全 文 ：Received 21 Jul. 2003 Accepted 3 Dec. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (2001AA211111, 2002AA212071).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (7): 854-861
Production and Analysis of Transgenic Maize with Improved Salt
Tolerance by the Introduction of AtNHX1 Gene
YIN Xiao-Yan, YANG Ai-Fang, ZHANG Ke-Wei, ZHANG Ju-Ren*
(School of Life Sciences, Shandong University, Jinan 250100, China)
Abstract: The AtNHX1 gene and hygromycin phosphotransferase (hpt) gene were introduced into the
embryonic callus cells of maize (Zea mays L.) elite inbred lines DH4866, Qi 319 and Luyuan 16106 by
Agrobacterium-mediated method. Plantlets were obtained from the hygromycin-resistant calli after screening.
PCR analysis and Southern blot hybridization indicated that there were 22.8% transgenic plants among the
regenerated plants. The transformation frequencies were obviously affected by Agrobacterium concentration,
the genotype of callus and co-cultivation duration. The segregations of AtNHX1 gene in the progeny were
not easily to be elucidated, and some lines showed Mendelian segregations. The screening of salt tolerance
indicated that the salt tolerance of some transgenic plants and their progenies were improved significantly
and the seeds of some lines were able to germinate and grow in the presence of 0.8%-1.0% sodium chloride.
Northern blotting analysis of T2 transgenic plants indicated that the salt tolerance of plants was correlative
with the transcription level of AtNHX1 in the leaves.
Key words: maize; AtNHX1 gene; Agrobacterium ; transgenic plant; salt tolerance
It is one of the salt-tolerant mechanisms in plants that
the overmuch Na + in cytoplasm is accumulated in vacuoles
(Nass et al., 1997; Martinoia et al., 2000). Some species of
non-halophytes lacked or minified the activity of Na+/H+
antiport. It was possible that the non-halophytes had lost
or attenuated the mechanisms of ion compartmentation in
cells. In 1997, Nass et al. isolated the NHX1 gene from
yeast. The NHX1 encoded the Na+/H+ antiport in
prevacuolar membrane. In 1999, Gaxiola et al. cloned the
AtNHX1 gene from Arabidopsis thaliana that was homolo-
gous to the NHX1 in yeast and restored the salt-sensitive
yeast mutant phenotypes after introducing the AtNHX1
into yeast. In this year, Apse et al. (1999) introduced the
AtNHX1 gene with strong promoter into A. thaliana and
obtained the transgenic plants overexpressing the AtNHX1.
The transgenic plants could grow and develop continu-
ously in soil when being watered with up to 200 millimolar
sodium chloride, and the AtNHX1 gene product was mainly
localized to vacuolar membrane fraction. In 2001, Zhang
and Blumwald reported that the salt tolerance of tomato
was improved by the transformation of AtNHX1. Up to now,
there were no reports about introducing the AtNHX1 into
monocotyledonous plants.
Maize is a crop that is moderately sensitive to salinity,
and exhibits low activity of Na+/H+ antiport. We have intro-
duced the AtNHX1 gene with CaMV35S promoter into cul-
tured cells of maize and obtained transgenic plants. The
salt tolerance of some transgenic plants and their prog-
enies were well improved. In this paper these results were
reported.
1 Materials and Methods
1.1 Plant materials
About 10-12 d after self-pollination, the immature maize
embryos of inbred lines DH4866, Qi 319 and Luyuan 16106
were isolated and cultured on modified MS medium to in-
duce callus. After 10 d, embryonic calli were produced, then
were selected for subculture at an interval of 10-15 d. Two
months later the embryonic calli were used for genetic
transformation.
1.2 Agrobacterium strain and plasmid
Agrobacterium used in transformation experiments was
LBA4404/pCAMBIA1300:: AtNHX1. There were a
hygromycin phosphotransferase (hpt) gene and a multiple
cloning site (MCS) in the T-DNA region of the plasmid
pCAMBIA1300. The AtNHX1 gene was inserted in the
MCS.
