Japanese Society of Grassland Science ISSN1744-6961

ORIGINAL ARTICLE
Blackwell Publishing Asia

Evaluation of tissue culture response from mature seeds of

Panicum spp.
Mi-Suk Seo, Manabu Takahara, Masumi Ebina and Tadashi Takamizo
Forage Crop Biotechnology Research Team, National Institute of Livestock and Grassland Science, Nasushiobara, Tochigi, Japan

Keywords
Callus induction; genotype; germination;
Panicum spp.; regeneration.
Correspondence
Tadashi Takamizo, Forage Crop Biotechnology
Research Team, National Institute of Livestock
and Grassland Science, 768 Senbonmatsu,
Nasushiobara, Tochigi 329-2793, Japan.
Email: takamizo@affrc.go.jp
Received 12 November 2007;
accepted 8 May 2008
doi: 10.1111/j.1744-697X.2008.00115.x

Abstract
The genus Panicum contains important warm-season forage grasses and species
with potential as biomass crops. We selected Panicum genotypes with high response
to tissue culture for genetic improvement. The highest frequency of callus induction
from mature seed of Panicum maximum cultivar Natsukaze was obtained on MS
medium containing 4.0 mg L1 2,4-dichlorophenoxyacetic acid and solidified with
0.3% Gelrite. We compared germination frequencies and callus induction capacities
among 24 genotypes of 11 Panicum species on this medium. Callus induction
frequencies varied among genotypes. Those with high germination frequencies
generally had high callus induction frequencies. On the other hand, especially in
P. maximum, the callus induction ratio (callus induction frequency/germination
frequency) depended on the reproductive mode and ploidy. The callus induction
ratio of three sexual accessions of P. maximum were very low compared to apomictic
accessions, and besides, a tetraploid sexual accession Noh PL1 had very low
germination and callus induction frequencies. Callus induction and regeneration
capacities were independent of each other. For shoot regeneration, we transferred
callus derived from the 24 genotypes onto MS medium supplemented with
1.0 mg L1 kinetin and 0.4% Gelrite. Six of the genotypes regenerated plantlets.
Among them, Panicum meyerianum produced the highest shoot regeneration
frequency of 61.6% and the maximum number of shoots callus1 in the shortest
time. The callus of P. meyerianum also showed vigorous proliferation. We thus
selected high-response genotypes of P. meyerianum.

Introduction
Panicum is one of the largest genera of the Poaceae, with
approximately 450 species. Many species grow in tropical and
subtropical regions, but some are well adapted to temperate
regions (Zuloaga et al. 1987). Most of these are large
perennial grasses, growing to 13 m tall, including several
warm-season species grown for forage production, such as
guineagrass (Panicum maximum Jacq.), switchgrass (Panicum
virgatum L.) and Kleingrass (Panicum coloratum L.)
(Patamalai et al. 1990; Carvalho et al. 2006; Sarath et al.
2007). Guineagrass has both facultative apomictic and sexual
reproductive genotypes in the same species (Nakajima &
Grassland Science 54 (2008) 125130 2008 Blackwell Publishing Ltd

Mochizuki 1983). Therefore, molecular approaches can be

used to reveal the mechanisms controlling apomixis (Chen
et al. 1999; Ebina et al. 2005).
Switchgrass has also come into the spotlight as a biomass
crop recently (Sanderson et al. 1996; McLaughlin et al. 1999).
Biomass crops are expected to make a significant contribution to
renewable energy sources, but available crops are limited to a
few species so far, such as sugarcane (Lakshmanan et al. 2006).
Therefore, the application of biotechnology to the genus Panicum
is very important, not only for understanding of apomictic
reproduction, but also for creating novel biomass crops.
An efficient tissue culture system is necessary to enable
successful use of biotechnology in crop improvement. Tissue
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Tissue culture responses of Panicum spp.

M.-S. Seo et al.

Table 1 Germination and callus induction from mature seeds of 24 accessions in Panicum

Panicum

Common name

Accession

Ploidy level
(reproductive mode)

Germination
frequency %

Callus induction
frequency % (Y/X)

Callus
induction ratio

P. antidotale Retz.
P. bergii Arechav.
P. capillare L.
P. coloratum L.

Blue panicgrass
Bergs panicgrass
Witchgrass
Kleingrass

P. dregeanum Nees

Wild type

P. hallii Vasey

Halls panicgrass

P. longijubatum Stapf
P. maximum Jacq.

Wild type
Guineagrass

P. meyerianum Nees
P. stapfianum Fourc.

Wild type
Stapfs buffalograss

P. virgatum L.

Switchgrass

TO7307
PI. 285216
PI. No. GEE
PI. 253241
PI. 277963
(D-9)R-1
(D-9)85 L-9
CPI. 68864
85 B-1
85 D-10
Natsukaze
Natsuyutaka
Natsukomaki
Gatton
Soil clean
Noh PL1
GR297
GR297 (84I-4)
PI. 299000
PI. 198589
85 Q-2
Blackwell, GR-59
Blackwell, GR-61
Blackwell, GR-63

