CA2210526A1 - Transgenic grasses - Google Patents

Abstract

The present invention relates generally to transgenic grass and to a method of producing same. More particularly, the present invention is directed to transgenic grass of the group Monocotyledoneae. The transgenic grass of the present invention exhibits the potential to express a range of beneficial traits such as reduced allergenicity, enhanced nutritional content and increased disease resistance.

Description

WO 96/2201~; PCT/AU96/00016 TRANSGENIC GRASSES

The present invention relates generally to l~ sgel~ic grass and to a me~od of producing same. More particularly, the present invention is directed to ~ .cgr~lic grass of the group Monocotyledoneae. The l~ sgenic grass of the present invention exhibits the potential to express a range of beneficial traits such as reduced allergenicity, enh~ncecl 10 nutritional content and i.lcreased dise~e re~ict~nre Bibliographic details of the publications mln~Pric~lly referred to in this speçific~tion are collected at the end of the descl;l)tion.

15 Throughout this specifil~tion, unless the context le.luil~s othe.~vise, the word "colnpri~e", or variations such as '~comrri~esll or "co...~ g", will be understood to imply the inclusion of a stated el~ment or integer or group of elemPnt~ or i--lege,~ but not the exclusion of any other element or integer or group of el~-tn~nt~ or illlege~.

20 The rapidly increasing sophi~tiration of recombinant DNA techniques is greatly f~r.ilit~ting the researcll and development of a range of biotechnology-related in~ TieS
The agricultural industry has been a particular bPnpfici~ry of recombinant technology especially in crop hllp~ovement and in floriculture.

25 Grasses are one of the most illl~oll~ll agricultural plants in the world.

Plants of the group Monocotyledoneae (plants of this group are hereinafter l~,f~ ,d to as a "Monocots") are particularly hll~ol~ll and include members of the family Poaceae (Cl,....il-eae) such ~ food crops (for example maize, wheat, rice) and forage grasses (for 30 ~x~mrle, Lolium, Festuca and Dactylis). They are, however, very difficult to manipulate in vi~ro and to genetically transform (Pollyku~, 1990, 1991). Direct DNA injection into young floral tillers and ~y~ s involving a llli~lul~ of pollen and exogenous DNA to obtain genetic Ll~r~,.l..alion (Ohta, 1986) have not proven generally applicable in monocots.

Transfer of foreign genes into plants using ~grobacterium tumefaciens is routine for 5 many dicotyledonous (dicot) plants. However, this procedure is not routinely applicable in monocots (De Cleene and DeLey, 1976). One reason for this may be the way monocot plants respond to wounding. In dicots, the cells ~ cPnt to wounding de-dirr~,c.l~iale and, after hlco.po.~Lulg the pl~qmi~l DNA, are inrl~ kle to form regenerable callus tissue. However, in monocots, wounding leads to the death of the 10 wound~ c~nt cells making infection very difficult (Pc llykus, 1991). There is a need, ~-..cir ,ic, to develop suitable protocols for l.,....~r,J,...i"~ cells of monocot plants with a c~aciLy to ~~;gen~dle into whole plants.

Direct gene transfer into protoplasts using ele.~ ,.alion or polyethylene glycol has 15 been succe~rul in reg~n~l~lion of cereals such as rice (Rhodes et al, 1988) and maize (Shim~moto et al, 1989). However, this m~tho~l has only been s~lcce~;~rul in a few cultivars of these crops and regeneration of cereal plants from protoplasts appears to be strongly genotype and culture depen~lPn~ In case of r.ve grass (Lolium multiforum), c~ ~.r....-ed stably l~ rollllcd callus clones from protoplasts have been obtained using 20 neol,lycil- phosph-)transferase (npt II) chimeric gene collsll u~ . However, plants could not be regenerated from these calli (Potrykus et al, 1985).

Regeneration of plants from single cells or protoplasts is eqq~nti~l in the genetic manipulation of plants using direct gene ~d..sr~. technology.
Regeneration of plants from non-l~al~ro~ned callus colonies derived from protoplasts have been reported for the monocot forage grasses, Lolium and Festuca arundinacea (Dalton, 1988a;b), however, such systems are generally not con~ cially applicable.
Genotypic variation (Dale, 1980; Lorz et al, 1985) is also a particular problem in 30 oull,reeding species such as C~ mmnnly oC~ seq bec~u~e each seed-derived embryo pos~e~es a unique genotype. It has been shown that standard culture con~litinnq to induce embryogenesis are not optimal for every genotype (Dale, 1980). In addition, protoplast and cell s~ ;on colonies of Lolium lines formed virtually only albinoshoots (Dalton, 1988a;b) hi~hlightin~ the difficulty of obtaining fimrtion~l re~nrr~ted plants.

S Thus, the gen~alion of plants from cells le~ nl~ a ~i~nifie~nt furt~er problem in the prodllrtion of genetically l.d~r~ ed plantswith desirable c~ L~ ;cs. In ~d~litiol~
sel~ction of llA~.~r~....A"~ is often difficult since non~ ro....~od cells are frequently to the selectio~ agents now commnnly employed.

10 Use of microprojectile bomba~ lll allows leco~ of ll~lsgel~ic plants without the col.e~ of protoplast culture and Agrobacterium host-specificity. Microprojectilebombardment employs high velocity met~ particles to deliver biologically active DNA
into plant cells. The conc~t was first described by Klein et al (1987) and has become a succe~rul DNA delivery method in a number of plants.
However, despite its a~a~ l sllrce~ee~ the technique h~ not until the advent of the present invention, been routinely s~lccç~rul in a broad range of monocot grasses.

