Post on 01-Mar-2023
The rooting ability of the dwarfing pear rootstock BP10030(Pyrus communis) was significantly increased by introduction
of the rolB gene
Li-Hua Zhu *, Xue-Yuan Li, Annelie Ahlman, Margareta Welander
Department of Crop Science, Swedish University of Agricultural Sciences, Box 44, SE-230 50 Alnarp, Sweden
Received 17 June 2003; received in revised form 17 June 2003; accepted 18 June 2003
Plant Science 165 (2003) 829�/835
www.elsevier.com/locate/plantsci
Abstract
The pear rootstock BP10030 (Pyrus communis ) is dwarf, frost hardy and compatible with most pear varieties, but very difficult to
propagate due to its poor rooting ability. In order to improve its rooting ability, we transformed this rootstock with the rolB gene.
Six transgenic clones were obtained and named 1-6. PCR and Southern hybridisation confirmed that all the transgenic clones
contained the rolB and nptII genes, but only two of them contained the gus gene. The in vitro rooting results showed that the
transgenic clones rooted from 67 to 100% without auxin, while the untransformed control did not root at all on the hormone free
rooting medium. To further confirm the increased rooting ability of these clones, cutting experiments were carried out on four
transgenic clones (1-4) and the untransformed control in the greenhouse. The rooting percentage ranged from 71 to 100 for the
transgenic clones and only five for the untransformed control. Moreover, the root number was also greatly increased ranging from
11 to 17 for the transgenic clones, but only 3 for the untransformed control. Another important alteration by the insertion of the
rolB gene was the root morphology. For the transformed clones, the roots were distributed evenly on the cut surface, but only on
one side of the cut surface of the untransformed control. Growth analysis in the greenhouse, conducted on four transgenic clones (1-
4) and the untransformed control, showed that three of the transgenic clones had a shortened stem length. The transgenic clones did
not show any other visual alteration in shoot phenotype compared with the untransformed control plants.
# 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cutting; Dwarfing rootstock; Pyrus ; rol B; Rooting; Transformation
1. Introduction
Pear (Pyrus communis ) is one of the most cultivated
fruit trees in the world. In modern pear production
dwarfing rootstocks are commonly used for reducing
tree size, enabling high-density planting and easy
management, and thus achieving high production effi-
ciency. However, there are few dwarfing rootstocks of
Pyrus type available in commercial pear production. As
an alternative, quince (Cydonia oblonga Mill.) has been
used as pear dwarfing rootstocks, but it is incompatible
with some pear cultivars, sensitive to alkaline soil
conditions and not hardy enough [1,2]. Therefore, it is
of great importance to select new or improve the existing
rootstocks. BP10030, selected in Balsgard, Sweden, is of
Pyrus type. It is dwarf, cold hardy and compatible with
most pear varieties tested, but very difficult to root, thus
limiting its commercial use. Improving the rooting
ability of this rootstock will add a great commercial
value to it.
Genetic transformation offers a great potential for
improving fruit tree rootstocks. The greatest advantage
of genetic transformation is that the recipient gains only
one or a few foreign genes while the main genetic
framework remains unchanged. As a result, only one or
a few traits of a plant can be modified. Since fruit trees
are normally propagated vegetatively, it is easy to keep
the new desirable traits once a rootstock or a cultivar
has been improved. Besides, fruit trees have a long life
span and it usually takes many years to breed a new
* Corresponding author. Tel.: �/46-40-41-5373; fax: �/46-40-46-
0442.
E-mail address: li-hua.zhu@vv.slu.se (L.-H. Zhu).
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0168-9452(03)00279-6
rootstock or cultivar. By using genetic transformation
method, the breeding process can be greatly accelerated.
The rolB gene was isolated from the Agrobacterium
rhizogenes which in nature causes hairy root disease indicotyledonous plants after infection [3�/5]. It has been
reported that the rolB gene can improve the rooting
ability in different plant species [6�/10]. Therefore, there
is a great potential to use the rolB gene in improving the
rooting ability for important woody plant species. The
purposes of this study were to improve the rooting
ability of pear rootstock BP10030 by introducing the
rolB gene and to evaluate growth characteristics of thetransgenic plants.
