Plant Physiol. (1996) 110: 1097-1107
The Competence of Maize Shoot Meristems for lntegrative
Transformation and lnherited Expression of Transgenes'
Heng Zhong, Baolin Sun, Donald Warkentin, Shibo Zhang, Ray Wu, Tiyun Wu, and Mariam B. Sticklen*
202 Pesticide Research Center, Department of Crop and Soil Sciences, Michigan State University, East Lansing,
Michigan 48824-1 31 1 (H.Z., B.S., D.W., S.Z., M.B.S.); and Section of Biochemistry, Molecular and Cell
Biology, Cornell University, Ithaca, New York 14853 (R.W., T.W.)
mays L.) morphogenesis demonstrated that the maize meristem is morphogenetically plastic and can be manipulated
to produce multiple shoots, somatic embryos, tassels, or
ears in a relatively genotype-independent manner by simple variation of in vitro culture conditions (Zhong et al.,
1992a, 1992b). Based on this concept, we transformed
maize meristems via microprojectile bombardment with a
series of chimeric genes, including bar, pin2, and gus.
In this paper, we report the efficient recovery of fertile
transgenic maize plants via a shoot-multiplication system
after microprojectile bombardment of shoot tips. Maize
shoot apices were transformed with a plasmid incorporating bar driven by the CaMV 35s promoter and pin2 with the
wound-inducible pin2 promoter (Fig. l), either alone or in
combination with another plasmid containing gus driven
by the 5' region of Actl (Fig. 1). The co-integration and
co-inheritance of linked and unlinked genes in transgenic
R,, RI,and R, maize plants were confirmed. The functional
activity of Actl-gus in transgenic R, and RI plants was
extensively analyzed.
We have developed a nove1 and reproducible system for recovery
of fertile transgenic maize (Zea mays L.) plants. The transformation
was performed using microprojectile bombardment of cultured
shoot apices of maize with a plasmid carrying two linked genes, the
Strepfomyces bygroscopicus phosphinothricin acetyltransferase
gene (bar) and the potato proteinase inhibitor II gene, either alone
or in combination with another plasmid containing the 5' region of
the rice actin 1 gene fused to the Escbericbia coli p-glucuronidase
gene (gus). Bombarded shoot apices were subsequently multiplied
and selected under 3 to 5 mg/L glufosinate ammonium. Co-transformation frequency was 100% (146/146) for linked genes and
80% (41/51) for unlinked genes. Co-expressionfrequency of the bar
and gus genes was 5 7 % (29/51). The co-integration, co-inheritance,
and co-expression of bar, the potato proteinase inhibitor II gene,
and gus in transgenic R,, R,, and R, plants were confirmed. Localized expression of the actin 1-CUS protein in the R, and R, plants
was extensively analyzed by histochemical and fluorometric assays.
The shoot tip, or shoot apex, consists of the shoot apical
meristem, a region in which lateral organ primordia form,
a subapical region of cell enlargement, and severa1 leaf
primordia (Steeves and Sussex, 1989). The meristem region
contains apical initial cells and subepidermal cells from
which the gametes are derived (Medford, 1992). Theoretically, there are two possibilities for recovering transgenic
plants via transfer of DNA into the shoot apical meristem.
One possibility is that transgenic progeny may be directly
produced via transformation of the subepidermal germline cells followed by the development of a partially transgenic reproductive organ. In this case, the primary transformants will always be chimeric. An alternative
possibility is to multiply transgenic apical meristem cells
and/or germ-line cells, which can be reprogrammed in the
developmental direction under in vitro conditions. Transgenic plants can be regenerated from these cells with or
without selection. Our previous research on maize (Zea
MATERIALS A N D M E T H O D S
Plant Materiais
Following our previous work on shoot multiplication of
20 genotypes of maize (Zea mays L.) (Zhong et al., 1992a),
another 16 genotypes were tested. A11 of them responded to
form multiple-shoot-tip clumps (data not shown). We randomly selected 12 genotypes (HNP, IGES, B73, VA22, and
FR632 from Illinois Foundation Seeds, Champaign, IL; 509,
5922, and 482 from the Michigan Agricultural Experiment
Station, East Lansing, MI; K1 from the Institute of Botany,
Academy of Science, Beijing, China; A188 from the Crop
Breeding Project, Department of Agronomy, University of
Minnesota, St. Paul; and L6 and L9 from the Agricultural
Abbreviations: Actl, rice actin 1 gene; BA, N6-enzyladenine; bar,
bacterial phosphinothricin acetyltransferase gene; CaMV, cauliflower mosaic virus; CSM, MS medium containing 2 mg/L BA;
CSMD, CSM containing 0.5 mg/L 2,4-D; CSMDSG, CSMD containing 3 mg/L G; CSMD5G, CSMD containing 5 mg/L G; gus, bacteria1 GUS gene; HNP, Honey N Pearl; IBA, indole-3-butyric acid;
IGES, Illinois Golden Extra Sweet; G, glufosinate ammonium
[monoammonium 2-amino-(hydrooxymethyl-phosphiny)butanoate]; MS, Murashige and Skoog basal medium; nos, nopaline synthase gene; PAT, phosphinothricin acetyltransferase; pin2, potato
proteinase inhibitor I1 gene.
This research was supported by the Office of USAID/CAIRO/
AGA/A, under Cooperative Agreement No. 263-0152-A-003036-00 and Michigan State University Bridging Funds to M.B.S.
The construction of plasmids was performed at Cornell University.
The development of the transformation system, the transformation, and the analysis of transformants were performed at Michigan State University.
* Corresponding author; e-mail sticklelQpilot.msu.edu; fax
1-517-353-1698.
1097
Zhong et al.
1098
H
B
v
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X*
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S B
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v
B,H
EV*;E
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0.8
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B E
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27
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Figure 1. Schematic representation of plasmids. pTW-a contains the
pin2 promoter (pin2 5'), the rice Actl 5' intron (As'l), the pin2
coding sequence (pin2), and the pin2 terminator (pin2 3') followed
by the CaMV 35s promoter (35S), the barcoding sequence (bar), and
the nos terminator (nos) in the vector pUC 19. pActl-F contains the
5' flanking sequence (A 5' FS), the noncoding portions of Actl exon
(open box), the Actl intron (A5'I), the 5' coding portions of Actl
exon (open box), the gus coding sequence (pus), and the nos terminator (nos) in the vector pBluescript KS (pBlu. KS). The restriction
enzyme sites are abbreviated as follows: B, BamHI; E, EcoRI; EV,
EcoRV; H, Hindlll; N, Nod; S, Sacl; and X, Xhol. *, Unique site in
plasmids. Numerals below each region are length of fragments in kb.
