The Plant Journal (1994) 6(6), 941-948

TECHNICAL ADVANCE

Production of fertile transgenic maize plants by silicon

carbide whisker-mediated transformation
Bronwyn R. Frame 1, Paul R. Drayton 2, Susan V.
Bagnall 1, Carol J. Lewnau 1, W. Paul Bullock 1,
H. Martin Wilson 1, James M. Dunwell 2, John A.
Thompson 2 and Kan Wang 1,*
11Cl Seeds, 2369 330th Street, Box 500, Slater, IA
50244, USA, and
2ZENECA Seeds, Jealott's Hill Research Station,
Bracknell, Berkshire, RG12 6EY, UK

Summary
A simple and inexpensive system for the generation
of fertile, transgenic maize plants has been developed.
Cells from embryogenic maize suspension cultures
were transformed using silicon carbide whiskers to
deliver plasmid DNA carrying the bacterial bar and
uidA (gus) genes. Transformed cells were selected
on medium containing the herbicide bialaphos.
Integration of the bar gene and activity of the enzyme
phosphinothricin acetyl transferase (PAT) were confirmed in all blalaphos-resistsnt callus lines analysed.
Fertile transgenic maize plants were regenerated.
Herbicide spraying of progeny plants revealed that
the bar gene was transmitted in a Mendelian feshion.
Introduction
In recent years the direct introduction of plasmid DNA into
plant cells has been accomplished by a number of
methods, of which three have been used to generate
fertile transgenic maize plants (Wilson et al., 1994). The
approach most frequently used is high-velocity microprojectile bombardment--a method now routine in many
laboratories (Christou, 1992) following the pioneering
work of Sanford et al. (1987). Fertile transgenic plants
have been produced through bombardment of embryogenic suspension culture (Fromm et al., 1990; GordonKamm et al., 1990) and immature zygotic embryos
(Koziel et aL, 1993). Wounding of such embryos followed
by electroporation has also been used successfully to
transform maize (D'Halluin et al., 1992). Both microprojectile bombardment and electroporation require

Received 28 April 1994; revised 5 August 1994; accepted 12 August

1994.
*For correspondence (fax +1 515 685 2548).

sophisticated and expensive equipment and supplies. A

third approach, direct gene transfer to protoplasts, has
also resulted in fertile, transgenic maize plants (Golovkin
et al., 1993). However, regeneration of plants from
protoplasts in maize is difficult, being at present limited to
certain tissue culture-adapted genotypes (M0rocz et aL,
1990).
In contrast to the technically demanding methods
described above, there have also been some reports on
the use of silicon carbide whiskers to transform plant
cells. Transformation of cell lines of maize (Kaeppler et
aL, 1990, 1992), tobacco (Kaeppler et aL, 1990), Agrostis
alba (Asano et aL, 1991), and most recently Chlamydomonas reinhardtii (Dunahay, 1993) cells has been
demonstrated using such whiskers. The method involves
the mixing (e.g. by vortex treatment) of cells in liquid
medium with whiskers and plasmid DNA. The resulting
collisions between cells and whiskers appear to lead to
cell penetration and DNA delivery (Kaeppler et aL, 1990).
Although stable transformation of maize cultures has been
achieved (Kaeppler et aL, 1992), the cell line used--Black Mexican Sweet com--was non-regenerable.
In this paper we report the production of fertile transgenic maize plants using the silicon carbide whisker
transformation method. Regenerable suspension cultures
of A188 x B73 were treated with whiskers and plasmid
DNA incorporating the bar and uidA (gus) genes, and
stable transformants were selected on medium containing bialaphos. PAT assays and Southern hybridization
analysis confirmed the transgenic status of the regenerated plants. Segregation data for the progeny of
several transformants are also presented. This represents the first report of transgenic plant production, in any
species, with the silicon carbide whisker-mediated transformation approach.

Results
Transient GUS expression in regenerable maize cells
Silicon carbide whisker-mediated transformation of A188
x B73 (A x B) suspension cells was achieved using the
procedure described in the Experimental procedures.
The components of the plasmid, pBARGUS (Fromm et
aL, 1990), used for transformation, are shown in Figure 1.
Cells vortex-treated for 60 sec with whiskers and
pBARGUS exhibited transient GUS expression at a mean
941

942

Bronwyn R. Frame et al.

woo

Figure 1. Schematic representation of pBARGUS.