2. Material and methods
2.1. Plant material and shoot culture conditions
Shoot cultures of the pear rootstock BP10030 used for
transformation were grown under the conditions as
described by Zhu and Welander [9]. Later, in order to
enhance the shoot elongation, shoot cultures of the
transformed and untransformed control were grown on
the medium containing Lepoivre [11] macro nutrients,
MS [12] micro nutrients and vitamins, sorbitol 30 g l�1,
BAP 2.2 mM, GA3 1.44 mM, solidified with 6 g l�1 agarat pH 5.5.
2.2. Agrobacterium strain and vector and transformation
Agrobacterium tumefaciens strain C58C1 (pGV3850)
was used for transformation. The strain harbours the
binary vector pCMB-B:GUS that contains the nptIIgene under the nopaline synthase (nos ) promoter and a
tandem gene construct consisting of the gus gene under
the rolB promoter and the rolB gene under its own
promoter (Fig. 1) [13]. The rolB construct was kindly
provided by Dr C. Maurel. For transformation, the
youngest 1-3 expanding leaves from 2-week-old shoots
were gently wounded by the clinical forceps with
carbide-coated tips and inoculated in the bacterialsolution for 20 min. The infected leaf explants were
then moved to the medium consisting of basal salts
according to Chevreau and Leblay [14], 15 mM TDZ
(thidiazuron), 1 mM NAA (naphtalene acetic acid), 30 g
l�1 sorbitol, 2.5 g l�1 Gelrite. The detailed transforma-
tion procedure, culture conditions of leaf explants and
selection of transformed clones have been previously
reported by Zhu and Welander [9].
2.3. PCR and Southern hybridisation
Plant DNA minipreparation was carried out as
described by Dellaporta et al. [15]. PCR primers and
analysis were according to Zhu and Welander [9]. For
Southern hybridisation, plant genomic DNA was iso-
lated from in vitro grown 5-week-old shoots using the
method according to Aldrich and Cullis [16]. BeforeDNA extraction, the shoot cultures were placed in cold
for 24 h for de-starching. Southern hybridisation was
carried out according to Holefors et al. [17]. Due to the
limitation of the in vitro plant materials for clones 5-6,
analyses concerning Southern hybridisation, the cutting
experiment and growth analysis were only carried out
on clones 1-4.
2.4. Rooting in vitro
Thirty shoots each of the six transgenic clones and
untransformed control were rooted in vitro. The rooting
method was according to Welander [18], but without
IBA (indole-3-butyric acid) in the rooting medium. The
rooting percentage and the root number per rooted
shoot were recorded after 3 weeks.
2.5. Ex vitro rooting by cuttings in the greenhouse
Micropropagated plants of the transgenic clones 1-4
and untransformed control were planted in pots with a
mixture of soil: perlite (1:1) in the greenhouse. The
growing conditions in the greenhouse were: a tempera-ture of 23/16 8C (day/night), a photoperiod of 16 h and a
light intensity of 200�/250 mmol m�2 s�1 with high-
pressure sodium lamps. When the plants were about 30
cm in height, cuttings were taken from them. Three
softwood cuttings from the top of each plant were taken.
Each cutting had 2-3 nodes and the shoot tip of the first
cutting was removed. Only one top leaf was remained on
each cutting. Twenty to thirty cuttings were taken fromeach type of plants depending on the availability of
plant material. The cuttings were placed in a container
with a mixture of soil: perlite (1:1) and the container was
covered with plastic film during the rooting period.
Manual watering was supplied when necessary. The
container was placed in the greenhouse with the same
Fig. 1. A schematic drawing of the T-DNA of the binary vector pCMB-B:GUS showing the location of the genes and the restriction sites. The coding
regions of the npt II, rol B and gus genes are indicated with the solid bars; arrows show the direction of translation. RB and LB represent right and left
borders of the T-DNA. Restriction sites: B, BamHI; E: Eco RI; H, HindIII; S, Sal I.
