CA2195206A1 - Transgenic cereal plants - Google Patents

Abstract

To obtain a transgenic cereal plant which is stably transformed, an exposed cereal meristem is subjected to biolistic bombardment in order to target nondifferentiated meristem cells for transformation. Immature embryos at the early proembryo, mid proembryo, late proembryo, transitional or early coleoptilar stage are harvested for biolistic bombardment. The meristem tissue or cells fated to contribute to the meristem then are manipulated in order to enlarge transgenic sectors, either through selection and/or through effecting a proliferation from the tissue of shoots or multiple meristems per se. The shoot population thus obtained then is screened, by means of a nonlethal enrichment assay, to identify either chimeric sectors that will contribute to germline transmission, or non-sectored, L2 periclinal chimeras that will by definition transmit to progeny. Increased time in culture, under selection, enhances the prospects for sectoral-to-periclinal conversions, and also selects for L1-to-L2 conversions which, through a shift in position, ultimately contribute to the germline. Transgenic sectors also are stabilized during the step of tillering.

Description

~ W096/04392 2 1 9 52 0~ 5~

m~ Tqr=~ Tc ~:EREAL P~ANTS
BACKGRorJND OF THE INVENTION
The present invention relates to obtaininy plants by a m-thn~nloyy that entails the biolistic bombardment of meristem tissue, at a very early staye of deve~: , and the selective ~nhAn~ of t~ y-~10 sectors, toward yenetic h, ; -~;ty, in cell layers that contribute to yermline trAnPm; P8i nn, Production of trAr~gPn;" plants first became routine through the use of Agrobacterium, and the use of this vector with totipotent tissues has become the method of choice for many dicotyledonous species. While steady progress has been made in ~Yp~n~;ng the y~uoLy~e and species range of this method, Agrobacterium-r~ ted transformation has not been widely nt;l; 7~ for ,,.~.lo~oLyle~n~ml~ species, including cereals, and is likely in the near term to remain restricted to specific ~"uLy~es. Similarly, protoplast-based methods are not widely applicable for monocots.
The first reports which appeared on biolistics-r- '; Ated production of fertile, transgenic maize were restricted to a specific hybrid, A188 x B73. See Gordon-Kamm et al., Plant Cell 2: 603 (1990), and Fromm et al., Bio/Tenhnnlogy 8: 833 (1990). Since then the tenhnl~le has been ~t~n~d to many important monocot crops, includiny barley, wheat, rice and oats, and the useful ranye in maize hag been ~YrAn~d slowly to include a handful of yenotypes, for example, the commonly used A188xB73, ~99, FR16 and Pa91 yenotypes. This work generally has revolved around a common theme, which is the initiation of reyenerable callus from the scutellum ~ of the embryo. In particular, all the reports in this context have hiyhlighted a prerequisite of initiating regenerable callus from the snutPll-lm of the immature embryo, regardless of whether there is } ' ' (i) of the scutellum just after embryo ;~n]Atinn, followed by selection of the callus grown from the scnt~llA~ cells, (ii) of freshly initiated callus after a short pre-W096l04392 2 1 9~206 - 2 - .~

culture of the scutellum or ~ of long-term calius or cell suspension cultures.
Progress in P~rAnrl;ng the callus-based approaches to new geno~ypes or species has occurred via ~AptAt;nnq Of she basic method to ~n~ tP differences in morphology and growth patterns tha~ typify different forms of immature, embryo-derived callus, i.e., friable callus versus compact callus, also referred to as Type II and Type I, respectively. Genotype restrictions remain, however, because some y~ r- does not produce an ay~Lu~Liate callus response.
With the advent of biolistics-mediated transformation, numerous groups have explored the pn~s;h;lity of using microprojectile-delivery methods with meristem tissues. It has L~ ;nPd "an open question,~ however, as to "whether integrative tran8formation in cells of the shoot apical meristem of [a] monocotyledonous species i8 [even] possible.~ silang et al., Plant ~. 4: 735 (1993).
The literature is marked by spPcnl~t;rn rnnrPnn;nrJ
barriers to t~nc~forming meristem target cells which may explain the lack of success in this area. It has been observed, for example, that cereal ~shoot meristems are tiny ~about lO0 ~m) and...biolistic particles hit large target areas at random," and that "meri8tematic cell8 may [have] molecular ~ nir~ which prevents [sic]
integration of foreign DNA...." Potrykus, Nature 355:
568, 569 (1992). More generally, the fact that monocot plant species tend to display less devPl ~ -Al plasticity than dicot species has engendered an P~rPrt~t;nn that monocotg are less amenable to stable transformation by biolis~ic and other techniques.
Given the lack of devpl~: Al plasticity in cereals, therefore, the historical focus of transformation efforts in these crops has been on callus derived from one of the few genotype8 that produce ~ype I or Type II ~ yuyellic callus. These transfn~r~t;nn targets were subject to ea8y use because a large ~ W096l04392 2 1 9 52 06 y~ C~ "

population of undetermined, proembryogenic cells could be selected. Accordingly, many research groups have taken advantage of this approach and have not pursued alternative target tissues. In particular, no one to date has reported germline transformation via meristem bombardmeno of maize, a key cereal crop. Lack of succe6s in this regard has been ascribed to the rigid deVell-L t~l fate of the cells ~ing the meristem.
SUMMARY OF THE INVENTION
It is therefore an objec~ of the present invention to provide a methn~nlogy for the repro~lcih~e prodl~ctinn of stably transformed cereal plants.
In ac~ ~l; Rh; ng this object and others, there has been provided, in accordance with one aspect of the present invention, a method for producing transgenic cereal plants, e.g., maize, sorghum, wheat, barley, oat or rice plants, that will transmit introduced DNA to progeny, comprising the steps of (A) introducing foreign DNA into cells selected from the group consisting of (i) cells of a meristem that is not enclosed by Ah~Arhinr; leaves and ~ii) cells fated to contribute to said meristem; then tB) ;n~l1r;ng reorganization of said meristem to increase transgenic sector size, whereby the l; kDl; hnod that a transgenic sector will contribute to germline transmission is increased; and thereafter ~ C) PYpn~;ng said meristem to conditions under which it differ~nt;stes to form a ~l~ntl~t, wherein said plantlet nnnt~;n~ said transgenic sector or is ~ , 0ol1Aly transformed by said foreign DNA, such that said plantlet can be grown into a transformed cereal plant that will transmit said foreign DNA to progeny.
The foreign DNA can be introduced into a plurality of meristems, at least some of which differentiate in step (C) to form a plurality of plantlets. The foreign DNA is introduced into a meristem that is not enclosed by ~h~Athing leaves ;nr~ ;ng meristems from early . , . , . . _ ... . _ . ...... .. ...

W096l04392 2 ~ 9 5 ~ 0 ~ "

proembryo, mid pLU~I~Ly~, late pLO_~Ly~, tranBitiona and early coleoptilar ssage embryos.
n one preferred ~ , reorgAn;7~t;nn is ~ffe~t~d through at least one ~-n;p~ At;nn selected from the group consJsting of (i) imposition of a nonlethal selective pressure on the meristems, (ii) ~ ' ;rAlly-induced meristem reorgAn;7~t;n~, and (iii) h~ Ally-induced shoot multiplication. In another preferred embodiment the conditions in step (C) are such that the meristems undergo maturation and plant dif~eront;At;nn to form shoot apices, and the method further compri8es offort;ng reorg~n;7~t;nn of merigtem tissue in the shoot apices to enlarge transformed sectors or to produce periclinal L2 chimeras. The recrgAn;7Atinn in this regard can be effected, for example, by o~pos;ng the shoot apices to nonlethal sol~ct~nn pL~8~uLe such that . tran8formed cells have a competitive growth advantage over nontransformed cells in the shoot apices, and the proportion of tran~f d cells in the shoot apices is increased. In yet another preferred : ' '; , the method further comprises a step be~ore step (B), e.g., before step (A), of wounding the apical dome selectively.
A method of the present invention also can comprise the further steps of (i) dissecting out an axillary bud from above the base of a leaf of a plAnrlet when a chimeric sector is observed in a substantial portion of the leaf, and then (ii) germinating the axillary bud into a whole plant or subjecting the axillary bud to shoot multiplication.
In yet another preferred : ~~;r~nt, the transgenic sector of a plantlet is stAh;l;70d by ;n~nn;ng tillers.
The apex of a transgenic p~Antl~t is removed, the wounded plantlet is grown to induce forr~tinn of a plurality of tillers, and transgenic tillers then are selected ~rom that plurality.
In arcnr~n~o with another aspect of the present invention, a transgenic cereal plant is provided that (A) is the product of a method as described above, _ W096/0~92 r~
~ _ 5 _ 2 ] 9 5 2 0 6 (B~ transmiss introduced DNA to progeny and (C) belongs to a cereal line chas is recalcitrant to callus-based ~ransformation. In a preferred ' '; , the tr~nrgrn; r cereal is a maize plant that is not produced by transformation of a ~noLy~e selected from the group consisting of A188, A188 x B73, H99, Pa91, FR16 and a genotype obtained from a cross involving any of the foregoing.
According to still another aspect of the present invention, a maize plant is provided that transmits introduced DNA to progeny and that haa a pedigree ;nrlnA;ng a line selected from the group consisting of PHT47, PHP02, PHV78, PHK05, PHW20, PHR62, PHN37, PHM10, PHV37, PHJ65, PH3W8, PHK29, PHJ33, PHP60, PHN73 and PHHV4.
Other objects, features and advantages of the present invention will become a~alrllt from the following detailed description. It should be understood, however, that the ~PtAil~ description and the specific examples, while ;n~;cAt;ng preferred ~hod; R of the invention, are given by way of illustration only. Indeed, various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a series of line drawings depicting the structure of a typical cereal embryo (a) at the coleoptilar stage, which in maize occurs appr~Yi~-t~ly 8 to 14 days after p~ll;nAti~n~ and (b) at the later third stage (about 22 to 28 days after poll;nAt;~n in maize~, respectively; (c) the model shoot tip of an angiosperm, - including cereals, shown in longitudinal section; and (d) the shoot and root structures which pertain in cereals ~ generally, with a unit phytomer of the shoot highlighted.
Abbreviations: c = coleoptile; cn = roleopt;1Ar node; cp = rnleopt;1Ar pore; cr = coleorhiza; m = mesocotyl; r =
primary root primordium; s = snqp~nR~r; sa - shoot apex;

_ _ _ _ _ _ _ _ _ , , , ... _ _ . .. . . .