Genetic Engineering Research Institute, Giza, Egypt) for
this research. Mature kernels of 12 genotypes were surfacesterilized with 70% ethanol and 2.6% sodium hypochlorite
and germinated on MS (Murashige and Skoog, 1962) basal
medium (Sigma) as described (Zhong et al., 1992a).
A 5-mm-long section of a 7-d-old seedling containing a
shoot tip, leaf primordia, and a portion of young leaves and
stem proximal to the shoot tip was excised, cultured in
darkness for 4 weeks, and then maintained in light (24 h, 60
pmol [quanta] m-* s-') at 4-week intervals on shoot multiplication medium CSMD or CSM (Zhong et al., 1992a).
Initiation of shoot-tip clumps from the original shoot tips
occurred 2 to 4 weeks after culture. Repeated subculture of
divided shoot-tip clumps resulted in a higher frequency of
shoot-tip formation and more compact shoot-tip clusters.
Morphologically, greater numbers of adventitious shoot
tips but less leaf and stem elongation were produced in
CSMD than in CSM, although both CSM and CSMD favored the axillary and adventitious bud formation (Zhong
et al., 1992a) for all genotypes tested. Shoot tips from the
multiplied clumps at the end of each subculture (4 weeks)
usually have severa1 germinated leaves. The shoot tips
with one to three visible leaf primordia were selected for
bombardment under a stereomicroscope (Carl Zeiss).
Because the sweet corn genotypes HNP and IGES were
our model materials for morphogenesis studies, shoot multiplication cultures of these two genotypes initiated on July
15, 1990, were also used for this research.
Plant Physiol. Vol. 'I 1 O, 1996
pin2 coding region was driven by the wound-inducible pin2
promoter and rice actin 1 (Actl) 5' intron and terminated
with the pin2 3' region (Fig. 1).
The plasmid pActl-F (6.5 kb; McElroy et al., l990) contained the Esckerichia coli u i d A gene (gus) (Jefferson, 1987)
driven by a region 1.3 kb upstream of the rice A c t l translation codon (McElroy et al., 1990) and followed by the nos
3' terminator in the pBluescript KS vector (Fi,g. 1). The
plasmid DNA was suspended in 10 mM Tris-HC1and 1 mM
EDTA buffer (pH 8.0) at a concentration of 1 pg/pL.
Microprojectile Bombardment
Plasmid DNA was precipitated onto gold particles (1.0
and 1.6 pm in diameter; Bio-Rad) or tungsten particles
(0.9-1.2 pm in diameter; GTE Sylvania, Towanda, PA)
following a modification of the original protocol described
by Bio-Rad. Briefly, 30 mg of particles were sterilized in 1
mL of 100% ethanol with vortexing for 30 min. A 50-pL
aliquot of the particle-ethanol suspension was pipetted into
a microcentrifuge tube while vortexing continuously. After
washing twice with sterile-distilled water, the particles
were resuspended in 332 pL of sterile-distilled water. A
total of 15 pL of plasmid DNA (15 pg of plasmid or a 1:l
mixture of two plasmids), 225 pL of CaCI, (2.5 M), and 50
pL of spermidine (0.1 M) were successively added and
continuously vortexed for 5 min at room tempel-ature. The
mixture was then incubated on ice for 10 min. The DNAcoated particles were pelleted by centrifuging at 13,000
rpm for 1 min. After discarding the supernatant, the particles were washed with 500 pL of absolute ethanol by
vortexing for 30 s, centrifuging for 1min, and removing the
supernatant and were finally resuspended in 100 or 200 pL
of absolute ethanol. For each bombardment, 10 pL of the
particle suspension (150 or 75 pg of particles per shot) was
pipetted onto the center of the macrocarriers. The prepared
macrocarriers were used as soon as the ethanol evaporated.
Prior to bombardment, shoot tips or shoot-tip clumps
from cultures of various stages were physically exposed by
remova1 of the coleoptile and leaves if necessary. The shoot
tips (placed in a circular area of about 1.5 cm diameter)
were positioned on multiplication medium solidified with
0.5% Phytagel (Sigma) in a Petri dish below the microprojectile stopping screen.
Bombardments were carried out using a Biolistic particle
acceleration device (PDS 1000/He, Bio-Rad) under a chamber pressure of 26 mm of Hg at distances of 1.5,2.0, and 6.5
cm from the rupture disc to the macrocarriers to the stopping screen to the target, respectively, with three helium
pressures (1100, 1550, and 1800 p.s.i.) and single or multiple shots per plates.
Plasmids
Selection and Regeneration of Transformants
The plasmid pTW-a (7.4 kb) contained the Streptomyces
kygroscopicus bar gene (Thompson et al., 1987) and the gene
pin2 (Keil et al., 1986) in the pUC19 vector. The bar coding
For analysis of the variation in stable expression in shoot
tips among different genotypes, bombarded explants were
immediately transferred to fresh multiplication medium
without selection for 6 weeks with one subculture. The
explants used for bombardment were at least 1-month-old
region was driven by the CaMV 355 promoter and was
terminated with the nos 3' region (Bevan et al., 1983).The
Maize Transformation via Bombardment of Shoot Tips
cultured shoot tips due to the intensive labor required to
dissect shoot tips from young maize seedlings.
For recovery of the transformants, bombarded shoot tips
were cultured on multiplication medium CSMD for 4
weeks. The clumps then were divided and subcultured on
CSMD3G for 4 weeks. Subsequent subculture at 4-week
intervals was carried out by selecting, dividing, and culturing green clumps on CSMD5G.
Regeneration of plants was obtained by transfer of
CSMD5G-selected shoot-tip clumps to MS medium containing 0.5 mg/L BA, 0.5 mg/L IBA, and 5 to 10 mg/L G.
Developed shoots were rooted on MS medium containing 1
mg/L IBA and 10 mg/L G. Plantlets then were transplanted to a soil mixture composed of 1:l (v/v) peat:perlite
and grown to maturity.