1.3 Media
The maize calli were induced and cultured on modified
MS medium (Li et al., 1990). Co-cultivation was on modi-
fied MS medium supplemented with 100 mmol/L
acetosyringone (AS), and the concentration of glucose was
10 g/L, while sucrose was 20 g/L. Agrobacterium was elimi-
nated on modified MS medium containing 200 mg/L
YIN Xiao-Yan et al.: Production and Analysis of Transgenic Maize with Improved Salt Tolerance by the Introduction of AtNHX1
Gene 855
cefotaxime or 200-300 mg/L carbenicillin. The selection
medium was the modified MS medium supplemented with
20 mg/L hygromycin B, sometimes including 200 mg/L
cefotaxime. The differentiation medium was the modified
MS medium containing 0-1 mg/L 6-benzylaminopurine (6-
BA). The rooting medium (pH 5.8-6.0) consisted of N6 in-
organic salts, N vitamins, 0.2 g/L casein hydrolysate (CH),
0-1 mg/L indolebutyric acid (IBA), 5 g/L active carbon, 20
g/L sucrose and 7 g/L agar.
Yeast extract peptone (YEP) medium (pH 7.0) containing
10 g/L yeast extract, 10 g/L Bacto-peptone and 5 g/L NaCl
was used for Agrobacterium culture. Collected bacteria
were suspended in liquid modified MS medium (pH 5.2)
supplemented with 100 mmol/L AS.
1.4 Culture of Agrobacterium
The single clone of Agrobacterium LBA4404/
pCAMBIA1300::AtNHX1 was picked out and grown in YEP
medium supplemented with 50 mg/L rifampicin and 50 mg/L
kanamycin at 28 ℃ with agitation 150-200 r/min. When the
value of OD600 of the bacterial suspension was up to 0.8-
1.0, Agrobacterium was harvested by centrifugation at
4 000 r/min for 5 min at 4 ℃. The bacterium pellets were
resuspended in liquid-modified MS medium (pH 5.2) supple-
mented with 100 mmol/L AS, and diluted to the required
concentration for infection.
1.5 Transformation of maize calli and regeneration of
plantlets
The embryonic calli in logarithmic phase were selected
and smashed into pieces of 2-3 mm in diameter, then im-
mersed in Agrobacterium suspension for 5 min with good
shakes. The infected calli were cultured on medium from 3 d
to 5 d in the dark at 25 ℃ for co-cultivation, then were
washed with sterile water for three times, and transferred to
the medium containing cefotaxime or carbenicillin to con-
trol the overgrowth of bacterium. After the cultures were
kept in the dark at 25 ℃ for 15-20 d, the proliferation of the
bacterium was inhibited. Then the calli were cultured on
the selection medium for three generations continuously in
the dark at a subculture interval of 15 d. Hygromycin-resis-
tant calli were selected and cultured on the medium without
hygromycin B for one generation under 16 h light and 8 h
dark, and then were transferred to the differentiation me-
dium for the regeneration of plantlets. The plantlets with
healthy roots were transplanted to flowerpots to grow up,
and then were planted in the fields.
1.6 PCR analysis and Southern blot of transgenic plants
and their progenies
The genomic DNA of leaves was extracted following
CTAB protocol. The PCR primer sequences of AtNHX1 gene
were as follows: 5-GATTCTCTAGTGTCGAAACTGC-3; 5-
TGGAACAAAGGGTACAAAGC-3. The amplified product
was 1 566 bp. PCR reactions were carried out in a 25-mL
volume. Reaction conditions were as follows: initial dena-
turation at 95 ℃ for 3 min, followed by 95 ℃ for 30 s, 56 ℃
for 45 s, and 72 ℃ for 1 min for a total of 35 cycles. The
products were electrophoresed in 1.0% agarose gels.
Plasmid DNA and the genomic DNA of PCR-positive
plants were digested with restriction enzyme EcoRⅠ/Hind
Ⅲ. The 1.6 kb AtNHX1 fragment in the plasmid was recov-
ered and labeled as probe. After digestion with EcoRⅠ/
HindⅢ, the DNA of transgenic plants should indicate hy-
bridization bands about 1.6 kb in size. The labeling of probe
and molecular hybridization were accomplished using the
DIG system (Roche Applied Science, Germany).
