Bull. Minamikyushu Univ. 40A: 59-63 (2010)
Approach to establishment of plant regeneration and
transformation system in sweet potato
(Ipomoea batatas) by culture of leaf segments
Lanzhuang Chen1*, Zhaosheng Du2, Yoshiko Nishimura1, Takuro Hamaguchi3,
Toru Sugita3, Ryutaro Nagata3, Hiroyuki Terao4 and Eiji Tsuzuki4
Labaratory of Vegetable Science, Department of Horticulture, Faculty of Horticulture, Minami Kyushu University,
11609, Minami Takanabe, Takanabe-cho, Miyazaki 884-0003, Japan; 2Institute of Agricultural Biotechnology,
Henan Academy of Agricultural Science, Nongye Road, Zhenzhou, Henan Province, China;
3
Department of Biotechnology, Miyazaki Prefecture Agricultural Experimental Station,
Sadowara, Miyazaki 880-0212, Japan; 4Faculty of Agriculture,University
of Miyazaki, Nishi 1-1,Gakuenkibanadai, Miyazaki 889-2192, Japan
1
Received October 7, 2009; Accepted January 27, 2010
Reprinted from
BULLETIN OF MINAMIKYUSHU UNIVERSITY
40A, 2010
Bull. Minamikyushu Univ. 40A: 59-63 (2010)
59
Approach to establishment of plant regeneration and
transformation system in sweet potato
(Ipomoea batatas) by culture of leaf segments
Lanzhuang Chen1*, Zhaosheng Du2, Yoshiko Nishimura1, Takuro Hamaguchi3,
Toru Sugita3, Ryutaro Nagata3, Hiroyuki Terao4 and Eiji Tsuzuki4
Labaratory of Vegetable Science, Department of Horticulture, Faculty of Horticulture, Minami Kyushu University,
11609, Minami Takanabe, Takanabe-cho, Miyazaki 884-0003, Japan; 2Institute of Agricultural Biotechnology,
Henan Academy of Agricultural Science, Nongye Road, Zhenzhou, Henan Province, China;
3
Department of Biotechnology, Miyazaki Prefecture Agricultural Experimental Station,
Sadowara, Miyazaki 880-0212, Japan; 4Faculty of Agriculture,University
of Miyazaki, Nishi 1-1,Gakuenkibanadai, Miyazaki 889-2192, Japan
1
Received October 7, 2009; Accepted January 27, 2010
A simple and efficient culture system of plantlet regeneration has been established from the
leaf segments of sweet potato (Ipomoea batatas). When the vegetative leaf segments were excised
from vine of sweet potato plants cultivated in growth chamber controlled, and sterilized with
sodium hypochlorite solution of 0.2 ~ 0.8% for 15 min, 100% of surviving rates was achieved in
the treatments of 0.3% and 0.4%. And then, they were cultured for callus formation on MS
(Murashige and Skoog 1962) and KN (Komamine and Nomura 1998) media supplemented with
2, 4-Dichlorophenoxyacetic acid (2, 4-D), Benzyladenine (BA) and 1-Naphthylacetic acid (NAA)
(Table 2), and the surviving rates of 100% were obtained from all of the treatments. After 2
times of subcultures of the calli on the same medium, they were placed onto KN medium with
different phytohormone combinations for somatic embryogenesis. As a result, somatic embryogenesis was obtained with white color and grain-like tissue, and somatic embryos with green color
were differentiated. The ultrastructures of the somatic embryos were observed by scanning electron microscopy (SEM), indicating embryogenic structures with nodular tissues (NO), scutellum,
coleoptile (CO) and germinated plumule (P). When a GUS gene set in pSMA35H2-NG was introduced into the Agrobacterium strain GV3101/PMP9, and a suspension solution of the Agrobacteria
was used to infect the calli derived from leaf segments
of "Miyazakibeni" and the rice calli of
。
"Nipponbare". After co-culture of the calli at 28 C in dark, the calli showed blue spots gradually
on the surface. When the embryogenic calli were transferred onto KN medium supplemented
with kinetin and giberellic acid for regeneration, they developed and formed multiple shoots.
Key words: GUS gene, Ipomoea batatas (L.) Lam., leaf segment, multiple shoot, somatic embryogenesis, transformation.
INTRODUCTION
As an important crop in tropical, subtropical and temperate areas, and an efficient biomass-producing plant
for starch, especially for bio-alcohol, recently, sweet
*Corresponding author: E-mail, [email protected]; Tel&Fax:
+81-986-46-1601.