As stated above, monocot grasses are h,lpoll~ll agriculturally bo~ at the co,ll.llercial 20 and ~llVh'U---~ levels. Grass pollen, on the other hand, is a ~eignifi~nt contributor of re~ildluly disorders such as hdyrt;~,~,r and other allergies with conconlil~ll major dowl~LI~idlll health costs including lost pro~l~lctic)n time.

There is a need, lll~lefolc, to develop genetic tran~rollllalion and plant leg~;ll..dlion 25 systems for mo~locots and in particular monocot grasses so that these plants can be generally n~ani~ulated to introduce beneficial traits or to reduce ul~wa~lled r.l~ tir.e In accordance with the present invention, an effective lln..ero~ nl;orl and legt;ll~dlion system has been developed for monocot grasses by the in~,~nlor ~. ".;1l;"~ the production of lldl~Sg~lliC forms of these plants.

Accordingly, one aspect of the present invention relates to a hdllsge",c nlol oco~ grass ~lrhihitin~ at least one altered cll~r3Gtrri~tic when coln~ d to a non-l~ g~..;cmonocot grass of the same species ,, More particularly, the present invention is dil-cted to a l,~s~ ic mnnnCQt grassexhibiting at least one altered ch~rl~ tic when co,l,~aled to a non-L, .~sge..ic monocot grass of the sarne species whc.e"~ said l~ sgr..ic monocot grass is l~ge~ d from a callus whelem cells of said callus are subjected to microparticle bombalLlle~l and/or 10 Agrobacterium-me~ trd llal~r~. of genetic m~tr.ri~ s~ul~ible for said altered cl~l'tP- ;~tic Even more particularly, the present invention provides a ~ ;gt~l~ir, monocot grass exhibiting at least one altered cl,~<.clt;- ;.~tic when con,~ed to a non-~ .;c monocot grass of the same species wh~.e;" said ~ sgel~ic monocQt grass is ~ led from a callus wherein cells of said callus are subjected to microparticle boml~ "e"l and/or Agrobacterium-me~ te~ ~r~ of genetic m~trri~ onsible for said altered charaçter1~tic and wl,~.ei" the callus is subjected to genetic ~ ;r -- ,,,AI;Oll and regeneration on a solid support.
In accorda.lce with these ~pects of the present invention and further aspects described and disclosed hel~eindrL~l, plC~ d altered ç~l~r~rt~ tics include but are not limited to altered biochemical or physiological pr~. .lies, altered nutritional pr~ .Lies of the grass or seeds thereof and altered allergenicity of the grass or its seeds or pollen. For P~mr~le, ch~ ~I~-;ctirs co"lt;,ll~lated herein include, but are not limited to, the following:

(a) resict~nce to plant pathogens;
(b) inl~ vcd nutritional value such as increased protein content or lower lipid content in grass seeds, (c) rnh~nred sulphur conlelll, (d) enh~nced C;~ ion of ~rolei"ase inhibitors, S
(e) cA~ ion of pi~m~nt genes, (f) ~;A~ ion of regulatory genes to control intligenous genes; and (g) re~ ction in levels of allergenic ploleills in, for ~Y~mrle, pollen.

S An "altered ch,uAçl~ ;~c" is readily ~ 1 by C~ PA~ g a l~ s~,~l~ic monocot grass with a non-L.a"sgel~ic grass of the same spe~;es. The C~ A- ;qon may be at the biochemicAl, physiological or visual level.

A monocot grass of the present invention in~ des any grass falling within the major 10 subf~miliçs, namely the Bambosoideae which incllldes bamboo, Arundinoideae which in-~ludes p~nl)as grass and reeds, Pooideae which inrln~lrs oats, barley, rye, wheat, corn and the forage grasses fescue (Festuca) and 1~ 1A~ (Lolium), Chloridoideae whichinrl~ q millet, t'ef, Mitchell grass, Rhodes grass, R~rmll-lA grass and Kallar grass and Panicoideae which inrlud-oq so,~hu"" maize, ~ A~ grass, Pangola grass, Buffel grass, 15 Bahia grass, sugar cane and Vetiver grass. Although the present invention is described and exemplified herein in relation to l~gl~, the techniques of tr~n~ro...~AI;on and regeneration are broadly applicable to any of the grasses as co.~le~..plAted above. In a p.eL~.led embodiment, the present invention does not extend to barley, wheat or corn.

20 In a particularly prere.led embodiment, the present invention colll~ )lates a llAI~.qg~ iC
plant having altered charactetiqtics by the introduction of non-in~ligent~us DNA. A "non-indigenous" DNA is one not n-rtnAlly resident in the plant before trAn~r~.. Al;Qn or is not notmAlly present in more than one copy. A further copy of an indigenous gene or genetic sequence may be introduced for the purposes of co-~u~l~,s~ion. A non-25 indigenous gene is also referred to as a foreign gene and inrludes genetic se.luellces Ç1A~ 'A1lY coll~,~onding to a "gene" or a part thereof or may be a nucleic acid molecule which in some way effects a change in the Llal~sg~;lllc plant. Examples of foreign genes ~ inrlu(le:

30 (a) a rçqiqtAnre gene against plant viruses, b~ctçriA fungi, n~tnAto~Ps and other pathogens;