Histochemical and Fluorometric Assay of CUS
The modified histochemical assay buffer consisted of
100 mM NaPO, buffer, 100 mM Na,EDTA, 50 mM
K,Fe(CN), 3H,O, and 0.1% Triton X-100 (pH 7.0). 5-Bromo4-chloro-3-indolyl-/3-~-glucuronic
acid (Clontech Laboratories, Palo Alto, CA) was dissolved in 50% ethanol, stored at
-2O"C, and added to buffer to a final concentration of 0.5
mg/mL prior to assay.
The fluorometric assay buffer consisted of 50 mM NaPO,
buffer, 10 mM P-mercaptoethanol, 10 mM Na,EDTA, 0.1%
sodium lauryl sarcosine, and 0.10/, Triton X-100. 4-Methylumbelliferyl /3-D-glucuronide was added to buffer at a
final concentration of 1 mg/mL prior to assay.
Whole plantlets and organs from the transgenic and
untransformed control plants were immersed in GUS substrate mixture immediately followed by vacuum treatment,
then incubated at 37°C. The histochemical and fluorometric
localization of GUS activity was carried out using freehand cross-sections. The stained materials were examined
under a Zeiss SV8 stereomicroscope or a Zeiss Axioskop
routine microscope.
Based on our studies, transient expression of gus in shoot
tips of maize 1 d after bombardment was comparable to
that in type-I and type-I1 embryogenic calli and immature
embryos of maize. A preliminary study using multiple
shoot tips of maize genotype HNP showed that the number
of blue sectors in shoot-tip clumps was similar 3 weeks and
3 months after bombardment. Therefore, expression of gus
in shoot tips 6 weeks after bombardment was considered to
be stable. The expression of gus in bombarded shoot-tip
clumps was observed under a stereomicroscope. A single
isolated cell or an aggregate of cells exhibiting dark blue
color in shoot meristems other than in leaves was considered as one GUS-expressing unit. The efficiency of stable
expression in shoot tips was relative to the number of shoot
tips bombarded. To localize the position of cells with stable
expression in the shoot tips, tissues with blue foci were
carefully dissected and examined under the microscope.
Chl was extracted by successive incubation in 70% ethano1 for 2 h and 100%ethanol overnight before the samples
were photographed.
1 099
PAT Activity Assay
The PAT activity was determined indirectly by resistance
of plants to herbicide application. Two applications of Ignite nonselective herbicide (also known as Basta; HoechstRoussel Agri-Vet Company, Somerville, NJ) containing 200
g/L G, the active ingredient, were performed. For R,
plants, a 1%solution of herbicide (v/v) was sprayed on
whole plants at the three-leaf and six-leaf stage in the
greenhouse. For RI and R, plants, the herbicide (1%) was
also locally painted on the youngest leaf at the three-leaf
stage and sprayed on whole plants at the six-leaf stage.
D N A C e l Blot Analysis
Genomic DNA was isolated from G-resistant multiple
shoot-tip clumps and from leaf tissues of untransformed
plants, primary transformants, and their progeny using the
hexadecyltrimethylammonium bromide method (Rogers
and Bendich, 1985). Ten micrograms of DNA per sample
were digested with restriction enzymes, fractionated in a
1% agarose gel, and transferred to Nytran membranes
(Schleicher & Schuell). Following the procedure recommended by Schleicher & Schuell, after prehybridization,
hybridizations were carried out with an [a-32P]dCTPprobe
labeled with T7 Quick Prime (Pharmacia) as described by
the manufacturer. Filters were analyzed by autoradiography using X-Omat film (Kodak).
Production and Analysis of Progeny
Transgenic plants were self-pollinated. Screening of
transgenic progeny containing bar was done either through
germination of immature embryos or mature caryopses on
MS basal medium with 5 mg/L G to minimize the time
required to obtain transgenic R, and R, plants, or through
treatment of greenhouse-germinated progeny with the herbicide Ignite. To detect the transgenic progeny containing
gus, roots, immature embryos, young leaf sections, and
pollen grains of RI and R, progeny were subjected to
histochemical assay. The segregation data were statistically
analyzed by
test (Strickberger, 1985).
2
RESULTS
Stable Expression of gus in Shoot Tips after Bombardments
Bombardment of undissected shoot tips using GUS as a
visible marker revealed that most expression events occurred on the leaves that covered the shoot tip or that
developed from leaf primordia. No expression was observed in shoot meristems after further development of
shoot-tip explants without multiplication (data not shown).
The efficiency of both transient and stable expression in
shoot tips was greatly affected by the penetration of particles, which depended on the size, quality, density, and
velocity of the particles. The larger gold particles (1.6 pm)
incorporated with high density, multiple bombardments
with 1800 p.s.i. acceleration pressure showed the lowest
transient and stable expression, but reducing the particle
density and/or acceleration pressure improved the effi-
1100
Zhong et al.
ciency of stable expression. Using smaller gold particles,
the most efficient level of stable expression was obtained
with a medium velocity (1550 p.s.i.), multiple shots (up to
four), and a low density of particles (75 /xg/shot). Tungsten
particles and gold particles gave comparable results. The
low density of particles (75 jag/shot) with multiple shots
gave a higher ratio of stable expression than the high
density of particles (150 jug/shot) regardless of single or
multiple shots.
The efficiency of stable expression in shoot tips of HNP
varied from 2% (bombarded twice with 150 ^,g of 1.6-jj.m
gold particles at 1800 p.s.i.) to 32% (bombarded four times
with 75 pig of 1.0-jum tungsten particles at 1550 p.s.i.). The
majority of blue sectors were observed out of the center of
the target 6 weeks after bombardment. From 530 shoot tips
examined, the percentage of blue foci below the epidermal
layer of the meristem was about 65% with gold and 72%
with tungsten with 1550 p.s.i. pressure, and about 22%
with gold and 10% with tungsten with 1100 p.s.i.
Shoot tips from 1-month-old cultures showed lower efficiency of stable expression after bombardment than older
cultures, although the level of transient expression was
similar. However, the efficiency of stable expression was
much higher in shoot tips bombarded at 1 or 2 weeks (30%)
than in shoot tips bombarded at 4 weeks after subculture
(8%). There was no significant difference in transient or
stable expression between the two media, CSM and CSMD.
