J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

Antisense Expression of MdTFL1, a TFL1-like
Gene, Reduces the Juvenile Phase in Apple
Nobuhiro Kotoda1, Hiroshi Iwanami, Sae Takahashi, and Kazuyuki Abe
Department of Apple Research, National Institute of Fruit Tree Science, Incorporated Administrative
Agency, National Agriculture and Bio-oriented Research Organization (NARO), Shimo-kuriyagawa,Morioka 020-0123, Japan
ADDITIONAL INDEX WORDS. CETS, fruit breeding, genetic transformation, juvenility, Malus×domestica, precocious ﬂowering
ABSTRACT. Because fruit trees such as apple (Malus ×domestica Borkh.) ﬂower and set fruit only after an extended
juvenile phase lasting several years, efﬁcient breeding of fruit trees is limited. We previously suggested that MdTFL1(Malus ×domestica TFL1) functions analogously to TERMINAL FLOWER 1 (TFL1) and that MdTFL1 is involved in
the maintenance of the juvenile/vegetative phase in apple. To clarify the function of MdTFL1 in apple, we produced
transgenic ʻOrinʼ apple trees expressing MdTFL1 antisense RNA. One of them ﬂowered only 8 months after the transfer to the greenhouse, whereas the nontransformed control plants have not ﬂowered in nearly 6 years. As expected,
the expression of endogenous MdTFL1 was suppressed in the transgenic lines that showed precocious ﬂowering. In
addition, the expression level of the transgene was correlated with the reduction of the juvenile phase. These ﬁndings
conﬁrm that MdTFL1 functions like TFL1 and that MdTFL1 maintains the juvenile and vegetative phase in apple.
Flower organs of the transgenic apple trees were normal in appearance, and a precocious ﬂowering transgenic line set
fruit and seeds. Interestingly, some ﬂowers of the transgenic apple trees developed without undergoing dormancy. The
expression of MdTFL1 in apple may affect ﬂower development as well as ﬂower induction.

In the development of all woody plants from seed, there is a
so-called juvenile phase, lasting up to 30 to 40 years in certain
forest trees, during which ﬂowering does not occur and cannot
be induced under normal conditions (Hackett, 1985). In the fruit
industry, it is important to accelerate ﬂowering by reducing thejuvenile or vegetative phase of the trees after planting in order tofacilitate the earliest possible production of fruit. Breeding fruit
trees such as apple often takes more than 20 years, including
periods of cross pollination, seedling selection, and regional trials
to produce varieties that meet consumer demands. For example,
the ʻFujiʼ apple, one of the most popular apple cultivars in theworld (OʼRourke et al., 2003), ﬁrst set fruit 12 years after being
sowed, and it was 23 years before it was released as a cultivar(Sadamori et al., 1963). Generally, the juvenile phase in apple
lasts 6 to 12 years (Visser, 1964; Zimmerman, 1972).
In fruit breeding, various practical techniques have been considered to accelerate the ﬂowering and fruiting of seedlings. The
basic idea is to grow the seedlings rapidly from the germination
stage to the transition to ﬂowering (Aldwinckle, 1975; Visser,1964). In apple, grafting the seedlings onto dwarﬁng rootstocks
such as ʻMalling 9ʼ (ʻM.9ʼ) and ʻMalling 27ʼ (ʻM.27ʼ) will usually bring earlier ﬂowering by 1 or 2 years (Visser, 1964, 1973).
Grafting seedlings onto bearing trees (top grafting), ringing,scoring, bark inversion, root pruning, and spraying with growth
retardants may stimulate earlier ﬂowering when applied to older
seedlings, but in these conditions, the juvenile phase probably
has already passed, and the seedlings are in transition (Kender,

Received for publication 31 Mar. 2005. Accepted for publication 2 July 2005.
We thank Hiroaki Ichikawa, the National Institute of Agrobiological Sciences,for providing the binary vector pSMAK251, and Tomoko Sekita, the National
Institute of Fruit Tree Science, for technical assistance. This work was supportedby the “Development of innovative plants and animals using transformation andcloning” project of MAFF and the Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN). This publication constitutes
contribution number 1369 of the National Institute of Fruit Tree Science.
1Corresponding author; e-mail address: koto@affrc.go.jp

74

1974; Zimmerman, 1972). Based on our experience, it would,
however, be difﬁcult to reduce the juvenile phase of apple seedlings to <4 years under normal growing conditions even if they
are grafted on the dwarﬁng rootstock. The length of the juvenile
phase is inﬂuenced more by genetic factors than by environmental
factors. Signiﬁcant correlations have been found between thelength of the juvenile phase and parent characteristics such as
growth rate and the length of the vegetative phase (Lavi et al.,
1992; Visser, 1965).
The most striking advances in our understanding of the genetic
control of the timing of ﬂowering have come from the study ofarabidopsis [Arabidopsis thaliana (L.) Heynh.] (Levy and Dean,
1998; Piñeiro and Coupland, 1998), and several genes such as
LEAFY (LFY), APETALA1 (AP1), TERMINAL FLOWER 1(TFL1), and FLOWERING LOCUS T (FT), which control ﬂowering time, have been isolated from arabidopsis (Bradley et al.,
1997; Kardailski et al., 1999; Kobayashi et al., 1999; Mandel et al.,
1992; Ohshima et al., 1997; Weigel et al., 1992). Over-expressionof LFY, AP1, or FT shortens the vegetative phase, causing early
ﬂowering (Kardailski et al., 1999; Kobayashi et al., 1999; Mandel
et al., 1995; Weigel and Nilsson, 1995), whereas over-expressionof TFL1 causes late ﬂowering in transgenic arabidopsis (Ratcliffe
et al., 1998). TFL1 plays a key role in the maintenance of theinﬂorescence meristem by preventing the expression of LFY and
AP1 (Ratcliffe et al., 1999). Apple orthologues of several of thesegenes such as AFL1, AFL2, MdAP1 (MdMADS5), and MdTFL1
have been isolated and characterized (Kotoda et al., 2000, 2002;
Kotoda and Wada, 2005; Sung et al., 1999; Wada et al., 2002;
Yao et al., 1999).
With the goal of elucidating the long juvenility of fruit trees,
we are interested in regulatory genes, such as arabidopsis TFL1,
snapdragon (Antirrhinum majus L.) CENTRORADIALIS (CEN),
and tomato (Lycopersicum esculentum Mill.) SELF-PRUNING(SP) genes, which maintain the vegetative phase and promote theemergence of shoots rather than ﬂowers (Bradley et al., 1996;
Pnueli et al., 1998), as well as genes such as LFY and AP1, which
promote ﬂowering. Therefore, we isolated and characterized
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