(b) a gene to illl~)l'OV~ rlutritional value of plants such as sunflower high sulphur gene SF8;
(c) a bloat rç~i~t~nce gene;
S (d) an antibody gene (also ~eLl-~d to as a "plantabody");
(e) a cereal thionin and ribosome hla~;liv~Lillg protein gene;
(f) an insect re~ nce gene inchl-ling BT toxin gene and plol~illase inhibitor gene from Nicotiana alata;
(g) a select~ble marker gene such as those colIfcllillg ~ ce to k~al.ly~ih"
pho~hil~cLlL;cin, spectinomycin and hy .~ yc~
(h) a re~olt l gene such as GUS, CAT and pigm~nt genes;
(i) a gene encoding a regulatory protein which mo~ tes ~ A~les~ion of a gene in plant cells;
(j) genetic sequences for co-~u~le~ion or having ~nti~n~e ~lopc;llies; and 15 (k) genetic sequences for re~ cin~ ~A~ression or tr~n~l~tion of allergenic ~.oteills such as Lol l,loteills including but not limited to LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX or LolpXI.
.
The genetic techniques of the present invention also co..lr~ le mo-lified plants having 20 indigenous genetic sequences deleted or otherwise mllt~ted For cc,--vellience, such a plant is ~llco...~ e~l hereby by the term "ll~lsgellic" in relation to monocot grasses.
Such a l.~sgel ic monocot grass includes grasses having 1~ duced or modified allergenic proteins such as LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX or LolpXJ..

25 Another aspect of the present invention contemplates a method for producing atransgenic monocot grass, said mlo.thod compri~ing deriving callus from a monocot grass and subjecting same to micl~Licle bombardment and/or Agrobacterium-m~ ted genetic L a.~r~l and then placing said callus under con~lition~ sllfflci~nt to permit l~gen~laLion of said callus into plantlets.
More particularly, the present invention is directed to a metho~ for producing asgel~ic monocot grass having at least one altered chalncl~ tic colll~Led to a non-L~ sgcllic monocot grass of the same speciec, said methocl co.~ i.-g obtaining callus from a m- n~ cot grass on a solid support, introducing into cells of said callus on said ~, solid support genetic m~tçri~l effecting said altered cl~c~ ;c via microparticle bombardment and/or Agrobacterium m~di~tçd hd~ srtl, subjecting said callus to selection S con-litione and then subjecting callus to ,egell~la~ion conflitione to forrn pl~ntlçtc In a particularly ~l~rclred embodiment, the s~lloction con-liti~nc c~ .. ;ees antibiotic or ~h~n ir~l re~iet~n~e where a gene encoding ~ e is on the introduced genetic sequence or vector carrying same. Most preferably, the s~l~oct;on con~itiQnc co...p.;ce 10 resi~t~nr~e to at least two antibiotics or at least one antibiotic and at least one rh~mic~l agent.

Preferably the callus is ~l~ed from mature and/or developing embryos obk.ille1 from seeds. More preferably the callus is grown on a callus intlllction mP~ m which is based 15 on that of Mllr~hige and Skoog (1992) [MS me~ m] but colll~ s 2,4-dichlol~hcl~y acetic acid (2,4D) in a range from about 1 to about 10 mg/L, preferably about 5mg/L, L-asparagine (at about l50mg/L) or equivalent amino acid or chPmic~l entity and preferably thi~mine HCl (at about 0.5 mg/L) or equivalent. The m.orlillm may also contain MS medium minerals and/or, in a particularly ~l~r~ d embo~lim~nt po!~
20 iodide or equivalent or functional analogue at 0.83 mg/L.

Preferably, the cells selected for ~ rol~ ;on are the healthy cells on the callus and this is f~ilit~ted by excision of the embryo and ~dj~cçnt cells. More preferably, the healthy cells are selecte(l for fr~n~form~tion at an age of belwccn 1 to 10 weeks, more 25 preferably 3 to 6 weeks and most preferably at about 4 weeks old. A particular advantage of the present invention is that the genetic ll~..srcl and l~r-.--,-l;cn is con(lllctç~l with the callus on solid medium such as agar and/or filter paper. An culture is not first plep~ed as commonly occurs in the prior art.

The callus is generally from cells which have been either dile~ lly or illdile~ lly obtained from the monncot grass tissue. This may be from embryo tissue, root, shoot inl~hltling leaf tissue and inflol~,sce ~ce tissues. Preferably the callus is derived from mature and/or 5 developing embryos or seeds.

Pl~relled monocot grasses include forage grass such as but not limited to lyc~s Lolium (l,~Cgld:iS), Agaropyron, Phalaris, Poa, Festuca, Dactylis, Aleopecuris and Phleum.
Genetic material co~ ted herein include vectors suitable for intro~lction into cells by microparticle bombardment and/orAgrobacterium medi~te~ a~ro~ iOn Although Agrobacterium meAi~ted l,~iroll.lalion on its own has yet to be widely used in monocots, in accor~ce with the present invention, the combination of microparticle 15 bomba~dmcllt and Agrobacterium meAi~ted tr~n~ro..~ ;on is col.lr~ tçd to be particularly useful as well as microparticle boml~al~llclll on its own. Suitable vectors will be well known by those skilled in the art and are described, for ~A~ll~lc in Croy R.R.D. (Ed) Plant Molecular Biology, Bios Scientific PUblic~ti~ ne, Blackwell SçiPntific Publication (1993).
A particularly useful vector for microparticle bombardment is pBINl9 or related vector or a pUC-baeed vector such as pUC18 or pUCl9 or equivalent or functional analogue.

Preferably, the genetic m~teri~l effects a red~lcti~ n in levels of l~egld~s pollen allergens 25 such as LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX or LolpXI (see, for ~Y~mple, Tnt~n~tional Patent App~ ti()n No. PCT/AU89/00123).