The 1.4 kb
fragment was used as a probe for Southern analysis.
P35S, cauliflower mosaic virus 35S promoter; I1, intron 1 of maize alcohol
dehydregenase f (Adhl); bar,
ceding region; 3', nopaline synthase
polyadenylation region;
coding region; PAdhl, promoter
of maize alcohol dehydregenase 1.

BamHI

bar
uidA,uidA(gus)

8oo

6oo

=
o

4oo

200

frequency of 150 GUS expression units (one unit = one

blue cell or group of adjacent blue cells) per treatment of
0.25 ml packed cell volume (p.c.v.). Cells treated without
DNA or whiskers did not express GUS activity. We
observed, however, that when cells were mixed with DNA
and whiskers solely by tapping a finger against the side of
the Eppendorf tube, a few blue spots could be detected
(data not shown). Premixing of DNA with whiskers was
not required to achieve transformation and did not enhance the efficiency of DNA delivery.
Effect of pretransformation treatments on transient GUS
expression
It has been reported that there is an increase in transient
expression and stable clone recovery from particle bombardment when cells are exposed to medium containing
a high concentration of sorbitol and mannitol prior to
transformation (Russell et al., 1992; Vain et al., 1993).
To investigate the effect of this treatment on whiskermediated transformation, A x B suspension cells (0.25 ml
p.c.v.) 1 day after subculture, were transferred into sterile
1.5 ml Eppendorf tubes and incubated for 30 min with
1 ml of liquid N6 medium containing equal molarities of
sorbitol and mannitol. Figure 2 summarizes the effect of
duration and molarity of this treatment on transient GUS
expression. As can be seen, incubation in 0.5 M S/M
medium (0.25 M sorbitol and 0.25 M mannitol) for 15-60
min increased GUS expression three- to fivefold compared with the non-treated control. Treatments with
higher (1 M S/M) or lower (0.25 M S/M) concentrations of
sorbitol and mannitol did not increase expression (Figure
2 and data not shown). All the experiments described
hereafter included pretransformation treatment with 0.5 M
S/M medium. No obvious effect on subsequent cell
growth was seen following incubation in this medium.
Effect of mixing method on transient GUS expression
and cell regrowth
Two different mixing methods were tested with this transformation approach. Tubes containing cells, whiskers and
plasmid DNA were placed either on a Vortex Genie II

0
16

80

80

Durstlon of treatment (rain)

Figure 2. Effect of pretransformaUon sorbitol/mannitol treatment on
transient GUS expression in A x B suspension cells.
0 M, 0.5 M and 1 M are different concentration of sorbitol/mannitol
solution.

vortex mixer or in a Mixomat reciprocating mixer. The

effectiveness of the two methods on transient GUS expression and cell growth after transformation is illustrated
in Figure 3. Although numbers of GUS expression units in
cells treated with the Mixomat for 3 sec or 5 sec were
higher than all other treatments, subsequent growth from
these cells was reduced. As shown in Figure 3(a),
transient expression was highest after 5 sec mixing
duration with the Mixomat, but the cell regrowth was
reduced to approximately 35% of the control value. After
10 sec of Mixomat treatment transient expression was
significantly decreased. This was likely due to severe cell
damage caused during prolonged mixing treatment. In
general, cell regrowth from cultures treated with the
Vortex Genie was better than that from those treated with
the Mixomat. Cell regrowth following 30 sec of vortex
treatment was equivalent to that of untreated control
tissue. Cell regrowth following 60 sec was about 75% of
the control value. Although the longer mixing duration
using the Vortex Genie (3 min) slightly increased the
number of GUS expression units, cell regrowth dropped
to approximately 60% of the control value (Figure 3b).
The association of whiskers and suspension cells after
mixing treatment and the transient expression of GUS in
transformed cells are illustrated in Figure 4(a) and (b).
Effect of time after subculture on transient GUS
expression
The A x B suspension culture was subcultured every 3.5
days. When transformation was carried out at different
times after subculture, transient GUS expression varied.
Figure 5 displays the comparison of GUS expression
units obtained using two cryopreserved lines, C8 and

Whisker-mediated maize transformation

(a)
tO0o

100

!
76 w

i-

H
0
0

i
i,
8
10
MlxlnO Duration (second8)

