[PDF] Fertile Transgenic Maize

16 Pages·2002·5.05 MB·English

The Plant Cell, Vol. 2, 603-618, July 1990 O 1990 American Society of Plant Physiologists
Transformation of Maize Cells and Regeneration of
Fertile Transgenic Plants
William J. Gordon-Kamm, T. Michael Spencer, Mary Lou Mangano, Thomas R. Adams,
Richard J. Daines, William G. Start, James V. O’Brien, Sheryl A. Chambers, Whitney R. Adams, Jr.,
Nancy G. Willetts, Thomas 6. Rice, Catherine J. Mackey,’ Roger W. Krueger, Albert P. Kausch, and
Peggy G. Lemaux
Discovery Research, DEKALB Plant Genetics, Eastern Point Road, Groton, Connecticut 06340
A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from
embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile
bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos.
lntegration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were
confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R,) were regenerated, and of 53
progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of
herbicide to leaves of bar-transformed Ro and R1 plants resulted in no necrosis, confirming functional activity of
PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one
encoding PAT and one containing the nonselected gene encoding B-glucuronidase. Ro plants regenerated from co-
transformed callus expressed both genes. These results describe and confirm the development of a system for
introduction of DNA into maize.
INTRODUCTION
Over the past decade, the value of introducing foreign various dicotyledonous crop species (Comai et al., 1985;
genes into plants has been well documented. In dicotyle- De Block et al., 1987; Haughn et al., 1988). Extending
donous species, genetic transformation has been used to transformation techniques to monocotyledonous plants, in
study plant processes such as the action of transposable particular the Gramineae, is of great importance for applied
genetic elements (Finnegan et al., 1989; Spena et ai., as well as basic research because members of this family
1989), the impact of manipulating metabolic pathways (e.g., maize, rice, and wheat) constitute a substantial por-
(Last and Gray, 1990; van der Krol et al., 1990), tissue- tion of the world’s food crop.
specific gene expression (Stockhaus et ai., 1987; Benfey Despite prolonged and substantial effort by many labo-
and Chua, 1989), as well as other aspects of gene regu- ratories, development of genetic transformation tech-
lation (Matzke et al., 1989; Rocha-Sosa et al., 1989). The niques for the major cereal crops has been slow. The
application of genetic transformation techniques to answer recalcitrance of maize and most other monocot species to
basic research questions will increase in frequency and Agrobacteriurn-mediated transformation has led to the de-
sophistication as methods continue to improve. In addition, velopment of direct DNA delivery methods. Electroporation
introduction of potentially useful agronomic traits such as of maize protoplasts (Fromm et al., 1986) has produced
insect, viral, and herbicide resistance has been accom- transgenic plants (Rhodes et al., 1988); however, these
plished in a number of dicotyledonous crop species (Gas- plants were not fertile. Despite published success in re-
ser and Fraley, 1989). For example, transgenic tobacco generating fertile maize plants from protoplasts (Prioli and
and tomato producing modified Bacillus thuringiensis toxin Sondahl, 1989; Shillito et al., 1989), regeneration of fertile,
display enhanced resistance to lepidopteran pests (Fisch- transgenic maize plants has not been reported. Presently,
hoff et ai., 1987; Vaeck et ai., 1987). Tomato transformed rice is the only cereal grain for which a protoplast-based
with the tobacco mosaic virus coat protein gene proved transformation system has been developed that can pro-
resistant to viral infection in both greenhouse and field duce fertile, transgenic plants (Shimamoto et al., 1989).
tests (Nelson et al., 1988). Genes conferring resistance to The problems surrounding cereal transformation have
a number of herbicides have also been introduced into been difficult to elucidate. A number of potential impedi-
ments to successful monocot transformation have been
’
To whom correspondences hould be addressed. suggested (Potrykus, 1989). For cell cultures, these in-
604 The Plant Cell
clude the lack of DNA delivery and selection methods that A
are compatible with regeneration of fertile plants.
Recently, we have been able to overcome these obsta-
cles by bombarding embryogenic maize suspension cells
H B S S E
with tungsten particles coated with the selectable marker
gene bar. The Streptomyces hygroscopicus bar gene en-
35s
codes phosphinothricin acetyltransferase (PAT), an en-
zyme that inactivates the herbicidal compound phosphin-
othricin (PPT) by acetylation (Murakami et ai., 1986;
Thompson et ai., 1987). PPT inhibits glutamine synthetase pDPG165
(Tachibana et ai., 1986a), causing rapid accumulation of
ammonia and cell death (Tachibana et ai., 1986b). This
gene has been used in Agrobacterium-mediated transfor-
mation of tobacco, tomato, potato (De Block et ai., 1987),
and rape (De Block et ai., 1989). The resulting transgenic
plants were protected against the broad-spectrum herbi-
cides glufosinate (the ammonium salt of PPT) and biala-
phos (a compound containing PPT). Previously, we used
bar as a selectable marker for the identification of nonem-
L L
bryogenic Black Mexican Sweet (BMS) maize cells trans-
formed by microprojectile bombardment (Spencer et al.,
1990). Here we describe bialaphos selection of stable
pDPG208
transformants from various embryogenic maize suspen-
sion cultures of A1 88 x 873 and A1 88 x 884, including
cryopreserved suspension cultures. Fertile transgenic 1 -___I _.___..