W096/04392 2 1 95206 ~ ( "

sc = scutellum: scn = scutellar node, sr = seminal root primordium.
FIGURE 2 is a schema~ic reprPqPntat;nn Of transformation methnt~nlogy according to the present invention.
DETATn~n DESCRIPTIO~ OF TR~ S~ K~4~ EMBODIMENTS
It has been discovered that the tl; ffi t'lll ties discerned previously in relation to applying a meristem-based transfnrr-t;nn strategy to cereals can be ~v~., by (A) biolistically targeting cells of the shoot apical meriatem under conditions such that the apical dome of the apical meristem is not Pnt-lost~d by qhPath;ng leaves, as APpicted in FIGURE 1; and (B) using a nonlethal s~lpct;nn regimen to effect an enrichment of transformed cells, for example, by giving transformed cells a competitive advantage over nontransformed cells, and thereby to facilitate an increase in sector width. The nonlethal sPlertinn also can promote the devP1t~ of a short-lived, mericlinal L2 event into a stable, periclinal event in which most or all of the cells contributing to the germ line (i.e., the L2 layer) are transformed. In addition, it has been found that selective pressure can promote Ll- to-L2 conversion events, thus increasing the prn~h;l;ty of germ line transmission.
The shoot apical meristem in cereals is highly variable between species. In most species, however, a stratified meristem exists that is ~ of two or three visible layers which generate the entire shoot: a superficial Ll, a subsurface L2 and, in some cases, a deeper ~3 layer. The outer layer(s) comprise the tunica, which are nh~ractPrized by anticlinal cell divisions. In contrast, divisions in the i~eLI ~ layer, the corpus, occur randomly, both anticlinally and periclinally. In maize the meristem is believed to be composed of only two layers, Ll and L2, and possibly a third L3 layer. See Poethig, ~o..~ o~ARY PROBLEMS I~ ~LANT AXATOMY 235-39 (1984). Cell differPnt;~t;nn to delimit the major ~ W096/04392 2 1 q52~6 P~
_ 7 -tissues of the shoot is position-dependent rather than lineage-dependent.
For example, in most species the Pp;~Prm1~ is generated almost exclusively by the ~1 layer, with the L2 layer contributing to the germ line. In the process of introducing a foreign gene into a subset of apical meristem cells, one creates a plant that nPCP~s~rily is ~chimeric," i.e., a plant in which portions have been altered in genetic composition. There are three major categories of chimeric plants, based on the characteristic pattern of gene~ic differences:
(1) sectoral chimeras, in which a portion of the plant is ~genetically distinct" through all cell layers by virtue, for example, of displaying a mutant somatic ph~--uLy~e, a change in ~ number, or the presence of transformed cells; (2) periclinal chimeras, in which an entire cell layer (~1 alone or ~2 alone, for instance) is different from the rest of the planti and (3) mericlinal chimeras, which repre8ent an ; nt~ te between the other two types, i.e., a genetic difference characterizes only a portion of one layer.
In this description, the terms ~biolistic~' and ~biolistically~ denote an approach to genetic transformation described, for example, in U.S. patents No. 4,945,050 and No. 5,141,131, the respective c of which are hereby incorporated by reference.
Pursuant to a biolistic approach, force is transmitted to small particles that carry DNA. for example, coated on particulate surfaces or ~hsorhe~ into the particles, in such a way that the exerted pressure forces particles into a targeted cell or tissue ("biological sample~); the particles thus are called ~microprojectiles" or "microcarriers." In other words, the microprojectiles are propelled at the biological sample, accelerating to such speed that, upon impact, they penetrate cPl 11ll ~r surfaces and are incorporated into the interior of a cell or cells in the sample.

_ _ _ _ _ _ _ _ _ _ _ _ _ . .. ..

w096/~4392 2 1 9 5 2 0 6 The microproiectiles should have an average ~; tPr sufficiently small to permit penetration of and retPnr;~n by cells of the biological sample without ~illing the cells. Particles of gold or tungsten, in the size range of about 0 1 to 4 microns, are illustrative of microprojectiles that are suitable for delivering -~-y ~ q nucleic acid into a host. Other types of biolistic delivery vehicles are disclosed, for example, by U.S. patents No. 5,120,657 (electrical discharge propels a carrier sheet toward target) and No. 5,240,842 lnucleic acid delivered via aerosol droplets), and in PCT
~ppl;~t;nn WO 92/01802 (ice particles as carrier).
In relation to aspect (A) I ;~nP~ above, the present invention cont ~l~rpR the biolistic targeting of apical meristem cells at an early devPl~ 1 stage.
In a preferred ' '; ', meristem cells are bombarded at a devP~ l pha8e that is no later than the coleoptile-ring stage, when the apical dome is fully exposed, lacking protection from leaf primordia, and is composed of fewer cells in the meristem than are present at later stages. The stages of maize ~..~Lyuy-llesis are described in detail by Poethig et al., Developmental Biologv 117: 392-~04 (1986), the ~nntPntq of which are incorporated by reference.
More specifically, the transformation method of the present invention focuses on coleoptilar and earlier stage embryos, namely, early proembryo, mid p~_..~Ly~, late ~,~ y~, and the transitional stages of embryo development. At the earliest stages of devPl~ ~ t, the meristem is not defined; instead, a group of cyr~pl~;cally dense cells undergo more rapid division and, ultimately, form the apical meristem.
In targeting these various embryo stages, therefore, DNA is introduced (i) into cells that make up the meristem proper (i.e., at the coleoptilar stage) or, (ii) in the earlier stages of dev~ , into cells that are destined, by position or fate, to contribute to the meristem. Biolistic ~ ' I according to the present ~ W096/04392 2 1 ~ 52 06 r~ "
g invention is effec~ed by orienting the embryo so that cells that are within a meristem or that are destined to contribute to the meristem are exposed directly to the biolistic projectiles.
In late ~ru. ' yu8, the axis side of the embryo is slightly flattened. allowing this side of the embryo to be placed face up (away from the agar) for ~ ' -d~ L.
Transition stage and coleoptilar stage embryos are similarly oriented. There is no such or;~nt~t~nn, however, for mid and early ~LU. ' yus on agar after isolation (i.e., before shooting). Rather, when plC yu8 are placed in a random ori~ntatinn on the agar medium, the meristem ~are~-Lly develops on the upper side of the embryo (away from the medium). Thus, pl~ ~ on the medium may be St; l~ting the embryo to re-orient its growth axis, for example, by virtue of the in vitro conditions which are provided (i.e., the new hoLI 1 gradient that is being est~hl;~h~d within the embryo).
A convenient and, hence, preferred source of meristems for use in the present invention are coleoptilar stage embryos. At the coleoptilar stage of cereal embryo dev~ , the coleoptile is visible as a ring of leaf primordium surrounding an exposed meristem. In maize, the early coleoptilar stage can generally be obtained 10 to 12 days after poll;n~jnn.
(The days-after-pollination criterion, or "DAP," is affected by embryonic environment and genotype, and therefore is an adjunct to develc~ ~1 staging based on morphology, which is an important criterion for timing of t~ LuL,.,~tion in the present invention.) At the early - coleoptilar stage the boundary of the meristem is distinct, with a visible tunica and corpus (L1 and L2 layers, respectively).
A particularly preferred source of target cells for use in the present invention are present in the early pl, ' yu, mid ~L~ ' yu, late pLu_..~Lyu and the transitional stages in embryo dev~l~ ~. In maize, the W096/0~92 .~
7 ~ 9~2 ~6 - lo early proembryo, mid ~LU~ Lyu, late ~LU~.~Lyu and the transitional stages can generally be i~nl~ted 2, 4, 7-8 and 8-10 DAP, respectively (see Poethig et al. (1986), cited above). Again, the devPl ~ 1 stage is the S important criterion. Rate of development and, hence, DAPs at which embryos are iqol~ted vary with growth environment and genotype.
At the mid ~L U. ' yu stage there i8 no distinction between the L1 and L2 layers. The distinrtinn between L1 and L2 progresses until it is well-defined by the time the embryo reaches the transitional stage.
Alternatively, immature stAmin~t~ inflor~qcPnr~q (tassels) and pistillate inflorPqcPnrPq (ears) can serve as sources of meristemg for tr~n~fnr~-t;rn in ~ rP
with the present invention. "Immature" here denotes a devPll~ ~1 state when floral meristems still are devPl,~ Ally plastic, i.e., are capable of shoot differ~nt;~t;nn. This devPl~ 1 plasticity should be exploitable, pursuant to the present invention, for transformation of many ~ m;n~rPm-q species, given the rerrr~ni7~d 3imilarities in inflorescence devPl~
among the grasses.
A trained technician can isolate 200 to 600 mid ~LU_..~Lyu, late ,ULO. ' yu, transitional or coleoptilar-stage embryos per day, the ease of isolation and numberof isolated embryos increasing with embryo size. On the order of ten times as many meristem PYpl~ntq can be isolated from immature tassels and~or ears, and a large percentage of~these can be induced to ~ollow a vege~ative pattern of devPl~, . Another important advantage or;~t~ with using floral PYrl~ntq as meristem sources is that many y~-lotyues exhibit better meristem growth and shoot multiplication when the floral P~pl~ntq are the starting material. This advantage is prnnrllnr~ for example, with respect to an inbred maize line with a lineage that includes line PHV78. Conversely, immature embryos are the preferred explant for some genotypes, such as maize inbreds having a lineage including line ~ W096/04392 2 1 9 5 2 0 6 ~ ~5" "

