~ion of a prp~plp-ctpcl DNA SG~,JI'I- ~t in plants, when those plants are grown under relatively non-stress con(1itions or typical Cl)n~1ition~ can result in a yield p~r~ e over plants which do not express the preselected DNA se~nent, or do not express the DNA at altered, increased or elevated levels.
Con~tructior of n~ID Vectors One embodiment of the invention is a vector co.~llu~Led to direct col.~liL~,Iive ~ ,cs:iion of the prç~PlçctP~ DNA seE;~ l For ~Y~Tnrle a ~.ef~ ,d embodiment of the illVGllLiOl- iS an CA~lG~ iOIl cassette co...~ ;..g the 25 Cauliflower Mosaic Virus 35S promoter (Odell et al., ~ (1985)) 5' to the mtlD gene. ~ l;vely the rice actin gene promoter (Wang et al., Mol Cell ~QL~ L~, 3399 (1992)) is placed 5' of the mtlD gene. It is ~nticir~tP~l that allpromoters which direct co..~ ;ve gene e,~ ;.sion in maize will be useful when operably linked to a mtlD gene. !~e~l~r~ es which direct polyadenylation are 30 preferably linked 3' to the mtlD gene. These sequences incl~ e, but are not 6~ PCT~US97/00978 limited to, DNA SC~lUellCCs isolated from the 3' region of Agro~acterium tumefaciens nopaline ~ylllh~e, octopine synth~e or L~ SC~i~L 7, or potato proteinase inhibitor II genes. It is anticipated that co~ ;v-e eA~ ion of the mtlD gene in all tissues of a monoeot plant, such ~ mai~, will t~nh~nr~e the 5 ability of the plant to ~ water turgor under ct n-l;t;ons of decre~ed water availability.
It is further eo-ll- ~pl~t~d that tissue specifie ~ ,lc;.~ion of a pr selc~,Lcd DNA segment, e.g., mtlD, will t nh~nce the agronomie ~- ~ r~ e of a monoeot plant, sueh as maize. Vectors for use in tissue-speeifie targeting of mtlD genes in 10 transgenie plants will typieally inelude tissue-speeifie promoters and may also include other tissue-speeific control ClC.11G11~ such as t ..l-~..t-. . se~.lces.
Promoters which direet speeific or c~ rc~l -x~l~ssion in eertain plant tissues will be known to those of skill in the art in light of the present rl;cclosllre~ These ;n~ 1e for example, the rbcS promoter, specifie for green tissue; the ocs, nos 15 and mas promoters whieh have higher aetivity in roots or wounded leaf tissue; a f~d (-90 to +8) 35S ~ molc. whieh direets enh~nee~l t"~lc~sion in roots, an a-tubulin gene that directs G~r~ ,ion in roots and promoters derived from zein storage protein genes which direet t;~ ion in endosperm. It is partieularly eollL~,~lated that one may adv,ulL~geously use the 16 bp ocs 20 enhancer el~m~nt from the octopine synthase (ocs) gene (Ellis et al., FMPO J., ~, 3203 ~1987)); Bouchez et aL, F~O J., ~, 4197 (1989)), especially when present in multiple eopies, to achieve ~nh~nced w.~ iion in roots.
ssion of mtlD in l~ sgell-e plants may be desired under speeified cnnr1;tionc. For example, the ~ ~ylcssion of mtlD genes may be desired only 25 under actual stress con~iitionc It is known that a large number of genes exist ~at respond to the cnvi~ ,...ent For example, ~ ession of some genes sueh as r~cS, eneoding the small subunit of ribulose bisphosphate ~b~3~ylase, is regulated by light as m~ ted throngh phytoehrome. Other genes are in-1rlced by secondary stimuli. For example, synthesis of abscisie aeid (ABA) is in~ ed by 30 certain t;llvho~ .ont~l factors, including but not lirnited to water stress. A

W O 97/26365 PCT~US97100978 .

number of genes have been shown to be inf11lrecl by ABA (Skriver et al., Cell, 2, 503 (1990)). Promoter regions that regulate G~lession of these genes will be useful when operably linked to mtlD.
It is ~ yosed that in some embot~;m~nte of the present invention 5 ~ ion of mtlD in a L~ g~-~ic plant will be desired only in a certain time period during the development of the plant. Devel~ timing is frequently correlated with tissue specific gene e~ ,ssion. For ~mple, ~ cs~ion of zein storage ~lol~ 3 is initi~te~d in the endosperm about 15 days after pollination.
To provide a L~ sgc,lic monocot plant that is ~ Y resistant or 10 tolerant to a reduction in water availability, several vectors were constructed co~ -i-,g a gene that encodes an enzyme which catalyzes the synthesis of an osmoprotectant. Such genes include, but are not lirnited to, the mtlD gene from E. coli and the HVA-l gene from barley. The ...~ 1 operon was originally cloned and ~h~ract~ri7P~ by Lee et al. (J. Racteriol., 153, 685 (1983)). The mtlD
15 gene has been shown to confer water stress rG~iet~nrG on LL~lsgenic tobacco plants (Tarczynski et al., Sci~nce, ~2, 508 (1993~.
C O~ ion of vector pDPG451. The .~ l;lol operon (mtlC, mtl~, mtlD) was obta~ned as a plasmid in C600 ~. coli from Malthius Muller, Univ. oi~ Freiburg ~pDPG409). To isolate the plasmid DNA, the plasrnid was first amplified using 20 chloramphenicol and then isolated ueing Qiagen large-scale plasmid p~ p~ n The mtlD gene was excised from the pDPG409 plasmid by digesting the r)NA
with restriction enzyrnes NsiI and PstI. The ~ et~l DNA was run on a 1.1%
SeaKem agarose gel in TAE bu~er (see Sambrook et al., Mol~ r Clo~;n~: A
Laboratory Manual (1989)) to separate the l;~m--.nt~ by si~ and the ~l~Lupl;ate 25 fragment was isolated ffom the gel using S&S NA45 m~mhr~n.o (Schleicher &
Schuell, Keene, NH).
~ e mtlD gene fragment was next cloned into the maize ~ ssion vector pDPG431 (35S promoter-adhl Intronl-Tr7 3' end). Vector pDPG431 DNA was digested with restriction enzymes NsiI and PstI to open up the backbone and the 30 mtlD fragment inserted by ligation. The ligated DNA was transformed into DHSa _ cells and the resulting colonies screened by mini-preps to identified those co..l;.;..i,.g the correct gene construct. The new vector was ~lçei~nRted pDPG451.
A map of the plRemicl is shown in Figure 1.
Conetnlr-tion of vector pDP&480. The NsiI-Pstl frRgm~nt from vector pDPG409 S contRinin~ the mtlD gene used to cu~ u-il pDPG451 was cut with restriction enzymes AvaI and HindIII. This removed about 122 bp of lmtrRnelRt~i sequence from the 5' end of the mtlD fragment and about 69 bp of Imtr~nelRte~l se~uence from the 3' end of the frR~m~nt The AvaI - HindIII fragment was ligated into pUCl9 DNA that had previously been digested with Aval and HindIII to open up 10 the plasmid backbone in the region of the ml-ltiple cloning sites. The pUCl9/mtlD construct was then digested with restriction enzyrnes SacI and HindIII to release a fragment co.~ the mtlD gene. This frR~m~nt was isolated by running the digestion reaction on an agarose gel and the ap~lu~,l;aLely-sized frR~m~nt t;~ ed from the gel using a S&S Elu-Quik DNA
15 purification kit, per the m~nnfRt~tllrer's il~L.u(;Lions.
The DNA frRgrn~nt was next ligated into pcDNAII DNA that had previously been fii~cterl with SacI and HindIII to open up the plasmid backbone in the region of the multiple cloning sites. The pcDNAIlJmtlD vector was then digested with restriction enzymes Bam~II.and PstI to release a fragrnent 20 co..~ ;"g the mtlD gene. This frRgment was isolated by running the digestion reaction on arl agarose gel and the a~plu~.ial~ly-si~d frR~nent ~ Y~ l from the gel using a S&S Elu-Quik DNA pllrifis~tiQn kit, per the mRmlf~*lrer's u~;lions. The DNA frR~m~nt was next ligated into pDPG431 DNA that had previously been digested with the rest~irtion enzymes Bam~II and PstI and the 25 baclcbone fragment col,ls.;..;"~ tne 35S promoter-adhI In~ronl and Tr7 3' endisolated by gel purification. The resulting maize ~ ,ssion vector was ~le~i~n~t~cl pDPG480. A map of the plR~mi~ is shown in Figure 3.
Con~truction of vector pDPG493. DNA from vector pDPG480 was modified to remove a~ ely 120 bp of Ull~ Rt~ DNA from the 3' end of the mtlD
30 gene fragm~nt To modify the 3' region, two oligonucleotides were made (DNA

W O 97/2636S PCT~US97/00978 Tntern~tional, Inc.) to anneal together and then used to replace about 150 bp ofthe 3' end of the mtlD gene fr~gmçnt The first oligQnllf leotide (mtlD-BI) had aseq~ nre of: 5' GTA ACC GCT TAT AAA GCA ATG CAA TAA TGA GTA
CTC TGC AG 3' (SEQ ID NO: 1). The second oli~omlrleotide (mtlD-B2) had a S sequence of: S' GAG TAC TCA TTA TTG CAT TGC l-l-l ATA AGC G 3' (SEQ ID NO: 2). The ~nn~lecl oligos duplicated the last twenty base pairs of themtlD gene starting at the BstEII restriction site and running up to and including the stop codon and created a new sequence after the stop codon. This new sequence created new ScaI and PstI sites.
The new vector was col~L.u-;Led in the following manner. Vector pDPG480 pl~micl DNA was fligested with restriction ~yll.es BstEII and NsiI to remove the 3' end of the gene fi~gment The ~iig~stec~ DNA was run on an agarose gel to size s~al~; the fr~gm~nt~ and the a~ ,ately-sized vector fi~gment was ~ n~ l~d from the gel using a S&S Elu-Quik DNA pllrific~tion kit, 15 per the m~mlf~ctllrer~s instructions. O~ig-n~ leotides mtlD-Bl and mtlD-B2 were ~nnt~ l together and ligated into the fiiges~ pDPG480 DNA fi~rn~nt The res~ltin~ vector was ~iesipn~te~l pDPG493. A map of the pl~mi~1 is shown in Figure 4.
Co.,~-l, u~;lion of vector pnPG586. A DNA r.~...G~.I CO..~5.;....~g the mtlD gene 20 was removed from vector pDPG480 by digesting the pl~mitl DNA with restriction enzymes BamHI and PstI. The DNA fr~gm~nt cu.lli~;..;..g the gene wasisolated by gel purification and e~3rti~ n from the gel using a S&S Elu-Quik DNA pll~ific~tion kit per the .. ,.. r~ s i~ ions. A DNA ~n.-nt c~ the Glbl promoter and Glbl t~ was isolated by ~i~estin~
25 vector pDPG423 DNA with restriction enzymes BamHI and PstI to open up the backbone in the polylinker region. The two fi~grnent~ were then ligated togetherto create vector pOPG586. A map of the pl~cmitl is shown in l~igure 5.
Con~lruction of vector pDPG587. Vector pDPG411 was digested with restriction enzymes XhoI and SacI to release a DNA fragment con~ g the 35S promoter 30 and a maize transit peptide sequence (MZTP). This DNA fi~ment was isolated W O 97/2636~ PCT~US97/00978 _ by gel purification and extraction from the gel using a S&S Elu-Qui~ DNA
purification kit per the m~nllfaehlrer~s instructions. A DNA backbone fr~m~n~
cu,.t~i..;,.g the mtlD gene was ~n~;laled by digesting the pcDNAII/mtlD vector described above with restriction el,~y~ XhoI and NsiI to open up the vector in 5 the polylinker region. These two fr~gm~ntc along with a Nsi-StuI-SacI linker (Keystone Laboratories, Inc.) were ligated together to create a vector c~,ei~n~t.od MZTP/mtlD. Plasmid DNA of this vector was digested with restriction e.~ylllc PstI to open up the vector at the 3' end of the mtlD gene sequence.
A DNA fr~ment co~ -g the Tr7 t~ .lor was isolated from plasmid 10 DNA of vector pDPG527 by digesting with restriction C.~ylllC PstI. This DNA
fragment was isolated by gel purification and extraction from the gel using a S&S Elu-Quik DNA purification kit per the m~mlf~r,tllrer's instructions. The MZTP/mtlD and Tr7 t. .,.,i..s~ r DNA fr~gm~ntc were ligated together to create the maize ~,A~r~s~ion vector pDPG587. The region from the end of the 35S
15 promoter, through the MZTP sequence and into the mtlD gene was sequenced by dideoxy DNA sequlo-nrin~ to confirm the correct composition of this region and to ensure that the MZTP and mtlD gene are in frame with one another. A map of the plasmid is shown in Figure 6.
An additional ~A~lG;>:~ion vector for the mtlD gene was created by 20 removing the bar gene from pDPG18~ using SmaI. After blunting the ends of the mtlD gene, it was ligated into the pUC-based vector; b~,L~,L,.. the maize AdhI
promoter/AdhI, intron and the I~ S~ 7 3' end from ~grobacterium tumefaciens (provided in pCEV5 from Calgene, Inc., Davis, CA). This plasmid vector was clecipn~tecl pDPG469.

