Plant Cell Rep (2002) 20:1181–1187

DOI 10.1007/s00299-002-0459-7

GENETIC TRANSFORMATION AND HYBRIDIZATION

L. Tian · H. Wang · K. Wu · M. Latoszek-Green

M. Hu · B. Miki · D.C. W. Brown

Efficient recovery of transgenic plants through organogenesis

and embryogenesis using a cryptic promoter
to drive marker gene expression
Received: 7 September 2001 / Revised: 6 February 2002 / Accepted: 12 February 2002 / Published online: 16 April 2002
© Springer-Verlag 2002

Abstract Our previous studies have shown that tCUP, a Keywords Constitutive expression · Cryptic promoter ·
cryptic promoter from tobacco, functions in all living Plant transformation · Selectable marker
plant cell types in a wide range of plant species. This led
us to investigate if an enhanced derivative, EntCUP∆, Abbreviations BA: N6-Benzyladenine ·
could be used to drive the neomycin phosphotransferase CaMV: Cauliflower mosaic virus ·
II (nptII) gene and select for kanamycin resistance in 2,4-D: 2,4-Dichlorophenoxyacetic acid ·
crop species that regenerate by organogenesis or embry- GUS: β-Glucuronidase · NAA: α-Naphthaleneacetic acid ·
ogenesis. Tobacco (leaves), cauliflower (hypocotyls) and nptII: Neomycin phosphotransferase II gene
alfalfa (leaves, petioles, stems) explants were co-culti-
vated with Agrobacterium containing either EntCUP∆-
nptII-nos or 35S-nptII-nos to compare the efficiency of Introduction
selection for kanamycin resistance. The infected alfalfa
explants were placed in somatic embryo induction me- Only a small number of cells in a plant tissue or organ
dia, whereas tobacco and cauliflower explants were explant can be genetically transformed using the meth-
placed in shoot induction media with kanamycin at con- ods that are currently available; therefore, selective
centrations that normally inhibit regeneration. Transgen- growth of the transformed cells is crucial for the recov-
ic plants were recovered from all of the explants with ery of transgenic plants. The usual strategy adopted to
both selectable marker gene constructs. The transforma- increase the likelihood of recovering transgenic plants
tion efficiencies using tCUP∆-nptII-nos were compara- from a variety of species and tissue types is that of con-
ble to or higher than those using 35S-nptII-nos in all stitutive or non-specific expression of a selectable mark-
three species tested. This study demonstrated that pro- er gene. The CaMV promoter of the 35S transcript
moters which are not associated with expressed plant (Odell et al. 1985) and promoters of the Agrobacterium
genes can be used as alternatives for the expression of T-DNA genes (Bevan et al. 1983) are the most frequent-
selectable marker genes in a broad range of tissues and ly used promoters for this purpose. The recent discovery
species for the generation of transgenic plants. of a plant cryptic promoter, tCUP, with strong constitu-
tive activity (Foster et al. 1999) raised new and interest-
ing possibilities. tCUP is cryptic by nature, as it is inac-
tive at its natural location in the tobacco genome where
Communicated by M.C. Jordan it is not associated with an expressed gene (Foster et al.
L. Tian · H. Wang · M. Latoszek-Green · D.C.W. Brown (✉) 1999). Yet it is very active as a promoter when fused to
Southern Crop Protection and Food Research Centre, cloned genes in a wide range of plant species, including
Agriculture and Agri-Food Canada, 1391 Sandford St, angiosperms and gymnosperms (Foster et al. 1999;
London, Ontario, Canada N5V 4T3 Malik et al. 2002). Furthermore, the activities of en-
e-mail: browndc@em.agr.ca
Tel.: +1-519-4571470, Fax: +1-519-4573997 hanced tCUP promoters are comparable to or exceed the
activity of the 35S promoter in most plants that have
K. Wu · M. Hu · B. Miki been tested (Malik et al. 2002; Wu et al. 2001).
Eastern Cereal and Oilseed Research Centre,
Agriculture and Agri-Food Canada, Ottawa, Ontario, The use of cryptic promoters to direct the expression
Canada K1A 0C6 of a selectable marker gene for the generation of trans-
Present address: genic plants is one of the first applications of cryptic
K. Wu, Biology Department, West Virginia University, promoter technology. It also reveals the great value of
P.O. Box 6057, Morgantown, WV 26506-6057, USA DNA sequences within the plant genome for which bio-
1182