L.-H. Zhu et al. / Plant Science 165 (2003) 829�/835830
conditions as stated above. The rooting percentage and
the root number were recorded after 4 weeks. The
cutting experiments were repeated once. The three
cuttings were mixed in the experiments because nodifferences in rooting ability were observed among the
three cuttings in a preliminary test.
2.6. Growth analysis in the greenhouse
Growth analysis was carried out on micropropagated
plants of the transgenic clones 1-4 and untransformed
control to evaluate growth characteristics of the trans-
genic clones. In vitro rooted plantlets were transferred to
pots containing a mixture of soil: perlite (1:1) and placedin the greenhouse with the same conditions as stated
above. The plant number of each type ranged from 10 to
30, depending on the availability of plant materials. At
the beginning, the plantlets were covered with plastic
cups for about 2 weeks and the cups were removed when
the plants had acclimatised. The plants were watered
with nutrients regularly after acclimation. After 4
months, the stem length and node number wererecorded.
2.7. Statistic analysis
All data were statistically analysed with Duncan’s
multiple range test using the Statgraphics program.
3. Results
3.1. Putative transformed clones and PCR
Six putative transformed clones were obtained on the
kanamycin selection medium. PCR analysis showed that
the rolB and nptII genes were inserted into the plant
genome for all the six clones, but only two clones (1-2)
showed the gus insertion (data not shown).
3.2. Southern hybridisation
Southern hybridisation was carried out only on the
transgenic clones 1-4 and the untransformed control.When the digested DNA was hybridised with the rolB
probe or nptII probe, one band was observed for clone
1, three bands for clones 3-4, and five bands of the rolB
gene and four bands of the np tII gene for clone 2 (Fig.
2). This indicates that one copy of both rolB, and nptII
genes was inserted into the plant genome for clone 1,
while three copies of both rolB and nptII genes inserted
for clones 3-4. For clone 2, four copies of the nptII geneand five copies of the rolB gene were inserted. When the
digested DNA was hybridised with the gus probe, one
band was observed for clones 1-2, but no bands for
clones 3-4 (Fig. 2), indicating one gus insertion for
clones 1-2, but no gus insertion for clones 3-4.
3.3. Rooting in vitro
In vitro rooting was conducted on all six transgenic
clones and the untransformed control. The transgenic
clones rooted on the hormone-free rooting medium,
while no roots were obtained for the untransformed
control (Fig. 3a). The rooting percentage ranged from
67 to 100 and the root number was 4.6�/9.0 for the
transgenic clones (Table 1). Clone 2 had a lower rooting
percentage compared with the other clones, while the
root number was greater for clones 3-6 compared with
the other two clones (Table 1).
3.4. Rooting ex vitro by cuttings
The cutting experiment in the greenhouse was carried
out on the transgenic clones 1-4 and the untransformed
control to further confirm the increased rooting ability
of the transgenic clones. The experiment was repeated
once and the results were combined as they were similar.
All transformed clones showed a significantly increased
rooting percentage compared with the control (Fig. 3c).
The rooting percentage ranged from 71 to 100 for the
transgenic clones, while only five for the control.
Meanwhile, the root number increased greatly for the
transgenic clones ranging from 11 to 17, but only 3 for
the control (Table 2). Moreover, the roots were dis-
tributed evenly on the cut surface for the transgenic
clones, but only on one side of the cut surface for the
untransformed control (Fig. 3d). The root length did not
significantly alter when comparing the transgenic clones
with the untransformed control (Table 2).
3.5. Growth analysis
Growth analysis was carried out on the transgenic
clones 1-4 and the untransformed control in the green-
house to evaluate the influence of the rolB gene on
growth characteristics of the transgenic clones. Stem
length decreased significantly for clones 2-4 compared
with the untransformed control, while no alteration in
stem length was found between clone 1 and the
untransformed control (Table 3). The node number
was decreased in clones 2 and 3, whereas the internode
length was also reduced in clone 2 compared with the
untransformed control (Table 3). The transgenic plants
did not show any other visual changes in shoot
phenotype compared with the control plants (Fig. 3b).