Materials and Methods
PLANT MATERIALS. Micropropagated tissues of the apple cultivar
Orin were used for Agrobacterium-mediated [A. tumefaciens(Smith & Towns.) Conn.] transformation. The ʻOrinʼ shoots were
subcultured every 4 weeks in a proliferation medium [MurashigeSkoog (MS) medium containing B5 vitamin (1.0 mg·L–1 nicotinicacid, 10 mg·L–1 thiamine chloride, 1.0 mg·L–1 pyridoxin, and 100
mg·L–1 myo-inositol), 1 mg·L–1 (4.4 µM) 6-benzyl-aminopurine(BA), 0.1 mg·L–1 (0.49 µM) indole-3-butyric acid (IBA), 3% (w/v)
sucrose, 0.7% (w/v) agar (pH 5.8)] at 23 to 25 ºC under long-day(LD) conditions (16 h light/8 h dark). Transformed and nontransformed shoots of tissue culture were grafted to apple seedlings orʻJM8ʼ [ʻMarubakaidoʼ (Malus prunifolia Borkh. var. ringo Asami)× ʻM.9ʼ], an easy-cutting apple dwarﬁng rootstock (Soejima etal., 1998). Grafted apples were grown in an isolated greenhousewith the temperature set at 20 to 25 ºC during the growing season(from April to November). Thereafter, the temperature was shiftedto below 7 ºC in the dormant season (from December to March).
Liquid fertilizer was sprayed once per month.
CONSTRUCTION OF TRANSFORMATION VECTORS. The plasmid
vector pUMDTFL1- (Kotoda and Wada, 2005) was cut with
Xba1 and SacI, after which the released MdTFL1 cDNA fragment was ligated to the binary vector pSMAK251 cut with thesame restriction enzymes in an antisense-oriented manner (Fig.
1). The characterization of pSMAK251, which contains genes
for ß-glucuronidase (GUS) and neomycin phosphotransferase
II (NPTII) in its T-DNA region, will be published elsewhere(H. Ichikawa, unpublished). The product of the ligation wasnamed pSMDTFL1.1- (35S::MdTFL1 antisense construct). The
MdTFL1 antisense gene was driven by the cauliﬂower mosaic
virus (CaMV) 35S promoter, and the vector conferred kanamycin
resistance (Fig. 1A).
TRANSFORMATION OF APPLE. The apple cultivar Orin was transformed with A. tumefaciens strain EHA101 bearing pSMDTFL1.1.
Agrobacterium tumefaciens was cultured overnight on a shaker in
20 mL liquid LB medium (10 g·L–1 tryptone, 5 g·L–1 yeast extract
and 5 g·L–1 NaCl, pH 7.0) with 100 mg·L–1 trobicin (Pﬁzer Japan,
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

A

MdTFL1 probe

nptII probe
0.2

1.8

0.3

0.8

Pnos

nptII

Tnos

P35S

0.7

BamHI

LB

HindIII

BamHI

0.7

XhoI

XbaI

RB

TrbcS

SacI

EcoRI

HindIII

T-DNA RegionRB

35S::MdTFL1-AS
rep

Kanamycin R

pSMDTFL1.1sta

ColE1 ori

B

(kb)

KpnI

MdTFL1LB

SpR

the apple gene MdTFL1, which is homologous to TFL1, andconcluded that MdTFL1 functions like TFL1 and is involved in
the maintenance of juvenility in apple (Kotoda and Wada, 2005).
TFL1/CEN/SP are closely related members of the CETS family,a small gene family that encodes 23-kD proteins, and they sharesequence similarity with a group of mammalian phosphatidylethanolamine-binding proteins (PEPBs) (Bradley et al., 1996,
1997; Oshima et al., 1997; Pnueli et al., 1998). The FT gene also
belongs to the CETS family, but it mediates signals for ﬂowering
antagonistically with its homologous gene, TFL1, and promotes
ﬂowering with LFY (Kardailsky et al., 1999; Kobayashi et al.,1999). In other species, a number of TFL1 homologues fromtobacco (Nicotiana tabacum L.2), oilseed rape (Brassica napusL.), cabbage (Brassica oleracea L. var. capitata L.), ryegrass(Lolium perenne L.), rice (Oryza sativa L.), pea (Pisum sativumL.), and navel orange [Citrus sinensis (L.) Osbeck] have beencloned and characterized (Amaya et al., 1999; Foucher et al.,
2003; Jensen et al., 2001; Mimida et al., 1999; Nakagawa et al.,
2002; Pillitteri et al., 2004).
We describe here the precocious ﬂowering of transgenic apple
trees caused by the suppression of MdTFL1 in the ﬁrst report on
the extreme reduction of the juvenile phase in deciduous fruit
trees.

The region used for vector construction(596 bp)
MdTFL-2SprimerMdTFL-2A
primerATG

TAG

poly (A)

MdTFL1 cDNA coding region (516 bp)
-8 +1

+516

+587

sense primer
for RT-PCR+690
antisense primer
for RT-PCR

The region corresponding to RT-PCR productof endogenous MdTFL1 (699 bp)

Fig. 1. Schematic representation of the transformation vector pSMDTFL1.1- and
MdTFL1 cDNA used for vector construction. (A) MdTFL1 cDNA was inserted
in the antisense orientation between the XbaI and SacI sites of the binary vector
pSMAK251, giving rise to pSMDTFL1.1-. Regions of the probes to detect
the nptII or MdTFL1 gene are indicated by the bold lines above each gene;
Pnos = nos promoter, Tnos = 3´ region of nos, P35S = cauliﬂower mosaic
virus 35S promoter, TrbcS = 3´ region of arabidopsis rbcS-2B gene, nptII =neomycin phosphotransferase II gene, LB = left border, RB = right border, SpR= spectinomycin/streptomycin resistance gene from Tn7, ColE1ori = ColE1
replication origin from pBR322, sta = region involved in plasmid stability, rep= region essential for plasmid maintenance. (B) Region between the positions–8 and +587 of MdTFL1 cDNA used for vector construction. This DNA region(596 bp) can be ampliﬁed by a PCR with gene-speciﬁc primers MdTFL-2S
and MdTFL-2A. For RT-PCR analysis; sense primer (position = -8 ~ +15) andantisense primer (position = +670 ~ +690) were used to detect endogenous
MdTFL1 transcripts. The 699-bp DNA fragment was obtained as a reverse
transcription (RT)-PCR product for endogenous MdTFL1; ATG = initiation codon,
TAG = stop codon. A box represents a coding region and a bold line including
a box represents the region inserted into the binary vector pSMAK251.

Tokyo) at 28 ºC. After centrifugation (2000 gn, 5 min), the pellet
was resuspended in MS medium with 3% (w/v) sucrose and 150µM acetosyringone (3´,5´-dimethoxy-4´-hydroxyacetophenone;
Acros Organics, Geel, Belgium) and further diluted with thismedium until an optical density (OD) of 0.5 to 0.8 at 600 nm wasreached. Leaf explants of the apple cultivar Orin were infectedwith the inoculum for 30 min, and then the leaf explants weretransferred to a co-cultivation medium consisting of MS salt, 3.3
mg·L–1 (15 µM) thidiazuron (TDZ), 0.93 mg·L–1 (5 µM) naphthaleneacetic acid (NAA), 29.4 mg·L–1 (150 µM) acetosyringone,
and 0.25% (w/v) gelrite. After co-cultivation for 5 to 7 d at 22
to 25 ºC in the dark, explants were transferred to an antibiotic
selection medium consisting of MS salt, 3.3 mg·L–1 (15 µM) TDZ,
0.93 mg·L–1 (5 µM) NAA, 25 mg·L–1 kanamycin monosulfate,
500 mg·L–1 Claforan (Pharmacia and Upjohn Ltd., Tokyo), and
0.25% (w/v) Gelrite. When adventitious shoots appeared, cultures
were transferred to an antibiotic selection medium containing
50 mg·L–1 kanamycin monosulfate and then incubated at 25 ºC
with a 16-h photoperiod (30 µmol·m–2·s–1). When the shoots had
produced several leaves, they were excised and transferred to a
proliferation medium containing 50 mg·L–1 kanamycin mono75