P~BW~. Access to both options significantly extends the genotype range for meristem transformation pursuant to the invention.
Whatever explant or tissue is used as a source of target cells for biolistic treatment in ac~u~d~.ce with the present invention, the b~ ' ded cells are subjected to a first, nonlethal selection pressure in the course of generating plantlets which are grown out directly (course I in ~IGUR~ 2) or, alternatively, are subjected to meristem reorg~ni7at;~n/ induced I -hcn;c~lly or ho-, Ally, in advance of a second nnnleth~l selection (course II in FIGURE 2). The aspects of nonlethal selection and induced meristem reorg~n;7~t;~n are addressed in greater detail below.
The deve~ 1 fate of cells within the meristem normally is rigidly determined. Thus, transformation of a particular cell within the meristem typically will result in a small tr~ncg~n;c sector made up only of the ~cc~n~ intq of that cell. Without further m-n;p~ ti~n, such sectors rarely if ever overlap ~ -tophytic tissue during normal development. But by targeting cells at earlier devPl~ ~1 stageg, as described above, and then applying mild selective conditions in accordance with the present invention, i.e., the pressure provides a growth advantage to transformed cells but is not severe enough to impede the overall development of the meristem, then the consequent faster division rate of transformed cells results in the ~sr~n~n~ cells comprising a greater portion of the meristem. Accordingly, the transgenic sector contributes to a larger portion of the mature plant, and there is a greater l;k~l; h~od that the - sector will contribute to germline tr~n~ si~n.
According to one preferred : '-'; , a selective growth advantage is imparted to transformed cells in the form of NPTII-encoded resistance to tobramycin, kanamycin or a related ~. It is acceptable, however, to confer resistance to another "bleaching~ antibiotic (by means of a streptomycin-resistance gene, for instance) or wog6/04392 21 ~2~6 - 12 - P~

herbicide, for eYample, by transformation with the crtI
gene, which imparts resistance to norflurazon. 3y the same token, the present invention rnnr~mrl~tes similar non-lethal strategies which entail the use of other selective agents, such as b;~l~rhns and hyyL~ y~in, with a corresponding, resistance-imparting gene, so long as the resulting selective pressure retards the growth of non-transformed cells relative to cellg in the tr~nRg~n;c sector.
A model experiment in this regard would involve PYrn~;ng samples of iRol~ted meristem tissue to a graded series of ~ lt;nnR of the selection agent in the medium of choice, and then determining a rnnrrntration threshold below which the selective pressure iavoring transformed cells is not so stringent as to be detrimental to general meristem dev~ . While this approach typically would result in rnnt;rll~ meristem growth during sPlert;nn~ the present invention also envisages esr~hl;Rh;nr, conditions of little or no meristem growth ("static conditions") which are punctuated by brief exposure(s) to a higher cnnr~ntration of 5~1~rt;nn agent ("pulsed selection") which otherwise would adversely affect overa~1 meristem development.
As noted above, cereal transformation according to the present invention optionally involves a reorr~n;7~t;on of the meristem, for example, by wounding of the apical dome. While other methods of wounding also result in reor~n;7~tinn~ a preferred method is to pierce the apical dome using a mi~L, ;p~ tion needle. T h e reorg~n;7pt;n~ thu8 effected alters growth in the apical dome and, it Eas been discovered, prompts a proliferation of multiple meristems which, in turn, ~nh~nr~
transfnrr-tinn frequency and sector size. For example, mechanically-induced meristem proliferation in conjunction with selective pressure results in an increase in frequency and size of the tr~n~g~n;r sectors observed in subsequent leaves.

W096l0439~
~ - 13 - 2 1 9 52 ~6 Meristem reorg~ni~Ation may precede biolistic treatment, followed by germination and 6election leading to the production of chimerically transformed plants (course I). Alternatively, mechanical w ~;ng can be performed after bombardment of the meristems in order to effect a proliferation of meristems. When applied in this manner on chimeric meristems, the sectors can enlarge because the reorganized meristems are derlved from a smaller number of cells and, hence, the percentage of transformed cells in the meristems is increased.
Pursuant to course II (see FIGURE 2), a reorgAn;~atinD is brought about by h- -lly-induCed shoot multiplication with respect to the developing shoot meristem of a plan~let selected for the presence of a transformed sector. The h~-, Ally induced reorg~n;7at;nn need not be exclusive of the optional, , -hin;~A1ly induced reorgAn;7At;nn ~ n~d above, and brings about meristem proliferation via shoot mult;E)licAt;nn.
To effect l1OL lly induced reorgAn;7~t;on, the developing shoot meristem first is lorAl;7e~, typically in a swelling that occurs in the germinated plantlet at the junction between the mesocotyl and the epicotyl (see FIGURE 2). A section of 2 to 3 mm in size which cnnrA;n~
25 the meristem then can be excised at the ~ll;nrJ point and cultured on a shoot proliferation medium of the sort described, for example, by Lowe et al., Plan~ Science 41:
125 (1985), and by Zhong et al., Planta 187: 483 (1992), respectively. To this end, meristems typically are cultured on MS medium with 2 mg~l BAP (6-benzyl-Am;nnpnrine), 3t sucrose and 9 mg/l agar. More generally, a shoot multiplication medium will utilize a cytokinin, such as Kineti~, BAP, Th;~;A7llron or Zeatin, at a nnnr~nrration between 0.5 and 10 mg/l. A low level of auxin also may be required in some genotypes.
Murashige and Skooge (MS) salts are adequate but probably not optimal, in that prPl;m;nAry experiments using media with ammonium levels higher than those in MS resulted in 21 ~5206 14 -an improved culture response. ~;t;on~l additives such as the auxin transport inhibitor, TIBA, and ethylene inhibitors like silver nitrate and cefotaxime also appear to be bPn~f; r; ~1 .
sy virtue of its hormonal constituency, the shoot proliferation medium forces the generation of a few to hundreds of shoots from each excised shoot meristem, thereby increasing the l;kPlihno~ of nht~;n;ng a snhpopnl~r;nn of shoots, some of which may arise from a transformed sector. Unlike mericlinal and sectoral chimeras, which exhibit a lower probability of g~rml;nP
tr~n~;cc;nn, a significant and repro~lln;hle percentage of the resulting shoots are periclinal chimeras and, hence, are "stabilized" in the sense that genetic hl ,~..eity is promoted within a cell layer, such as the L2 layer, that ultimately contributes to germline tr~np~;csinn~
To identify the af~ innPd shoot 5llhroplll~rlnn, the large population of induced shoots is screened to identify non-sectored, periclinal chimeras. This is a ~ chpd via a nonlethal asaay which brings about an enrichment of transformed cells through the use of selective agents (i) that bleach normally green tissue at levels that do not inhibit growth or (ii) that inhibit growth of non-transformed meristem sectors without . sign;~;r~ntly reducing viability of the meristems.
Use of ~n appropriate selective agent at nonlethal levels, as described, also provides the ~ L~.ity to assess visually the extent of hl , -;ty within a transformed -meristem layer. Increased time in culture under selection, pursuant to the present invention, Pnh~nrPs the prospect of mericlinal-to-periclinal conversions and of sectoral-to-homogeneously transformed conversions, and also selects for L1-to-L2 conversions which, through a shift in position, ultimately contribute to the germline.
From the preceding c~ ~ry it is apparent that one aspect of the present invention relates to forcing ~ WO96/0439Z 2 1 95206 meristem reorganization, before bombardment, after bombardment or both, by suppressing cell growth through selective wounding of the apical dome, prompting rJ~nPrat~on of multiple meristems, or by exposing excised meristems to h~ A 1 stimuli likewise leading to multiple meristems, albeit in the form of proliferated shoots. According to yet another preferred embodiment, the axillary bud of a transformed pl~ntl~t can be dissected out, from just above a leaf base, when a chimeric sector is observed in a substantial portion of that leaf. The iRolat~d axillary bud L~p' esenLs an additional meristem that can be grown into a whole plant, or taken through a brief cycle of shoot mult;~l;c~t;rn as described, thereby to obtain a more ~ , ?ously trirRf~ ~ plant.
The purpose of this approach, as for the others ~;Rcucsed above, is to increase the frequency of g~rml ;n~
tr~n~;Rs;~n. Thus, if a transformed sector runs through more than one leaf, it should be possible to "capture"
that tr~nRf~rr-t;r~n event in a axillary bud, i.e., convert a transformed mericlinal or sectoral chimera into a perirl;n~lly or homogeneously transformed shoot.
Another method of st~hil;~;nr, transgenic sectors is to induce tillers in the transformed plant. In those cases in which transgenic sectors are limited to the lowermost leaves or domains of maize plants, tillering is induced to st~h; 1; 7e these transgenic sectors.
~ y means of the present invention, a wide range of cereal varieties can be transformed stably, in a ~noLy~e-;n~rrn~nt manner, for the first time. In maize, for example, this means that elite lines which - were previously ;n~cr~rsible to transformation characterized by tr~nr-; RS; rn of imparted trait(s) to seed progeny now can be g~nrt;r~lly ~nr~;n~red to express various phenotypes of agronomic interest. The gene3 implicated in this regard include but are not limited to those categorized below.

_ _ _ _ _ _ ... .. . .... _ . . ..

