Strategies for the production of maize-derived pharmaceuticals using cytoplasmic male sterile lines: In vitro tissue culture/transformation and field breeding approaches

Abstract

Plant-made pharmaceuticals (PMPs) offer promise as efficient and cost-effective products for the treatment of human and animal diseases. An advantage of producing pharmaceuticals in maize is the large storage capacity and stability for proteins and starch in seed, al- lowing for manufacturing recombinant proteins such as antigens and antibodies. Other advantages of the maize system include safety, high yields, and scalability of pro- duction and processing. However, the benefits of this technology must be balanced against potential health and environmental risks that may be associated with its use. Because PMPs presently have no provision for regulatory tolerance, their inadvertent occurrence in foods and feeds remains an important economic consideration, even when the health and environmental risks are low. Pollen drift is considered a source of potential contamination of maize- made pharmaceuticals in the food chain. In addition to physical and temporal isolation requirements, open field pharmaceutical maize production also calls for controlled pollen release. Here, we describe two strategies to ad- dress the issue of transgenic pollen drift. First, we de- scribe the development and genetic transformation of a tissue culture-amenable male-sterile line using biolistic- or Agrobacterium-mediated transformation methods. Sec- ondly, we describe the introgression of a transgene from male-fertile transgenic maize to male-sterile germplasm by conventional breeding. After six seasons of breeding, this second strategy allows us to obtain 100% transgenic seeds from an open-field production using a non-transgenic line as the pollinator.

Full text

ABSTRACT - Plant-made pharmaceuticals (PMPs) offer
promise as efficient and cost-effective products for the
treatment of human and animal diseases. An advantage of
producing pharmaceuticals in maize is the large storage
capacity and stability for proteins and starch in seed, al-
lowing for manufacturing recombinant proteins such as
antigens and antibodies. Other advantages of the maize
system include safety, high yields, and scalability of pro-
duction and processing. However, the benefits of this
technology must be balanced against potential health and
environmental risks that may be associated with its use.
Because PMPs presently have no provision for regulatory
tolerance, their inadvertent occurrence in foods and feeds
remains an important economic consideration, even when
the health and environmental risks are low. Pollen drift is
considered a source of potential contamination of maize-
made pharmaceuticals in the food chain. In addition to
physical and temporal isolation requirements, open field
pharmaceutical maize production also calls for controlled
pollen release. Here, we describe two strategies to ad-
dress the issue of transgenic pollen drift. First, we de-
scribe the development and genetic transformation of a
tissue culture-amenable male-sterile line using biolistic- or
Agrobacterium-mediated transformation methods. Sec-
ondly, we describe the introgression of a transgene from
male-fertile transgenic maize to male-sterile germplasm by
conventional breeding. After six seasons of breeding, this
second strategy allows us to obtain 100% transgenic seeds
from an open-field production using a non-transgenic line
as the pollinator.
KEY WORDS: Genetic transformation; Male-sterile; Plant-
made pharmaceuticals; Zea mays.
INTRODUCTION
Maize grain is used as feedstock for many large
volume industrial products (e.g. ethanol, biodiesel,
poly-lactic acid, sweeteners) and has been demon-
strated to be an effective expression system for
functional proteins of prokaryotic (WITCHER et al.,
1998; STREATFIELD et al., 2001; CHIKWAMBA et al.,
2002a; BAILEY et al., 2004); viral (STREATFIELD et al.,
2001) and eukaryotic (HOOD et al., 1997; ZHONG et
al., 1999; YANG et al., 2002) origins. Previously we
have successfully produced a transgenic maize line
expressing a B-subunit protein of Escherichia coli
heat labile enterotoxin (LT-B) (CHIKWAMBA et al.,
2002a). Mice fed with maize-derived LT-B were pro-
tected against diarrhea-inducing E. coli toxins and
showed reduced symptoms compared to control
mice fed with non-transgenic maize meal (CHIKWAM-
BA et al., 2002a).
There are several benefits of producing antigens
for use as edible vaccines in maize (STREATFIELD,
2007; RAMESSAR et al., 2008). Maize is a major crop
world wide used for food, feed and fuel. As food
and feed, maize is well tolerated by both humans
and animals since it is non-toxic and non-allergenic.
Compared to bacterial and mammalian systems, the
use of maize for producing recombinant proteins
greatly reduces the possibility of contamination with
mammalian pathogens. Maize yields are high and its
seed is a natural protein storage site that can be har-
nessed as an efficient protein production factory.
According to H
OOD and coworkers (HOOD et al.,
1999), maize can be used to produce foreign pro-
teins at rates that are in excess of 2 kilograms per
acre at a cost of a few cents per milligram. Breeding
techniques can be used in this crop to enhance for-
Maydica 54 (2009): 199-210
STRATEGIES FOR THE PRODUCTION OF MAIZE-DERIVED PHARMACEUTICALS
USING CYTOPLASMIC MALE STERILE LINES: IN VITRO
TISSUE CULTURE/TRANSFORMATION AND FIELD BREEDING APPROACHES
K. Wang
1,2,*
, B. Frame
1,2
, X. Xu
1,3
, L. Moeller
1,3
, K. Lamkey
1
, R. Wise
4
1
Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA
2
Center for Plant Transformation, Plant Science Institute, Iowa State University, Ames, Iowa 50011, USA
3
Interdepartmental Plant Biology Major, Iowa State University, Ames, Iowa 50011, USA
4
Corn Insects and Crop Genetics Research, USDA-ARS, and Department of Plant Pathology,
Iowa State University, Ames, IA, 50011-1020, USA
Re ceived May 14, 2009
* For correspondence (fax +1-515-294-2299; e.mail: kanwang
@iastate.edu).