27

sulfate. The transgene insertion was conﬁrmed by a polymerase
552
553
chain reaction (PCR) with speciﬁc primers for nptII (NPTII-3S
554
sense primer, 5´-GCC CTG AAT GAA CTG CAG GAC GAG
555
GC-3´; NPTII-3A antisense primer, 5´-GCA GGC ATC GCC ATG
556
557
AGT CAC GAC GAG A-3´; PCR product, 411 bp) or MdTFL1
558(MdTFL-2S sense primer, 5´-CTC TTA AAA TGA AAA GAG
559
CC-3´; MdTFL-2A antisense primer, 5´-TTC TCA CAT GTC AAT
560
561
AAG TT-3´; PCR product, 596 bp). The region ampliﬁed with
562
primers (MdTFL-2S and -2A) for MdTFL1 is shown in Fig. 1B.
563
Conditions of ampliﬁcation for each primer set were as follows;564
565
1 cycle of 300 s at 94 ºC, 30 cycles of 15 s at 94 ºC, 15 s at 65 ºC
566
and 30 s at 72 ºC for nptII; 1 cycle of 300 s at 94 ºC, 40 cycles567
of 30 s at 94 ºC, 30 s at 50 ºC, and 60 s at 72 ºC for MdTFL1.
568
Putatively transformed shoots were excised and transferred to 569
a570
proliferation medium free of antibiotics.
DNA BLOT ANALYSIS. Genomic DNA was obtained from transgenic and nontransgenic ʻOrinʼ apple leaves using the CTAB
method as described by Kotoda et al. (2002). The DNA (10 µg)
was digested individually with either BamHI, EcoRI, HindIII, or
XbaI according to the manufacturerʼs instructions (Takara Biomedicals, Tokyo) and then separated on 0.8% (w/v) SeaKem GTG
agarose gel (Cambrex Bio Science, Rockland, Maine). The DNA
bands were transferred to Hybond-N+ (Amersham Pharmacia
Biotech, Buckinghamshire, UK) and hybridized with a digoxigenin (DIG)-labeled PCR probe encoding the nptII or MdTFL1
gene (Fig. 1A). Primer sets used for DIG-labeled PCR probes of
NPTII and MdTFL1 were NPTII-3S and -3A, and MdTFL-2S and-2A, respectively (Fig. 1). DIG-labeled DNA Molecular-Weight
Marker II (Roche Diagnostics, Mannheim, Germany) was used
as a size marker. Hybridization was performed in DIG Easy Hyb(Roche Diagnostics) at 42 ºC for 16 h, followed by two rinses
in 2×SSC containing 1% (w/v) SDS at room temperature for 5
min and two washes in 0.5×SSC containing 1% (w/v) SDS at
68 ºC for 20 min. The detections were performed according to
the manufacturerʼs protocol (Roche Diagnostics). The resulting
materials were analyzed in a LAS-1000 image analyzer (Fuji
Photo Film Co., Tokyo).
RNA BLOT ANALYSIS. For Northern blot analysis, apple leaves
were collected from greenhouses at our research center in Morioka, Japan. Total RNA was isolated from leaves of transgenic
and nontransgenic ʻOrinʼ apples in the growing season using
the CTAB method as described by Kotoda et al. (2000). Twenty
micrograms of the total RNA was separated on 1.2% SeaKem
GTG agarose gel containing 5.0% (v/v) formaldehyde, after
which the gel was blotted on Hybond-N+. Hybridization was
performed in DIG Easy Hyb at 50 ºC for 16 h with a DIG-labeled
PCR probe using MdTFL1 cDNA as a template. The primer set
used for preparing the probe was MdTFL-2S and -2A (Fig. 1B).
This probe detects only transcripts of the transgene because no
expression of endogenous MdTFL1 was detected in nontransgenic
ʻOrinʼ apple leaves examined in RNA blot analysis. As an internal
control, the expression of the histone H3 gene (Histone H3) was
measured. The wash and detection was performed in the samemanner as that used for DNA.
REVERSE TRANSCRIPTION (RT)-PCR ANALYSIS. Total RNAs
were isolated from cultured tissues of each transgenic line and a
nontransgenic control using the SV total RNA Isolation System(Promega, Madison, Wis.). A speciﬁc primer (5´-TTG TGG CAT
ACA TTG TAA ATA-3´, 21 bp) for MdTFL1 (Fig. 1B; Table 1)
and a random primer (nonadeoxyribonucleotide mixture) for
AFL1, AFL2 (apple orthologues of LEAFY; Wada et al., 2002),
MdAP1 (an apple orthologue of APETALA1; Kotoda et al., 2000,
76

Table
1.1. Gene-speciﬁcprimer sets used for reverse transcription PCR analyses
Table
Gene-specificof transgeniclines. primer sets used for reverse transcription PCR analyses oftransgenenic lines
PrimerMdTFL1
Sense primer
Antisense primer
AFL1
Sense primer
Antisense primer
AFL2
Sense primer
Antisense primer
MdAP1
Sense primer
Antisense primer
Histone H3
Sense primer
Antisense primer

Oligonucleotide
5´-CTC TTA AAA TGA AAA GAG CCT CGG-3´ (24 mers)
5´-TTG TGG CAT ACA TTG TAA ATA-3´ (21 mers)
5´-GCA CCC GTT CAT TGT GAC GG-3´ (20 mers)
5´-GCC CAA AAT CTC TCG CCC-3´ (18 mers)
5´- GCA CCC GTT CAT TGT GAC GG -3´ (20 mers)
5´-CTT ATC AGT TAT ATT GAA GCG-3´ (21 mers)
5´-ATG GGG AGA GGT AGA GTT CAG CTT-3´ (24 mers)
5´-TTA GAC ACA TGG AAG TGG CTG TGG-3´ (24 mers)
5´-TGA AGA AGC CCC ACA GAT A-3´ (19 mers)
5´-ACA CAA GAA ACT ATA AAC C-3´ (19 mers)

