ORIGINAL PAPER

Effects of transgenic rootstocks on growth and developmentof non-transgenic scion cultivars in apple

Anders Smolka • Xue-Yuan Li • Catrin Heikelt •

Margareta Welander • Li-Hua Zhu

Received: 3 September 2009 / Accepted: 18 January 2010 / Published online: 5 February 2010

� Springer Science+Business Media B.V. 2010

Abstract Although cultivation of genetic modified

(GM) annual crops has been steadily increasing in the

recent 10 years, the commercial cultivation of GM

fruit tree is still very limited and reports of field trials

on GM fruit trees are rare. This is probably because

development and evaluation of GM fruit trees require

a long period of time due to long life cycles of trees. In

this study, we report results from a field trial on three

rolB transgenic dwarfing apple rootstocks of M26 and

M9 together with non-transgenic controls grafted with

five non-transgenic scion cultivars. We intended to

investigate the effects of transgenic rootstock on non-

transgenic scion cultivars under natural conditions as

well as to evaluate the potential value of using the

rolB gene to modify difficult-to-root rootstocks of

fruit trees. The results showed that all rolB transgenic

rootstocks significantly reduced vegetative growth

including tree height regardless of scion cultivar,

compared with the non-transgenic rootstocks. Flow-

ering and fruiting were also decreased for cultivars

grown on the transgenic rootstocks in most cases, but

the fruit quality was not clearly affected by the

transgenic rootstocks. Cutting experiment and RT-

PCR analysis showed that the rolB gene was stably

expressed under field conditions. PCR and RT-PCR

analyses displayed that the rolB gene or its mRNA

were not detectable in the scion cultivars, indicating

no translocation of the transgene or its mRNA from

rootstock to scion. Our results suggest that rolB

modified rootstocks should be used in combination

with vigorous scion cultivars in order to obtain

sufficient vegetative growth and good yield. Alterna-

tively, the rolB gene could be used to dwarf vigorous

rootstocks of fruit trees or produce bonzai plants as

it can significantly reduce the vegetative growth of

plants.

Keywords rolB � GM apple rootstock �Scion cultivar � Growth � Flower � Fruit quality

Introduction

Genetic engineering is becoming more and more

important in modern plant breeding as it can modify

specific traits that are not easily reached using

conventional breeding methods. Commercial cultiva-

tion of genetically modified plants (GMPs) has

steadily increased worldwide in the past 10 years.

By 2008, the cultivation of GMPs had reached over

100 million hectares in 25 countries. The cultivated

GMPs are predominantly annual crops with increased

insect and pesticide resistance or herbicide tolerance,

A. Smolka � X.-Y. Li � C. Heikelt � M. Welander (&) �L.-H. Zhu (&)

Department of Plant Breeding and Biotechnology,

Swedish University of Agricultural Sciences, Box 101,

230 53 Alnarp, Sweden

e-mail: Margareta.Welander@ltj.slu.se

L.-H. Zhu

e-mail: Li-Hua.Zhu@ltj.slu.se

123

Transgenic Res (2010) 19:933–948

DOI 10.1007/s11248-010-9370-0

while the commercial cultivation of GM fruit tree is

very limited. So far, the only GM tree species that are

commercially cultivated are GM poplar with insect

resistance grown in China and GM papaya with virus

resistance grown in USA and China (James 2008).

Fruit trees are commonly propagated by grafting a

scion cultivar onto a rootstock. Rootstocks have a

special value for fruit production as they can

effectively control tree size, influence growth and

development of cultivars grafted onto them and

tolerate adverse soil conditions (Drake et al. 1988;

Fallahi et al. 1985; Ferree et al. 2001a, b; Hirst and

Ferree 1995, 1996; Lauri et al. 2006; Tubbs 1974;

Tworkoski and Miller 2007; Webster et al. 1985).

However, there are no ideal rootstocks available for

commercial production and genetic improvement of

existing rootstocks or breeding new rootstocks is thus

a continuous goal for fruit tree breeding. Genetic

modification has been proved to be efficient for

improving currently available rootstocks where only

one or a few traits need to be improved (Zhu et al.

2001, 2003). Studies on GM fruit trees under field

conditions are still very scarce with the only reports

on fire blight resistance by Norelli et al. (2003) and

Aldwinckle et al. (2003), fruit firmness by Hrazdina

et al. (2003) and fruit flavour by Dandekar et al.

(2004). However, there is little information about the

field trials and evaluation of field-grown trees

regarding growth and development in these reports.

The information about effects of transgenic root-

stocks on non-transgenic scions of fruit trees under

field conditions is not available since there is no field

trial on GM rootstocks of fruit trees. Clearly,

systematic evaluation of GM fruit trees concerning

growth and development is necessary to increase our

knowledge on GM trees under field conditions.

Another issue related to GM rootstocks is that,

unlike GM scion cultivars, the use of GM rootstocks

in combination with non-transgenic scion cultivars

may circumvent the food safety issue if transgenes or

their products are not present in scion fruits. The

results reported so far have been inconsistent in this

regard. Dutt et al. (2007) and Bortolotti et al. (2005)

have reported that transgene proteins were found in

sap and phloem of non-transgenic scions grafted onto

transgenic rootstock in grape and tobacco, respec-

tively, but Youk et al. (2009) reported that no

transgenic products were translocated from transgenic

rootstock to non-transgenic scion in watermelon.

Since reports on this issue are very limited, further

studies are needed to facilitate the future application

of GM rootstocks in fruit production.

In modern apple production, dwarfing rootstocks

are commonly used for achieving high production

efficiency. Dwarfing rootstocks are often propagated

vegetatively, but they are often difficult-to-root,

especially when they reach the adult phase. Improve-

ment of the rooting ability is thus necessary for these

rootstocks. The rolB (rooting locus B) gene, isolated

from the soil bacterium Agrobacterium rhizogenes

(Cardarelli et al. 1987; Vilaine and Cassedelbart

1987), is a well documented rooting related gene and

has been proved to stimulate rooting in different plant

species when overexpressed (Capone et al. 1989; Dai

et al. 2004; Feyissa et al. 2007; Geier et al. 2008;

Rugini et al. 1991; Spena et al. 1987; Tzfira et al.

1996; Welander et al. 1998). Except for its effect on

adventitious rooting, the rolB gene can also stimulate

flowering in transgenic tobacco plants (Altamura et al.

1994). The rolB gene has been used for modifying

difficult-to-root dwarfing apple and pear rootstocks

and showed a great increase in rooting ability both in

vitro and ex vitro as well as reduced plant size under

greenhouse conditions (Zhu et al. 2001, 2003). It has

also been reported that, under non-limiting nutrient

conditions, the relative growth rate of the transgenic

apple rootstock was not altered by the rolB gene

compared to the untransformed control (Zhu and

Welander 1999). It is however unknown how the rolB

transgenic rootstocks will affect growth and develop-

ment of non-transgenic scion cultivars under field

conditions. The answer to this question would facil-

itate the potential use of the rolB gene in breeding of

fruit tree rootstocks in the future.

In this study we evaluated the effects of rolB

transgenic apple rootstocks on growth, flowering and

fruit quality of non-transgenic scion cultivars grafted

onto these rootstocks, the stability of the rolB gene

expression under natural conditions, possibility of

translocation of the rolB gene or its mRNA from

rootstock to scion. Our results show that the rolB

transgenic rootstocks reduced growth and flowering

of the scion cultivars, while the fruit quality was not

apparently affected. These effects varied among

different cultivars with the strongest effect on the

cultivar Discovery. The rolB gene is stably expressed

under field conditions. No translocation of the rolB

gene or its mRNA was detected in the scion cultivars.

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Materials and methods

Plant material

The field trial was located in the Southern part of

Sweden and initiated by planting in vitro rooted

rootstocks in a nursery in 2001. Rootstocks used in

the trial consisted of non-transgenic M26, transgenic

M26-rolB (clone F), non-transgenic M9, transgenic

M9-rolB1 (clone ARB3) and M9-rolB2 (clone

ARB9). These transgenic rootstocks have previously

been described by Welander et al. (1998) and Zhu

et al. (2001). The reasons for choosing these clones

are because they do not contain the gus gene and have

either single or two copies of the rolB gene in order to

maintain the stable gene expression under field

conditions. The clone F of M26 contains one copy

of the rolB gene and rooted better than clone C.