eign protein expression (HOOD et al., 1997; WITCHER
et al., 1998; STREATFIELD et al., 2001; CHIKWAMBA et al.,
2002b). Moreover, the infrastructure for large scale
grain production and seed processing are well es-
tablished for the crop in the United States.
Since maize is one of the most important crops
globally, there are many issues that arise regarding
its use as a source of biopharmaceuticals. Major
concerns, especially in the case of vaccine-produc-
ing maize plants, are whether the antigens will in-
advertently enter the food chain and what measures
are in place to prevent this (R
IPPLINGER et al., 2009).
There are three broad categories of grain handling
systems used in the U.S.: commodity, identity-pre-
served, and seed (S
TRAYER, 2004). These systems
vary in their purity standards, which are primarily
based on end-user requirements. The commodity
system is used when there are no requirements for
differentiation by end users and the identity-pre-
served system is used when end-users require spe-
cific grain characteristics. The seed system is used
specifically for producing the seed to be planted for
commodity and identity-preservation systems
(S
TRAYER, 2002, 2004).
One major concern with regard to cross-contam-
ination in these systems is the potential for trans-
genic maize pollen to drift to surrounding non-
transgenic maize fields. Several measures can be
taken to prevent pollen contamination from a vac-
cine-producing maize plant. First, any APHIS (Ani-
mal and Plant Health Inspection Service, USA) ap-
proved pharmaceutical maize field release currently
requires a separation distance of 1 mile (5280 feet
or 1609 km) from the nearest maize plant (B
RS,
2006). This physical isolation is eight times greater
than the distance required for production of founda-
tion maize seed or identity preservation. Second,
confinement strategies for transgenic maize pro-
duced under permit include conditions that prevent
transgenic maize pollen from pollinating surround-
ing fields. Current APHIS rules stipulate that non-
pharmaceutical maize may be grown within one
half mile (2640 feet) of the test site if the regulated
pharma-maize is control-pollinated by detasseling or
bagging procedures. For temporal isolation, phar-
ma-maize must be planted no less than 28 days be-
fore or after the surrounding non-pharma maize is
planted (A
PHIS, 2008).
In addition to spatial and temporal isolation, it is
also possible to prevent contamination from trans-
genic pollen drift by producing vaccines in maize
varieties that are male-sterile. A number of cytoplas-
mic male-sterile varieties producing near 0% viable
pollen can be used for this purpose (L
EVINGS III,
1993; G
ABAY-LAUGHNAN and LAUGHNAN, 1994). Be-
cause transgenic male-sterile maize does not pro-
duce viable pollen, its female flowers can be polli-
nated by non-transgenic maize pollen to set seed.
According to R
IPPLINGER et al. (2009), if pollen pro-
duced from pharmaceutical maize is controlled, the
risk of accidental release and food supply contami-
nation is very close to the target tolerance level of
zero.
Here we describe two strategies that can be used
to produce male-sterile transgenic maize lines suit-
able for open-field pharmaceutical production. One
strategy is to directly introduce the transgenes into
transformable male-sterile germplasm. A fertile and
transformation-amenable Hi II genotype (A
RMSTRONG
et al., 1991) was converted into a male-sterile, trans-
formation-amenable group of lines by crossing to
cytoplasmic male sterile (CMS)-T (Texas) maize.
This is the first report of successful transformation
of such male-sterile lines using the biolistic- (gene
gun-) or Agrobacterium-mediated methods.
The second strategy involves transfer of the sub-
unit vaccine gene from a fertile transgenic line to a
cms-T B37 background. After six seasons of breed-
ing, male-sterile transgenic maize seed can be used
for open-field production using a non-transgenic
pollen donor to produce seeds for large scale re-
combinant protein recovery, 100% of which contain
the subunit vaccine gene.
MATERIALS AND METHODS
Germplasm
Development of the tissue culture amenable (T) germplasm
used in this study was previously described (W
ISE et al., 1999b).
Briefly, T-cytoplasm A188 [(T) A188] was pollinated by N-cyto-
plasm Hi II (high Type II, or HT), the standard germplasm for
maize transformation (A
RMSTRONG and GREEN, 1985; ARMSTRONG et
al., 1991). (N) HT is originally a tissue-culture line derived from
A188 and B73, hence the selection of (T) A188 as the T-cyto-
plasm female. Immature embryos were excised from the result-
ing F
1
seed, cultured on N6E30 medium (FRAME et al., 2000), and
selected based on Type II callus response. Callus was then trans-
ferred to regeneration medium (F
RAME et al., 2000), and T cyto-
plasm plantlets were grown to maturity. Since the resulting ears
were T cytoplasm, they were pollinated by (N) HT. Male-sterile,
(T) HT plants were maintained by pollinating with the (N) HT
stock (P
EI, 2000). Genotype nomenclature and crosses made for
seed increase and embryo production for transformation purpos-
es are detailed in Table 1. For introgression experiments, cms-T
B37 seed was increased via isolation plot in our 2000 Ames, IA
summer nursery.