(b)
|o0

coo

400

78

60
~0
t80
Vortoxlng Durntlon (oooondo)
--m-Qu8 oxpro881on unit8
Rogrowth

Figure 3. Effect of mixing duration of Mixomat (a) and Vortex Genie II

(b) on transient GUS expression and cell regrowth.
Cell regrewth of each treatment was measured as dry weight after 4
weeks in embedding medium without selective agent,

C12, on different days after subculture. The numbers of

transient GUS expression units 1 day after subculture
were significantly higher than any other day for both C8
and C12 (P = 0.0002 at (z = 0.05). The values for days 2,
3 and 4 after subculture did not differ significantly from
each other. For line C8, the value at day 0 was significantly higher than at day 4, whereas there was no such
difference for line C12. These data suggest that the
efficiency of DNA delivery is related to culture age (in
terms of days following subculture).
Production of stably transformed callus and transgenic
plants
Table 1 summarizes the results from nine independent
stable transformation experiments. The efficiency of
stable clone recovery ranged from 0.2 to 1.7 clones per
millilitre p.c.v, of treated cells. The embedding selection
scheme described in Experimental procedures in conjunction with the use of bialaphos as selective agent
enabled efficient recovery of transformed callus (Figure
4c). Non-transformed tissue rarely survived in this selection scheme.

943

A total of 311 plants were regenerated from 22 independently transformed callus lines. Both the phenotype
and fertility of plants regenerated from bialaphos-resistant callus were similar to plants regenerated from nontransformed callus (Figure 4d and e). The transformed
status of callus and plants was confirmed either by PAT
assay (Figure 6), PCR, or leaf painting with IgniteTM
herbicide (data not shown). Of the nine putative transformants included in Figure 6, eight were PAT positive.
While tmnsformant AAG was negative in the PAT assay
and leaf painting, PCR results indicated that it did contain
the bar gene (data not shown).
Figure 7 shows the Southern hybridization analysis of
several primary transformants (Re). Genomic DNA was
digested with BamHI and hybridized with a 1.4 kb BamHI
fragment containing the bar gene (Figure 1). The nontransformed plant control showed no hybridization (lane
1), whereas the digested DNA samples from six of eight
transformants (lanes 4, 6-9 and 11) showed a 1.4 kb
hybridizing band co-migrating with the plasmid control
(lanes 12 and 13), indicating the presence of the bar
gene. The estimated copy number ranged from one to
five, although in some cases more than 10 copies were
observed. Clones RU (lane 5) and hal (lane 10) showed
hybridizing fragments at positions higher than 1.4 kb.
Since both clones exhibited PAT activity (Figure 6, lanes
5 and 11), this suggested that the bar gene remained
intact. The hybridizing fragments greater than 1.4 kb in
the two clones may be the products of plasmid/plant
junctions which could be due to the loss, during integraUon, of the BamHI site between the uidA gene and
the 3' region (Figure 1).
Inheritance of the bar gene
Progeny from several independently transformed Re
plants were analysed to determine the inheritance of the
bar gene. Its expression was assessed by the spraying of
herbicide on 7- to 9-day-old plantlets. Figure 4(e) shows
that plants expressing the bar gene developed no
symptoms, whereas the non-transformed plants (the
middle row) showed necrosis and died 1-2 weeks after
treatment. The herbicide spray allowed us to monitor
the segregation of PAT expression in large numbers of
progeny. For example, Table 2 presents segregation data
from the R1 and R 2 progenies of four transformed plants.
Bialaphos-resistant R1 plants were either selfed or used
as male or female parent in crosses with non-transformed
B73 plants. The data in Table 2 were not significantly
different from a one-to-one segregation in reciprocal
crosses and a three-to-one segregation in selfings, as
estimated by ~2 analysis, and indicate that the PAT
activity was encoded by, and transmitted as, a single,
dominant allele. To test further the inheritance of PAT

944

Bronwyn R. Frame et

al.

Figure 4. Whisker-mediated maize transformation.