W096/04392 2~ /. 9,. ." ~

I. Genes ~hat Confer Resi3tance To Pesta or Disea~e And That E~code:
(A) A Bacillus ~huringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48: 109 (1986), who disclose the cloning and nucleotide se~uence of a Bt ~-endotoxin gene.
IluLeuve-, DNA molecules Pnro~inr~ ~-endotoxin genes can be purchased from American Type Culture Collection (Rockville, MD), under ATCC
C~q~irn Nos. 40098, 67136, 31995 and 31998.
~B) A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24: 825 (1994), who disclose the nucleotide SPr~llPnrP~
of several Clivia miniata mannose-binding lectin genes.
(C) A vitamin-binding protein such as avidin. See U.S. patent application serial No. 07/911,864, the rrnrpnts of which are hereby incorporated by reference. The rrrl;r~t;rn teaches the use of avidin and avidin homologues as larvicides against insect pests.
~D) An enzyme inhibitor, for example, a protease inhibitor or an amylase inhibitor See, for example, Abe et al ., J. Biol. Chem. 262: 16793 (1987) (nucleo~ide CPrlllPnrP of rice cysteine proteinase inhibitor), Huub et al., Plant ~olec. i3iol. 21: 985 (1993) (nucleotide se~uence oi cDNA Pnrr~i ng tobacco proteinase inhibitor I), and Sumitani et al., Biosci.
Biotech. Biochem. 57: 1243 (1993) (n--rleo~;~e 6equence of Streptomyce6 niLLo~o.~s ~-amylase inhibitor).
(~) An insect-speci~ic hormone or pheromone such as an ecdysteroid and juvenile hormone, a vari~nt thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., 2 ~ 9 5 2 0 6 Nature 344: 453 (1990), of baculovirus expression of cloned juvenile hormone esterase, an ~nactlvator of juvenile hormone.
(F) An insect-sp~if;~ peptide or nuuLuu~Lide s which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor), and Pratt et al., Bioche~. Biophys. Res. Co~m. 163:
1243 ~1989) (an allostatin is i~nt;~ in Diploptera puctata). See also U.S. patent No.
5,266,317 to T~- lck; et al., who disclose genes onro~;ns insect-specific, paralytic neurotoxins.
(G) An insect-specific venom produced in nature by a snake, a wasp, etc. For eample, see Pang et al., Gene 116: 165 (1992), for ~;Cclos~~re of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.
(H) An enzyme r~cp~ncihle for an hyperaccn~-~lat;~n of a monterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.
(I) An enzyme involved in the modification, including the post-translational modification, of a biologically active r~l~c~ ; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nucleaae, a cyclase, a trAnq~-n~ce, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glllc~n~ce, whether natural or synthetic. See PCT application W0 93/02197 in the name of Soott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-~n~o~l;ns se~uences can W096l04392 _ _ r~ .W ., ~ q~2 ~ - 18 -be obtained. for e~ample, from the ATCC under accession Nos. 39637 and 67152. See also Kramer~o~ al., rnseCs Biochem. Molec. Biol . 23:
691 (1993), who eeach the nucleotide sequence of a cDNA ~nrn~;n~ tobacco hookworm rh;t;n~Re, and ~ rk es al., Pla~t Molec. Biol. 21:
673 (1993), who provide the nucleotide sequence of the parsley uoi4-2 polyubiquitin gene.
(J~ A molecule that st;m~ t~q signal t~nc~llrtir,~, ~or example, see the ~iqnlnqllre by Botella et al , Plant Molec. Blol . 24: 757 (1994), of nucleotide s~lrnroq for mung bean n~l l;n cDNA clones, and Griess et al., Plant Physiol.
104: 1467 (1994), who provide the nucleotide sequence of a maize r~l ~ 1;n cDNA clone.
(Kj A hydrophobic moment peptide. 5ee ~.S. patent ~prlir~t;nnq serial No. 08/168,809 (~;qrloqllre of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and serial No.
08/179,632 (teaches synthetic antimicrobial peptides that confer disease resistance), the respective nnnt~ntq of which are hereby incorporated by reference.
(L) A , ' dne permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes et al., Plant Scl. 89: 43 (1993), of heterologous expression of a cor~r~rl n -~ lytic peptide analog to render transgenic tobacco plants resistant to pgonAnm~nAq ~o7~n~re~rum (M) A viral-invasive protein or a complex toxin derived therefrom. For example, the ~t;nn of viral coat proteins in transformed plant cells imparts resistance to viral infec~ion and/or disease dev~
Pffect~ by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. Rev.

~ ~096/~392 2 ~ 95206 ~ ' ."

Phytopathol. 28: 451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.
(N) An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical ~ hn,l; C function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON
~nn~ R PLANT-MICROBE INTERACTIONS (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).
(o) A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993), who show that tr~n~gon;~ plants expressing le~c 'nn~nt antibody genes are protected from virus attack.
(P) A devel., t~l-arre5tive protein produced in nature by a pathnson or a parasite. Thus, fungal endo a-1,4-D-polygalacturonases facilitate fungal ~nl nnl 7~t; on and plant nutrient release by 8olllh; 1; 7lng plant cell wall homo-a-1,4-D-galacturonase. See Lamb et al., Bio/Technology 10: 1436 (1992). The cloning and charPoto~; 7~t; nn of a gene which encodes a bean endopolygalac-turonase-;nh;h;t;ng protein is described by Toubart et al., Plant ~. 2: 367 (1992).
(Q) A devel~ ~l-arrestive protein produced in nature by a plant. For example, I~ J et al., Bio/~echnology 10: 305 (1992), have shown t~at transgenic plants expressing the barley ~096/04392 2 1 9 5 2 0 6 ~ "

ribosome-inactivating gene have an increased resistance to fungal disease.
II. Gene~ That Confer Resi~tance To A ~h; ~ , For Exa~ple:
(A~ A h~rhi ~; ~P that inhibits the growing point or meristem, such as an im;~7~1 in~n~ or a sulfonylurea. Exemplary genes in this category code for mutant ALS and A~AS enzyme as ~s~r;he~, for example, by Lee et al., ~30 J.
7: 1241 (1988), and Miki et al., Theor. Appl.
Genet. 80: 449 (1990~, respectively.
(B) Glypho8ate (re5i8tance imparted by mutant EPSP
synthase and aroA genes, respectively) and other rh~Rrh~n~ ~ ~- J~ such as glufosinate (PAT and bar genes), and pyridinoxy or phenoxy proprionic acids and cy~lo~hp~ne~ (ACCa8e inhihjt~r-~nro~;ng genes). See, for example, U.S. patent No. 4,940,835 to Shah et al., which discloses the nucleotide sequence of a form of EPSP which can confer glyphosate resistance.
A DNA molecule ~n~o~; ng a mutant aroA gene can be nht~;r~d under ATCC ~r~csi~n No. 39256, and the nucleotide sequence of the mutant gene is ~ o~e~ in U S. patent No. 4,769,061 to Comai. European patent app1;ca~i~n No. 0 333 033 to Kumada et al. and U.S. patent No 4,975,374 to Goodman et al. disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-ph~sph;nothricin~ The nucleotide sequence of a ph~5rh;nnthricin-acetyl-transferase gene is provided in European application No. o 242 246 to Eeemans e~ al. De Greef et al., ~io/~e~n~lo~y 7: 61 (1989), describe the pro~ tion of transgenic plants that express chimeric bar genes coding for ph~ph;n~thriCin acetyl transferase activity. F , l~ry of genes . .. . _ _ _ _ _ _ _ _ ~ W096/04392 2 1 952~6 .~ . ."

conferring resistance to phenoxy proprionic acids and cynlnPhPYnnpq~ such as sethoxydim and haloxyfop, are the ACC1-Sl, Acc1-52 and Accl-S3 genes described by Marshall et al., Theor. Appl. Genet. 83:
435 (1992).
(C~ A herbicide that inhibits photosynthesis, such as a triazine (p8bA and g5+ genes) and a benzonitrile (nitrilase gene). Przibilla et al., Plant Cell 3: 169 (1991), describe the nrmation of Chl~..~G c with plasmids Pn~o~;ng mutant p5bA genes. Nucleotide sequences for nitrilase genes are ~;crlosP~ in U.S. patent No. 4,810,648 to Stalker, and DNA
molecules cnnt~;n;ng these genes are available under ATCC AncPcs;nn Nos. 53435, 67441 and 67442. Cloning and expression of DNA coding for a glut~th;nnP S-transferase is described by ~ayes et al., Biochem. J. 285: 173 (1992).
20 III. Genes That Con~er Or Contri~ute To A
Value-Added Trait. Such As:
(A) Nutritional PnhAn, - , as illustrated by (1) Uigher lysine content: A cereal such as maize could be transformed with a gene that increases lysine content, making the cereal nutritionally more complete and thereby Pl;m;nAt;ng need ~or added lysine, for example, in poultry and swine feeds.
(2) ~igher me~h;~n;n~ content: A gene would be added to increase meth;on;nP
levels in a cereal crop to offset an overall low - rhinninP content, for example, in a poultry feed which ~ ;nra lower- and higher-meth;nnlnP
cnmrnnPntc such as soybean and maize, respectively.

W096l04392 21 952 06' - 22 - r~

(B) Decrea3ed phytate content (1) Intro~ tinn oi a phytase-encoding gene would enhance hreakdown of phytate, adding more free phosphate to the transformed cereal. For example, see Van ~artingsveldt et al., Gene 127: 87 (1993), for a digclogure of the nucleotide R~ n of an Aspergillus niger phytase gene.
(2) A gene could be introduced that reduces phytate content. This could be ac ,];Rh~d~ for example, by cloning and then re-introducing DNA
~qRoci~ted with the single allele which is responsible for maize mutants characterized by low levels of phytic acid. See Raboy et al., Maydica 35: 383 (1990).
(C) Modified carbohydrate composition ~f~ct~, for example, by transforming maize with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al , ~.
Bacteriol. 170: 810 (1988) (nucleotide secuence of Streptococcus mutans fructosyltransferase gene)~ St~i tz e~ al., ~ol. Gen. Genet. 200:
220 (1985) (nucleotide se~uence of Bacillus subtilis levansucrase gene), Pen et al., Bio/T~rhnn~ogy 10: 292 (1992) (production of transgenic plants that express Bacillu3 lirh~n;formiq ~-amylase~, Elliot et al., Plant ~olec. ~3iol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes), S0gaard et al., ~. Biol~ Chem. 268: 22480 (1993) (site-directed mnr~rJ~n~clq of barley amylase gene), and Fisher et al., Plant Physiol. 102: 1045 (1993) (maize endosperm starch hr~nrhing enzyme II) ~ W096/04392 2 1 9 5 2 0 6 ~ W5 ~

Synthesis of genes suitably employed in the present invention can be effected by means of mutually priming, long oligonucleo~ides. See, for example, Ausubel et al.
(eds.), CURRENT PROTOCOLS IN MOT~ BIO~OGY, pages 8.2.8 to 8.2.13 (Wiley Interscience 1990), and Wosnick et ~ al., Gene 60: 115 ~1987). MOLe~veI, current techniques which employ the polymerase chain reaction permit the synthesis of genes as large as 1.8 kilnh~qPq in length.
See Adang et al., Plant Molec. Blol~ 21: 1131 (1993), and Bambot et al., PCR Methods and Applications 2: 266 (1993).
Maize lines that can be t~nqformed via the present invention include, among others, inbreds that are employed in producing commercial hybrids. ~hese inbreds, both proprietary and publicly-available, span many heterotic families. The pLefelled Le~ ,t~tives within the heterotic groupings, and the relative use of entire heterotic patterns, vary with the market in question.
For example, different y~ is favored when breeding for the n~nt;n~nt~l United States, including different geographic areas of adaptation (for example, the South, the East, the West, the North and the Central Corn Belt), for Europe and for South America, as well as for other international markets.
Callus-m~ te~ - ~hn~nlogy is unsuitable for many inbreds which do not produce the required callus response or which provide callus that grows in a manner rendering the methn~nlogy unusably inefficient ("recalcitrant"
inbreds). Accordingly, such methr~nlogy has been limited to a large extent to tr~nqfnrr~t;on of a few genotypes, such as, in maize, A188, A188 x B73, H99, Pa91, FR16 and genotypes nht~ir~d via a cross involving one of these genotypes. By contrast, meristem transformation ~U~UallL
to the present invention is applicable to any line, regardless of how that line responds to callus-~ t~d transformation. Thus, even cereal lines heretofore deemed recalcitrant to transfnrr~t;nn can be transformed stably via the present invention. Illustrative of the r~ _ W096/04392 2~ 9~206 r~ l ."