200 K. WANG, B. FRAME, X. XU, L. MOELLER, K. LAMKEY, R. WISE

Plant materials
For genetic transformation: Immature embryos (1.5-2.0 mm)
were aseptically dissected from 10-13 day old field or green
house-grown ears of male-sterile (T) and male-fertile (N)
germplasm. Male-sterile phenotypes were assessed for each
donor plant used or transgenic R
0
and R
1
plant produced in this
study by monitoring the presence or absence of pollen shed
from mature plants. Structural anatomy of (T) and (N) cytoplasm
transgenic plants (Fig. 1) was compared and imaged using an
Olympus SZ stereo-microscope (Leeds Precision Instruments,
Minneapolis).
DNA plasmid constructs and Agrobacterium strain for
plant transformation
Construct pAHC25 (CHRISTENSEN and QUAIL, 1996), containing
the maize ubiquitin promoter driving both bar and gus-intron
gene cassettes (Pubi-bar/Pubi-gus intron), was used for all biolis-
tic gun-mediated transformation experiments.
Agrobacterium tumefaciens strain EHA101 (H
OOD et al.,
1986) harboring the standard binary vector pTF102 (FRAME et al.,
2002), was used in all Agrobacterium-mediated transformation
experiments. This vector carries a P35S-bar/P35S gus-intron cas-
sette. A fresh bacteria culture was initiated from -80°C every four
weeks on solid YEP (AN et al., 1988) containing antibiotics (100
mg/L spectinomycin, 50 mg/L kanamycin, 25 mg/L chloram-
phenicol) and stored at 4°C. Cultures for weekly experiments
were initiated from this 4°C “mother” plate and grown at 19°C
for 3 days before use. Final OD
550
in all experiments ranged
from 0.3-0.4. All other bacteria manipulations, maintenance and
pre-infection preparation steps were identical to the protocol de-
tailed in F
RAME et al. (2002).
Culture media
Biolistic transformation: All media components and prepara-
tion steps were detailed in F
RAME et al. (2000) as adapted from
SONGSTAD et al. (1996). Petri plates (100x15 mm) were used for all
bombardment and selection media.
Agrobacterium-mediated transformation: All N6 media (CHU
et al., 1975) components were described in FRAME et al. (2002).
Co-cultivation media was modified from Z
HAO et al. (2001) to
contain 300 mg/L cysteine; resting and selection media contained
a combination of cefotaxime (100 mg/L) and vancomycin (100
mg/L) for counter selection. All solid media tissue culture steps
used 100x25 mm Petri-plates and all stocks were prepared as de-
scribed in F
RAME et al. (2002).
Regeneration Media: Independent, bialaphos resistant Type
II callus events generated from either transformation protocol
were sub-cultured to a Petri-plate (100x25 mm) containing MS
salts (MURASHIGE and SKOOG, 1962) and modified MS vitamins
(FRAME et al., 2006), 6% sucrose, 100 mg/L myo-inositol, no hor-
mones (ARMSTRONG and GREEN, 1985), 0.3% gelrite, pH 5.8 and
supplemented with 4 mg/L glufosinate (Sigma, St. Louis). Cefo-
taxime (250 mg/L), added to this media after autoclaving, was in-
cluded for Agrobacterium-derived putative events. Three weeks
201MALE-STERILE MAIZE FOR PHARMACEUTICAL PRODUCTION
TABLE 1 - Description of germplasm used for genetic transformation*.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Base Phenotype
Cross for seed Cross for
Transformation
germplasm Pedigree
––––––––––––––––––––
increase transformation
genotype
ID Female Male nomenclature **
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
(T) pA (T) A188/(N) HT pA x (N) HT pA BC6 fertile sterile (T) pA x (N) HTpA none NT
(T) F
1
(T) A188/(N) HT pA x (N) HT pB BC
0
fertile sterile (T) pA x (N) HTpB (T) F1 x (N) HTF1 (T) F
2
(T) PeiA(BC
0
) [F98 (T) A188/HT A/B x (N) fertile sterile (T) PeiA x (N) HTpA (T) PeiA(BCn) (T) A(BC
0
) in 2003/4
A188/HT A/B] x (N) HT pA x (T) A(BC
1
) in 2004
Tissue culture regenerated plants from (N) HTF
1
the greenhouse
(T) PeiB(BC
0
) [F98 (T) A188/HT A/B x (N) fertile sterile (T) PeiB x (N) HTpB (T) PeiB(BC
n
) (T) B(BC
0
) in 2003/4
A188/HT A/B] x (N) HT pB x (T) B(BC
1
) in 2004
Tissue culture regenerated plants from (N) HTF
1
the greenhouse
(N) HTpA HT parent A fertile fertile (N) HTpA x (N) HTpA none NT
(N) HTpB HT parent B fertile fertile (N) HTpB x (N) HTpB none NT
(N) HTF
1
HT pA x HT pB fertile fertile (N) HTpA x (N) HTpB (N) HTF
1
x (N) HTF
1
(N) HTF
2
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
* Male-sterile (T) pA, (T) PeiA or (T) PeiB seed was increased in 2003 by pollinating with the male-fertile (N) HTpA or (N) HTpB or to
make the F
1
hybrid cross (T) pA x (N) HTpB. Embryo production for transformation purposes was achieved by crossing these stock plants
with (N) HT F
1
pollen in all cases. In 2003 and 2004, pollinations were carried out on plants grown from the original pedigree [(T) BC
0
x
(N) HTF
1
]. As well, in 2004, pollen from (N) HTF
1
was taken to one generation advanced (T) A(BC
1
) and (T) B(BC
1
) plants to produce (T)
A or (T) B(BC
1
) immature embryos for transformation experiments. (N) HTF
1
plants were sib or self pollinated to produce (N) HTF
2
em-
bryos for transformation of control material in this study.