Figure 2. Recovery of transgenic maize plants.
A, Shoot tips prior to bombardment (X40). B,
Untransformed controls after 1 month of selection (1) and recovery and multiplication of Gresistant multiple shoot-tip clumps bombarded
with pTW-a after 1st month (2), 2nd month (3),
and 3rd month (4) of selection (X0.4). Arrows
show some of the G-resistant clumps. C, A tassel
of an herbicide-resistant R0 plant (X0.1). D, Ears
of an herbicide-resistant R0 plant (X0.1). E, R,
progeny 10 d after foliar spray of 1% Ignite. All
of the nontransformed control plants (1 and 2)
are sensitive, whereas transformed plants (3-6)
segregated into resistant and sensitive individuals (marked by arrows) (X0.25).
Plant Physiol. Vol. 110, 1996
Different genotypes did not show any significant difference
in either transient or stable expression of gus.
Thus, the optimal delivery of DNA into subepidermal
cells of shoot tips was achieved by the bombardment of
more than 2-month-old shoot-tip clumps that were subcultured every 2 weeks, with a low density (75 fig/shot) of
1.0-nm gold or tungsten particles with multiple shots (four)
and 1550 p.s.i. acceleration pressure.
Recovery of Stably Transformed Shoot-Tip Clumps and
Regeneration of Transformants
HNP and IGES were chosen for recovery of transformants because they were model materials for our morphogenesis study. Their shoot-tip clumps were regularly initiated and continuously subcultured for more than 4 years
without loss of regeneration ability or fertility. The shoot
tips were carefully selected and gathered under a stereomicroscope as shown in Figure 2A prior to bombardment
under the optimal conditions described above. As many as
five blue sectors showing stable expression were found in
a single shoot tip 1 month after bombardment (Fig. 3A).
Further development of the bombarded shoot tips without
multiplication gave rise to different patterns of chimeric
shoots after GUS histochemical assay (Fig. 3B). The blue
sectors at the leaf tips or on the leaf blade were most
probably from the transformed cells of the leaf primodium,
Maize Transformation via Bombardment of Shoot Tips
which could not produce multiple shoot tips under our
culture conditions, whereas the development of transformed meristematic cells could result in the successive
formation of blue sectors through the leaf blades and
sheaths. The 1-month multiplication of bombarded shoottip clumps without selection pressure gave similar developmental patterns.
Production of PAT- and CUS-Positive Shoot-Tip Clumps
The transformation results with plasmid pTW-a (which
contains the bar gene) from eight independent experiments
with a total of 22 plates are summarized in Table I. The
efficiency of recovery of G-resistant shoot-tip clumps after
the first round of selection (1month) with 3 mg/L G varied
from O to 6.7% under different culture periods prior to
selection.
Six independent co-transformation experiments with
two unlinked genes (bar and gus in plasmids pTW-a and
pAct2-F, respectively) gave a slightly lower efficiency of
recovery of G-resistant shoot-tip clumps after the first
round of selection, ranging from O to 3.4% (Table 11), compared to the efficiency of two linked genes, bar and pin2, in
plasmid pTW-a (Table I).
Our results indicated that 1 month of multiplication of
bombarded shoot-tip clumps without selection pressure is
necessary to achieve a high efficiency of transformation.
More than 1 month of multiplication did not improve the
recovery of resistant shoot-tip clumps. During the 1st
month of selection on CSMD3G, most of the bombarded
and unbombarded (control) shoot-tip clumps turned
brown and necrotic within 2 weeks. A small number of
G-resistant shoot tips emerged clearly with a healthy green
color from these necrotic tissues near the end of the 1st
month of selection (Fig. 28). The shoot-tip clumps from
control experiments rarely survived in this selection
scheme. The size of the G-resistant clumps varied from less
than 0.1 to 0.3 cm’. Each clump contained 5 to 20 visible
shoot tips. Since the multiple shoot-tip clumps were very
compact, intimate signal communication and substance exchange between cells in the clumps provided a great possibility of cross-protection between transformed cells and
nontransformed cells. Chimeric clusters of shoots without
selection pressure were produced at such a stage (Fig. 3C).
Further selection of transformed shoot-tip clumps devoid
of nontransformed cells was carried out by dissecting, dividing, and culturing of the surviving shoot tips on
CSMD5G until a11 divided shoot-tip clumps uniformly survived in CSMD5G. Clumps resistant to 5 mg/L G were also
resistant to 10 mg/L G. In both HNP and IGES, stable
uniform growth clumps were obtained after three or four
rounds of selection (1 month in CSMD3G and 3 months in
CSMD5G) (Fig. 28). One independent shoot-tip clump (one
transformation event) from the 1st month of selection was
multiplied to about 15 shoot-tip clumps at 5 months after
bombardment and was capable of regenerating more than
1500 transgenic plantlets.
A11 independent G-resistant clumps were further subjected to DNA hybridization analysis for the presence of
bar, pin2, and gus (Fig. 4). Hybridization to a11 three trans-
1101
genes in high-molecular-weight DNA in a11 independent
G-resistant clumps, but not in controls, confirmed that the
transgenes bar, pin2, and/or gus were integrated into the
chromosomal DNA of a11 resistant transformants. The
transformation efficiency of two linked genes (bar and pin2)
was 100% in both transformation (95/95) and co-transformation (51/51) experiments (Tables I and 11).The co-transformation frequency of two unlinked genes (bar and gus)
was 80% (41/51) (Table 11). Histochemical assays of GUS
activity in a11 independent G-resistant transgenic clumps
(Fig. 3D) revealed a co-expression frequency of 57% (29/
51) for two unlinked genes (gus and bar). High co-transformation frequency with lower co-expression frequency of
unlinked genes suggested that the expression of gus in
some PAT-positive events did not occur or occurred at a
histochemically undetectable level.
Regeneration and Analysis of Primary Transformants (R,)
Over 2000 plantlets regenerated from 26 independent
transgenic events of both genotypes, HNP and IGES, were
transferred to the greenhouse for further development.
About 90% of them survived to maturity in the greenhouse.
