The Break-through in the Reduction of Juvenile Phase in Apple
Using Transgenic Approaches
N. Kotoda, M. Wada, T. Masuda and J. Soejima
Department of Apple Research, National Institute of Fruit Tree Science, National
Agricultural Research Organization, Shimo-kuriyagawa, Morioka 020-0123, Japan
Keywords: Malus x domestica, juvenility, early flowering, flower induction
Abstract
In contrast to herbaceous plants, fruit trees such as apple [Malus x domestica(Borkh.)] flower and set fruit only after an extended juvenile phase that lasts several
years. In the course of studying juvenility in apple, we have cloned the MdTFL gene
homologous to Arabidopsis TERMINAL FLOWER1 (TFL1) gene, which suppresses
floral meristem identity genes, LEAFY (LFY) and APETALA1 (AP1). The expressionof MdTFL (Malus x domestica TFL1) mRNA was strong in sepals and mature leaves,and highest in early July, about two weeks before flower induction, in apical buds inapple. Transgenic apples expressing antisense MdTFL gene flowered at about eight to
15 months after grafting onto rootstocks. On the other hand, non-transformed
control plants have not flowered in five years. The flower organs of the transgenic
apples were normal and fertile. These results demonstrate that the suppression of theexpression of MdTFL gene reduces the juvenile phase and can induce precocious
flowers in apple.
INTRODUCTION
In the development of all woody plants from seed, there is a so-called juvenile
phase lasting up to 30-40 years in certain forest trees, during which flowering does notoccur and can not be induced by normal conditions (Hackett, 1985). Therefore, thebreeding of fruit trees such as apple (Malus x domestica) often requires more than 20 years,
including the periods of cross pollination, seedling selection and regional trials to produce
varieties that meet the demands of consumers. For example, the ‘Fuji’ apple, a leading
cultivar in Japan, Korea, and China, set the first fruits 12 years after sowing, and took 23
years before it was released as a variety (Sadamori et al., 1963). Generally, the juvenile
period in apple (Malus x domestica) can last four to eight years, but a certain Malus sp.
used as root stock neither flowers nor sets fruits for many years. Thus, the greatest factor
that limits breeding efficiency of fruit trees is their long juvenile phase. It is important tounderstand the mechanism of flower induction and development, and apply it to theimprovement and production of the fruit, because apple is one of the most commercially
important tree fruits in the world. However, the genetic factors controlling flower induction
in apples have not been investigated in detail. To investigate the possible factor responsible
for the maintenance of juvenility in apple, we have cloned the AFL, MdAP1(MdMADS5),
and MdTFL genes, putative apple homologs of LFY (Weigel et al., 1992, 1995), AP1(Mandel et al., 1992, 1995), and TFL1 (Bradley et al., 1997; Oshima et al., 1997),
respectively (Kotoda et al., 2000, 2002; Wada et al., 2002; Yao et al., 1999). In addition, we
produced transgenic apples expressing sense or antisense of these genes for early flowering.
In this paper, we describe isolation and characterization of MdTFL, and precocious
flowering of apple by expressing antisense MdTFL.
MATERIALS AND METHODS
Plant Materials
The apple [Malus x domestica (Borkh.)] cultivar, Jonathan, was used in this study toisolate and characterize the MdTFL gene. The apple cultivar Orin, and Arabidopsis
thaliana (ecotype Columbia) were used for Agrobacterium-mediated transformation.
Proc. XXVI IHC – Biotechnology in Hort. Crop Improvement
Eds. F.A. Hammerschlag and P. Saxena
Acta Hort. 625, ISHS 2003
Publication supported by Can. Int. Dev. Agency (CIDA)

