Full text of DOI:10.1007/BF02901907

transgenes are always distributed in the genome random-
ly[5,6]. In most cases of micro-projectile bombard-
ment-mediated transformation, different transgenes car-
ried on two separate plasmids were co-introduced into
plant cells, one of the plasmids carries selectable marker
gene, while the other is interest gene. The two plasmids
usually co-segregate as a locus, which suggests that they
are co-integrated into very close genomic loci[7, 8]. As for
the specific arrangement style of the two plasmids, it is
still not clear.
FISH analysis of the integra-
tion patterns in transgenic
rice co-transformed by micro-
projectile bombardment
JIN Weiwei1, LI Zongyun1, NING Shunbin1,
LING Dinghou2, LI Lijia1 & SONG Yunchun1
Fluorescence in situ hybridization (FISH), which can
identify the physical position of gene within a genome,
has become one of the most powerful tools to study
cyto-genetics[9,10]. The new developed Fiber FISH has
greatly improved the resolution (2ü3 kb) and sensitivity
(500 bp target sequence could be located effectively) of
1. Key Lab of the Ministry of Education for Plant Developmental Biol-
ogy, Wuhan University, Wuhan 430072, China;
2. South China Institute of Botany, Chinese Academy of Sciences,
Guangzhou 510650, China
Correspondence should be addressed to Song Yunchun (e-mail: ycsong@
whu.edu.cn)
Using multi-color fluorescence in situ hybridi-
FISH[11]
.
Abstract
zation (FISH), we localized transferred barnase-ps1 and
pHctinG DNA sequences onto chromosomes of two trans-
genic rice plants, named Q12 and Q13, both of which were
produced by micro-projectile bombardment. In both Q12
and Q13, each detected cell showed 2ü3 signal spots on their
chromosomes respectively. The signals of both barnase-ps1
and pHctinG were mostly detected in the adjacent chromo-
somal sites in which their signals were overlapped and could
be recognized by the signal color on the metaphase chromo-
somes. Fiber FISH further demonstrated that the multiple
copies in each of the two DNA sequences distributed adja-
cently on the DNA fiber in Q13. Combined with the results of
Southern hybridization, the possible integration patterns in
transgenic rice co-transformed by micro-projectile bom-
bardment have been discussed.
In this study, sequences barnase-ps1 (about 4.8 kb)
and pHctinG (about 8.0 kb) have been physically local-
ized to the chromosomes in 2 tested transgenic rice plants
by multi-color fluorescence in situ hybridization (FISH).
Meanwhile, combined with the results of Fiber FISH and
Southern blot, the integration patterns of transgenes are
discussed.
1
Materials and methods
(ν) Plant materials. The tested materials included
2 rice transgenic plants, named Q12 and Q13, both were
produced by micro-projectile bombardment. All the tested
materials were obtained by co-transferring the recipient,
Oryza sativa japonica cv Qiu Guang, with barnase-ps1
and pHctinG, and they belong to T0 generation.
Keywords: FISH, transgenic rice, co-transformation, integration
pattern.
(ξ) Southern blotꢀand histochemical assay of Gus.
The DNA isolations and the Southern hybridization
were performed as described by Sambrook[12]. The probes
were the coding sequence of barnase which was cloned in
the barnse-ps1 plasmid, and the restriction loci were EcoR
ĉ. They were labeled with 32P-dCTP following the pro-
cedure of nick translation kit manual (Promega, USA).
The genomic DNA of transgenic rice was digested with
BamHĉ, EcoRĉ and Pstĉ, respectively. The detection
of Gus activity was performed as described by Jeffer-
It was first reported in 1987 that the foreign genes
were delivered into living cells by micro-projectile bom-
bardment. This technique has been developed rapidly in
plants, especially in those which were recalcitrant to
Agrobacterium-mediated transformation methods. A lot of
researchers suggested that the expression levels of trans-
genes depend on their integration patterns[1,2]. So to study
the integration patterns is very important. It would help us
understand the integration mechanisms of foreign genes
better. Intensive studies on integration pattern of trans-
genes have been carried out with genetics and molecular
biology methods in transgenic plants including transgenic
rice[3,4]. But, up to date, no FISH analysis on the integra-
tion patterns of transgenes in rice has been reported. Most
researches show that micro-projectile bombardment-me-
diated transformation could usually result in 3 kinds of
integration patterns: the full integration of the alien plas-
mid(s), partial integration, and occasional concatenation
integration. Generally, the copy numbers of reported
transgenes range from 1 to 20. The integration sites of
son[13]
.
