Plant Science 162 (2002) 679 /687
www.elsevier.com/locate/plantsci

Overexpression of MdMADS5, an APETALA1-like gene of apple,
causes early flowering in transgenic Arabidopsis
Nobuhiro Kotoda a,*, Masato Wada a, Shinnosuke Kusaba b, Yuriko KanoMurakami b, Tetsuo Masuda a, Junichi Soejima aaDepartment of Apple Research, National Institute of Fruit Tree Science, National Agricultural Research Organization, Shimo-kuriyagawa, Morioka
020-0123, Japanb
National Institute of Fruit Tree Science, National Agricultural Research Organization, Tsukuba 305-8605, Japan
Received 10 August 2001; received in revised form 28 December 2001; accepted 2 January 2002

Abstract
An apple (Malus /domestica ) gene MdMADS5 , a putative homolog of Arabidopsis APETALA1 (AP1 ) and Antirrhinum
SQUAMOSA , was introduced into Arabidopsis using Agrobacterium tumefaciens EHA101 to examine how the gene affects
flowering and whether it may be effective in producing transgenic apple that flowers early. Five of the 15 primarily transformed (T1)plants flowered 5 /10 days earlier than wild-type plants and produced only two to three rosette leaves when they flowered. The
terminal flowers on the transgenic plants resembled transgenic Arabidopsis expressing 35S::AP1 or tfl1 mutant plants. Thisphenotype observed in T1 transgenic plants was inherited in the following generation. Based on these results, it was suggested thatthe function of the MdMADS5 gene was similar to that of the AP1 gene. Although the mechanism of flower-bud formation in theapple might be different from that in Arabidopsis , the MdMADS5 gene might be involved in flower-bud formation of the apple.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: APETALA1 ; Arabidopsis thaliana ; Early flowering; MADS-box; Malus /domestica

1. Introduction
Arabidopsis thaliana has been widely used in studies
on basic plant physiology and molecular biology
because of its small genome, short regeneration time,
self-compatibility, and ease in efficient regeneration andtransformation procedures. The most striking advancesin our understanding of the genetic control of the timingof flowering have come from work on Arabidopsis [1 /3].
Genes that control flowering time have been isolated
and they include LEAFY (LFY ) [4], APETALA1 (AP1 )[5], CAULIFLOWER [6], and TERMINAL FLOWER
1 (TFL1) [7 /9] in Arabidopsis . Overexpression of LFY[10] and AP1 [11] shortened the juvenile period andcaused early flowering, whereas overexpression of TFL1
delayed flowering [12].

* Corresponding author. Tel.: 81-19-641-3164; fax: 81-19-6413819.
E-mail address: koto@affrc.go.jp (N. Kotoda).

Genetic studies on morphogenesis in apple havestarted now that these genes related to plant development, including flower initiation, have been isolated in
Arabidopsis . In apple, MdMADS1 [13] which has a
MADS-box domain [14] was isolated based on work in
Arabidopsis . As for genes homologous to AP1 ,MdMADS2 [15] and MdMADS5 [16,17] were cloned
and characterized. Overexpression of MdMADS2 wasfound to cause early flowering in tobacco (Nicotiana
tabacum ) [15]. In our previous study, we cloned afragment of MdMADS5 and analyzed it to determine
the relationship between floral bud formation and its
expression. In apple, MdMADS5 was expressed in
sepals 2 months after flower-bud differentiation [17].
Recently, Yao et al. [18] cloned the apple MdP1
homologous to PISTILLATA (PI) [19] and identified
that MdPI has been mutated by a retrotransposon
insertion in apetalous mutants such as ‘Rae Ime’ and‘Spencer Seedless’. Although several apple genes including MADS-box genes have been isolated, few of them0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 0 2 4 - 9

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N. Kotoda et al. / Plant Science 162 (2002) 679 /687

are focused on flower induction and how they functionin plants.
In this paper, we describe the effect of the apple
MdMADS5 gene, a putative homolog of AP1 , onflowering time in transgenic Arabidopsis and discuss
the possibility of involvement of MdMADS5 on flower
development in apple.