~Y~ple IT
Prep~ration of Type ~I Callus for Transformation Tniti~tion ~n~i M~int~n~ne~ of Cell ~ine ~Tg24 TmmRtTlre embryos (0.5-1.0 mm) were excised from the B73-derived 5 inbred line AT and cultured on N6 mediurn with lO0 ,uM silver nitrate, 3.3 mg/L
dicamba, 3% sucrose and 12 mM proline (Medium 2004, see Table l). Six months after initiation, type I callus was ~ rt;l-ed to Medium 2008. Two months later type I callus w~ Ll~lsr~.led to a medium with a lower corl~ontration of sucrose (Medium 279). A sector of tvpe II callus w~ Identified 17 months 10 later and was llcul~re~l~d to Mediurn 279. This cell line is ~ r~l." in nature, unorg~ni7e~1 rapid growing, and embryogenic. This culture is e~ily adaptable to culture in liquid or on solid m~flillm The first suspension cultures of AT824 were initi~tPcl 31 months after culture initiation. Suspension cultures were initi~t~"l in a variety of culture media 15 including media C~ ig 2,4-D as well ~ ~ nTh~ as the auxin source, e.g., media tle~ign~tecl 210, 401, 409, 279. Cultures were ~ n;l~fcl by transfer of a~lv~ ly 2 rnl packed cell volume (PCV) to 20 ml fresh culture medium at 3.5 day intervals. AT824 w~ routinely L~al~r~ ,d betw~;ell liquid and solid culture media with no effect on growth or morphology.
Sus~el~ion cultures of AT824 were initially Clyu~-~s~ved 33-37 months after culture initiation. The survival rate of this culture was i~ vved when it was ~lyv~l~s~,.ved following three months in ~ ..,sion culture. AT824 .~ion cultures have been ~,Iyo~es~ ed and reiniTi~t~cl from c~yv~ ,.v~lion at regular intervals since the initial date of freezing. P~p~te~l25 cycles of L~,~,~lg have not aLrt;cLed the growth or ~ r ~ hility of this culture.

_ W O 97/26365 PCT~US97/00978 Table i~ r.sliv~: Embotlimçnt~ of Tissue Culture Media Which are Used for Type II Callus Developme~t? ~evelopment of S~ n~i~n Cultures and Rcgcn~.~Ltion of Plant Cells (Spe~;fi~lly Maize Cells) BASAL OTHER
MEDIA NO. MEDIUM SUCROSE pH COMPONENTS
(AmountlL) 101 MS 3% 6.0 MS ~dl~ lS
100 mg myo-inositol Rsl-~.to~r 189 MS - 5.8 3 mg BAP
.04 mg NAA
.5 mg niacin 800 mg L-~ n~
100 mg C~ --;--o~
20 g sorbitol 1.4 g L-proline 100 mg myo-inositol Gelgro - 201 N6 2% 5.8 N6 Vi~LilS
2 mg L-glycine 1 mg 2,4-D
100 mg casein hydrolysate 2.9 g L-proline Gelgro 210 N6 3% 5.5 N6 ~ikulLllS
2 mg 2,4-D
250 mg Ca pantothenate 100 mg myo-inositol 790 mg I~ p~rz-gin~
100 mg casein hydrolysate 1.4 g L-proline 2 mg glycine Hazelton agar W O 97/26365 PCTrUS97/00978 6g BASAL OTMER
MEDIA NO. MEDIUM SUCROSE pH COMPONENTS~
(Amount/L) r 223 N6 2% 5.8 3.3 mg dicamba I mg thiamine 0.5 mg niacin 8Q0 mg L~:~cp~r~?gine 100 mg casein hydrolysate 100 mg myo inositol 1.4 g proline Gelgro 3 mg bialaphos 227 N6 2% 5.8 2 mg L-glycine 100 mg casein hydrolysate 2.9 g L-proline Gelgro 279 N6 2% 5.8 3.3 mg dicamba 1 mg thi~mine 0.5 mg niacin 800 mg L-a~a,a~ e 100 mg casein hydrolysate 100 mg myo-inositol 1.4 g proline Gelgro 401 MS 3% 6.0 3.73 mg Na,LDTA
0.25 mg thi~min~
1 mg 2,4-D
2 mg NAA
200 mg casein hydrolysate 500 mg K2SO4 400 mg KH,PO, 100 mg myo-inositol W O 97/26365 PCT~US97/00978 BASAL OT~ER
MEDIA NO. MEDIUM SUCROSE pH COMPONENTS
(Amount/L) 409 MS 3% 6.0 3.73 mg Na2EDTA t 0.25 mg th;~mine 9.9 mg ~iic~mh~
200 mg casein hydrolvsate 500 mg K2SO4 400 mg KH2PO4 100 mg myo-inositol 425 MS 3% 6.0 3.73 mg Na2EDTA
0.25 mg thi~minç
9.9 mg dicarnba 200 mg casein hydrolysate 500 mg K2SO4 400 mg KH2PO, 100 mg myo-inositol 3 mg bialaphos 50I CIark's 2% 5.7 Medium 607 0.5x MS 3% 5.8 0.5 mg thi~mine 0.5 mg niacin Gelrite 734 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1.5 mg 2,4-D
14 g Fe sequestrene 200 mg casein hydrolysate 0.69 g L-proline Gelrite W O 97/26365 PCTrUS97/00978 BASAL OTHER
MEDIA NO. MEDIUM SUCROSE pEI COMPONENTS~
(Amount/L) 735 N6 2% 5.8 1 mg 2,4-D
0.5 mg niacin 0.91 g L-~p~r~ginf~
100 mg myo-inositol I mg thi~min~
0.5 g ~ES
0.75 g MgC12 100 mg casein hydrolysate 0.69 g L-proline Gelgro 739 N6 2% 5.8 1 mg 2,4-D
0.5 mg niacin 0.91 g L-asp~r~gin~
100 mg myo-inositol 1 mg thi~mint-0.5 g MES
0.75 g MgCl2 100 mg casein hydrolysate 0.69 g L-proiine Gelgro 1 mg bi~ h-~s 750 N6 2% 5.8 1 mg 2,4-D
0.5 mg niacin 0.91 g L-~d.a~ e 100 mg myo-inositol 1 mg thi~min~
0.5 g MES
0.75 g MgC12 100 mg casein hydrolysate 0.69 g L-proline Gelgro 0.2 M ~
1 mg bi~l~phos W O 97/26365 PCTrUS97/00978 BASAL OTHER
MEDIA NO. MEDIUM SUCROSE pH COMPONENTS~
(Amount/L) 758 N6 2% 5.8 I mg 2,4-D J
0.5 mg niacin 0.91 g L-~p~rA~ine 100 mg myo-inositol 1mg ~ ni,~
0.5 g MES
0.75 g MgCI7 100 mg casein hydrolysate 0.69 g L-proline Gelgro 3 mg bialaphos 2004 N6 3% 5.8 I mg thi~mine 0.5 mg niacin 3.3 mg ~ic~m}~
17 mg AgN03 1.4 g L-proline 0.8 g L-asparagine 100 mg casein hydrolysate 100 mg myo-inositol Gelrite 2008 N6 3% 5.8 1 mg ~hi~mine Q.5 mg niacin 3.3 mg tlic~mh~
1.4 g L-proline O.X g L-~ IA~;II~.

Basic MS mt?riillrn described in Murashige et al., (cited supra (1962)). This me~iTIm is typically modified by decreasing the NH4NO3 from 1.64 g/l to 1.55 g/l, and omitting the pyridoxine HCI, nicotinic acid, myo-inositol and glycine.
S N6 m~lium described in Chu et al., Scientia Sinica. 18, 659 (1975).

W 097/26365 PCT~US97/00978 NAA = Napthol Acetic Acid IAA = Indole Acetic Acid 2-IP = 2, isopentyl adenine 2,4-D = 2, 4-Dichlorophenoxyacetic Acid S BAP = 6-benzyl aminopurine ABA = abscisic acid *Basic m~ m described in Clark, J. Pls~nt Nutrition 5, 1039 (1982) Tniti~tion and M~inten~nce of Type II ~',~111l~ of the ~e~otype Hi-IT
The Hi-II genotype of' corn was developed from an A188 x B73 cross.
This genotype was developed sperifir~lly for a high frequency of initiation of type II cultures (100% le~ollse rate, Armstrong et aL., ~i7~: Genetics Coop Newsletter, 65, 92 (1991)). Tmm~tllre embryos ~8-12 days post-pollination, 1 to 1.2 rnm) were excised and cultured embryonic axis down on N6 me~ lm 15 c~-"~ i"g 1 mg/L 2,4-D, 25 mM L-proline (Medium 201) or N6 me~ m cont~ining 1.5 mg/L 2,4-D, 6 mM L-proline (Medium 734). Type II callus was initiRt~cl either with or without the presence of 100 ,uM AgNO3. Cultures initi~tecl in the presence of AgNO3 were transferred to medium lacking this compound about 14-28 days after culture initiation. Callus cultures were 20 ;n~lb~t~l in the dark at about 23-28~C and LL~l~r~ d to fresh culture medium at about 14-21 day intervals.
Hi-II type II callus was m~int~in~d by manual selection of callus at each transfer. Alternatively, callus was ~ ended in liquid culture m~-lillm, passed through a 1.9 mm sieve and replated on solid culture medium at the time of 25 transfer. This sequence of manipulatiQns enriches for recipient cell types.
Regenerable Type II callus that is suitable for tran~rollll~lion was routinely developed from the Hi-II genot,vpe and hence new cultures were developed every 6-9 months. Routine generation of new cultures reduces the period of time over W O 97/26365 PCTrUS97/00978 which each culture is m~int~in~d and hence insures reproducible, highly regenerable, cultures that routinely produce ~ertile plants.
Initi~tiorl of ~mhryos of ~h.o genotSype Hi-II.
T...,..~ embryos of the Hi-lI genotype (8-12 days post pollination, 1.0-5 2.5 mm) were excised and cultured embryonic axis down on Medium 201, or other equivalent or similar medias, with or without the addition of 100,uM
AgNO3. r.. n~.. ,G embryos were cultured in the dark at about 23-28~C for about 0-14, ~lGÇ~,.ably about 2~, days prior to ll~r~ ion.

~ ple ~
Tran~form~tion of C~ll Cultures Microprojectilç Bombardment: AT824.
AT824 :jus~Gl~ion culture cells were sllb~ ,d to fresh Medium 401, at about 0-3, p.~ bly at about 2, days prior to particle bombardment. Cells were plated on to solid Medium 279, or other similar medias, at about 0-24, preferably about 4, hours before boml)ar~ ,.-L of about 0.5-1.0 ml packed cell volume per filter. Tissue can be treated with or without the ~ c n of about 200 mOsm sorbitol or m~nnitol for about 0-5, preferably about 3, hours prior to bombal.ll~ L.
DNA was ~l~ci~iL~led on to gold particles as follows. A stock solution of gold particles was prepared by adding 60 mg of 1 ~lm gold particles to 1000 ~Ll absolute ethanol and ;"~ h,.l;-,g for at least 3 hours at room le~ followed by storage at about -20~C. Twenty to thirty-five 1ll sterile gold particles are centrifuged in a mic~uce..Lliruge for 1 minute. The ~ l is removed and 25 one ml sterile water is added to the tube, followed by cPnlrifilg~fi~n at 2000 rpm for S i,~ s Microprojectile particles are ~~u~ -ded in 30 ~Lg total DNA
cc)~ ;..g a selectable marker, such as bar, EPSPS, or deh, and the m~'D gene W O 97126365 PCTrUS97/00978 which is operably linked to a promoter. Approximately 220 ~11 sterile water, 250~112.5 M CaCl2, and 50 ul spermidine stock are then added. The mixture is thoroughly mixed and placed on ice, followed by vortexing at 4~C for 10 minutes and cG.iL.;I..p~tiQ~ at 500 rpm for 5 minl~tes The sllp~m~t~nt is removed and the 5 pellet re~u~ ded in 600 111 absolute ethanol. Following cPntrifiJg~tion at ~00 rpm for S ",i,...l~c the pellet is resuspended in 36 ~Ll of absolute ethanol.
Approxim~t~.ly 5-10 ~1 of the particle IJL~dLdlion was dispensed on the surface of the flyer disk and the ethanol was allowed to dry completely. DNA
was introduced into cells using the DuPont Biolistics PDSlOOOHe particle 10 bombardment device. Particles were accelerated by a helium blast of ,p~ hllately 1100 psi. Zero to seven, preferably about 1-4, days following bombardment, cells were transferred to 10-20 mls liquid Mediurn 401, or other similar merli~c Tissue was subcultured twice per week. In most cases, during the first week there was no selection ,~)l'CS:iUlG applied.
15 Microprojectile Bomb~ Type II .~ us firom th~ genotype Hi- IT.
Hi-II callus cultures are bombarded similarly to AT824 suspension cultures. A~plo~illlalely 0.5-1.0 ml packed cell volurne was plated on to Whatman filters after a brief liquid phase. Cells were either plated on to solidmedia or left on a bed of wet filters prior to bonlb~llent. Cells can be 20 bombarded with or without the :~AAitir)n of an osmoticum before bc~lllbalLllent (liquid or solid) in a manner similar to that described above for AT824.
Following particle boml)~ elll cells r~m~into~l on solid Medium 201, or other similar mt~ c, in the absence of selection for about 0-2 weeks, preferably for about 1 weelc At this time cells were removed from solid m~inm, re~ ended 25 in liquid Mediurn 201, or other similar m.oAis~c, replated on Whatrnan filters at about 0.1-1.0 ml PCV per filter, and transferred to Medium 201, or other similarm~Ai~c, co~ i,-g about 0.5-3.0 mg/L bialophos.