Abbreviations: BA, Benzyladenine; BAP, 6-Benzylaminopurine; 2, 4D, 2, 4-Dichlorophenoxyacetic acid; GA3, Giberellic acid; GUS, Betaglucuronidase; IAA, Indoleacetic acid; Kin, Kinetin; KN, Komamine
and Nomura (1998); MS, Murashige and Skoog (1962); NAA, 1Naphthylacetic acid; ZR, Zeatin riboside.
potato plays a key role and has become attracted target.
As known as that, in general, the propagation of sweet
potato is kept by vegetative organism, like tubers, the viral
diseases could not be removed easily unlike seeds. In
other words, the viral diseases will live with the host
plants forever, except they are eliminated by apical culture, resulting into virus-free ones. Therefore, the viral
diseases, which include russet crack disease (Cali and
Moyer 1981) and 'obizyo-sohi' (Sweet potato feathery
mottle virus (SPFMV)) disease, usually cause serious
economic losses in sweet potato in Japan (Usugi et al.
1994).
To produce virus-free plants, some attempts of shoot
apex culture have been carried out using MS (Murashige
and Skoog 1962) medium (Nagata 1984, 1990, 1991).
60
Approach to establishment of plant regeneration and transformation system in sweet potato (Ipomoea batatas) by culture of leaf segments
Fig. 1. Callus formation, somatic embryogenesis and germination of somatic embryos from culture of leaf segments of
sweet potato. A) Tubers of "Miyazakibeni" of sweet potato used in this study; B) New plants growing from the tubers in growth
chamber; C) Leaf segment was cut and cultured on KN and MS media; D) Initial callus like white frost observed on rear surface of
the leaf segment, after three days of culture; E) Callus formation after two weeks of culture; F) Somatic embryogenesis with white
color from callus derived from leaf segments after moved on KN medium; G) Somatic embryos with green color after another two
weeks of culture; H) Germination of somatic embryo with elongation of tissue, which initiates plantlet, cultured on modified KN
medium.
And recently, a modified and efficient clonal propagation
by shoot apex culture and quick detection of virus-free
plants of sweet potato have been established (Chen et al.
2008). However, the shoot apex culture is costly, and the
initial virus-free sweet potato plants can be often infected
after cultivation. Therefore, it would be worthwhile to
breed SPFMV resistant varieties using transgenic techniques, either with the resistant genes of sweet potato or
other genes conditioning the virus resistance.
International Potato Center (CIP), where the sweet
potato genetic resources collected from the world were
kept and conserved, hold several varieties which were
found to be resistant to the russet crack disease (Annual
Report CIP 1989). Recently, Okada et al. (2001) reported
that they had produced transgenic sweet potato with virus
resistance of SPFMV-S, using culture of mesophyll protoplasts and eletroporation method.
As described above, either the shoot apex culture (Nagata
1984, Chen et al. 2004, 2008) for virus-free plants or mesophyll protoplasts culture (Murata et al. 1994) for plant
regeneration or transgenic plants (Okada et al. 2001) needs
higher handy techniques to get the materials for starting
culture. On the other hand, a simple plant regeneration
system has been tried, for example, by culture of leaf
discs in sweet potato (Otani et al. 1996) and in wild species
of sweet potato (Pido et al. 1995). When we used the protocols, the complete plant could not be obtained, except
the recovery of shoot-like organism, in the preliminary
experiments.
In our laboratory, ASG-1, an apomixis-specific gene has
been cloned from apomictic guinea grass (Panicum maximum) (Chen et al. 1999, 2005). As the ASG-1 showed
homologies to seed- or embryo-specific genes (YamaguchiShinozaki and Shinozaki 1993; Baumlein et al 1991), it
is expected to help to form seeds from vegetative reproduction plants, like sweet potato and potato, which can
form immature seeds in some cases. Our laboratory's aim
is not only to produce transgenic plants of virus resistance but also to create seed-reproduction plants from
today's vegetative reproduction ones by ASG-1 gene
transformation. To realize the aim, the establishment of
an efficient and simple plant regeneration system in
sweet potato is very important for us.
In this study, as the preliminary step to get transgenic
plants, we have made the approach to establishment of a
simple and efficient plant regeneration system via somatic embryogenesis from culture of leaf segments that can
be collected in any season, at any time, and then be used
to produce transgenic plants.
MATERIALS AND METHODS
Plant materials. Sweet potato, Ipomoea batatas (L.)