The total RNA of leaves was extracted following guani-
dine isothiocyanate protocol (Gu et al., 1995). The probe
was 1.6 kb AtNHX1 fragment. The labeling of probe and
molecular hybridization were accomplished using the DIG
system (Roche Applied Science, Germany).
1.7 Salt tolerance of transgenic plants and their prog-
enies
The transgenic plants and their progenies were self-
pollinated or pollinated with pollens from the non-transgenic
plants of identical genotype. The seeds were planted in
little buckets full of sand and watered with different con-
centrations of NaCl solutions until the plants grew out of
three leaves. Then the plants were watered with 1/2 MS
medium inorganic salts solution supplemented with differ-
ent concentrations of NaCl solutions.
2 Results
2.1 The optimization of parameters for maize callus
transformation
Calli and Agrobacterium were grown competitively on
medium in co-cul t iva t ion. The production o f
hydroxybenzene compounds from callus cells increased
after the infection of Agrobacterium. It was important to
control the production of hydroxybenzene compounds
because the high concentration of hydroxybenzene com-
pounds could cause the callus cells to turn brown or die.
When the concentration of Agrobacterium for infection
was reduced, the production of hydroxybenzene com-
pounds could be relieved. The concentration of
Agrobacterium suspension also influenced the transfor-
mation frequency of maize callus (Table 1). When infected
with low concentration of Agrobacterium suspension, the
calli showed low death rate and low rate of the hygromycin-
resistant calli. The peak rate of the hygromycin-resistant
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004856
calli (7.2%-8.0%) was achieved at the appropriate concen-
tration of Agrobacterium (OD600 was 0.6), accompanying
the increased death rate of callus. When the calli from dif-
ferent inbred lines were infected with the same concentra-
tion of Agrobacterium, a remarkable difference in the rate
of the hygromycin-resistant calli was seen (Table 1).
The length (days) of co-cultivation affected the trans-
formation frequency obviously. With longer co-cultivation
duration, the transformation frequency decreased, and the
rate of brown calli and dying calli increased. In initial ex-
periments good results were obtained when co-cultivation
was done for 2–5 d. The peak rate was obtained following 4
d co-cultivation in farther experiments. The rate of
hygromycin-resistant calli was obviously different among
different genotype of callus when the length (days) of co-
cultivation was altered. The rate of the hygromycin-resis-
tant calli of DH4866 and Luyuan 16106 fluctuated within a
relatively narrow range, but that of Qi 319 fluctuated in a
larger scale. The peak rate of hygromycin-resistant calli of
Qi 319 was 8.3% (Table 2).
2.2 The differentiation of resistant calli and the trans-
plant of plantlets
After selected for three generations the hygromycin-
resistant calli were cultured on the medium without
hygromycin B for one generation, then were induced for
the regeneration of plantlets on differentiation medium.
About 3.5 plantlets were produced in one bottle (about 60
pieces of calli in each bottle). Plantlets with a height of 2–3
cm were transferred to the rooting medium to develop roots.
The plantlets with developed roots were transplanted in
flowerpots. The surviving rate was 70%–85%. When the
plantlets grew up above 10 cm high, they were planted in
the fields with above 85% surviving rate.
2.3 Traits of T0 (regenerated) plants and molecular analy-
sis
The surviving regenerated plants in the fields usually
did not grow well. Most of them showed short and less
leaves, abnormal male and/or female organs. The tassels
and ears of the regenerated plants usually developed
disjointedly. The tassels born fewer branches and pollens
than the controls did. Only part of the plants produced a
smaller number of seeds by self-pollinated. Among the 158
normal plants, there were 43 PCR-positive ones by PCR
analysis (Fig.1). Among the 43 plants, 36 plants were con-
firmed to be transgenic ones by Southern blot (Fig.2).
Namely, there were 22.8% regenerated plants that were
transgenic. The hybridization bands of a few plants were
not on the expected position (the expected position of hy-
bridization bands was 1.6 kb) (Fig.2). It was possible that
structural alterations of exogenous gene had taken place in
the process of integration into maize genome. There were
32 transgenic plants that were self-pollinated or pollinated
with pollens from non-transgenic plants. The number of
seed on an ear varied from 3 to 241.