337

Apple leaves and flowers were collected from the experimental field or greenhouses at our
research center in Morioka, Japan.
Construction of Transformation Vectors
The plasmid vector pUMDTFL12.1+ containing MdTFL cDNA was cut with Xba1and SacI, and the MdTFL cDNA was ligated to a binary vector pSMAK251 (Yamashita etal., 1995), cut with the same restriction enzymes, in a sense oriented manner to yield
pSMDTFL12.1.2+. The plasmid vector pUMDTFL1- was cut with Xba1 and SacI, andligated to a binary vector pSMAK251 in an antisense oriented manner to yield
pSMDTFL1.1-.
Transformation of AppleAn Agrobacterium strain EHA101 containing pSMDTFL1.1- was used fortransformation of apple leaves. The MdTFL genes were controlled by the CaMV 35S
promoter. Agrobacterium was grown overnight and induced by acetosyringone. Apple
leaves were inoculated with 0.5 OD inoculum for 30 min. The leaf explants weretransferred to a co-cultivation medium and incubated in the dark. After 1 week, the cultures
were transferred to antibiotic selection plates containing Murashige and Skoog (MS)medium, 25 mg L-1 kanamycin, and 500 mg L-1 claforan. When adventitious shootsappeared, cultures were transferred to antibiotic selection plates containing MS, 50 mg L-1
kanamycin, and 500 mg L-1 claforan and incubated in the light. When shoots had produced
several leaves, they were transferred to proliferation medium. After checking the transgene
insertion by PCR using specific primers for MdTFL, shoots were transferred to MS
medium free from antibiotics.
RNA Blot Analysis
RNA was isolated from ‘Jonathan’ apple at various developmental stages. RNA wasseparated on a 1.2% agarose gel containing 5.0% (v/v) formaldehyde. Total RNA (20 µg)
was loaded per lane. The gels were blotted onto Hybond-N+ (Amersham). Hybridization
was done with DIG-labelled sense and antisense RNA probes, which were synthesizedfrom pBMDTFL12+ cut with EcoRI and SalI by using T7 and T3 RNA polymerases,
respectively. Hybridization and washing were performed according to a standard protocol(Boehringer Mannheim). The resulting images were analyzed by a LAS-1000 image
analyzer (Fujifilm).
RESULTS
Gene Cloning and Expression Pattern of MdTFL
To investigate the genes possibly related to juvenility in apple, we have cloned the
MdTFL gene, a putative homologue of Arabidopsis TFL1, by RACE methods. The primers
used in cloning are shown in Fig. 1A. The predicted protein of the MdTFL with 172
amino-acid residues had a greater similarity to TFL1 rather than to CEN: 74% amino-acidresidues were shared with TFL1, 72% with CEN (Bradley et al., 1996), 72% with SP(Pnueli et al., 1998), and 56% with FLOWERING LOCUS T (FT) (Kardilsky et al., 1999;
Kobayashi et al., 1999) which acts in an antagonistic manner to TFL1. Therefore, this gene
was designated MdTFL after Malus x domestica TFL1 homolog. DNA blotting was
performed using apple DNA digested with BamHI, EcoRI, HindIII, NcoI, XbaI, and XhoI
probed with MdTFL. Two to four bands were detected in DNA blotting analysis, suggestingthat there is an unknown gene homologous to MdTFL. In fact, it is reported that
Arabidopsis genome contains six TFL1-like genes, forming a gene family (Mimida et al.,2001). The coding region of MdTFL cDNA was used to analyze the expression of MdTFL
both in various living tissues and in the apices of current shoots in apple at different
developmental stages by generating an antisense digoxigenin (DIG)-labeled RNA probe.
The MdTFL mRNA was expressed preferentially in sepals and mature leaves, but very
weakly in petals, apical buds and cotyledons of seedlings. There was no expression in338