2. Materials and methods
2.1. Plant materials
The apple (Malus /domestica var. Jonathan) andArabidopsis ecotype Columbia (Col) were used in thisstudy. Apple leaves and flowers were collected from theexperimental field at our research center in Morioka,
Japan.
2.2. Extraction of DNA from apple
DNA was isolated from the ‘Jonathan’ apple bycetyltrimethylammonium bromide (CTAB)-based methods [20]. Approximately 3 g of young leaves were frozen
in liquid nitrogen and ground into fine powder. The
powder was mixed with 20 ml of isolation buffer (IB)(10% polyethylene glycol 6000, 0.35 M Sorbitol, 0.1 M
Tris /HCl pH 7.5, and 1% 2-mercaptoethanol) in a 50ml centrifugation tube on ice for 2 min. The tube wascentrifuged at 12 000gn for 5 min at 25 8C in a swing
bucket rotor KS-5000P (Kubota Co., Tokyo, Japan).
The pellet was then resuspended in 9 ml of lysis buffer(LB) (0.35 M Sorbitol, 0.1 M Tris /HCl pH 7.5, and 1%
2-mercaptoethanol), and 1 ml of 10% sarcosine wasadded. The solution was mixed gently (about 50 rpm) at
25 8C for 10 min using a hybridization oven (Iwaki
Glass Co., Ltd, Tokyo, Japan), and 10 ml of 2 /CTAB(2% CTAB, 0.1 M Tris /HCl pH 9.5, 20 mM EDTA, 1.4
M NaCl, and 1% 2-mercaptoethanol) was added to it,followed by gentle mixing at 56 8C for 20 min. The
solution was extracted twice with chloroform/isoamyl
alcohol [24:1 (v/v)], and DNA in the aqueous phase was
precipitated with an equal volume of 2-propanol at
25 8C. Nucleic acids were collected with an inoculationloop, then redissolved in 5 ml of 1 M NaCl containing
RNase at a concentration of 10 mg ml 1 in a 15 ml
tube, and incubated at 56 8C for 2/3 h. The DNA was
precipitated with 10 ml of ethanol and washed twicewith 2 ml of 70% ethanol for 5 min. After air drying, thepellet was dissolved in TE (10 mM Tris /HCl pH 8.0,
and 1 mM EDTA) and stored at 4 8C.
2.3. DNA blotting analysis
Ten micrograms of genomic DNA was digested withrestriction enzymes, Bam HI, Eco RI, and Hin dIII andelectorophoresed on 0.8% agarose gel (FMC Bioproducts, Rockland, ME, USA). The gel was blotted onto
Hybond N  (Amersham, Buckinghamshire, UK) andhybridized with a digoxigenin (DIG)-labeled PCR
probe. A 567-bp fragment of MdMADS5 cDNA
amplified between squa/ap1-1S and -5A primers wasused as a template of PCR probes for DNA gel blot
analysis (Fig. 1A and Table 1). Hybridization was
performed at 65 8C in a 0.5 M Na /Pi buffer (0.5 M
Na2HPO4, pH 7.2), 7% SDS, and 1 mM EDTA for 16 h.
2.4. Gene cloning of MdMADS5 and vector construction
for the expression of the MdMADS5 gene
Since MdMADS5 was expressed exclusively in sepals[17], an apple cDNA library from sepals was used as
template to clone a full length MdMADS5 cDNA by
TaKaRa LA PCRTM in vitro cloning kit (Takara
Biomedicals, Tokyo, Japan). After the cDNA synthesis,
an Eco RI cassette was ligated to the cDNA. Then, 5?
rapid amplification of cDNA ends (RACE)- and 3?
RACE-PCR were performed using a set of adaptor
primers and two antisense primers, and a set of adaptor
primers and two sense primers, respectively (Fig. 1A and
Table 1). Because the fragment of AP1 -like gene wecloned was found to be identical to MdMADS5 [16], thecorresponding 1.0-kb cDNA was amplified by using
long and accurate (LA)-PCR [21]. Specific sense primer
1S, derived from information of gene bank dataregistered by Yao et al. [16] and a specific antisense
primer 7A, derived from a 600-bp fragment amplified by
3? RACE-PCR were used (Table 1). The PCR products
were cloned into a t-tailed SmaI site of pUC119 vectors(Takara Biomedical). The sequences of several clones ofinserts in pUC119 were analyzed by dideoxy methods
using a Thermo Sequenase pre-mixed cycle sequence kit
according to the manufacturer’s instructions (Amersham, Buckinghamshire, UK) and universal primers in
the pUC119 vectors on a Hitachi SQ5500 automatedsequencer (Hitachi, Tokyo, Japan).
The pUC119 plasmid vector, pUMDAP1.2/, containing MdMADS5 cDNA, was digested with Xba I and
Sac I, ligated to the binary vector pSMAK 251 [22] in asense-oriented manner under the CaMV 35S promoter,resulting in pSMDAP1.1/ (Fig. 1B). It was thenintroduced into a disarmed strain of Agrobacterium
tumefaciens , EHA101 [23]. Selectable marker genes
encode kanamycin resistance for plants and spectinomycin for bacteria.
2.5. Arabidopsis transformation
An A. tumefaciens strain EHA101 was used to transform Arabidopsis Columbia plants by a floral-dip
method [24]. To select transformed plants, the sterilized
seeds were suspended in 0.1% sterile agarose, plated on