Diploid (S)
Tetraploid (S)
Diploid (S)
Tetraploid (S)
Tetraploid (S)
Unknown (A & S)
Unknown (A & S)
Diploid (S)
Diploid (S)
Unknown (S)
Tetraploid (A)
Tetraploid (A)
Tetraploid (A)
Tetraploid (A)
Tetraploid (A)
Tetraploid (S)
Diploid (S)
Diploid (S)
Unknown (S)
Diploid (S)
Diploid (S)
Octoploid (S)
Octoploid (S)
Octoploid (S)

4
2
7
2
22
18
44
58
95
55
21
83
59
89
76
3
68
33
47
54
27
80
95
97

1.7 (6/350)
3.5 (12/345)
6.9 (24/350)
0.0 (0/350)
21.1 (74/350)
12.4 (45/363)
21.9 (76/347)
49.9 (179/359)
96.7 (298/308)
31.9 (110/345)
22.2 (64/289)
62.5 (143/229)
61.6 (213/346)
33.3 (116/348)
50.0 (78/156)
0.3 (1/290)
8.9 (31/350)
3.7 (13/350)
26.3 (92/350)
64.2 (147/229)
10.3 (36/350)
68.9 (241/350)
69.1 (242/350)
94.3 (197/209)

0.43
1.75
0.99
0.00
0.96
0.69
0.50
0.86
1.02
0.58
1.06
0.75
1.04
0.37
0.66
0.10
0.13
0.11
0.56
1.19
0.38
0.86
0.73
0.97

Number of germinated seeds/100 of seeds inoculated 100. (Number of seeds inducing callus [Y]/number of seeds inoculated [X]) 100. Callus
induction ratio was calculated for callus induction frequency/germination frequency. A, apomictic reproduction mode; S, sexual reproduction mode.

culture of Panicum has been reported in only two species,

guineagrass (Lu & Vasil 1981, 1982; Akashi & Adachi 1991;
Chen et al. 2002) and switchgrass (Alexandrova et al. 1996).
However, in those cases, calli were derived only from
immature embryos or node culture. Tissue culture of such
explants is more complicated than from mature seed, which
offers several advantages, being very easy to handle and
available year round (Huang and Wei 2004).
Herein, we report the culture responses of various
accessions of the genus Panicum using mature seeds as
explants. The genotypes with high culture response will be
used for genetic improvement.

Materials and methods

Plant materials
Twenty-four accessions in 11 Panicum species, which are
listed in Table 1, were used in this study. The seeds have been
preserved at 4C at the National Institute of Livestock and
Grassland Science (NILGS), Tochigi, Japan.
126

Germination
To test the germination abilities, we cultured 100 seeds per
accession. Dehusked mature seeds were surface-sterilized in
2% sodium hypochlorite for 20 min and then rinsed three
times with sterile distilled water. The seeds were placed on
1% agar containing 20 mmol L1 NaH2PO42H2O solution
adjusted to pH 6.5 with 1 mol potassium hydroxide (KOH),
and incubated at 25C in the light for 2 weeks.

Callus induction
Panicum maximum cultivar Natsukaze was used to test the
optimum concentration of 2,4-dichlorophenoxyacetic acid
(2,4-D) for callus induction. Dehusked mature seeds were
surface-sterilized in 70% ethanol for 1 min and in 2%
sodium hypochlorite for 25 min, and then rinsed three times
with sterile distilled water. The seeds were placed on sterile
filter paper on MS medium (Murashige & Skoog 1962)
supplemented with 0, 1, 2, 4 or 8 mg L1 2,4-D and solidified
with 3.0 g L1 Gelrite [Wako Pure Chemical Industries, Ltd.,
Grassland Science 54 (2008) 125130 2008 Blackwell Publishing Ltd

M.-S. Seo et al.

Tissue culture responses of Panicum spp.

Osaka, Japan]. Seeds were incubated at 25C in the dark for

4 weeks. The experiment was repeated three times with 350
580 seeds in each treatment.
We then investigated the callus induction and shoot
regeneration frequencies of the 24 genotypes. Mature
seeds (~363 genotype1) were surface-sterilized and rinsed as
above. For callus induction, the seeds were placed on MS
medium containing the optimal 2,4-D concentration
determined as above, and incubated at 25C in the dark
for 4 weeks. Calli were subcultured at 4-week intervals onto
the same medium.