2002), and the apple Histone H3 (internal control) were usedin RT reactions (30 ºC for 10 min, 42 ºC for 50 min, 99 ºC for
5 min) with 1 µg of total RNA. Because the speciﬁc antisense
primer for MdTFL1 was designed between the positions +670
and +690 of MdTFL1 cDNA, ≈100 bp downstream the regionused for vector construction, the primer speciﬁcally anneals tothe endogenous MdTFL1 mRNA to synthesize the ﬁrst strand
cDNA in a RT reaction. RT-PCR products of MdTFL1 (699 bp),
AFL1 (583 bp), AFL2 (564 bp), MdAP1 (613 bp), and Histone H3(371 bp) were identiﬁed using gene-speciﬁc primer sets shownin Table 1. Conditions of ampliﬁcation for each primer set wereas follows: 1 cycle of 300 s at 94 ºC, 40 cycles of 30 s at 94 ºC,
30 s at 53 ºC, and 60 s at 72 ºC for MdTFL1 and Histone H3;
1 cycle of 300 s at 94 ºC, 40 cycles of 30 s at 94 ºC, 30 s at 50ºC, and 60 s at 72 ºC for AFL1 and AFL2; 1 cycle of 300 s at 94ºC, 40 cycles of 30 s at 94 ºC, 30 s at 55 ºC, and 60 s at 72 ºC
for MdAP1. Ampliﬁed products were separated on 1.5% (w/v)
SeaKem ME agarose gel (Cambrex Bio Science) in 1×TAE buffer. DNA was then stained in an ethidium bromide solution at
1.0 µg·mL–1 for 20 min.
POLLEN VIABILITY TEST. Anthers from the transgenic lines andthe nontransgenic control ʻOrinʼapple were collected in a 1.5-mL
tube and incubated at 25 ºC with silica gel for the dehiscence ofanther. Thereafter, pollen was plated on 1% (w/v) agar containing 17% (w/v) sucrose and then incubated at 25 ºC for pollen
germination. The pollen germination was photographed using
light microscopy (Nikon Corp., Tokyo).
Results
TRANSFORMATION OF APPLE WITH 35S::MdTFL1 ANTISENSE

GENE. To clarify the function of MdTFL1 (GenBank accession

no. AB052994), a putative orthologue of arabidopsis TFL1, we
produced transgenic apple trees with the MdTFL1 antisense gene
driven by the cauliﬂower mosaic virus (CaMV) 35S promoter forthe purpose of suppressing the expression of endogenous MdTFL1.
The transformation vector contained neomycin phosophotransferase II gene (nptII), which detoxiﬁes antibiotics such as neomycin,
kanamycin, G418, and paromomycin. Thus, kanamycin sulfate
was used as a selective agent to obtain the transformed shootsof the apple cultivar Orin. About 2000 leaf disks of the ʻOrinʼapple were used in the Agrobacterium-mediated transformation.
Kanamycin-resistant shoots regenerated from ﬁve independent
leaf disks were cultured in the proliferating medium after selection. The putatively transformed ﬁve kanamycin-resistant shoots
were micropropagated in the proliferating medium and then eachline was multiplied by grafting individual shoots in tissue culture
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

28

to ʻJM8ʼ dwarﬁng rootstocks, giving rise to one to four clones Table 2. Phenotype of transformed ʻOrinʼ apples with the MdTFL1 antisense
gene.
571
Table
2. Phenotype of transformed ‘Orin’ apples with the MdTFL1 antisense gene
per line (Table 2). Numbers following the line (e.g., 303-1 and
572
Time to first
303-4 in Table 2) mean clones multiplied by greenwood grafting
573
Transgenic
nptIITransgeneflowering
GrowthGraftedzyx
as described above. The nontransformed ʻOrinʼ apples were also
574
line
gene
RNA
(months) w
habitv
year
ND
ND
69
N
1997
grafted onto ʻJM8ʼ or seedlings as controls in the same way575
as Control-1
576
Control-2
ND
NT
–
N
2000
transformed lines. The grafted transformed lines and nontrans577
303-1
+
+
11
SB
2000
formed control plants started to be grown in the greenhouse for
578
303-4
+
+
11
SB
2000
further analysis at the same time for the greenwood grafting.
579
705-1
+
+
8
W, SB
2000
580
705-2
+
+
13
W, SB
2000
Insertion of the transgene was conﬁrmed by a PCR with speciﬁc
581
705-3
+
+
11
W, SB
2000
primers for nptII and MdTFL1, revealing that three independent
582
705-4
+
+
11
W, SB
2000
transformants (lines 303, 614, and 705) and two escapes (lines
583
614-1
+
+
25
W
2001
584
837-1
ND
NT
–
N
2002
837 and 850) were produced (Table 2).
585
837-2
ND
NT
–
N
2002
DNA BLOT ANALYSIS OF TRANSGENIC LINES. DNA blot analysis
586
850-1
ND
NT
–
N
2002
was performed for transgenic lines using DIG-labeled nptII
587
850-2
ND
NT
–
N
2002
ND
NT
–
N
2002
and MdTFL1 PCR probes to demonstrate the copy number 588
of z 850-3
zThree589
Three independently
independentlyregenerated
regeneratednontransformed
non-transformed‘Orin’
apples
were
grafted on seedli
ʻOrinʼ
apples
were
graftedthe transgene. DNA blot probed with an nptII cDNA fragmentonseedlingsor(control-1)
or ʻJM8ʼ rootstocks(control-2).
the 303-1 and 3(control-1)‘JM8’ rootstocks(control-2).
Numbers Numbers
followingfollowing
the line (eg.
590
showed that line 614 has one copy and lines 303 and 705 haveline
(e.g.,clones
303-1 multipliedand 303-4)bymean
clones multiplied
bytissuegreenwood
graftingof4)
mean
greenwood
graftingofculturedshoots.
591
tissue-cultured
shoots.
two copies of the transgene (Fig. 2). Every line has a common
y
yDNA
592
DNA waswas extracted
extractedfrom
fromthetheleaves,leaves,andandPCR
PCRwaswasperformed
performedtotodetect
detectnptII
nptII genes.
x2.9-kb fragment derived from the region between two BamHI
2 RNA was extracted from the leaves, and Northern blot analysis was performe
genes.
593 Fig
Total
Total RNA was extracted from the leaves, and Northern blot analysis was per2, xformedsites of T-DNA on the lane of BamHI digestion (Fig. 1A; Fig.594
detect thetransgene
RNA. RNA.
to detect
the transgenew
w
arrowhead). DNA blot probed with an MdTFL1 cDNA fragment
Months595Months counted
counted fromfrom grafting
graftingand
andtransfer
transfertoto thethe greenhouse.
greenhouse.—, not ﬂowv
N, normal
normalgrowth
growthhabit;
habit;SB,SB,short
shortbranch;branch;W, weak tree vigor.
reﬂected the same result as that with nptII (data not shown). 596 ered.
vN,W, weak tree vigor.
597
ND, not
not detected;
detected;NT,NT,notnottested;
tested.+, detected.
ND,
PRECOCIOUS FLOWERING OF TRANSGENIC APPLE TREES. Transgenic line 705-1 produced a solitary ﬂower only 8 months after
303
705
614
the transfer to the green house (Fig. 3A; Table 2), despite theprevious observation that apple seedlings ﬂower naturally after
B E H X B E H X B E H X
M
7 to 8 years (Zimmerman, 1972). Other lines also showed preco(kb)cious ﬂowering, ranging from 11 to 25 months after the transfer
to the greenhouse (Table 2). In contrast, no ﬂowering took place
23.1
in the nontransgenic controls grafted onto seedlings for ≈6 years
9.4(Table 2, control-1). In addition, nontransgenic controls grafted
6.5
onto ʻJM8ʼ dwarﬁng rootstocks have not ﬂowered for 5 years
in the greenhouse (Table 2, control-2). After breaking the ﬁrst
4.3
dormancy, six transgenic apple trees, including lines 303 and2.9 kb705, produced some solitary ﬂowers on their leaf axils and at
2.3
the tops of 2-year-old or current shoots (Fig. 3B). Normal apple
2.0
trees produce many clusters comprised of about ﬁve ﬂowers on
their fruit-bearing shoots. These transgenic lines also produced
clusters comprised of a normal number of ﬂowers from thesecond leaf (Fig. 3 B and C). Interestingly, line 705 showed an Fig. 2. Southern blot analysis of transformed ʻOrinʼ apples with the MdTFL1
antisense gene. Genomic DNA (10 µg) was isolated from the leaves of three
ever-ﬂowering habit as well as reduced juvenility (Fig. 3C). As
transformants (lines 303-1, 614-1, and 705-1), digested individually with BamHI,
for leaf morphology, line 303 had round, more serrated leaves,
EcoRI, HindIII, or XbaI, and then separated on 0.8% (w/v) agarose gel. The
DNA bands were transferred to Hybond-N+ and hybridized with a digoxigenin
and line 705 had leaves with much smaller stipules than those(DIG)-labeled PCR probe encoding the nptII. BamHI, EcoRI, HindIII, and XbaIof the controls (Fig. 3D).
were the enzymes with no recognized positions in the region used for nptII probe
In line 303, the branches were upright and relatively short due(Fig. 1A). Hybridization was performed in DIG Easy Hyb (Roche Diagnostics,
to retarded growth compared to the other lines and nontransgenic
Mannheim, Germany) at 42 ºC for 16 h, followed by two washes in 0.5×SSC
controls (Table 2). The tree vigor of lines 614 and 705 was weakercontaining 1% (w/v) SDS at 68 ºC for 20 min. A DIG-labeled DNA Molecular
Weight Marker II (Roche Diagonistics) was used as a size marker; M = molecular
as a whole, and the branching was a little more complicated thanweight marker, B = BamHI, E = EcoRI, H = HindIII, X = XbaI.
those of the nontransgenic controls because foliar buds were
often converted to ﬂoral buds. A diagram of the growth habitsof the nontransgenic control (WT) and the transgenic line (35S:: 705-1, had no stamens (Fig. 5A), whereas most of the ﬂowers
MdTFL1-AS) is shown in Fig. 4. In apple, ﬂowers are produced emerging after the following season were normal in appearance
on terminal inﬂorescences (Fig. 4B) after going through the (Fig. 5B), like those of the nontransgenic control ʻOrinʼ apple
juvenile phase (Fig. 4A). In contrast, the transgenic line with a (Fig. 5C). To conﬁrm the fertility of transgenic apple trees that
35S::MdTFL1 antisense construct ﬂowered within 1 year after showed precocious ﬂowering, we carried out pollination between
the transfer to the greenhouse (Fig. 4C). The conversion of foliar transgenic apple trees and other nontransgenic apple cultivars.
buds (shown in brown arrowheads) into ﬂoral buds (shown in pink Line 705-1, pollinated with the Japanese apple cultivar Sansa, setarrowheads) leads to the cessation of shoot growth, resulting in a some fruit (Fig. 5D). On the other hand, the representative line,
change in the plant architecture to shorter and more complicated 705-1, was used to pollinate apple cultivars Sansa and McIntosh
branches (Fig. 4 D).
Wijcik because pollen from the precocious lines (Fig. 5K) had
FLOWER AND FRUIT DEVELOPMENT OF THE MOST PRECOCIOUS an ability to germinate on agar plates as the controls (Fig. 5L),
FLOWERING LINE. The ﬁrst ﬂower of the most precocious line,
resulting in the setting of some fruit (Fig. 5E). The development
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