L.-H. Zhu et al. / Plant Science 165 (2003) 829�/835 831
4. Discussion
In the present study, it was clearly demonstrated that
the integration of the rolB gene into the plant genome
significantly increased the rooting ability of the dwarfing
pear rootstock BP10030. Both the rooting percentage
and the root number were increased, as shown from the
results of in vitro rooting and ex vitro rooting by
cuttings. Increased rooting ability caused by the rolB
gene has been previously reported in different plant
species [3�/8,10]. The rolB gene has also been shown to
reduce the size of some transgenic plants [8,10,19]. In
this study, the transgenic clones 2-4 had a shortened
stem length compared with the control, which may
further reduce the tree size compared with the control
plant and thus be of interest to fruit growers.
Reports on the correlation between the copy number
of transgenes and the gene expression have not been
consistent [10,17,20�/23]. In the present study, the copy
number of the rolB gene ranged from 1-5 for different
transgenic clones, while the rooting percentage was
similar among the clones with 1-3 copies of the rolB
Fig. 2. Southern hybridisation of the rol B, npt II and gus genes, digested with BamHI and hybridised with the rol B probe, npt II and gus probes,
from the four transformed and untransformed control plants of the dwarfing pear rootstock BP10030. Figures 1�/4: the four transgenic clones. C:
untransformed control. M: markers.
Fig. 3. (a) In vitro rooted plants of the transgenic clones (1-3) and the untransformed control (C); (b) Greenhouse-grown plants of the four
transgenic clones (1-4) and the untransformed control (C) from the growth analysis experiment. (c) Rooted plants from cuttings of the four
transgenic clones (1-4) and the untransformed control (C); (d) Root distribution on the cut surface of the two transgenic clones (1-2) and the
untransformed control (C) of the dwarfing pear rootstock BP10030 from the cutting experiment.
L.-H. Zhu et al. / Plant Science 165 (2003) 829�/835832
gene insertion. However, for clone 2 with 5 copies of the
rolB gene, the in vitro rooting percentage decreased
significantly and the ex vitro rooting percentage by
cuttings also tended to decrease although the difference
was not significant compared with the other clones.
Meanwhile, stem and internode lengths of clone 2 were
significantly lower than those of clone 1 with only one
rolB gene insertion. This suggests that the multiple
copies of the rolB gene insertion into the plant genome
impose a negative effect on rooting. Sedira et al. (2001)
have reported that more than two copies of the rolB
gene insertion decreased the rooting ability of the apple
rootstock Jork [24]. Other reports suggest that the
phenotype is probably more associated with the position
of the transgene on the plant genome [10,17,25,26].
However, it is more likely both copy number andposition of the transgene influence the phenotype.
In this study, the transgenic clones 2-4 had multiple
copies of the rolB and nptII gene insert, but only one
copy of the gus gene for clone 2 and no gus insert for
clones 3 and 4. This indicates a deletion of the gus gene
from the T-DNA. The loss of the gus gene may have
been caused by deletion of the left part of T-DNA
during the integration as the gus gene is located nearestto the left border. The mechanism of a foreign DNA
insertion into the plant genome is not fully understood.
The occurrence of transgene deletion has been pre-
viously observed in transgenic rice [27], tobacco [28] and
apple [10] plants.
Agrobacterium -mediated transformation results in
both single and multiple copies of transgene inserts at
different locations in the plant genome [7,10,18,25,29].Multiple copies of transgenes are often revealed as
repeat structures or inverted repeats, which are probably
associated with gene silencing of the transgene-encoded
sequences [30,31] due to increased DNA methylation
[32]. Therefore, screening transgenic plants with single
transgene inserts would be of valuable practice to avoid
the gene silencing or instability of transgene expression.
In our case, it is better to choose clone 1 with one rolBgene insert for future commercial production to avoid
possible gene silencing or instability of the transgene
expression.