(a) Association of silicon carbide whiskers (needle-like material) with A x B suspension cells visualized under light microscopy (Axioskop, Zeiss); Bar =
50 gm; (b) transient GUS expression in cells after transformation; (c) bialaphos-resistant callus; (d) tassel and (e) ear of Ro plant produced by whiskermediated transformation; (f) R1 progeny test recorded 2 weeks after herbicide spray. The non-transformed control plants exhibited severe necrosis within 4
days and died soon after (the middle row). The transgenic plants remained green and healthy.
1000

[] C8

C12

400 -

Tlme ifter subculture ( d s y s )

Figure 5. Effect of time post-subculture on transient GUS expression in
two cell lines C8 and C12.

Table 1. Summary of clone recovery from nine whiskermediated transformation experiments

Exp.
no.

Cells
treated
(ml p.c.v.)

Clones
recovered

PCR
(+/total)

Ratio
(clone per ml
p.c.v.)

1
2
3
4
5
6
7
8
9
Total

5
8
20
10
6
10
5
4
7
75

4
3
4
4
3
17
1
1
3
40

4/4
2/3
4/4
4/4
3/3
17/17
1/1
1/1
3/3
39/40

0.80
0.38
0.20
0,40
0.50
1.70
0.20
0.25
0.43
0.53

Whisker-mediated maize transformation

945

Table 2. Segregation of bargene activity based on leaf spraying

assays in R2 and R3 progenies of transgenic maize plants
Herbicide
sensitive

Herbicide
resistant

Z2c

pd

Self
Female
Male

9
22
56

30
28
37

0.01
0.50
1.65

0.93
0.48
0.19

RU

Self
Female
Male

23
37
34

56
30
36

0.51
0.53
0.01

0.47
0.46
0.90

UA

Self
Female
Male

27
47
56

64
48
37

0.82
0.00
3.48

0.36
1.00
0.06

UI

Self
Female
Male

24
43
46

57
52
50

0.70
0.67
0.09

0.40
0.41
0.76

Self-1
Self-2

0
27

97
66

Homozygous
0.61 0.44

RU

Self-1
Self-2

14
31

61
64

1.28
2.56

0.26
0.11

UA

Self-1
Self-2

28
27

64
56

1.17
2.12

0.28
0.14

Ul

Self-1
Self-2

25
0

65
69

0.24
0.63
Homozygous

Transformant
R2a
RR

Figure 6. PAT assay.

PAT activity in a non-transformedcontrol (lane 3) and nine independent
transformants (lanes 4-12). PAT activity, as indicated by acetylated
phosphinothricin (arrow), is seen in the calli and leaves of clones RR
(lanes 1 and 4) and RU (lanes2 and 5), leavesof clonesAAC, AAD, AAE,
AAF, AAI and AAJ (lanes 6-8 and 10-12). Twenty-fivemicrograms of
proteinextractwere loadedper lane.

R3b
RR

Cross

aBialaphos-resistant R1 plants were either selfed or used as

male or female parent in cresses with non-transformed B73
plants.
bBialaphos-resistant R2 plants were selfed.
cz2, chi-square values with Yates (continuity) correction.
dp, ;(2 probability with 1 degree of freedom.
Figure 7. Southernblot analysisof maizegenomicDNA.
Autoradiographof Southern blot of genomic DNA extracted from leaf
tissue of a non-transformedcontrol(lane 1) and eight independenttransformants. Lanes 2 and 3, undigestedDNAs from transformantsRR and
RU; lanes 4-11, the BamHI-digested DNAs (12 pg per lane) from
transformed lines RR, RU, AAC, AAD, AAE, AAF, AAI and AAJ. The blot
was hybridizedwith a digoxigenin(DIG)-dUTPlabelled 1.4 kb bar gene
fragmentfrom pBARGUS.Lanes 12 and 13, one copy and five copiesof
the bar gene fragmentcontrols, refer to the diploid genomeand contain
5.2 pg and 26 pg, respectively,of the 1.4 kb bar expression unit released
from pBARGUSwith BamHI. The arrow indicatesthe 1.4 kb BamHI bar
genefragment.Molecularmarkers (kb) are shownon the left.

activity in the following generations, 16 bialaphosresistant R 2 plants representing each of the four transformation events were self-pollinated. Plants from two R 3
populations of each transformant were germinated and
sprayed for PAT activity. As shown in Table 2, the populations of clones RU and UA showed the expected 3:1
segregation ratio. One population of clones RR and UI
showed a 3:1 ratio, while the other population of these
two clones was 100% bialaphos-resistant, indicating they
were homozygous for the bar gene. These segregation

results confirm that the bar gene was stably transmitted

to the R 3 generation in a Mendelian manner.