Approach to establishment of plant regeneration and transformation system in sweet potato (Ipomoea batatas) by culture of leaf segments
Lam. cv. "Miyazakibeni", cultivated in southern Kyushu
in Japan was provided kindly from Horticulture branch,
the Miyazaki Prefectural Agricultural Experimental
Station (Miyokonojo, Miyazaki, Japan). The
tubers were
。
planted in vermiculite sterilized under
120
C,
15
min. and
。
grown in a growth chamber at 25 C, 3.3 μmol m-2s-1 on
16 h photoperiod.
Leaf segment culture. After the 3rd to 5th expanded
leaves far from the growing point (meristem) of the
young runner were cut and treated with 0.2~0.8% concentration of sodium hypochlorite solution for 15 min,
they were washed with sterilized water. Then, the leaves
were put onto the glass laboratory dish, and after the
edges and veins were carefully removed with a knife and
tweezers, the remains of the leaves were cut into segments in size of about 0.5~0.7×0.3~0.5 cm. For callus
formation, the leaf segments were cultured on MS and
KN (Komamine and Nomura 1998) media supplemented
with 1- naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP) and 2, 4-dichlorophenoxyacetic acid (2, 4D) with different concentrations, respectively, in dark
condition. For embryogenesis and germination of somatic embryo, the calli formed on MS and KN media were
transferred onto KN medium supplemented with zeatin
riboside (ZR), indoleacetic acid (IAA), giberellic acid
(GA3), 2, 4-D, NAA, kinetin (Kin) and benzyladenine
(BA)。with different combinations, in a growth chamber
of 25 C, 3.3μmol m-2s-1 on 16 h photoperiod.
Microscopic studies. Embryogenic calli (Fig. 1F)
were prepared for scanning electron microscopy (SEM)
by fixing in FAA solution (ethanol 45%, acetic acid
2.5% and formalin 2.5%) for 24 h. Sample fixation was
followed by tissue dehydration by an ethanol series
(50%, 60%, 70%, 80%, 90%, 99.5% and 100%) and
twice in 100% 3-methylbutyl acetate. Thereafter, the
samples were critically point dried, and sputtered with
gold prior to SEM (HITACHI S4100).
61
Table 1. Effects of sterilizing concentrations of sodium
hypochlorite solution on culture of leaf segments of sweet
potato (Observed after culture of two weeks, sterilizing time
is 15 min. each treatment)
Sterilizing concentration(%)
No. of explants
Surviving rates (%)
0.2
0.3
0.4
0.6
0.8
30
90
30
100
30
100
30
0
30
0
Table 2. Effects of concentrations of photohormones on
callus formation of sweet potato derived from in vivo the
leaf segments of sweet potato in different media (Observed
after four weeks of culture; Referring to M & M)
No. explants Media
30
30
30
30
30
MS
MS
KN
KN
KN
Combinations of photohormones
2, 4-D
BA
NAA
Rates of callus
(%)
1
2
1
2
2
0.5
0.5
0.5
0.4
0.5
0.01
0.01
0.01
0.01
0.01
100
100
100
100
100
pSMA35H2-NG and an Agrobacterium strain GV3101/
PMP9 were provided kindly by Dr. Ichikawa (NIAR,
Japan). For the protocol of infection and removing of
Agrobacterium, and GUS dyeing was according to that
of rice (Dr. Toki NIAR, Japan, by personal communication).
RESULTS AND DISCUSSION
GUS gene expression in calli mediated by Agrobacterium.
For GUS gene transformation, calli from culture of leaf
segments were used in this study. A GUS gene set in
Effect of the concentration of sodium hypochlorite
solution for surviving rates of leaf segments. Up to
Fig. 2. Scanning electron microscopy of somatic embryos. A) Nodular embryogenic tissue (NO) of somatic embryos of Figure
1F; B) Scutellum (SC), coleoptile (CO) and germination of plumule (P) of somatic embryos of Figure 1G. Bar = 100μm in Fig. 2B.
62
Approach to establishment of plant regeneration and transformation system in sweet potato (Ipomoea batatas) by culture of leaf segments
Fig. 3. GUS gene expression in calli of sweet potato after co-cultivation with Agrobacterium tumefaciens strain
GV3101/PMP90 (pSMA35H2-NG Hm). A) Calli derived from leaf segments before GUS staining; B) Calli derived from rice seeds
of "Nipponbare" before GUS staining; C) Calli with blue color from leaf segments (Upper) and those from rice seeds (Lower), respectively, after GUS staining.