ARB3 and ARB9 contain two copies of the rolB gene

(all clones with one copy of the rolB gene were

unfortunately lost). Both clones had significantly

increased rooting compared to the control, while

clone ARB9 is dwarfer than ARB3. Five types of

apple cultivars (Aroma, Discovery, Elise, Elstar and

Jonagold) were budded onto the five rootstocks in

July 2002 in the nursery. The budded trees were

allowed to grow two seasons in the nursery before

being planted in the field trial. Well-developed

budded trees were then planted in the field trial in

May 2004. Every scion-rootstock combination con-

sisted of 10 trees which were randomly distributed in

the field to avoid potential soil effects. The trees were

planted in a distance of 2.5 9 3 m and managed with

a general maintenance including spraying with pes-

ticides, pruning and fertilization. The spaces between

the trees were covered with grass. The pruning was

carried out every spring in a way to obtain crown

trees and the degree of pruning may differ from year

to year as well as from cultivar to cultivar based on

their growth habits. In order to secure vegetative

growth of small trees, flowers were removed during

the first 2 years of the field trial. The non-grafted rolB

rootstocks and non-transgenic control rootstocks

were also planted in the field in 2004.

Registration of growth data, flowers and fruits

Tree height, trunk diameter at about 10 cm above the

ground and annual shoot growth were registered from

all trees after growth cessation from 2003 to 2007

(the registration was started when the trees were still

in the nursery). Flower buds and the number of

flowers from all trees were counted before full bloom

from 2005 to 2007 and the number of fruits from all

trees was recorded in autumn from 2007 to 2009.

Fruit quality analysis

Fruits were harvested when they were fully ripen and

stored in a paper bag covered with a plastic bag at

4�C before fruit quality analysis. Fruit quality

parameters analysed include fresh weight, diameter,

firmness, colour, total soluble solids (TSS), titratable

acidity (TA), total phenols and vitamin C content.

Depending on the available amount of fruits at

harvest time (some were damaged due to insects and

diseases), fruit quality analysis was carried out only

on three cultivars, namely Elise, Elstar and Jonagold

in 2008. Fruit fresh weight, diameter, firmness and

colour were measured on 10 randomly picked apples

from 5 trees for each combination. The measurements

were carried out after 1 month of storage for Elise

and Elstar, and after 6 months of storage for Jona-

gold. For analysing TSS, TA, total phenols and

vitamin C, 2 apples were made into one biological

sample, and 5 biological replicates were made for

each parameter with 3 measurements from each

biological replicate.

Fruit firmness and colour measurements

Firmness was measured with a penetrometer FT 327

with 11 mm plunger. The measurements were made

on two sides (sun-side/shade-side) of apples and the

means were presented as kg/cm2. Fruit colour was

measured on three positions of a fruit with a Minolta

Chroma Meter CR-200 according to manufacturer’s

instructions. The results were expressed as hue angle,

which is determined as H = tan - 1(b/a) where a

represents chromaticity on a green (-) to red (?) axis

and b represents chromaticity on a blue (-) to yellow

(?) axis. H ranges from 0 (red colour) to 90 (yellow

colour).

Analysis of TSS and titratable acidity

About equally large sectors from the sun-side and

shade-side of apples were used to prepare juice in a

Transgenic Res (2010) 19:933–948 935

123

food mixer. TSS was analysed using Precision

Instrument’s digital refractometer RFM 80. Titratable

acidity (TA) was titrated as malic acid with 0.05 M

NaOH to pH 8.

Analysis of total phenolics

Total phenolics from flesh and peel were analysed

separately using a modified Folin-Ciocalteu colorimet-

ric method according to Dewanto et al. (2002). In brief,

chopped apple flesh or peels were extracted in 50%

ethanol for 10 min. After centrifuging at 13,000 rpm for

15 min, 63 ll of the supernatant was added to a 1.0 ml

cuvette together with 250 ll water and 63 ll Folin–

Ciocalteau’s reagent. After reaction for 6 min, 625 ll

Na2CO3 7% was added into the cuvette to raise the pH

for phenols to be oxidized to phenolates (Dewanto et al.

2002). The samples were then allowed to stand for

75 min before being measured at 765 nm with a

Shimadzu Recording Spectrophotometer UV-240

Grapicord. A standard curve of gallic acid at the

concentrations of 0, 5, 10, 20 and 40% was made to

calculate the content of total phenols in the samples.

Total phenol content was expressed as mg gallic acid

equivalent per g fresh weight.

Vitamin C analysis

The content of vitamin C was analysed in form of

L-ascorbic acid using HPLC. Tissues of about 5 g

fresh weight were homogenised with 25 ml 1.5%

metaphosphoric acid with an Ultra-Turrax homoge-

nizer. The homogenised mixture was centrifuged at

13,000 rpm at 4�C. 500 ll of the supernatant was

added to an Eppendorf tube, followed by addition of

550 ll 7 mM DTT to reduce dehydroascorbic acid

(DHA) to ascorbic acid. After 30 min of reaction, the

sample was centrifuged for 5 min at 10,000 rpm at

4�C. The whole extraction process was executed

under green light conditions to minimize degradation

of vitamin C. Supernatant of 600 ll was transferred

to a HPLC vial for HPLC analysis (LaChrome Merck

Hitachi with the data program D-7000 HSM HPLC

and the column Phenomenex Synergi 4u polar RP).

The mobile phase was single, containing 20 mM

KH2PO4 buffer and 4% methanol with pH adjusted to

2.3 with H3PO4. Ten microliters were injected and

the detection wavelength was 248 nm with the flow

rate of 1 ml/min. The content of ascorbic acid was

calculated based on a standard curve of known

concentrations of ascorbic acid.

Cutting experiment

In order to confirm that the rolB gene is stably

expressed and can still stimulate adventitious rooting

under field conditions, a cutting experiment was

carried out in greenhouse. Cuttings were made of

annual shoots taken from non-grafted non-transgenic

and transgenic rootstocks grown in the field trial in

July of 2007. About 2–3 nodes were taken for each

cutting with two half leaves. If leaves were small, two

full leaves were kept. The prepared cuttings were

then planted into a mixture of soil and perlite (1:1) in

a plate covered with plastic film to maintain high

humility. About 30 cuttings were taken for each

rootstock. The temperature in the greenhouse was 23/

18�C (day/night) with natural light. The rooting result

was recorded after 2 months.

Expression of the rolB gene under field conditions

To investigate the stability of the transgene expression

under field conditions, growing shoot tips of the non-

grafted rolB rootstocks and non-transgenic control

rootstocks were taken in July 2007 (when shoots grew

vigorously) for RT-PCR analysis of the rolB gene.

Total RNA extraction and RT-PCR were carried out

according to Feyissa et al. (2007) and Zhu et al. (2008).

The primers were 50-ATGGATCCCAAATTGCTAT

TCCTTCCACGA-30 and 50-TTAGGCTTCTTTCTT

CAGGTTTACTGCAGC-30, yielding a 776 bp product.

Detection of the rolB gene in scion cultivar

Leaf and flower samples were taken from the scion

cultivars grafted onto the transgenic rootstocks.

Genomic DNA extraction and PCR analysis were

carried out according to Zhu et al. (2001, 2003).

Detection of rolB mRNA in scion cultivar

In order to verify if the rolB mRNA could be

translocated from rootstock to scion, growing shoot

tips were taken from the scion cultivars grafted onto

the different rootstocks. Total RNA was extracted

and RT-PCR was performed according to Feyissa

et al. (2007).

936 Transgenic Res (2010) 19:933–948

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Statistic analysis

Data was subjected to analysis of variance (ANOVA)

with Duncan’s multiple range test using the Stat-

graphics program.