It has been reported that the increased rooting ability
caused by the rolB gene is associated with the increased
sensitivity of plants to auxins [33]. Filippini et al. (1996)
have reported that the rolB protein is a tyrosine
phosphatase, suggesting the involvement of the rolBprotein in the auxin signal-transduction pathway [34].
Although the rolB gene is mainly expressed in the root
meristems [35], its function is not only restricted to roots
as it can promote organ formation from the rolB
transgenic tobacco thin-cell-layer cultures [36]. This
suggests that expression of the rolB gene not only
influences rooting, but also affects other growth and
developmental processes, such as, changes in growthcharacteristics of some transgenic clones observed in this
study. Nevertheless, the significant improvement in the
rooting ability of the transformed clones of BP10030
makes mass production of this rootstock possible at a
low cost. Since BP10030 is a dwarfing pear rootstock of
Pyrus type, the use of the rolB transgenic plants of this
rootstock will bring a bright perspective to commercial
pear production in the near future. One concern in usingtransgenic plants is the stability of the altered trait in
offspring. However, this may not apply to our case
because fruit tree rootstocks are propagated vegeta-
tively. The risk to lose the transgene (rolB) is thus very
Table 1
Results of in vitro rooting from six transgenic clones (1-6) and the
untransformed control of the dwarfing pear rootstock BP10030
Plant Rooting % Root number
BP10030 (control) 0 a 0 a
Clone 1 93 c 5.6 b
Clone 2 67 b 4.6 b
Clone 3 100 c 9.0 c
Clone 4 94 c 8.7 c
Clone 5 94 c 8.7 c
Clone 6 90 c 8.1 c
Figures followed by different letters differ significantly at P�/0.05
(n�/30).
Table 2
Results of ex vitro rooting by cuttings from the transgenic clones (1-4)
and the untransformed control of the dwarfing pear rootstock
BP10030
Plant Rooting % Root number Root length (mm)
BP10030 (control) 5 a 3 a 85 a
Clone 1 89 b 11 b 62 a
Clone 2 71 b 11 b 59 a
Clone 3 100 b 17 b 96 a
Clone 4 100 b 15 b 81 a
Figures followed by different letters differ significantly at P�/0.05
(n�/20�/30).
Table 3
Results of growth analysis from the transgenic clones (1-4) and
untransformed control of the dwarfing pear rootstock BP10030 in
the greenhouse
Plant Stem length
(cm)
Internode length
(mm)
Node
number
BP10030 (control) 36 c 7.8 b 46 b
Clone 1 31 bc 7.8 b 40 ab
Clone 2 16 a 4.7 a 34 a
Clone 3 23 ab 6.6 ab 35 a
Clone 4 22 ab 5.9 ab 37 ab
Figures followed by different letters differ significantly at P�/0.05
(n�/10�/30).
L.-H. Zhu et al. / Plant Science 165 (2003) 829�/835 833
low, as compared with annual crops where the segrega-
tion occurs after crossing and selection. Nevertheless,
before any commercialisation of these transgenic clones,
we will further evaluate the influence of these clones ongrowth and development of pear cultivars grafted onto
them and to evaluate the stability of transgene expres-
sion in greenhouse and in the field.
Acknowledgements
The financial support from The Swedish Research
Council for Environment, Agricultural Sciences and
Spatial planning to this research is highly acknowledged.
References
[1] H.B. Jacob, A new clonal rootstock for high density pear
orchards, Acta Hortic. 475 (1998) 169�/177.
[2] S.J. Wertheim, P.S. Wagenmakers, D. Sugar, How to increase
productivity of future pear orchards, Acta Hortic. 367 (1994)
138�/151.
[3] F.F. White, B.H. Tayor, G.A. Huffman, M.P. Gordon, E.W.
Nester, Molecular and genetic analysis of the transferred DNA
regions of the root-inducing plasmid of Agrobacterium rhizogenes ,
J. Bacteriol. 164 (1985) 33�/44.
[4] M. Cardarelli, D. Mariotti, M. Pomponi, L. Spano, I. Capone, P.