maize inbred_ thus affected are PHT47, PHP02, PHV78, PHK05, PHW20, PHR62, P~N37, PHMI0, PHV37, P~J65, PH;3W8, PHK29, PHJ33, PHP60, PHN73, and PHHV4. By the same token, the present invention should be applicable to newly-developed inbreds and to new heterotic groups which are created through the combination of existing gPrmplAQm, including "exotic" ~-t~riAl brought into breeding p, ~Ld~l~S from sources in the tropics and elsewhere.
10According to a preferred ~mho~ therefore, the present invention rnnt lAtes a trAncg~nic plant that belongs to a cereal line that is recalcitrant to callus-ba8ed method transformation. Conversely, another preferred: '; t ~nr ,-qY~Y tr~nqg~n;r maize plants 15that are not produced by transformation of A188, A188 x B73, H99, Pa91 or FR16. In this context, the phrase "cereal line~ denotes a group of grAm;n~onq plants of the sub-family PoAni~R~ which display relatively little variation between individuals with respect to more than one distinctive trait, g~n~r~lly although not exclusively by virtue of several g~n~rat;ong of self-pnll;n~t;nn.
(In addition, the term "line~' here is used sufficiently broadly to include a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques.) A plant is said to ~belong" to a particular line if it (A) is a primary trangformant (To) plant regenerated irom material of that line or (B) has a pedigree comprised of a To plant of that line. In this context, the term ~pedigree~ denotes the lineage of a plant, e.g., in terms of the sexual crosses effected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
The present invention is further described in the ~ollowing ~Y~mpl~Y, which are illustrative only. In carrying out the ~ Y, a general procedure was ~ollowed ~or ~violistic transformation. According to this p.~cedu~, 60 mg of 1.0 to 1.8 ~m tungsten ~ Wo96/OJ392 21 95206 r~ ."

microprojectile3 (source: General Electric) were suspended in 2 ml of 0.1 M HNO3 and ~n; C~tP~ for twenty minutes on ice. After centrifugation at 10,000 rpm to remove HNO3, 1 ml of sterile deionized water was added, followed by a brief sonication and further centrifugation. This water rinse was repeated twice, after which the water was removed and 1 ml of 100~ EtOH
was added. The particles were resnqr~n~P~ by sonication, and the EtOH rinse was repeated. After the addition of 1 ml of sterile, ~Pi~n;7~d water and a further sonication, four aliquotes of the resulting sU~p~n~i ~n ~250 ~l each) were pipetted into separate tubes ~2 ml volume). Sterile, ~ n;7ed water (750 ~1) was added to each tube, which then could be stored at -20~C. For purposes of DNA ~e~L~Lion, 50 ~1 of the s~n;~ated tungsten microprojectile suspension were pipetted into a 1.5 ml tube, to which wa6 added 1 to 10 ~g of the foreign DNA. After mixing, 50 ~1 of 2.5 M CaCl2 golution were added and, with further mixing,20 ~l of 0.1 M spermidine also were introduced.
The resulting composition was mixed, sonicated, and then centrifuged for about ten seconds. After the supernatant was withdrawn and 2s0 ~1 of 100~ EtOH were added, the composition again was sonicated and centrifuged, and the supPnnAt~n~ was withdrawn. Finally, 30 ~1 of 100~ EtOH
solution were added to the composition, which thereafter was used, in a s ~1 aliquot per shot, with rupture disks ranging from 200-llOo p.s.i.

W096/04392 21 q5206 26 - r~l,. s~( ."

Exam~le l. TRUNSFO~M~TION WITH NON-LET~AL SRr.R~T~N
(A) Evaluation of M~ize ~istone Promoter Linked to NPTII
Ears of a proprietary maize y~ Ly~e~ designated "N10000" for purposes of this description, were harvested seven days after poll;nAtinn at the early coleoptilar stage of dev~l ~ . Harvested ears were surface-sterilized in 50~ Chlorox with Tween 20 for 20 minutes, and then rinsed three times with sterile ~p;rni7~d water.
Rernel tops were removed with a scalpel and embryos were dissected from ~n~n~_"". Sixty-seven embryos were placed axis side up, 10 embryos per plate, onto maturation medium ~MS salts, 0.1 g/L myoinositol, MS
vitamins, 0.5 mg/L zeatin, 150 g/L sucrose, and 6 g/L
Sea-Kem agarose; pH 5.6 prior to autoclaving). Embryos were inrllh~t~ overnight at 28~C in the dark before bombardment.
In these studies, embryos were transformed with plAr-;~R DP6212 and DP3953. DP6212 contain~ the 2xhistone-143 promoter, the ~irst intron of the maize ADHl gene, the nptII gene rnrC~ing neomycin pho~hoL,allsferase ~NPTII), and a 3' transcript procPr~ing region from the Proteinase Inhibitor II
(PinII) gene of potato. DP3953 rnnt~in~ the ubiquitin promoter, the first intron of the uoi gene, the gene ~nnn~;ng ~-glucuronidase (GUS), and a 3~ transcript processing region from the PinII gene. Embryos were bombarded with DP6212 and DP3953, mixed at a 1:1 ratio, at a rnnr~ntration of one 1 ~g of total D~A per tube of acid-washed tungsten particles. This conr~nt~ation, at ten times less than the standard, was optimal for yielding transformants with more uniform G~S staining patt~n~ and yet having no detrimental effect on the function of the selectable marker or the frequency of trAn~fnrr-tinn Pursuant to the above-~;~cn~sPd biolistics protocol, the particles were delivered as five 5-~l shots per tungsten tube, using a PDS-1000 Helium gun with 1100 ~ W096/04392 21 952~6 r~

p.s.i rupture disks. All embryos received one bombardment per plate.
After bombardment, embryos were --int~inPd at 28~C
in the dark for seven days on Maturation Medium. Embryos then were transferred to 272~ shoot elongation medium (MS
salts, o.l g/L myoinositol, MS vitamins, 30g/L sucrose, and 4g/L gelrite) which rnrt~;nPd 150 mg/L to~L~.~.y~in sulfate as the selection agent. Embryos were ;nrllh~teA
in the light at 28~C. At the time of transfer, embryos had Plnrg~ted cotyledons.
At two, three and four weeks after bombardment, recovered plantlets were sampled and analyzed for GUS
expression via r ~hn~nlo~y described by McCabe et al., 9io/Technology 87: 923-26 (1988). Leaf tips were placed in about 200 ~1 of histochemical stain and allowed to ;nrnh~te at 37~C overnight in the dark to m~Y;m;~e GUS
expression. Data from the first and second leaves are summarized in the following table.
T~ble 1 G~S Actlvity in Tissues of Trarsformed N10000 Plate #Plants Analvzed #GUS + GUS Sector Tv~e 1 9 6 half leaves 2 8 5 half leaves or 5pOtty 3 9 4 linear sectors 4 9 6 half leaves or spotty 7 3 linear sectors 6 11 2 complete staining Any plantlets showing positive sectors were transferred to culture tubes cnnt~;n;ng shoot elongation medium that did not contain tobL~I~ly~in. Each new leaf was P~minP~ for GUS expression. Pl~ntlet~ that were consistently positive for GUS were transferred to the greenhouse for maturation when root development was well est~hli~hPd Plants that stopped expressing GUS were observed for phenot-ype changes, i.e., necrosis, bleaching W096/04392 ~ 28 -or general lack of growth, which indicated that an escape from selection had occurred. Plants with normal phenotypes were:analyzed for NPTII protein by means of a NPTII ELISA kit available from 5'--~3', Inc., 5603 Arapahoe Road, Boulder, C0 80303 (catalog No. 5307-661-~14). Positives were transferred to the grePnhnnqe.
Transgenic plants maturing in the grppnh~nqe were sampled for GUS activity or NPTII protein in each new leaf and in tassel and ear tissues to characterize the expression pattern in each plant. Pcll;n~t;nnq were completed as selfs or as sibs. Eight to ten days after pol1in~t;nn, embryos were rescued by harvesting and surface sterilizing the ears, P~r;q;ng the embryos and placing the embryos on shoot P1nnrJ~t;nn medium for germ;n~t;n~. (This ~Lu~eduLe was not required but was preformed to arrplprnte the analysis process.) T1 leaf tis8ue was sampled for GUS historhPm;c~l assays and painted with 2% kanamycin sulfate in 0.2~ SDS buffer to verify transmission of the tr~nRgPnPq. Samples of mature leaves were harvested from the T0 transformant for Southern analysis to further characterize the transformation.
~istochemical analysis of one N10000 plant, designated "2-4," demonstrated that GUS was e~le~sed in leaves, silks tprimary ear), primary ear husks and cob, and central spike and branches of tassel. In addition, So~thPrn analysis confirmed the presence of the NPTII
structural gene in leaf tissue harvested from the mature ~ plant. Segregation of this hybridizing band correlated with NPTII-positive E~ISA results in this plant. Analysis of the central stalk also showed G~S
expression in epidermal layer of adventitious roots and in a vascular bundle of the central stalk.

~ W096io4392 2 1 9 ~ 2 ~ 6 A ~ I / V ~. . ' I /

(B) rL~fu~tion of M~ize Lines with Non-Lethal selection Experiments were performed to determine the efficacy of non-lethal selection in a variety of maize y~noLy~es. Genotype N10000 and several other proprietary genotypes, designated "P10000," "W20000," "ElOooO,"
"P~P02" and "R20000," respectively, for purposes of this description, were transformed with plasmids DP6212 and DP3953 as described above. Table 2 below enumerates data showing that the non-lethal selection method is applicable to various maize lines, based on co-transfor~-ti~n experiments where the expression of the second n~n~Plertp~ gene was used to assess stable sector frequency and slze.