Five plants from each of 10 independent transformed
clumps were analyzed for the presence of bar, pin2, and/or
gus using Southern blot hybridization. DNA hybridization
analysis of genomic DNA from each plant digested with
EcoRV (which has a unique site in pTW-a) and/or EcoRI
(which has a unique site in pAct2-F) revealed that the
patterns of hybridization bands with probes containing the
bar, pin2, or gus coding region were identical in the five
plants from the same clump, confirming that they were
from the same transformed cell, and varied from one
clump to another, suggesting that they were independent
transformation events (data not shown). A comparison of
the copy number and insertion site of a11 of the transgenes
in each different transformation event was also performed
(eg. see Fig. 5). Genomic DNA digested with EcoRI gave a
0.9-kb bar-hybridizing band that comigrated with the
0.9-kb fragment, containing the bar and nos 3’ terminator,
from the EcoRI digestion of pTW-a (Fig. 5A). Genomic
DNA digested with HindIII showed a 3.0-kb pin2-hybridizing band that comigrated with the 3.0-kb fragment, containing the pin2 promoter, the Actl 5’ intron, the pin2
coding region, and the pin2 terminator, from the HindIII
digestion of pTW-a (Fig. 5B). The EcoRV-digested genomic
DNA showed different hybridization band patterns, indicating a different integration site for both bar and pin2 in
different transformation events with various copy numbers
(Fig. 5, A and B). Following the digestion of genomic DNA
with EcoRI and NotI, a 3.3-kb gus-hybridizing band was
seen that comigrated with the 3.3-kb fragment, containing
a portion of the rice Actl promoter, the gus coding region,
and the nos 3‘ terminator, from the EcoRI-NotI digestion of
pAct2-F. Following the digestion of genomic DNA with
EcoRI (there is only one site in the plasmid pAct2-F), the
pattern of mobility and intensity of the EcoRI hybridization
bands in Figure 5C suggested that the intact gus was integrated into different sites in different independent trans-
1102
Zhong et al.
Plant Physiol. Vol. 110, 1996
Figure 3. Histochemical and fluorometric assay of Acf/-gus expression in transgenic maize plants. A, SUiblr expression of
GUS in a shoot meristem 6 weeks after bombardment without selection (X300, 30 min of staining); I, leaf; m, meristem. B,
Shoots directly developed from control (1) and transformed (2-4) shoot tips without selection 6 weeks after bombardment
with pActl-f (X0.2, 1 h of staining). Arrows show successive formation of the chimeric blue sectors through the entire
leaves. C, Shoot clumps regenerated from control (1) and 1-month G-resistant (2 and 3) shoot-tip clumps (X0.2, 1 h of
staining). D, Shoot-tip clumps from controls (column 1) and from gus-positive, G-resistant (columns 2-8) independently
transformed events (X0.35). E, Cross-section of a G-resistant multiple-shoot-tip clump 4 months after bombardment ( X 2 5 ,
1 h of staining). F, Plantlets regenerated from untransformed control (1) and G-resistant (2) shoot-tip clumps (X0.4, 3 h of
staining); sr, secondary lateral root. G, Shoot tips, TA10 (XI 20, 10 min of staining); st, shoot tip. H, Transverse section of
leaves, TA10 (X10, 20 min of staining); Ib, leaf blade; Is, leaf sheath; Iv, lateral vein; iv, intermediate vein. I, Transverse
Maize Transformation via Bombardment of Shoot Tips
1103
Table 1. Summary o f transformation experiments with pTW-a
Genotype
HNP
IGES
Subtotal
No. of
Plates
Shoot Tips
No. of
Age of
Cultures"
4
2
2
3
4
3
2
2
22
622
181
284
291
560
41 2
21 9
260
2829
50
2.5
2.5
52
23
45
3
21
Days
Postcultureb
3
3
30
30
30
30
30
30
No. of lndependent
bar+/pinPd
O
1 (0.6)
19 (6.7)
16 (5.5)
21 (3.8)
15 (3.6)
9 (4.1)
14 (5.4)
95 (3.4)
1/I
1 911 9
16/16
21/21
1 5/15
9/9
14/14
95/95
EventsC
Months after the initiation of shoot tip multiplication from the 7-d-old seedlings. All of the shoot tips for bombardment were selected from
The
the shoot-multiplication cultures 2 weeks after subculture.
Days of multiplication after bombardment before first-round selection.
number of independent events was the total number of independent G-resistant clumps obtained at the end of the 1st month of selection. The
number in parentheses is the relative efficiency (%) of stable transformation measured as total G-resistant clumps divided by total number of
bar+/~in2+shows the number of indeDendent c l u m m with each gene.
bombarded shoot tiix, X100%.
a
-
formants. The estimated copy number varied from approximately 4 to more than 10.
To assess the expression of bar, Ignite solution (200 g/L
[16.22%]G, the active ingredient) was sprayed on a11 of the
putatively transformed plants. A11 of the plants were resistant to 1%Ignite, whereas untransformed control plants
showed necrosis in 2 d and died in 10 d after the application of Ignite.
More than 90% of R, transformants produced one normal tassel (Fig. 2C) and one to three fertile ears per plant
(Fig. 2D) 2 to 3 months after transfer to the greenhouse.
About 50% of them showed reduced stature compared to
seed-derived plants. Morphologically abnormal characteristics such as pistillate flowers on tassels and curly leaves
were observed in less than 5% of the plants. Less than 1%
of the plants that survived failed to develop to maturity.
The yield of kernels per ear varied from 6 to 200 after
self-pollination, although most of the plants yielded 50 to
80 kernels. Mature kernels were harvested 9 months after
bombardment.
Histochemical and Fluorometric Localization of CUS in
R,, R,, and R, Plants
The visualization of the A c t l - p s activity pattern in transgenic R, plants is summarized in Figure 3, E to S. High GUS
activity was observed in the multiplicationareas of shoot-tip
clumps after histochemical assay (Fig. 3E). Whole plantlets
regenerated from in vitro transgenic and untransformed
shoot-tip clumps were immersed in 6 mL of 5-bromo-4chloro-3-indo~y~-~-~-g~ucuro~c
acid assay buffer mixture.