W O 97126365 PCT~US97/00978 Ronlbarrltnent of I~ lc Fmhryos.
Tmm~tllre embryos (1.0 - 2.5 mm in length) were excised from surface-st~ril;7to-1, greenhouse-grown ears of Hi-II about 10-12 days post-pollin~tion A~-u~imately 30 embryos per petri dish were plated axis side down on Medium 5 201, or other similar medias. Embryos were cultured in the dark for about 1-14 days at about 23-28~C.
Approximately four hours prior to bomb~l.c.lt, embryos were r~ d to Medium 201 with the sucrose conr~ntr~3tion increased from about 3% to 12%. When embryos were transferred to the high osmoticum ".~.1;".., 10 they were arranged in concentric circles on the plate, starting 2 cm f~om thecenter of the dish, pocitic-~lod such that their coleorhizal end was orientated toward the center of the dish. Usually two concentric circles were formed with about 25-35 embryos per plate.
Pl~ ~aLiOn of gold particles carrying plasmid DNA was p~ .ro..lled as described above. The plates CU~ ;tl~ embryos were then placed on the third shelf from the bottom, at about S cm below the sLo~g screen. The 1100 psi rupture discs were used. Each plate of embryos was bombarded once. Embryos were allowed to recover about 0-7, preferably about 1, days on high osmotic strength Illediulll prior to initiation of sel~ct;on St~hle T~ rù~ nl;on of SC716 ~nt1 AT824 Cells Usir~ pDPGl65 ~ncl pnP(~T~08 by Flectropor~tion Maize ~ cl~ion culture cells were enzyme treated and ele~L.u~oldltd using conditions described in Krzyzek et al. (PCT Publication WO 92J12250, incol~u.dled by l~r~ .~ .lce herein). SC716 or AT824 ~ n culture cells, three days post sllbc~llhlre, were sieved through 1000 ,um sf~inlP~ steel mesh and washed, 1.5 ml packed cells per 10 ml, in incubation buf~er (0.2 M l~n~ ;tvl, 0.1% bovine serum albumin, 80 mM calcium chlorid~7 and 20 mM 2-(N-W O 97/26365 PCTrUS97/00978 morpholino)-ethane sulfonic acid (MES), pH 5.6). Cells were then treated for 90 min~ltrs in incubation buffer cont~inin~ 0.5% pectolyase Y-23 (Seishin ph~rm~relltic~l, Tokyo, Japan) at a density of 1.5 ml packed cells per 5 ml of enzyme solution. During the enzyme tre~trn~nt~ cells were incllh~t.od in the dark 5 at approximately 25~C on a rotary shaker at 60 rpm. Following pectolyase tre~tmrnt, cells were washed once with 10 mI of incubation buffer followed by three washes with electroporation buffer (10 mM 4-(2-Hydroxyethyl)-l-pi~c.,,~ eth~nesulfonic acid (HEPES), 0.4 mM m~nnitol). Cells were ~,.u.~ ded in ele~iLIo~oldLion buffer at a densit,v of 1.5 ml packed cells in a total 10 volume of 3 ml.
T.inrs~ri7rd plasmid DNA, 100 ,ug of EcoRI digested pDPG165 and 100 ~Lg of EcoRI digested pDPG208, was added to 1 ml aliquots of elecllo~o~dlion buffer. The DNA/elec;Llu~olaLion buffer was inrub~t~l at room lelllp~laLulc for oxil~ t;ly 10 ,~j"~ c To these aliquots, 1 ml of ~us~ension culture 15 cells/ele-;LI~olaLion buffer (c~ ;..ing ~pl~x;~ ly 0.5 ml packed cells) were added. Cells and DNA in cle~iLI~,~uldLion buffer were inrllh~tef~ at room lG111~,.alUIG for approxim~trly 10 III;I~ S One half ml aliquots of this m-ixture were ~.fell~d to the ele.;~-ol)o-~ion chamber (Puite, Plant Cell Rep., 4,274 (1985)) which was placed in a sterile 60 X 15 mm petri dish. Cells were elec~lopoldLed with a 70,100, or 140 volt (V) pulse discharged from a 140 microfarad (,uf) c~r~ritor Approximately 10 minlltes post-electroporation, cells were diluted with 2.5 ml Mediu~n 409 co~ lg 0.3 M "~ ells were then sG~aled from most of the liquid meflj~lm by drawing the ..u~.~ .ion up in a pipet, and ~relling 25 the mr~ lm with the tip of the pipet placed against the petri dish to retain the cells. The cells, and a small amount of m~ium (~pl~ dl~ly 0.2 ml) were dispensed onto a filter (Whatman #1, 4.25 cm) overlaying solid Medium 227 W O 97/26365 PCT~US97/00978 (Table 1) U~ 0.3 M mannitol. After five days, the tissue and the supporting filters were transferred to Medium 227 Cf)nt~;nin~ 0.2 M m~nnitol.
After seven days, tissue and ~UplJU~ lg filters were L~ ar~ d to Medium 227 without m~nnitol.
5 F1ectroporation of Tmm~i -re embryos Tmm~tllre embryos (0.4 - 1.8 mm in length) were excised from a surface-sterilized, greenhouse-grown ear of the genotype H99 11 days post-pollination.
Embryos were plated axis side down on a modified N6 m~ lm CO-I/~;n;-.g 3.3 mg/l dicamba, 100 mg/l casein hydrolysate, 12 mM L-proline, and 3% sucrose 10 solidified with 2 g/l Gelgro~), pH 5.8 (Medium 726), with about 30 embryos per dish. Embryos were cultured in the dark for two days at about 24~C.
Tmmç~ tely prior to ele.;l~ o,dlion, embryos were enzym~tie~lly treated with 0.5% Pectolyase Y-23 (Seishin ph~rm~eutical Co.) in a buffer co..l~.;,.;"~
0.2 M m~nn;tol~ 0.2% bovine serum albumin, 80 mM calcium chloride and 20 15 mM 2-(N-morpholino)-ethane sulfonic acid (MFS) at pH 5.6. El~ylllalic digestion was carried out for 5 ".1.,..l~ at room Ic~ cldlulc. A~ u;~ lately 140embryos were treated in batch in 2 ml of en_yme and buffer. The embryos were washed two times with 1 ml of 0.2 M m~nnitol, 0.2% bovine serum albumin, 80 ~ m chloride and 20 mM MES at pH 5.6 followed by ~ree rinses with 20 cle~ upoldlion buffer consisting of 10 mM HEPES and 0.4 M m~nnitol at pH

7.5. For the electroporations, the final rinse of ele~ upoldlion buffer was removed and the embryos were in~lb~t~d with 0.33 mg/ml lin~ri7~d pDPG165, 0.33 mg/ml :lu~ coiled pDPG215, or 0.33 mg/ml 1;~F~ 1 pDPG344 in cle~ ol)olalion buffer. One half ml aliquots of DNA in elc~,LLo~o~ nn buffer 25 and twenty embryos were transferred to the cle~ alion chamber that was placed in a sterile 60xlS mm petri dish. An electrical pulse was passed through i.c.

W O 97/26365 PCTrUS97/00978 the cells from a 500 ,uf ç~r~citor that was charged to l00 volts (400 V/cm fieldstrength, 160 ms pulse decay time; expi~npnt~ pulse).
Tmm~ t,Dly following ele-i~u~cldlion, embryos were diluted l:10 with Medium 726 COII~ g 0.3 M .~ 1. Embryos were then tr~n~ferred to 5 Gelgro~) solidified Medium 726 CO~ g 0.3 M .~h~ ol. Embryos were incubated in the dark at about 24~C. After five days embryos were transferred toGelgro solidified Medium 726 cont~ining 0.2 M m~nnitol. Two days later embryos were llcu~r~ d to selection m~-linm F,Y~nUPIe rV
Identification of Tr~n~formed Cells U.ein~ .~eiectable Markers In order to provide a more efficient system for i~nt;fi~tic-n of those cells receiving DNA and ;~ g it into their gen-)mPs it is desirable to employ a means for selecting those cells that are stably ~ r~.",.~ One lS exemplary embodiment of such a method is to introduce into the host cell a marker gene which confers le~ e to some nor,n~lly in~1;bi~o.y agent, e.g., an antibiotic ûr herbicide. The l ot~l-1ially l.~rulllled cells are then exposed to the agent. In the population of surviving cells are those cells wLc.~ generally the re~ict~nre-cvl~r~ lg gene has been ;III~t~ and ~r~sed at sufficient levels 20 to perrnit cell survival. Cells may be tested further to c~nfir,-n stable integration of the exogenous DNA. Using embryogenic ~ icn c.lll"l~s, stable trarnsfo",.s..,l~ are recovered at a fic4~ cy of ~u~lv~;"~te1y 1 per l000 tr~n~ nt1y c~ cs:~mg foci.
One of the difficulties in cereal ,l,",~;r~...",.1;on, e.g., corn, has been the 25 lack of an effective selective agent for ~ ro",-,-~ cells, from to~ ol~lll cultures (Potrykus, Tr~n(l~ P.iot~ h 1, 269 (1989)). Stable l~ro...~ ; were recovered from bombarded nonembryogenic Black Mexican Sweet (BMS) maize suspension W O 97/26365 pcTrus97/oo978 culture cells, using the neo gene and selection with the aminoglycoside, kanamycin (Klein et al., Pl~nt Phvsiol., 91, 440 (1989). This approach, while applicable to the present invention, is not ~lef~Ll~,d because many monocots are;ne~neitive to high conr~ntr~tions of aminoglycosides ~Dekeyser et al., ~1~
S Physiol.. 2Q, 21-7 (1989); Hauptmann et al., Pl~nt Phy.eiol., 86,602 (1988)). The stage of cell growth, duration of exposure and con~çn1r~tion of the antibiotic, may be critical to the sllçcçe~ful use of arninoglycosides as selective agents to identify L.,u~.,rul~ u.L~, (Lyznik et al., Pl~nt Mol. Riol 1~,151 (1989)), Yang et al., pl~nt Cell Rep., 7, 421 (1988); Zhang et al., Pl~nt Ceil Rep.. 7, 379 (1988)).
10 For exarnple, D'Halluin et al. (The Pl~nt Cell, 4, 1495 (1992)) demonetr~t~A that using the neo gene and selecting with ~ a,l.y-ii.lLIcu~ l~lL., could be isolated following ele~tlupoldlion of imm~3tllre embryos of the genotype H99 or type I
callus of the genotype PA91. In addition, use of the aminoglycosides, kanamycin or G418, to select stable l"...~r~ nt~ from embryogenic maize cultures can 15 result in the isolation of 1.,.7i~1L calli that do not contain the neo gene.
One herbicide which has been ,.~g~>e~"~ d as a desirable selection agent is the broad .,~e~iLluLl. herbicide bi~l~rh~-s B~ rhns is a tripeptide antibiotic produced by Streptomyces hygroscopicus and is co.nposed of phosphinothricin (PP17, an analogue of L-gint~mic acid, a~id two L-alanine rç~ 7~ Upon 20 removal of the L-alanine residues by intracellular pepti~e~, the PPT is released and is a potent inhibitor of ~l..l7....il)~ synthetase (GS), a pivotal enzyme involved in am nonia ~imil~ti~n and nitrogen metabolism (Ogawa et al., Sci. Rep.
~ik~, 13, 42 (1973)). Synthetic PPT, also known as Gluro~ the active ingredient in the herbicides Basta~ or Liberty~ is also eLr~ iYe as a selection 25 agent. Inhibition of GS in plants by PPT causes the rapid ~c~lm~ tic-n of ammonia and death of the plant cells.