logical functions have not yet been assigned. As a step Cauliflower transformation
towards demonstrating the usefulness of cryptic promot-
The transformation of cauliflower followed the protocol of Ding et
er technology, we show here that a promoter derived al. (1998). Seven- to ten-day-old in vitro-seedlings of cauliflower
from tCUP (EntCUP∆) was able to effectively drive se- (Brassica oleracea L. var. botrytis) were harvested. Whole hypo-
lectable marker gene expression in different plant tis- cotyls were excised and placed on PTM medium (MS ingredients,
sues, leading to the efficient recovery of transgenic 1 mg/l 2,4-D, 0.1 mg/l kinetin) for 3 days for pretreatment.
Pretreated hypocotyls were dissected into segments of 3–5 mm
plants through different pathways of plant regeneration. in length and placed on CM1 medium (MS ingredients, 2 mg/l
The transformation efficiencies using EntCUP∆ was BA, 0.01 mg/l NAA). A drop of diluted Agrobacterium-culture
found to be comparable to or to exceed that using a 35S was placed on the surface of each explant. The explants were co-
promoter. cultured with Agrobacterium in the dark in a foil paper-covered
box for 4 days. After the co-culture period, explants were trans-
ferred to CM2 medium (CM1 medium containing 2 mg/l AgNO3
and 300 µg/ml timentin) and maintained on this medium for
7–10 days. Twenty explants were placed in each petri dish
Materials and methods (15×90 mm). After 7–10 days on CM2 medium, the explants were
transferred to CM3 selection medium (CM2 medium supplement-
Vectors ed with 25 µg/ml kanamycin) where they were maintained with
transfer to fresh CM3 medium every 2 weeks. Green shoots were
Three constructs were used in the study. EntCUP∆ (Malik et al. excised and transferred to CMR medium (CM3 medium lacking
2002) replaced the 35S promoter in pCAMBIA2300 (CAMBIA, growth regulators and containing 30 µg/ml kanamycin) for root
Canberra, Australia; personal communication) to generate the Ent- development. The number of green shoots that formed roots was
CUP∆-nptII-nos construct. As a control, pCAMBIA2300 that car- recorded. Two experiments were conducted for cauliflower trans-
ried an nptII gene driven by a 35S promoter (35S-nptII-nos) was formation with a total of ten petri dishes for 35S-nptII-nos and 11
used in all transformation experiments. In addition, EntCUP∆- petri dishes for EntCUP∆-nptII-nos.
GUS-nos (Malik et al. 2002) was used separately to evaluate con- All of the media for cauliflower transformation contained 2%
stitutive expression of EntCUP∆. sucrose with a pH of 5.7.