plants were regenerated from transformed embryogenic 0.5 kb
callus lines, and transgenic Ro and R, plants produced
Figure 1. Schematic Representation of Plasmids Used in Bom-
sufficient functionally active PAT to protect leaf tissue from
bardment Experiments.
the herbicidal action of PPT.
(A) pDPGl65.
(B) pDPG208.
E, EcoRI; H, Hindlll; B, BamHI; S, Smal
RESULTS
Bombardment and Selection in the bombardment procedure, such as the use of screens
to disperse tungsten particles and modifications in the
Cells from embryogenic maize suspension cultures were precipitation protocol.
bombarded with the bar-containing plasmid pDPGl65 After bombardment, cells on filters were resuspended in
alone or in combination with a plasmid encoding p-glucu- nonselective liquid medium, cultured for 1 to 2 weeks, and
ronidase (GUS), pDPG208. Schematic maps of these plas- transferred to filters overlaying solid medium containing 1
mids are shown in Figure 1. In selection experiments in or 3 mg/L bialaphos. The degree of inhibition of tissue
which a GUS plasmid was included, two of the filters with growth during selection was dependent upon the distri-
bombarded cells were histochemically stained to deter- bution of the cells on the filter and on the concentration of
mine the number of foci (clusters of transiently expressing bialaphos used. At the density plated [O5m L packed Cell
cells) with GUS activity 48 hr post-bombardment. The total volume (PCV)/filter], the growth of the cells cultured on 1
number of foci was at least 1000 per filter in ali experi- mg/L bialaphos was only partially inhibited (30% to 50%
ments. In two separate experiments designed to quantitate of nonselected growth as determined by visual observa-
transiently expressing cells (using an A1 88 x B73 suspen- tion). After 3 to 4 weeks, much of this tissue was trans-
sion culture designated SC82), the mean number and ferred as discrete clumps (-5 mm in diameter) to identical
standard deviation of GUS-staining foci per filter was 1472 medium. On medium containing 3 mg/L bialaphos, the
? 211 and 2930 f 390 (n = 3 and 4, respectively). The growth of cells on the original selection filter was severely
number of cells in a focus ranged from one to 10 and inhibited (-1 0% of nonselected growth) and selection was
averaged two to three. In these experiments the numbers continued without removing the tissue from the original
of stained foci were at least fivefold greater than in previous filter. Using either selection protocol (1 or 3 mg/L biala-
experiments using BMS suspension cells (Spencer et ai., phos), resistant cell colonies emerged on the selection
1990). This difference is in part attributed to modifications plates approximately 6 to 7 weeks after bombardment, as
Fertile Transgenic Maize 605
shown niF iguo Nrr e.Ae2s istant colonies were recovered
from selection plates containing nonbombarded tissue.
Bialaphos-resistant callus lines were maintain-exe ddna
pann dseoedl ection med dmniuuaf mctoh h is tsisaswue
embryogs sehann oi. cFOwB ,iigbn u2sree rved grofwoth
selected callus lines maintained on bialaphos was similar
to that of control tissue on nonselective medium.
TAFAciPgtiuv n .ir3Eteiy mbryogenic SC82 Transforma1nEts
to E11 and a Nonselected Control (EO).
Analysis of Transformants
Twenty-five micrograms of protein extract were loaded per lane.
The suspension culture usen inid itial experiments, SC82, a B sMi S3-1bBar transformant (see Spenct eear l., 1990).