CA 02243269 l998-07-l6 W O 97/26365 PCTrUS97/00978 The organism producing bialaphos and other species of the genus Streptomyces also synthP~i7Ps an enzyme phosphinothricin acetyl transferase (PAT) which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogenes. The use of the herbicide reci~tRn~e 5 gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE
3642 829 A wLeLei,l the gene is isolated from Streptomyces viridochromogenes.
In the bacterial source org~ni~m, this enzyme acetylates the free arnino group of PPT p.~./ellLllg auto-toxicity (Thompson et al., Fh~O J., 6, 2519 (1987)). The har gene has been cloned (Murakami et al., Mol. Gen Genetics. 205, 42 (1986);
10 Th~mrson et al., supra) and t;~ re,,ed in ~ g~--ic tobacco, tomato and potatoplants (De Block, l~l~RO J.,6,2513 (1987)) and Brassica (De Block et al., Plant P~siol.~ ~1, 694 (1989)). In previous reports, some ~lSgcl~C plants which c~ ;,sed the rP~i~t~nce gene were completely resistant to commercial formulations of PPT and bialaphos in greenhouses.
EP patent 0 242 236 refers to the use of a process for protecting plant cells and plants against the action of ~ t~minP ~7y~ e inhibitors. This application also refers to the use of such of a process to develop herbicide rç~i~t~n-~e in ~ l plants. The gene encoding r~si~t~n~e to the herbicide LIBERTY (E~oechst, phosphinothricin or Gl~lfo~in~tP~)) or Herbiace (Meiji Seika,20 bialaphos) was said to be introduced by Agrobacterium infection into tobacco (Nicotiana fabacum cv Petit Havan SRl), potato (~olanum tuberosum cv Benolima) and tomato (Lycopersicum esculentum), and col~lled on these plants rP~i~t~n~e to application of herbicides.
~nother herbicide which is usefi~l for selection of l,d.lsr~,. ed cell lines in 25 the practice of this invention is the broad ~e~L~ herbicide glyphc-ss~tP
Glyphosate inhibits the action of the enzyme EPSPS which is active in the aromatic amino acid bio:~yllLl,~Lic pathway. ~nhibition of this enzyme leads to W O 97/26365 PC~rUS97/00978 starvation for the amino acids phenyl~l~nin~, tyrosine, and tr~Lophan and secondary metabolites derived thereof. Comai et al., U.S. Patent 4,535,060, issued August 13, 1985 describe the isolation of EPSPS mutations which infer glyphosate rç~i~t~nre on the Salmonella ~phimurium gene for EPSPS, aroA. The S EPSPS gene was cloned from Zea mc~s and mutations similar to those found in a glyphosate resistant aro~ gene were introduced in vifro. The mutant gene encodes a protein with amino acid changes at residues 102 and 106. Although these mutations confer resistance to glyphosate on the enzyme EPSPS, it is anticipated that other mutations confer the same phenotype.
An ~ pl*~ y embodiment of vectors capable of delivering DNA to plant host cells is the plasmid, pDPG165 and the vectors pDPG433, pDPG434, pDPG435, and pDPG436. The plasmid pDPG165 is illustrated in Figure 2. A
very hllpo~ t cOlllpO~ of this plasmid for yullJoses of genetic l~ ~lm~lion is the bar gene which encodes a marker for selection of L.~.~ru.l-led cells 15 exposed to b;~l~ph-)s or PPT. Plasmids pDPG434 and pDPG436 contain a maize EPSPS gene with mutations at amino acid residues 102 and 106 driven by the ~ctin promoter and 35S promoter-Adhl intron I, ~ c~ y~ The ~ led EPSPS gene enco~lçs a marker for selection of l.~rulllled cells.
Tr~n~form~tion of Cell T in~ AT824 Usir~ Ri~l~hns Sel~-~tion Pollowin~ p~rffcle 20 P~olnb~L,l,l~c~ -Selection in r iquid Mt ~ m A :iu~ ion culture of AT824 w~ l~.*;.l1~1;...?d in Medium 401. The bomba~ w~ done ~ described above, with a few variations. Four filters of AT824 :,u:~l,~ sion cultures were plated out at ~10~L;III~I~IY 0.75 ml PCV on toMedium 279. There were 4 filters bombarded with pDPG165 (Figure 2, 35S-bar-25 Tr7) and pDPG480 (Figure 3, 35S-mtlD-Tr7). The cells were left on the solid Medium 279 for 4 days and then put into liguid Mediull- 401. Liquid selection w~ started after one p~sage (3.5 days) using 1 mglL bialaphos. Cells were thin W O 97/26365 PCT~US97/00978 plated one week later at 0.1 ml PCV (2 weeks after bombardment) on to Medium 279 + 3mg/L bi~l~phns Putative transfo~ x were observed about 8 weeks later. A total of 46 bialaphos-resistant lines and 25 lines c...ll;1;..;.lg mtlD DNA, as det~rminPd by a polymerase chain reaction, were obtained.
5 Tr~n~form~tion of Cell T.ine AT824 Usin~ Rialaphos Selection Following Particle Rombar~lment--Solid Medillm Sele~ tion Cells were bombarded as described above, except the gold particle-DNA
plcpal~lion was made using 25 ~1 pDPG319 DNA (bar gene and aroA Gx~le~sion cassette co,~l~;..;..g the a-tubulin promoter). Following particle b(nllba~dlllent 10 cells rçm~ineri on solid Medium 279 in the ~hsenf~e of selection for one week.
At this time cells were removed from solid medium, rcsux~ ded in liquid Medium 279, replated on Whatman filters at 0.5 ml PCV per filter, and tr~nxfprred to Medi~rn 279 cn--~ ;llg 1 mg/L bialaphos. Following one week, filters were ~ llxr~lGd to Medium 279 c(l~ ;..;..g 3 mg/L bi~l~phns One week 15 later, cells were l~u~nded in liquid Medium 279 and plated at 0.1 ml PCV on Medium 279 co--~ g 3 mg/L bialaphos. Nine l.,...xrO,...~nt~ were ic1pntifiied 7 weeks following bombardment.
Tr~n~ro,...~lion of Hi-IT e~ in~ T~i~l~hns Selection Followin~ P~r~icle Romhi1"1,. e."
Hi-II callus was initi~t~-l and bombarded as described above. Four filters were bombarded with pDPG165 Figure 2, (35S-~ar-Tr7) and pDPG493 (Figure 4, 35S-mtlD-Tr7). After bomb~ll.,nl, cells were allowed to recover on solid media for 3 days. The four original bombarded filters were l~,...~r~ d to Mc(lium 201 col~ 1 mg/L bi~l~rhns for 2 weeks. After this time, cells 25 were removed from solid medium, ~c,~ p~n-l~d in liquid meflillm, replated on Whatman filters at 0.5 ml PCV per filter, and l~ d to Medium 201 co~ ,.;"g 1 mglL bialaphos. Following 2-3 weeks, cells were l~U~ ~tle~l in WO 97/26365 PCT~US97/00978 .

liquid medium and plated at 0.1 ml PCV on Medium 201 cn../~;ll"lg 3 mg/L
bialaphos. Putative L.al~r~ L~ were visible about 5-6 weeks after thin plating.
There were 8 bialaphos-resistant lines, and out of t_ese 4 Ll~folmal.L~ contained mtlD DNA, as rl~L~ e~ by PCR.
S Another consideration is that plants may need to have very high levels of osmoprotectant to show a .cignifir~nt change in stress resict~n~e. Thus, a combination of mtlD constructs with dif~erent promoters was transformed into Hi-II callus, and mtlD PCR+ ~ ro~...h.,l~ were obtained. Southern and PCR
analysis can rleterrnin~? which mtlD constructs have been incol~ul~Led into which 10 L~ sr~llnants.
Tr~n~rc,l."~lion of Jmm~hlre Fmhryos of the Genntype Hi-TT Usin~ Ri~l~hos ~c a Selective A~ent Followin~ Particle Bo~ h..e..l Tmm~hlre embryos of the genotype Hi-II were bombarded as described above using pDPG670 (H3C4-adhl-bar-Tr7~ and pDPG598 (Actl-mtlD-Tr7).
15 Embryos were allowed to recover on high osmoticurn me~ m (Medium 201 +
12% sucrose f lOOIlM AgNO3) for about 1-3 days, I~l..r~ .d~ly at least overnight, i.e., for about 16 - 24 hours, and were then Ll~r~ ,d to selection medium c~ hl;ll~ 1 mg/l bi~l~rhns (Medium 201 + 1 mg/l bi~l~rh-s +lOO~lM AgNO3).
Embryos were m~in~ine-l in the dark at 24~C. After two to four weeks on the initial selection plates about 50% of the embryos had formed Type II callus and were ~ sr~ d to selective me~i--m c~""S~;.,;,.g 3 mg/l bialaphos (Medium 201 +
3mg/L bialaphos). Re~ollding tissue was subcultured about every two weeks onto fresh selection me~ m (Medium 201 + 3mg/L b~ rh-s). Six bi~l~rhns-resistant lines were l.co~.Gd from this c"~.;,. .ent If cells are producing too much m~nnitol at the callus level~ there may be possible cell death due to swelling or bursting. Tmm~hlre embryo llcu~ lldLion c~ . . ".lent~ have been con~lllete~ using a low level of nl~ .l during selection.
;

W O 97/26365 PCT~US97/00978 .

It is possible that osmoticum in the medium may colmt~r~rt ...~....;l~l producing cells to make a more isotonic environment. It may be possible to obtain high mtlD expressing ~ ults by doing so.

h',~ ple V
pl~-ntg From Tr~ngfor~ned Cellc For use in agriculture, transf~rrn~tion of cells in vitro is only one step toward commercial utilization of these genotypically new plant cells. Plants must be regen~or~f~d from the ILd~rulllled cells, and the regen~or~t~1 plants must be10 developed into full plants capable of growing crops in open fields. For this ~ l,uose, fertile corn plants are required. The following protocol describes a method for re~ Gll~atillg plants, but one of skill in the art will be f~miii~3r with other equally efficient protocûls.
During ~ ;nn culture development, small cell ag~ tes (10-100 15 cells) are formed, a~p~uG.llly frûm larger cell clusters, giving the culture a dispersed a~?l)e~ e Upon plating these cells to solid media, somatic embryo development can be intln~e~, and these embryos can be matured, g~ ...;..~l~cl and grown into fertile seed-bearing plants. The ~'ll*l2 r,1t~ ics of embryogenicity,le~-.. ."hi1ity, and plant fertility are gr~rlll~lly lost as a function of time in 20 suspension culture. GYU~1GSG1 valion of ~ .e~.iion cells arrests development of the culture and plG~ ; loss of these ~'ht~ c~ ;eS during the ~;lyoplescl v~lion period.
Re.~.onerz~tion of ATg24 Tl,...~rul~ nt~ ~n~l HiTT e~llus Tla~r~ were produced as described above. For l~e .~.lion tissue 25 was first ~l~srGll~d to solid Medium 223 or Medium 201~1mg/L bialaphos and inrll~te~ for two weeks. T.~l:jrUll..~l~ can be initially subcultured on any solid culture that :iU~J~Ul~i callus growth, e.g., Medias 223, 425, 409, and the like.

W O 97/26365 PCT~US97/00978 Subsequently I d.l~rollllants were subcultured one to three times, but usually twice on Mediurn 189 (first passage in the dark and second passage in low light) and once or twice on Medium 101 in petri dishes before being L ~l~rell~,d to Medium 607 in Plant Cons~. V~ri~tion.~ in the reg~ner~ti~ n protocol are normal based on S the progress of plant regeneration. Hence some of the tran~rollll~ were first subcu3tured once on Medium 425, twice on Mediurn 189, once or twice on Medium 101 followed by transfer to Medium 501 in Plant Cons~. As shoots developed on Medium 101, the light hlh,l~iLy was increased by slowly adjusting the ~ t~nr.e of the plates from the light source located overhead. All subculture 10 intervals were for about 2 weeks at about 24~C. T..~ r~ nt~ that developed 3 shoots and 2-3 roots were L.~l~r~ d to soil.
Plantlets in soil were incubated in an iTI~ ed growth charnber and conditions were slowly ad3usted to adapt or condition the plslntl~t~ to the drier and more illllrnin~ted con-1ition~ of the greenhouse. After adaptation/conditiorling 15 in the growth ch~l-bc;l, plants were transplanted individually to 5 gallon pots of soil in the greenhouse.