Plant regeneration
Calli were next transferred to regeneration medium containing
MS salts and vitamins, 30 g L1 sucrose, 1.0 mg L1 kinetin, and
4.0 g L1 Gelrite. The pH of the medium had been adjusted to
5.8 before autoclaving. The cultures were maintained at 25C
under a 16-h photoperiod for 6 weeks. The regenerated
plants were potted and grown to maturity in a greenhouse.

Callus proliferation
To investigate the patterns of callus proliferation, we
subcultured 4 mg of callus of Panicum longijubatum Stapf,
Panicum meyerianum Nees, and Panicum stapfianum Fourc.
on MS medium containing 4.0 mg L1 2,4-D and incubated
it at 25C in the dark. Callus proliferation was assessed
by measuring the calli fresh weight once a week for 7 weeks.
Each genotype had three independent replicates.

Results

Figure 1 Effect of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration

on callus induction from mature seed of Panicum maximum (cv.
Natsukaze) on MS basal medium with 0.3% Gelrite. Vertical bars
represent standard errors of three replications.

calli of P. maximum were soft, watery and translucent. Those

of P. virgatum GR-59 and GR-61 were compact and yellow;
that of GR-63 was compact, non-friable and white. Accessions
with a high callus induction frequency tended to have a
high germination frequency also, and vice versa (r = 0.860,
P < 0.01). But in P. maximum, the callus induction frequency
of apomictic accessions was significantly higher than that
of sexual accessions. The sexual accessions GR297 and
GR297 (84I-4) had a low callus induction ability but a high
germination frequency. The sexual accession Noh PL1 had
both low germination ability and very low callus induction
ability. All accessions with high germination frequencies
showed high efficiencies of callus induction except P. maximum.

Optimal concentration of 2,4-D for callus induction

The induction frequency of primary callus ranged 027.3%
(Figure 1). The induction frequency was reduced at 8.0 mg L1,
so the optimal concentration for callus induction was
4.0 mg L1 2,4-D.

Genotypic differences in germination and callus

induction
Next, we determined the frequency of callus induction from
mature seeds of all accessions on MS medium containing
4.0 mg L1 2,4-D (Table 1). Panicum hallii Vasey (accession
no. 85 B-1) had an excellent callus induction frequency
(97%). The callus of P. hallii looked as white and soft, and
watery calli from mature seeds were also observed. Some
accessions of P. maximum and P. virgatum showed high
frequencies of germination and callus induction. Callus
morphology was genotype-dependent and ranged in texture
and color from friable and yellow to compact and white. The
Grassland Science 54 (2008) 125130 2008 Blackwell Publishing Ltd

Genotypic differences in plant regeneration

Ten of the 24 accessions showed green spots in callus culture,
and six of those formed shoots (Table 2). Three accessions of
P. virgatum produced a high green spot formation frequency,
but only GR-63 produced a high frequency of green spots
(53.3%), yet its shoot regeneration frequency was very low
(4.4%). P. meyerianum showed the highest shoot regeneration
frequency (61.6%) and produced several shoots callus1,
which appeared in only 4 weeks (Figure 2). P. longijubatum
also produced comparatively many shoots (42.5%). The
other eight accessions, however, showed shoot regeneration
frequencies below 10%.

Proliferation patterns of callus

We examined the proliferation pattern of callus of
P. meyerianum, P. longijubatum and P. stapfianum, all with good
shoot regeneration ability. In all the 3 species observed, the
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M.-S. Seo et al.

Table 2 Shoot regeneration frequency from mature seed-derived calli of 10 genotypes in Panicum

Panicum

Common name

Accession

Spot formation
frequency (%)

Shoot regeneration
frequency (%)

No. of
shoots callus1

P. capillare L.
P. coloratum L.
P. hallii Vasey

Witchgrass
Kleingrass
Halls panicgrass

P. longijubatum Stapf
P. meyerianum Nees
P. stapfianum Fourc.
P. virgatum L.

Wild type
Wild type
Stapfs buffalograss
Switchgrass

PI. No. GEE

PI. 277963
CPI. 68864
85 B-1
85, D-10
PI. 299000
PI. 198589
Black well, GR-59
Black well, GR-61
Black well, GR-63

18.4
28.3
4.8
6.0
42.5
63.3
23.5
40.5
95.0
53.3

8.0
0.0
0.8
0.0
42.5
61.6
5.9
0.0
0.0
4.4

5.0 1.11
0.0 0.00
4.0 0.00
0.0 0.00
3.4 0.54
5.6 0.49
2.0 1.00
0.0 0.00
0.0 0.00
3.0 0.82

Mature seed-derived calli were cultured on MS medium containing 1 mg L1 kinetine and 0.4% Gelrite for 6 weeks. Data shown are the mean of
average standard error. Number of calli indicated spots/number of calli inoculated 100. Number of calli producing shoots/number of calli
inoculated 100.