Results

Effect of transgenic rootstock on success

of budding

The budding union between rootstock and scion was

formed smoothly for all cultivars grown on the

transgenic and control rootstocks, observed by naked

eyes. The success of budding was almost 98% in

general for all combinations, indicating that the rolB

gene did not have any negative effect on budding

(data not shown).

Effect of transgenic rootstock on tree growth

The bud breaking time was not affected by the

transgenic rootstocks, as observed by naked eyes each

spring. However, the rolB transgenic rootstocks

significantly reduced tree growth in most cases, as

measured by tree height, stem diameter, annual shoot

length and annual shoot number as shown in Tables 1,

2, 3 and 4. From 2005 onward, this reduction is

significant for all combinations when compared with

Table 1 Plant height (cm) of five apple cultivars grafted onto transgenic and non-transgenic apple rootstocks

Combination

(cultivar/rootstock)

2003

(Mean ± SE)

2004

(Mean ± SE)

2005

(Mean ± SE)

2006

(Mean ± SE)

2007

(Mean ± SE)

Average of

5 years

(mean ± SE)

Aroma/M26 85 ± 2.7b 110 ± 3.2a 130 ± 2.9a 160 ± 4.8a 170 ± 2.9a 131 ± 1.5a

Aroma/M26-rolB 69 ± 2.2c 102 ± 3.3a 115 ± 2.9b 129 ± 4.8b 149 ± 2.9b 113 ± 1.5b

Aroma/M9 95 ± 2.2a 109 ± 3.3a 135 ± 2.9a 152 ± 5.1a 180 ± 2.8a 134 ± 1.5a

Aroma/M9-rolB1 73 ± 2.2c 111 ± 3.1a 117 ± 2.9b 121 ± 5.1b 153 ± 2.9b 115 ± 1.5b

Aroma/M9-rolB2 60 ± 2.2d 92 ± 3.2b 107 ± 2.9b 107 ± 4.8c 139 ± 2.9c 101 ± 1.5c

Discovery/M26 91 ± 2.2a 95 ± 3.7a 116 ± 2.9a 132 ± 3.9a 136 ± 2.9a 114 ± 1.6a

Discovery/M26-rolB 73 ± 2.2c 72 ± 3.7b 92 ± 2.9b 99 ± 4.1b 119 ± 2.9b 91 ± 1.5b

Discovery/M9 92 ± 2.2a 95 ± 3.3a 116 ± 2.9a 129 ± 3.9a 136 ± 2.6a 114 ± 1.5a

Discovery/M9-rolB1 80 ± 2.2b 88 ± 3.8a 87 ± 2.9b 88 ± 3.9b 105 ± 3.0b 90 ± 2.0b

Discovery/M9-rolB2 62 ± 2.3d 90 ± 3.1a 96 ± 2.9b 95 ± 3.9b 112 ± 2.9b 91 ± 1.6b

Elise/M26 96 ± 2.1a 111 ± 3.5a 130 ± 2.9a 151 ± 4.0a 149 ± 2.9a 127 ± 1.7a

Elise/M26-rolB 70 ± 2.1b 85 ± 3.0b 108 ± 2.9b 115 ± 4.0c 113 ± 2.9bc 98 ± 1.6c

Elise/M9 95 ± 2.2a 95 ± 3.2b 136 ± 2.9a 137 ± 4.0b 149 ± 2.7a 122 ± 1.6a

Elise/M9-rolB1 72 ± 2.2b 99 ± 3.2b 111 ± 2.9b 115 ± 4.3c 123 ± 2.9b 104 ± 1.7b

Elise/M9-rolB2 57 ± 2.3c 83 ± 3.2b 97 ± 2.9c 97 ± 4.0d 110 ± 2.9c 89 ± 1.7d

Elstar/M26 78 ± 2.2b 108 ± 3.2b 148 ± 2.9a 182 ± 4.1a 165 ± 2.9b 136 ± 1.6b

Elstar/M26-rolB 85 ± 2.2b 112 ± 3.1b 132 ± 2.9b 154 ± 4.3b 146 ± 2.9d 126 ± 1.7c

Elstar/M9 92 ± 2.2a 125 ± 3.1a 146 ± 2.9a 186 ± 4.4a 179 ± 2.6a 146 ± 1.6a

Elstar/M9-rolB1 83 ± 2.1b 116 ± 3.1b 125 ± 2.9b 132 ± 4.1c 156 ± 3.0c 122 ± 1.6d

Elstar/M9-rolB2 68 ± 2.2c 112 ± 3.2b 116 ± 2.9c 128 ± 4.4c 145 ± 2.9d 114 ± 1.5e

Jonagold/M26 78 ± 2.2b 119 ± 3.2ab 136 ± 2.9a 164 ± 5.5a 158 ± 2.9b 131 ± 1.5b

Jonagold/M26-rolB 77 ± 2.2bc 121 ± 3.1ab 120 ± 2.9b 130 ± 5.5c 145 ± 2.9c 119 ± 1.5c

Jonagold/M9 101 ± 2.2a 125 ± 3.6a 138 ± 2.9a 150 ± 5.5b 175 ± 2.9a 138 ± 1.5a

Jonagold/M9-rolB1 72 ± 2.2c 120 ± 3.3ab 120 ± 2.9b 126 ± 5.5cd 142 ± 2.7c 116 ± 1.5c

Jonagold/M9-rolB2 63 ± 2.3d 114 ± 3.2b 119 ± 2.9c 111 ± 5.5d 138 ± 2.9c 109 ± 1.5d

Data were collected from 10 trees for each combination. Duncan’s multiple test was carried out among five different rootstocks

within one cultivar. Different letters in each column within one cultivar indicate significant differences at P = 0.05

Transgenic Res (2010) 19:933–948 937

123

their control rootstocks. In most cases, the above

parameters were lower for M9-rolB2 compared with

M9-rolB1. Of all tested cultivars, Discovery showed

the lowest tree growth. All combinations with this

cultivar showed very limited growth over the years,

especially on the transgenic rootstocks. The second

weakest cultivar is Elise, while the other three

cultivars had similar growth vigour.

Effects of transgenic rootstocks on flowering

and fruit setting

Flowering time, observed by naked eyes, was not

affected by the transgenic rootstocks. The results on the

number of flower buds and flowers are presented in

Tables 5 and 6. In most cases, both flower bud number

and flower number were reduced for Discovery and

Jonagold grafted onto the M26-rolB rootstock com-

pared with M26, while irregular changes were observed

for the cultivars Aroma, Elise and Elstar. For the

rootstock M9, flowering was significantly reduced for

all the cultivars on M9-rolB2 for year 2006 and 2007,

while it varied among cultivars on M9-rolB1. The fruit

number was significantly reduced for Discovery grafted

on M26-rolB compared with M26 in most cases. For

M9, the fruit number was reduced for all the cultivars

grown on M9-rolB2, but only for Discovery, Elstar and

Jonagold grown on M9-rolB1 (Table 7).

Table 2 Stem diameter (cm) of five apple cultivars grafted onto transgenic and non-transgenic apple rootstocks

Combination (cultivar/

rootstock)

2003

(Mean ± SE)

2004

(Mean ± SE)

2005

(Mean ± SE)

2006

(Mean ± SE)

2007

(Mean ± SE)

Average of

5 years

(mean ± SE)

Aroma/M26 1.2 ± 0.2a 1.6 ± 0.2a 2.0 ± 0.2a 2.2 ± 0.6a 3.1 ± 0.1a 2.0 ± 0.06a

Aroma/M26-rolB 1.0 ± 0.1bc 1.3 ± 0.1b 1.6 ± 0.1c 1.7 ± 0.1b 2.3 ± 0.1c 1.6 ± 0.06b

Aroma/M9 1.0 ± 0.1bc 1.2 ± 0.2c 1.8 ± 0.2b 2.1 ± 0.1a 2.8 ± 0.1b 1.8 ± 0.06a

Aroma/M9-rolB1 1.0 ± 0.1bc 1.2 ± 0.2c 1.6 ± 0.2c 1.6 ± 0.2c 2.0 ± 0.1d 1.5 ± 0.06b

Aroma/M9-rolB2 0.9 ± 0.1c 1.2 ± 0.2c 1.5 ± 0.2c 1.6 ± 0.1c 1.7 ± 0.1e 1.4 ± 0.06b