** Nomenclature of immature zygotic embryo donor plant for genetic transformation.
(T): Texas male sterile cytoplasm.
(N): Normal male fertile cytoplasm.
NT: not tested.

202 K. WANG, B. FRAME, X. XU, L. MOELLER, K. LAMKEY, R. WISE
FIGURE 1 - Tassels of gun-derived pAHC25 transgenic (T) F
2
(left) and (N) HTF
2
(right) plants at anthesis in greenhouse. No anther extru-
sion is visible on tassel of (T) F
2
transgenic event (b) while (N) HTF
2
event (c) is undergoing anthesis. Florets of transgenic (T) F
2
plants
contain degenerated anthers (d and f) while those of (N) HTF
2
transgenic plants are filled (e) with pollen grains (h, bottom). No pollen
grains are visible in (T) germplasm transgenic anthers (h, top). GUS expressing, viable pollen grains are visible in X-Gluc stained anthers
of (N) transgenic germplasm (i, bottom) but not (T) transgenic germplasm (i, top) transformed with pAHC25 using the biolistic gun.

203MALE-STERILE MAIZE FOR PHARMACEUTICAL PRODUCTION
FIGURE 2 - (N) HTF
1
(right) and (T) F
1
(left) greenhouse grown donor ears (a). (T) F
2
immature embryo stained for transient GUS expres-
sion 5 days after Agro-infection with pTF102 (b). Putative transgenic events (arrows) emerging from 3 mg/L bialaphos selection of Agro-in-
fected (T) A(BC
1
) field 2004 embryo infections (c). Stable GUS expression in putative callus events from field 2004 Agrobacterium-derived
(N) and (T) events (d). Seed set on 3 (T) F
2
(top) and 3 (N) Hi II F
2
(bottom) pAHC25 gun-derived transgenic events (e).

later, mature somatic embryos were germinated in the light on
MS Salts and modified MS vitamins, 3% sucrose, 100 mg/L myo-
inositol and 0.3% gelrite (pH 5.8) without glufosinate or cefo-
taxime. Petri-plates were wrapped with vent tape.
Transformation protocols
Biolistic-gun mediated: Transformation and selection proce-
dures were as previously described for immature zygotic embryo
transformation (FRAME et al., 2000) except that DNA quantity per
bombardment was reduced and embryos were bombarded 3
rather than 4 days after dissection in the present study. This pro-
tocol is used for routine production of transgenic (N) HT maize
in our laboratory. Briefly, 30 zygotic embryos per plate were dis-
sected, scutellum side up, in a 2 cm
2
grid on the middle of a
Whatman No 5 filter paper laying on the surface of callus initia-
tion medium. These plates were incubated at 28°C (dark) for 3
days. The morning of bombardment, an osmotic pretreatment of
targeted embryos was begun by carefully transferring each grid
of embryos (still on the filter paper) to the center of a Petri plate
(100x15 mm) containing maintenance media supplemented with
0.2 M sorbitol and 0.2 M mannitol (VAIN et al., 1993). Four hours
later, each plate of embryos on osmotic medium was bombarded
once at 650, 6 cm target, ¼ inch gap. For bombardment, 10
macro-projectiles (10 µl each) were prepared from one tube of
0.6 micron gold particles (3 mg) coated with 0.075 µg plasmid
pAHC25. The following morning, embryos were transferred,
scutellum side up, to the surface of maintenance medium (28°C,
dark) and 10 days later sub-cultured to selection medium (28°C,
dark) containing 2 mg/L bialaphos. Embryos were subcultured to
this medium a second time three weeks later. Six to 10 weeks af-
ter bombardment, each bialaphos resistant putative transgenic
callus event was harvested to its own plate of selection medium
for bulking up and regeneration as described above.
Agrobacterium-mediated: Infection, co-cultivation and selec-
tion procedures were all according to FRAME et al. (2002). This
protocol is used routinely in our laboratory for production of
transgenic maize using an Agrobacterium standard binary vector
to transform (N) HTF
2
germplasm. Briefly, embryos from a single
ear were dissected to an eppendorf tube (2 ml) filled with bacte-
ria-free infection medium and washed once with the same before
infecting with diluted, pre-cultured (2-5 hours) Agrobacterium
suspension. After 5 minutes infection (without vortexing), em-
bryos were plated scutellum side up on 4-day old co-cultivation
media containing 300 mg/L cysteine, and cultured at 20°C (dark).