Where the selectable marker encoded by the construct confers resiet~nce to an antibiotic it preferably encodes rçeiet~nre to a class of antibiotics. This feature allows selection 30 with two or more antibiotics which is a particularly pief~.~ed feature of the present invention. This provides a way of o~.col~ g the problem of untransformed plant cells being able to grow on the selected medium. For eY~mple the construct may encode the _ 9 _ gene for neolllycin rhnsFhn~ r~ ~ -e~, npt II,or other antibiotic reei~nre genes such as lly~ y~ rÇ~ict~nre aph 3'II or aph IV ~monp;~t others. P~ Ç~.ably, the gene is coupled to an a~lo~liate promoter such as pEmu, Ubi I, Act I or the CaMV 35S
promoter.
Where neolllyei~ phosl)h~.L~ r. . .ce is Pn~ o~ by the c(s~L.~ then grn~r~lly l~ullycill and gçnrtirin are used in the selection m~ lm Preferably, t_ese antibiotics are present in the amount 15 to 50 mg/L l~lallly~il- and 15 to 50 mglL gf-n~ticin 10 A further aspect ofthe present invention co..l~ tçs a m~t~lncl of legen~lillg monocot grass plants from 1.~ rO..,~rd plant cells, said m-othnd co...~ c~ cl~..g cells on a solid SU~ulL upon which said cells are subjected to l-i--..~rol...~tion means on a me~lillm and under ap~ opl;ate conditions and time ~Lel~ m said me~ m co.~ shooting and rooting hormc)nes and then cnltllrin~ the reslllt~nt shoots on a ...e~ ..ll in ~e ~bs~nr-e of 15 biologically active horm~n~s.

The mto~lillm which has an absence of hormonrs may be any suitable m~ m and inrllldes soil or potting mix.

20 A rooting hormone is a class of colll~oullds known as auxins which include indoleacetic acid (IAA), n~rhth~lçnr~cetir, acid (NAA) and indolebutyric acid (IBA).

A shooting hormone is a class of compounds known as cytc-kinin~ which inr.hl-lçssynthetic shooting hormones such as 6-benzylall~illu~ ~ (BAP), kinPtin, zeatin, 2iP
25 (N~ (~2-iso~ llyl)-~ oninp trans-6-(4-hy~iLo~y-3-methylbut-2-enyl) amino purine.

The ill~t;llkJl~ have ~lel~ pd that callus cultures are particularly suitable for leE ~ .dlion bec~ e green pl~ntlet~ or plants can be regenc.dled efficiPntly from callus.
This is in cc/llllasl to the albino or weak, non-viable plants which have been reported in 30 the prior art. This aspect of the present invention avoids the use of protoplasts or cell ,e~.~ions, and hence, gP.nPtir~lly ....ir~.... cells can be used as a st~rting m~tPri~l In addition, this avoids the problem of using physiologically fragile and nutritionally fastidious protoplasts or cell su~pellsions of the prior art.

Preferably the cells are from forage grasses, more preferably from the genus Lolium, most preferably ryegrass.
The plant cells may be llal~rolllled by any a~lo~llate method. Most ~,er~,lably the cells are L allsrc,lllled by the microprojectile bombardment technique or a comhin~tion of this technique with Agrobacterium-merli~tç~l ~d,lsr~ ic)n 10 Preferably, the transformed cells are callus cells produced from mature and/or developing embryos. Preferably the callus is l,r~al~,d from mature and/or developing embryos obtained from seeds. More preferably the callus is grown on a callus in~ ction m~tlinm which is based on that of Murashige and Skoog (1962) but collLaills 2,4-D in a range from about 1 to about 10 mg/L and more preferably about Smg/L, L-asparagine 15 at a~fox;..-~tely 150 mg/L and thi~min~ HCl at a~lo-x;.~.~tely 0.5 mg/L.

Preferably, the cells chosen for transformation are the healthy cells on the callus and this is .f~r.ilit~t~?tl by eYci.cil n of the embryo and ~ c~nt cells. Generally, the healthy cells are selected for transformation at an age of bc;lw~ell about 1 to about 10 weeks, more 20 preferably about 3 to about 6 weeks such as at about 4 weeks old from cultures grown in the ~bs~n~e of light. However, the present invention extends to cultures earlier than 3 weeks (e.g. 1 week) or ollder than 6 weeks (e.g. 10 weeks).

Any suitable system may be used to identify the transfo....~ ;. This may be done25 through a selection process by, for çY~mpl~, antibiotic or herbicide reci~nr-e selection or, ~ v~ly~ direct analysis of the DNA co~ od in IJuL~Live l~.cro. .~ may becarried out. The methods used for direct DNA analysis will be known by those skilled in the art. Herbicide resict~n~e selection in~hl-les but is not limited to the Bar gene and the Basta selectioIl system (Hoechst), the aroA gene or EPSP gene (through 30 overprodl~ction of the EPSP gene) and glyphosate or the ALS gene and ~hl~r.ca~lfuron.

WO ~6122015 PCT/AU96100016 In one particular embotlimPllt, the reg~ on m~ m co,lkLills a shooting:rooting hnrmnn~ ratio of from about 1:0 to about 10:1 and is preferably about 2:1. For eY~mrle the m~ lm may contain from about 0.05 to 2.0 mg/L shooting hl)nnonr and 5 from about zero to about 0.075 mg/L rooting hnrmon~

In another ç~mrle ~e ...e~ .. clJ"l~ s about 0.5 mg/L shooting hnnnnne and 0.025mg/L rooting hormone. An rY~mrle of sllit~hle shooting hnrmone is BAP and of a rooting hormone is IAA. If only a shooting hnrmon~ is used, then BAP at 10 a~>~ro,~ ely 0.2 mg/L to d~ o~ ely 4.0 mg/L and more preferably at about 2 mg/L is a~opliale.