was derived from embryogenic typeI I callusf o A188x
B73. Bialaphos-resistant callus lines selected from SC82
were analyzed for activity of the bar gene product PAT by
thin-layer chromatography. Protein extracts from 11 callus
oEt1 11) EcliT no(aenAcst s atisPivanhinetoyd w,i n
F danigacu t,ri3ve ity levels varied approximately 10-fold
among the isolates. Isolates E1 to E11 were further ana-
lyzed for the presence of bar. Chromosomal DNA from E1
to E11 was digested with EcoRI and Hindlll to release the
1.9 rea-kxbbp ression unit (35S promoter-toar-T' r37e nd,
Figure 1A). Ge1 nbA 1foiraN molllDaamipc hos-resistant
isolates contained bar-hybridizing sequences,s a shownni
Figure 4. The hybridization in all isolates to a fragment
migratingo ctso leinguthadtm ley-b l ayragmer btkh a2n
inating pUC19 sequences containedn i thi rsab probe
preparationo ns; uch hybridization occurredn is ubsequent
experiments using the same genomic DNA and a different
preparation of the bar probe. Hybridization to a 1.9-kb
fragmenn ti eig1 1h eihts fto olates indicated that these
isolates contained intact copies of the 1.9-kb bar expres-
sion unit. The estimated copy numbers of the intact unit
ranged from one or two (E1, E7, E8, E10, and E11) to
B approximately0 2 (E3,d na ,4E E6). Hybridization witheht
bar proben i isola5 tEo ednac s2Ec urred onla y ost ingle,
higher molecular weight fragment (~3 kb). To establish
that the PAT structural gene was intact in these isolates,
g seadwnig oAemNsDitce d with Smal, which releaseas
559-bp fragment containing the PAT structural gene (Fig-
ure 1Ad nh)a,y bridizeo dt3 2P-labeled bar. This analysis
confirmed the presence of a single intact copy of bar (data
t sohonwn).
The hybridization patterns of some of the SC82 isolates
dna ,4E ,3E dna ;8E dnwa e7Ere ; 5iEd ednnat i2cEa(l
E6); therefore,s i ti probable that some isolateston did
arise from independent transformation events but repre-
sent transformants that were separated during selection.
Seven hybridization patterns were unique, likely repre-
senting seven independent single-cell transformation
Figure. 2 Bialaphos Selectionf o Embryogenic Callus. events. The patterns and intensities of bar hybridization
for the seven transformants were unchanged during 4
(A) SC82 bialaphos-resistant colony selected on 1 mg/L bialaphos
(7 weeks post-bombardment). months in culture (data not shown), providing evidence for
) B(Embryogenic SC82 bialaphos-resistant callus selectednda the stability of the integrated sequences. The seven inde-
maintained on 1 mg/L bialaphos (bar = 1 mm). pendent transformants (E1, E2/E5, E3/E4/E6,, E97E/E8 ,
606 The Plant Cell
events. These 20 transformants were from two experi-
ments (4 and 5) and a total of 16 bombarded filters
- OE W M1"1(E> k01bE) 9E 8E 7E 6E 5E 3E 1W EM (4Ek b2E) .)1(Ta ble
Using other embryogenic suspension culn tSurCae8s 2,
culture thas awrte initiated from cryopreserved cells (ex-
periment 6) or an A188 x B84 (SC94) suspension culture
-9.6 (experiment 3), numerous independent transformants were
-6.6 recovered (19 and 8, respectively; Table 1). All transform-
ants contained the bar gene and expressed PAT. The copy
numbe forb ar-hybridizing sequence eshdt, iversitf yob ar-
I
hybridizing patterns, and levels of PAT expression (data
V t osnhown) were comparable e ewhxittph erim-eendts
scribed abov dnwea ith those performed previously with
BMS (Spencer et al., 1990).
Cotransformation
Transformants from SC716 experiments (4 and 5) were
analyzed for integration and expression of the nonselected
gene encoding GUS. As determined by histochemical as-
sa eySh, Ctf o7fou1r 6 transformants (, R7RdR5n1, a6,
R21d ad)h etectabS laUeG ctivity3 months post-bombard-
Figure 4. Integration of the bar Gene in Bialaphos-Resistant
SC82 Isolates E1 toE11. ment (T. aE)b1xlep ression patterns o ebfohsuetrr vneid
coexpressing callus lines vare inehudTm. bf eocr ells with
DMA gel blot of genomic DMA (4 /ig/lane) from E1 to E11 and a
GUS activity withiy nnga iven transformant sampled ranged
nonselected control (EO) digested with EcoRI and Hindlll. Blot