stamens, carpels, stems and roots. MdTFL mRNA from the apices of apple was looked atthroughout the season. MdTFL mRNA was expressed strongly in early July, about two
weeks before the occurrence of flower induction in apple. It decreased gradually from Julyto mid-December, with an increase to April concurrent with sepal and leaf development.
Interestingly, the seasonal expression pattern of MdTFL in the apices of apple appeared to
be opposite to that of AFL gene (Kotoda et al., 2000).
Transgenic Arabidopsis with MdTFL
To determine whether MdTFL gene functions as TFL1, we constructed a binary
vector pSMDTFL12.1.2+ or pSMDTFL1.1- where full-length MdTFL cDNA was inserted
in sense or antisense oriented manner, respectively, under the control of 35S CaMV
promoter. The vector carrying MdTFL cDNA (pSMDTFL12.1.2+) was introduced into
wild type Arabidopsis plants (Columbia). Thirty-four independent transgenic plants thatsurvived on kanamycin were identified. Eight of thirty-four primary transformants (T1generation) flowered later than wild-type plants and showed the similar phenotype to thatof transgenic Arabidopsis overexpressing TFL1(Ratcliffe et al., 1998). The phenotype
observed in the T1 generation was inherited in the following generations. Quantitative
characteristics of six independent T2 transgenic lines are shown in Table 1. In the T2 plants,
the first flower opened 39.4 days after sowing and the latest flower opened at day 59,
compared to 30.3 for the control plants (Table 1). Rosette leaf number, a measure ofdevelopmental time to flowering, was higher in transgenic plants and was as many as 23.0
for the seedlings from T-S22 as compared to 8.0 for the controls. The transformants had
fifteen to twenty rosette leaves and neither flower buds nor bolting were seen, whereas thecontrol plant had many flowers and brown pods at day 35. In addition, there was little
difference between Arabidopsis plants transformed with a binary vector pSMDTFL1.1carrying MdTFL antisense gene and control wild-type plants in flowering time and rosette
numbers at flowering (data not shown). These results suggest that MdTFL functions like
TFL1 and it is an apple ortholog of TFL1.
Expression of MdTFL in Transgenic Apples
Based on these results described above, MdTFL was thought to function as thedevelopmental switch between the vegetative and reproductive phase in apple like TFL1 in
Arabidopsis. If this is the case, there is a possibility that the suppression of the MdTFL genewould trigger flower induction during the vegetative phase of apple. To investigate thisspeculation, we produced transgenic apples overexpressing the MdTFL antisense gene.
About two thousand leaf discs of apple variety ‘Orin’ were used for Agrobacteriummediated transformation. After regeneration of transformed shoots from three independent
leaf discs, the shoots were cultured for a month and the insertion of transgenes was
confirmed by PCR using specific primers for nptII and MdTFL genes. The transformed
shoots from each line were grafted onto rootstocks to produce 11 transgenic lines (Table 2),
and these were transferred to an isolated greenhouse. In lines 303-1, 2, 3, and 4, the numberof branches was relatively low and the branches were upright as compared to the other lines(Table 2). The lines 705-3 and 4 showed a weak growth habit. Under in vitro culture
conditions, no flowers were produced in transgenic or control plants. The expression of thetransgene was analyzed in leaves of transformants by RNA blot hybridization. The MdTFL
antisense mRNA was detected by hybridizing with sense RNA probe, confirming that all
the transgenic lines tested express MdTFL antisense mRNA in their leaves (Table 2).
Especially, it was expressed strongly in transgenic lines 303-1, 303-4, and 705-1, although
the expression level was relatively low in lines 705-3 and 705-4. To examine the effect of
MdTFL antisense gene expression on the other apple genes, expression analysis of AFL and
MdMADS5 was performed using reverse transcription (RT) – PCR. Unexpectedly, in leaves,no difference in the gene expression was observed between transgenic and non-transgenic
plants (data not shown).