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681

Fig. 1. Sequence comparison of MdMADS5 and vector construction. (A) Comparison of the MdMADS5 sequence with its homolog AP1 [5] inArabidopsis and SQUA [29] in Antirrhinum . Amino acids at positions of identity between two or three of these proteins are blocked in black. Arrows
above sequences indicate primer sites. (B) Representation of the transformation vector pSMDAP1.1. The MdMADS5 cDNA was inserted in senseorientation between the Xba I and Sac I sites of the binary vector pSMAK251, giving rise to pSMDAP1.1. pNos, Nos promoter; pAnos, the 3?
region of nos ; p35S, cauliflower mosaic virus 35S promoter; pArbcS, the 3? region of rbcS ; NPTII, neophosphotransferase; LB, left border; RB, rightborder; Sa, Sal I; B, Bam HI; H, Hin dIII; X, Xba I; Sm, Sma I; Sc, Sac I; E, Eco RI; Se, Spe I; sta, region involved in plasmid stability; rep, essential
region for plasmid maintenance.

kanamycin selection plates, and transferred to a growthchamber BIOTRON (Nippon Medical and Chemical
Instruments Co., Ltd, Tokyo, Japan) set at 22 8C underlong day conditions (16 h light/8 h dark). The selection
plates contained one half strength of Murashige andSkoog (MS) medium (Wako Pure Chemicals Co., Ltd,Tokyo, Japan), 0.8% agar (Difco Laboratories, Detroit,
MI, USA), 25 mg ml1 kanamycin monosulfate (Meiji
Seika Kaisha Ltd, Tokyo, Japan) and 50 mg ml 1
claforan (Pharmacia and Upjohn Ltd., Tokyo, Japan).
Transformants were identified as kanamycin-resistantseedlings that produced green leaves and well-estab-lished roots in the medium. Transformants were transplanted into moistened potting soil consisting ofvermiculite and perlite [1:1 (v/v)] after they developed
2 /5 adult leaves. The day of sowing was counted as day
0. Morphological analyses were performed in the
primary (T1) and subsequent generations.
2.6. RNA analysis by RT-PCR
Detection of MdMADS5 transcripts by RT-PCR was
performed by using the RT-PCR kit (Toyobo, Tokyo,
Japan). As an internal control, the actin AAc1 gene [25]

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682
Table 1
Primers used for PCR cloning of MdMADS5
PrimerDegenerate primers
squa/ap1-1S sense primer
squa/ap1-5A antisense primer
Cassette primers
C1 primer

C2 primer

5? RACE primers
R1A antisense primer
R2A antisense primer
3? RACE primers
R1S sense primer
R2S sense primer
MdMADS5 specific primers
1S sense primer
7A antisense primer

Oligonucleotide

5?-AAA/GGGIAAA/GT/CTITTT/CGAA/GTA-3?
5?-GCIGTA/GTCIAA/GT/
CTGT/CTC-3?
5?-GTACATATTGTCGTTAGAACGCGTAATACGACTCA-3?
5?-CGTTAGACGCGTAATACGACTCACTATAGGAGA-3?
5?-AAGCAGGTCAAGGCCATGGTTCTG-3?
5?-TTAGACACATGGAAGTGGCTGTGG-3?
5?-CCTGGATTCCTTGACTCTCAAGG-3?
5?-TTTGGAGCAACAGCTTGACACCG-3?
5?-CAGTTTCTGGGTTGTCTTTC-3?
5?-ACTCAATATTTCTCAGGTTT-3?

was employed. RNA was isolated from whole plants ofArabidopsis by a method using CTAB described previously [17]. MdMADS5 - and AAc1 -specific transcripts
were identified using 1 mg of total RNA as a template
and the following primers: a sense primer 1S and an
antisense primer 7A for MdMADS5 (Table 1), and asense primer Aac1-S: 5?-GTG CTC GAC TCT GGA
GAT GGT GTG-3? and an antisense primer Aac1-AS:
5?-CGG CGA TTC CAG GGA ACA TTG TGG-3? for
Aacl, giving rise to a 1037- and 457-bp long PCR
product, respectively. PCR reactions were run for 40
cycles at 50 8C for MdMADS5 and at 58 8C for AAc1 .
The PCR products were run on 1.5% (w/v) agarose gelsstained with ethidium bromide.