Agrobacterium culture Alfalfa transformation

Agrobacterium tumefaciens (GV3101/pMP90) was grown at 28°C Alfalfa (Medicago sativa L., clone N4) plants were propagated in
to OD600=1.0 in LB medium containing 150 µg/ml kanamycin, vitro. Petioles and stems were cut into about 0.8-cm fragments,
100 µg/ml gentamycin and 150 µg/ml rifampicin. The Agrobacte- and the leaves were cut into pieces of approximately 5 mm2. The
rium culture was then diluted to 50% using LB medium and used explants were pre-cultured for 2 days on SH2K medium [Schenk
for the transformation of tobacco and alfalfa. For cauliflower and Hildebrandt medium (1972), 1 mg/l 2,4-D, 0.2 mg/l kinetin,
transformation, the Agrobacterium culture was centrifuged for 25 mM proline, 0.4 mM thioproline, 50 mM potassium sulfate and
5 min at 5,000 rpm and the cells resuspended in 10 ml CM1 liquid 1.5% sucrose, pH 5.8]. After a 2-day pre-culture, the explants
medium (see below). were infected with Agrobacterium. The infected explants were
then cultured on SH2K medium for 2 days, following which they
were transferred to SH2K medium supplemented with 50 µg/ml
Tobacco transformation kanamycin and 300 µg/ml timentin. Petiole and stem fragments
were cultured on 20×60-mm plates and leaf explants on 15×90-
In vitro-maintained tobacco (Nicotiana tabacum cv. SR1) plants mm plates. Five petiole and stem explants were usually placed on
were used for the transformation. The transformation essentially each plate and ten leaf explants on each plate, all on SH2K medi-
followed the procedure described by Horsch et al. (1985). Leaves um. Three to six plates were used for each type of explant for each
were cut into about 3-mm2 disks that were then co-cultured with construct. The explants were transferred to fresh induction medi-
Agrobacterium for 2 days on MSS medium [MS salts (Murashige um after 2–3 weeks. After 6 weeks on SH2K induction medium,
and Skoog 1962), B5 vitamins (Gamborg et al. 1968), 1 mg/l BA, the explants were then transferred to BOi2Y medium for embryo
0.1 mg/l NAA, 3% sucrose, 0.25% Gelrite, pH 5.7]. After 2 days, development. BOi2Y medium consists of Blaydes medium
the tissues were transferred to MSS medium containing 300 µg/ml (Bingham et al. 1975) modified to contain 3% sucrose, 0.2% yeast
timentin and 100 µg/ml kanamycin. Ten leaf disks were cultured extract, 100 mg/l myo-inositol and 0.25% Gelrite, pH 5.9, plus
in a petri dish (20×60 mm) with a total of six dishes for each con- 300 µg/ml timentin and 50 µg/ml kanamycin. The number of em-
struct. The number of shoots formed on each leaf disk and the bryos formed and the number of responsive explants were record-
number of responsive explants were recorded at different times. ed. Alfalfa transformation was conducted once.
Observation and comparison were conducted on the same date for Elongated mature embryos with well-formed cotyledons were
each construct. The experiment was repeated three times, and as selected and transferred to half-strength MSO (MS ingredients,
all gave similar results data from only one experiment is present- 2% sucrose, 0.25% Gelrite) supplemented with 100 µg/ml kana-
ed. mycin and 300 µg/ml timentin for germination. The germinated
Well-formed shoots were transferred to Majenta boxes (Majenta embryos were first transferred to half-strength MSO in Majenta
Corp, Chicago) containing MSR medium for root induction. MSR boxes, and well-established plants were later transferred into soil
and MSS are basically the same medium only in the former the in the greenhouse.
growth regulators have been removed and the medium modified to Controls – explants that were not infected with Agrobacterium
contain 1.5% sucrose and 150 µg/ml kanamycin. Once the roots – were included in each of the transformation experiments with to-
were well developed, the plantlets were transferred to soil in a bacco, alfalfa and cauliflower. All cultures were maintained at
greenhouse. 25°±2°C under a 16/8-h (day/night) photoperiod with light sup-
T0 seeds from transgenic tobacco plants were sterilized first in plied by fluorescent Sylvania “Cool-White” light at a photosyn-
70% ethanol and then in 1.3% sodium hypochlorite followed by thetic photon flux of about 50 µmol/m2/s.
three washes in sterile distilled water. The seeds were placed on a
filter paper on MSR medium containing 200 µg/ml kanamycin for
germination evaluation.
 1183
Fig. 1A–C Constitutive ex-
pression of the reporter GUS
gene driven by the EntCUP∆
promoter in different transgenic
plant species stained with the
GUS substrate, X-GLU.
A GUS gene expression in a
T1 seedling of a tobacco plant,
B GUS gene expression in a T0
seedling of an alfalfa plant,
C GUS gene expression in a T0
cauliflower plantlet