After 3 days co-cultivation, all embryos were transferred to rest-
ing media containing cefotaxime (100 mg/L) and vancomycin
(100 mg/L) but no bialaphos and incubated at 28°C (dark) for 7
days after which all embryos were transferred to selection medi-
um containing 1.5 mg/L bialaphos and antibiotics. Selection was
enhanced to 3 mg/L bialaphos for 2 two-week intervals after
which individual, transgenic events could be seen emerging as
actively growing Type II callus lobes on otherwise brown and
dying zygotic embryo explants (Fig. 2c). At this time, each puta-
tive event was sub-cultured to its own plate of selection medium.
Putative events that continued to produce vigorous, embryogenic
callus on selection medium containing 3 mg/L bialaphos were re-
generated for 3 weeks (25°C, dark) on media containing 4 mg/L
glufosinate, germinated in the light, and grown to maturity in the
green house as previously described (FRAME et al., 2002).
Histochemical GUS assays of callus and leaves
from R
1
progeny
GUS histochemical assays (JEFFERSON, 1987) were carried out
on all bialaphos-resistant putative transgenic calli recovered from
selection, and on leaf pieces of male-sterile and control R
1
proge-
ny plants to confirm expression of the gus transgene in segregat-
ing offspring of these germplasms. A single, 2 cm leaf piece was
cut from each progeny plant, grouped by event in 60x20 mm
Petri plates, and submerged in 4 ml X-Gluc solution. Plates were
vacuum-infiltrated for 10 minutes at 15 in Hg then wrapped with
204 K. WANG, B. FRAME, X. XU, L. MOELLER, K. LAMKEY, R. WISE
TABLE 2 - Biolistic gun transformation frenquency (TF), seed set, and pollen shed for Hi II (N) and (T) germplasms.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Donor # Bialaphos % Stable % Events % Events
Source plant # Ears # Emb bb
a
resist % TF
b
events GUS with with pollen
genotype events positive
c
>50K
d
shed
e
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
2003 gh (N) HTF
2
15 945 93 10 65 50 87
(T) F
2
19 1400 232 17 60 82 0
2003 field (N) HTF
2
16 1110 136 12 61 55 70
(T) F
2
11 600 87 15 46 58 0
(T) A(BC
0
) 13 881 116 13 54 75 0
(T) B(BC
0
) 15 930 228 25 58 70 0
2004 field (N) HTF
2
9 800 101 13 74 NT NT
(T) A(BC
0
) 2 272 48 18 69 NT NT
(T) A(BC
1
) 2 138 7 5 57 NT NT
(T) B(BC
0
) 6 382 50 13 80 NT NT
(T) B(BC
1
) 3 81 23 28 65 NT NT
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
a
all embryos bombarded (bb) with pAHC25.
b
# independent bialaphos (2 mg/L) resistant events / total # embryos bombarded (X100).
c
all callus events were assayed for histochemical GUS expression.
de
# independent events to seed (1 or 2 plants per event) ranged from 8 -30 events.
NT (not tested).

parafilm and incubated at 28°C for 20 hours. Chlorophyll de-
staining was done using a 6 hour 70% ethanol wash followed by
an overnight 95% ethanol wash. A stereo-microscope was used
to identify the number of blue and non-blue staining leaves for
each segregating event.
R1 progeny screening for bar gene expression
After sampling progeny for GUS assays, R
1
progeny of male-
sterile and control germplasm were screened for bar gene ex-
pression by spraying 9 day old seedlings with a 250 mg/L solu-
tion of glufosinate prepared from the herbicide Liberty® (Bayer,
Research Triangle Park, NC). The herbicide resistant/sensitive
scores were recorded 4 days after the spray.
RESULTS AND DISCUSSION
Biolistic gun transformation
of male-sterile germplasm
Field and green house derived immature embryos
of (N) HTF
2
and (T) male-sterile germplasms were
targeted for biolistic transformation in 2003 and 2004
(field only). Non-transgenic (T) F
2
, and (T) B(BC
n
)
embryo donor ear phenotypes were similar, with up
to 200 zygotic embryos per ear (Fig. 2a), however
(T) A(BC
n
) embryo donor ears were consistently
small with poor seed set (not shown). Transient GUS
expression in (T) germplasm embryos was observed
(Fig. 2b), and the average transformation frequency
(TF) for the (T) germplasms was equal to or higher
than for the (N) HTF
2
control material (Table 2).
Across the 2003 and 2004 field seasons, the highest
average TF was achieved for (T) B(BC
0
) material
(19%) compared with the (N) HTF
2
control (12%).
Histochemical GUS expression was observed in 62%
of the bialaphos resistant callus events assayed in
this study and expression of the unselected gene did
not appear to depend on germplasm (Table 2).
Except for the male-sterile tassel phenotype, all
(T) germplasm transgenic plants resembled those of
(N) germplasm control plants and transgenic ear
sizes were similar (Fig. 2e). Average female fertility
(events with > 50 kernels) for transgenic events de-
rived from (T) germplasm was higher (71%) than
for (N) HTF
2
control events (52%, Table 2). Finally,
none of the 97 (T) cytoplasm transgenic plants (rep-
resenting 67 independent transgenic events) taken
to maturity shed pollen, while an average of 78% of
the 35 (N) HTF
2
transgenic events in this study shed
pollen at maturity (Table 2, Fig. 1). Furthermore, all
32 R
1
progeny plants (representing 16 gun-derived
events) taken to maturity after progeny tests were
male sterile (not shown).