Tnble 2 r,~ Ldge of Plantlets Expressing GUS Activity After ~rmin~ti~n on Nonlethal Selection Medium R~".j . NlOCOO P10000 W20000 ~10000 PE{pO2 R20000 A 65.6 --- --- --- --- ---B 23.1 --- --- --- --- --- --C 16.7 D --- --- --- - 71.1 --- ---E oo.0 --- 38.5 --- --- ---F16.4 --- --- --- 7.3 ---G12.3 --- --- --- --- 20.0 H39.1 --- --- --- --- ---I--- --- --- 20.0 --- 15.5 _ -_- 7.2 K-~~ ~~~ ~~~ 4.3 --- -~~
~ --- 5.0 --- --- --- _--M --- --- --- 5.0 --- 1.3 Avera~es 26.1 5.0 38.5 20.7 7.3 12.3 W096J04392 2 ~ 9~2~ "

(C) Evaluation of Tr~ r~ tiOZl FLe4ueuey When Meristem Reorganization I8 Effected by M~rhAnirA7 Di~ruption of Apical Dome Prior to F ~ 7 ' Ears of~gênotypes E10000 and W20000 were harvested at the early col~opt;lAr stage of dev~
and at 11 and 9 dayg, regpectively, after poll;nAtinn.
One hundred and sixty embryos were isolated and ;nrnhat~ on maturation medium, as described above.
The apical dome of several embryos was disrupted prior to b ' ~ to force the meristem to reorganize and form new meristematic areas.
~rh~ni r~l di8ruption wa8 performed by means oi miuLl ;pn1Atinn needles, ranging from 0.5 ~m to 5 ~m in diameter, which were attached to a World Precision In~LL, c M3301 mi-L, ;pll1~tor. Needle penetration of each embryo was effected in the center of the apical dome to a depth ranging from a few microns to a few hundred microns, ~p~n~;nrj on the morphology of the embryo (embryos with a larger sc~lt~ m will tolerate deeper penetration). The preferred targeted depth of penetration was between 50 ~m and 150 ~m. Embryos then were bombarded with NPTII and G~S constructs, as described above.
Embryos were m~;ntA;n~d in the dark at 28~C for seven days on Maturation Medium and then trans~erred to 272K medium rnnt~;n;nrj 150 mg/L TubL~ yuin sulfate. At time of transfer, embryos had multiple meristem formation with elongated cotyledons. Embryos were ;nrllhat~ in the light at 28~~.
At two, three and four weeks post-bombardment, first and second leaves of reoovered plantlets were analyzed for G~S expression by histo~h~m;rcl assay.
Table 3 shows the results of G~S assays, as well as observations on merigtem fnrm~t;nn.

~ W096/04392 2 1 95206 .~ . ."

Table 3 Effect of M~h~nin~7 Disruption on rr~ rn,'- ticn Efficiency and Meristem Fn~-tinn MAn1m~l~tion _ # New ~ GUS-Positive Meristems ~lan~~
150 ~M 80 89 45.9~
[17/37 analyzed]
none 60 0 37.5~
[3/8 analyzed]

These data d -LL~e that the r--hnn;ril disruption of the apical dome resulted in new meristem formation and a higher frequency of transfnrr-t;nn. In addition, mechanical disruption provided a more ~nf;nlln~lq GUS expression pattern in relation to non-~-n;plllat~d plants, which displayed a narrower, more spotty pattern of GUS expression. Thus, meristems not disrupted frequently exhibited leaf tip GUS expression only, whereas most of the meristems that were disrupted showed wide, cnnt;nllnl1q sectors in leaves.

Exam~le 2. TRANSFn~ WIT~ S~OOT MULTIPLICATION
(A) General M~fhn~nl~y Embryos at the coleoptilar stage were ;col~t~d and cultured scutellum side down on an embryo maturation medium ~10-20 embryos/plate~. Since there can be considerable s~Acnn~l and genotypic variation affecting embryo ontogeny, embryo stage rather than size or days after p~ll;n~t;n~, was monitored carefully.
Embryos typically were matured on MS-based medium cnnt~;n;ng 0.5 mg/L zeatin, 1 mg/L indoleacetic acid, and elevated sugar levels which serve as an osmoticum. The embryos were cultured for a period ranging from 0-48 hours post-isolation, with 12-24 hours being optimal. Meristems then were bombarded w096l0439~ ~l 9 5206 32 - r~ ."

with genes conferring kanamycin or streptomycin resistance, along with other nonselected genes, such as agronomic or visual marker genes.
After bombardment, the embryos were cultured in the dark to promote gprmin~t;nn. After one to two weeks, the embryos were moved to a germ;n~t;nn medium, such as hormone-~ree or low-hormone MS medium. The germinated plantlets generally had a ~,_ll;ng at the junction between the mesocotyl and epicotyl. Thi8 swelling occurred in the region rrnt~in;nrJ the developing shoot meristem.
Two to three ~;ll; tPr sections ;~rl nA; ng the meristem were excised and cultured on a shoot proliferation medium which rnnt~;n~d the appropriate l30L -~ and a 5~1 ~ct;nn agent. The sections were regularly trimmed of ~lnnrJate~ leaves and transferred to fresh medium every 10 to 14 days. Cultured meristems were ;nrnhat~ at 28~C in the dark. After three to nine weeks, the proliferating meristems were transferred to an ;llll~;n~t~ culture room.
Transformed sectors were i~Pnr;f;rd one to two weeks after culture in the light, ba~ed on their green phenotype, i.e., nontransformed ti88ue l~ ;n~
bleached upon selection. In general, plants were regenerated by lowering the hormone rnnr~ntration~
although in some ye~l~Ly~es cytokinin rnnr~n~rations were increased to promote plant regeneration. Since regenerated plants ~ r;--~ have difficulty rooting, rooting was promoted by culture on SH medium with 30 l mg/l NAA, or by nicking the base of the stem and dipping the shoot8 in a l mg/ml NAA solution.
(B) NPTII ~ rv,.l~tiOn of ~oney l~7d Pearl One hundred eighty coleoptilar-stage maize embryos of the ~oney and Pearl variety were harvested nine days after pnll;n~t;rn. The srut~ of the ;Rol~t~ embryos averaged 0.48 mm in length. These embryos were placed on embryo Maturation Medium (10 ~ W09~04392 2 l 9 ~ ~ 0 6 . ~ . . . "

embryos per plate) and cultured overnight in the dark at 28~C.
Sixteen plates of the embryos were bombarded twice according eo the above-described method with plasmid DP551, using 1.8 ~m tungsten particles at a DNA
ronr~ntration of 10 ~g DNA/tube of tungsten. Plasmid DPS51 rnnt~;nc ADH intron 1, GUS gene, and nos terminator, as well as ADH intron l, NPTII gene, PlnII
terminator. Both GUS and NPTII genes are regulated by 35S CaMV Sr~l~nr~q Plates cont~;n;nr; these embryos were cultured and matured in the dark at 28~C. Eight days later, a few of the embryos were placed in X-Gluc h;~torhrm;cAl stain. All embryos rnntA;n~d intense blue staining, indicating GUS activity.
Most of the embryos had grrm;nAtr~ n;nrt~n days after particle bombardment. At this time, the region cnrtAin;ng the meristem and leaf primordia was excised as described above and cultured on agar Snl; ~; f;ed MS
medium with 2 mg/L BAP and 50 mg/L kanamycin. Leaf tissue was stained and chimeric blue staining sectors were observed in eight of the sixteen plates. The region cnntA;n;ng the meristem was trimmed of elongated leaves and transferred to fresh medium every 10 to 14 days. Twenty six days after ~ . t, the level of :
2S kanamycin was increased to 100 mg/L. Proliferating meristems were transferred to the light a week later.
These experiments produced three ;n~rr~n~nt transformation events. Two of the transformants have been characterized by PCR, GUS staining, NPTII ELISA
assay and South~rn analysis. One of these events exhibited strong GUS activity and high levels of NPTII
protein. The T1 and T2 generations from this event were used for subse~uent analysis. Progeny displayed a co-segregating, l-to-1 ratio after outcrossing, based on both GUS activity and NPTII ELISA results (see Table 4) consistent with M~n~l;An inheritance of the integrated genes. Integration and segregation of the NPTII gene, which correlated with positive NPTII ELISA

W096/04392 2~95206 34 ~

results, were -demonstrated through Southern analysis of ~rl plants.

~C) aadA Trarsfo~zation of Honey and Pearl Coleoptilar ssage Honey and Pearl embryos were isolated and cultured on 288B medium (MS medium with 0.5 mg/l zeatin. 1mg/l IAA, 0.25M sorbitol, and 4~
sucrose snl;~;fied with 3 g/l gelrite). Eight plates with ten embryos per plate were bombarded once, ag described above. Each particle preparation (enough for six shots~ employed a c 'in~d total of lO ~g of DNA
(5 ~g DP4790 + 5 ~g DP460 or DP3536). Plates 1 to 4 were 1 ' d~d with plasmid DP4790, which ~nnt~inq a 35S CaMV promoter, omega', aadA and ocs terminator (provided by Dr. Jonat~an Jones, John Innes Institute), and with plasmid DP460, which rnnt~;nc a 35S CaMV
promoter, AD~ intron, GUS gene, and nos t~rmin~tnr.
Plates 5 to 8 were l ' -ded with pl~ C DP4790 and DP3536. The latter plasmid contains a cab promoter, AD~ intron 6, GUS gene, and ocs t~rmin~tnr. All embryos were grown and g~rmin~t~d as described in part (B) of this example, supra. After g~r~in~tion, the regions nnnt~ining the meristems were cultured on agar-solidified MS medium cnnt~;n;ng 2 mg/L ~3AP and 100 mg/~
streptomycin sulfate.
After cultured meristems were moved to an illuminated culture room, a green sector was observed on a proliferating meristem on plate 6. All other cultured meristems were white due to ssreptomycin hl~nh;ns. GUS staining at this time revealed a mix of sectored and non-sectored blue staining leaves.
About seven weeks a~ter bombardment, sorting out was observed in the leaves from the transformation event on plate six. Some leaves were non-sectored GUS+ while others were still mericlinal. Transformation was confirmed using PC~, GUS staining and qonth~rn analysis.