In addition to the above, the regeneration mr-lillm prere.ably also ço~ C L-acp~r~ginr at about 50 to about 750 mg/L and thi~min~ HCl at about 0.05 to about 5 mg/L.
The shoots may be L~ r~ d directly to soil or potting mix and grown under d~ lialt; con~liti~nC. Pl~;r lably, the pl~ntl-otc are grown at a t.elll~,ld~ of about 15-30~C although 25~C is pl~L..ed and the cells are allowed to regr-~ Ir in light.

20 A particularly prere.l~d aspect of the present invention c~ tes a method of producing a lldllsroll.lcd monocot grass comrricing bombarding callus cells derived from a mature embryo of a monocot grass with a microprojectile Co,,~ a nucleic acid consl~u~;l encoding a desired trait under the control of an a~ropliate promoter and carrying a select~kle marker, under d~ro~;dle conditions to allow lld~ro,...n~;on, 25 optionally further subjecting cells to Agrobacterium-m~ t~l transfer of a similar nucleic acid construct, selecting transformed cells on a selection mP~ lm under conditions and for a time sufficient to permit re~ t;ll. .alion of plants from the lla.~roll.,ed cells wh~ said regeneration conditions comprice shooting hormone and rooting h~rmf~ne or shooting hormone alone and then c~llt~ring the res~h~nt shoots on a mf?~ lm 30 in the ~bsenre of biologically active hormonrs The above con-lition.~ lead to rege~ ;on of green plantlets and plants with photosynthetic ability.

S The invention also eYt~n-lc to 1, ~ g~--.;c plants made by the m~thofl~ described above.

The mPthorl~ of the present invention permit for the first time, the pro~lction of monocot grasses eY~ibiting altered ch~n~ . ;ctics such that the modified plants have desired pl'~c~lies. For ~Y~mple~ ulsg~,lic ~ye~,l~s can now be co~LIu;led having10 pollen with reduced allergenic plopc.lies. In this regard, ~nti~n~ cosu~p,e~ion, ribozymes, regulatory genes or other suitable means may be employed to reduce cA~.e~ion of allergenic proteins such as LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX
or LolpXI or i~of~.. ~ thereof. The present invention eYt~n~l~, therefore, in a particularly ~lcr~l~d embo~im~nt, to pollen from l~ s~elfic monocot gr~es, such as ly~,css, 15 eYhibiting useful p.~ lies, such as exhibiting reduced allergenicity.

Accordingly, another aspect of the present invention provides a seed or seeds or pollen from a lldllsgt;mc monocot grass wherein said lLdllsgtil~iC monocot grass exhibits at least one altered ch~<~ tic when co~ a~ed to a non-l,~lsg~llic monocot grass of the same 20 species.

Preferably, the LLa.lsgenic monocot grass exhibits altered biorh~mic~l or physiological pro~c.lies, altered nutritional properties of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.

Preferably, the lld-lsgenic monocot grass exhibits at least one of the followingcha,~c~ tiCs:

(a) resi~t~n~e to plant pathogens;
30 (b) improved nutritional value such as increased protein content or lower lipid content in grass seeds;
(c) ~nh~n-~ed sulphur colll~lll, CA 02210~26 1997-07-1~

(d) ~nh~nre~ ,res~ion of proteillase inhibitors, (e) t;A~le3~ion of pigment genesj (f) cA~ession of regulatory genes to control in~lig~n-us genes; and (g) reduction in levels of allergenic ~lUltillS.
S
Preferably, the seed or seeds or pollen which exhibits reduced allergenic ~)lup~Les.

Preferably, the seed or seeds or pollen which PYhi~it~ reduced levels of one or more of LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX or LolpXI.
Preferably, the seed or seeds or pollen is from forage grass such as lye~51oSS.

The invention will be filrther described with ~r~lce to the following non-limitin~
Figures and FY~mples.
In the figures:

Figure 1 is a photographic lepl~se~ ion showing cell cultures and pl~ntlet~ Panel A
shows embryos on callus induction me~ lm Panel B shows cultured callus from 20 embryos and shoot initi~tion on callus. Panel C shows pl~ntlet~ forming shoots; these plantlets are derived from transformed callus cells. Panel D shows a regen~Led plant derived from Ll~ulsroll~led callus.

Figure 2 is a photographic l~les~ m of DNA gel blot analysis of ryegrass 25 tran~r~ t~. Each lane colllilills 2011g of total leaf DNA which was ~ stecl with EcoR~ and BamHI and hyhri~li7~cl to a 32p labelled NptII gene probe. Lanes 1 and 2 contain digested DNA from non-lld,lsrc,lllled control plants. Lanes 3 to 9 contain DNA
from lld.~r,lllled plants derived from single callus.

CA 022l0~26 lgg7-o7-l~
wo 96/22015 PCT/AUg6/00016 Embryo production 5 Embryos from Lolium rigi~um and or Lolium ~ c seeds were used to induce callus.
Mature seed is surface stPrili7~1 in 5% v/v hypochlorite and soaked from 8 hours to 48 hours in sterile water. Mature embryos are ~ sectecl and placed on MS me~ m (Mur~hige and Skoog basal salt and minerals supplemPnt with L-~ ;..e 150 mg/L, thi~minP HCl 0.5 mg/L, 5mg/L 2,4D,2% w/v sucrose and 0.8% w/v agar at pH5.8) 10 [Murashige and Skoog, 1962]. po~ .. iodide may also be used at a~ru~ ely 0.83 mglL. Callus was initi~te(l during a culture period of four weeks in dark at 25~C.
Embryonic callus cultures were found to be ideal targets from microprojectile-mP~ t~P~
lldllsrolllli1~;on because l~gel~ldble cells are not ~ce~ively ~hiel~le~l and can be arranged to occupy most of the target area.