Costantino, Agrobacterium rhizongenes T-DNA genes capable of
inducing hairy root phenotype, Mol. Gen. Genet. 209 (1987) 475�/
480.
[5] A. Spena, T. Schmullin, C. Koncz, J. Schell, Independent and
synergistic activity of the rol A , B and C loci in stimulating
abnormal growth in plants, EMBO J. 6 (1987) 3891�/3899.
[6] I.L. Capone, L. Spano, M. Cardarelli, D. Bellincampi, A. Petit, P.
Constantino, Induction and growth properties of carrot roots
with different complements of Agrobacterium rhizogenes T-DNA
genes, Plant Mol. Biol. 13 (1989) 43�/52.
[7] M. Welander, N. Pawlicki, A. Holefors, F. Wilson, Genetic
transformation of apple rootstock M26 with rol B gene and its
influence on rooting, J. Plant Physiol. 53 (1998) 371�/380.
[8] M. Welander, L.H. Zhu, The rooting ability of rol B transformed
clones of the apple rootstock M26 and its relation to gene
expression, Acta Hortic. 521 (2000) 133�/138.
[9] L.H. Zhu, M. Welander, Adventitious shoot regeneration of two
dwarfing pear rootstocks and the development of a transforma-
tion protocol, J. Hortic. Sci. Biotechnol. 75 (2000) 745�/752.
[10] L.H. Zhu, A. Holefors, A. Ahlman, Z.T. Xue, M. Welander,
Transformation of the apple rootstock M.9/29 with the rol B gene
and its influence on rooting and growth, Plant Sci. 160 (2001)
433�/439.
[11] M. Quoirin, P. Lepoivre, P. Boxus, Un premier bilan de dix
annees de recherche sur les cultures de meristemes et la multi-
plication in vitro de fruitiers ligneux (in French), Compte rendu
des recherches , Station des Cultures Fruitieres et Marrıcheres de
Gembloux 1976�/1977 (1977) 93�/117.
[12] F. Murashige, F. Skoog, A revised medium for rapid growth and
bioassys with tobacco tissue cultures, Physiol. Plant. 15 (1962)
473�/492.
[13] C. Maurel, N. Leblanc, H. Barbier-brygoo, C. Perrot-Rechen-
mann, M. Bouvier-Durand, J. Guern, Alterations of auxin
perception in rolB-transformed tobacco protoplasts, Plant Phy-
siol. 105 (1994) 1209�/1215.
[14] E. Chevreau, C. Leblay, The effect of mother plant pretreatment
and explant choice on regeneration from in vitro pear leaves, Acta
Hortic. 336 (1993) 263�/266.
[15] S.L. Dellaporta, J. Wood, J.B. Hicks, A plant DNA miniprepara-
tion: version II, Plant Mol. Biol. Rep. 1 (1983) 19�/21.
[16] J. Aldrich, C. Cullis, RAPD analysis in flax: optimization of yield
and reproductivity using KlenTag1 DNA polymerase, Chelex 100
and gel purification of genomic DNA, Plant Mol. Biol. Rep. 11
(1993) 128�/141.
[17] A. Holefors, Z.T. Xue, M. Welander, Transformation of the apple
rootstock M26 with the rol A gene and its influence on growth,
Plant Sci. 136 (1998) 69�/78.
[18] M. Welander, In vitro rooting of the apple rootstock M26 in adult
and juvenile growth phase and acclimatization of the plantlets,
Physiol. Plant. 58 (1983) 231�/238.
[19] L.H. Zhu, M. Welander, Growth characteristics of the untrans-
formed and transformed apple rootstock M26 with rol A and rolB
genes under steady-state nutrient supply conditions, Acta Hortic.
521 (2000) 139�/146.
[20] E. Gendloff, B. Bowen, W. Buchholz, Quantitation of chloram-
fenicol acetyltransferase in transgenic tobacco plants by ELISA
and correlation with gene copy number, Plant Mol. Biol. 14
(1990) 575�/584.
[21] S.L.A. Hobbs, T.D. Warkentin, C.M.O. DeLong, Transgene copy
number can be positively or negatively associated with transgene
expression, Plant Mol. Biol. 21 (1993) 17�/26.
