Biologia 66/2: 251—257, 2011
Transgenic lines of Begonia maculata generated by ectopic
expression of PttKN1
Quan-le Xu1,2, Jiang-ling Dong1,NanGao1,Mei-yuRuan1, Hai-yan Jia1,LiangZhang1,
& Chong-ying Wang1*
1Institute of Cel l Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000,P.R.China;e-mail:
2College of Life sciences, Northwest A&F University, Yangling 712100,P.R.China
Abstract: KNOX (KNOTTED1-like homeobox) genes encode homeodomain-containing transcription factors which play
crucial roles in meristem maintenance and proper patterning of organ initiation. PttKN1gene, isolated from the vascular
cambium of hybrid aspen (Populus tremula ×P. tremuloides), is a member of class I KNOX gene family. In order to under-
stand the roles of PttKN1gene in meristem activity and morphogenesis as well as to explore the possibility to generate novel
ornamental lines via its ectopic expression, it was introduced into the genome of Begonia maculata Raddi by Agrobacterium
tumefasciens–mediated gene transformation here. Four types of transgenic plants were observed, namely coral-like (CL)
type, ectopic foliole (EF) type, phyllotaxy-irregular (IP) type and cup-shaped (CS) type, which were remarkably diﬀerent
from corresponding wild type and were not also observed in the regenerated plantlets of wild type plant. Among these
four types of transgenic plants, the phenotype of coral-like was observed for the ﬁrst time in the transformants ectopically
expressed KNOX genes. The observation of scanning electron microscope (SEM) showed ectopic meristems on the adaxial
leaf surface of the transformants. Interestingly, the plantlets with ectopic foliole could generate new ectopic folioles from the
original ectopic folioles again, and the plants regenerated from the EF-type transformants could also maintain the original
morphology. The same speciﬁc RT–PCR band of the four types of transgenic plantlets showed that PttKN1was ectopically
expressed. All these data demonstrated that the ectopic expression of PttKN1caused a series of alterations in morphology
which provided possibilities producing novel ornamental lines and thatPttKN1played important roles in meristem initiation,
maintenance and organogenesis events as other class I KNOX genes.
Key words: Begonia maculata; ectopic expression; morphogenesis; KNOX;PttKN1
Plant developmental and morphological traits were
greatly related to the roles and regulations of tran-
scription factors (Peng et al. 1999; Wang et al.1999;
Zhang 2003). Among all transcription factors, KNOT-
TED1(KN1)-like homeobox genes (KNOX) were well
studied since they had been widely identiﬁed in mono-
cot and dicot species (Scoﬁeld & Murray 2006, Hay
et al. 2009), especially the class I KNOX genes,
such as KNOTTEDI (KN1), SHOOTMERISTEMLESS
(STM), knotted1-like homeobox gene from Arabidopsis
thaliana1(KNAT1), Oryza sativa homeobox 1 (OSH1),
Oryza sativa homeobox 15(OSH15) and Potato home-
obox 1 (POTH1). Their functions in morphogenesis
have been extensively studied by analyzing transgenic
plants. Sinha et al. (1993) found that the leaves of
KN1-transgenic tobacco produced numerous shoots.
Chuck et al. (1996) found that the ectopic expression of
KNAT1was able to transform simple leaves into lobed
leaves in Arabidopsis. In transgenic potato, the overex-
pression of POTH1produced dwarf plants with abnor-
mal leaves (Rosin et al. 2003). These reports conﬁrm
that KNOX gene is a key player in plant morphogen-
esis which derived from the balance of the stem-cells
proliferation and maintenance in SAM.