Regeneration of plants from competent cells after bombardment .
t~omr~ct embryonic callus is transferred to MS mP~ m CO,.~ 2% w/v sucrose, 0.5 20 mg/l BAP, 0.025 mg/l IAA and 0.8% w/v agar at 25~C in light. Roots were regene.dled from shoots on MS m~ m with no h~lrmonPs and c~ g 0.6% w/v agar. Figure 1 shows shoots regenerated from callus of l~eglass. These shoots are green.

An Effective microprojectile delivery system Microprojectile bombardment conditions were optimised on the basis of frequency of transient ~ ression in callus tissues. Parameters like microprojectile velocity, mllltiple 30 bombardments and age and si~ of embryogenic callus were evaluated in order toachieve high efficien-~.y of transient t;~re~lng cells. P~ used was 24 to 29 inches Hg, ~ t~nce from stopping screen was 6-13 cm and rupture disk ~ glh was 1100 psi.

The strong moncot promoter pEmu, coupled to the GUS ~ e~ gene (Last et al, 1991)was used to optimise bombardment con~ ion~ in l~ ;e-ll assays. The GUS
histoAh~mie l assay for callus tissue is ~ c ccu.di..g to Klein et al (1988).

For stable kàl~rol,llalion ~ nt~ two col~ku~L~ were used: the pEmu promoter couple to the npt-II (neoll,ychl phosphollallsr~dse) gene in order to allow selection on geneticin, ka~ ycill and neomy~in and anther specific promoter Bgp 1 coupled to GUS
in addition to the 35S promoter coupled to npt II. This pl emid also has the CaMv 35S
promoter coupled to the npt-II (neol,lyci.l phnsp~ r~ e) gene in order to allow selection on an antibiotic.

Se!~_t'A of stably transformed tissue and regenerated plants The concellkdlion of the antibiotics, g~n~ticin, and k;~la~lly-;ill le.lui~ed to inhibit the growth and to kill control u~lk~rolmed callus was ~ ecl There were found to be in the range of 15 to 50 mg/L geneticin and 15 mg/L to 50 mg/L kall~ullycill. Plants were regenerated from the callus lines which grown in the plei,~llce of a sel~cti~ n m~ lm c~ g gen~oticin C-)n~A~ntrA~tion of g~n~ti~Ain was the amount required to kill control (untransformed) tissue-cultured. The l~ t;(1 shoots were again exposed to m~ lm co"l;l;"i"g gen~tiCin and k~l~ly~;in. Sul~/ivillg shoots were considered as the ~uLdliv~ kdl~ro,ll,ant shoots.

EXAMPLE S
Confirmation of stable transformation NPT-II ELISA assay:
The ~lualllily of NPT-II in tissue of ~ r~""il~lki was be measured using an NPT-II
ELISA assay kit (5 prime-3 prime Inc.) Leaf tissue of lldllsr~Jlllled tissue-culture plants 30 was used. Leaftissue from ll,..l~l~l ".~(1 tissue-culture plants showed ~ignific~nt illclease of NPT-II levels over ullll~.xr~ll..e~l control plants.

-CA 02210~26 1997-07-1~

PCR analysis:
The gemonic DNA from leaf tissues of transformed plants were used from RAPD PCR
analysis. Npt-II gene coding region se~,.n~ was chosen from PCR ~mrlific~tion Tran~ro,llled plants showed t,he ~re3ence of ~mplified band of DNA which 5 untransformed plants fail to show col..,*,onding DNA fr~gment So..lLe~ analysis:
Integration of the introduced construct was also colll;....~cl using southern analysis.
Genomic DNA from leaf tissue was extract using a method by Dellaporta et al (1983).
Genomic DNA was digested with EcoRI and/or BamE:~. The tlige~sted DNA resolved and was t;Y1...;..erl for completenee~ of digestion by electrophoresis on a 0.7% w/v agarose gel. After transferring to a nylon m~,mhr~ne it was be probed with npt-II coding se~luence labelled with 32p. The membrane was then analysed using ~lltor~lio~,ld~hy.
15 DNA from transformed plants displayed hybridisation with a single a,u~ ox;n-~lely 3.1-3.5 kb band while no signal was obtained from the DNA of u~ dl~r --lled plants (Figure 2).
.

Basta resistance s e~ r~i~n The construct pAHC25 which çn~ode,s the bar gene cl-nf~ E phosrhinn~ricin re~ nce and GUS is used to bombard 4 week old callus cells as described above. The callus cells are then selected on a suitable ",~ .." c~-nl~in;..g Basta (glufosinate 25 ~mmor~i~) belw~ll 0.1 to 5.0 mg/L, preferably 0.5 to 5.0 mg/L and most preferably 1.0 to 3.0 mg/L. The callus cells are tld~r~ d to fresh Basta col-l~;--;--~, rne~ lm at two week intervals over a period of six weeks. Shoots are lcgen~dled on s~ hle me~ lm wi~luul herbicide or suitable medium co~ ;n;n~ 1 mg/L Basta. Regen~,.dled shoots are then exposed to Basta to conrll.ll tolerance. Roots are then l~g~ dl~d from the shoots.
30 ~,st;llce of the introduced DNA is then conl~ by Southern analysis.