Discovery/M26 1.1 ± 0.2a 1.6 ± 0.1a 1.9 ± 0.1a 2.1 ± 0.2a 2.9 ± 0.1a 1.9 ± 0.03a

Discovery/M26-rolB 0.9 ± 0.2b 1.4 ± 0.1b 1.6 ± 0.1b 1.6 ± 0.1b 2.0 ± 0.1c 1.5 ± 0.02c

Discovery/M9 1.1 ± 0.3a 1.4 ± 0.2b 1.8 ± 0.2a 2.0 ± 0.2a 2.7 ± 0.1b 1.8 ± 0.02b

Discovery/M9-rolB1 1.0 ± 0.1a 1.1 ± 0.1d 1.4 ± 0.2c 1.5 ± 0.1b 1.7 ± 0.1d 1.3 ± 0.03d

Discovery/M9-rolB2 1.1 ± 0.2a 1.2 ± 0.2c 1.6 ± 0.2b 1.6 ± 0.2b 1.8 ± 0.1d 1.5 ± 0.02c

Elise/M26 1.4 ± 0.3a 1.6 ± 0.1a 2.1 ± 0.2a 2.1 ± 0.1a 2.9 ± 0.1a 2.0 ± 0.03a

Elise/M26-rolB 1.0 ± 0.3b 1.4 ± 0.1b 1.5 ± 0.2c 1.6 ± 0.1b 2.2 ± 0.1b 1.5 ± 0.04c

Elise/M9 1.0 ± 0.3b 1.3 ± 0.1c 1.8 ± 0.2b 2.0 ± 0.1a 2.7 ± 0.1a 1.8 ± 0.03b

Elise/M9-rolB1 1.1 ± 0.2b 1.2 ± 0.1c 1.5 ± 0.2cd 1.6 ± 0.2b 2.0 ± 0.1bc 1.5 ± 0.03c

Elise/M9-rolB2 1.1 ± 0.2b 1.2 ± 0.1c 1.4 ± 0.1d 1.4 ± 0.2c 1.8 ± 0.1c 1.4 ± 0.03d

Elstar/M26 1.2 ± 0.2b 1.5 ± 0.1a 1.9 ± 0.1b 2.2 ± 0.1b 3.5 ± 0.1a 2.0 ± 0.03a

Elstar/M26-rolB 1.3 ± 0.2a 1.4 ± 0.1b 1.7 ± 0.1c 2.0 ± 0.2c 2.8 ± 0.1b 1.8 ± 0.04b

Elstar/M9 1.2 ± 0.2b 1.2 ± 0.1c 2.1 ± 0.1a 2.4 ± 0.1a 3.5 ± 0.1a 2.1 ± 0.03a

Elstar/M9-rolB1 1.2 ± 0.2b 1.3 ± 0.1b 1.9 ± 0.1b 2.3 ± 0.2b 2.6 ± 0.1b 1.9 ± 0.04b

Elstar/M9-rolB2 1.3 ± 0.1a 1.4 ± 0.1b 1.8 ± 0.1bc 1.8 ± 0.2c 2.2 ± 0.1c 1.7 ± 0.03b

Jonagold/M26 1.1 ± 0.3b 1.4 ± 0.2a 1.8 ± 0.1b 2.0 ± 0.2a 2.9 ± 0.1a 1.8 ± 0.03b

Jonagold/M26-rolB 1.2 ± 0.1ab 1.3 ± 0.1b 1.6 ± 0.1c 1.6 ± 0.1bc 2.2 ± 0.1b 1.6 ± 0.03c

Jonagold/M9 1.3 ± 0.4a 1.4 ± 0.2ab 2.1 ± 0.2a 2.1 ± 0.2a 2.8 ± 0.1a 1.9 ± 0.03a

Jonagold/M9-rolB1 1.2 ± 0.2ab 1.2 ± 0.1c 1.7 ± 0.1b 1.7 ± 0.1b 2.2 ± 0.1b 1.6 ± 0.03cd

Jonagold/M9-rolB2 1.2 ± 0.1ab 1.3 ± 0.1c 1.5 ± 0.1c 1.6 ± 0.1c 1.8 ± 0.1c 1.5 ± 0.03d

Data were collected from 10 trees for each combination. Duncan’s multiple test was carried out among five different rootstocks

within one cultivar. Different letters in each column within one cultivar indicate significant differences at P = 0.05

938 Transgenic Res (2010) 19:933–948

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Weight, diameter, firmness and colour

Results for fruit weight, diameter, firmness and

colour are presented in Table 8. In general, there

were no consistent changes in fruit weight and

diameter when comparing the transgenic rootstocks

with their control counterparts. In some cases, the

fruit weight and diameter did not differ or were

inconsistent between the transgenic and control

rootstock. This is also true for firmness. For M26,

the fruit colour tended to be more reddish for trees on

the transgenic rootstock for all cultivars, but no

significant difference was found for Elise. For M9,

there was almost no difference in fruit colour in most

cases. Compared with the other cultivars, Jonagold

fruits had much lower firmness and this is apparently

due to the much longer storage period for this cultivar

before analysis was carried out.

TSS, TA, Vitamin C and phenolics

For Jonagold, a significantly increased TSS was

detected in fruits grown on both transgenic M9

rootstocks compared with non-transgenic M9 root-

stock. For Elstar, TSS was significantly increased in

fruits grown on the transgenic rootstocks of both M26

and M9. There was no difference in TSS for Elise

between the transgenic rootstocks and non-transgenic

ones (Table 9). There was no significant difference in

TA in most cases for all cultivars when comparing

the transgenic rootstocks with the non-transgenic

control for both M26 and M9 (Table 9). There was no

significant difference in the content of ascorbic acid

for Jonagold when comparing the transgenic root-

stocks with the non-transgenic control. For Elstar, the

Vc content was lower in fruits on M26-rolB com-

pared to M26, but higher on M9-rolB1 compared

Table 3 Shoot length (cm)

of five apple cultivars

grafted onto transgenic and

non-transgenic apple

rootstocks

Data were collected from 10

trees for each combination.

Duncan’s multiple test was

carried out among five

different rootstocks within

one cultivar. Different

letters in each column

within one cultivar indicate

significant differences at

P = 0.05

Combination

(cultivar/rootstock)

2004

(Mean ± SE)

2005

(Mean ± SE)

2006

(Mean ± SE)

Average of 3 years

(mean ± SE)