Constitutive expression of GUS gene in transgenic plants

Results and discussion
Primary regenerated plants or T1 seedlings transformed with Ent-
CUP∆-GUS were stained with X-GLU according to Jefferson In this study, the efficiencies of EntCUP∆-nptII-nos and
(1988). 35S-nptII-nos as selectable markers were compared un-
der culture conditions used for the induction of organo-
Statistical analysis genesis in tobacco or cauliflower and the induction of
embryogenesis in alfalfa. The transformation of tobacco
Data were analyzed using computer software, Minitab (Minitab, leaf, cauliflower hypocotyl and alfalfa leaf, petiole or
Reading, Mass.). stem was conducted by Agrobacterium-mediated trans-
formation. Use of the GUS reporter gene indicated that
NPTII ELISA the EntCUP∆ promoter was active in each of these or-
gans (Fig. 1a–c). EntCUP∆ differed from tCUP in that
The presence of NPTII in transgenic tobacco plants was analyzed an upstream translational initiation codon in the leader
using the PathoScreen NPTII kit (Agdia, Indiana) following the
instructions of the manufacturer. sequence (Foster et al. 1999) was eliminated, a Kozak
consensus sequence (Kozak 1984) was added to the
translational start site and the size of the amino-terminal
1184
Table 1 Comparison of transformation efficiency in cauliflower
using EntCUP∆-nptII-nos and 35S-nptII-nos. Cauliflower hypo-
cotyls were used as explants in Agrobacterium-mediated transfor-
mation, and shoots were induced in shoot induction medium in the
presence of 25 µg/ml kanamycin. The transformation efficiency
(the number of shoots per plate) with EntCUP∆-nptII-nos is com-
parable to that with 35S-nptII-nos (P=0.94, t-test)