77

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�������

�����

Fig. 3. Precocious ﬂowering of transgenic ʻOrinʼ apples by constitutive expressionof the MdTFL1 antisense gene. (A) Eight-month-old transgenic line 705-1
grafted onto ʻJM8ʼ dwarﬁng rootstock; terminal ﬂower (tf) and graft union (gt)are indicated by arrowheads. (B) Twelve-month-old transgenic line 705-1 after
the breaking of the ﬁrst dormancy; solitary ﬂower (sf) and ﬂower clusters (fc)
are indicated by arrowheads. (C) Three-year-old transgenic line produced bygrafting a scion of line 705-1; an ever-ﬂowering characteristic is observed. (D)Leaves from transgenic lines 303-1 (left), 705-1 (center), and a nontransgenic
control (right) [scale bars: (A) 10 cm, (B) and (C) 5.0 cm, (D) 2.0 cm].

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Fig. 4. Schematic representation of the phenotype of a wild-type and a transgenic
apple with a 35S::MdTFL1 antisense gene (AS). The expression of 35S::
MdTFL1-AS causes changes in the plant architecture. (A) Two-year-old wildtype (WT) plant; no ﬂowers are present on shoots during the juvenile phase.
(B) Seven to 8-year-old wild-type (WT) plant; ﬂowers are produced on terminal
inﬂorescences after the end of the juvenile phase; no ﬂowers are present onvegetative shoots growing upright (water sprout) even in the reproductive
phase. (C) One-year-old precocious line expressing 35S::MdTFL1-AS; ﬂowers
are present at the top of the shoot (terminal inﬂorescence) and in leaf axils,
resulting in the termination of the shoot growth. (D) Two-year-old precocious
line. The tree vigor becomes weak and the plant architecture is changed dueto the replacement of shoots by ﬂowers. New growth is initiated from axillary
meristems proximal to the terminal inﬂorescence. Foliar buds and ﬂoral buds
are shown in brown and pink arrowheads, respectively.