Agrobacterium-mediated transformation
of male-sterile germplasm
Greenhouse and field immature embryos of (N)
HTF
2
and (T) germplasms were transformed with
the Agrobacterium standard binary vector pTF102
(EHA101) (FRAME et al., 2002) in the spring and sum-
mer, respectively, of 2004. On average, the three (T)
male-sterile lines transformed at higher frequencies
than did the (N) HTF
2
male-fertile control, demon-
strating that this T-cytoplasm material is readily
transformable using the Agrobacterium-mediated
205MALE-STERILE MAIZE FOR PHARMACEUTICAL PRODUCTION
TABLE 3 - Agrobacterium-mediated transformation frequency, seed set, and pollen shed for Hi II (N) and (T) germplasms.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Donor # Bialaphos % Stable % Events % Events
Source plant # Ears # Emb inf
a
resist % TF
b
events GUS with with pollen
genotype events positive
c
>50K
d
shed
e
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
2004 gh (N) HTF
2
2 286 5 2 100 93 100
(T) F
2
18 1903 27 1 91 86 0
(T) A (BC
0
) 7 579 25 4 100 65 0
(T) B (BC
0
) 9 748 12 2 83 66 0
2004 field (N) HTF
2
12 659 44 7 88 NT NT
(T) F
2
5 290 12 4 92 NT NT
(T) A (BC
0
) 15 907 82 9 90 NT NT
(T) A (BC
1
) 8 466 102 22 84 NT NT
(T) B (BC
0
) 9 448 21 5 100 NT NT
(T) B (BC
1
) 12 661 28 4 78 NT NT
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
a
all embryos infected (inf) with pTF102(EHA101) standard binary vector and co-cultivated on medium with 300 mg/L L-cysteine.
b
# independent bialaphos (3 mg/L) resistant events / total # embryos infected (X100).
c
all callus events were assayed for histochemical GUS expression.
de
# independent events to seed (1 or 2 plants per event) ranged from 12 -23 events.
NT (not tested).

method described (Table 3). In field 2004 experi-
ments, a TF of 22% was achieved using (T) A(BC
1
)
germplasm compared with 7% for the (N) HTF
2
control. (N) HTF
2
, (T) F
2
and BC
0
of (T) A and (T)
B were all targeted in both the greenhouse and field
experiments in 2004 and the average TF for field-de-
rived embryos (6%) was higher than that for the
greenhouse study (2%). In histochemical GUS assays
carried out on all bialaphos resistant callus events
recovered from Agrobacterium-mediated experi-
ments, 90% of events also expressed the gus gene
(Table 3, Fig. 2d). An average of 72% of the 56 (T)
male-sterile transgenic plants (representing 41 inde-
pendent transgenic events) grown to maturity in the
green house were female-fertile and none shed
pollen. In contrast, the five (N) HTF
2
control events
(1 plant/event) in this study shed pollen at maturity
and all were female-fertile (Table 3).
Progeny analysis of transgenic events
Analysis for expression of the bar and gus trange-
nes in gun-derived R
1
progeny plants demonstrated
Mendelian inheritance of the bar gene in 14 of 15 (T)
germplasm derived events and 2 of 3 (N) HTF
2
events (Table 4). In contrast, gus marker gene expres-
sion in segregating progeny did not follow the pre-
dicted pattern for Mendelian inheritance at a single
locus in almost half of the events (8 of the 18 events).
These abnormal segregation ratios were observed in
both male-sterile and male-fertile genetic back-
grounds (Table 4). Expected segregation ratios were
observed for bar gene expression in all R
1
progeny
plants derived from Agrobacterium-mediated trans-
formation events (Table 4), and only 2 of these 12
events segregated abnormally for the gus gene.
Introgressing of transgenic maize expressing
subunit vaccine into male-sterile
germplasm via breeding
One strategy proposed for the safe production of
pharmaceutical maize seed is to express the phar-
maceutical proteins in a male sterile genetic back-
ground (LAMKEY, 2004). A breeding scheme using 3
genotypes was designed for transferring the trans-
gene into a T-cytoplasm male-sterile (CMS) back-
ground. In order for a T-cytoplasm line to produce
pollen, it must carry dominant alleles of two nuclear
genes, Rf
1
and Rf
2
(SCHNABLE and WISE, 1998; WISE et
al., 1999a,b). Plants with the genotype Rf
1
/rf
1
,
Rf
2
/rf
2
can also be fertile as they carry the required
copy of the dominant Rf
1
allele plus 1 copy of the
dominant Rf
2
allele.