~ W096/04392 2 1 9 52 ~6 ~ "

(D) T~du~ru~ dtion of sn elite i~bred Eight days after pn~l;n~tion. coleoptilar stage embryos of an elite inbred, designated "B30000" for purposes of this description, were isolated and cultured on 288L medium in fifteen plateg crnt~lning ~wenty embryos per plate. Twelve plates were bombarded, using standard protocols. Briefly, particle bombardment was performed with six 3hots using 650 psi rupture disks and 1 ~m tungsten particles, which were coated with plA~ c DP5397 (proprietary agronomic gene) and DP5606 (Ubi promoter/Ubi-intron/ NPTII/pin II
terminator linked to cab promoter/AD~ intron 6/GUS/ocs termlnator~ at a rnnrrntration of 5 ~g DNA/particle preparation tube for each plasmid.
lS Plasmid DP5397 is a proprietary agronomic plasmid which ront~in~ a ~t gene, while plasmid DP5606 crn~in~
the Ubi promoter, Ubi intron, NPTII gene, and PinII
terminator, which is linked to a cab promoter, AD~
intron 6, GUS gene, and ocs tr~min~tr~
After bombardment the meristems were cultured on agar solidified MS medium rnnt~ining 2 mg/L BAP, 0.25 mg/L, 2,4-dichluLu~henu~y acetic acid and 3~
sucrose. Five weeks after bombardment, meristems were placed on kanamycin selection (100 mg/L). To avoid irreversible bleaching of the meristems, this tissue was cycled between selective and non-selective media.
Five months after bombardment, a large green sector was removed from a b3 ~rh~ shoo~ culture. Three small leaves were removed from the sector and stained with X-Gluc. The leaves were found to express GUS
activity in non-epidermal cells.
A single plant was regenerated from this series of experiments. The plant produced copious amounts of pollen and several ears. The pollen was found to be segregating for GUS expression, which was surprising since this gene was under the control of the cab promoter. All leaves of this plant exhibited strong, non-sectored GUS activity. The tassel glumes also were W096l0~92 2~ 9~ a~ r.l . " ~

positive ~or GUS activity. Samples of leaf tissue from ~his T~ plant cnnr~inFd the NPT-II and Bt proteins (as verified by ~heir respeclive ELISA's~ and exhibited s~rong GUS activity ~fluorometric analysis). The ~S
histochemical assay verified tr~nFm;csion to progeny in 42 of 106 seedlings sampled to date, which is consistent with ~n~ n inheritance.
Exam~le 3. TRANS~7M~ REGI~EN E~PLOYIXG rN~AT~RE
EAR AND m~'ASSEL M~ L~
(A) ~ Rinn of immature ears ~ rom plants harvested seven to nine weeks after r1~nting, leaveg were removed aseptically, one at a time, and the ears were exposed. The ears were dissected out of the husks under a ~;qq~ting microscope. Longitudinal bisection of the ears increased the response and exposed the meristems more fully to bombardment.
~B) StagLng and 8~7~Ctinn of l~uJive ~r7~nt~
The size of the whole excised ear and the dev~ 1 stage of the meristems were found to be reliable indicators of proper timing of harvest.
Smaller ears are less dev~ lly ~ot~rmin~d and more re8ponsive to hormonal stimuli, but fewer meristems survive resulting in fewer targets for transformation. Although smaller inflorescences have been used, two millimeters was used as the practical lower size limit for transformation experiments. The upper limit for selection of responsive targets was determined by meristem stage; developmental plasticity decreased dramatically once the glumes began to be obvious and approached the sides of the meristematic dome.
(CJ Tnitial culture mecium Various media have been used, and inbreds respond differently to these variations. A preferred medium used in the initial stage of floral meristem culture (used for various genotypes) consisted of Murashige and Skoog salts, MS vitamins, 0.1 mg/l 2,4-D, 0.5 gm/1 6-~ w0~6/04392 2 1 9 5 2 0 6 r~

BAP, l-proline at 12.2 ~M, 8~ sucrose, and silver nitrate ac 30 mg/l. A preferred gelling agent is GELRITE (product of Merck and Co, Inc./Kelco division, Rahway, NJ) at 3.5 g/l.
.; (D) ~ ~
Imma~ure ear ~rlcnts were bombarded using 650 psi rupture disks and a stainless steel screen (100 um mesh size) sllcrpn~p~ approximately 0.5 to 1.0 cm above the tissue. DNA precipitation and other b '--d~lldllL
parameters were as described in Example 1.
(~J S-lh~ectinn, subculture and ~7~rfinn M~intPn~nre of rapid growth and survival of individual meristems was achieved by subsecting the ears four to SlX days after icol~i nn, into pieces with four to eight meristems each. These pieces were cultured onto shoot multiplication medium, which has the same basal composition as the initial culture medium (above) but with 1 mg/l BAP and 3~ sucrose.
Meristem tissue was subcultured rPre~te~ly, at two week intervals on the shoot multiplication medium.
Tncllhat i n~ Of } ' ded ear meristems in X-gluc consistently resulted in high frequencies of transient GUS express1on two days after bombardment. Stable sectors in leaves produced by multiple shoot clumps 2s were found to express 5US one month after bombardment, At this stage, leaves were apprn~i~tply 1 to 2 cm in length, and transformed sectors were found that extended more than half the length of the leaf. In addition, one meristem sacrificed at this stage expressed high levels of GUS in a histochemical assay.
One month of shoot multiplication was followed by one month of selection using 100 mg/l streptomycin.
After this treatment, all material was subcultured once more onto medium without the selective agent, and were additionally moved into the light. Leaves and shoots in non-selected cultures quickly turned green. ~eaves in selected cultures L~ in~d hle~hPd (white).

_ _ _ _ _ _ _ , ... . , .. . .. _ _ . _ _ W096l04392 2 1 9 5 2 0 6 (F) Plant regeneration Putatively transformed shooss clumps were sransferred ~o medium lacking plant growth regulators.
Varying degrees of leaf dev~N ~ occurred on 1 mg/l 3AP, and shoots soon formed and elongated in the absence-of hormones.
(G) Rooting Rooting at high frequency was effected via several days of exposure to MS- or SH-based media with 1-5 mg/l NAA.
EY~nle 4. TRA~NsF~TJ~TInN OF EARLY ~Aur~-~A~u~ ~ID
~A~SU, LATE ~"~ YU, TT~'Yll~ T- AND
EARLY COLEOPTI~AR-STAGE E~3RYOS
Imma~ure embryos at the mid proembryo, late ~u ' yu, transitional and early coleoptilar stage were harvested and cultured on culture medium 610A, c~nt~;n;ng high ~ tions of cytokinin and osmoticum. The 610A culture medium comprised MS 6alts, MS vitamins, 100 mg/~ myo-inositol, 0.4 mg/L ~h;~;n~-HCl, 1 mg/L zeatin riboside, 0.1 mg/L ~AF, 60 g/L
sucrose, 400 mg/L asparagine, and 7 g/L Hazelton TC
agar. After one day of recovery, the embryos were bombarded with DNA, by means of the particle gun as described above, and punctured in the center of the 2S area of where the apical meristem will develop with a 0.5~m mi.~, n;pnlAti~n needle.
Embryos were allowed to ma~ure for 7 days in the dark and then transferred to a hormone-free medium ~nt~ining 1 mg/l bi~l~phm5 Following another 7 days of culture on hormone-free medium in the dark, the embryos were transferred to germination medium, and cultured in the light for mmntinll~d germinatjmn~ As leaves developed, plant phenotype was observed and samples were taken to check for sector formation by histochemical assay (GUS) as described above.

Healthy plants with normal phenotype and/or reporter gene activity were transferred to the ~ W096/04392 21 95206 P~l/t,~ . ."

greenhouse for maturation The data shown in Table 4, which were generated v1a the above-discussea protocol, demonstrate f or ln~red N10 0 0 0 the sector f requency obtained across several similar experiments, using embryos staged at m1d proembryo, late proembryo, transltional r and early coleoptilar .
Tal ~le 4 Sector FL eyueu~,y Obtai~2ed Wi th Nid PL~, Later PL~ ' YtJ, TraJ2sitional and Early Coleoptilar Stage En!bryos Embryost.8c N Tr n.~5erles GUS frec,uer~cy GUS p-tten~of Sec~or .,tprcssion P~er~en~
Euty 3D B~R/GUS 14.2% l~f tps~ ftlel l~vet I ~d 2 Coleomil r nf 1-3 r~s Tr nstttor~ 250 B~RIGUS U.5% s~uc ~d Setton rD t ~
L~r ~ftf l, L-tcProembryo 200 B~IUGUS 34% S~ddleuld Sr~tonsrltt~
tr L~f I or ~f 5 Mid 110 BAR/GUS 3% Lu,Qr~d Sectonsttttlt Prcembtyo t~,hcl. I~f Luf I

The WS f requency observed af ter targeting mid 20 ~Lo_..~Ly- s rPflect~d, at the time these data were collected, a relatively poor survivial rate after bombardment and selection But the addition of 1 mg/l zeatin to medium 610A, an increasing of the agar rrrrPntration (12 g/l), and the use of lower rupture 25 disc pressure (200 p.s.i.) during particle delivery increases survival of mid proembryos after isolation and DNA delivery.

W096l04392 6 40 _ ~xam~le s. ~K~ . TRANSF~M~TTnN -- DIRECT
~MTN~TIr~- APP~OAC~
Genotype N10000 plants were pnl 1; n~tP~ and, eight days later, embryos were placed into culture. The harvested embryos thus were late-pLue-l~Lyu stage.
More speclfically r embryos were cultured at day 0, axis up, onto modified 610A medium, cnnt~1n;ng 150 g/l sucrose, 1 mg/l zatin. and 12 g/l agar, and incubated at 28~C overnight in the dark. At day 1, ~ollowing the overnight inrnh~tinn, the apical meristems of all embryos were disrupted in the center of the apical dome using a 0.5~m Femtotip mi~L~ ;rnl ~tiOn needle. A11 embryos were LeLuL..ed ~or an overnight incubation at 28C in the dark. At day 2 ~ was effected lS with the PDS-1000 ~elium particle gun, one shot per plate, using 650PSI rupture disks. DNA employed in this regard was DP3528+DP3953 [2x35S::BAR+UBI::G~S] at 1 ~g/tube of 1-~m tungsten. At day 2 all embryos were ~-;nt~;n~ in the dark at 28~C for 7 days on 610A
medium to allow meristem maturation to occur. At day 7 (after 7 days on 610A), embryos were transferred to 612 medium rnnt~;n;ng MS salts and vitamins, 0.001 mg/l kinetin, 0.1 mg/l adenine sulfate, 20 g/l sucrose, 6 g/l agar and 0.5 mg~L h;~l~rhn5, for grrmin~inn and selection. At day 14 embryos were kept in the dark at 28~C ~or 7 days before transfer to the light for further g~rm;n~t;nn. On days 21-49 GUS hi5torhrmir~l assays on developing leaves were cnn~nrt~d~ and on day 35 growing plantlets were transferred to tubes r~nr~;n;ng MS medium with no l1OLI nS and 5 mg/~
h;~1~phnq. On day 56 plant 6-l (SID 180741) and plant 2-7 (SID 180742) were trans~erred to the gre~nhnnqe~
The total number of embryos cultured and bombarded was 48, of which 37 developed normally. The number o~
embryos that grew beyond leaf 1 was 17, with four plants showing GU3 expression. Two plantq survived 5 mg/L-h;~l~rhnq selection with normal root ~ W096/04392 2 1 9 5206 ~ "