F~ ple VI
I:~eterrnin~Tiol- of IVI~ Activity 20 M~nnitol-l-P ne~y lrog~n~e (;~n~ sp.~~TrOph.-tu..~ c ~
The MDH assay has been used to det~rrnin~ if there is ~ ion of the mtlD gene in transforrned callus or leaf tissue. The spectrophotoll.cl~. measures dirrelc~llces at the 340 nrn wavelength, looking for a change from NAD+ to NADH, a result of ~r~s:jion of the mtlD gene fh~n~ing .. i.. ;l~l-l-phosphate to 25 fructose-6-phosphate.
Bacterial extracts are used as controls. An ali~uot of the glycerol stoclcs of b~cteri~ c~ the bar gene (pl65) or C.,.. /i1;.. ;.. g the mtlD gene (p480) W O 97126365 PCTn~S97/00978 was put into LB media (100 mg/L ~mpicill;n). These cultures are grown overnight at 37~C. The next day cultures are spun down at 5,000 rpm for 5 minlltes The pellet is rinsed with either Tris-citrate (0.1 M Tris-citrate, pH 8.5) or PAT buffer ~50 mM Tris-HCI, pH. 7.5, 2 mM EDTA, 0.15 mg/rnl leupeptin, 5 0.15 mg/ml PMSF, 0.3 mg/ml BSA, 0.3 mg/ml DTT)and spun down again. Then the pellet, about 200 ,ul of glass beads, and 500 ~1 of buffer are put into a 1.5 ml eppendorf tube and shaken twice for 20 seconds on "high" (MINI-BEADBEATERTM, Biospec Products). The tubes are then spun down and the sUpern~t~nt is used for the assay. All tubes are kept on ice.
For callus or plant eYtr~ctc about 0.5 g of tissue is used. Tissue is homogenized with a~p.oxi...~t~ly 250 ~1 of Tris-citrate or PAT buffer. Extracts are spun down in the microfuge at 14,00b rpm for 5 minnt~s Protein is quantified using the BioRad assay.
For the MDH assay, a master assay mix is made to be used for all the 15 samples. The mix inrllldes: 2.5 ml 0.1 M Tris-citrate, pH 8.5, 0.1 ml of 4 mMNA~' (dissolve one 20 mg vial of SIGMA, ~-nico~ ...;de ?~ ninlo dinucleotide, in 7.15 ml ddH20), and 0.1 ml of 6 mM ...~ ol-1-phosphate (SIGMA)).
The :,I,c~,L~u~h-.tc-m~tric readings were done as follows: 1 ml of assay rnix was put into a cuvette. Then 2-100 ,ug of protein was added. The cuvette was 20 inverted about 3 times and then the reading was initi~te~i Mea~ ents were taken for up to S .,.;~ s at 340 nm.
Rioassays for ~ ;lUl Callus assays were con~ cted on transformants derived from AT824 (S80HO-52) and Hi-II callus (HC05II-SS), as well as on controls. Callus growth 25 assays were started by plating 0.1-0.5 g callus fresh weight on to Whatman filters. Filters were then put on to media with ~flrliti~nsll cOI~ l;ons of WO 97/26365 PCT~US97/00978 osmoticum. The osmoticum includes mannitol (0, 0.3, 0.6, 0.9 M) and NaCl (0, 50, 15û, 250 mM). Fresh weight gains were taken after 2-3 weeks in culture.
To ~lPtprmin~ if there is a significant arnount of ~ nilol being produced at the callus level, osmotic potential readings can be con~ cte~l on 0.1 g callus 5 samples using the Psychrometer (Wescor Inc. C-52 sarnple ch~mhPre) by meth~e well known to the art.

h'.Y~ le vr~
Tr~7n~forln~nt pl~nt.~ into the GrP- nhouse ~nd Ch~ i~n of ~0 pls~nt~
Once plants are regenerated, hardened off in the growth charnber, plants are transferred to the greenhouse to obtain seed. Leaf sarnples are taken of theR~ plants as well as subsequent generations and crosses and endogenous m~nnitol levels are ~etPrminP~l Phenotypic changes in the plants posePe~;ng the ~Llsg~ e 15 were docnment~P~1 To ~letPrminP the m~nnitol content of these plants, a~ lely 30 grams (fresh weight) of the tip of mature, healthy leaves are e~mrlec~ The leaf sarnples are placed in 50 ml poly~io~ylene test tubes in a -70~C freezer. Frozenleaf samples are then dried in a freeze drier and stored until analysis. In s~
20 50 ml polypropylene test tubes, 1.0 to 1.5 gram qll~ntitiPe of dried leaf sarnple are weighed. The samples are then homogenized in 40 mls of 80% ethanol (v/v) using a Polytron. The reslllting solutions are incubated in a 72~C water bath for 30 ~ es, with a brief vo~ g step at a~p-~ ,ately 15 31~;~.11~t''/: Following the incubation, the solutions are heated in a boiling water bath for 2 mimltPs 25 The samples are then c~..l.;r.g~-l at 3000xg for 15 mimltPe The rPslllting sllpPrn~r~nte are then lcco~ ,d and taken to dryness overnight in a 40~C nitrogen evaporator. The 1~ g paste is frozen then freeze-dried for ~PI~JX;II~ 1Y 2 . CA 02243269 1998-07-16 W O 97/2636S PCT~US97100978 hours. The dried material is dissolved in 0.5 mls of distilled, deionized water to for n the aqueous simple carbohydrate extract. The extract is purified prior to HPLC sep~r~tion techniques by passing it through a C-18 solid phase extraction column (Varian Bond Elut(~)) and a 1.2 micron acrodisc filter.
Mannitol content of the simple carbohydrate extracts are det~rmin~fl using HPLC separation techniques. An RCM m~n~s~r~l.,..ide column (Phenomenex~) is used, with water as the mobile phase. The st;~ualdled simple sugars are detectecl with an Errna~) ERC-7512 refractive index detector. The resulting sample chromatograms are analyzed using l~ mr~ peak integr~tion software 10 and coll~d.~,d to cl~umd~ograms of m~nnit-~l standards.
The above procedure for m~nnitl~l extr~(~tic~n and qu~ntific~tion from corn leaf m~teri~l was tested using a plant species which was known to possess naturally occurring endogenous levels of ~ ol. F.xtr~ct~ were prepared from leaves, roots, small stems, and large staL~cs of the celery plant. All four extracts 15 were found to possess fl~ect~hle levels of m~nnitol. Based on chromatograms obtained from standards, the amount of ...~ ol in the tissue was estim~te(l to be bcL~,e.l 20 mg (roots) to 112 mg (stems) per gram of dry weight.
During the mid vegetative stage of development, greenhouse grown Ro maize plants were sampled for leaf m~nnitol cont~nt, according to the above 20 described procedure. Over a 10 month period, leaf samples from one hundred four Ro plant clones from s~;vt;nle~ll callus cell lines were assayed. Carbohydrate extracts of Ro clones from several cell lines were found to exhibit HPLC
cl~ulll~Lograms which cu..l~ d peaks with retention times similar to m~nnitol ~lda.ds. Although leaf samples from most of the cell lines expressed relatively 25 small amounts of leaf tissue m~nnit- l, those derived from two cell lines were -found to express pukL~ , levels of m~nnitol which were over 3.0 milligr~m~ per gram of dry weight (mg/g dry wt.). Addition of ~ 1 to the extracts resulted W O 97/2636S PCT~US97100978 in an increase in the area of the "~ nilul" peak without the production of any new peaks. Levels of leaf tissue msmnitr~l in Ro clones ranged from 19.31 mg/g dry wt. for the cell line HCOSII-55 (derived from Hi-II callus) to 3.63 mg/g drywt. for the cell line S80HO-52 ~derived from AT824).
S T~ sro~ n Using the Glbl promoter T~ ~.ll,ed plant cell lines derived from AT824 suspension (S87KM) and imm~ re embryos (HI68KM) which were PCR~ for the pDPG586 construct have been in regeneration. The pDPG586 vector is potentially sensitive to ABA
induction at the callus level due to the presence of the Glbl promoter. Moreover, 10 levels of ABA are increased in drought st;nsilive plants during a period of drought ~Landi et al., Mavdica. 40 (1995)), inrlic~ting that an ABA inducible promoter is also drought inducible.
Droughted pDPG586-c~ g transgenic plants are tested for the production of ABA and for increased levels of m~nnitol HPLC analyses showed 15 low levels of ~-.~ 1 in leaf tissue from these plants. Young LdllSgCl)lC
see-ilin~ are exposed to ABA and di~,.cllces in mannitol w~y~ ion d~t .. ;.-at later plant stages. MDH assays are c~n~ cteA on ABA treated callus from tissue transformed with this col~llu~l~ Seed viability after drought is also tested to dete~nin~ wh~,Lllel Illn~ is ci~-y~e;~:ied in the embryo.
20 Tln.l.~irullllnlion Usir~ the M~i7~ Trs~n.cit Peptide ~S7.TP) The MZTP was used to express mtlD in the chloroplast. Increased mtlD
~,cssion in the ehlolopla~l can give protection to the chloroplastic photosynthetic system under reduced water availability cor~-litinn~ The e~lession of mtlD thus allows the chloroplast to osmotically adjust to the 25 cellular conditions that change as a result of changes in the water relations in the plant. In addition, if Illnl..l;Lol is expressed exclu.,i~,~ly in the cytosol, some disruption of chloroplast function eould oecur due to the imbs~ nre of osmotic .

W O 97126365 PCT~US97/00978 .

relations b~ the co~ cuL,nents of the cell. Moreover, increased mtlD
G~.e;,~ion in the chloroplast may also provide anti-oxidant activity.
One construct, pDPG587 (35S-MZTP-mtlD-Tr7 3'), has been tested using AT824 ~u~ellsion (S85KN, S87KN, S88LG), Hi-II callus (HS06LG, HZ04LG), S and imm~t lre embryos (HI88LG, HI89LG, HI9OLG, IH07LW, DL04LW, IH16LW, CS12LW, DTOlLW). PCR+ Ll~sÇ.. ,.. ,I~i with the construct were obtained. Furthermore, the presence of m~nnit-~l was clet~ctkd in transforrnantsC(J~ g the (35S-MZTP-mtlD-Tr7 3') ~ rG;,~ion ç~ccette Chloroplast viability assays, m~gn~tic isolation of chloroplasts, and greenhouse and field studies of the rt~elllt~nt t~ncfiorm~l plants under a range of water stress or non-stress conditions are ~c.r3lllled, by methods described herein or by other methods well known to the art.

F,Y~-nlple vm ~h~racterizatior~ of Rl Tr~n~forlnants Seed were lGco~.Gd from several outcrosses of S80HO-52 and HCO5II-55 R" plants. The first R, seed became available from the outcrosses involving S80HO-52 X AW. The R, seed was evaluated in three SG~dLG greenhouse pl~ntingc The first planting of twenty-two R, seeds r~snlting from the cross of S80HO-5207 X AW were planted in the greenhouse to eulll~c results from HPLC ~lel~....i.-k~l leaf tissue ...z~ ol levels to PCR-derived data. The carbohydrate profiles obtained from the twenty-two plants revealed twelve as G~ G;,~ing levels of m~nnitol cu~ ~dble to the R~,plant. The results were found 25 to agree with the PCR data developed from the sarne set of plants.
A second, larger, planting of R, populations was made in the greenhouse after additional seed became available. The pl~nting inrlllc~k~l eight populations from various outcrosses of S80HO-52 Ro plants to AW. Twenty seeds were planted per population among ten 15-gallon pots, two seeds from the lldllSg~lliCpopulation per pot plus the common tester, AW. Therefore, a total of three plants per pot were planted. During the mid-late vegcl~ e stage of development 5 a drought episode was imposed on the plants for 33 days. During midday and predawn sampling periods, several whole plant physiological measurements were collected when a~prop,i~le, inclllrling the following: 1. water relations pararneters (under water stress and r~ d con~l;tiorie)~ 2. gas ç~crh~n~e measurements, 3.Ieaf t~ c.~ c~ 4. leaf m~nnitcl .e~mrl~, 5. plant height, 6. flowering 10 synchrony, and 7. Glufosinate~) scn~ilivi~y test.
All populations exhibited approximately a 1:1 segregation for Glufosinate~ sensitivity. No visual, morphological differences were observed between plants which were resistant to Glufosinate~) (and ~le.,~.llably possessed the preselected DNA segm~nt) and those which were scll~iLivc~ This indicates 15 that no deleterious effect on plant growth and development occurred with the mtlD gene at this level of cA~l~s~ion.
Data from the twenty plants evaluated arnong each population were sorted by r~i.et~nce versus sGl~iliVily to Glufo.~ , then mean values were g~ ed In all pop~ til~n~ cA~l~,ssion of leaf tissue ...i1....il.-1, as ~c~t..~;"rd 20 by HPLC, co-segregated with cA~lc~ion of Glufosinate(~ reci~t~n~e Levels of leaf tissue ...s~l...iLol were found to a~plox;..~ levels ~A~i.,sed in the Ro plants.
A time course of leaf osmotic potential values collected from the S80HO-5201, -5205, and -5208 populations was assembled from the water relations data (Figure 7). With the exception of I sampling period in the -5205 population, 25 plants which exhibited r~ t~nc.e to Glllfa~in~te(g~ applic~tion~ were found to express more negative predawn osmotic potential values when compared to plants which were Glufosinate~ scnsiLivc. However, to fully understand the influence W O 97/26365 PCT~US97/00978 of increased leaf tissue solutes (such as m~nnit~l) on osmotic potential, the influence of tissue dehydration due to drops in total water potential must be ~Yzlmine~l During the first predawn sarnpling period, total water potential values S were between -1.0 to -2.0 bars. No differences were observed between plants with and without the gene. Plants were rewatered prior to the second and third predawn sampling period, which brought the total water potential values to -0.2 bars. When the total water potentials approach zero, the osmotic potentials are directly collly~able since the water content is similar. This indicates that 10 differences in osmotic potential values were infl~l~rlred only by differences in ~rcllmlll~t~d cell solutes and not by dehydration. During the midday sampling period, total water potential values were between -10.0 to -15.0 bars, in(l;f~ting that tissue dehydration occurred. As shown in Figure 7, there were no differences in leaf osmotic potential among any of the poplll~tion~ during the 15 rnidday period.
Midday gas CY~ h~ c data, leaf rolling observations, plant heights, and ~nth~ to siLIcing intervals were also collected in all of the eight R, popllls~tio~
No st~ti~ticz~lly si~nific~nt di~erences were observed between plants with and without the gene for any of these measurements. This in~ ~t~os that at this level 20 of ~ ol c~yl~ ion, no deleterious effect on the Ll~gcnic plants was noted.
The osmotic potential finciingc in this c,~y. ,;..- .1 lcyl~,scllLed the first direct link between gene in-1llce~ leaf ~ ul c~Lylc~ion and a significant whole plant physiological trait related to drought tolerance.