The first line, designated (T) B37 rf
1
/rf
1
, Rf
2
/Rf
2
,
b/b, was inbred B37 that contained male-sterile cyto-
plasm, was male-sterile, and did not contain the LT-
B gene (designated by b/b). The second line, desig-
nated (N) B37 rf
1
/rf
1
, Rf
2
/Rf
2
, b/b, contained normal
cytoplasm, was male-fertile, and did not contain the
LT-B gene. This line is referred to as a maintainer
line because it is needed to produce seed from the
male-sterile line. The third line, designated (N) HT
rf
1
/rf
1
,Rf
2
/Rf
2
, B/B, carried the transgene, and was
male-fertile. In this line, the transgene cassette con-
tains a gene encoding the B-subunit of the E. coli
heat labile enterotoxin (LT-B) under control of the
27 kD gamma zein promoter (an endosperm-specific
promoter). The transgene was introduced into (N)
HT germplasm (CHIKWAMBA et al., 2002a) and the LT-
B expressing homozygous line was generated after
several generations of self pollination. B is the trans-
gene we wanted to transfer to T cytoplasm, and
throughout this study, we assumed that HT
germplasm was a non-restoring genotype.
Figure 3 illustrates the breeding strategy used for
transferring the LT-B gene into male-sterile (T) B37.
Note that females are always listed first in a cross.
In Steps 1 and 2, we generated a BC
1
population
that contained 50% heterozygous transgenic seeds
(N) B37
2
rf
1
/rf
1
,Rf
2
/Rf
2
, B/b and 50% non-trans-
genic segregants (N) B37
2
rf
1
/rf
1
,Rf
2
/Rf
2
,b/b, by
crossing the male-fertile restorer line (N) B37
rf
1
/rf
1
,Rf
2
/Rf
2
,b/band transgenic heterozygous line
(N) HT rf
1
/rf
1
,Rf
2
/Rf
2
, B/b (produced in Step 1).
In Step 3, any non-transgenic segregants were
effectively eliminated at the seedling stage by spray-
ing with herbicide Liberty®. Pollen from herbicide
resistant heterozygous transgenic seeds (N) B37
2
rf
1
/rf
1
,Rf
2
/Rf
2
, B/b was used to cross with the male-
sterile (T) B37 rf
1
/rf
1
,Rf
2
/Rf
2
,b/b. This step resulted
in a male-sterile population containing 50% het-
erozygous transgenic seeds, (T) B37
3
rf
1
/rf
1
,
Rf
2
/Rf
2
, B/b, and 50% non-transgenic segregants, (T)
B37
3
rf
1
/rf
1
,Rf
2
/Rf
2
,b/b.
Step 4 (a, b and c) demonstrates how we selected
and bred a homozygous transgenic maintainer line
(N) B37
2
rf
1
/rf
1
,Rf
2
/Rf
2
, B/B from the BC
1
maintainer
line harvested in Step 2. Once the homozygous trans-
genic maintainer line was achieved, it was used in
Step 5 to pollinate the heterozygous sterile BC
2
gen-
erated from Step 3. Seeds from Step 5 were male-
sterile and all carried the LT-B transgene, 50% (T)
B37
4
rf
1
/rf
1
,Rf
2
/Rf
2
, B/B and 50% (T) B37
4
rf
1
/rf
1
,
Rf
2
/Rf
2
, B/b. This seed was then used in field produc-
tion (Production 1) for LT-B maize, in which a non-
206 K. WANG, B. FRAME, X. XU, L. MOELLER, K. LAMKEY, R. WISE

transgenic line was used as the pollen donor. After
this four-season breeding cycle, 75% of the seeds
harvested were expected to express the LT-B protein.
To achieve 100% LT-B seed from field production
(Production 2), the homozygous transgenic sterile
line (T) B37
5
rf
1
/rf
1
,Rf
2
/Rf
2
, B/B) was generated over
an additional two growing seasons (Step 6).
This breeding strategy was accomplished using
both greenhouse facilities and field nurseries. Open-
field containment procedures followed strict USDA-
APHIS regulations. Transgenic pollen remained con-
tained at all times by bagging tassels, or by using
male sterile material. Tassel production and pollen
viability in field conditions were monitored after the
207MALE-STERILE MAIZE FOR PHARMACEUTICAL PRODUCTION
TABLE 4 - Segregation analysis for gus and bar gene expression in R
1
generation progeny plants
a
.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Segregation Ratio
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Genotype Event ID Herbicide Gus
––––––––––––––––––––––––––––––– X
b
––––––––––––––––––––––––––––––– X
2f
Res
b
Sen
c
Pos
d
Neg
e
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Biolistic events
g
HTF
2
187 7 8 0.1 6 9 1
(T) F
2
753 13 12 0.0 0 25 25
(T) F
2
771 4 21 11.6 3 22 14
(T) F
2
777 11 18 1.7 13 16 0
(T) F
2
760 12 17 0.9 0 29 29
(T) F
2
752 11 18 1.7 15 14 0
HTF
2
193 8205.1 02828
(T) A(BC
0
) 1057 17 13 0.5 0 30 30
(T) A(BC
0
) 1066 13 15 0.1 5 23 12
(T) A(BC
0
) 1028 15 14 0.0 15 14 0
(T) A(BC
0
) 1024 8 12 0.8 9 11 0
(T) A(BC
0
) 1018 12 16 0.6 0 28 28
HTF
2
186 12 18 1.2 0 30 30
(T) B(BC
0
) 1252 14 11 0.4 17 8 3
(T) B(BC
0
) 1108 16 14 0.1 15 15 0
(T) B(BC
0
) 1207 10 15 1.0 16 9 2
(T) B(BC
0
) 1176 9 15 1.5 13 12 0
(T) B(BC
0
) 1204 18 11 1.7 19 10 3
Agro events
g
HTF
2
63 12 15 0.3 16 11 1
(T) F
2
468 16 14 0.1 15 15 0
(T) F
2
465 13 12 0.0 12 13 0
(T) F
2
405 15 12 0.3 0 27 27
HTF
2
72 14 15 0.0 15 14 0
(T) A(BC
0
) 453 16 11 0.9 14 13 0
(T) A(BC
0
) 446 10 16 1.4 9 17 3
(T) A(BC
0
) 426 17 12 0.9 15 14 0
HTF
2
64 20 10 3.3 15 15 0
(T) B(BC
0
) 429 14 16 0.1 18 12 1
(T) B(BC
0
) 444 12 15 0.3 5 22 11
(T) B(BC
0
) 445 13 17 0.5 16 11 1
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
a
Transgenic plants were crossed as the female parent with pollen from non-transformed B73 plants or Hi II (Agro event #’s 64, 72, 444).