J. AMER. SOC. HORT. SCI. 131(1):xxx–xxx. 2006.
78

an theability
to germinate
on agar plates
the controls
5L),oftransgenic
fruit appeared
normalas(Fig.
5 D and(Fig.
E) comresulting
in thesetting of somefruit grown(Fig. 5E).
The development
paredto thenontransgenic
controls
normallyin the ﬁeldof theF).
transgenic
fruit
normal(Fig. 5about
D and6 E)com(Fig.
Fruit fromlineappeared705-1 were
harvestedmonthsparedpollination
to the nontransgenic
controls
normallyin theofﬁeld
after(Fig. 5G). The
skingrowncolor andthe aromathe(Fig. F). Fruit
705-1
6 monthstransgenic
fruitfromwerelinealmostthe weresame harvested
as those ofabout
nontransgenic
after pollination(Fig. the5G).size
Theofskinand thearoma
theʻOrinʼ
fruit, although
the colortransgenic
fruit
was ofabout
transgenicthat
fruit
almostthe same as thoseof nontransgenic
one-thirdofwerefruit ofthe nontransgenic
controls
grown in theʻOrinʼSome
fruit,transgenic
although fruit
the size
the transgenic
fruitothers
was aboutﬁeld.
wereofseeded(Fig. 5H), andwere
one-thirdthat ofInterestingly,
fruit of the nontransgenic
controls
grown
in theoften
seedless.
the precocious
line hada markedﬁeld. Somefruit wereseeded(Fig. 5H),(Fig.
and others
tendency
totransgenic
set some fruit
without
fertilization
5I). Inwere
line
oftentheseedless.
Interestingly,
precocious
lineﬂoral
had abuds
marked705,
transition
to ﬂoweringthe
often
occurred and(fb)tendency
to setonsomefruit without
fertilization
5I). occurs
In linewere
producedgrowing
current shoots
where(Fig.
it hardly
705,
transition to ﬂowering
often(Fig.
occurredinthethenontransgenic
control plants
5J). and ﬂoral buds (fb)
wereproduced ANALYSES
on growing
current shoots
where
it hardlyexpresoccurs
OF TRANSGENIC
LINES. Transgene
EXPRESSION
in theinnontransgenic
plants (Fig.
5J).
sion
the leaves of control
transformants
was analyzed
by RNA blot
ANALYSES
OF MdTFL1
TRANSGENIC
LINES. Transgene
expresEXPRESSIONprobed
(Fig.
6A).
The probe
washybridization
withsion in theof transformants
was analyzed
blot
MdTFL1preparedby leavesdirect labelingof ampliﬁcation
productsbyofRNA
6A).
Thepreparedprobe washybridization
probedwithinMdTFL1
with
DIG-dUTP
in a PCR
the same (Fig.
manneras thatin
prepared
direct labelingof ampliﬁcationof MdTFL1
DNA
blotbyanalysis.
This probe
detected onlyproducts
the transcriptsof thewith DIG-dUTP
inleaves
a PCR because
in the samemanner as that
prepared intransgenein appleno expressionof endogenous
DNA blotwas
analysis.
Thisinprobe
detected only control
the transcriptsof theMdTFL1detected
the nontransgenic
ʻOrinʼ apple
transgenein applebecauseno (Fig.
expressionof endogenous
leavesexaminedin leaves
RNA blot
analysis
6A, cont),
although it
MdTFL1the nontransgenic
controlʻOrinʼ
apple
candetectwas
bothdetectedsense andinantisense
MdTFL1 RNA.
In ourprevious
leavesitexaminedin RNA blot analysis(Fig. 6A,
althoughinit
wascont),
not expressedwork,
was also demonstrated
that MdTFL1
can detectboth sense
and antisense
MdTFL1RNA.expression
In our previous
leaves(Kotodaand Wada,2005). The
transgenewas
was705-1
not expressedinwork, it wasalso demonstrated
relatively
strongin transgenic thatlinesMdTFL1
303-1 andcomparedleaves(KotodaWada, 2005).
Thewithtransgene
expressionwastoline 614-1
andandcorrelated
inversely
the time
to ﬂowering
relatively
strong (Fig.
in transgenic
lines
303-1
andthe705-1
comparedintransformants
6A; Table2). Todetectexpressionof
to line 614-1MdTFL1,
and correlated
inverselywith the time
to ﬂowering
endogenous
RT-PCR
was performed
as shown
in Fig.
in transformants(Fig.tissue
6A; Table2). Todetectshootsthe expressionof
6B.
Total RNAs from
culturesof somein each line
endogenous
MdTFL1,
RT-PCR
was performed
as shown
in Fig.
MdTFL1
is normally
expressed
in vegetative
were
used because
6B. TotalRNAstissue(Kotoda
cultures andof someshootsin eachline
shoot
apices,notfromin leavesWada,
2005).
Becausevegetative
were
used because
MdTFL1expressed
was usedinfor
the ﬁrst
anantisense
primerMdTFL1
speciﬁc istonormallyshoot apices,
not in leaves
and Wada,2005).
BecausestrandcDNA synthesisin a(Kotoda
RT reaction,
the primer
would
anfor the ﬁrst
an antisense
primersense
speciﬁcto MdTFL1
MdTFL1
mRNA,wasnotusedto antisenseone
neal
to endogenous
strand cDNA
synthesis
in aInRT
reaction,primerwould
anderivedfrom thetrangene.
addition,thetheprimersite
was out
antisense one
neal
endogenous
sense MdTFL1
cDNAmRNA,
used fornot
theto35S::MdTFL1
ofthetoregionof the MdTFL1
derived fromthe trangene.
addition,
the bpprimersite productwas out
antisense
construct(Fig. 1B).InThus,
the 699
RT-PCR
cDNA As
used
for the the
35S::MdTFL1of theregiontoof
the MdTFL1
MdTFL1.
expected,
expression
was
speciﬁcendogenous
antisense
construct(Fig. was1B). suppressed
Thus, the 699
bp RT-PCR
product
MdTFL1in thetransgenic
linesof
endogenous
MdTFL1.
As expected,
the expression
was
speciﬁc toascompared
toendogenous
the nontransgenic
controlʻOrinʼ apple,
although
MdTFL1
suppressedlines
in thetransgenic
endogenous
itofwasstill detectablein wasthe transgenic(Fig.
6B). Tolines
exas comparedto thenontransgenic
control ʻOrinʼapple, although
on theamine
the effectof theantisense suppressionof MdTFL1
it wasgenes
still detectabletransgeniclines (Fig.
6B). To exother
involved ininthethe
ﬂoweringof apple,the expression
of
onwas
the
amine theeffectthe antisense
suppressionof MdTFL1
AFL1,
AFL2,
andofMdAP1(MdMADS5)
in transgenic
lines
other genes
the ﬂoweringof fromapple,thethetissue
expressionofanalyzed
byinvolved
RT-PCRinusing
the RNAs
culture
AFL1,
AFL2,
MdAP1
transgeniclines was
AFL1 (MdMADS5)and AFL2 areinapple
orthologues
ofofshoots(Fig.and6C).
analyzed by RT-PCR using
the etRNAs
fromWada
the tissue
FLORICAULA/LEAFY(Kotodaal., 2000;
et al., culture2002),AFL1
and AFL2
are apple
orthologues
ofof shoots
(Fig.
and
MdAP1
is an6C).
apple
orthologueof AP1(Kotodaet al., 2000,(Kotodaal., 2000; Wadaet al.,FLORICAULA/LEAFY
wasetup-regulated
in line3032002),
(Fig.
2002;
Yao et al., 1999). AFL1
and MdAP1
an appleorthologue(Kotoda ettissuesal., 2000,
is notexpressedofinAP1
the vegetative
but
6C),
althoughisAFL1
AFL1
wasin
up-regulated
in line303et(Fig.
2002; Yao etinal.,
1999).
speciﬁcally
ﬂoral
shoot
apicesnormal apple
(Wada
al.,
AFL1
is notisexpressedthe vegetative
tissuesbut6C), althoughwhichnormallyinexpressedin bothvegeta2002).
For AFL2,
speciﬁcallyin shoot
ﬂoral apices,shoot apices
in normal
apple
(Wada
et theal.,tive
and ﬂoral
the gene
expression
wasseen in
AFL2,
is normally
inof
both
vegeta2002). Forlines
AFL2
wastransgenic
as whichwell (Fig.
6C). Theexpressed
expression
tive and ﬂoral
the gene
expression
was seenin thetherelatively
high shootin lineapices,
705, which
ﬂoweredthe earliestof allAFL2 wastransgenic linesTheas well(Fig. 6C).
The expression
was notofdetectedintransformants.
expressionof MdAP1
relatively
high in lines
line 705,
ﬂoweredcontrol
the earliest
all theeithertransgenic
or a which
nontransgenic(Fig.of6C).
In
was(Histone
not detectedintransformants.
expressionof MdAP1
H3) of
thisexpression The
analysis,the histone
H3 geneeither wastransgenic
a nontransgenic
apple
adoptedlines
as anorinternal
control. control (Fig. 6C). In
this expression analysis, the histone H3 gene (Histone H3) ofapple was adopted as an internal control.
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.5