from -5% to -90% and, in addition, the level of GUS
was hybridized with 32P-labeled bar from pDPG165 (25 x 106
activity in those cells varied. Cointegration frequency was
Cerenkov cpm). Lanes designat5 ec1 cdod opnpiaey s reofetr
determined by washing the genomic blot hybridized with
the diploid genome and contain 1.9 pg and 9.5 pg, respectively,
of the 1.9-kb bar expression unit released from pDPG165 with bar (Figure 5A) and probing with 32P-labeled GUS-coding
EcoRI and Hindlll. sequence. EcoRI and Hindlll, which excised the bar
expression unit from pDPG165, also released from
PDPG2a 028 .1-kb fragment contae iGnhiUntgS -coding
sequence and the nos 3' end (Figure 1B). Seventeen of
E10, and E11) were derived from two separate bombard- the independenr atb transformants contained sequences
ment exa pt 0od1bent aorafo i)mml 2 dbenaan r1tds( ed that hybridizS eUGp edhtr ot obe, 2;RR 1did 941R dn,a
filters. These data are summarized in Table 1. t on(Figur; eB5 Table .)1 Transformantsn i whicSUhG
Bombardment experiments were also performed using activity was detected (R5, R7, R16, and R21) contained
a suspension culture, SC716, derived from a separate the 2.1-kb EcoRI/Hindlll fragment encodinS gUG( Figure
initiationf o typeI I callusf o A188x B73.e hT SC716 5B). Transformants that contained large numbers of bar-
suspension culture was cryopreserved 5 months after the hybridizing fragmd eRnna2ts1 ,)(5 RaRl1s ,o contained
suspension was initiated and then reestablished after 8 large nuf mforabgemr sente sg ethhnaett h yboridtize d
months' storagn elii quid nitrogen be nbif oeosrmue bard- ed n5 Scn(BFoaU)di.g iGnuASrgie 5ms ilar observations
ment experiments. The SC716 cells were bombarded with using independent plasmids were reported using PEG-
separate plasmids eSnT cU(ApoGDdPiPn gG d1n6a5) mediated transformatiof on AS 1MpB8r 8xo toplasts
(pDPG208). Ss edalee ssccatirowibn r SoefCd 82, usin1g (Lyznik et al., 1989) and bombardment-mediated transfor-
mg/L bialaphos. Twenty-one bialaphos-resistant callus mation of BMS suspension cells (Spencer et al., 1990).
lines were isolated; all expressed PAT at levels comparable Cotransformation analyses were also performed on 19
wie tShhC tth8 o2fos ies olateles g(t dsoAahntoNa wDn) . independent bialaphos-resistant isolates selected froma
blot analyses of the SC716 isolates demonstrated that all suspension culture reinitiated from cryopreserved SC82
bialaphos-resistant isolad o tbeRatas2 rh 1-1te )Rst(ed cells (experimen, 6tT ae hfbTr el.)e1q uencf yoc ointegra-
hybridizing sequencess ,a shownn i Figure. A5T hirteen tiod cnnao expression nti hese isolats easwsim ilao rtt hat
isolates contained intact copies of the 1.9-kb bar expres- describedr of SC716 isolates (Table eht saw sa ,)1 heter-
sion unit. Twenty unique hybridization patterns were ob- ogeneous pa SstUtteaGr ifnno inr egoFx. ampe ltenroa, ns-
served among the 21 SC716 isolates (R8 and R10 were formant, Y13, which contained intaS UccGot din-esg
identical), suggesting 20 independent transformation quence (dat ostnah own), exhibited chimS aeUcrGitci vity
Fertile Transgenic Maize 607
Table 1. Summary of Maize Transformation Experiments
lndependent No. with
bar lntact bar No. with GUS No. with Cointegration Coexpression
Culture Filters Transformants Expression Coding GUS Frequency Frequency
Exp. Bombarded Bombarded Recovered Units Sequence Activity (“4
(“/O)
1 SC82 4 4 3 NA”
2 SC82 6 3 2 NA
3 sc94 10 8 6 NA
4 SC71 6b 8 13 8 11 3 85 23
5 SC71 6b 8 7 4 6 1 86 14
6 SC82b 4 19 17 13 3 68 16
Totals 40 54 40 30 7 77 (30/39) 18 (7/39)
a NA, not applicable; only pDPGl65 DNA used or cotransformation analysis not done.
Culture reinitiated from cryopreserved cells.
in callus tissue, as shown in Figure 6. This type of expres- each independent transformant was analyzed by DNA gel
sion pattern has been described previously in cotrans- blot hybridization. Hybridization to bar was observed only
formed BMS cells (Klein et al., 1989; Spencer et al., 1990). in high molecular weight DNA (data not shown).