Table 2 Co-cultivation duration influenced the rate of hygromycin-resistant calli
Co-cultivation duration
Genotype 2 d 3 d 4 d 5 d
a b (%) a b (%) a b (%) a b (%)
DH4866 285 4.2 358 5.0 401 6.0 254 3.5
Qi 319 263 3.7 361 4.7 386 8.3 262 2.7
Luyuan 249 4.8 317 5.4 300 5.7 238 2.5
16106
The conditions of the experiment: the Agrobacterium concentration was 0.6 (the value of OD600). Calli were infected for 5 min. After co-
cultivation, the calli were cultured on selection medium containing 200 mg/L cefotaxime and were screened continuously for three generations.
The hygromycin-resistant calli were counted after three-generation selection. a, No. of calli infected; b, rate of hygromycin-resistant calli.
Table 1 Effects of Agrobacterium concentrations on the rate of the hygromycin-resistant calli
Agrobacterium concentration (OD600)
Genotype 0.2 0.4 0.6 0.8 1.0 1.2
a (%) b (%) a (%) b (%) a (%) b (%) a (%) b (%) a (%) b (%) a (%) b (%)
DH4866 68.4 1.3 64.2 2.9 55.7 7.4 49.5 6.6 47.5 4.3 46.7 0.6
Qi 319 72.7 1.7 68.1 3.8 61.8 8.0 48.5 5.7 44.2 2.0 43.5 0.9
Luyuan 67.3 1.4 64.7 1.9 60.8 7.2 54.3 7.4 53.8 2.6 50.6 1.2
16106
Conditions for transformation: calli were infected for 5 min, co-cultivated for 4 d and cultured on Agrobacterium-inhibited medium containing
200 mg/L cefotaxime for 10 d. The data were obtained after screening continuously for three generations. a, rate of surviving calli after
infection. b, rate of hygromycin-resistant calli.
YIN Xiao-Yan et al.: Production and Analysis of Transgenic Maize with Improved Salt Tolerance by the Introduction of AtNHX1
Gene 857
2.4 Molecular analysis and salt-tolerance assay of the
progenies of transgenic plants
By PCR analysis, positive plants appeared in 19 lines
among the 21 self- pollinated lines, and appeared in 10 lines
among 11 backcross lines of the transgenic progenies (T1
generation). All plants in three lines that consisted of 35, 7
and 5 individuals respectively did not show expected bands.
The ratios of PCR-positive to negative plants of 14 self-
pollinated lines and seven backcross lines that consisted
of more than 24 individuals in one line are shown in Table 3.
From Table 3, it was concluded that the transgene was
segregated as a single-locus in transgenic lines DH4866-
04, DH4866-05, DH4866-08, DH4866-13, DH4866-19,
DH4866- 20, DH4866-22, Qi 319-01 and Qi 319-09, and seg-
regated as two unlinked loci in lines DH4866-02, DH4866-
09 and Qi 319-03. Non-Mendelian segregations were ob-
served in the rest lines.
PCR-positive T1 plants of some lines were confirmed by
Southern blot hybridization (Fig.3). It was found that the
transgenic plants of most transgenic lines had the same
hybridization bands as their parents, while a few of plants
showed altered bands (Fig.3, the hybridization band of lane
7 differed from that of the parent) that might be caused by
unequal exchange of homoeologous chromosomes in
meiosis, or gene rearrangement, or base mutation of en-
zyme cutting site. The salt tolerance of the plant of which
DNA showed hybridization band on lane 7 of Fig.3 re-
sembled the control one in salt-tolerance assay. Namely,
the plant lacked salt tolerance.
There were two self-pollinated lines that were eliminated
at pollination stage for their abnormal development among
the 29 T1 transgenic lines. The plants of the other 27 lines
were grown normally, and had some traits different from
that of the non-transgenic controls. According to PCR re-
sults and agricultural traits, a part of T1 plants were se-
lected and self-pollinated for breeding.