Ten minutes after incubation at 37"C, the root hairs presented
the first sign of GUS activity, followed by the emergence of
blue coloration in the root tips, secondary roots, and root
elongation zones within 30 min. The appearance of the blue
color on the younger leaves began from the base of the leaves
and gradually extended to the entire leaves and the older
leaves. The entire plantlets turned to intense blue after 2 h of
incubation at 37°C (Fig. 3F). Shoot tips, including meristems
and leaf primordia, showed the strongest GUS activity after 5
min of exposure to the assay buffer (Fig. 3G).
A series of hand-cut cross-sections from control and
transgenic plants was subjected to histochemical and fluorometric staining to localize the cellular pattern of GUS
activity. After 10 min of incubation, the sections were
observed under the microscope. The younger the leaves,
the more intense the blue (Fig. 3H). Stems proximal to
shoot tips had more intense blue staining than internodes
(Fig. 31). The highest intensity of GUS staining was located
in the shoot and root tip areas, axillary buds, and vascular
system (Fig. 3J).Fluorometric localization of GUS revealed
that the high GUS activity existed in phloem cells of the
vascular system of transgenic plants (Fig. 3K), whereas no
fluorescence was observed in the sections of the control
plants. The pattern of GUS intensity in cells was phloem >
epidermis > mesophyll cells in leaves, and phloem > root
hair > endodermis > epidermis > cortex cells in roots.
Reproductive meristems showed blue color as intense as
that of the vegetative meristems, particularly in a11 spikelet
primordia of reproductive organs (Fig. 3, L and M). Silks
over 6 cm long from most independent transformants dis-
Figure 3. (Continued from previous page.) section of leaves and a stem proximal to the shoot tip, TA10 (XlO, 20 min of
staining); Is, leaf sheath, sm, stem. J, Transverse section of a stem with an axillary bud, TAlO (XlO, 20 min of staining); ab,
axillary bud; Ih, leaf hair; ls, leaf sheath; sm, stem. K, Fluorometric assay of GUS in a transverse section of leaf and stem,
TAlO (X10, l 0 . m i n of staining); Ih, leaf hair; ls, leaf sheath; pl, phloem; sm, stem; sv, stem vascular bundle. L, An ear
primordium, TA10 (X32, 15 min of staining); sp, spikelet-pair primordium. M, lmmature ear, TAlO (X10, 1 h of staining);
s, spikelet primordium. N, lmmature ear, TA34 (X0.5, 1 h of staining); SI,silk. O, Mature pollen, TAlO (X20, 30 min of
staining). P, lmmature tassel, TA1 O ( X 12, 1 h of staining); br, lateral branch; cr, central rachis. Q, lmmature florets, TA1 O
(X3.5, 1 h of staining); an, anther; gi, inner glume; go, outer glume. R, lmmature embryos from heterozygote, TA 3-1 1 (X6,
30 min of staining). S, Mature pollen from homozygote, TA 10-27 (X25, 30 min of staining).
Zhong et al.
1104
Plant Physiol. Vol. 110, 1996
Table II. Summary of co-transformation experiments with pTW-a and pActl-F
Genotype
No. of
No. of
Age of
Days
No. of Independent
Plates
Shoot Tips
Cultures"
Postcultureb
Events'7
33
26
2.5
2.5
53
29
30
30
30
30
HNP
ICES
Subtotal
2
3
2
5
3
2
312
421
194
607
377
201
17
2112
3
30
faar+/pm2+/gus+/GUS+d
0
13/13/11/10
5/5/4/2
21/21/16/11
9/9/7/4
3/3/3/2
51/51/41/29
13(3.1)
5 (2.6)
21 (3.4)
9 (2.4)
3(1.5)
51 (2.4)
a
Months after the initiation of shoot tip multiplication from the 7-d-old seedlings. All of the shoot tips for bombardment were selected from
h
c
the shoot-multiplication cultures 2 weeks after subculture.
Days of multiplication after bombardment before first-round selection.
The
number of independent events was the total number of independent G-resistant clumps obtained at the end of the 1st month of selection. The
number in parentheses is the relative efficiency (%) of stable transformation measured as total G-resistant clumps divided by total number of
d
bombarded shoot tips, X100%.
bar+/pin2+/gus+/G\JS+ shows the number of independent clumps with each gene or enzyme activity.
played no detectable GUS activity, but a strong blue coloration was observed throughout the entire development
of silks in several independent transformants containing
multiple GUS transgenes (Fig. 3N). Mature pollen grains
showed relatively high GUS activity, but were variable
(Fig. 3O). Florets, glumes, young anthers, and young silks
displayed intense GUS staining (Fig. 3, P and Q). Mature
flowers except for pollen did not exhibit GUS activity even
with overnight incubation. No reproductive organs from
the control untransformed plants showed any blue staining.
Histochemical assays of hand-cut cross-sections of vegetative organs from in vitro plantlets from different trans-
formation events and their R: and R2 progeny (containing
single or multiple copies of transgenes) revealed that the
pattern of GUS activity was similar with the exception of
about 2 to 4% of plantlets, in which the elongation zones of
roots showed higher intensities of GUS staining than the
root tips (data not shown).
GUS activity in immature embryos of transformants varied
depending on their developmental stage (Fig. 3R). However,
all immature embryos exhibited a low intensity of GUS staining in the coleoptile and the center of scutellum tissues.
Mature pollen grains from primary transformants (R0)
from most of the independent transformation events had a
Probes
bar
.
R
: «M
1*1 **>
«-,
kh - - ' l ' - ' - : - f - f
7.2 —
pin2
bar
gus
Figure 4. DNA gel blot analysis of undigested genomic DMA (10 /u-g/lane) from independent G-resistant (T1-T56) and
GUS-positive (TA1-TA19) shoot-tip clumps. pTW-a and f>Act1-f were linearized with fcoRV or fcoRI, respectively. 1, 2,
and 5 each represents reconstruction with number of copies of the plasmid pTW-a or pAct1-f to correspond to that copy
number of transgenes in transformed plants. CO, Untransformed control. A and B, Transformed with pTW-a; C and D,
transformed with pTW-a and pAcfJ-F. Each filter shown was hybridized with the indicated coding region probe.