Aroma/M26 75 ± 18a 39 ± 5a 57 ± 12a 57 ± 1.1a

Aroma/M26-rolB 57 ± 15b 21 ± 5c 38 ± 10c 39 ± 1.1c

Aroma/M9 80 ± 18a 32 ± 3b 47 ± 11b 53 ± 1.1b

Aroma/M9-rolB1 54 ± 14b 22 ± 4c 27 ± 8d 34 ± 1.1d

Aroma/M9-rolB2 46 ± 15c 13 ± 4d 19 ± 5e 26 ± 1.2e

Discovery/M26 48 ± 13a 30 ± 5a 39 ± 9a 39 ± 1.1a

Discovery/M26-rolB 31 ± 12b 17 ± 6c 26 ± 9b 25 ± 1.1c

Discovery/M9 42 ± 13a 24 ± 4b 30 ± 12b 32 ± 1.1b

Discovery/M9-rolB1 21 ± 11c 14 ± 5c 14 ± 5c 16 ± 1.2d

Discovery/M9-rolB2 25 ± 12bc 6 ± 4d 8 ± 4d 13 ± 1.3d

Elise/M26 49 ± 17a 28 ± 10a 39 ± 11a 39 ± 1.1a

Elise/M26-rolB 29 ± 15b 20 ± 6b 28 ± 8c 26 ± 1.1b

Elise/M9 50 ± 15a 30 ± 8a 33 ± 10b 38 ± 1.1a

Elise/M9-rolB1 32 ± 13b 20 ± 7b 21 ± 6d 24 ± 1.2b

Elise/M9-rolB2 28 ± 13b 12 ± 4c 15 ± 6e 18 ± 1.2c

Elstar/M26 69 ± 17b 46 ± 7a 62 ± 10a 59 ± 1.1b

Elstar/M26-rolB 51 ± 16d 31 ± 8c 43 ± 13b 42 ± 1.1c

Elstar/M9 85 ± 15a 39 ± 9b 65 ± 8a 63 ± 1.1a

Elstar/M9-rolB1 59 ± 13c 25 ± 8d 34 ± 11c 39 ± 1.1d

Elstar/M9-rolB2 50 ± 13d 13 ± 5e 20 ± 9d 28 ± 1.1e

Jonagold/M26 62 ± 18b 41 ± 12a 50 ± 16a 51 ± 1.1a

Jonagold/M26-rolB 44 ± 16cd 18 ± 6b 36 ± 13b 33 ± 1.1b

Jonagold/M9 74 ± 16a 34 ± 10a 48 ± 15a 52 ± 1.1a

Jonagold/M9-rolB1 48 ± 13c 23 ± 7b 28 ± 13c 33 ± 1.1b

Jonagold/M9-rolB2 41 ± 11d 12 ± 5b 17 ± 5d 23 ± 1.2c

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123

with M9. For Elise, there was no consistent difference

in ascorbic acid when comparing the transgenic

rootstocks with the non-transgenic rootstocks

(Table 9). Total phenolics did not differ significantly

in either flesh or peels of Jonagold when comparing

the transgenic and non-transgenic rootstocks, while

the results on Elise and Elstar did not give a

consistent trend in the content of total phenolics

(Table 9).

Rooting of cuttings taken from the non-grafted

rolB transgenic rootstocks

In order to verify if the rolB effect on rooting is

stable under natural conditions, we took cuttings

from non-grafted transgenic rootstocks that were

planted together with the other field trial material

(Fig. 1). The results showed that the rooting

percentage was 73% for M26-rolB compared with

0% for M26, while 67 and 43% for M9-rolB1 and

M9-rolB2, respectively, compared to 3% for M9.

The rooting percentage of transgenic rootstocks was

significantly higher than their control counterparts

(Table 10).

Stability of transgene expression under field

conditions

In order to verify stability of transgene expression

under field conditions, RT-PCR was performed to

detect mRNA of the rolB gene in non-grafted

transgenic rootstocks that were planted together with

the other field trial material. The result showed that

the gene was expressed in all the three transgenic

clones grown in the field for several years, indicating

that the rolB gene is stably expressed (Fig. 2).

Table 4 Shoot number of

five apple cultivars grafted

onto transgenic and non-

transgenic apple rootstocks

Data were collected from 10

trees for each combination.

Duncan’s multiple test was

carried out among five

different rootstocks within

one cultivar. Different

letters in each column

within one cultivar indicate

significant differences at

P = 0.05

Combination

(cultivar/rootstock)

2005

(Mean ± SE)

2006

(Mean ± SE)

Average of 2 years

(mean ± SE)

Aroma/M26 17 ± 0.7a 26 ± 1.5a 22 ± 0.8a

Aroma/M26-rolB 12 ± 0.7b 15 ± 1.5b 14 ± 0.8b

Aroma/M9 18 ± 0.7a 22 ± 1.6a 20 ± 0.9a

Aroma/M9-rolB1 11 ± 0.7b 9 ± 1.6c 10 ± 0.8c

Aroma/M9-rolB2 9 ± 0.7c 7 ± 1.5c 8 ± 0.8d

Discovery/M26 14 ± 0.7a 18 ± 0.8a 16 ± 0.8a

Discovery/M26-rolB 10 ± 0.7b 9.4 ± 0.9c 10 ± 0.9b

Discovery/M9 13 ± 0.7a 15 ± 0.8b 14 ± 0.8a

Discovery/M9-rolB1 8 ± 0.7c 4 ± 0.8d 6 ± 0.8c

Discovery/M9-rolB2 7 ± 0.7c 0.9 ± 0.8e 4 ± 0.8c

Elise/M26 10 ± 0.6a 16 ± 1.2a 13 ± 0.8a

Elise/M26-rolB 6 ± 0.6b 8 ± 1.2b 7 ± 0.8bc

Elise/M9 9 ± 0.6b 14 ± 1.2a 12 ± 0.8a

Elise/M9-rolB1 5 ± 0.6b 7 ± 1.2bc 6 ± 0.9c

Elise/M9-rolB2 4 ± 0.6b 4 ± 1.2c 4 ± 0.8c

Elstar/M26 17 ± 0.8b 27 ± 1.3a 22 ± 0.8a

Elstar/M26-rolB 13 ± 0.8d 17 ± 1.3b 15 ± 0.8b

Elstar/M9 20 ± 0.8a 28 ± 1.4a 24 ± 0.9a

Elstar/M9-rolB1 15 ± 0.8c 18 ± 1.3b 17 ± 0.8b

Elstar/M9-rolB2 11 ± 0.8d 10 ± 1.4c 11 ± 0.9c

Jonagold/M26 14 ± 0.9a 23 ± 1.1a 19 ± 0.8a

Jonagold/M26-rolB 10 ± 0.8b 12 ± 1.1b 11 ± 0.8b

Jonagold/M9 15 ± 0.8a 19 ± 1.1a 17 ± 0.8a

Jonagold/M9-rolB1 9 ± 0.8b 10 ± 1.1b 10 ± 0.8b

Jonagold/M9-rolB2 7 ± 0.8b 5 ± 1.1c 6 ± 0.8c

940 Transgenic Res (2010) 19:933–948

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Detection of the rolB gene for its presence

in the scion cultivars

PCR analysis was performed to investigate if the

transgene could be translocated from rootstock to

scion cultivar. No rolB band was detected in leaves or

flowers of the scion cultivars, suggesting that the

transgene was not translocated from rootstock to

scion (data not shown).

Detection of rolB mRNA in the scion cultivars

RT-PCR was conducted to check if the rolB mRNA

could be detected in the scion cultivars. No rolB

mRNA was detected in growing shoot tips of the

scion cultivars, indicating no translocation of the rolB

mRNA from rootstock to scion (Fig. 3).

Discussion

The rolB transgenic rootstocks significantly affect

growth of scions, but not the fruit quality

The effect of dwarfing rootstocks on scions has

already been studied extensively. In general, dwarfing

rootstocks can effectively control scion vigour and

reduce plant height, thus resulting in smaller trees

(Lauri et al. 2006; Tworkoski and Miller 2007). It has

also been reported that the growth rate of scion

cultivar can be independent of rootstock (Bulley et al.

2005). In this study, all growth parameters of the

scions grafted onto the transgenic rootstocks were

lower than those on the non-transgenic rootstocks

regardless of scion cultivar, indicating that the rolB

gene could reduce the growth rate of scion cultivars.

Table 5 Number of flower

buds of five apple cultivars

grafted onto transgenic and

non-transgenic apple

rootstocks

Data were collected from 10

trees for each combination.