The salt tolerance of T2 transgenic plants was assessed
(Fig.4). The seeds were sowed in sand buckets and wa-
tered with different concentrations of NaCl solutions. The
germination capacity and the number of plants at 5-leaf
stage were counted. When the non-transgenic plants
(controls) were watered with 0.5% NaCl solution, the
germination capacity and the rate of 5-leaf plant of DH4866
Fig.1. PCR analysis of the T0 transgenic plants (DH4866).
Lanes 1-7, PCR-positive plants. Lane M, DNA marker DL2000.
Lane NT, a non-transformed control. Lane P, PCR result of plas-
mid DNA.
Fig.2. Southern blotting analysis of the T0 transgenic plants
(DH4866). Lanes 1-7, PCR-positive plants. Lane M, DNA
marker λ/HindⅢ. Lane NT, a non-transformed control. Lane P,
AtNHX1 fragment from plasmid DNA.
Fig.3. Southern blotting analysis of the T1 transgenic plants.
Lanes 1-3, plants originated from the line corresponding to the
T0 transgenic plants in lane 2 of Fig.2; lanes 4-6, plants origi-
nated from the line corresponding to the T0 transgenic plants in
lane 4 of Fig.2; lanes 7-9, plants originated from the line corre-
sponding to the T0 transgenic plants in lane 3 of Fig.2, and the
hybridization band of lane 7 differs from that of the parent. Lane
NT, a non-transformed control. Lanes A and B, one copy and five
copies of the AtNHX1 fragment from plasmid DNA. Lane M,
DNA marker l /HindⅢ.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004858
were 57% and 23% respectively, and those of Qi 319 were
38% and 13% respectively. The lower leaves of surviving
plants withered and the top of the upper leaves dried. Un-
der the same condition, the transgenic plants showed ob-
viously improved salt tolerance, and the germination ca-
pacity was above 80% except DH4866-081. When non-
transgenic seeds (DH4866 and Qi 319) were watered with
the NaCl solutions of 0.8% and 1.0%, germination capacity
of them was nearly zero, while some transgenic plants from
16 T2 transgenic lines (DH4866 11 lines, Qi 319 5 lines) were
grown well, some plants from 10 transgenic lines could tol-
erate 0.8% NaCl and some plants from 6 transgenic lines
could tolerate 1.0% NaCl. Although the salt tolerance of
the transgenic plants was improved greatly, the degrees of
salt tolerance were varied. Part of plants in three lines of
DH4866-02 could tolerate 0.8% NaCl, but almost died when
watered with 1.0 % NaCl solution (Table 4). The salt toler-
ance of three lines of DH4866-04 was excellent, and some
plants of them could tolerate 1.0% NaCl, and the germina-
tion capacity under salinity stress was also high. The three
lines of DH4866-08 showed a remarkably difference in salt
tolerance. The salt tolerance of DH4866-081 was similar to
that of the control ones, while the DH4866-082 and DH4866-
083 showed improved salt tolerance. The germination ca-
pacity and the rate of 5-leaf plants of DH4866-082 and
DH4866-083 did not show significant differences and were
both higher. Some plants of these two lines could tolerate
1.0% NaCl (Table 4). The three lines from DH4866-09 were
also excellent in salt tolerance. PCR and Southern blotting
analysis indicated that the transgene still existed in the
plant genome in the T2 individuals of DH4866-02, DH4866-
04, DH4866-08 and DH4866-09 though the salt tolerance of
them was different obviously.
Northern blotting analysis of T2 transgenic plants (Fig.
5) indicated that the salt tolerance of DH4866-081, DH4866-
082, DH4866-083, DH4866-023, DH4866-022, DH4866-042
and DH4866-043 was correlative with the transcription level
of AtNHX1 in the leaves. For example, the salt tolerance of
DH4866-042 and DH4866-043 was significantly improved
and those plants exhibited strong Northern hybridization
bands, while DH4866-022 had poor salt tolerance and the
corresponding band was feeble, the DH4866-081 lacked salt
tolerance and there was no signal in the corresponding
lane.
Vigorous T2 plants suffered from the screening of salin-
ity stress were transplanted in the fields to grow up and be
self-pollinated. The transgenic lines with steady salt toler-
ance were selected from their progenies.