Discussion
This paper represents the first report of transgenic plant
production, in any higher plant species, using silicon
carbide whiskers as the DNA delivery system. Whilst this
method has been used to transform Chlamydomonas
reinhardtii (Dunahay, 1993), previous work with whiskermediated transformation in higher plants described only
transient gene expressions (Asano et al., 1991; Kaeppler
eta/., 1992) or stable transformation of non-regenerable
tobacco and maize cell-suspension cultures (Kaeppler el
al., 1992).
Our success in producing fertile transgenic plants with
this approach is due, in part, to the use of treatments preand post-whisker transformation which enhance DNA
delivery and the subsequent recovery of transformed

946

Bronwyn R. Frame et al.

tissue. Exposure of cells to a medium containing a high

molarity of sorbitol and mannitol was previously found to
be beneficial with microprojectile bombardment of maize
cells (Russell et al., 1992; Vain et al., 1993). The use of
this pretreatment clearly enhanced DNA delivery in the
present system.
The length of time between subculture and treatment
also influenced transient expression, with treatment I day
post-subculture giving the highest number of GUS expression units. A similar effect has been noted with
bombardment and electroporation (data not shown).
In addition, mixing method and duration clearly influenced DNA delivery. The published reports of whisker
transformation involved the use of vortex mixing (Asano
et al., 1991 ; Dunahay, 1993; Kaeppler et aL, 1990, 1992).
Our results demonstrate that rapid oscillation of cells and
whiskers (Mixomat treatment for 1 sec) can also result
in stable transformation provided that cells survive in
sufficient number for adequate regrowth. Other methods
of mixing cells with whiskers and DNA are currently being
investigated.
The selection strategy used in these experiments
allowed rapid growth of transformed tissue as a result of
'surface embedding' treated cells. Very few non-transformed callus lines grew through this selection scheme.
As with clones derived from particle bombardment,
ease of plant regeneration and fertility of regenerants
were cell line and clone dependent. Generally, plants
regenerated from independently transformed clones were
similar in phenotype to non-transformed control plants.
The reduced seed set sometimes observed in these
plants was most likely due to the effects of cell culture
age and in vitro selection rather than the transformation
process itself. This was confirmed by the complete fertility
of transgenic plants in subsequent generations.
The transformed status of the callus and plants produced was determined by a combination of PCR and
Southern analysis, together with assays demonstrating
functional gene product (PAT and GUS) activity. Transmission and expression of the introduced bar gene in the
progeny of transgenic plants was as expected for a single
dominant allelle, providing further evidence of integrative
transformation. The fate of genes introduced into maize
cells via whisker transformation does not appear to differ
from other direct DNA delivery approaches.
The exact mechanism for whisker-mediated transformation is not known. Silicon carbide has great intrinsic
hardness and fractures to give sharp cutting edges
(Greenwood and Earnshaw, 1984) and it is believed that
cell penetration by whiskers occurs during vortex mixing
(Kaeppler et aL, 1990). In the present experiments,
premixing whiskers and DNA, as reported elsewhere
(Kaeppler et aL, 1990, 1992), were not required to
achieve efficient DNA delivery. This could suggest that

whiskers do not 'carry' DNA into the treated cells but

function as numerous needles facilitating DNA delivery
into cells during the mixing process. We have also found
that other materials with similar characteristics to silicon
carbide whiskers (e.g. silicon nitride whiskers) can deliver
DNA into plant cells (unpublished data).
Transformation efficiency in these experiments, based
on treated cell volume, is estimated at one fifth to one
tenth that achieved in our laboratories with microprojectile
bombardment of A188 x B73 cell-suspension cultures.
We believe, however, that the efficiency of whisker transformation can be improved further through experimentation and that even at its current efficiency it can
be considered as a practical alternative transformation
method for maize, requiring no expensive equipment
or consumables, especially for those laboratories
which do not have access to other more sophisticated
technologies.
Our results suggest that whisker transformation of any
species where regenerable suspension cultures exist
should be possible once DNA delivery parameters have
been established. Recently, we have obtained stably
transformed lines from whisker treatment of embryogenic
callus derived from an elite stiff stalk maize inbred variety,
indicating that the method can be extended to target
tissues other than suspension cells.