A

E

lf

tf

F

303
614
705
Cont
Blank

MdTFL1transgene
Histone H3

EtBr

C

Cont
12WAFB
Blank
PC

D

lf

MdTFL1

Histone H3

303
614
705

lf

tf

705-1

B
Cont

C

614-1

B

303-1

A

AFL1
AFL2
MdAP1

Histone H3

G

H

seed

I
pf

J

fb

K

L

Fig. 5. Flowers, fruit, and pollen viability of the transgenic ʻOrinʼ apples with a
35S::MdTFL1 antisense gene. (A) to (C) Flowers of transgenic and nontransgenic
ʻOrinʼ apples. (A) Close-up of the solitary ﬂower of the transgenic line 705-1
shown in Fig. 3A. (B) Close-up of the ﬂower cluster of the transgenic line 705-1
shown in Fig. 3B. (C) Close-up of ﬂowers from a control ʻOrinʼ apple grownin the ﬁeld; tf and lf in (B) and (C) indicate terminal ﬂower and lateral ﬂower,
respectively. (D) to (F) Fruit of transgenic and nontransgenic ʻOrinʼ apples.
(D) Four-week-old young fruit of transgenic 705-1 crossed with ʻSansaʼ. (E)Four-week-old young fruit of ʻMcIntosh Wijcikʼ crossed with line 705-1. (F)Four-week-old young fruit of the control plant. (G) to (I) Fruit developmentwith and without pollination. (G) Mature fruit with pollination. (H) Inside of
the fruit in (G); left = fruit having a seed (arrowhead), right = seedless fruit.
(I) Young fruit of line 705 that developed without pollination; arrowheads
indicate parthenocarpic fruit (pf). (J) to (L) Pollen germination of transgenic
and nontransgenic control plants on 1% (w/v) agar containing 17% (w/v)
sucrose. (J) Floral bud (fb) produced on the top of the growing current shootbefore the dormant period in the transgenic precocious line, although ﬂoral bud
differentiation normally occurs after the cessation of shoot growth. (K) Pollen
from line 705-1. (L) Pollen from nontransgenic ʻOrinʼ apple [scale bars: (A) to(C) 0.5 cm, (D) to (I) 2.0 cm, (J) 3.0 cm, (K) and (I) 100 µm].

Discussion
ANTISENSE EXPRESSION OF MdTFL1 INDUCED THE PRECOCIOUS

FLOWERING AND DEVELOPED FRUIT IN APPLE. There are two ways

to produce plants with precocious ﬂowering. One is the constitutive expression of genes that promote ﬂowering, such as LFY,
AP1, and FT. The other is the suppression of genes that delay
ﬂowering, such as TFL1, by antisense expression, co-suppression, or RNA interference (RNAi). We have shown here that theantisense expression of MdTFL1 effectively induces precocious
ﬂowering in transgenic apple. The transgenic lines 303 and 705
ﬁrst ﬂowered 8 to 13 months after the transfer to the greenhouse,and line 614 ﬁrst ﬂowered 25 months after the transfer to the green
house, in contrast to the nontransgenic controls, one of whichﬁrst ﬂowered at 69 months after the transfer to the greenhouse(Table 2). It is certain that the precocity of the transgenic lines
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

Fig. 6. Expression analyses of transgenic ʻOrinʼ apples with a 35S::MdTFL1
antisense gene by Northern blot and reverse transcription (RT)-PCR. (A)Northern blot analysis of transgene expression in the leaves of each transgenic
line (lines 303-1, 614-1, and 705-1); hybridization was performed in DIG
Easy Hyb (Roche Diagonistics, Mannheim, Germany) at 50 ºC for 16 h with adigoxigenin (DIG)-labeled PCR probe using MdTFL1 cDNA as a template. (B)
RT-PCR analysis for the expression of the endogenous MdTFL1 in the shootsof tissue culture of each transgenic line (lines 303, 614, and 705); a speciﬁc
antisense primer for MdTFL1 was used in a RT reaction, and then PCR was
performed with primers speciﬁc to endogenous MdTFL1. (C) RT-PCR analysis
of apple genes involved in ﬂowering using RNAs from shoots of tissue cultureof each transgenic line (lines 303, 614, and 705). AFL1 and AFL2 are apple
homologues of FLORICULA/LEAFY and MdAP1 is an apple homologue of
APETALA1. In the expression analyses, nontransgenic ʻOrinʼ apple (cont) andhistone H3 gene (Histone H3) were used as a control plant and an internal
control of gene expression, respectively; EtBr = ethidium bromide, PC = positive
control (plasmid containing each gene).

was due to the effect of the antisense expression of MdTFL1
because the nontransgenic control ʻOrinʼ apples grafted onto
seedlings (control-1) or ʻJM8ʼ dwarﬁng rootstocks (control-2)
did not initiate ﬂowering for at least 5 years after the transfer tothe greenhouse (Table 2). The expression level of the transgene
was relatively high in lines 303 and 705, which have two copiesof the transgene, as compared to line 614, which has one copy
(Fig. 2, Fig. 6A). These results show that the expression levelof the transgene is correlated with the precocity of the transformants. As expected, the expression of endogenous MdTFL1 inthe shoots was suppressed in transgenic lines as compared to thenontransgenic control apple (Fig. 6B), indicating that MdTFL1
intrinsically represses the transition to ﬂowering and maintains
the juvenile phase in apple. The knockdown of the MdTFL1 geneby co-suppression or RNAi would induce much more precocity,
based on the ﬁnding that the endogenous MdTFL1 in the transformants was not suppressed completely by the antisense method.
Some defects in the ﬂower organs of transgenic lines, such as thelack of stamens and pistils or a decreased number of ﬂowers per
cluster, can be attributed to the underdevelopment of ﬂoral buds
but not directly to the function of MdTFL1 because such defects
or unusual solitary ﬂowers were rarely observed as they grew.
Most ﬂowers of the transgenic lines had functionally normal
reproductive organs, resulting in normal fruit that contained
several seeds (Fig. 5 G and H). In fact, it was found by crossing
tests that line 705 had both pollen and seed viability. The apple
cultivar Sansa was crossed by transgenic line 705, and thenseedlings from the crossing were obtained and the inheritanceof the transgene was preliminarily conﬁrmed by a PCR (datanot shown). These results show that the precocious line with a
35S::MdTFL1 antisense construct could be both a pollen andseed parent of apple in cross breeding. The precocious line had
79