Most of the KNOX genes that have been exten-
sively studied so far are isolated from annual herbaceous
plants such as maize, Arabidopsis,Oryza sativa and
potato, however, KNOX genes from woody plants (tree)
have been scarcely investigated. A class I KNOX gene
from the palm species Elaeis guineensis was thought
to be associated with meristem function and a distinct
mode of leaf dissection (Jouannic et al. 2007). Later,
Barth et al. (2009) found that overexpressionof KNAP1
(KN1-like class I homologues from apple tree) in Koh-
leria drastically altered the leaf shape. Although there
have been several researches, it is still necessary to ex-
plore whether class I KNOXgenes from woody plants
play the same role as those from herbaceous plants in
PttKN1is a class I KNOX gene isolated from
the vascular cambial region of a hybrid aspen (Pop-
ulus tremula×tremuloides). To understand whether the
PttKN1gene from the aspen functions as the class I
KNOX genes from herbaceous plants in meristematic
2011 Institute of Botany, Slovak Academy of Sciences
252 Q. Xu et al.
initiation and maintenance, it was transformed into
petunia (Hu et al. 2005) and cockscomb (Meng et al.
2009). The PttKN1transgenic petunia showed altered
phenotypes such as dwarf and bushy, a loss of apical
dominance, lobed leaves and changes of ﬂower colour
(Hu et al. 2005). Similar phenotypes were observed
in PttKN1transgenic cockscomb (Meng et al. 2009).
Those results demonstrated that PttKN1played a sim-
ilar role as the class I KNOX genes from herbaceous
plants in morphogenesis.
In order to develop new ornamental lines and fur-
ther investigate the role of PttKN1in meristem activ-
ity and morphogenesis, PttKN1was introduced into
Begonia maculata, which is one of the best evergreen
perennial cultivars with cane-like stem, bold foliage and
large clusters of red pendulous ﬂowers, and belongs to
the ﬁbrous rooted type of Begonia. Begonia is one of
the most popular ornamental plants grown in gardens,
pots, hanging baskets and greenhouses. Meanwhile, ge-
netic engineering has provided a valuable means of ex-
panding the horticulture gene pool and promoting the
generation of new ornamental varieties. In this study,
we reported for the ﬁrst time that the ectopic expres-
sion of PttKN1in B. maculata caused the alteration of
leaf shape and plant architecture, including four novel
Material and methods
Plant species, bacterial strain and vector
Begonia maculata Raddi, purchased from a garden com-
pany as small plants, were grown in pots ﬁlled with mixed
soil containing vermiculite, perlite and peat moss (1:1:1) in
growth chamber with photoperiod of 16 h light of 24 µmol
m−2s−1. The used bacterial strain and vector were Agrobac-
terium tumefasciens GV3101 and pPCV702, respectively,
supplied friendly by Dr. Olof Olsson (G¨oteberg University,
Sweden). The pPCV702, containing a copy of CaMV 35S
and neomycin phosphotransferase gene (NPT II ), was intro-
duced into A. tumefasciens GV3101 by electroporation. Pt-
tKN1gene was integrated under the downstream of CaMV
Callus induction and plant regeneration from B. maculata
20-day-old young leaves were surface-sterilized with 70%
(v/v) ethanol for 1 min and then with 0.1 % (w/v) mer-
curic chloride for 8 min. After being rinsed thoroughly
with sterile distilled water, they were cut into 0.5 ×0.5
cm2pieces and cultured on Murashige & Skoog (MS)
medium supplemented with 6-benzylaminopurine (BA) 4.0
mg L−1,α-naphthaleneacetic acid (NAA) 0.2 mg L−1and
2,4-dichlorophenoxy acetic acid (2,4-D) 0.1 mg L−1under
the cool white ﬂuorescent light of 24 µmol m−2s−1with a
photoperiod of 16 h light and 8 h dark in growth chamber.