Experimental procedures
Suspension cell culture
Immature zygotic embryos of A188 x B73 from greenhousegrown ears 10-12 days postpollination were aseptically excised
and plated scutellum surface uppermost on modified N6 medium
(Armstrong and Green, 1985; Chu et al., 1975) containing 6 mM
L-proline, 2% (w/v) sucrose, 2 mg 1-1 2,4-dichlorophenoxyacetic
acid (2,4-D) and 0.3% (w/v) Gelrite (Clinical Standards Laboratories, West Warwick, RI) at pH 6.0. To initiate suspension
culture, 3 g of proliferating type II callus from a single embryo
was added to 20 ml of MS-based liquid medium (Murashige and
Skoog, 1962) containing 100 mg 1-1 myo-inositol,2 mg 1-12,4-D,
2 mg 1-1 1-naphthaleneacetic acid (NAA), 6 mM L-proline, 200
mg 1-1casein hydrolysate (Difco Laboratories),3% (w/v) sucrose
and 5% (v/v) coconut water (Gibco Laboratories) at pH 6.0.
Suspension cultures were maintained in this medium in a 125 ml
Erlenmeyer flask at 28C in darkness on a rotary shaker at 125
r.p.m. Suspensions were subcuitured every 3.5 days by addition
of 3 ml packed volume of cells and 7 ml of conditioned culture
medium to 20 ml of fresh culture medium. The cell suspensions
were cryopreserved according to Shillito et al. (1989) and
thawed by submerging cryo tubes (Nunc, Inc., Naperville, IL) at
45C in a water bath. Cells were immediately plated to stedle
filter paper disc (Whatman No. 4, 5.5 cm) in a 60 x 20 mm petri
dish using a 5 ml disposable pipet. Excess cryoprotectant was
removed by repeated blotting with filter paper. Cells on the filter
paper disc were placed on solid N6 medium and subcultured
weekly for 3 weeks after which the suspension cultures were
reinitiated as above.

Whisker-mediated maize transformation

Plasmid DNA

947

Plasmid DNA was purified using a QIAGEN plasmid Maxi kit

(QIAGEN Inc., Chatsworth, CA).

to maturity in 3 gallon pots containing equal parts of peat:pedite:

soil. Plants were fertilized twice a week by watering with an
amended Peters' mix (Hummert International, St Louis, MO,
USA).

Transformation procedure

Herbicide spray assays

Whisker preparation. A sterile 5% (w/v) suspension of silicon

carbide whiskers (Silar SC-9 whiskers, Advanced Composite
Materials Corp., Greer, SC) in water was prepared immediately
prior to use (Kaeppler et aL, 1990).
Tissue preparation. Two hundred and fifty microlitres of
packed cell volume (p.c.v.) of suspension cells 1 day after subculture were dispensed into a 1.5 ml Eppendorf tube. The cells
were then dispersed in 1 ml of liquid S/M medium (N6 with
0.25 M sorbitol and 0.25 M mannitol) and maintained for 30 min
at room temperature. After treatment, 750 ILl of S/M medium was
withdrawn leaving 250 I~1of medium with the cells.
DNA delivery. Forty microlitres of 5% whisker suspension and
25 I~1of plasmid DNA (1 ~g p.I-~) were added to the S/M-treated
suspension cells. Tube contents were first finger tapped to mix
and then placed either upright in a multiple sample head on a
Vortex Genie II vortex mixer (Scientific Industries Inc., Bohemia,
NY) or horizontally in the holder of a Mixomat dental amalgam
mixer (Degussa Canada Lt.d, Burlington, Ontario). Transformation was carried out by mixing at full speed for 60 sec (Vortex
Genie II) or shaking at fixed speed for 1 sec (Mixomat).

