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.

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