About one month later, the regenerated adventitious
buds were inserted into 1/2 MS with NAA 0.3 mg L−1for
rooting. Another month later, the regenerated shoots with
well-developed roots were transplanted into a 1:1:1 mixture
of vermiculite, perlite, and peat moss to be cultured under
dim light for one week and then under natural light and
Transformation of PttKN1 gene to B. maculata
20-day-old young leaves were surface sterilized by sequential
soaking in 70% (v/v) ethanol (1 min) and 0.1% (w/v) mer-
curic chloride (8 min). After being rinsed thoroughly with
sterile distilled water, they were cut into 0.5 ×0.5 cm2pieces
and then soaked in the cultures of A. tumefasciens GV3101
harbouring pPCV702 with diﬀerent optical density (0.205,
0.220 and 0.340 OD600 ) for 1 min. Then they were trans-
ferred onto MS solid medium containing 4.0 mg L−1BA,
−1NAA and 0.1 mg L−12,4-D and co-cultured in
the dark for 2 d, followed by culturing on MS solid medium
−1BA, 0.2 mg L−1NAA, 0.1 mg L−12,4-
D and 500 mg L−1cefotaxime. Seven days later they were
transferred to MS solid medium with 4.0 mg L−1BA, 0.2
mg L−1NAA, 0.1 mg L−12,4-D, 500 mg L−1cefotaxime
and 100 mg L−1kanamycin for transformant selection, cal-
lus induction and plant regeneration. The newly generated
buds and shoots were treated following the same method as
Total RNA was isolated from leaves of putative trans-
genic and wild type plants using Trizol Isolation Reagent
of Invitrogen. RT-PCR was performed with one step
RNA PCR kit (TaKaRa Biotechnology, Dalian, China).
A 300bp fragment of PttKN1gene was ampliﬁed using
primers: forward 5’-gctgctcgtcaagagtttgg-3’ and reverse 5’-
aatctcaggtagttcagtctccc-3’ (Hu et al. 2005) under the fol-
lowing conditions: one cycle of 50◦
C for 30 min; one cycle
C for 2 min; 30 cycles of 94 ◦
C for 30 s, 55◦
C for 30
C for 1 min; and ﬁnally elongated at 72 ◦
min. RT-PCR products were electrophoresis-separated on
1% agarose gel and photographed with Alpha ImagerTM
2000 Documentation Analysis System. All above kits were
used according to the manufacturer’s protocol.
Scanning electron microscopy
Leaves of transgenic and wild type plants were ﬁxed with
4% glutaraldehyde (v/v) (Sigma) dissolved in phosphate
buﬀer, dehydrated in ethanol series, desiccated in critical
point dryer, coated with gold, and ﬁnally observed under
JEOL1600 scanning electron microscope (Japan).
Induction and plant regeneration from the transgenic plants
To understand whether the traits of the PttKN1transgenic
plant above could be transmitted through micropropaga-
tion, the leaf segments of the transgenic plants grown in
pots in a greenhouse were re-cultured on MS agar medium
with BA 4.0 mg L−1, NAA 0.2 mg L−1and 2,4-D 0.1 mg L−1
under the same illumination conditions as above. About one
month later, the morphology of the regenerated adventitious
buds and shoots were observed and also photographed.
Callus induction and plant regeneration of B. maculata
The leaf explants of B. maculata which produced white
and loosen callus on their surface after about 15 days
being cultured on MS medium supplemented with 4.0
mg L−1BA, 0.2 mg L−1NAA and 0.1 mg L−12,4-
D (Fig. 1A). Another 30 d later, these calli diﬀerenti-
ated into adventitious buds (Fig. 1B). 20-day-old ad-
ventitious buds, about 2 cm high, were transferred to
rooting medium (1/2 MS with NAA 0.3 mg L−1), sub-
sequently well-developed roots were formed (Fig. 1C).
Transformation of Begonia maculata 253
Fig. 1. Plantlet regeneration of Begonia maculata from leaf explants. A – callus formed on the surface (cut ends) of leaf explants;
B – adventitious buds formed on the callus; C – regenerated plantlet with roots; D – regenerated plantlet growing in mixed soil.
Table 1. Inﬂuence of A. tumefasciens density (OD600) on transformation eﬃciency of leaf explants in Begonia maculate.
Rate of bud
0.205 579 13 10 2.25 76.92 1.73 5.53 48
0.220 579 33 25 5.70 75.76 4.32 3.11 63
0.340 413 39 24 9.44 61.54 5.81 2.65 57
The shoots with roots could survive and grow well after
being transplanted into a mixture of vermiculite, perlite
and peat moss (1:1:1) (Fig. 1D).