Variable activity detected in the cells from a single trans- Analysis of Ro plants regenerated from SC716 trans-
formant may be attributed to differential expression of the formants is not complete; however, two plants from each
genes, absence of the gene in some cells, or variable of the 1O regenerable SC716 callus lines have been tested
penetration of the GUS substrate. for PAT activity (data not shown). For seven SC716 callus
lines both plants contained PAT activity; for three callus
lines one plant was PAT-positive, the other was PAT-
Regeneration and Analysis of R, Plants
negative. The reason for the lack of PAT activity in some
SC716 plants has not yet been determined but may be
Using several regeneration protocols, a total of 76 plants due to inactivation of, or the absence of, bar in some
were regenerated from four of the SC82 transformants
plants.
selected in experiments 1 and 2; 73 plants reached ma-
turity. A total of 219 plants were regenerated from 10
SC716 transformants selected in experiments 4 and 5; to Phenotypes of Ro Plants
date, 35 have reached maturity. Regeneration from callus
lines selected in other experiments is in progress. A sum- Plants regenerated from SC82 transformants (E2/E5, E3/
mary of regeneration efforts is presented in Table 2. E4/E6, E1 O, and E1 1) were similar to seed-derived A1 88
Twenty-one Ro plants representing each of four regen- x 873 plants in that they were comparable in height with
erable, transformed SC82 callus lines (E2/E5, E3/E4/E6, A1 88 (3 feet to 5 feet) and exhibited some characteristic
Elo, and Ell) selected in experiments 1 and 2 were 873 traits such as upright leaves and anthocyanin accu-
analyzed for PAT activity. Protein extracts from leaves of mulation in stalks and prop roots. Severa1 abnormal phe-
each of these plants contained PAT activity, and the activ- notypic characteristics were common to many SC82 Ro
ity levels were comparable with levels in callus lines from plants, such as palmate leaves, split leaves, wilting, coarse
which the plants were regenerated (data not shown). The silk, and underdeveloped tassels. Nontransformed control
activity in extracts from one plant regenerated from each plants were not regenerated from this culture and, there-
callus line is shown in Figure 7. Genomic DNA from these fore, cannot be compared phenotypically. Pistillatef lowers
four callus lines and two Ro plants derived from each callus developed on tassels of one E1 1 (1/ 6), several E1 O (3/22),
line was analyzed for the presence of bar, as shown in and almost one-third of the E2/E5 (12/37) plants with a
Figure 8. All Ro plants analyzed contained sequences that range of three to approximately 20 ovules per tassel.
hybridized to bar. Furthermore, in all instances the hybrid- Primary and secondary ears developed frequently on most
ization profiles observed in plant DNA were identical in E2/E5, Elo, and E11 plants; a mature E2/E5 plant is
pattern and intensity to the hybridization profiles of the shown in Figure 9A. Anthers rarely extruded from the
corresponding callus DNA. To confirm that the bar-hybrid- tassels of plants regenerated from SC82 transformants,
izing sequences were integrated into chromosomal DNA, and the limited number of anthers which were extruded
undigested genomic DNA from one plant derived from did not dehisce pollen. Some phenotypic characteristics
608 The Plant Cell
differences that characterized the individual callus lines of
SC82. These plants were more uniform and abnormalities
less frequ eephnThte. notyf poteh ese plants clos-eelry
sembled that of control plants regenerated from an SC716
cryopreserved culture that was not bombarded. Plant
MW (kb)
height ranged from 3 feet to 6 feet, with the majority of
•
-9.6 the plants between 5 feet and 6 feet. Most mature plants
649.6..3-6 -- f|:c J i --64..63 produced large, multibranched tassd enplasr imdanray
1 secondary ears. Pistillate flowers also developed on tas-
• sel foss everal SC716 plants. Although anther extrusion
2.0- » -2.0 occurred ta approximatelye ht samew ol frequencyni sa
the SC82 plan atssm , all amo foupno ltlen dehisced from
some extruded anthers. For most of the SC716 plants that
reached maturity, senescence did not commence until at
least 30 days after anthesis.
B
s
a
!So8
MW (kb)
9.6- -9.6
6.6- »• -6.6
4.3- * -4.3
.1
2.0- -2.0
Figure 5. Integration of Introduced Genes in Bialaphos-Resistant
SC716 Isolates R1 to R21.
6 l /ebi f(ggo lgoA/el atAnMnoMemD)(D iAcf r )om oisto l1aRtes
R21 digested with EcoRd HnaI indd lhnlal ybridizedo 3t 2P-labeled
bar probe (10 x 106 Cerenkov cpm). Two copies of the bar
expression unit per diploid genome is 5.7 pg of the 1.9-kb EcoRI/
Hindlll fragment from pDPG165.
) B(Blot froms aw )wA( ashedo t removr abe ehpt robedna
hybridized with 32P-labe6 S 0Clp1eUreG dox r 5be3en( kov cpm).
Two copies of the 2.1-kb GUS-containing EcoRI/Hindlll fragment
from pDPG208 is 6.3 pg.
observed were uniquo etp lants regenerated froma spe-
cific callus line, such as the lack of ears on E3/E4/E6
plantsa dna "grassy" phenotyp1 2e ot plu( ong, narrow
leaves) exhibite 11E lldap yb lantsl .lA SC82 plants-es
nesced prematurely; leaf necrosis began approximatel2y Figur. e6 Histochemical DeterminaS AUtGioc ftnoi vitn yi Biala-
weeks after anthesis. phos-Resistant Callus Line Y13.