~,y~ntplc IX
Fvaluation of the Two F~hest ~,~press-~ Mannitol Cell ~,ines, S80HO-5201~W and T~COSI~-5503 ~AW
In a third greenhouse planting one R, population was included from each of the two highest m~nnitol G~ s~ lg cell lines. Twenty seeds from each of the populations S80HO-5201 X AW and HCOSII-5503 X AW were grown to the mid vegGLali~e stage of development. Plant production and arrangement in the greenhouse was similar to that described above in Example VIII. I,H132 was used as the common tester. Therefore a total of three plants per pot were 10 planted. A drought episode was then irl~osed on half of the twenty plants from each population. During midday and predawn sampling periods, the sarne whole plant physiological measurements used in the previous R, G~ nt were collected. All populations exhibited ~pr~xi...~t~oly a 1:1 segregation for Glufosinate~ sensitivity. Data from the ten plants evaluated among each 15 population/L.~,-/.--f~ l cc-mhin~tion were sorted by reei~t~nr-e versus sensitivity to Glufo.cin~tf ~, then mean values were g~ ..f .,.1~
Leaf tissue ...~....;1~.1, ~ ~lettorminec~ by HPLC, was llleasulGd once during the predawn sampling period. In both populations, GA~l~,s~ion of ~ lol co-se~ lGd with G~r~sion of <~]nfo~in~t~ r~ t~nr~ As was found in the E~o 20 plants, Rl plants from the cell line HCOSII-55 expressed leaf tissue m~nnitollevels which were a~pl.~ 1y 8 times higher than R~ plants from the cell line S80HO-52 CIable 2). Among both pop~ tion~> plants which were exposed to the drought stress conditions expressed higher levels of leaf tissue ...~ ol than plants grown under well watered conditions.

W O 97/26365 PCTrUS97/00978 Table 2. Predawn leaf mannitol content (mg/g dry wt.) in two cell lines.
GLUFOSINATE~ GLUFOSINATE(~
TRF~TMFl~T I~F!~ISTA~T SFNSITIVF.

Stressed 39.8 0.0 Watered 8.1 0.0 Stressed 5.1 0.0 ~5 Watered 1.1 0.0 Water relations data collected at 2 time periods prior to the rcwatc,;llg of plants for the HCO5II-55 and S80HO-52 pop~ tic-n~ are shown in Tables 3-6.
In the HCO5II-55 population, significant (P<0.05) differences were observed between plants which were resistant to Glufosinate~) compared to plants which 25 were not for both predawn and midday osmotic and turgor potential values.
Among the S80HO-52 population, ~ignific~nt (P<0.05) di~ lces were observed between Glufosinate~ resistant and ~el~iLivt; plants for predawn osmotic potential values, but not during the midday period.

W O 97/26365 PCT~US97/00978 Table 3. Leaf water relations during the predawn period for HCO5II-5503 X
AW Glufosinate~) resistant and susceptible plants grown under water stress and watered conditions.
GLUFOSINATEt~ PRFT~AWN P~.RTOD
TRF.~T~F.NT RF~IST~NCF. TOTAT. OSMOTIC TURGOR Y
-ka~-Stress No - 12.49 - 14.99 2.51 Stress Yes -11.73 -16.93* 5.20*
P(<) ns 0.05 0.05 Watered No - 0.20 -11.68 11.48 Watered Yes - 0.20 -12.70 12.50 P(~) ns ns ns Table 4. Leaf water relations during the midday period for HCO5II-5503 X AW
Ghlfosin~t.o~ resistant and ~us~ible plants grown under stress and watered conditions.
GLUFOSINATE~ MTr~I~AY PF.RTOD
TRF.~.TMF.NT F~F~T!~T.~CF. TOTAL OSMOTIC TURGOR
-k~-Stress No-16.21 -16.14 -0.07 Stress Yes -14.93 -18.94* 4.01*
P(<) ns0.050.05 Watered No- 4.90 -13.57 8.67 Watered Yes - 5.13 -14.25 g.l2 P(~ ns ns ns W O 97/26365 PCTrUS97/00978 Table 5. Leaf water relations during the predawn period for S80HO-5201 X AW
Glufosinate~ resistant and susceptible plants grown under watered stress and " watered conditions.
S GLUFOSINATE~) p~FnAWN pFRTOD
TRF~TMFNT ~F~;IsTAl~cETOTAT OSMOTIC TURGOR
Skess No -13.00 -14.94 1.94 10 Stress Yes -15.60* -16.40* 0.80 P(S) 0.05 0.05 ns 15 Watered No - 020 -12.58 12.38 Watered Yes - 0.20 -13.08 12.88 P(<) ns ns ns Table 6. Leaf water relations during the midday period for S80HO-5201 X AW
Glufo~in~t~q~ resistant and susceptible plants grown under stress and watered period.
GLUFOSINATE~) MTnnAY PFRIOD
TREATMFNT RF~ISTANCE TOTAT OSMOTIC TURGOR
_~
Stress No -15.01 -15.30 0.29 Stress Yes -16.37 -15.62 -0.74 P(5) ns ns ns Watered No - 6.30 -15.87 9.57 Watered Yes - 6.45 - 16.62 10.17 P(c) ns ns ns W O 97/26365 PCTrUS97/00978 After rewatering of the drought stressed plants, Glufosinate~ resistant HCO5II-55 plants continued to m~int~in more negative osmotic potential values than Glllfosin~te~) sensitive plants for up to S days (Tables 7-8). Osmotic 5 ad31lctment, as calculated by the difference between rewatered and watered plants was over 4 bars for both sample periods. These are si~nific~nt changes in osmotic potential levels compared to the plants not having the mflD gene or expressing m~nnitol. For the lower m~nnit~l e~E,..,ssil~g line S80HO-5201, no significant differences were observed for changes in osmotic potential between the 10 Glufosinate~) resistant and susceptible plants (Tables 9-10). The contrast between the higher and lower mannitol e~ lg lines may indicate the range of expression needed to work with in crop improvement.

Table 7. Differences in osmotic potential of HC~OSII-5503 X AW Glufosinate~
resistant and susceptible plants 12 hours after lc;w~h,.illg.
TIME = 12hrs Rewatered GLUFOSINATEt~) REWATER_D WATERED nTFFFRF~cF
RFSISTA~rCF OSMOTIC OSMOTIC
-bars-No - 11 .70 - 1 0.800.90 Yes -17.30**~ -11.70 5.6 P(<) 0.001 ns W O 97/26365 PCT~USg7/00978 Table 8. Di~erences in osmotic potential of HCO5II-5503-X AW Glufosinate~
resistant and susceptible plants 5 days after rewatering.
TIME = S days Rc;w~Lel~d GLUFOSINATE(~ REWATERED WATERED DIFFFR F~CF
p~F.~ ,4NC~. O~;MQTIGQSM~TI~--bars-No -12.90 -11.80 1.1 Yes -16.40** -12.10 4.3 P(<) 0.01 ns Table 9. Differences in osmotic potential o~ S80HO-5201 X AW Glufosinate~
resistant and ~ sc~ ible plants 12 hours after rewatering.
TIME = 12hrs R~w~ d GLUFOSINATE~) REWATEREDWATERED nI~FFRFNCF.
~F~IsTANcF~ OSMOTICOSMOTIC
-bars-No -11.73 -13.02 -1 .29 Yes -12.20 -12.89 -0.69 P(~ ns ns W O 97/2636S PCTrUS97/00978 .
- 10,0 Table 10. Differences in osmotic potential of S80HO-5201 X AW Glufosinate~
resistant and susceptible plants 5 days after l~w~L~;llg.

TIME = S days Rewatered GLUFOSrNATE(~ REWATERED WATERED DJFFFRFI~CF
E~F~ISTA~CF OSMOTIC OSMOTIC
-bars-No -12.36 -12.44 -0.08 Yes -13.26 -12.94 0.32 P(<) ns ns Among both populations, no statistically ~ign;fic~nt differences were 20 observed between plants with and without the gene for midday gas PY~h~nge data, leaf rolling observations, plant hei~ht~, and ~nth~si~ to silking intervals.
On the Gll.r.~ fe~ resistant HC05~I-55 plants, at flowering and fi~er developing during the ~infill, a leaf spe~L Iinp: which developed into a leaf clorosis followed by necrosis was observed. This abnorrn~lity was observed 25 mainly on plants grown under the well w~L~.~d tre~tn~ent The droughted plantsdid not eY~ibit this leaf ~ iion in the upper most leaves after lcw~,~hlg.
Due to drought in~lucecl leaf firing and s~ ,cç~ e it was not able to read the lower leaves of the stressed plants for the chlorosis or ~e--L li..g The s~ Lu~ls first ~ ,d on the oldest leaves of the plant and progressed to younger leaves 30 prior to the onset of physiological maturity. Other than the leaf chlorosis, the plants were morphologically normal and set seed. This chlorosis may disappear with plants where mannitol accumulation is targeted to the chloroplast.

W O 97/26365 PCT~US97/00978 Seed planted from this cell line has confirrn~d that this chlorosis first starts in the lower most (oldest) leaves and progresses up the plant as the leaves become older. To clctc..";..e what the pattern of m~nnitol ~-~c--m~ tion is and if there is a correlation to the occurrence of the chlorosis, leaf sarnples of the plants S from oldest to newest leaves will be analyzed. Also ultrastructural studies are being done through tr~n~mie,ciQn microscopy to see the cellular ultrastructure in the chlorotic areas co.l,p~h~,d cellular ultrastructure in leaf samples from plants without the gene and to green sectors on leaves of plants having the gene.

~.Y~nlple X
~F,valuation of lvl~nnitol F~press;n~ Transformants ;n a Field FnVironm~nt Ullder Water Stress and Irr~tf~i Conditions Under field conditions, it is n~cç~.~n. y to evaluate the phenotvpe of plants having dirr~,.e.l~ levels of m~nnitol G,~ ,ion under i~rig~t~fi and water stressed 1 5 Gont1ition.
Germplasm evaluated were the following: (S80H05201X AW) X BK R2 g~ ,.dlion with the mtlD gene; (S80H05201X AW) X BK R2 gen~-~tio~ without the gene; (HCOSII5503XAW) X BK R2 generation with the gene; and BK, a standard inbred line.
The contribution of different levels of m~nnitol t;~ple;,~ion to stress tolerance among R2 generation plants from two mannitol W~lGSsil~g cell lines, S80EIO-52 and HCO5II-55, was evaluated. The two segle~ g populations were derived from crosses of greenhouse grown R1 generation plants, ,.,..~r~ with co"~l,ucl~ co~ ;llg the mtlD gene and the bar gene, crossed 25 to the elite stiff stalk inbred d~eipn~t~od BK. Stable tran~ro..~an~ were ~i~t~min~(i by r~eiet~nre to the herbicide Glufoein~t~9. Leaf tissue of R1 ge.,~,ldlion plants cont~in~d m~nnitf~l conct~-ntr~tions from at least about 5.0 mg/g W O 97/26365 PCT~US97/00978 , dry weight, for the low e~ illg cell line, and up to about 40.0 mg/g dry weight for the high c;~les~ g cell line. The R2 generation populations were planted in a modified r~n~ mi7t-d complete block design with 4 repetitions nested within areas of low and high water supply.
A drought stress episode was c~ ces~irully m~ in the low water supply plot for a period of 12 days at the mid to late vGg~LalivG growth stage.
The two populations were treated with a 2% Glllfc-~in~te~) solution and both populations seg.~ LLed 1:1 for Gl..r~ ~ reCi~t~n~e Within each plot, data were collected from both Gl~ si-la1e(~) resistant and se.~iLi~ plants. On eight 10 sGyaLdLe dates, during the midday sarnpling period, me&~ul..--ents of leaf t~ peLdlu e, and associated environmental data, were collected. The eight dates ranged from the early stages of the ~Lvua;LI stress to seven days after ..,~ .;..g.
Water relations data were collected on ten s~ualG dates during both predawn and midday sampling periods. During several stressed and l~aleled sampling 15 dates, leaf samples were collected for m~nnit-)l analysis.
HPLC ~let~rtnin~tion~ of leaf tissue ...~ l from samples collected 6 days after the drought irnposition are shown in Table 11. In both populations, G~lGs:jion of m~nnitol co-se~-e~ alGd with ~ ssion of Glufosin~t~ resi~t~n~e Glufosinate~) resistant plants from the cell line HCO5II-55 G~L~ssed leaf tissue20 mannitol levels which were approxim~t~ly 6-8 times higher than plants from the cell line S80HO-52. In previous greenhouse ~ ; with R1 plants, Glnfosin~te~ resistant plants which were exposed to drought stress conditions G~ ,s~ed higher levels of leaf tissue l..~ .;lf-l than plants grown under well watered conditions. In this t..~- ..llent the drought stress episode had little effect 25 on ...~ l levels. In general, the levels of rn~nnitol t;~ s:ied among Glufosinate~ le:,is~-L plants in this ~ nt were less than 20% of the levels observed among the sarne cell lines grown in the greenhouse. This difference CA 02243269 l998-07-l6 W O 97/26365 PCTrUS97/00978 may be the result of the c~ sed ~shortened) growth period associated with the el~ lle~ll employed in this t~JGlilllGl~L Expression of the mtlD gene in these ,.~ r..,..,t,.,~ was under the ll~u~s~ ional control of the Cauliflower Mosaic Virus 35S promoter.
S The levels of other plant carbohydrates in these lines was also ~let~rrninpr Glucose was the only carbohydrate to exhibit si~nific~nt(P<0.05) differences bdw~ll Glufo~ le~) sensitive and resistant plants, i.e., Glufosinate g) resistant plants contained higher levels of glucose relative to Glllfos;n~teq~ se.~ilive plants.
~ce~use glucose is known to have pleiotropic effects in plant cells, it is c~ klllplated that the levels of glucose may need to be moclto-r~ted in these plants.