Construct Total no. Average no. Responsea

of explants of shoots (in percentage)
recovered
per plate

EntCUP∆-nptII-nos 220 3.1 17

35S-nptII-nos 200 3.2 16
a Percentage of explants that showed shoot regeneration

The shoots were excised and transferred to growth

regulator-free MSR medium containing 150 µg/ml kana-
mycin, and roots subsequently formed at the base of the
regenerated shoots (Fig. 3b). Shoots regenerated via the
standard in vitro protocol without a transformation pro-
cedure survived in the presence of kanamycin, but no
roots developed (Fig. 3b). The seeds (T1) from transgen-
ic tobacco plants (T0) transformed with the EntCUP∆-
nptII-nos gene germinated and developed on medium
containing 200 µg/ml kanamycin, whereas seeds from
untransformed plants did not (Fig. 3c). As expected, not
all of the T1 seeds from transgenic plants germinated due
to segregation. In the resistant plants, NPTII protein was
detected by ELISA at levels of 0.1–0.6% of total protein.
The regeneration of cauliflower from hypocotyls of
germinated seeds occurs through organogenesis (Ding et
al. 1998). Following co-cultivation with Agrobacterium,
shoots developed 5–6 weeks after the hypocotyl explants
Fig. 2A, B Comparison of transformation efficiency in tobacco were introduced into culture with 25 µg/ml kanamycin
using EntCUP∆-nptII-nos and 35S-nptII-nos. Tobacco leaf disks
were infected with Agrobacterium. The shoot regeneration from (Fig. 3d). Shoot formation continued for a further
leaf disks on shoot induction medium containing kanamycin is 4–6 six weeks. In contrast to organogenesis in tobacco
shown during progressive stages of tissue culture. A Percentage of leaf explants, shoot initiation on cauliflower hypocotyls
leaf-disk explants that showed shoot regeneration, B average num- was slower but it continued for a longer period of time.
ber of shoots per leaf disk. Bars: Standard error of the mean The shoots produced roots when transferred to rooting
medium containing 30 µg/ml kanamycin, whereas con-
peptide that was fused to the GUS gene was reduced trol untransformed shoots did not (data not shown). As
(Malik et al. 2002). These modifications enhanced tCUP shown in Table 1 and Fig. 3d, the transformation effi-
activity in all organs (Malik et al. 2002). ciencies using EntCUP∆-nptII-nos and 35S-nptII-nos
Plant regeneration from tobacco leaves is typically by were similar.
organogenesis. Following co-cultivation with Agrobacte- Alfalfa was used as a host to study selection in em-
rium, shoots began to emerge around 10 days after the bryogenic cells because it is a model plant for studies on
leaf disks were cultured on the induction medium in the somatic embryogenesis (Saunder and Bingham 1972; re-
presence of 100 µg/ml kanamycin. The percentage of viewed by McKersie and Brown 1997). The leaves, peti-
leaf disks which produced shoots and the number of oles and stems were used as tissue explants for co-culti-
shoots that formed per leaf disk transformed with Ent- vation with Agrobacterium. Callus formed on petioles
CUP∆-nptII-nos and 35S-nptII-nos, respectively, were and stems, and somatic embryos subsequently formed on
very similar over the next 9 days in culture (Fig. 2a, b). the callus (shown for petioles in Fig. 3e). Some callus
Following this period, fewer new shoots developed. development was also observed on the stems and peti-
Control untransformed leaf disks gradually turned yel- oles of control explants on induction medium containing
low or white and did not produce shoots in the presence kanamycin, but no embryos differentiated on the callus
of kanamycin (Fig. 3a). In leaf disks infected with Ent- (Fig. 3e). Little callus developed on leaf explants, but so-
CUP∆-nptII-nos, shoots developed in the presence of matic embryos appeared to develop directly from the leaf
kanamycin levels as high as 500 µg/ml (data not shown). tissues (Fig. 3f). Direct somatic embryogenesis on alfalfa
 1185
Fig. 3A–F In vitro regenera-
tion and plant recovery with
different organ explants from
different species under kana-
mycin selection. A Shoot re-
generation from tobacco leaf
disks infected with Agrobacte-
rium containing EntCUP∆-
nptII-nos after 4 weeks in cul-
ture. No shoots formed on the
control uninfected explants
(left). B The plants transformed
with the EntCUP∆-nptII-nos
construct produced roots (ar-
row) in the medium containing
kanamycin, but no roots
formed from untransformed
shoots that regenerated via the
standard regeneration proce-
dure (left). C T1 seeds of Ent-
CUP∆-nptII-nos-transgenic
plants showed vigorous germi-
nation, and germinated seeds
continued to grow and develop
on a medium containing
200 µg/ml kanamycin (right).
Seeds from untransformed con-
trol plant showed initial signs
of germination, but no further
development was observed in
the presence of kanamycin
(left). D Shoot regeneration
from cauliflower hypocotyl ex-
plants infected with EntCUP∆-
nptII-nos and 35S-nptII-nos af-
ter 9 weeks in culture. E So-
matic embryo formation on the
calli induced from petioles of
alfalfa infected with EntCUP∆-
nptII-nos after 7 weeks in cul-
ture (right plate). No embryos
developed on the control calli
(left plate). F Somatic embryo
formation directly on the leaves
of alfalfa infected with Ent-
CUP∆-nptII-nos after 7 weeks
in culture

Table 2 Comparison of trans-

formation efficiency in alfalfa Type of explants EntCUP∆-nptII-nos 35S-nptII-nos
using the EntCUP∆-nptII-nos
and 35S-nptII-nos selectable Leaves Number of explants 39 30
marker genes. Alfalfa leaves, Responsea (in percentage) 100 90
stems, and petioles were infect- Average no. of embryos per explant 18.6 10.9
ed with Agrobacterium contain- t-testb P=0.0039
ing the EntCUP∆-nptII-nos or Stem Number of explants 27 30
35S-nptII-nos construct. So- Responsea (in percentage) 63 53
matic embryos were induced Average no. of embryos per explant 3.4 1.4
under the selection of 50 µg/ml t-test P=0.022
kanamycin
Petioles Number of explants 39 40
Responsea (in percentage) 69 60
Average no. of embryos per explant 2.8 2.1
t-test P=0.16
a Percentage of explants that showed embryogenesis
b Comparison of the number of embryos formed on explants with EntCUP∆-nptII-nos and 35S-nptII-
nos transformation
1186

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