a marked tendency to set parthenocarpic fruit [pf (Fig. 5I)], suggesting that the antisense expression of MdTFL1 may reinforce
parthenocarpy in ʻOrinʼ apple since the original ʻOrinʼ apple
occasionally produces parthenocarpic seedless fruit. Several
apple mutants, such as ʻNoblowʼ, ʻWellington Bloomlessʼ, andʻSpencer Seedlessʼ, produce apetalous ﬂowers that develop into
parthenocarpic seedless fruit (Stout, 1929). However, ﬂowers fromthe transgenic line or nontransgenic control ʻOrinʼ apple had four
normal ﬂoral organs (sepals, petals, stamens, and pistils) (Fig. 5
B and C), unlike the apetalous ﬂowers of those mutants.
Floral bud differentiation in transgenic line 705 occurred
several times over a period of at least 4 months, and ﬂowers were
produced sequentially during the period (Fig. 3C). In addition,line 705 produced ﬂoral buds and ﬂowers on the growing current
shoots (Fig. 5J). Shoots that produced ﬂowers ceased vegetative
growth, causing the tree to become less vigorous and to develop
shorter branches than the controls (Fig. 4 C and D). In contrast,
normal apple trees neither induce ﬂoral bud differentiation on
the growing vegetative shoot (Fig. 4B, water sprout) nor comeinto ﬂower even on reproductive shoots growing after anthesisin the current season; rather, they ﬂower naturally after breaking
dormancy the following spring season. Antisense expression of
MdTFL1 frequently shortened the period of ﬂower development,
possibly by abolishing the repression of ﬂower development bydormancy. The observation is consistent with the ever-ﬂowering characteristic of the line 705. These ﬁndings suggest that
MdTFL1 affects ﬂower development and plant architecture aswell as ﬂower induction in apple.
FACTORS CONTROLLING THE TRANSITION FROM THE JUVENILE
TO THE REPRODUCTIVE PHASE. At our institute, ﬂoral bud differentiation begins in early July; at this time, MdTFL1 mRNA isexpressed strongly in apices of current apple shoots (Kotoda andWada, 2005). The expression of MdTFL1, however, decreases
gradually until mid-December, paralleling the development of
ﬂower primordia. Previously, we reported that the expression of
the AFL1 gene was detected after the onset of ﬂoral bud differentiation and that MdAP1 mRNA appeared concurrent with sepal
formation (Kotoda et al., 2000; Kotoda and Wada, 2005; Wadaet al., 2002). AFL1 or AFL2 was up-regulated in the precocious
line 303 or 705, respectively, as compared to the nontransgenic
control, whereas MdAP1 was not expressed in those transgeniclines (Fig. 6C). Thus, the down-regulation of MdTFL1 might
trigger the up-regulation of AFL1 and/or AFL2 in apical buds,
resulting in ﬂower induction in apple, although the up-regulationof AFL1 or AFL2 was transient in the transgenic lines with a 35S::
MdTFL1 antisense construct. It is interesting that the juvenile
and/or vegetative phases can be regulated by only a TFL1-likegene even though their maintenance is one of the most important
events in the early stage of development in woody plants. Basedon these results, MdTFL1 must be one of the factors controlling
the transition from the juvenile/vegetative to the reproductive
phase in apple.
We produced several transgenic apple trees that ectopically
expressed genes possibly involved in ﬂowering, but there were
few transgenic apple trees that markedly induced precocious
ﬂowering other than those expressing MdTFL1 antisense RNA.
A decade ago, it was found that the juvenile phase of an aspen
hybrid of two poplar species (Populus tremula L. x P. tremuloides
Michx.) could be reduced using transgenic procedures by introducing arabidopsis LFY under the CaMV 35S promoter (Weigel
and Nilsson, 1995). Five-month-old shoots of transgenic aspen
hybrids grown in tissue culture produced solitary ﬂowers in the80

axils of normal leaves. However, Rottmann et al. (2000) reported
that multiple lines of poplar over-expressing either LFY or PTLF
(a poplar homologue of LFY) did not show the early ﬂowering
phenotype that was expected. In fruit crops, transgenic citrus[C. sinensis x Poncirus trifoliata (L.) Raf.] expressing LFY or
AP1 initiated ﬂowering 12–20 months after the transfer to thegreenhouse, resulting in normal, fertile ﬂowers that set fruit andseeds, despite the observation that regenerated nontransformed
plants initiated ﬂowering only after 5 years of growth in thegreenhouse (Peña et al., 2001). However, it is not clear whether
there is a common mechanism of ﬂower induction in citrus andapple because citrus, an evergreen fruit tree, is very different
from apple, a deciduous fruit tree, in terms of growth and fruiting
habits. In fact, unlike that of citrus, the constitutive expressionof MdAP1 or AFL1 in apple does not cause precocious ﬂowering
except for the single instance of apple that expresses arabidopsis
AP1 (N. Kotoda, unpublished data). Therefore, the competenceof apple to respond to the constitutive expression of AP1- or
LFY-like genes might be lower than that of citrus. Together
with the fact that only a few instances of precocious ﬂowering
in deciduous woody plants by over-expression of LFY or AP1
have been reported, LFY and AP1 may not be effective factors forbreaking the juvenile and/or vegetative phases of deciduous fruit
trees such as apple. Like TFL1, CEN, SP, and FT, the putative
protein encoded by the MdTFL1-shared sequence is similar to themammalian phosphatidylethanolamine-binding proteins (PEBP),
originally named for their ability to bind phospholipids in vitro(Schoentgen and Jolles, 1995). In mammals, PEBP is an inhibitorof Raf-1 kinase activity (Yeung et al., 1999). Several years ago,the crystal structure of CEN was determined, suggesting that thebiological effects of CEN arise from its ability to form complexes
with phosphorylated ligands (Banﬁeld and Brady, 2000). In the
Raf-1 system, RKIP, an SP homologue protein from mammals,
binds to Raf1 as tomato SP binds to SPAK (SP-associated kinase)in the SP system (Pnueli et al., 2001; Yeung et al., 1999). MdTFL1
may also function by interacting with proteins like SPAK or byother factors involved in ﬂowering.
In citrus, the over-expression of CiFT, a mandarin orange(Citrus unshiu Marc.) homologue of FT, induced precocious
ﬂowering 3–22 months after the transfer of the transgenic plants
to the greenhouse, which indicates that CiFT has a key role in
the regulation of ﬂower induction (T. Endo, in preparation). FT
is a member of the CETS family and has conserved motifs that
contribute to the conformation of the ligand-binding site, but its
function is antagonistic to TFL1. Studies of factors that interact
with CETS genes are needed to clarify the mechanism that underlies the transition to ﬂowering in woody plants.
Conclusion
For the ﬁrst time in the study of deciduous fruit trees, wehave shown how effectively the antisense expression of MdTFL1
induces the precocious ﬂowering of apple. In addition, we haveshown that MdTFL1 has a key role in the regulation of juvenility, ﬂower induction, and ﬂower development in apple. The
reduction of the generation time by the use of this transgenic
approach to suppress endogenous TFL1-like genes may be
applicable not only to fruit trees but also to the other woody
plants that have a long juvenile period. The future use of thesetechniques should be advantageous in breeding, crop production,and basic research, e.g., molecular studies on woody plants,
including fruit trees.
J. AMER. SOC. HORT. SCI. 131(1):74–81. 2006.

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