Of 219 plants regenerated from 10 independent SC716 Y13 was selected from SC82 reinitiated from cryopreserved cells
transformants, approxima5 teh3lay ve reached maturity (experiment 6) and tested for GUS activity 3 months post-bom-
(Tae ShbT Cl.)e27 16 plat oenn xdtisdh ibe hpitt henotypic bard.mmm 5e.0 =n ratB.
Fertile Transgenic Maize906
Table 2. Regenerated Plants (R) and Progeny (R,)
0
Independent
bar Regenerable No. No. of Ro
Culture Transformants Transformed Ro Reaching Producing Kernels R,
Exp. Bombarded Recovered Callus Lines Plants Maturity Kernels Recovered Plants
1,2 SC82 7 4 76 73 28 184 50
4,5 SC716 20 10 219 (35) (9) (51) (31)
3 SC94 8 2" 11* (0) (0) (0) (0)
6 SC82 19 4" 23" (0) (0) (0) (0)
Parentheses indicate that experims esn nipti itlr logress, e dsratailtla being collected.
• Regeneration in progress.
Back-Crossing Transgenic R Plants with nontransformed B73 pollen. Kernels on transformed
0
SC82 Ro plants developed normally for approximately 10
Progeny (R,) were obtained from transformed R playnbts days to 14 days post-pollination. After this period, which
0
back-crossing transformed SC82 plants with seed-derived corresponded to the initiation of premature senescence in
B73 plants. Anthers that extruded on the tassels of 10 E2/ most of the plants, kernels ceased development and often
E5 and one E10 R plants were collected, macerated to collapsed. A total of 184 kernels developed on the SC82
0
release pollen,d na appliedo t nontransformed, seed-de- Ro p7 lE3a2n f/toEs 51(T 20pa 1l:ab)E2nle t 2s,2 ffiove
3 sr7iivlkBe.d Examinatf iopoon llen removed from extrud- plants, and two of six E11 plants. Viable progeny were
ing anthers of SC82 plants revealed that only a small recov yeebrmedb ryo rescue2 Efr12o/mE 5d pnlaants
percentage had reached the binucleate stage. Mature, one E10 plant. Embryos were excised from kernels that
starch-filled pollen grains were rarely observed and failed developed on SC82 R0 plants and ranged in length from
to germinate when placed on germination medium (Pfahler, about 0.5 mm to 4 mm. Fifty viable progeny have thus far
1967)o N.c onfirmed transformed progeny were recovered been recovered from SC82 R0 plantfso o(Twat b;)2le
from back-crossing SC82 R pollen3 pw7laBitnh ts. Polli- which are shown in Figure 9B.
0
nation of transgenic SC82 R ears with nontransformed SC716 Ro plants were also back-crossed with seed-
0
B73 pollen, however, did result in kernel development. I5 nm3ao 3 tdpu alrT7adeetene ,BtrSsh i vfC.reot7d m1 6
addition, several kernels developed from pistillate flowers R0 plants, nine plants (representing five independent callus
on male inflorescences of SC82 plants that were pollinated lines) yielde1 d5k ernef los w13, hich produced vigorous
R, seedlings (Tab. )M2le ost kernels that developneod
SC716 plants did not require embryo rescue. Kernels often
devee 0 l0 pdoh3d4pltad aa sr nneyooynoaotsfdsm t e
were germinated in soil. The remaining seed was germi-
RO Plants nated on MS-based medium (Murashige and Skoog, 1962)
o tmonitor germinatiod ntnar ansferreo dts oil afwteef ar
days.n I additione ht ot improved kernel development
observn SeodC 716 R plants relatio vSte C82 R plants,
0 0
pollen dehisced from antherf oss everal SC716 plandntas
some of this pollen germinated in vitro (Pfahler, 1967).
Ears treated with this pollen havet ey ton reached maturity.
Analysis of Progeny
Figure 7. PAT Activity in R Plants.