Maize Transformation via Bombardment of Shoot Tips
Figure 5. Southern blot analysis of five indepen-
pTW-a
A
kh
3 3 5 1
c
b
a
a
CO
a
b
TA10
a
b
c
TA23
a
b
TA34
c
a
b
TA41
c
a
b
TA50
c
a
b
c
pT\V-a
B
kh
3 3
10 4
c
d
b
1105
d
CO
d
b
TA10
d
b
c
TA23
d
b
TA34
c
d
h
c
e
c
TA41
r
i
h
TA50
t
d
b
c
dent transformed R0 plants. 1, 3, 4, 5, and 10
each represents the amount of the plasmid
pTW-a or pActl-f that would correspond to that
number of the transgenes in transformed plants.
Each lane contains 10 ^g of genomic DMA from
a nontransformed control maize plant (CO) or
putatively transformed maize plants (TA10,
TA23, TA34, TA41, and TA50). Lanes a, DMA
digested with fcoRI; lanes b, DNA digested with
fcoRV; lanes c, undigested DNA; lanes d, DNA
digested with H/ndlll; lanes e, DNA digested
with fcoRI and Noti. A, Filter was hybridized
with a-32P-labeled bar coding region probe. B,
Filter was hybridized with a-32P-labeled pin2
coding region and terminator probe. C, Filter
was hybridized with a-32P-labeled gus coding
region probe.
14.1 —
oAcil-¥
C
kh
3 3 in 4
c
a
e
c
CO
e
a
TA10
e
a
c
TA23
e
a
TA34
TA41
TA50
3.3 —
ratio of GUS positive:negative of approximately 1:1. Mature pollen grains from single anthers of RQ, Rj, and R2
transformants displayed different intensities of GUS staining. All mature pollen grains from homozygous Rj and R2
progeny showed GUS activity (Fig. 3S).
Genetic and Molecular Analysis of R, and R2 Progeny
The phenotypes of R, and R2 plants were identical to
those of plants derived from native self-pollinated kernels
in the greenhouse. To determine the inheritance of selected
and unselected genes, bar and gus, in self-pollinated R a and
R2 progeny, expression of gus and/or bar in progeny from
13 independent primary transformants was analyzed. GUS
histochemical assay of roots, immature embryos, young
leaf sections, and pollen grains was used to assess the
presence and expression of gus in progeny. The presence of
bar, corresponding to PAT activity, in progeny was determinated by resistance of the plantlets to 5 mg/L G in vitro
and resistance of the plants to 1% Ignite in the greenhouse.
Figures 2E and 3R present the segregation of expression of
bar in Rj progeny and gus in immature embryos of Rt
progeny, respectively. The results of segregation of both
bar and gus expression in R a and R2 progeny are summarized in Table III.
The x2 analysis indicates that the inheritance of bar and
gus expression followed Mendelian inheritance for a single
dominant locus (3:1 segregation ratio) in all of the R2
progeny tested but not in all of the Rj progeny tested. The
segregation ratio for PAT activity in the progeny of transformant T93 was significantly different from the expected
ratio of 3:1. The segregation ratio of PAT and/or GUS
activity in transformants T77 and TA50 fit a 15:1 model for
the presence of two independent dominant loci. Segregation ratios for plants T8, T20, TA3, TA10, and TA34 fit a 1:1
model, consistent with lack of either male or female transmission.
Southern blot analysis of selected events revealed the
presence of transgenes in Ra and R2 progeny, concordant
1106
Plant Physiol. Vol. 110, 1996
Zhong et al.
Table III. Segregation of expression of bar and gus in self-pollinated R , and R, progeny
Generation
RI
R2
Transformant
HNP T8
HNP T16
ICES T20
HNP T44
ICES T63
ICES T77
HNP T93
HNP TA3
HNP TA10
HNP TA23
HNP TA34
ICES TA41
ICES TA50
HNP T8-4
HNP T8-6
HNP T44-3
IGES T77-9
HNP TA3-11
HNP TA1 0-1 3
HNP TA23-7
Total
Assayed
43
36
51
16
147
78
74
18
32
69
44
93
22
53
86
41
46
13
50
21
PAT(+/-)"
GUS(+/-)'
In vitro
In vivo
15113
813
31/20
1313
1114
1716
9 215 5
7414
20154
511 3
1917
30114
713
68/25
1614
815
016
1511O
2011 4
2111
4111 2
59/27
31/10
3011 6
1013
2 515
810
XZh
4.1d
O
4.76d
0.08
11 .43d
1 5.38d
88.2gd
18.96d
3.37d
3.02
3.67
0.09
3.88d
0.06
1.55
0.01
1.86
0.02
0.96
0.02
In vitro
In vivo
1018
1719
3311 1
614
65/28
O16
1511 O
19115
NDe
1218
815
XZh
1814
2.67
7.04d
0.82
6.82d
1 .O4
0.24
2011 o
810
2.67
0.02
a Number of individual progeny resistanvsensitive to 5 mg/L C i n vitro or 1% lgnite in the greenhouse (in vivo).
x2 values calculated using
Number of individual progeny staining positivelnegative by GUS histochemical assay on the roots or immature
Yates' correction factor.
embryos of progeny germinated in vitro or on the young leaves and pollen grains of progeny grown in the greenhouse (in vivo).
Values are
significantly different from the expected ratio of 3:l at P < 0.05 with 1 degree of freedom.
e ND, Not determined.
with the corresponding enzyme activity of PAT and GUS
(data not shown).
DISCUSSION
In this paper, we describe a nove1 transformation system
that we believe will be a useful tool for both basic and applied research on maize. The competence of in vitroproliferated maize shoot meristems for integrative transformation and inherited expression of transgenes was
demonstrated. Explants previously used for maize transformation are immature embryos, their derived type-I and
type-I1 calli, or suspension-cultured cells (Rhodes et al.,
1988; Fromm et al., 1990; Gordon-Kamm et al., 1990;
D'Halluin et al., 1992; Golovkin et al., 1993; Koziel et al.,
1993; Frame et al., 1994; Laursen et al., 1994; Lowe et al.,
1995; Wan et al., 1995). Both dissection of zygotic coleoptile-stage embryos and maintenance of donor plants in the
greenhouse or field are very time consuming and labor
intensive. In contrast, the explants (shoot tips) we used
were easily and simply obtained from in vitro-regenerated
shoot tips derived from germinated seedlings. Uniform
explant sources can be obtained at any time of the year. In
our system, the cycle from mature kernels of donor plants
to the mature transgenic kernels was completed within 10
months.