Of a total of 540 inoculated leaf explants, 438 could
form callus. All of callus produced adventitious buds;
13.50 buds per explant on average, and the adventitious
buds all could root in rooting medium. After being ex-
planted to soil, about 93% of the shoots survived and
exhibited normal development.
Introduction of PttKN1 gene to B. maculata
The leaf explants were infected with cultures of A.
tumefasciens at diﬀerent densities (0.205, 0.220 and
0.340 OD600) to get high transformation eﬃciency. The
ﬁnal result showed that the explants infected with 0.340
OD600 of A. tumefasciens cultures shared the highest
survival rate of 9.44% on the selection medium (Ta-
ble 1). In addition, it could be seen that the number
of survived explants of A. tumefasciens infected on the
selection medium increased with increased OD600 value
(Table 1). From 1571 leaf explants infected by A. tume-
fasciens, we obtained a total of 85 explants/callus re-
sistant to kanamycin.
Morphological features of PttKN1-transgenic plants
Normal B. maculata plants have strong main stem and
alternate phyllotaxy. Their leaves are simple, ovate and
entire with oblique base. Leaf blade is not eudipleu-
ral and its adaxial surface is dark green with regularly
arrayed white speckles, but the dorsal one is dark red
without speckles (Figs 2A, 2H).
Of a total of 168 putative PttKN1-transgenic plants
obtained here, 72 showed no visible diﬀerences from
wild type plants, and 96 showed abnormality to diﬀer-
ent extent. Based on morphological features, the plants
with visible abnormality were roughly divided into four
groups, i.e. coral-like (CL) type, ectopic foliole (EF)
type, phyllotaxy-irregular (IP) type and cup-shaped
(CS) type. These diﬀerent types counted for 17.26%,
21.43%, 1.79% and 0.89% of total, respectively.
The CL-typed transformants showed dwarﬁsm and
had no clear stem and apical dominance, both stem and
leaves were not easily distinguishable. The leaves (or
stems) appeared needle-like, had multilevel branches
(2–4) and lost original dorsoventrality (Figs 2B, 2C).
The EF-typed transformants had distinctive stems
and roughly normal leaves, but 1–8 small ectopic folioles
(EFs) were formed on the leaf surface. The folioles were
round and mostly located at the centre or close to the
main vein of leaves. Interestingly, these EFs could pro-
duce smaller folioles on their surface again compared to
original EFs (Fig. 4D). All EFs were mostly curled, and
254 Q. Xu et al.
Fig. 2. Phenotype of wild type Begonia maculata and its transformants. A – wild-type plant; B – CL-type transformant; C – local
magniﬁcations of B showed multilevel needle-like branches; D – EF-type transformant; E – local magniﬁcations of D showed the EFs
produced smaller folioles that were located at the centre or close to the main vein of round leaves; F – IP-typed transformant, with
two leaves at single node (arrows); G – IP-type transformant, showing fused stem (arrows); H – young leaf of wild-type plant; I –
CS-type leaf; J – regenerative ectopic foliole producing leaves from ectopic foliole. CL – coral like; EF – ectopic foliole; IP – irregular
phyllotaxy; CS – cup shaped.
had dark green colour, unclear dorsoventrality and ir-
regular white speckles compared to the wild type. Some
EFs also displayed a petiole-like structure (Figs 2D,
The phyllotaxy of the IP-typed transformants dif-
fered from the normal one, and there were two leaves on
each side. The two leaves exhibited identical size and
shape, and did not show notable diﬀerence from nor-
mal leaves. In addition, the stem of the IP-type trans-
formants seemed to consist of two stems fused together
(Figs 2F, 2G), thus it looked ﬂatter and thicker than
the normal one.
The transformants of the fourth type, CS type, dis-
played nearly completely the same phenotype as the
wild type, but a few leaves were changed into cup-
shaped (Fig. 2I).