Figure 2 Stages of plant regeneration in Panicum meyerianum. (a) Callus derived from mature seed. (b) Green spots regenerated from callus after
1 week. (c) Shoots regenerated from callus after 3 weeks. (d) Shoots regenerated after 4 weeks. (e) Regenerated plants grown in the greenhouse. Plants
were regenerated from callus cultured on MS medium containing 1 mg L1 kinetin and 0.4% Gelrite. Bars: (ad) 1 cm; (e) 30 cm.

morphological characterization of callus were mostly yellow,

compact and very friable. Figure 3 shows the callus proliferation
patterns of the three species. The callus weight of P. meyerianum
quadrupled between 2 and 3 weeks (F = 11.34, P < 0.01),
then increased more slowly thereafter. P. stapfianum and
P. longijubatum showed similar proliferation patterns as
P. meyerianum, though the weight of P. longijubatum was low.

Discussion
It is generally recognized that the response of plants to
tissue culture is controlled genetically, as reported for barley
128

(Sharma et al. 2004), sugarcane (Lakshmanan et al. 2006)

and rapeseed (Kennedy et al. 2005). Therefore, the selection
of a genotype with excellent culture response ability is very
important for plant genetic improvement.
We examined germination, callus induction and shoot
regeneration of 24 accessions of Panicum (Table 1). Germination is affected by both internal and external stimuli (Bewley
1997; Koornneef et al. 2002), and treatment with plant
hormones can stimulate germination (Sarath et al. 2006).
Therefore, the low germination frequencies of some of these
accessions might be improved by the addition of gibberellic
acid, nitric oxide or other chemicals.
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M.-S. Seo et al.

Tissue culture responses of Panicum spp.

To our knowledge, this is the first report in which genotypic

differences of tissue culture responses from mature seed of
various Panicum spp. were investigated. There are strong
genotypic influences on callus induction, proliferation and
shoot regeneration capacity in Panicum. We could thus select
responsive genotypes of P. meyerianum which should be
amenable to genetic transformation. Studies of transformation with those genotypes and screening of various culture
conditions to get more elite genotypes in other species
are currently being carried out. The tissue culture system
reported here can provide protocols that are very easy to
handle and efficient for genetic improvements of Panicum
using the mature seeds.

Figure 3 Proliferation patterns indicated by callus weight in three

accessions of Panicum. Calli were subcultured every week on MS
medium containing 4 mg L1 2,4-dichlorophenoxyacetic acid and 0.3%
Gelrite. Points and bars show the average standard error of three
independent samples.

Accessions with high germination frequencies tended to

have high callus induction frequencies, indicating a correlation between the germination and callus induction capacities
of mature seeds. On the other hand, the callus induction
ratios of three sexual accessions of P. maximum were very low
compared to apomictic accessions, and besides, a tetraploid
sexual accession Noh PL1 had both very low germination and
callus induction frequencies. These results indicate that
the culture responses in P. maximum might be influenced by
the reproduction mode or ploidy of accessions. However, in
Paspalum, no dramatic differences were detected among in
the apomictic and sexual genotypes used for either callus
induction or plant regeneration (Molinari et al. 2003).
Only six genotypes regenerated shoots among the 24 with
callus induction capacity. Therefore, callus induction and
regeneration capacity are independent of each other. Notably,
P. meyerianum showed a high frequency of shoot regeneration and vigorous callus proliferation in spite of a low callus
induction frequency (26.3%). This independence has been
reported also in wheat (Sears & Deckard 1982; Chowdhury
et al. 1991). In addition, the regeneration ability of the
genotypes was related to the morphological characteristics of
the callus: P. meyerianum and P. longijubatum, with yellow,
compact and very friable calli, had a comparatively high
regeneration capacity, whereas P. maximum, with translucent,
soft and watery callus, did not regenerate. Such a correlation
between the morphological characteristics of callus and
regeneration ability has been reported for various other
species (Armstrong & Green 1985; Ke & Lee 1996; Toyama
et al. 2003).
Grassland Science 54 (2008) 125130 2008 Blackwell Publishing Ltd

Acknowledgments
We thank Mr Makoto Kobayashi and Mr Masahito Inafuku
for valuable suggestions and Mrs Etsuko Kaneda, Mrs Yko
Nakada and Mrs Ritsuko Chiba for assistance in the seed
culture. This work was funded by a MAFF research project,
Development of innovative crops through the molecular
analysis of useful genes.

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