Table 3 The segregation ratios of the T1 individuals of transgenic plants
Lines Line origin
No. of PCR- No.of PCR-
Ratio
Ratio tested c2 Ppositive negative
(PCR+:PCR-)
plantlets plantlets
DH4866-01 T0 self-pollination 5 24 0.2/1 3/1 51.60 <0.005
DH4866-02 T0 self-pollination 27 3 9/1 15/1 0.72 >0.25
DH4866-03 T0 self-pollination 4 28 0.1/1 3/1 66.67 <0.005
DH4866-04 T0 self-pollination 20 6 3.3/1 3/1 0.05 >0.75
DH4866-05 T0 self-pollination 17 8 2.1/1 3/1 0.65 >0.25
DH4866-08 T0 self-pollination 20 10 2/1 3/1 1.11 >0.25
DH4866-09 T0 self-pollination 27 1 27/1 15/1 0.34 >0.50
DH4866-12 T0 self-pollination 7 19 0.4/1 3/1 32.05 <0.005
DH4866-13 T0 self-pollination 16 8 2/1 3/1 0.89 >0.25
DH4866-14 T0 self-pollination 8 18 0.4/1 3/1 27.13 <0.005
DH4866-19 T0 backcross 13 18 0.7/1 1/1 0.81 >0.25
DH4866-20 T0 backcross 16 12 1.3/1 1/1 0.57 >0.25
DH4866-21 T0 backcross 3 25 0.1/1 1/1 17.29 <0.005
DH4866-22 T0 backcross 15 11 1.4/1 1/1 0.62 >0.25
DH4866-24 T0 backcross 7 22 0.3/1 1/1 7.76 <0.01
Qi 319-01 T0 self-pollination 21 5 4.2/1 3/1 0.46 >0.25
Qi 319-02 T0 self-pollination 3 22 0.1/1 3/1 52.92 <0.005
Qi 319-03 T0 self-pollination 26 3 8.7/1 15/1 0.83 >0.25
Qi 319-04 T0 self-pollination 7 23 0.3/1 3/1 42.71 <0.005
Qi 319-08 T0 backcross 9 20 0.5/1 1/1 4.17 <0.05
Qi 319-09 T0 backcross 18 12 1.5/1 1/1 1.20 >0.10
When degree of freedom is 1, c20.05=3.84；c20.10=2.71；c20.25=1.32；c20.50 =0.45；c20.75=0.10.
YIN Xiao-Yan et al.: Production and Analysis of Transgenic Maize with Improved Salt Tolerance by the Introduction of AtNHX1
Gene 859
Table 4 Salt tolerance determination of T 2 transgenic plants
Salt concentration
Lines 0.5% NaCl 0.8 % NaCl 1.0% NaCl
a b c d a b c d a b c d
DH4866(CK) 60 34 14 ++++ 60 3 0 60 0 0
DH4866-021 30 28 26 + 30 22 14 +++ 30 4 0
DH4866-022 30 27 18 +++ 30 10 2 ++++ 30 4 0
DH4866-023 30 28 21 +++ 30 25 20 +++ 30 6 1 ++++
DH4866-041 30 29 29 + 50 43 17 ++ 30 16 11 +++
DH4866-042 30 30 29 + 50 41 38 ++ 30 23 17 ++
DH4866-043 30 28 27 + 50 44 36 ++ 30 20 15 ++
DH4866-081 40 23 9 ++++ 30 3 0 30 0 0
DH4866-082 40 37 37 + 30 22 16 ++ 30 14 7 +++
DH4866-083 40 38 36 + 30 14 12 ++ 30 16 12 +++
DH4866-091 30 26 25 + 30 20 17 ++ 30 12 8 +++
DH4866-092 30 25 25 +++ 30 21 11 +++ 30 4 3 +++
DH4866-093 30 28 26 + 30 23 15 ++ 30 13 9 +++
T2 transgenic plants came from 4 T0 transgenic plants（DH4866-02, DH4866-04, DH4866-08, DH4866-09）. a, No. of seeds; b, No. of seeds
germinated; c, No. of 5-leaf plants; d, degrees of injury on 5-leaf plants. +, light symptom of injury: the plant showed normal appearance, but the
tip of the leaves turned yellow or necrotic; ++, moderate symptom of injury: the plant grew relatively slowly, the upper part of the leaves were
necrotic; +++, serious symptom of injury: the plant grew slowly, the majority of leaves became yellow or dying; ++++, nearly to death.