Duncan’s multiple test was

carried out among five

different rootstocks within

one cultivar. Different

letters in each column

within one cultivar indicate

significant differences at

P = 0.05

Combination

(cultivar/rootstock)

2005

(Mean ± SE)

2006

(Mean ± SE)

2007

(Mean ± SE)

Average of 3 years

(mean ± SE)

Aroma/M26 3.3 ± 0.7a 3.3 ± 0.8c 113 ± 11a 40 ± 3.2a

Aroma/M26-rolB 2.2 ± 0.9a 3.8 ± 0.7b 81 ± 11bc 29 ± 3.9b

Aroma/M9 2.4 ± 0.8a 5.6 ± 0.8a 85 ± 12b 31 ± 3.8ab

Aroma/M9-rolB1 3.1 ± 0.7a 1.0 ± 0.8d 67 ± 11bc 24 ± 3.5bc

Aroma/M9-rolB2 2.2 ± 0.9a 0.7 ± 0.7d 48 ± 10c 17 ± 3.6c

Discovery/M26 5.1 ± 0.8a 6.3 ± 0.8a 91 ± 11a 34 ± 3.6a

Discovery/M26-rolB 2.8 ± 0.7b 3.1 ± 0.7c 63 ± 12b 23 ± 3.6b

Discovery/M9 5.8 ± 0.7a 3.6 ± 0.8b 72 ± 11b 27 ± 3.6ab

Discovery/M9-rolB1 2.0 ± 0.9b 0.6 ± 0.8d 45 ± 11c 16 ± 3.8c

Discovery/M9-rolB2 1.8 ± 0.7b 0.3 ± 0.7d 46 ± 11c 16 ± 3.4c

Elise/M26 5.7 ± 1.0a 4.2 ± 0.7a 77 ± 11a 29 ± 4.1a

Elise/M26-rolB 1.0 ± 1.2b 2.2 ± 0.8b 57 ± 11ab 20 ± 4.4b

Elise/M9 2.6 ± 0.9b 3.6 ± 0.8a 69 ± 11ab 25 ± 3.8ab

Elise/M9-rolB1 1.6 ± 0.9b 1.7 ± 0.7b 48 ± 12bc 17 ± 3.9bc

Elise/M9-rolB2 1.7 ± 0.8b 0.4 ± 0.7c 37 ± 10c 13 ± 3.5c

Elstar/M26 2.3 ± 1.0a 1.2 ± 0.7d 110 ± 11a 38 ± 3.5a

Elstar/M26-rolB 1.5 ± 1.2a 2.2 ± 0.8c 83 ± 11b 29 ± 3.5bc

Elstar/M9 3.0 ± 1.1a 2.7 ± 0.7b 108 ± 11a 38 ± 3.6a

Elstar/M9-rolB1 4.0 ± 1.0a 3.1 ± 0.8a 95 ± 11ab 34 ± 3.8ab

Elstar/M9-rolB2 2.5 ± 1.1a 1.0 ± 0.8e 57 ± 11c 20 ± 3.8c

Jonagold/M26 6.2 ± 1.0a 1.9 ± 0.7a 133 ± 11a 47 ± 3.5a

Jonagold/M26-rolB 2.5 ± 0.9ab 2.0 ± 0.8a 79 ± 11c 28 ± 3.8bc

Jonagold/M9 4.0 ± 0.9ab 2.7 ± 0.7a 107 ± 11b 38 ± 3.5ab

Jonagold/M9-rolB1 6.0 ± 0.8a 1.7 ± 0.7a 70 ± 11cd 26 ± 3.9c

Jonagold/M9-rolB2 0.1 ± 0.8b 0.3 ± 0.8b 30 ± 10d 10 ± 4.1d

Transgenic Res (2010) 19:933–948 941

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This result is in line with the previous reports about

the rolB gene under greenhouse conditions (Zhu et al.

2001, 2003). A separate study carried out in a growth

unit with well-controlled climate conditions showed

that, under non-limiting nutrient conditions, the

relative growth rate of the rolB transformed apple

rootstock was not altered compared to the untrans-

formed control (Zhu and Welander 1999). The

explanation of this inconsistency could be that the

rolB transgenic rootstocks are likely to be more

sensitive to soil conditions than non-transgenic ones,

probably due to more hairy roots caused by the rolB

gene, which are mainly distributed on the soil

surface. Our current study clearly showed that the

flowering and fruiting were significantly reduced for

the cultivar Discovery grown on all the transgenic

rootstocks and the tendency is most dramatic for the

transgenic M9 rootstocks. This is likely due to the

significantly reduced tree growth. It has been reported

that yields are lower for scions grown on dwarfing

rootstocks compared with those on vigorous root-

stocks (Di Vaio et al. 2009).

Some studies have showed that dwarfing root-

stocks can affect the fruit quality (Autio et al. 1996;

Daugaard and Callesen 2002; Drake et al. 1988;

Fallahi and Kilby 1997; Fallahi et al. 1985), while

others reported that the fruit quality was not obvi-

ously affected by the rootstocks (Antognozzi et al.

1993; Riesen and Monney 1996). This inconsistency

may be attributed to differences in yield, choice of

cultivar and rootstock combinations, climate and

cultivation. In this study, the fruit quality was

generally not affected by the transgenic rootstocks,

indicating that the rolB transgenic rootstocks do not

clearly affect the fruit quality of non-transgenic scion

cultivars. Since our results on fruit quality analysis

Table 6 Number of

flowers of five apple

cultivars grafted onto

transgenic and non-

transgenic apple rootstocks

Data were collected from 10

trees for each combination.

Duncan’s multiple test was

carried out among five

different rootstocks within

one cultivar. Different

letters in each column

within one cultivar indicate

significant differences at

P = 0.05

Combination

(cultivar/rootstock)

2005

(Mean ± SE)

2006

(Mean ± SE)

2007

(Mean ± SE)

Average of

3 years

(mean ± SE)

Aroma/M26 7.1 ± 2.1ab 14 ± 2.3b 567 ± 56a 196 ± 16.4a

Aroma/M26-rolB 5.7 ± 3.1ab 17 ± 0.7ab 406 ± 56bc 143 ± 19.7ab

Aroma/M9 5.1 ± 2.7b 27 ± 0.7a 436 ± 60b 156 ± 18.8a

Aroma/M9-rolB1 9.4 ± 2.4a 7 ± 0.7bc 338 ± 56bc 118 ± 17.6bc

Aroma/M9-rolB2 5.7 ± 2.8ab 3 ± 0.7c 243 ± 50c 84 ± 18.5c

Discovery/M26 24.4 ± 2.5a 31 ± 0.7a 458 ± 56a 171 ± 18.0a

Discovery/M26-rolB 13.9 ± 2.3b 14 ± 0.7b 323 ± 60b 117 ± 17.8bc

Discovery/M9 32.6 ± 2.6a 17 ± 0.7b 359 ± 56b 136 ± 17.8ab

Discovery/M9-rolB1 11.0 ± 2.3b 2.9 ± 0.7c 232 ± 56c 82 ± 19.0c

Discovery/M9-rolB2 9.3 ± 0.7b 1.5 ± 0.7c 232 ± 53c 81 ± 17.0c

Elise/M26 18.7 ± 3.5a 18.8 ± 0.7a 385 ± 56a 141 ± 20.7a

Elise/M26-rolB 4.7 ± 4.2b 11 ± 0.7ab 287 ± 56bc 101 ± 21.9ab

Elise/M9 10.3 ± 2.9b 15.6 ± 0.7a 343 ± 56ab 123 ± 18.8a

Elise/M9-rolB1 7.9 ± 2.9b 6.5 ± 0.7ab 237 ± 60cb 84 ± 19.7b

Elise/M9-rolB2 6.5 ± 2.5b 0.8 ± 0.7b 182 ± 50c 63 ± 17.5b

Elstar/M26 6.4 ± 2.4a 6.4 ± 0.7b 554 ± 56a 189 ± 17.6a

Elstar/M26-rolB 4.0 ± 2.4a 10.2 ± 0.7ab 412 ± 56b 142 ± 17.6ab

Elstar/M9 7.3 ± 2.5a 14.1 ± 0.7ab 543 ± 56a 188 ± 18.0a

Elstar/M9-rolB1 7.9 ± 2.8a 15.7 ± 0.7a 477 ± 56ab 167 ± 19.1b

Elstar/M9-rolB2 5.0 ± 2.8a 5.2 ± 0.7b 278 ± 53c 96 ± 19.1b

Jonagold/M26 18.0 ± 2.4a 8 ± 0.7ab 661 ± 56a 229 ± 17.6a

Jonagold/M26-rolB 9.7 ± 2.8ab 9.3 ± 0.7ab 398 ± 56c 139 ± 19.1bc

Jonagold/M9 11.0 ± 2.4ab 11.7 ± 0.7a 526 ± 56b 183 ± 17.8ab

Jonagold/M9-rolB1 16.7 ± 2.8a 6.1 ± 0.7ab 352 ± 56cd 125 ± 19.4c

Jonagold/M9-rolB2 0.7 ± 4.1b 1.5 ± 0.7b 154 ± 50d 52 ± 20.6d

942 Transgenic Res (2010) 19:933–948

123

are based mainly on 1 year study, more studies may

be needed for a final conclusion. To our knowledge,

this study is the first report regarding the effect of

GM rootstocks on non-transgenic scion cultivars in

fruit trees under field conditions.