0
To date, a total of 36 SC82 E2/E5 progeny have been
One plant derived from eace hhtf foo ur transformed regenerable
08 w11e rT eeadA chnptiPtova sitftiy tOeirvsoete f.d
callus lines (E10, E11, E2/E5d nEa, 3/E4/E6s atw)e stTeAP drof
activity. Protein extracts from a nontransformed B73 plant and a progeny analyzed for PAT activity in Figure 10A, six were
BMS cell culture transformant (Spenct aeelr ., 1990) were included positive. GenA ofrMmoDimc the 0sp1er ogs eaanwnya -
as controls. Approximf a 0otnot5eQt lay l s prueraospwteedi n l eblgot Ahe ypMhbrertisDde fizrnoa oclytyei fozbne d
reaction. bar, as shown in Figure 10B. The six progeny that had
ePhlaTnt Cell 016
E3/E1 1E4/E6 01EE2/E5 FunctionalT AAcPti vfioty
C RO RO C RO RO C RO RO C RO RO u •*" Functional activity of PAT was assessed by localized ap-
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plication of a commercial herbicide formulation containing
PPT to leaves of SC82 R and Ft, plants. No necrosis was
9.4 — — 9.4 0
observed on leaves of R plants containing either high
6.6 — — 6.6 0
levels (E2/E5) or low levels (E3/E4/E6) of PAT. Herbicide-
treated E3/E4/E6 and control leaves are shown in Figure
1. AH1 erbis caaidwlse o applieo ltde avef osE 2/E5 prog-
eny segregating for bar. As demonstrated in Figure 11 B,
— 2.0 leaves of R, plants expressing bar exhibited no necrosis 6
days after application of the herbicide, whereas R! plants
without bar developed necrotic lesions. No necrosis was
observed on transformed leaves up to 30 days post-
application.
Coexpressi aoN fnoo nselected Gene
Plants were regenerated from the coexpressing callus line
Y13, shown in Figure 6. Plants regenerated from Y13
Figure. 8 Comparisor abnS fo equencesn i Transformed Callus
(experiment 6, Table 2) were assayed for GUS activity,
dCnaorresponding R Plants.
0 and histochemically stained leaf tissue from one plant is
Genomics d DiagMewsA ted with d EHncinoadRl lIl, which release shown in Figure 12. Numerous cell types including epider-
e ht1.9-kbr ab expression unit from pDPG165. Hybridizationsaw mal, guard, mesophyd nlbla, undle sheath cells stained
a 5 ot59-bp 32P-labeled Smal DMA fragment containx 2in4( rgab S aUpctoGivsi ittryiov.fe Staining inte sngasrwietya test
106 Cerenkov cpm). The DNA fragments were separated by gel
in the vascular bundles (data not shown). Although all leaf
electrophoresis, blottedo t nylon membraned n,a hybridizedot samples from the regenerated plants tested (5/5) ex-
bar. Lanes designated E3/E4/E6, E11, E2/E5, and E10 contained
pree snshoetnd selected gene, some nonexpressing leaf
5 ng of either callus (C) or R plant (R) DNA. Control lane
0 0 sectors were also observed. Leaf tissue extracts from
contained 5 /*g of DNA from a nontransformed A188 x B73 plant.
three Y13 and three control plants were also assayed for
Lane designated1 c opy cone htt1a fo.i n9gpe- 3k.d2b EcoRI/
Hindlll fragment from pDPG165, representing one copy per diploid GUS activy ibftlyu orometric analysis (Jefferson, 1987).
genome. Activity detectedo wt ni opposing leaves from eachfo
th3 rp1elYae nts tt eales sataeswdt 100-fold higher than
that in control leaves (data not shown).
PAT activity each containea sd ingler ai dcbeo nfpotyic al
DISCUSSION
in mobility to that detected in E2/E5 callus and R plants;
0
the four PAT-negative pt ocroongn detaindiny bar-hybrid-
izing sequences. The ratio of PAT-positive to PAT-negative The generatiof nos tably transformed fertile plants sci rucial
progeny of E2/E5 R plants is consistent with a 1:1 seg- for both the study of basic plant processes and the intro-
0
regation ratio of a dominant, single-copy gene. The single duction of new traits into crop species. For maize, the
progeny recovered from an E10 R plant contained PAT utilif toyp rotoplast transformation methos badhes en lim-
0
activity (data ton shown). Segregatio tonn did rcab fo or- itedo t monitoring transient expression fo introduced genes
relate with the phenotypic variability of R, plants, such as (Frommt e al., 1986), suchs a studying aspectsf o tissue-
plant hed tiangsahst el morpholoe pn ghFlaIyti g .n,Butr9e specific expression (Let eea l., 1989). Recoverf yot rans-
ehrti gnoht contains bae hrpe t, llhaet nnfott does nontI. formed fertile maize plants from electroporated protoplasts
addi! tRipolna e,n htmst ofwose tre more vigorous than ht oabnse en documented despite independent reporftos
the RO plants. infertile protoplast-derived transgenic plants (Rhodetes
I R s13eO eehtf dlings recovereo dt date fromeht al., 1988) and of regeneration of nontransformed fertile
SC716 transformants, 10 of 16 tested had PAT activity. plants from protoplasts (Prioli and Sondahl, 1989; Shillito
PA0 pTs r1 doaagece ttewifnn vcfoyiottie y ufRd rrom et al., 1989). Various alternativeso t protoplast transfor-
0
plax npirtsso g refeonpyr exsiesn tidngn aca l8lu1s Rline mation have been suggested, such as microinjection (Po-
from RO plants representing callus line R9 (data not shown). trykus, 1989d )na laser-mediated ANdD elivery into intact
Fertile Transgenic Maize 611
Figure 9. Mature R Plant and R, Progeny.