Fig.4. Salt-tolerance test of T2 progenies of the transgenic plants.
A. The seeds were sowed in sand buckets and watered with 0.8%
NaCl solution. The left bucket, DH4866 (non-transformed
control). A few seeds could germinate, but no one was able to
grow. The right bucket, the transgenic plants DH4866-093 at 3–
4 leaf stage could still grow normally. B. The seeds were sowed in
sand buckets and watered with 1.0% NaCl solution. From left to
right in the first row, the first bucket was the control (non-trans-
formed DH4866), while the second, third and fourth buckets
were DH4866-041. The second row (four buckets) was DH4866-
042. The third row (four buckets) was DH4866-043.
3 Discussion
Ishida et al. (1996) elucidated that the transformation
frequency could be affected by the factors such as geno-
type of maize, type of callus tissues, physiological state
and developmental period of callus tissues, composition of
plant culture media, Agrobacterium concentration, selected
marker gene and type of vectors. Moreover, Agrobacterium
strains, antibiotics, co-cultivation duration and resting pe-
riod (Zhao et al., 2001) also influenced the transformation
frequency. To achieve an overall high level of transforma-
tion all the factors should be considered carefully and or-
ganized in an optimal combination. In this study, the exog-
enous gene was introduced into the callus cell of maize
eli te inbred l ines by Agrobacterium-mediated
transformation, and transgenic plantlets were regenerated
from the hygromycin-resistant calli, and the parameters of
Fig.5. Northern blotting analysis of the T2 transgenic plants.
Lane 1, a non-transformed control. Lanes 2–8, DH4866-081,
DH4866-082, DH4866-083, DH4866-023, DH4866-022,
DH4866-042, DH4866-043.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004860
transformation were optimized. But after regeneration the
rate of the transgenic plantlets was lower. Probably it was
caused by the different responses of maize cells to
hygromycin B. When the concentration of hygromycin B
in medium was increased, it was very difficult to regenerate
plantlets from the hygromycin-resistant calli.
In this study, the segregations of the transgene in the
progenies of transgenic plants were not easily to be
elucidated. Only in some lines Mendelian segregations were
observed. Moreover, in the progenies of some transgenic
plants the transgene could not be found by molecular
analysis. Probably those transgenic plantlets were chimeric
and transgenic cells did not take part in the production of
gametophytes. The segregation ratio of transgene in the
progenies of some transgenic lines did not accord with
Mendelian segregations. It was probable that the transgenic
plantlets were chimeric, or they produced the transgenic
gametophytes that had the inferior ability to compete for
fertilization. According to the segregation ratio of transgene,
we found that the T-DNA was usually integrated into plant
genome with low copy (1-2 loci). So the stabilized and
uniformed transgenic plants could be obtained easily.
This paper reported that the AtNHX1 coming from di-
cotyledonous A. thaliana could well improve the salt toler-
ance of monocotyledonous maize. In this work the salt tol-
erance of the majority of transgenic plants carrying AtNHX1
was obviously improved in contrast to the non-transgenic
control ones, but the levels of the improvement showed
significant differences. Only the progenies of a few
transgenic plants exhibited outstanding improvement in salt
tolerance. The salt tolerance of the sister lines coming from
one transgenic plant was usually similar. But there were
some exceptions, for example, the salt tolerance of DH4866-
081 was obviously different from that of its sister lines.
Considering the progenies and transgenic parent were both
PCR-positive, possible explanations would be transgene
silencing. Moreover, there were some differences in the
degree of salt tolerance among the individuals in one line,
and the possible reasons would include the segregations
of AtNHX1 gene and/or other traits of the plants.
Acknowledgements: The authors thank Prof. ZHAO Yan-
Xiu and Prof. ZHANG Hui (Shandong Normal University)
for providing plasmid pCAMBIA1300:: AtNHX1.
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