Expression of the rolB gene under field conditions

Stable expression of transgenes under field conditions

has been demonstrated in various species, such as

poplar (Li et al. 2008; Meilan et al. 2002), sunflower

(Rousselin et al. 2002), pineapple (Sripaoraya et al.

2006) and soybean (Padgette et al. 1995). However,

loss of transgene expression does occur, as has been

demonstrated in aspen (Kumar and Fladung 2001),

tall fescue (Bettany et al. 1998), oilseed rape (Metz

et al. 1997), oat (Pawlowski et al. 1998) and many

other species. The loss of transgene expression can be

due either to deletion of the transgene itself (Cherd-

shewasart et al. 1993; Heberlebors et al. 1988;

Tencate et al. 1990), or to epigenetic silencing of

the transgene. Silencing can occur at the transcrip-

tional level or at the posttranscriptional level (for

reviews, see Baulcombe 2004; Brodersen and Voin-

net 2009). Since apple trees have a long life cycle, the

stable expression of transgenes is particularly impor-

tant for potential commercialisation of GM root-

stocks. The results on cutting and RT-PCR in this

study showed that the rolB gene is stably expressed in

the transgenic rootstocks under field conditions over a

period of several years. Since this result was only

based on three transgenic clones, further studies with

more transgenic clones over even longer period are

required to draw final conclusion.

The rooting percentage of cuttings of the trans-

genic rolB rootstocks is lower than the rooting

Table 7 Number of fruits

of five apple cultivars

grafted onto transgenic and

non-transgenic apple

rootstocks

Data were collected from 10

trees for each combination.

Duncan’s multiple test was

carried out among five

different rootstocks within

one cultivar. Different

letters in each column

within one cultivar indicate

significant differences at

P = 0.05

Combination

(cultivar/rootstock)

2007

(Mean ± SE)

2008

(Mean ± SE)

2009

(Mean ± SE)

Average of

3 years

(mean ± SE)

Aroma/M26 20 ± 5.4a 14 ± 5.8c 47 ± 7.8a 27 ± 4.9ab

Aroma/M26-rolB 5.8 ± 5.8a 16 ± 5.8c 32 ± 7.0ab 18 ± 5.1ab

Aroma/M9 13.7 ± 5.9a 56 ± 5.8a 23 ± 7.5ab 31 ± 5.2a

Aroma/M9-rolB1 7.3 ± 5.9a 48 ± 5.8ab 22 ± 7.0ab 26 ± 5.1ab

Aroma/M9-rolB2 5.6 ± 5.9a 31 ± 5.8bc 11 ± 7.5b 16 ± 5.1b

Discovery/M26 2.4 ± 5.9c 32 ± 5.8ab 85 ± 7.8a 40 ± 5.1a

Discovery/M26-rolB 1.6 ± 5.9c 15 ± 5.8c 56 ± 7.5bc 24 ± 5.4b

Discovery/M9 13 ± 5.9a 34 ± 5.8a 78 ± 7.5ab 42 ± 5.1a

Discovery/M9-rolB1 3.2 ± 5.9c 34 ± 5.8a 30 ± 7.6cd 22 ± 5.2bc

Discovery/M9-rolB2 6.9 ± 5.9b 20 ± 5.8bc 20 ± 5.9d 16 ± 5.1c

Elise/M26 26 ± 5.9ab 43 ± 5.8b 44 ± 6.7a 38 ± 5.1ab

Elise/M26-rolB 14 ± 5.9b 37 ± 5.8b 18 ± 6.7b 23 ± 5.1c

Elise/M9 35 ± 5.9a 56 ± 5.8ab 56 ± 6.7a 49 ± 5.1a

Elise/M9-rolB1 9 ± 6.2b 79 ± 6.1a 13 ± 6.7b 34 ± 5.4bc

Elise/M9-rolB2 8.5 ± 5.9b 44 ± 5.8b 11 ± 6.7b 21 ± 5.1c

Elstar/M26 66 ± 6.2a 22 ± 5.8ab 57 ± 6.5a 48 ± 5.4a

Elstar/M26-rolB 41 ± 5.9b 13 ± 5.8b 30 ± 6.5a 28 ± 5.1cd

Elstar/M9 65 ± 5.9a 34 ± 5.8a 42 ± 6.5a 47 ± 5.2ab

Elstar/M9-rolB1 26 ± 5.9bc 33 ± 5.8a 39 ± 6.5a 33 ± 5.1bc

Elstar/M9-rolB2 13 ± 6.2c 18 ± 6.1b 15 ± 6.5a 15 ± 5.3d

Jonagold/M26 27 ± 5.9ab 19 ± 5.8a 60 ± 7.8a 35 ± 5.1a

Jonagold/M26-rolB 17 ± 5.9abc 12 ± 5.8ab 41 ± 7.8ab 23 ± 4.9b

Jonagold/M9 36 ± 5.9a 19 ± 5.8a 64 ± 7.8a 40 ± 5.1a

Jonagold/M9-rolB1 14 ± 5.9bc 17 ± 5.8ab 27 ± 7.8bc 19 ± 5.2bc

Jonagold/M9-rolB2 7 ± 5.9c 10 ± 5.8b 9 ± 7.8c 9 ± 5.1c

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Table 8 Fruit quality parameters of three apple cultivars grafted onto non-transgenic and transgenic rootstocks in the field trial

(2008)

Combination

(cultivar/rootstock)

FW

(mean ± SD, g)

Diameter

(mean ± SD, mm)

Firmness

(mean ± SD, kg/cm2)

Colour

(mean ± SD, H0)

Elise/M26 254 ± 39a 9.1 ± 1.34a 7.5 ± 0.49a 16.1 ± 4.5a

Elise/M26-rolB 279 ± 50a 9.2 ± 2.12a 6.9 ± 0.42a 16.6 ± 6.5a

Elise/M9 212 ± 19a 8.0 ± 0.35a 7.9 ± 0.98a 21.9 ± 8.6a

Elise/M9-rolB1 199 ± 68a 7.7 ± 0.09a 7.6 ± 0.21a 24.6 ± 17.6a

Elise/M9-rolB2 190 ± 47a 8.5 ± 1.70a 7.4 ± 0.07a 28.2 ± 19.6a

Elstar/M26 194 ± 11.9a 7.0 ± 0.24a 5.6 ± 0.35a 27 ± 4.3b

Elstar/M26-rolB 202 ± 11.8a 7.1 ± 0.91a 5.1 ± 0.53b 45 ± 12.8a

Elstar/M9 185 ± 11.8ab 6.8 ± 0.41ab 5.3 ± 0.51ab 37 ± 13.9ab

Elstar/M9-rolB1 156 ± 11.9b 6.2 ± 0.64c 5.1 ± 0.39ab 42 ± 12.0a

Elstar/M9-rolB2 152 ± 11.9b 6.4 ± 0.45bc 4.6 ± 0.42c 46 ± 14.0a

Jonagold/M26 195 ± 29.0b 7.8 ± 0.43b 3.9 ± 0.46ab 37 ± 15.6b

Jonagold/M26-rolB 228 ± 29.6a 8.3 ± 0.41a 3.5 ± 0.27b 53 ± 11.7a

Jonagold/M9 192 ± 37.0b 7.7 ± 0.43b 3.3 ± 0.26b 46 ± 14.0ab

Jonagold/M9-rolB1 197 ± 21.8b 7.8 ± 0.35b 4.1 ± 0.32a 58 ± 12.1a

Jonagold/M9-rolB2 162 ± 18.9c 7.4 ± 0.36b 3.8 ± 0.57ab 58 ± 11.9a

Duncan’s multiple range test was carried out among five different rootstocks within one cultivar. Different letters in each column

within one cultivar indicate significant differences at P = 0.05

Table 9 Internal fruit quality parameters of three apple cultivars grafted onto non-transgenic and transgenic rootstocks in the field

trial (2008)

Combination (cultivar/

rootstock)