now, there have been many methods established concerning the shoot apex culture (Chen et al. 2004; Nagata
1984, 1990, 1991), petiole protoplast culture (Murata et
al. 1987), ovule culture (Murata and Fukuoka 1994) and
mesophyll and cell suspension protoplast culture (Murata
et al. 1994), in sweet potato. There were also some
papers reported concerning leaf segment culture for
embryogenic callus formation (Otani et al. 1995), and
plant regeneration in sweet potato (Otani et al. 1996) and
in wild species of sweet potato (Pido et al. 1995). However,
when the above protocols were used for culture of leaf
segments in sweet potato, all of them could not be
repeated with plant regeneration, except the recovery of
shoot-like organism. Therefore, a practical and detailed
procedure is needed to bridge the blank of leaf segment
culture, as it is very simple and valuable culture system
for propagation and gene transformation in sweet potato.
Here, we firstly checked the effect of sodium hypochlorite solution concentration on leaf segment culture (Table
1). Among the range of 0.2~0.8% for 15 min, surviving
rates of 100% were achieved in the treatments at 0.3% and
0.4%. From the result, it is clear on leaf segment culture
that the treatment at a 0.3% or 0.4% sodium hypochlorite
solution for 15 min. can stop the infection of unwanted
bacteria perfectly when using the young runner cultivated in growth chamber.
Callus formation on different media with different
combinations of phytohormones. We compared the
effects of MS and KN media supplemented with 2, 4-D,
BA and NAA on callus formation (Table 2). Callus always
occurred around the leaf segments of 0.9cm× 0.7cm in
size (Fig. 1C, 1D) at culturing of two weeks. When we
measured the size of callus at interval of two weeks, the
sizes were 1.4×1.0 cm, 2.0×1.4 cm and 2.5×1.9 cm,
respectively. The callus formation rates of 100% were
obtained from all of the treatments used (Table 2). In
general, there were no differences between the media and
combinations of phytohormones. Phytohormones are needed for callus formation from leaf segments, as there is
nothing formed in leaf segment culture when phytohormone-free MS and KN media were used.
Somatic embryogenesis and germination of somatic
embryo. After 2 times of subcultures of calli on the
same medium, they were placed onto KN medium with
different phytohormone combinations for somatic
embryogenesis. After two weeks of culture in the KN
medium, somatic embryogenesis, firstly, occurred on the
surface of the original callus placed, with white color
(Fig. 1F). At the same medium, the culture was continued for 4 weeks, and grain-like somatic embryos were
differentiated around the original callus. And then, the
calli with white color were transferred onto the same
medium, and after two weeks of the culture, the somatic
embryos with green color were differentiated (Fig. 1G).
The ultrastructures of the somatic embryos were
observed with SEM (Fig. 2). Embryogenic structures
with nodular tissues (NO) (Fig. 2A), and somatic embryo
with scutellum, coleoptile (CO) and germinated plumule
(P) (Fig. 2B) were observed. These structures were similar to those previously reported in Panicum maximum
(Lu and Vasil 1985, Chen et al. 2002), Paspalum notatum (Marousky and West 1990, Chen et al. 2001).
The rates of 100% were obtained for somatic embryogenesis on KN medium supplemented with 1.0mg/l IAA and
1/0mg/l Kin, and 0.01mg/l NAA and 2.0 mg/l Kin, respectively (Table 3). On the other hand, no somatic embryogenic calli were obtained on KN medium supplemented
with 1.0 mg/l IAA, 1.0mg/l BA and 2.0mg/l 2, 4-D, and
1.0mg/ l IAA and 4/0mg/l 2, 4-D, respectively. From the
result of somatic embryo differentiation, the phytohormone
combination affects the rates of somatic embryogenesis.
Germination of somatic embryo was observed on the
green callus derived from leaf segments after moved on
modified LS regeneration medium with half sucrose
(15%) for the culture of two weeks (Fig. 1H). The shootlike tissues were regenerated from the germinated somatic
embryos after another two weeks culture. Now the
plantlets with shoot-like tissues were kept by subculture
on the same medium. The difficult point is that shoot-like
tissues could not grow into complete plant. However,
media and phytohormone combinations are being used
for overcoming the difficult point in our laboratory.