CA 02210S26 1997-07-lS

WO 96t2201S PCT/AU96100016 Those skilled in the art will appreciate that the invention described herein is susceptible to v~ri~tion~ and mo~lific~tion~ other than those specific~lly described. It is to be lm~ rstc-od ~at the invention inchldPs all such v~ri~ti~n~ and mo(lifir,~tion~ The S invention also includes all of the steps, r~ es, compositinn~ and colll~)uu~ s referred to or in-lir~ted in this specific~tic-n, individually or collectively, and any and all combin~tion~ of any two or more of said steps or fe~ es.

CA 02210~26 1997-07-1~

REFERENCES:

Dale, P.J. (1980) Z Pfanzenphysiol. 100: 73-77.
Dalton, S.J. (1988a) Plant Cell Tissue Organ Culture 12: 137-140.
D~ton, S.J. (1988b) ~ Plant Physiol. 132: 170-175.
De Cleene M and De Ley J. (1976) Bot. Rev. 42: 389-466.
Dellaporta et al (1983) Plant Miol Biol Rep 4: 19.
Jones, M.G.K. and Dale P.J. (1982) Z. Pflur~erl~h~siol. 105: 267-274.
Klein T.M., Fromm M.E. and Sanford J.C. (1988) BioTechnology 6: 559-563.
Klein T.M., Wolf E.D., Wu R. and Sanford J.C. (1987) Nature 327: 70-73.
Last D.I., Breffel R.I.S., Chamberlain D.A., C'h~ h--ry, A.M. Larkin, P.J, Marsh E.L., Peacock W.J. and Dennis E.S. (1990) Theor. Appl. Genet. 81: 581-588.
Lorz H., Baker B., and Schell J. (1985) Molec. Gen. Genet. 199: 178-182.
Murashige and Skoog (1962) Physiol. Plant 15: 473.
Ohta, Y. (1986) Proc. Natl. Acad. Sci. USA 83: 715-719.
Potrykus 1., Saul M.W., r~u~ J., Paszkowski J. and Shillito R. (1985) Mol. Gen.
Genet. 199: 183-188.
Pollykus 1. (1990.) BioTechnology 8: 535-542.
Pollykus 1. (1991) Ann. ~ev. Plant Physiol. Plant Mol. Biol. 42: 205-225.
Rhodes, C.A., Pierce, D.A., Mettler, I.J., Mascarenhas, D and Detmer, J.J. (1988) Science 240: 204-206.
~him~moto, K., Terada, R., Izawa, T. and Fujimoto, H. (1989) Nature 338: 274-276.

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Claims (40)

CLAIMS:

1. A transgenic monocot grass exhibiting at least one altered characteristic when compared to a non-transgenic monocot grass of the same species.

2. A transgenic monocot grass exhibiting at least one altered characteristic when compared to a non-transgenic monocot grass of the same species wherein said transgenic monocot grass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium-mediated transfer of genetic material responsible for said altered characteristic.

3. A transgenic monocot grass exhibiting at least one altered characteristic when compared to a non-transgenic monocot grass of the same species wherein said transgenic monocot grass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium-mediated transfer of genetic material responsible for said altered characteristic and wherein the callus is subjected to genetic transformation and regeneration on a solid support.

4. A transgenic monocot grass according to claim 1 or 2 or 3 exhibiting altered biochemical or physiological properties, altered nutritional properties of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.

5. A transgenic monocot grass according to claim 1 or 2 or 3 exhibiting at least one of the following characteristics:

(a) resistance to plant pathogens;
(b) improved nutritional value such as increased protein content or lower lipid content in grass seeds;
(c) enhanced sulphur content;
(d) enhanced expression of proteinase inhibitors;
(e) expression of pigment genes;
(f) expression of regulatory genes to control indigenous genes; and (g) reduction in levels of allergenic proteins in, for example, pollen.

6. A transgenic monocot grass according to claim 1 or 2 or 3 wherein said grass is from a subfamily selected from Bambosoideae, Arundinoideae, Chloridoideae, Pooideae and Panicoideae.

7. A transgenic monocot grass according to claim 1 or 2 or 3 wherein said grass is from the subfamily Pooideae.

8. A transgenic forage grass exhibiting at least one altered characteristic whencompared to a non-transgenic forage grass of the same species.

9. A transgenic forage grass exhibiting at least one altered characteristic whencompared to a non-transgenic forage grass of the same species wherein said transgenic monocot grass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium-mediated of genetic material responsible for said altered characteristic.

10. A transgenic forage grass exhibiting at least one altered characteristic when compared to a non-transgenic forage grass of the same species wherein said transgenic forage grass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium-mediated transfer of genetic material responsible for said altered characteristic and wherein the callus is subjected to genetic transformation and regeneration on a solid support.

11. A transgenic monocot grass according to claim 8 or 9 or 10 exhibiting altered biochemical or physiological properties, altered nutritional properties of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.

12. A transgenic forage grass according to claim 8 or 9 or 10 exhibiting at least one of the following characteristics:

(a) resistance to plant pathogens;
(b) improved nutritional value such as increased protein content or lower lipid content in grass seeds;
(c) enhanced sulphur content;
(d) enhanced expression of proteinase inhibitors;
(e) expression of pigment genes;
(f) expression of regulatory genes to control indigenous genes, and (g) reduction in levels of allergenic proteins in, for example, pollen.

13. A transgenic forage grass according to claim 8 or 9 or 10 wherein said forage grass is from Lolium, Agaropyron, Phalaris, Poa, Festuca, Dactylis, Aleopecuris and Phleum.