Selection of transformed tissue and plant regeneration

Selection. After treatment, cells were plated on filter paper
(Whatman No. 4, 5.5 cm) overlaying N6 medium (2 mg I 1 2,4-D,
3% sucrose, 0.3% Gelrite, pH 6.0) in 60 x 20 mm plastic petri
dishes and wrapped with Urgopore tape (Sterilco, Brussels).
One week later, the filter paper and cells were transferred to the
surface of N6 selection media with 1 mg 1-1 bialaphos, obtained
by deformulation of the herbicide HerbiaceT M (Meiji Seika,
Japan). After a further 2 weeks of subculture, the cells were
removed from the filter paper and suspended in 5 ml of selection
medium (held at 37C) containing 0.6% (w/v) low-gelling temperature agarose (Sea PlaqueTM, FMC Corporation). The suspension was divided into two equal aliquots and each aliquot
was evenly plated over 20 ml of selection medium solidified with
0.3% Gelrite in two 100 x 25 mm petri dishes. These plates were
kept at 28C in darkness. After 4-5 weeks, rapidly growing,
putatively transformed calli were picked and transferred to the
surface of fresh selection medium.
Plant regeneration. Plants were regenerated from bialaphosresistant callus by first transferring type II callus (Armstrong and
Green, 1985) on to MS-based medium containing 1 g 1-1 myoinositol, 6% sucrose, 1 mg 1-1 N,'~J~, 0.3% Gelrite (pH 6.0) at
25C in darkness. After 2-3 weeks, opaque, mature somatic
embryos were transferred to MS medium containing 3% sucrose
and 0.25 mg 1-1 NAA in the light (~ 200 i~Em-2 S 1, 16 h photoperiod). Developing plantlets were transferred to glass vials
containing 15 ml of half-strength MS containing 3% sucrose,
0.3% Gelrite (pH 6.0) and no phytohormones. After 1-2 weeks,
plants were then transferred to soil in the greenhouse and grown

Screening of R 2 and R3 segregating progeny was accomplished

by spraying 9- to 12-day-old seedlings with IgniteT M herbicide.
Ignite TM herbicide contains 16.22% monoammonium 2-amino(hydrooxymethyl-phosphiny) butanoate as active ingredient
(Hoechst-Roussel Agri-Vet Company, Somerville, NJ). Each 40
x 52 cm soil bed containing four rows of transgenic (25 seeds
per row) and one row of untransformed control seedlings were
sprayed with 40 ml of a 2% Ignite solution (in H20) with a handheld sprayer. The response was recorded 6 days later.

Analysis of transformed callus and plants

Histochemical GUS assays were conducted following published
protocols (Jefferson, 1987; McCabe eta/., 1988). PAT activity
was determined using a thin-layer chromatographic assay
(De Block et aL, 1987; Spencer et al., 1990). Genomic DNA was
isolated from leaf tissue for Southern analysis essentially as
described by Saghai Maroof et al. (1984) and as described by
Edwards et al. (1991) for polymerase chain reaction (PCR)
amplification. The probe used in Southern analysis was produced by gel purification of the BamHI restriction enzyme
fragment from pBARGUS and was labelled with digoxigenin
(DIG)-dUTP via random primed labelling (the Genius System,
Boehringer Mannheim Corporation). Standard procedures were
used for DNA blot analysis (Sambrook et al., 1989).

Acknowledgements
The authors thank David Peterson for work on the development
of the A x B suspension culture and selection scheme; Michael
Fromm for providing plasmid pBARGUS; lan Evans and Tony
Fentem for de-formulation of HerbiaceTM; ShuPing Jiao and
Miralba Agudelo for technical assistance.

References
Armstrong, C.L. and Green, C.E. (1985) Establishment and
maintenance of friable, embryogenic maize callus and the
involvement of L-proline. Planta, 164, 207-214.
Asano, Y., Otsuki, Y. and Ugaki, M. (1991) Electroporationmediated and silicon carbide whisker-mediated DNA delivery
in Agrostis alba L. (Redtop). Plant Sci. 79, 247-252.
Christou, P. (1992) Genetic transformation of crop plants using
microprojectile bombardment. Plant J. 2, 275-281.
Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu,
C.Y. and Bi, F.Y. (1975) Establishment of an efficient medium
for anther culture of rice through comparative experiments on
the nitrogen sources. Sci. Sinica, 18, 659-668.
De Block, M., Botterman, J., Vandewiele, M., e t al. (1987)
Engineering herbicide resistance in plants by expression of a
detoxifying enzyme. EMBO J. 6, 2513-2518.

D'Halluin, K., Bonne, E., Bossut, M., de Beuckeleer, M. and

Leemans, J. (1992) Transgenic maize plants by tissue electroporation. Plant Cell, 4, 1495-1505.