Examination for GUS gene expression in calli transformed with Agrobacterium. A GUS gene set in
pSMA35H2-NG was introduced into the Agrobacterium
strain GV3101/PMP9, and a suspension solution of the
bacterium was used to infect the calli derived from leaf
Approach to establishment of plant regeneration and transformation system in sweet potato (Ipomoea batatas) by culture of leaf segments
Table. 3. Effects of concentrations of photohormones on
somatic embryogenesis in callus derived from leaf segments
of sweet potato (Observed in three weeks after culture on
KN medium)
No. callus
cultured
Combinations of photohormones (mg/l)
BA
IAA
NAA
2, 4-D
Kin
40
40
48
48
--1.0
--
1.0
-1.0
1.0
-0.01
---
--2.0
4.0
1.0
2.0
---
No. somatic
embryogenesis (%)
40 (100)
40 (100)
48 (0)
48 (0)
KN medium was used in somatic embryogenesis (Referring to M &
M). ZR: zeatin riboside; NAA: 1- naphthaleneacetic acid; BAP:
6-benzy- laminopurine; 2, 4-D: 2, 4-dichlorophenoxyacetic acid;
IAA: indoleacetic acid; GA3: giberellic acid; Kin: kinetin; BA:
benzyladenine
63
Establishment of propagation method in large quantities to produce virus-free plants of sweet potato, Ipomoea batatas (L.)
Lam. by means of cultures of shoot apex and sucker. Bull. Fac.
Agri, Univ. Miyazaki 50: 1-9 (In Japanese).
Chen, L.Z., Okabe, R., Guan, L.M. and Adachi, T (2002) A
simple and efficient culture of leaflets for plant regeneration in
guineagrass (Panicum maximum). Plant Biotech. 19: 63-68.
Komamine, S. and Nomura, K. (1998) Entrance of plant cytological technology. Gakkai Publishing House Tokyo, pp.22-23
(In Japanese).
Lu, C. and Vasil, I.K. (1985) Histology of somatic embryogenesis in Panicum maximum (guinea grass). Am. J. Bot. 72:
1908-1913.
Marousky, F.J. and West, S.H. (1990) Somatic embryogenesis and plant regeneration from cultured mature caryopses of
bahiagrass (Paspalum notatum Flugge). Plant Cell Tissue
Organ Cult. 20: 125-129.
Murashige, T. and Skoog, F. (1962) A revised medium for
rapid growth and bioassays with tobacco tissue culture.
Physiol. Plant. 15: 473-479.
segments of "Miyazakibeni" (Fig. 3A) and rice calli of
"Nipponbare" (Fig. 3B). After 。three days of co-culture of
calli and Agrobacterium at 28 C in dark, a GUS dyeing
solution was added
onto the collected calli, and incubat。
ed for 4 h at 37 C. The calli showed blue spots gradually
on the surface. As the time passes, the blue spots are
increased, until the whole callus was dyed with blue
color after 12 h (Fig. 3C). As the control, rice calli also
dyed with blue color. From the above results, it is suggested that this transformation system described in this
study is usable and practical for transformation of sweet
potato, when the system of plant regeneration is established from leaf segment culture.
ACKNOWLEDGMENT
Murata T (2001) Virus resistance in transgenic sweetpotato
[Ipomoea batatas L. (Lam)] expressing the coat protein gene of
sweet potato feathery mottle virus. Theor Appl Genet 103: 743751.
Murata, T., Fukuoka, H., Kishimoto, M. (1994) Plant regeneration from mesophyll and cell suspension of sweet potato,
Ipomoea batatas (L.) Lam. Breed. Sc.i 44: 35-40.
Nagata, R. (1984) Transmission of russet crack-like symptom
by grafting to sweet potato grown from apical meristem tips (in
Japanese). Proc. Assoc P1 Protein. Kyushu 30:33-35.
Nagata, R. (1990) The technique to produce virus-free plants
of sweet potato, Ipomoea batatas (L.) Lam., and its effect. Bul.l
Miyazaki Agri. Exp. Sta. Japan 25: 77-90 (in Japanese).
Nagata, R. (1991) The utility of meristem culture plants as
controlling the russet-crack like symptom of sweet potatoes and
the prevention of re-infection of them. Agri. Tech. 46: 71-74 (in
Japanese).
The authors thank M. Shimokoori of Miyazaki Prefectural Agricultural Experimental Station (Horticulture
Branch, Miyokonojo, Miyazaki, Japan) for providing
tubers of sweet potato, and Dr. Ichikawa of NIRA for
providing transgenic vector in this study.
Okada, Y., Saito A., Nishiguchi, M., Kimura, T., Mori, M.,
Hanada, K., Sakai, J., Miyazaki, C., Matsuda, Y. and
Murada, T. (2001) Virus resistance in transgenetic sweetpotato [Ipomoea batatas L. (Lam)] expressing the coat protein gene
of sweet potato feathery mottle virus. Theor. Appl. Genet. 103:
743-751.
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