Table 11. Midday leaf ~ content of 2 I~2 populations grown under watered and drought stressed co~liti(-n~ in Kihei, HI.
GLUFOSINATE~ GLUFOSINAT_~
TRF~TM~T RESISTA~T SF~SII~VE
(mg/g dry wt.) Eigh Expressing Popula~ion Stressed 5.71 0.0 Watered 5.06 0.0 Prob (<) ns ns Low l~xp~ Population Stressed 0.84 0.0 Watered 1.36 0.0 Prob (<) 0.01 ns Table 12 shows the water relations results for both populations grown under water stress conditions. The results l~,~l.,s~llt the average of six midday periods collected prior to l~rd~ lg and indicated more favorable leaf turgor potential values among plants co,.,p,;~;"g the mtlD gene co,llp~ d to plants 5 which do not contain the gene. These differences were observed in botn the lowand high m~nnitol ex~..,,,~hlg populations. The improvements in turgor levels among Glufosinate~ resistant plants in both populations were the combined results of hll~)l'OV~;lllC.iL~ in total water potential (~w) and osmotic potential (~5)~
Since osmotic potential is infln~n~ed by bo~ cellular deliy~lld~ion and by the 10 active accurnulation of solutes, the less negative ~'w values in the Glufosinate resistant plants prevented the detection of si nifif~.~nt differences for ~5.
However, the combined changes among both Y'w and ~5 led to highly significant (P<Q.OI) h~plov~lllents in leaf turgor.

W O 97/26365 PCTrUS97/00978 Table 12. Midday water relations parameters for plants exhibiting resi.ctRn~e and sensitivity to Glufosinate(~ applications among low and high mRnnitQl t;~ esSillg R2 populations grown under water stress conditions in Kihei, HI. Results are ~e average of 6 dates.

., .
Low Expressillg High Expressing Population Population Glufosinate~ F~çcictRnce 10WRt~r RelRtio~c ~esis. ~ Resis. Sens (bars) Y~ -9.25 -9.73 -8.55 -9.87***
ns -13.03 -12.73 -12.99 -12.88 ns ns ~p 3.77 3.00** 4.44 3.01 ~**

** Prob. < 0.05 *** Prob. < 0.01 Table 13 shows the predawn water relations results for the plants grown under stress. Tn the high mRnnitol G~iC~ g population, plants which were resistant to Glufosinate~ applications G,~plt;ssed significantly (P< 0.01) more 30 favorable values for all three water relations parameters. DifrcL~ eGs among l~Si~ versus :~uscc~lible plants in the low G~ g population were not ci nificRnt W O 97/2636~ PCTrUS97/00978 .

Table 13. Predawn water relations parameters for plants exhibiting resi~t~nce and sensitivity to GlufosinateO applications among low and high ~ l expressing R2 populations grown under water stress conditions in Kihei, HI. Results are forone sample date.
s Low l~xpressing High Expressing Population Population Glufosinate~ ~eci~nre Water Rel~tion~ ~ç~ ~ Resis. ~n~

(bars) ~w -I .2 -1.2 -0.85 -I .05***

ns --10.69 -1 1.03 -10.57 -9.32**

ns Y'p 9.83 ~.49 9.72 8.27***

ns ** Prob. c o.o5 *** Prob. c 0.01 Table 14 shows the predawn water relations results for the same plot collected 24 hours after r~;w~L~ g. Total water potential di~ ces were elim;n~te~ by the .~;w~ g event, however significant di~ ces in osmotic 30 potential remain and, since turgor is calculated from total and osmotic water potenti~ the ~rCl~m~ tion of mannitol resulted in higher turgor values in the high ~ ssil~g population. Again, differences among plants in the low ssil~ population were not significant. The improvements in water rel~tion~

parameters associated with the ~l~,sellce of the mtlD gene in plants were smaller 35 in m~gni~ e than illl~lVVt;lll~,.lL~i observed in previous greenhouse studies and W O 97/26365 PCTrUS97/00978 may be the result of lower levels of leaf marmitol exl~r~ssion. Because water potential and higher turgor ~r~ e under water stress are correlated with a drought resistant phenotype (Morgan, Aust. J. ~ric. ~es., ~, 607 (1983)), changes in water relations ~oci~t~d with the presence of the mtlD gene in maize 5 can provide plants with an altered ability to utilize available water.

Table 14. Rewatered predawn water relations p~r~m~ttor~ for plants exhibiting resistance and sensitivity to Glufosinate g) applications arnong low and high mannitol ~lC~illg R2 populations previously grown under water stress I0 conditions in Kihei, HI. Results are for one sarnple date.

Low E~ , High E~cpressing Population pop~ fio~
Glufosinatet~ t~rlçe Water Rel~til-n~ Resis. Sens. Resis. ~n~
(bars) ~w -0.2 -0.2 -0.2 -0.2 ns ns Y'5 -10.77 -10.82 -10.61 -9.70**
ns 1~.57 ~0.62 10.41 9.50**
ns ** Prob. < 0.05 During~the coliection of midday water relations data, observations of drought-in~lced leaf rolling were recorded. In previous field e~ ents, more favorable water relations p~ramet~r~ among hybrids and inbreds grown under 35 drought stress conditions were correlated with decreases in leaf rolling (flatter W O 97/2636S PCTrUS97/00978 leaves). ~d~liti~n~lly, less leaf rolling arnong hybrids have been correlated with higher relative yield under stress. In this study, Glufos;n~t~ resistant plants in both the high and low ~ "~ilol ~rc;,~ g populations exhibited highly significant (P< 0.01) decreases in leaf rolling (Table 15.3.
s Table 15. Midday leaf rolling scores for plants exhibiting r~ t~nce and sensitivity to Glufosinate~) applications among low and high m~nnitQI e~ as~llg R2 populations grown under water stress conditions in Kihei, HI. Results are theaverage of 5 dates.

Low E~ ; High E:~. c-Population Populatior Cilufosinate~ ~ t~nre Resis. ~ . Resis.
3.8' 3.6*** 4.1 3.6***

~Score: I= Severely rolled leaves to 5 = Flat leaves *** Prob. < 0.01 More favorable water relations l~ l.,.",- t~ i have also been correlated with 25 higher rates of leaf ~ dLion and, as a result, cooler leaf t~lly~dlulc:s. In one study, leaf ~ dlllre data was collected on eight dates. Analysis of the results indicated si nific~ntly (P< 0.01) cooler leaf tel~ s among plants which possess the mtlD gene colllp~u~,d to plants which did not (Figure 8). These temperature differences were observed in both the low and high ~ lll;lol 30 t;~.essi,lg populations.
The leaf chlorosis :,ylll~loms, which were Qc~ori~teri with high levels of m~nnitol e~;yics~7ion in greenhouse studies, were observed arnong both popnl~titm~ in this study. The most severe syrnptoms were found arnong W O 97/26365 PCTrUS97/00978 .

Glufosinate~ resistant plants in the high GAyressillg population. The degree of leaf chlorosis was more severe than the chlorosis observed in greenhouse grown plants and may have been exacerbated by the high light hlLGllsilies which occur in the field environrnent employed in those studies.
Expression of leaf tissue .~ .. ;lnl among both cell lines, co-segregated with GA~L~s~ion of Glufosinate~ reCi~tRn~e and was lower than that observed in previous greenhouse c~lr~ entC with R1 plants. GlufocinRte~\ resistant plants from the high l-ldl~liLol GAylGsSillg cell line (HCOSII-55) exhibited more favora~le turgor potential levels during midday and yl~ dawll water stress 10 conditions, and during yl~ddw~ vdtc~d con-lition~ The low ..ln~.n;(ol CAylc~ g cell line (S80HO-52) exhibited more favorable turgor potential levels during midday stress conditions. Glufosinate~) lG~i~L~ll plants from both populations exhibited less leaf rolling and ,l~n.ll~ el1 cooler leaf tclllp~dL~uGs.
The occurrence of mRnnitol-in~ eed leaf chlorosis was more GxlGllsivG than in 15 previous greenhouse c,~l,clilnents, and is ~u~e;led to be light hll~.~iLy dependant. Thus, several improvements in whole plant L~v-lgllL tolerance traits were observed in plants co-se~GgdLil1g for Clllr~i..n~ reeict~noe and the mtlD
gene. The improvements in water relations par~m~t~rc, leaf rolling, and canopy L~llly~ldLulG (L~ i-dLion) are all illlyu~L~ulL factors in d.OU~I1L stress recict~n-e F-~ple X~
~osure of M~ Plants F~l~t..';.~ M~nnitol to Salt Stress R3 ~,~n~dLion seeds of the high ll.n.~ l GX~ g line (HCOSII-5503) were ge....i.~led in paper towels (12 seeds per towel) moistened with water 25 CO.~In;~ g 1% Glufosin~t~ and I.2 rnl/L of DOM.AIN (fimg;ci~le). Seeds were allowed to gennin~te at 25~ C for S days. The rG~..Ik...l ~ iving see~llingc were ~r~ d to a hy~pollics system for further evaluation. ~ nlively, seeds can be gerrn;nz~.tçd without Glufosinate~) and the segregating population ~ minP~I
The hydroponic system consist of tanks which individually hold approximately 4 liters of solution. The individual g~rrnin~terl plants were placed S in sponge like m~qt~:ri~i with slits cut to accept the plants and were inserted into holes in the lid of the tank. The planting density was twenty seelliing~ per tank.
The hydlopollic solution was described by Clark (J. Pl~nt Nutrition, 5, 1039 1982)). The solution was aerated for the duration of the ev~ ti/~n For the purposes of a see~iing assay generally 1/4 or 1/2 ~ lh Clarks solution is used.
l O The plants were grown for 6 days, which corresponds to the 2-3 leaf stageof growth in 1/2 strength Clark's solution in the absence of added NaCI. At th,spoint, the hydroponic solution was changed (1/2 strength Clark's) and salt (NaCI) added to the solution. Plants were assayed for resistance to 0, 50, 100, 150, 200 and 250 mM NaCI in 1/2 ~ n~ l Clarlc's solution.
At coî~ of NaCI less than 150 rnM, no di~ ces in a~e~dnce of plant growth (no wilting) were observed after a 24 hour exposure to salt. At 200 mM NaCl, and more particularly at 250 mM NaCl, wilting was observed in the control plants, i.e. same genotype that was used for l~al~ro~ lion and not in the ~..llcd m~nnitol-c~ ;..;..g plants. Upon harvest, which is 7 days after 20 the start of the salt stress, cl~,t~ ;on of osmotic potentials demonstrates that a favorable shift in osmotic potential is associated with the presence of m~nnitol, rçsl-ltin~ in the ...;.;"~ .re of turgor. Salt-stressed mannitol-e~-Gwillg transformants have ~ignifi~ntly more dry matter than controls.

W O 97/26365 PCTrUS97/00978 F,Y~-nrle XI~
osure of M~i7~ Pl~nts ~xpressi~ Mannitol to a ~e of Fnvironmental Stresses.
S~lt or osmotic stress. Transgenic seeds co..~ g the mtlD gene are S germin~tecl in the presence of various salt or osmotically active solutions toclPt~rmine whether Lldl,sgenic seeds demonstrate increased tolerance or resict~nre to salt stress. Alternatively, see~lings can be grown in hydroponic systems and challenged with salt or agents of differing osmotic potentials at different, or all, developmental stages in order to assess the response of m~nnitnl G~rei,sing 10 plants to these stresses. Growth and physiological me~ulGl-lents are used to document the differences.
Cold. To demonstrate whether m~nnitol G~rG~sion can confer increased g~rmin~tion ability under cool conditions, Llalls~ellic seeds co..~;..;..g the mtlD
gene are germin~tPd under conditions similar to the standard cold germin~tion 15 test used in the corn industry. ~lt~rn~tively, transgenic seeds are planted under cool seed bed collrlitir,n~ made cool by artificial en~,i.. ,.. 1~ or naturally cool seed beds in the field. Additionally, plants G2~ S~ g .~.~n~ ol are challenged during the grain filling period for cool night time ~llll~e~dlUlcS in order to demonstrate less inhibition of leaf or canopy activity as a result of cold stress during this time of crop development. Young l.d-lsg~- ic seedlings are grown at low telll~e~d~ , such as about 15 ~C, during the light and dark period. The expression of ~ ol in these seeflling~ allows for increased growth and allows the see-lling~ to become photosynthetic under such conditions, as well as to survive and grow.
Frost/Fr~.o7~. Mannitol ~ es~ g plants are assayed for increased freezing tolerance at the see~lling stage as well as late season periods. These assays are done in artificial environments to ~im~ t~ frost or freeze events.