948

Bronwyn R. Frame et al.

Dunahay, T.G. (1993) Transformation of Ch/amydomonas reinhardtii with silicon carbide whiskers. BioTechniques, 15,
452-460.
Edwards, K., Johnstone, C. and Thompson, C. (1991) A
simple and rapid method for the preparation of plant genomic
DNA for PCR analysis. Nuc/. Acids Res. 19, 1349.

Fromm, M.E., Morrish, F., Armstrong, C., Williams, R.,

Thomas, J. and Klein, T.M. (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize
plants. Bio/'l'echnology, 8, 833-839.
Golovkin, M.V., Abraham, M., M6rocz, S., Bottka, S., Feher,
A. and Dudlfs, D. (1993) Production of transgenic maize
plants by direct DNA uptake into maize protoplasts. Plant Sci.
90, 41-52.
Gordon-Kamm, W.J., Spencer, T.M., Mangano, M.L., et al.
(1990) Transformation of maize cells and regeneration of
fertile transgenic plants. Plant Cell, 2, 603-608.
Greenwood, N.N. and Earnshaw, A. (1984) Silicon carbide,
SiC. In Chemistry of the Elements. Oxford: Pergamon Press,
p. 386.
Jefferson, R.A. (1987) Assaying chimeric genes in plants. The
GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387-405.
Kaeppler, H.F., Gu, W., Somers, D.A., Rines, H.W. and
Cockbum, A.F. (1990) Silicon carbide fiber-mediated DNA
delivery into plant cells. Plant Cell Rep. 9, 415-418.
Kaeppler, H.F., Somers, D.A., Rinse, H.W. and Cockbum,
A.F. (1992) Silicon carbide fiber-mediated stable transformation of plant cells. Theor. Appl. GeneL 84, 560-566.
Koziel, M.G., Beland, G.L., Bowman, C., et al. (1993) Field
performance of elite transgenic maize plants expressing an
insecticidal protein derived from Bacillus thuringiensis. Bio/
Technology, 11,194-200.
McCabe, D.E., Swain, W.F., Martinelli, B.J. and Christou, P.
(1988) Stable transformation of soybean (Glycine max) by
particle acceleration. Bio/'l'echnology, 6, 923-926.
M6rocz, S., Donn, G., N6meth, J. and Dudits, D. (1990) An

improved system to obtain fertile regenerants via protoplasts

isolated from a highly embryogenic suspension culture. Theor.
Appl. Genet. 80, 721-726.
Mursshige, T. and Skoog, F. (1962) A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol.
Plant. 15, 473-497.
Russell, J.A., Roy, M.K. and Sanford, J.C. (1992) Major improvements in biolistic transformation of suspension-cultured
tobacco cells. In Vitro Cell. Devel. Biol. 28P, 97-105.

Saghai Maroof, M.A., So,man, K.M., Jorgensen, R.A. and

Allard, R.W. (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal
location, and population dynamics. Proc. Nat/Acad. Sci. USA,
81, 8014-8018.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Sanford, J.C., Klein, T.M., Wolf, E.D. and Allen, N. (1987)
Delivery of substances into cells and tissues using a particle
bombardment process. J. Particle Sci. Technol. 5, 27-37.
Shilllto, R.D., Carswell, G.K., Johnson, C.M., Di Mais, J.J.
and Harms, C.T. (1989) Regeneration of fertile plants from
protoplasts of elite inbred maize. Bio/'l'echnology, 7, 581-587.

Spencer, T.M., Gordon-Kamm, W.J., Daines, R.J., Start, W.G.

and Lemaux, P.G. (1990) Bialaphos selection of stable
transformants from maize cell culture. Theor. Appl. Genet. 79,
625-631.
Vain, P., McMullen, M.D. and Finer, J.J. (1993) Osmotic treatment enhances particle bombardment-mediated transient and
stable transformation of maize. Plant Cell Rep. 12, 84-88.
Wilson, H.M., Bullock, B.P., Dunwell, J.M., Ellis, J.R., Frame,
B.R., Register M, J.R. and Thompson, J.A. (1994) Maize. In
Transformation of Plants and Soil Microorganisms (Wang, K.,
Herrera-Estrella, A. and Van Montagu, M., eds). Cambridge:
Cambridge University Press, pp. 65-80.