0
(A) Mature transgenic plant regenerated from E2/E5 callus.
(B) R, progeny derived from E2/E5 plant by embryo rescue.
cells (Weber et al., 1987), but the most rapid progress has gene werew f rooelbq tauaei ntnaecdy (McCa tbeael .,
been made using microprojectile bombardment (Klein et 1988). Using anthocyanin biosynthesis as a visual marker,
al., 1989; Spencer et al., 1990). bombardmef nost hoot meristem foism mature ma-mizee
bryos wia trhe gulatory gene resultn eside ctof otrrsa ns-
formed, pigmented tissue (Tomes, 1990). Analysis of cell
Microprojectile Bombardmef nMot aize lineage patterns in maize (McDaniel and Poethig, 1989),
however, suggests that germline transformation fo maize
Given the goal of obtaining fertile, transgenic maize plants, by such an approach might be difficult. A third target for
there ersa everal attractive cellular targetr somf icroprojec- microprojectile bombardms eciun lttured cells. Transform-
tile bombardment-mediated DNA delivery. One of these is ants were isolated after bombardment of a nonembry-
pollen. Although transient expression of a reporter gene ogenic, nonregenera SbMmlBea ize suspension culture,
was detected in bombarded tobacco pollen (Twell et al., d knaaun soaienmng ycin selection (Klet iean l., 19d8n9a)
1989), stable transformation by microprojectile bombard- ba dnrab ialaphos selection (Spencert e al., 1990).
t ebe yyepnnla antrote pnroor tefsmf d opaeonhl lten Although microprojectile bombardment obviates proto-
specie Ass. econd potential targs meit eristematic tissue. plast isolation, another difficn uclitey real transformation
Bombardmenf ot soybean apical meristems with DNA- has n beae eflhf aeeftconc k tive selective agr eotonftt i-
coated gold particles resulted in chimeric plants containing potent cultures (Potrykus, 1989). Despite the successful
transgenic sectors; progeny containing the introduced f ok easunamya c ssiaen lective ager onfbt ombarded
612 The Plant Cell
B
R0a b cd
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„
4.4 -
2.0 - - 20
0.6 - fc
Figure 10. PAT Activity and DNA Gel Blot Analysis of Segregating
Progeny from Four E2/E5 R Plants.
0
(A) Analysis of PAT activity in 10 progeny (lanes a to j) and a
nontransformed control plant (lane k). Lanes designated a, b to h,
i, and j contained protein extracts from progeny of four separate
parental R plants. Lane designated callus contained protein ex-
0
tract from E2/E5 callus. Approximf to5 oa^tt9a2 ell y rperoptein
reaction.
l egb( ABlNoD)t analysif so genomA NiisDco lated fr0o1 meht
progeny analyzed in (A). Genomic DNA (5 ^g/lane) was digested
with Smal, which releases a 0.6-kb fragment containing bar from
pDPGd h1ny6ab5 6r ,0Cid1eizree dnx r k awpo0rbvi5othb (e
cpm). Lane designated R cont Aafre NinoRhDemtd parefont
0 0
progen. aLy ane designate 1cd opy contained pDPG165 digested
with Sm oatr lepresent approximate e1 hl5yct 5o f9po-yb p
Fig. 1uB1rea sta Resistann cTei ransformed dRn R,a Plants.
fragmer nedpti ploid genome (0.8 pg). 0
(A)A Basta* solutio snaaw ppliea d olta rge nai )mrc 8e x a4~(
e htcenterf o leavesa fo nontransformed A1883 7B x plant (left)
a t rdannasgenic R E3/E4/E6 plant (right).
0
(B) Basta was also applied to leaves of four R, plants: two plants
nonembryogenic BMS cultures (Klein et al., 1989) and o inpwlantt sd cwnr o(iatnlaehtr bfoae t( ha)riunihe gtbirnhTbgt i)- .
protoplast selection protocols (Rhodes et al., 1988), its s aca, piwRdppelal ieon n1dtti s -cm circo leftos ur locatinoons
use with cultured maize cells has been hampered by each leaf, two on each side of the midrib. Photographs were
endogenous resistance (Hauptmann et al., 1988; W.J. taken 6 days after application.