Columbia plants carrying the cDNA of MdMADS5
fused to the CaMV 35S promoter. By floral-dip method,
15 independent transgenic plants that survived onkanamycin were identified (Table 2). Five of the 15
primarily transformed plants (T1 generation) carrying
35S::MdMADS5 flowered earlier than the wild-type
plants by 5/10 days and produced only two to threerosette leaves when they flowered. The phenotype inlines 1 and 2 was so severe that it was difficult to obtain
their seeds. The terminal flowers on the transgenic
plants resemble those of tfl1 mutant plants [9]. Similar
results were obtained in T1 transgenic Arabidopsis with
35S::AP1 (data not shown). This phenotype observed in
T1 transgenic plants was inherited in the following
generation as a dominant Mendelian trait and cosegregated with kanamycin resistance. All transgenic
plants with early flowering contained a kanamycin
resistance gene. T2 Transgenic line No. 12-1 showed
early flowering although no differences in appearance
were seen between T1 transgenic line No. 12 and thewild-type plants. Quantitative characteristics of fiveindependent transgenic lines (T3 generation) are shownin Table 3. Typical 35S::MdMADS5 transformants (T4seedlings from the transgenic line No. 3-1-7) thatflowered 17 days after sowing are shown in Fig. 3A
and B. The wild-type plant had eight rosette leaves andno flower buds on day 20 (Fig. 3C). The inflorescence oftransformants with early flowering was shorter ( B/3 cm)
and numbers of rosette leaves were extremely reduced(2 /5) at flowering as compared to controls (Fig. 3B and
Table 3). In wild-type Arabidopsis , flower consists offour floral organs (four sepals, four petals, six stamens,

3. Results
DNA blotting was performed using apple DNA
digested with Bam HI, Eco RI, and Hin dIII probedwith a 567-bp long MdMADS5 cDNA fragment. Twobands for Bam HI and Eco RI, and four bands for
Hin dIII were detected in DNA blotting analysis,suggesting that there might be a gene similar to
MdMADS5 (Fig. 2).
In order to examine whether MdMADS5 functions
like AP1 , we produced transgenic Arabidopsis ecotype

Fig. 2. DNA blot analysis. Equal amounts (10 mg) of apple DNA were
blotted onto a Hybond N  filter. Restriction enzymes (Bam HI,
Eco RI, and Hin dIII, from left to right) used to digest apple genomic
DNA are indicated at the top. Hybridization was done with a DIGlabeled PCR probe lacking the MADS-box region of MdMADS5 . The
numbers to the right represent the size of the DNA in Kb.

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683

Table 2Comparison of flowering time, number of rosette leaves in T1 transgenic and wild-type Arabidopsis (Col) plants in LD conditions
Transgenic line