The phenotypes identical with that of the above
transformants were not observed among the regener-
ated plants from leaf explants of the same plant, which
were not infected by A. tumefasciens.
In order to conﬁrm that PttKN1was expressed in
Fig. 3. RT-PCR analysis of the putative PttKN1-transgenic
plants. M, marker; lane 1, non-transformed control; lane 2, coral-
like transformant; lane 3, ectopic foliole transformant; lane 4,
phyllotaxy-irregular; lane 5, cup shaped typed transformant. The
300 bp indicates PttKN1-speciﬁc bands.
the transgenic plants, at least two plants from each of
the four types of transformants were subjected to RT-
PCR analysis. As shown in Fig. 3, each of them dis-
played one completely identical speciﬁc band of about
300bp, indicating that PttKN1gene had been expressed
in transformed plants.
Transformation of Begonia maculata 255
Fig. 4. SEM micrographs of the adaxial leaf surface from wild and PttKN1-transgenic plants (ectopic foliole-type). A – wild type,
showing very ﬂat and smooth upper leaf surface; B-D – transgenic plants. B – numerous protuberances with diﬀerent size and shape
on leaf surface; C – single small protuberance on ﬂat leaf surface; D – several pieces of ectopic folioles on ﬂat adaxial leaf surface,
which had a very big outgrowth (white arrows) and smaller folioles generating from ectopic folioles (black arrows).
Histological and anatomical characteristics of the
To understand the origin of the ectopic foliole in the
EF-typed transformants, a SEM examination was per-
formed. The upper surface of the leaves of the wild
B. maculata appeared very ﬂat and smooth. Polyg-
onal cells with uniform size were regularly arranged
(Fig. 4A). For the EF-typed transgenic plants, how-
ever, some adaxial leaf surfaces were extremely acci-
dented, showing numerous protuberances with diﬀer-
ent size and shape. The cell shape and arrangement
were extremely irregular. And some adaxial leaf sur-
faces looked like that of the wild type, but single small
spherical protuberance could be observed (Fig. 4B-D);
The feature of the in vitro regenerated plants from the
PttKN1 transgenic plants
To understand whether the phenotypes of transgenic
plants could be transmitted by micropropagation, the
leaf explants from the EF-type were cultured on MS
medium with BA 4.0 mg L−1, NAA 0.2 mg L−1and
2,4-D 0.1 mg L−1. Similarly, white and loosen callus
formed on the leaf explants when cultured around 15
d, and they diﬀerentiated into adventitious buds 30 d
The plants regenerated from the EF-type transfor-
mants still maintained the original morphology: there
were many circular folioles across the adaxial surface of
large leaves, and on these circular folioles, smaller fo-
lioles in comparison to original EFs could be observed
(Fig. 2J). The initial folioles were produced at the top of
the petiole and the subsequent ones at the sites nearby
midveins and secondary veins, which was the same as
in the EF-type transgenic plants.
In the present study, four types of PttKN1-transgenic
B. maculata, i.e. coral-like (CL), ectopic foliole (EF),
phyllotaxy-irregular (IP) and cup-shaped (CS), were
obtained via gene introduction in B. maculata medi-
ated by A. tumefasciens. These transformants were re-
markably diﬀerent in morphology from the wild typeB.
maculata. RT-PCR assay conﬁrmed that PttKN1gene
was expressed at transcription level in each of the four-
typed transgenic plantlets.
Among the four types of PttKN1-transgenic plant-
lets, the EF type is notably interesting. The transfor-
mants have normal stems and leaves, but there are nu-
merous small round folioles across the adaxial surface
of the leaves. The folioles can produce smaller round
folioles on their surface again. Although it had been
well reported that ectopic meristems formed on the sur-
face of the transgenic leaves (Sinha et al. 1993; Lin-
coln et al. 1994; Chuck et al. 1996; Hu et al. 2005), EF
typed transgenic plants in this experiment could gen-
erate ectopic folioles from the original ectopic folioles
again. This has not been reported neither among the
class I KNOX transgenic plants nor among transgenic
plants of other genes, to our knowledge. This fact in-
dicated that PttKN1probably had quite a strong role
in meristematic activity and ability of morphogenesis.