To date, we have regenerated plantlets of 36 genotypes
tested via shoot-tip multiplication, but the frequencies of
fertile plant production in a11 genotypes other than HNP
and IGES are still unknown. The frequency of plantlet
production from multiple shoot tips varied from 24 to 97%
in 36 genotypes tested. To increase the transformation fre-
quency and apply the system to a wide range o1 genotypes,
further research is required on aspects such as developmental failure of shoot tips. Particularly, the failure of elite
inbred lines on both multiplication and regeneration has
been reported (Lowe et al., 1995). We have transformed
two sweet corn genotypes and observed stable expression
of gus in shoot meristems in 10 other genotypes tested.
Three keys to increase the transformation efficiency and to
make the co-transformation possible were the selection of
newly developed shoot tips, the multiplication of bombarded shoot tips for 1 month without selection pressure,
and the use of selectable marker genes, such as bar, with the
appropriate initial selection pressure. In combination with
the previous result that chimeric plants were produced in
a11 genotypes tested and stable transformants were recovered in two genotypes with shoot multiplicaticln responses
at opposite ends of the spectrum (Lowe et a]., 1995), we
believe our system could be developed to be truly genotype independent and applied to other cereals. The system
also has the potential to be used for studying the formation
and function of shoot meristems.
In our study, both CaMV 35s and the Actl 5' region were
used to control the expression of bar and gus. The activity
of the CaMV 35s promoter in our transgenic maize was
adequate to express bar for herbicide resistance in the vegetative stage and was stably transmitted to their progeny,
as was the case in transgenic rice (Cao et al., 1992). Results
from our extensive histochemical and fluorometric assays
of GUS activity in vegetative and reproductive organs of
primary transformants and their progeny demonstrated
activity of the rice Actl 5' region in different cells, tissues,
and organs throughout development. Combined with sim-
Maize Transformation via Bombardment of Shoot Tips
ilar results reported earlier i n other monocots (Zhang et al.,
1991; Zhong et al., 1993), o u r results indicate that the rice
Actl 5‘ region could be a good promoter f o r monocot
transformation even w i t h o u t modification of the expression structure with other features.
Received November 27, 1995; accepted January 10, 1996.
Copyright Clearance Center: 0032-0889/96/110/lO97/11.
LITERATURE ClTED
Bevan M, Barnes WM, Chilton M (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic
Acids Res 11: 369-385
Cao J, Duan XL, McElroy D, Wu R (1992) Regeneration of herbicide resistant transgenic rice plants following microprojectilemediated transformation of suspension culture cells. Plant Cell
Rep 11: 586-591
D’Halluin K, Bonne E, Bossut M, Beuckeleer MD, Leemans J
(1992) Transgenic maize plants by tissue electroporation. Plant
Cell 4: 1495-1505
Frame BR, Drayton PR, Bagnall SV, Lewnau CJ, Bullock JM,
Willson HM, Dunwell JM, Thompson JA, Wang K (1994) Production of fertile transgenic maize plants by silicon carbide
whisker-mediated transformation. Plant J 6 941-948
Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J,
Klein M (1990) Inheritance and expression of chimeric genes
in the progeny of transgenic maize plants. Bio/Technology 8:
833-839
Golovkin MV, Abraham M, Morocz S, Bottka S, Feher A, Dudits
D (1993) Production of transgenic maize plants by direct DNA
uptake into embryogenic protoplasts. Plant Sci 9 0 41-52
Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR,
Daines RJ, Start WG, O’Brien JV, Chambers SA, Adams WR,
Willetts NG, Rice TR, Mackey CJ, Krueger RW, Kausch AP,
Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603-618
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS
gene fusion system. Plant Mo1 Biol Rep 5: 387-405
Keil M, Sanchez-Serrano J, Schell J, Willmitzer L (1986) Primary
structure of a proteinase inhibitor I1 gene from potato (Solanum
tubeuosunz). Nucleic Acids Res 14: 5641-5650
Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R,
Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K,
Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E,
Rhodes R, Warren GW, Wright M, Evola SV (1993) Field per-
1107
formance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology
11: 194-200
Laursen CM, Krzyzek RA, Flick CE, Anderson PC, Spencer TM
(1994) Production of fertile transgenic maize by electroporation
of suspension culture cells. Plant Mo1 Biol 24: 51-61
Lowe K, Bowen B, Hoerster G, Ross M, Bond D, Pierce D,
Gordon-Kamm B (1995) Germline transformation of maize following manipulation of chimeric shoot meristems. Bio/Technology 13: 677-681
McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient
actin promoter for use in rice transformation. Plant Cell 2:
167-171
Medford JI (1992) Vegetative apical meristems. Plant Cell4: 10291039
Murashige T, Skoog F (1962) A revised medium for rapid growth
and bioassay with tobacco tissue cultures. Physiol Plant 15:
473-497
Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ
(1988) Genetically transformed maize plants from protoplasts.
Science 7: 204-207
Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram
amounts of fresh, herbarium, and mummified plant tissues.
Plant Mo1 Biol 5: 69-76
Steeves TA, Sussex IM (1989) Patterns in Plant Development, Ed
2. Cambridge University Press, New York
Strickberger MW (1985) Genetics. Macmillan, New York, pp
126-146
Thompson CJ, Novva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicideresistance gene bar from Streptomyces hygrascopicus. EMBO 6
2519-2523
Wan Y, Widholm JM, Lemaux PG (1995) Type 1 callus as a
bombardment target for generating fertile transgenic maize (Zea
mays L.). Planta 196: 7-14
Zhang W, McElroy D, Wu R (1991)Analysis of rice Actl 5’ region
activity in transgenic plants. Plant Cell 3: 1155-1165
Zhong H, Bolyard MG, Srinivasan C, Sticklen MB (1993) Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Rep
13: 1-6
Zhong H, Srinivasan C, Sticklen MB (1992a) In-vitro morphogenesis of corn (Zeu mays L.). I. Differentiation of multiple shoot
clumps and somatic embryos from shoot tips. Planta 187
483-489
Zhong H, Srinivasan C, Sticklen MB (1992b) In-vitro morphogenesis of corn (Zeu mays L.). 11. Differentiation of ear and tassel
clusters from cultured shoot apices and immature inflorescences. Planta 187: 490-497