(ο) DNA fibers and chromosome preparation for
FISH. Chromosome preparation followed the proce-
dures described by Song et al.[14]. Isolation of plant nuclei
and extension of DNA fibers followed the methods of
Jackson[11]. The nuclei were stored in the 1Ή1 NIBΉ
100% glycerol at ꢀ20ć. The quality and concentration of
nuclei can be detected under a microscope after being
stained with DAPI. Extending the fibers is a critical step
in Fiber FISH. Pipet 2 PL nuclei and mix in 100 PL NIB,
Chinese Science Bulletin Vol. 46 No. 23 December 2001
1965

NOTES
Table 1 Copy number deducted based on Southern blot analysis,
sterility and GUS activity of T0 plants
centrifuge them at 3000ü3600 r/min for 5 min, then pipet
the glycerol out. Resupend the nuclei in PBS, subse-
quently pipet 2 PL suspension on a clean poly-L-lysine
slide and air-dry for 5ü10 min. And then add 10 µL STE
lysis buffer on the top of the nuclei and incubate for 4 min.
Slowly drag the solution down the slide with the edge of a
clean coverslip helding just above the surface of the slide.
Air-dry for 10 min. Fix in fresh 3Ή1 100% ETOH: acetic
acid for 2 min. And then bake at 60ć for 30 min. The
slides can be used directly in FISH without further prepa-
ration. The quality of DNA fiber extending can be de-
tected under a microscope after being stained with
YO-YO (see Plateĉ-3).
(π) Probe preparation for FISH. The barenase-ps1
clone is about 6.5 kb in size and it contains the ps1 pro-
moter (1.7 kb), barnase gene and the rest of the clone. To
avoid homologous sequence influencing the results of
hybridization, the ps1 promoter was cut off before label-
ing, because the ps1 was cloned from rice. The pHctinG
clone is about 8.0 kb in size, which carries hph, Gus genes
and 35S promoter. They were labeled with digoxigenin
and biotin respectively, following nick translation protocol
offered by the kit (Sino-American Biotechnology Com-
pany, China).
Code of
plant
GUS
activity
Copy number of
barnase
Setting for
selfing (%)
Sterility
Q12
Q13
CK
+
4
5
0
high
5.3
+
complete
fertile
0
84.4
ꢀꢁ
(ξ) Multi-color FISH of transgenes. The tested
two plants probe showed both barnase-ps1 (ps1 promoter
was cut off) and pHctinG hybridization signals on their
chromosomes (Plateĉ-1, 2). The lowest detection rate
was 11.7% and the highest 17.5%, and it was not the same
in different detection sites. The karyotype analysis was
difficult based only on the chromosome morphology. The
experimental data including the detection rates, the arm
ratio of chromosomes located by signals, and the frac-
tion-length (FL) value which was the percentage of the
distance from detection site to the centromere divided by
the length of the arm detected, are listed in table 2. Three
signal spots were detected in transgenic rice Q12, two of
them were located in the close region of the same long
arm and the other one was on another chromosome. Plate
ĉ-1 shows that the two signals, which were closely lo-
cated (signals a and b), displayed mix-color of red (di-
goxigenin labeled and Rodamine detected) and green
(biotin labeled and FITC detected). This indicated that
barenase-ps1 and pHctinG were co-transformed into al-
most the same chromosomal sites. And the other signal
(signal c in Plateĉ-1) shows pure green. It means that
only pHctinG was integrated into this chromosomal site.
Two signal spots were detected on the different chromo-
somes respectively in Q13, one was on the long arm and
the other was on the short arm. Plateĉ-2 shows that the
two signals were mix colored, meaning that the two genes
were also integrated in the adjacent chromosome sites.
(ρ) In situ hybridization and fluorescence detection.
In situ hybridization followed the procedures de-
scribed by Song et al.[14]. Fluorescence detection was done
in the following steps: the slide was covered with goat
anti-biotin FITC conjugate (Sigma) and mouse anti-dig
(Boehringer Mannheim), and subsequently treated with
rabbit anti-goat biotin conjugate (Sigma) and Dig-anti-
mouse (Boehringer Mannheim). Then goat anti-biotin-
FITC and Rodamine anti-dig (Boehringer Mannheim)
were added. The signals were amplified by immunological
reaction, at 37ć for 30 min each and washed with PBS at
intervals. And finally the slides were counterstained with
1 Pg/mL DAPI containing. Chromosomes were observed
with an Olympus BX60 fluorescence microscope
equipped with Sensys 1401E CCD camera. Red, green,
and blue images were captured in black and white with
different filters. The images were combined and pseu-
docolored in the computer using software V++.