developmens, and were cransferred to the gree~house for maturation.
SID 1ao741 and 180742 both showed GUS expression at the time of greenhouse trangfer and had normal leaf and root dev~l ~ -, whereas all other plants died. SID 180742 showed GUS expression in leaves 1-8 only.
Table 5 Sector Placement by GUS histocAemical assay of SID780741 Structure GUS T:~h~LlO~Vue Leaf 1 negative Leaf 2 leaf tip Leaf 3 leaf tip Leaf 4 saddle sector: margin and midrib Leaf 5 midrib sector Leaf 6 saddle sector margin and midrib Leaf 7 midrib sector Leaf 8 sadd'e sector margin and midrib Lea.~ 9 midr~b sector Lea-~ lO sadd_e sector margin and midrib Lea-~' 11 mi~r-b sector Lea.''12 saddle sector margin and midrib Leaf 13 midrib sector Lea~ 14 ha:f leaf Leaf 15 ha_f leaf Leaf 16 ha_f leaf Leaf 17 ha_f leaf Leaf 18 half leaf Leaf lg half leaf Leaf 20 entire leaf Leaf 21 entire leaf Tassel To date: central stalk shows pollen staining; 5 tassel branches also positive Anthers Endothecium and epidermis positive Anther glumes Epidermis posisive Leaves were painted with l~ Ignite in lanolin paste at the V6-V8 stage of dev~ . SID 18074 showed resistance to Ignite in the sectored ~GUS
expressing) areas only. SID 180742 showed no resistance to Ignite. PCR analyses were done on sampled leaves and confirmed presence of both GUS and sAR genes in SID 180741 and GUS genes in SID 180742.

..... , _ W096l04392 2 1 9 5 ? 0 6 P~

One of the first ma~or differences observed after moving towards an earlier develu~ ns~l stage, i.e..
when targeting late proembryos, was the pro~ tinn of saddle sectors (see Poethig (1986), supra, for a description). Extant information ronrrrn;nr meristem orgAn;7~t;nn suggested to the present inventors that this might lead to germline transmission through the tassel. Saddle sectors extend from the leaf primordium up through the central portion of the apical dome and back into another portion of the leaf primordium. The ~trnr;~n Of tr~nr~n;c gectors into the central portion of the meristem greatly increa5es the probability of the sector contributing to the tassel and, ultimately, to pollen_ Previous research on maize anatomy and c7onal analysis has shown that maize cnnt~;nr an organized, layered apical meristem beginning at the transitional stage of devP7~ . See RAn~nlph~ ~. Agric. ~es. 53:
881-916 (1936), and Poethig (1986), supra.
Furthr- ~, a paper by Dawe and Ereeling, Developmental Biol. 142: 233-45 (1990~, regarding cell 1;r~Ag~C in the male flower of maize, ;n~;r~tP~ that the L1 and L2 layers of the apical meristem give rise to the two layers of the anther wall. Only the inner layer is derived from the same cell lineage as the male germ cells, the L2. It also was found that events that occurred (by irrA~iAtirn) before the organization of the shoot apical meristem rnntA;nrd sectors in both layers of the anther wall with inheritance through the pollen. Events that occurred after the transitional stage of devrl~ ~ were limited to only one cell lineage with inhr~;tAnro only when sectors occurred in the L2 layer Transformant 180741 was bombarded by the particle gun at the late ~LU~..~Lyu stage of devrl~ L ' I before meristem layer organization occurs. It rnnt~;n~d a saddle sector which, by definition, is a sector which traverses the apical dome and bisects the ~eristem, in ~ W096/04392 43 _ 21 9~205 a region of the meristem that will later develop into ~he ~assel (see Poethig (1986), supra). It also was wounded by a micro-manipulatlon needle, to encourage meristem reorganization, and exposed to h;~l ~phn5 as r; the selective agent. GUS his~ochemical data showed expression in both layers of the leaves, in the anther wall, and in about 50~ of the pollen from the central stalk.
Exam~le 6. S~RTTT~TT~ OF T~TC~T~TC SECTORS BY
NEANS OF TTTTTrTTl'~
As noted above, tillering of transformed plants is an alternative to shoot mult;plir~t;nn for 5t~h;l;7~t;nn of ~ranRg~n;c sectors. Accordingly, elite lines may be induced to tiller, pursuant to the present invention, thereby st~h;l; 7; ng transgenic sectors.
In this example, tillering was induced in control plants using the method described by De Wolff, ~uphytica 20: 524-26 (1971). A triangular ; nr; c; on was made with a number 11 scalpel blade at the approximate height of, or slightly above, the shoot apex of two week-old s~e~l;ngq. The ;nr; ~;n~ was made perpendicular to the plane of the leaves in order to avoid damage to the mldribs. The shoot apex was removed from P10000, PHP02, G30000 and E10000 sP~l; ngS , Each of these genotypes represents inbreds from sign;f; r~ntly different heterotic families.
Untreated plants of the same y~--uLy~e were used as controls. If the ;nr;cinn was too far above the apex the ~,uu~d~ was repeated just below the initial ; nr; qi r,n .
he wounded plants and untreated controls were --;ntain~d in 24 hour cnnt;nun~q light (greenhouse during the day, growth chamber by night) for two weeks.
A replicate treatment was grown under light/dark conditions.

W096/04392 2 1 952O6 ~ "

Significant tillerlng was obEerved in the plants ~rom which apices were removed. The in~luence of rrntinl1rllq light on tillering frequency, relative to normal light/dark conditions, was variable and may depend on genotype. The untreated controls did not tiller.
The hole made by the ;nricinn could have been plugged with lanolin and phytr~ ~c, such as TIBA
(1 mg/L) or 3AP (10 mg/L), to increase tillering frequency. In tke alternative, or in addition to the phy~f~ ~, selective agents such as kanamycin could have been added to the ;nr;qi~n to identify and select transgenic sectors.

Claims (16)

What Is Claimed Is:

1. A method for producing transgenic cereal plants that will transmit introduced DNA to progeny, comprising the steps of (A) introducing foreign DNA into target cells selected from the group consisting of (i) cells of a meristem that is not enclosed by sheathing leaves and (ii) cells fated to contribute to said meristem; then (B) inducing reorganization of said meristem to increase transgenic sector size, whereby the likelihood that a transgenic sector will contribute to germline transmission is increased, wherein said reorganization is effected through at least one manipulation selected from the group consisting of (i) imposition of a nonlethal selective pressure on said meristems, (ii) mechanically-induced meristem reorganization, and (iii) hormonally-induced shoot multiplication; and thereafter (C) exposing said meristem to conditions under which it differentiates to form a plantlet, wherein said plantlet contains said transgenic sector or is homogeneously transformed by said foreign DNA, such that said plantlet can be grown into a transformed cereal plant that will transmit said foreign DNA to progeny.

2. A method according to claim 1, wherein step (A) is effected at early proembryo, mid proembryo, late proembryo, transitional or early coleoptilar stage.

3. A method according to claim 1, wherein said foreign DNA
is introduced into a plurality of meristems, and at least some of said meristems differentiate in step (C) to form a plurality of plantlets.

4. A method according to claim 1, wherein said conditions in step (C) are such that said meristems undergo maturation and plant differentiation to form shoot apices, and wherein said method further comprises effecting reorganization of meristem tissue in said shoot apices to enlarge transformed sectors or to produce periclinal L2 chimeras.

5. A method according to claim 4, wherein said effecting of reorganization comprises exposing said shoot apices to nonlethal selection pressure such that transformed cells have a competitive growth advantage over nontransformed cells in said shoot apices, and the proportion of transformed cells in said shoot apices is increased.

6. A method according to claim 1, where step (A) comprises biolistic bombardment of an embryo at a developmental stage no later than the coleoptilar stage.

7. A method according to claim 1, wherein said effecting of reorganization comprises culturing said shoot apices under conditions such that multiple shoots proliferate therefrom through organogenesis.

8. A method according to claim 1, further comprising a step before step (C) of wounding said apical dome selectively.

9. A method according to claim 8, wherein said wounding is carried out before step (A).

10. A method according to claim 1, wherein step (A) comprises biolistic bombardment of meristems on immature ear or tassel explants.

11. A method according to claim 1, wherein said target cells are maize, sorghum, wheat, barley, oat or rice target cells.

12. A method according to claim 12, wherein said target cells are maize target cells.

13. A method according to claim 5, further comprising the steps of (i) dissecting out of an axillary bud from above the base of a leaf of a plantlet when a chimeric sector is observed in a substantial portion of said leaf, and then (ii) germinating said axillary bud into a whole plant or subjecting said axillary bud to shoot multiplication.

14. A method for producing transgenic cereal plants that will transmit introduced DNA to progeny, comprising the steps of (A) introducing foreign DNA into target cells selected from the group consisting of (i) cells of a meristem that is not enclosed by sheathing leaves and (ii) cells fated to contribute to said meristem; then (B) inducing reorganization of said meristem to increase transgenic sector size, whereby the likelihood that a transgenic sector will contribute to germline transmission is increased; and thereafter (C) exposing said meristem to conditions under which it differentiates to form a plantlet, wherein said plantlet contains said transgenic sector or is homogeneously transformed by said foreign DNA;
(D) removing the shoot apex from seedlings of said plantlet, whereby a wound is produced;
(E) growing said plantlets to induce formation of a plurality of tillers; and (F) selecting a transgenic tiller from said plurality, such that said transgenic tiller can be grown into a transformed cereal plant that will transmit said foreign DNA to progeny.

15. A method according to claim 14, wherein phytohormones that increase tillering are introduced, subsequent to step (i), into said wound.

16. A method according to claim 14, wherein a selective agent is introduced after step (i) into the wound, to identify and select transgenic sectors.