Days to floweringa

Rosette leaves at time of floweringb

Note

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
wtc

18
16
16
20
20
28
28
28
22
28
24
24
30
28
30
25.5d

3
2
3
2
2
8
7
8
8
8
9
10
7
8
6
9.5d

Early flowering
Early flowering
Early flowering
Early flowering
Early flowering(n 10)eaDays to flowering is defined as the time when flower primordia were first visible to the naked eye.
Rosette leaves were counted on the day that flower primordia were first visible.
c
wt  wild-type Arabidopsis columbia plants.
dMean number.
eNumber of plants.
band a pistil) (Fig. 3D, E). However, in transgenic lines
with a severe phenotype, abnormal floral organs wereobserved (Fig. 3F, G) and some were less fertile.
The time of flowering is regulated by endogenous
signals and environmental conditions such as daylength. As A. thaliana is a quantitative long-day (LD)plant, LD photoperiods accelerate flowering, althoughplants also flower under short-day (SD) after extended
periods of vegetative development. To study the effect of
day-length on flowering in transgenic plants with
35S::MdMADS5 , T3 transgenic Arabidopsis plants
were grown under both LD (16 h light/8 h dark) andSD (8 h light/16 h dark) photoperiods (Table 3). Ten to
15 seeds from each T2 transgenic line (Nos. 2-3, 3-1, 4-1,
5-3, and 12-1) derived from T1 lines (Nos. 2, 3, 4, 5, and12, respectively) were used in this study. With LD
photoperiods, seedlings from lines Nos. 2-3, 3-1, and12-1 showed severe phenotypes, and those from lines
Nos. 4-1 and 5-3 showed milder ones. Most plants fromseveral lines with a strong phenotype, such as line No. 23, died as a result of poor growth from the effects of thetransgene, resulting in few seeds being obtained fromthem. In SD photoperiods, on the other hand, alltransformants flowered earlier than the wild-type plants
as they did in LD photoperiods. Seedlings from line No.
2-3 showed an especially severe phenotype of alltransgenic lines with little difference in flowering time
and the number of rosette leaves compared to thecorresponding seedlings under LD photoperiods. In
this transgenic line, flowering time and amount ofvegetative development did not seem to depend on
day-length. The other transgenic lines were delayed inflowering under SD photoperiods compared to thosegrown in LD photoperiods, but they flowered 4/6
weeks earlier than the wild-type plants that required
about 9 weeks to flower.
The expression of the MdMADS5 mRNA in T1
plants was confirmed by RT-PCR (Fig. 4). All putativetransformed lines showed expected RT-PCR products of
1037-bp for MdMADS5 . No amplification was observed for the cDNA prepared from non-transformed
Arabidopsis plants (Fig. 4, wt). The expression of
MdMADS5 mRNA was detected in all of the kanamycin-resistant plants tested for expression analysis. The
MdMADS5 mRNA was expressed relatively weakly infive lines that flowered early (Fig. 4, lanes 1 /5),compared to other lines. It was detected only weakly
in the line No. 5 plant, although the line No. 5 plant
flowered earlier and produced far fewer rosette leaves
than the wild-type plants.

4. Discussion
We have produced transgenic Arabidopsis plants
expressing MdMADS5 . They flowered earlier and hada shorter inflorescence and reduced number of rosetteleaves compared to the controls (Fig. 3 and Table 3).
The MdMADS5 gene apparently caused early floweringin Arabidopsis , and our results showed that the
MdMADS5 gene might have a similar function to thatof AP1 . In our previous study, we showed that the
MdMADS5 gene was expressed in sepals and that it was
first expressed 2 /3 months after floral-bud differentia-

684Line LD conditions (16 h light/8 h dark)

2-3
3-1
4-1
5-3
12-1
wt
a

SD conditions (8 h light/16 h dark)Days to floweringa

Rosette leaves at time offloweringb

Length of inflorescence(cm)cNumber ofplantsDays to floweringa

Rosette leaves at time offloweringb

Length of inflorescence(cm)cNumber ofplants

17.0
16.691.2
21.890.8
20.894.2
17.592.7
23.690.5

2.0
2.090
3.090.7
4.590.5
2.190.3
7.390.5

1.9
2.290.9
6.793.6
4.392.0
2.491.5
12.691.7

1
9
4
4
8
7

20.892.4
30.393.4
35.097.3
27.998.5
24.192.0
63.691.5

2.090
5.791.4
6.091.9
5.293.5
4.992.0
18.193.9

1.790.5
3.592.1
3.193.2
4.193.2
7.092.3

4
7
4
11
7
9

Days to flowering is defined as the time when flower primordia were first visible to the naked eye. Values are mean9SEM.
Rosette leaves were counted on the day that flower primordia were first visible. Values are mean9SEM.
c
Length of inflorescence was measured a month after sowing. Values are mean9SEM.
b

N. Kotoda et al. / Plant Science 162 (2002) 679 /687

Table 3Comparison of flowering time, number of rosette leaves in T3 transgenic and wild-type Arabidopsis (Col) plants grown in LD and SD conditions