Table 2 The FISH of barnse-ps1 and pHctinG on transgenic ricea)
Arms of
signal
located
Code of No. of cells Detection Ratio of long
plant
FL (%)
detected rate (%) to short arm
2
Results
Q12
72
65
L
L
L
L
S
1.76f0.10** 83.3f4.9
1.76f0.10** 69.0f3.4
60
55
65
55
0
(ν) Southern blot and GUS activities. From the
results of the Southern blot[15], the copy number of
barnase gene could be estimated. They were 4 and 5 re-
spectively (table 1). Based on the research results reported,
the integration of multiple copies of the transgenes is
common. The two detected rice plants showed GUS ac-
tivities, but they were at different levels (table 1). The
control was the negative in GUS activities.
1.30f0.14*
93.0f5.0
Q13
CK
71
40
1.70f0.10** 38.4f4.1
1.50f0.08** 91.7f4.1
ꢀꢁ
ꢀꢁ
ꢀꢁ
a) S, Short arm; L, long arm; site for pHctinG;
*
barnse-ps1 and pHctinG.
site for both
**
1966
Chinese Science Bulletin Vol. 46 No. 23 December 2001

the copy number of barnase in Q13 was 5 based on
Southern blot. But there were 5 hybridization signals only
in one fiber region (Plateĉ-4). Obviously, the copy num-
ber inferred from Southern hybridization bands probably
was underestimated, especially in the condition that the
(ο) Fiber FISH of transgenes. The results of Fiber
FISH (Plateĉ-4) show that multiple copies of barnse-ps1
and pHctinG linked on the extending DNA fibers of Q13.
only red (barnse-ps1) and green signals (pHctinG) but
also yellow signals (mixed color) were detected. The hy-
bridization signals distributed sparely on the DNA fibers
in beads-shape as reported by other researcheres[11]. The
signal color was red to green or the reverse in the order
from one end to the other end. In other words, the integra-
tion order was barnse-ps1 to pHctinG or reverse. The
several different DNA fibers in Q13 basically followed the
multi-copy transgenes integrated into the same regions[17]
.
So, the true copy number of barnase may be more than 5
in Q13.
Our results showed that on the one hand, the re-
peated copies were not linked; on the other hand, the for-
eign genes were interspersed in the genome. Therefore, it
suggested that it was not true that the multiple copy for-
eign genes integrated closely resulted from replication and
repairing as suggested by Jergensen et al. and Gharti-
Chhertri et al.[18,19]. Our viewpoint was consistent with
that of Myrism et al.[16]. Due to multiple copy integration,
the different copy would be able to be not the same for
their template strands of transcription. As a result,
mRNAs transcribed with different strands would block
each other. This might be one of the reasons for transgene
silencing. Of course, it needs to be proved by further study.
As for the spaces between the transgenes, they probably
were the traces left by self-repair after integration.
same pattern in the signal arrangement (Plate ĉ-4), it
could be inferred that the multiple barnse-ps1and pHctinG
copies integrated closely on the same fiber instead of dif-
ferent ones.
3
Discussion
The integration position has been a focus in the field
of genetic engineering. In this study, the two kinds of
plasmids were co-transformed into rice by micro-proje-
ctile bombardment. The FISH on the two tested transgenic
rice plants showed that the integration sites of foreign
genes had been distributed on the 5 regions of one mem-
ber of each in 4 chromosome pairs (table 2). And they
were located in the middle and terminal regions of chro-
mosome long and short arms, some were located closely
and others separately. No obvious tendencies or patterns
were observed about integration position. Obviously, it is
necessary to study further with more transgenic plants in
order to corroborate the results obtained above. But it
could be suggested that the integration sites probably re-
lated to the position shot by micro-projectile. We think,
where it was shot, it might be the integrated. The integra-
tion was probably a random event with neither preferen-
tial donor sequences nor favorite recipient regions for
under micro-projectile bombardment. That the number of
integration sites in the long arms was more than that in the
short arms could also be explained by random event, be-
cause the bigger surface of long arms had more chances to
be bombarded.
Acknowledgements
We would like to thank Dr. Alice Cheung (Yale
University, USA) and Dr. Futterer (ETH, Switzerland) for kindly pro-
viding the Barenase-ps1 and pHcintG plasmids. And we are grateful to
Prof. Jiang Jiming (University of Wisconsim) for his warm guidance.
This work was supported by the National Natural Science Foundation of
China (Grant No. 39900083) and the Research Fund for the Doctoral
Program of Higher Education (Grant No. 207980112).
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