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Precocious Flowering in Transgenic Apples
Although apples flower naturally after four to eight years, the transgenic line 705-1
produced a solitary flower only eight months after grafting onto a rootstock. Normally,
apple trees require chilling for several months before developing leaves and flowers inspring, but this line flowered without undergoing a dormancy period. On the other hand,such an effect was not observed in non-transgenic controls. In addition, not a single
instance of precocious flowers has been seen in more than 100 other lines that were
produced with various constructs during the past 6 years at the National Institute of Fruit
Tree Science. The first flower of the line 705-1 was normal in appearance except that thestamens were much shorter than those of non-transgenic plants. After the break of the firstdormancy, six transgenic lines, including the line 705-1, produced solitary flowers in theaxils of leaves and at the top of two-year-old shoots or at the top of current shoots. But
normal apple trees produce many clusters which consist of about five flowers on fruit
bearing shoots (not on current shoots). In transgenic lines, some flowers appeared normal
and others showed homeotic conversions resulting in flowers with more petals or without
pistils. In the morphology of leaves, 303 lines showed round-shaped and more serrate
leaves and 705-1 and 705-2 lines had leaves with much smaller stipules than those ofcontrols.
Line 705-4 started to flower one month later than line 705-1. Surprisingly, it has
been producing flowers continuously for at least three months. In nature, flower inductionof apple occurs about two month after anthesis, and flower organ primordia are formed
during the period from summer to the next spring (Kotoda et al., 2000). In general, apple
trees neither produce flowers nor set fruits on any current shoots under normal conditions.
However, it is likely that flower induction in line 705-4 occurs at any time and the periodrequired for the development of flower organs is reduced extremely. Such a phenomenon
was also observed in the transgenic line 705-3.
To confirm the fertility of the transgenic apples showing early flowering, we carriedout crossing and pollen germination tests. Line 705-1 crossed with a Japanese apple
cultivar ‘Sansa’ set three fruits out of five flowers. One of the young fruits is developing.
Furthermore, pollen from line 705-1 was used to pollinate a columnar type apple ‘Wijcik’.
The development of these transgenic fruits appeared to be a little slower than those ofwild-type plants. Pollen from early flowering lines tested had the ability to germinate on aplate containing 1% agar and 17% sucrose, as did the controls (Table 2).
DISCUSSION
Environmental conditions that promote rapid, continuous growth generally reduce
the length of the juvenile period. In apple, seedling scions grafted onto dwarfing rootstocks
flower two to four years earlier than the seedlings from which they were taken (Hackett,1985). There is a report that apple seedling progenies grown continuously in optimal
greenhouse conditions flowered 16-20 months after germination by using defoliation tobreak dormancy but that seedlings from the same crosses grown in the field had not begun
to flower four years after germination (Aldwinckle, 1975). From our experience, however,
it would be difficult to reduce the juvenile period to less than two years in apple. In
producing transgenic plants for early flowering, there are two ways of thinking. One isoverexpressing the genes that promote flowering such as LFY and AP1, and second issuppressing the genes which delay flowering such as TFL1. We hypothesized thatsuppressing the late flowering genes could reduce the juvenile phase and induce precocious
flowers more effectively than promoting the early flowering genes in woody plants,
because only a few successes have been reported on precocious flowering in woody plants
by overexpressing LFY or AP1. In this study, we showed that the expression of MdTFL
antisense gene could induce precocious flowers in apple effectively. Although we produced
several transgenic apples overexpressing genes possibly involved in the flowering switch
including FLO, AP1, and their putative apple homolog genes AFL and MdMADS5,
respectively, there have been no transgenic apples other than those expressing the MdTFL
antisense gene that induces precocious flowering. In contrast with apple, citrus
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overexpressing LFY or AP1 produced precocious flowers 12-20 months after their transfer
to the greenhouse (Peña et al., 2001). In apple, it is likely that MdTFL plays an importantrole rather than AFL or MdMADS5 in regulating the transition to flowering. In woody
plants, the maintenance of juvenile phase should be one of the most important events at theearly stage of the plant development. However, it is interesting that the juvenile phase
could be regulated by a small number of genes such as TFL1-like genes.
ACKNOWLEDGEMENTS
We are grateful to Drs. F. Motoyoshi and H. Ichikawa for providing a lambda vecor
with TFL1 genomic clone and a binary vector pSMAK251, respectively. We thank Dr. N. C.
Oraguzie for critical reading of this manuscript, Ms M. Saito and T. Sekita for technicalassistance, respectively. This work was supported by a grant from the Ministry ofAgriculture, Forestry and Fisheries (MAFF) in the ‘Development of innovative plants andanimals using transformation and cloning’ project.
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Tables

Table 1. Phenotype of T2 transformed Arabidopsis with MdTFL sense gene.
_______________________________________________________________________________

Transgenic
linea

Days tofloweringb

Rosettes
No. ofat timeplantsof floweringc_______________________________________________________________________________

Control
T-S6
T-S10
T-S11
T-S21
T-S22
T-S28

30.3 ± 1.6
39.4 ± 5.1
59.0 ± 15.6
45.4 ± 6.0
44.7 ± 6.9
47.0 ± 0
53.0 ± 6.0

8.0 ± 1.0
11.8 ± 3.6
15.6 ± 4.3
11.8 ± 2.3
18.0 ± 4.3
23.0 ± 4.6
21.5 ± 1.5

10
5
5
5
3
3
2

_______________________________________________________________________________
a

Non-transgenic controls and seedlings from primary transformants (T1) with MdTFL gene
were grown under long-day (16hr light/8hr dark) conditions.
bDays to flowering is defined as the time when flower primordia were first visible to thenaked eye.
cRosettes were counted on the day that flower primordia were first visible.
b,cValues are means ± SEM.

Table 2. Phenotype of transformed apples with MdTFL antisense gene.
____________________________________________________________________
Transgenictransgene
Time toGrowthPollenGraftingline
mRNAa
flowerhabitcviability(yr/month)(months)b____________________________________________________________________
Controld
ND
N
1997/8
303-1
+++
11
LB
NT
2000/6
303-2++
LB
2000/6
303-3++
LB
2000/6
303-4
+++
11
LB
NT
2000/6
705-1
+++
8
N
+
2000/4
705-2
++
15
N
+
2000/4
705-3
+
11
W, MB
+
2000/6
705-4
+
11
W, LB
+
2000/6614-1
NTN
2001/6614-2
NTN
2001/6614-3
NTN
2001/6______________________________________________________________________________________________________amRNA was extracted from leaves and northern blot analysis was performed.
ND, not detected; NT, not tested.
b
The months were counted after grafting.
cN, Normal growth habit; LB, less branched; MB, more branched; W, weak growth habit.
dThree independently regenerated non-transformed plants were used as controls.

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