Furthermore, EF typed transgenic plants were beauti-
ful in shape and could be transmitted via micropropa-
gation, which suggested a potential ornamental value.
The phenotype with changed phyllotaxy appeared
in fasciata1(fas1), fasciata 2(fas2), tonsoku (tsk)and
pinoid (pid) mutants of Arabidopsis (Leyser & Furner
1992; Bennett et al. 1995; Suzuki et al. 2004), aber-
256 Q. Xu et al.
rant phyllotaxy (abphyll) of maize (Jackson & Hake
1999; Giulini et al. 2004), low auxin transport (lat)
mutant of tobacco (Naderi et al. 1997) and in some
transgenic plants expressing KNOX genes (Lincon et al.
1994; Chuck et al. 1996; Tamaoki et al. 1997; Nishimura
et al. 2000). In the present IP-type transformants, their
phyllotaxy still remained diﬀerent from the normal one,
but the number of leaves per node has doubled. The
pair of leaves looked similar in size and shape and had
a separated petiole. In addition, their stems were fas-
ciated and looked like a fusion of two stems (Fig. 2F,
2G). Phyllotaxy alteration was also observed in PttKN1
transgenic petunia (Hu et al. 2005). This phenomenon
suggested a stronger role of PttKN1in the remodelling
of phyllotaxy and plant morphogenesis.
Meristems are closely connected with the postem-
bryonic plant growth and initiation of organs. It has
been studied extensively that KNOX genes play vi-
tal roles in the shoot apical meristem (SAM) function
(Hake et al. 2004; Norberg et al. 2005; Wang et al.
2006) and lateral meristem activity (Ko & Han 2004;
Schrader et al. 2004; Groover et al. 2006) via maintain-
ing the stem cell population. Class I KNOX genes were
found to be necessary during all stages of the plant life
(Jasinski et al. 2006; Sakamoto et al. 2006). Primordia
of lateral organs, such as leaves, emerge from peripheral
regions of the SAM. Leaf shapes are highly correlated
with expression patterns of class I KNOX genes in leaf
promordia (Uchida et al. 2010). Ectopic expression of
class I KNOX genes resulted in morphologically altered
leaves and ﬂowers in transgenic Arabidopsis (Chuck et
al. 1996; Liu et al. 2008), tobacco (Nishimura et al.
2000), tomato (Kim et al. 2003; Kimura et al. 2008).
From our research, it could be concluded that as a
member of class IKNOX gene family, PttKN1played
a similar role in meristem as the other class I KNOX
genes. The morphological, histological and anatomical
analysis of EF-type transgenic plants indicated that Pt-
tKN1played a role in meristem initiation and mainte-
nance; ectopic expression of PttKN1could greatly en-
hance the meristematic activity not only in SAM but
also in other tissues, which further resulted in the mor-
phologic changes. Ectopic expression of PttKN1gene in
petunia (Hu et al. 2005), and cockscomb (Meng et al.
2009) also proved the function of the gene in meristems.
Taken together, ectopic expression of PttKN1gene
in B. maculata caused alterations of leaf shape and
plant architecture. Most importantly, it was the ability
of the ectopic foliole on the surface of the transgenic B.
maculata to generate new ectopic foliole again, as well
as the phyllotaxy alteration with two identical leaves at
each side of one internode. These data showed that the
PttKN1gene from vascular cambium of the tree played
an important role in meristem activity and morphogen-
esis, and its ectopic expression might have a possibility
to develop novel ornamental lines.
This work was supported by the National Natural Science
Foundation, P. R. China (30370087), by City gardening de-
partment of Xi’an, P. R. China (XA081023) and the Special
Foundation for Young Scholars of Northwest A & F Univer-
sity, P. R. China (Z111020912). The authors thank Dr. Olof
Olsson (G¨oteberg University, G¨oteberg, Sweden) for kindly
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Received September 7, 2009
Accepted June 22, 2010