N. Kotoda et al. / Plant Science 162 (2002) 679 /687

685

Fig. 3. Transgenic and wild-type Arabidopsis plants grown under LD photoperiods for 20 days. Arabidopsis plants were grown in one-half strengthof MS medium for 10 days and transferred to potting soil. (A) Transgenic Arabidopsis carrying a 35S::MdMADS5 gene (left) and wild-type
Arabidopsis Columbia (right). Arrows indicate terminal flowers. (B) Whole plants of transgenic (left) and wild-type (right) Arabidopsis . (C) Rosetteleaves of wild-type Arabidopsis . (D) Inflorescence of wild-type Arabidopsis . (E) Close-up view of a flower of wild-type plant. (F) Inflorescence oftransgenic Arabidopsis . (G) Close-up view of a terminal flower of the transformant. Scale bar 10 mm (B), 3 mm (C) and 2 mm (D, E, F, G). Photos
were taken 20 (A, B, C, F, G) and 30 days (D, E) after sowing.

tion concurrent with sepal formation of apple [17].
Therefore, it was suggested that the MdMADS5 geneis involved in flower development after floral-bud
differentiation, although the mechanism of flower-bud
formation in apple might be different from that in
Arabidopsis . To be more certain that the MdMADS5
gene functions similarly to AP1 , ap1 mutant-rescue
experiments will be required. Based on the result thattransgenic Arabidopsis with 35S::MdMADS flowered
earlier than the controls under both LD and SD
photoperiods, the MdMADS5 gene does not seem tohave an effect on a photoperiodic pathway in Arabidopsis. The MdMADS5 gene was not expressed strongly
in transformants showing early flowering relative tothose that were wild type in appearance. The result

suggests that the severity of phenotype in transformants
does not depend solely on the level of gene expression inthis case, although an exact answer to the cause was not
obtained.
The breeding of fruit trees such as apple (Malus /
domestica ) often requires more than 20 years, including
periods of cross pollination, seedling selection, andregional trials, to produce varieties that meet thedemands of consumers. For example, the ‘Fuji’ apple,a leading cultivar in Japan, Korea, and China, took 23
years before it was released [26]. Thus, one of factorsthat limits breeding strategies in tree fruits is the long
juvenile phase that lasts several years [27]. Several years
ago, it was found that transgenic approaches could
reduce the juvenile phase of Populus by introducing

Fig. 4. Analysis of MdMADS5 expression in transgenic Arabidopsis . Detection of MdMADS5 was performed by RT-PCR. MdMADS5 was
identified using 1 mg of the total RNA as a template and the specific primers, giving rise to a PCR product of 1037 bp. PCR products were run on
1.5% (w/v) agarose gel stained with ethidium bromide. The numbers above the lanes indicate the transgenic line No. Plants of lines Nos. 7 and 8 were
not examined. As an internal control, RT-PCR on the actin AAc1 gene was employed. wt, the wild-type Arabidopsis Columbia plant.

686

N. Kotoda et al. / Plant Science 162 (2002) 679 /687

LFY under the CaMV 35S promoter and the transgenic
poplar flowered 5 months after regeneration [10]. Therefore, these techniques could be applicable to fruit trees
in future years. We believe that the transgenic approach
would be a useful breeding strategy for reducing thetime required for generation among woody plants. In
addition to this study on MdMADS5 , we confirmedthat both AFL1 and AFL2, twin apple homologs of
LFY , also cause early flowering in Arabidopsis (Wada etal., unpublished results). Recently, Peña et al. [28]reported that citrus expressing LFY or AP1 were early
flowering and they were fertile. However, the efficiency
of producing early flowering transgenic lines with
Arabidopsis LFY or AP1 genes seems to be low inwoody plants mentioned above. This suggests that
regulatory genes such as LFY and AP1 do not always
function beyond species as well as expected. Based onthese results, we are producing transgenic apples expressing endogenous apple genes, AFL or MdMADS5 ,for early flowering. Since environmental and genetic
factors controlling flower development in apple have notbeen made clear so far, the MdMADS5 gene could, atleast, be one of the tools available for studying themechanism of flower development in tree fruits such as
the apple.

Acknowledgements
We gratefully acknowledge Dr H. Ichikawa, National
Institute of Agrobiological Resources, Tsukuba, Japan,
and Professor M. Yanofsky, the University of California, San Diego, for providing us a binary vector
pSMAK251 and pAM563 (35S::AP1 construct), respectively. We also thank Dr C. Honda, National Instituteof Fruit Tree Science, Tsukuba, Japan, for technicaladvice. This research was supported by a grant from the
Ministry of Agriculture, Forestry, and Fisheries (MAFF) for the ‘development of innovative plants and animals using transformation and cloning’. Contribution
No. 1244 of National Institute of Fruit Tree Science.

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