TSS

(mean ± SD, %)

Vc (mean ± SD,

mg ascorbic acid/

100 g FW)

Phenol-flesh

(mean ± SD,

mg/g FW)

Phenol-peel

(mean ± SD,

mg/g FW)

TA (mean ± SD,

mg malic acid/

100 g FW)

Elise/M26 14.2 ± 0.1b 6.7 ± 0.3b 0.4 ± 0.1b 0.6 ± 0.1b 473 ± 7.1a

Elise/M26-rolB 14.7 ± 0.2b 5.6 ± 0.3c 0.6 ± 0.1ab 0.8 ± 0.1a 400 ± 3.5b

Elise/M9 13.9 ± 0.3b 8.9 ± 0.8a 0.4 ± 0.1b 0.6 ± 0.1b 461 ± 15.9a

Elise/M9-rolB1 14.4 ± 0.2b 5.6 ± 0.5c 0.5 ± 0.1b 0.5 ± 0.1b 388 ± 2.3b

Elise/M9-rolB2 15.3 ± 0.3a 8.4 ± 0.1a 0.7 ± 0.1a 0.9 ± 0.1a 452 ± 21.3a

Elstar/M26 13.1 ± 0.1c 7.4 ± 0.5b 0.8 ± 0.1a 1.1 ± 0.1a 540 ± 74.0a

Elstar/M26-rolB 14.6 ± 0.0a 6.6 ± 0.6c 0.6 ± 0.1a 1.0 ± 0.1ab 543 ± 6.7a

Elstar/M9 13.4 ± 0.0b 6.0 ± 0.2cd 0.7 ± 0.1a 1.1 ± 0.1a 506 ± 16.9a

Elstar/M9-rolB1 14.8 ± 0.1a 9.0 ± 0.5a 0.9 ± 0.1a 1.1 ± 0.0a 561 ± 7.7a

Elstar/M9-rolB2 13.0 ± 0.0c 5.3 ± 0.3d 0.7 ± 0.1a 0.9 ± 0.1b 523 ± 10.3a

Jonagold/M26 13.4 ± 0.4a 5.2 ± 0.4a 0.5 ± 0.1ab 1.9 ± 0.1a 341 ± 39.4a

Jonagold/M26-rolB 12.4 ± 0.5ab 5.4 ± 0.5a 0.4 ± 0.3b 1.9 ± 0.2a 302 ± 20.4a

Jonagold/M9 12.1 ± 0.3b 5.2 ± 0.5a 0.5 ± 0.1ab 1.9 ± 0.1a 305 ± 22.1a

Jonagold/M9-rolB1 13.3 ± 0.6a 6.0 ± 0.7a 0.8 ± 0.2a 2.0 ± 0.1a 331 ± 58.1a

Jonagold/M9-rolB2 13.6 ± 1.0a 5.2 ± 0.3a 0.7 ± 0.1ab 1.7 ± 0.3a 308 ± 45.0a

Duncan’s multiple range test was carried out among five different rootstocks within one cultivar. Different letters in each column

within one cultivar indicate significant differences at P = 0.05

944 Transgenic Res (2010) 19:933–948

123

percentage of the same rootstocks reported earlier

from in vitro rooting (Welander and Zhu 2000; Zhu

et al. 2001). This is probably due to weak growth of

the transgenic rootstocks under field conditions.

When the cuttings were made, the annual growth

was limited, especially for the transgenic M9 root-

stocks. A weak growth would limit accumulation of

nitrogen and/or carbohydrates in stems, which in turn

might lead to a low rooting percentage as has been

demonstrated for many species (Hambrick et al.

1991; Rapaka et al. 2005; Zerche and Druege 2009).

No translocation of the rolB gene or its mRNA

from rootstock to scion

The results from investigations on translocation of

transgenes and their products from rootstock to scion

reported in the literature have been inconsistent. It

has been reported that graft-transmissible signals can

be translocated from stock to scion (Garcia-Perez

et al. 2004; Kudo and Harada 2007; Tournier et al.

2006). The influence between rootstock and scion is

in some cases mutual, for example a rootstock

transformed with a construct for gene silencing could

induce gene-specific silencing in an originally non-

silenced scion (Palauqui et al. 1997; Voinnet et al.

1998), while the silencing effect could also be

transmitted from scion to rootstock (Sonoda and

Nishiguchi 2000; Tournier et al. 2006). The trans-

ported products could be small RNAs (siRNAs;

Hewezi et al. 2005; Tournier et al. 2006; Yoo et al.

2004), mRNAs (Kim et al. 2001), and proteins

(Golecki et al. 1999; Gomez et al. 2005). However,

Ayre and Turgeon (2004) could not detect transmis-

sion of the CO (CONSTANS) gene or its transcripts

from stock to scion in Arabidopsis thaliana. In this

study, PCR and RT-PCR could not detect the rolB

gene or its mRNA in scion cultivar, suggesting no

Fig. 1 Rooting of cuttings of rolB transgenic apple rootstocks

and non-transgenic controls

Table 10 Rooting results of cuttings taken from the rootstocks

grown in field

Rootstock Total cuttings Rooted cuttings Rooting %

M26 30 0 0c

M26-rolB 30 22 73a

M9 30 1 3c

M9-rolB1 30 20 67a

M9-rolB2 30 13 43b

Cutting was carried out in greenhouse without auxin treatment.

Different letters in the root% column indicate significant

differences at P = 0.05

Fig. 2 RT-PCR of rolB mRNA in non-grafted rolB transgenic

rootstocks. M markers, P plasmid DNA, B blank, 1 M26-rolB,

2 M9-rolB1, 3 M9-rolB2

Fig. 3 RT-PCR of rolB mRNA in scion cultivars. M markers,

P plasmid DNA, B blank, 1–5 Jonagold and 6–10 Elise, grafted

onto M26, M26-rolB, M9, M9-rolB1, M9-rolB2 rootstocks,

respectively

Transgenic Res (2010) 19:933–948 945

123

translocation of the rolB gene and its mRNA from

rootstock to scion. However, it is unclear if small

RNAs could be translocated from rootstock and

sicon. It also remains uncertain if the ROLB protein

could be translocated from rootstock into scion. We

have made several attempts for producing ROLB

antibodies for Western blot analysis without success.

We have encountered great difficulties in purifying

the recombinant ROLB protein and ROLB antibod-

ies, probably because the ROLB protein most likely

is a membrane protein (Filippini et al. 1996). The

extremely low sensitivity of the ROLB antibodies

made detection of ROLB protein at nanogram levels

from plant material impossible (Smolka et al., unpub-

lished data). It has been reported that the phloem

provides a pathway for the transport of small and

macromolecules including soluble proteins (Bor-

tolotti et al. 2005). As mentioned above, the ROLB

protein is not water soluble and may not be possible

or easy to transport from rootstock to scion. However,

this needs to be proven.

The perspective of using the rolB gene

in modifying horticultural crops

Over the years, there have been many attempts to

utilise the rolB gene for improving the rooting ability

in a number of plant species and the results have been

satisfactory in most cases. However, there is so far no

report about how the rolB gene will affect growth and

development under field conditions over a long

period of time. It has been unclear if the rolB

transgenic plants can be of commercial interest for

fruit growers. Our current study shows that the rolB

gene indeed reduced growth and flowering as well as

fruiting of non-transgenic scion cultivars, especially

cultivars with weak growth vigour. This reduction

might be positive for vigorous cultivars in commer-

cial production where thinning is often required when

excessive flowering occurs. From our results, we

recommend using rolB modified rootstocks in com-

bination with vigorous scion cultivars or dwarfing

vigorous rootstocks with the rolB gene.

Another potential use of the rolB gene may be for

making bonzai plants of fruit trees and ornamentals

where extremely slow growth rates are desirable.

Acknowledgments We thank Annelie Ahlman for her

excellent technical support, Carin Emanuelsson and Miao

Zhou for collecting some data and Karl-Erik Gustavsson for

support with the HPLC analysis. FORMAS, The Royal

Physiographic Society in Lund, Nilsson’s fund are acknow-

ledged for financial support.

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