14. A transgenic forage grass according to claim 8 or 9 or 10 wherein said forage grass is from Lolium.

15. A transgenic ryegrass exhibiting at least one altered characteristic when compared to a non-transgenic ryegrass.

16. A transgenic ryeglass exhibiting at least one altered characteristic when compared to a non-transgenic ryegrass wherein said transgenic ryegrass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium-mediated transfer of genetic material responsible for said alteredcharacteristic.

17. A transgenic ryegrass exhibiting at least one altered characteristic when compared to a non-transgenic ryegrass wherein said transgenic ryegrass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium-mediated transfer of genetic material responsible for said alteredcharacteristic and wherein the callus is subjected to genetic transformation andregeneration on a solid support.

18. A transgenic ryegrass according to claim 15 or 16 or 17 exhibiting alteredbiochemical or physiological properties, altered nutritional properties of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.

19. A transgenic ryegrass according to claim 15 or 16 or 17 exhibiting at least one of the following characteristics:

(a) resistance to plant pathogens;
(b) improved nutritional value such as increased protein content or lower lipid content in grass seeds;
(c) enhanced sulphur content;
(d) enhanced expression of proteinase inhibitors;
(e) expression of pigment genes;
(f) expression of regulatory genes to control indigenous genes; and (g) reduction in levels of allergenic proteins in pollen.

20. A transgenic ryegrass according to claim 15 or 16 or 17 producing pollen exhibiting substantially reduced levels of at least one allergenic protein.

21. A transgenic ryegrass according to claim 20 wherein the allergenic protein is selected from LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX or LolpXI.

22. A method for producing a transgenic monocot grass, said method comprising deriving callus from a monocot grass and subjecting same to microparticle bombardment and/or Agrobacterium-medisted genetic transfer and then placing said callus under conditions sufficient to permit regeneration of said callus into plantlets.

23. A method for producing a transgenic monocot grass having at least one altered characteristic compared to a non-transgenic monocot grass of the same species, said method comprising obtaining callus from a monocot grass on a solid support, introducing into cells of said callus on said solid support genetic material effecting said altered characteristic via microparticle bombardment and/or Agrobacterium mediated transfer, subjecting said callus to selection conditions and then subjecting callus to regeneration conditions to form plantlets.

24. A method according to claim 22 or 23 wherein the monocot grass is from a subfamily selected from Bambosoideae, Arundinoideae, Chloridoideae, Pooideae andPanicoideae.

25. A method according to claim 22 or 23 wherein monocot grass is from the subfamily Pooideae.

26. A method according to claim 22 or 23 wherein the monocot grass is ryegrass.

27. A method according to claim 22 or 23 wherein the altered characteristic exhibited is altered biochemical or physiological properties, altered characteristic is of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.

28. A method according to claim 22 or 23 wherein the altered characteristic isselected from:

(a) resistance to plant pathogens;
(b) improved nutritional value such as increased protein content or lower lipid content in grass seeds;
(c) enhanced sulphur content;
(d) enhanced expression of proteinase inhibitors;
(e) expression of pigment genes;
(f) expression of regulatory genes to control indigenous genes; and (g) reduction in levels of allergenic proteins in pollen.

29. A method of regenerating monocot grass plants from transformed plant cells, said method comprising contacting cells on a solid support upon which said cells are subjected to transformation means on a medium and under appropriate conditions and time wherein said medium contains shooting and rooting hormones and then culturing the resultant shoots on a medium in the absence of biologically active hormones.

30. A method according to claim 29 wherein the monocot grass is selected from Lolium, Agaropyron, Phalaris, Poa, Festuca, Dactylis, Aleopecuris and Phleum.

31. A method according to claim 29 wherein said monocot grass is from Lolium.

32. A method of producing a transformed monocot grass comprising bombarding callus cells derived from a mature embryo of a monocot grass with a microprojectile containing a nucleic acid construct encoding a desired trait under the control of an appropriate promoter and carrying a selectable marker, under appropriate conditions to allow transformation, optionally further subjecting cells to Agrobacterium-mediated transfer of a similar nucleic acid construct, selecting transformed cells on a selection medium under conditions and for a time sufficient to permit regeneration of plants from the transformed cells wherein said regeneration conditions comprise rooting hormone and shooting hormone and then culturing the resultant shoots on a medium in the absence of biologically active hormones.

33. A method according to claim 32 wherein the monocot grass is selected from Lolium, Agaropyron, Phalaris, Poa, Festuca, Dactylis, Aleopecuris and Phleum.

34. A method according to claim 32 wherein said monocot grass is from Lolium.

35. A seed or seeds or pollen from a transgenic monocot grass wherein said transgenic monocot grass exhibits at least one altered characteristic when compared to a non-transgenic monocot grass of the same species.

36. A seed or seeds or pollen according to claim 35 wherein the transgenic monocot grass exhibits altered biochemical or physiological properties, altered nutritional properties of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.

37. A seed or seeds or pollen according to claim 36 wherein the transgenic monocot grass exhibits at least one of the following characteristics:

(a) resistance to plant pathogens;
(b) improved nutritional value such as increased protein content or lower lipid content in grass seeds;
(c) enhanced sulphur content;
(d) enhanced expression of proteinase inhibitors;
(e) expression of pigment genes;
(f) expression of regulatory genes to control indigenous genes; and (g) reduction in levels of allergenic proteins.

38. A seed or seeds or pollen according to claim 37 which exhibits reduced allergenic properties.

39. A seed or seeds or pollen according to claim 37 which exhibits reduced levels of one or more of LolpI, LolpII, LolpIII, LolpIV, LolpV, LolpIX or LolpXI.

40. A seed or seeds or pollen according to claim 39 wherein the monocot grass is ryegrass.