W O 97126365 PCTrUS97/00978 Alternatively, seeds are planted outside during times when the naturally OCCul~ g environrnent would impose tne stress.
Hi~h Heat. Mannitol G~ S~illg pla~ts are assayed in artificial cllvhol,.llents or in the field in order to demonstrate that the lla~lsgelle confers 5 r~ t~n~e or tolerance to heat.

~Y~I~pie X~T7 M~nnitol ~,~pressior Cau~es Yield }ncr~P
U~1P.r ~elatively Nor--Stress or More Typical Fnvironrnent~
Seeds of l~ln~llli~OI e~ ,S~ g corn plants are planted out in test lplots and their agronomic lJ~ rul ~ ce is colll~ed to standard corn plants using techniques familiar to those of skill in the art. Optionally included in this colllp~LIison are plants of similar genetic background without tne transgene. A
yield benefit is observed and plants exhibiting the increased yield are advanced15 for commerçi~li7sltiQIl Fu~ ,Llllore, transgenic plants with incleased levels of m~nn;tol are field tested for a~lollc,lllic ~,.Çul"~ e under con~ on~ including, but not limited to, limited and/or adequate water av~ hility. When colll~cd to subst~nti~ily isogenic nf~ g~,Llic plants, m~nnitol cUl~ g plants exhibit higher yield than 20 their nollL~IsgcL~ic cUu~-L~L~L j under non-optimal ~,~UWill~, corlriiticlns.
All publications and patents are illcol~uldLed by reference herein, as though individually incul~laLed by lef~,L~,nce. The invention is not limited to the exact details shown and described, for it should be ~ ..od that many 25 variations and modifications may be made while l~ within the spirit and scope of the invention defined by the claims.

CA 02243269 l998-07-l6 W O 97126365 PCTrUS97/00978 SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: DELALB Genetics Corp.

(ii) TITLB OF T~E INVENTION: TRANSGENIC MAIZE WITH INCREASED
MANNITOL CONTENT
(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ~n~R~-~S:
(A) ~nn~SEE: Schwegman, Lundberg, Woessner & Kluth, P. A.
(B) STREET: P. O. Box 2938 (C) CITY: Minneapolis (D) STATE: MN
(E) COUNTRY: USA
(F) ZIP: 55402 (V) CO~YU 1~K READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COM~UL~:~: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: EastSEQ Ver~ion 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Unknown (B) FIhING DATE: 17-JAN-1997 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/594,861 (B) FIhING DATE: 19-JAN-1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Vik~n;n~l, Ann S
(B) REGISTRATION NUMBER: 37,748 (C) REFERENCE/DOCKET NUMBER: 950.025WO1 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 612-359-3260 (B) TELEFAX: 612-359-3263 ~ (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs CA 02243269 l998-07-l6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

. .

Claims (58)

WHAT IS CLAIMED IS:

1. An expression cassette comprising a preselected first DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in a host cell, wherein the promoter is selected from the group consisting of the Glb promoter, the AdhI promoter, and the ActI promoter.

2. The expression cassette of claim 1 wherein the osmoprotectant is a sugar.

3. The expression cassette of claim 2 wherein the osmoprotectant is a sugar alcohol.

4. The expression cassette of claim 2 wherein the osmoprotectant is a sugar selected from the group consisting of fructose, erythritol, sorbitol, dulcitol, glucoglycerol, sucrose, stachyose, raffinose, ononitol, mannitol, inositol, methyl-inositol, galactol, hepitol, ribitol, xylitol, arabitol, trehalose, and pinitol.

5. The expression cassette of claim 1 wherein the osmoprotectant is selected from the group consisting of proline and glycine-betaine.

6. The expression cassette of claim 1 wherein the enzyme catalyzes the synthesis of a sugar.

7. The expression cassette of claim 6 wherein the enzyme catalyzes the synthesis of mannitol.

8. The expression cassette of claim 1 further comprising a second DNA
segment encoding an amino terminal chloroplast transit peptide which is operably linked to the preselected first DNA segment.

9. The expression cassette of claim 8 wherein the chloroplast transit peptide is a maize chloroplast transit peptide.

10. The expression cassette of claim 1 which further comprises an enhancer element.

11. The expression cassette of claim 10 wherein the enhancer element is subject to tissue-specific regulation.

12. The expression cassette of claim 1 which further comprises a selectable marker gene or a reporter gene.

13. An expression cassette comprising (a) a preselected first DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in a host cell; and (b) a second DNA segment that encodes an untranslated regulatory element, wherein the second DNA segment separates the preselected DNA segment from the promoter.

14. The expression cassette of claim 13 wherein the untranslated regulatory element is the AdhI intron 1.

15. The expression cassette of claim 13 wherein the promoter is turgor-inducible.

16. The expression cassette of claim 13 wherein the promoter is abscisic acid inducible.

17. The expression cassette of claim 13 wherein the promoter is developmentally regulated.

18. The expression cassette of claim 13 wherein the promoter is a constitutively expressed promoter.

19. The expression cassette of claim 13 wherein the promoter is subject to tissue-specific regulation.

20. The expression cassette of claim 13 wherein the promoter is water-stress inducible.

21. An expression cassette comprising (a) a preselected first DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in a host cell; and (b) a second DNA segment encoding a maize chloroplast transit peptide, wherein the second DNA segment is operably linked to the preselected first DNA
segment.

22. A method to increase water stress resistance or tolerance in monocot plant cells, comprising:

(a) introducing into cells of a monocot plant an expression cassette comprising a preselected first DNA segment enconding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in the monocot plant cells, to yield transformed monocot plant cells; and (b) expressing the enzyme encoded by the preselected first DNA
segment in the transformed monocot plant cells so as to render the transformed monocot plant cells substantially tolerant or resistant to a reduction in water availability that inhibits the growth of untransformed cells of the monocot plant.

23. The method according to claim 22 wherein the expression cassette is introduced into the plant cells by a method selected from the group consisting of electroporation, protoplast transformation, and microprojectile bombardment.

24. The method according to claim 22 wherein the cells of the monocot plant comprise cells of callus, immature embryos, gametic tissue, meristematic tissue or cultured cells in suspension.

25. The method according to claim 22 wherein the expression cassette further comprises a second DNA segment enconding an amino terminal chloroplast transit peptide which is operably linked to the preselected first DNA
segment.

26. The method according to claim 25 wherein the second DNA segment encodes a maize chloroplast transit peptide.

27. The method according to claim 25 wherein the enzyme is expressed in the cytosol of the cells of the transformed monocot plant.

28. The method according to claim 25 wherein the enzyme is expressed in the chloroplasts of the cells of the transformed monocot plant.

29. A transformed plant regenerated from the transformed plant cells obtained by the method of claim 25.

30. A transformed seed of the transformed plant of claim 29.

31. A method to increase water stress resistance or tolerance in a monocot plant, comprising:
(a) introducing into cells of a monocot plant an expression cassette comprising a preselected DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in the monocot plant cells, to yield transformed monocot plant cells; and (b) regenerating a differentiated fertile plant from said transformed cells, wherein the enzyme encoded by the preselected DNA
segment is expressed in the cells of the plant so as to render the transformed monocot plant substantially tolerant or resistant to a reduction in water availability that inhibits the growth of an untransformed monocot plant.

32. A transformed monocot plant, which plant is substantially tolerant or resistant to a reduction in water availability, the cells of which comprise a recombinant DNA segment comprising a preselected DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, wherein the preselected DNA segment is present in the cells of the plant and wherein the enzyme encoded by the preselected DNA segment is expressed in an amount effective to confer tolerance or resistance to the transformed plant to a reduction in water availability that inhibits the growth of the corresponding untransformed plant.

33. The transformed plant of claim 32 wherein the transformed plant has an improved osmotic potential when the total water potential of the transformed plant approaches zero relative to the osmotic potential of a corresponding untransformed plant.

34. A method for altering the sugar content in a monocot plant, comprising:
(a) introducing into cells of a monocot plant an expression cassette comprising a preselected DNA segment encoding an enzyme which catalyzes the synthesis of a sugar, operably linked to a promoter functional in the plant cells, to yield transformed plant cells, and (b) regenerating a differentiated fertile plant from said transformed plant cells, wherein the enzyme encoded by the preselected DNA segment is expressed in the cells of the differentiated plant in an amount effective to increase the sugar content in the cells of the differentiated plant relative to the sugar content in the cells of an untransformed plant.

35. The method according to claim 34 wherein the sugar is not detectable in the cells of the untransformed plant.

36. A transformed monocot plant having an altered sugar cellular content comprising a recombinant DNA segment comprising a preselected DNA
segment encoding an enzyme which catalyzes the synthesis of a sugar, wherein the enzyme encoded by the preselected DNA segment is expressed in an amount effective to alter the sugar content of the cells of said plant.

37. The transformed plant of claim 36 wherein the sugar content of the leaves.
seeds, or fruit of the cells of the transformed plant is greater than the sugar content of the leaves, seeds, or fruit of the cells of an untransformed plant.

38. A method for altering the mannitol content in a monocot plant, comprising:
(a) introducing into the cells of the monocot plant an expression cassette comprising a preselected DNA segment encoding an enzyme which catalyzes the synthesis of mannitol, operably linked to a promoter functional in the plant cell to yield transformed plant cells; and (b) regenerating a differentiated fertile plant from said transformed plant cells, wherein the enzyme encoded by the preselected DNA segment is expressed in the cells of the differentiated plant in an amount effective to increase the mannitol content in the cells of the differentiated plant relative to the mannitol content in the cells of an untransformed monocot plant.

39. The method according to claim 38 wherein the mannitol content of the transformed plant cells is greater than the mannitol content of the plant cells of step (a).

40. The method according to claim 38 wherein the mannitol content of the transformed plant cells during a reductin in water availability is at least about 1.1 to 50 times greater than the mannitol content in the transformed plant cells during water availability.

41. A transformed monocot plant having an altered mannitol cellular content comprising a recombinant DNA segment comprising a preselected DNA
segment encoding an enzyme which catalyzes the synthesis of mannitol, wherein the enzyme encoded by the preselected DNA segment is expressed so as to alter the mannitol content of the cells of said plant.

42. The transformed plant of claim 41 wherein the mannitol content of the seeds, leaves or fruit of the transformed plant is greater than the mannitol content of the seeds, leaves, or fruit of an untransformed plant.

43. A fertile transgenic Zea mays plant comprising a recombinant DNA
segment comprising a promoter operably linked to a first DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, wherein the level of enzyme expressed from the first DNA segment in the cells of the transgenic Zea mays plant is substantially increased above the level in the cells of a Zea mays plant which only differ from the cells of the transgenic Zea mays plant in which the recombinant DNA segment is absent, and wherein the recombinant DNA segment is transmitted through a complete normal sexual cycle of the transgenic plant to the next generation.

44. The fertile transgenic Zea mays plant of claim 43 wherein the recombinant DNA segment further comprises a second DNA segment encoding an amino terminal chloroplast transit peptide operably linked to the first DNA segment.

45. The fertile transgenic Zea mays plant of claim 43 wherein the osmoprotectant is a sugar.

46. A seed produced by the transgenic plant of claim 43.

47. A progeny transgenic Zea mays plant derived from the seed of claim 46.

48. A progeny transgenic Zea mays seed derived from the plant of claim 43.

49. A method to increase salt stress resistance or tolerance in a monocot plant, comprising:
(a) introducing into cells of a monocot plant an expression cassette comprising a preselected DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, operably linked to a promoter functional in the monocot plant cells, to yield transformed monocot plant cells; and (b) regenerating a differentiated fertile plant from said transformed cells, wherein the enzyme encoded by the preselected DNA
segment is expressed in the cells of the plants so as to render the transformed monocot plant substantially tolerant or resistant to an amount of salt that inhibits the growth of an untransformed monocot plant.

50. A transformed monocot plant, which plant is substantially salt tolerant or resistant, the cells of which comprise a recombinant DNA segment comprising a preselected DNA segment encoding an enzyme which catalyzes the synthesis of an osmoprotectant, wherein the preselected DNA segment is present in the cells of the plant and wherein the enzyme encoded by the preselected DNA segment is expressed in an amount effective to confer tolerance or resistance to the transformed plant to an amount of salt that inhibits the growth of the corresponding untransformed plant.

51. The method according to claim 31, 34, 38, or 49 further comprising (c) obtaining progeny from said fertile plant of step (b), which comprise said preselected DNA segment.

52. The method according to claim 51 wherein said progeny are obtained by crossing said fertile plant of step (b) with an inbred line.

53. The method according to claim 51 comprising obtaining seed from said progeny and obtaining further progeny plants comprising said preselected DNA segment from said seed.

54. The method according to claim 53 wherein seeds are obtained from said further progeny plants and plants comprising said preselected DNA
segment are recovered from said seed.

55. The method according to claim 52 comprising obtaining seed from said progeny and obtaining further progeny plants comprising said preselected DNA segment from said seed.

56. The method according to claim 55 wherein seeds are obtained from said further progeny plants and plants comprising said preselected DNA
segment are recovered from said seed.

57. The method according to claim 52 wherein the progeny obtained in step (c) are crossed back to the inbred line, to obtain further progeny which comprise said preselected DNA segment.

58. The method according to claim 57 wherein said further progeny are crossed back to the inbred line to obtain progeny which comprise said preselected DNA segment.