[22] J.D.G. Jones, D.E. Gilbert, K.L. Grady, R.A. Jorgensen, T-DNA
structure and gene expression in petunia plants transformed by
Agrobacterium tumefaciens C58 derivatives, Mol. Gen. Genet. 207
(1987) 478�/485.
[23] A. Shirsat, N. Wilford, R. Croy, Gene copy number and levels of
expression in transgenic plants of a seed specific gene, Plant Sci.
61 (1989) 75�/80.
[24] M. Sedira, A. Holefors, M. Welander, Protocol for transforma-
tion of the apple rootstock Jork 9 with the rol B gene and its
influence on rooting, Plant Cell Rep. 20 (2001) 517�/524.
[25] L.H. Zhu, A. Ahlman, X.Y. Li, M. Welander, Integration of the
rolA gene into the genome of the vigorous apple rootstock A2
reduced plant height and shortened internodes, J. Hort. Sci.
Biotechnol. 76 (2001) 758�/763.
[26] R.L. Bell, R. Scorza, C. Srinivasan, K. Webb, Transformation of
‘Beurre Bosc’ pear with the rol C gene, J. Amer. Soc. Hort. Sci.
124 (1999) 570�/574.
[27] M. Takano, H. Egawa, J.E. Ikeda, K. Wakasa, The structures of
integration sites in transgenic rice, Plant J. 11 (1997) 353�/361.
[28] Y.S. Kim, M.H. Lee, S.R. Min, O.J. Yoo, J.R. Liu, Frequent
occurrence of transgene deletion in transgenic plants, Mol. Cells 8
(1998) 705�/708.
[29] C. Grevelding, V. Fantes, E. Kemper, J. Schell, R. Masterson,
Single-copy T-DNA insertions in Arabidopsis are the predomi-
nant form of integration in rooted-derived transgenics, whereas
multiple insertions are found in leaf discs, Plant Mol. Biol. 23
(1993) 847�/860.
[30] M. De Neve, S. De Buck, A. Jacobs, M. Van Montagu, A.
Depicker, T-DNA integration patterns in co-transformed plant
cells suggest that T-DNA repeats originate from ligation of
separate T-DNAs, Plant J. 11 (1997) 15�/29.
[31] A. Depicker, M. Van Montagu, Post-transcriptional gene silen-
cing in plants, Curr. Opin. Cell Biol. 9 (1997) 373�/382.
[32] S.L.A. Hobbs, P. Kpodar, C.M.O. DeLong, The effect of T-DNA
copy number, position and methylation on reporter gene expres-
sion in tobacco transformants, Plant Mol. Biol. 15 (1990) 851�/
864.
[33] A. Delbarre, P. Muller, V. Imhoff, H. Barbier-Brygoo, C. Maurel,
N. Leblang, C. Perrot-Rechenmann, J. Guern, The rol B gene of
Agrobacterium rhizogenes does not increase the auxin sensitivity
L.-H. Zhu et al. / Plant Science 165 (2003) 829�/835834
of tobacco protoplasts by modifying the intracellular auxin
concentration, Plant Physiol. 105 (1991) 563�/569.
[34] F. Filippini, V. Rossi, O. Marin, M. Trovato, P. Costantino, P.M.
Downey, F. Lo Schiavo, M. Terzi, A plant oncogene as a
phosphatase, Nature 379 (1996) 499�/500.
[35] I. Capone, G. Frugis, P. Costantino, M. Cardarelli, Expression in
different populations of cells of the root-meristems is controlled
by different domains of the rol B promoter, Plant Mol. Biol. 25
(1994) 681�/691.
[36] M.M. Altumura, T. Archilletti, I. Capone, P. Costantino,
Histological analysis of the expression of Agrobacterium rhizo-
genes rol B-gus gene fusions in transgenic tobacco, New Phytol.
118 (1991) 69�/78.
L.-H. Zhu et al. / Plant Science 165 (2003) 829�/835 835