b
Res, resistant to glufosinate spray (bar-expresser).
c
Sen, sensitive to glufosinate spray (bar non-expresser).
d
Pos, GUS assay positive (gus-expresser).
e
Neg, GUS assay negative (gus non-expresser).
f
X
2
=3.8 (0.05, 1 df).
g
biolistic events transformed with pAHC25 and Agro events with pTF102 (EHA101).

208 K. WANG, B. FRAME, X. XU, L. MOELLER, K. LAMKEY, R. WISE
FIGURE 3 - Breeding schemes for achieving (a) 75% and (b) 100% LT-B maize seed production in the field. T, male sterile, N, male fertile,
rf
1
/rf
1
,Rf
2
/Rf
2
, nuclear restorer gene genotype, B, dominant transgene LT-B. Genotype with letter strikethrough represents non-transgenic
segregants eliminated by herbicide spraying at plantlet stage.
(a)
(b)

transgene was transferred into the male-sterile line.
All of these plants produced tassels, 75% of which
did not shed pollen. In the 25% of transgenic male-
sterile plants that did shed pollen, no viable pollen
was detected (X. Xu, unpublished), indicating that
no fertility was restored in these plants.
CONCLUSIONS
We describe possible strategies for using cytoplas-
mic male sterile (cms-T) germplasm in open-field
production of transgenic maize producing a pharma-
ceutical product. In the first approach, a male-sterile
germplasm amenable for tissue culture and transfor-
mation was developed from the widely used Hi
Type II genotype. Male-sterile transgenic events
(non-pharmaceutical) were generated using the bi-
olistic gun and Agrobacterium-mediated transforma-
tion methods and grown to maturity in the green-
house. While in vitro culture induced variation did
not alter the male sterile phenotype in this study, the
possibility that this could occur cannot be ruled out.
Secondly, a pharmaceutical transgene introduced
into (N) cytoplasm using the biolistic gun was trans-
ferred into a male-sterile genotype via conventional
breeding. Our transgene or the transgene insertion
site did not restore male fertility in transgenic maize
plants (although a non viable pollen was shed in
25% of plants), thereby affirming the applicability of
this technology. However, it is prudent to state that
any potential restoration or reversion should be ex-
amined on a gene-by-gene basis.
There may be a third strategy that could acceler-
ate production of male-sterile homozygous pharma-
ceutical maize seed. Both male-fertile and male-ster-
ile genotypes can be directly transformed with the
transgene of interest. The male-fertile transgenic
maize that serves as the maintainer line could be
made homozygous and its pollen could then be
used to cross to the male-sterile transgenic female.
Progeny would then carry two copies of the trans-
gene at different loci. While this practice may save
2-3 seasons of breeding, the presence of multiple
transgene copies may cause transgene silencing
(M
ENG et al., 2003; SHOU et al., 2004).
The outcome of using cms-T maize for large
scale open-field release of a pharmaceutical product
is untested. T-cytoplasm maize is highly sensitive to
the host selective pathotoxin HmT, produced by
Cochlobolus heterostrophus Drechsler race T (WISE
et al., 1999a). While no obvious pathogen damage
was observed in the small field experiments carried
out in our study, it is important to bear in mind that
recurrent large scale field release of cms-T genotype
may encourage reappearance of Southern maize
leaf blight, which could lead to yield loss in Texas
male-sterile, pharmaceutical maize.
ACKNOWLEDGEMENTS - The authors thank Deqing Pei for initi-
ating the Hi II T-cytoplasm lines for transformation, Tina Paque
for greenhouse and field assistance in preparing transformation
starting materials, Lise Marcell, Marcy Main, Jenifer McMurray and
Kyle Taylor for their assistance in transformation experiments
and progeny tests, Horan BioProduction, Kent Berns, Kevin
VanDee, Mark Honeyman for the breeding program. Partial fi-
nancial support for this project is from Iowa State University
Plant Science Institute BioPharmaceuticals and BioIndustrials Ini-
tiative, USDA-ARS CRIS project 3625-21000-035-00D, and Pioneer
Hi-Bred International.
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