Plant Prod. Sci.

9(1) : 65 ź 70 (2006)

Identification of Callus Induction Potential of 15 Indonesian

Rice Genotypes
Nono Carsono1 and Tomohiko Yoshida2

(1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology;
2
Faculty of Agriculture,Utsunomiya University, Utsunomiya 321-8505, Japan)

Abstract : The callus induction potential of 15 indica rice genotypes from Indonesia was examined in comparison
with that of the japonica rice Nipponbare. Callus was induced from embryos of mature seeds and root segments
on MS and CI media. There was genotype × medium × explant interaction for inducing white/cream/yellow
callus with an organized structure (callus type I and II) and for callus browning, but not for callus induction
ability and diameter of callus. Genotypes significantly differed in inducing high quality of calluses depending on
medium and explant used. Four indica types, Fatmawati, Ciapus, BP-23 and BP-360-3, had callus induction-related
traits similar to those of Nipponbare. These genotypes would be useful for tissue-culture based research and for
crop improvement, particularly for genetic transformation. Culturing seed explant on MS was more suitable for
callus induction than either root explant on MS or both explants on CI medium.

Key words : Callus browning, Callus induction ability, Embryogenic callus, Rice.

ǽCallus induction is one of the substantial steps for callus induction has been found among the different
selecting the suitability of genotypes for tissue-culture- genotypes of indica rice (Abe and Futsuhara, 1986;
based research and for plant improvement, particularly Peng and Hodges, 1989; Seraj et al., 1997) and japonica
for genetic transformation. In rice, different callus rice (Yoshida and Oosato, 1998; Ogawa et al., 1999).
types, i.e., type I, II, III and IV, can be induced (Visarada Indica rice cultivars generally show less induction of
et al., 2002). Type I callus is white and cream colored callus formation than japonica (Abe and Futsuhara,
compact organized callus, type II is a yellow organized 1986). Not only callus production, but also somatic
callus, type III is a yellow or brown unorganized callus, embryogenesis and subsequent plant regeneration
and the type IV is highly unorganized white, yellow or (Chu and Croughan, 1990) are also inferior in indica
brown callus. Type-I and-II calluses are embryogenic rice, which may limit the success in the transformation
and can be induced from tissues of various organs such for indica rice.
as immature seeds (Masuda et al., 1989), immature ǽCallus induction as well as regeneration potential is
embryos (Koetje et al., 1989), and roots (Abe and affected not only by genotype and the type of explant
Futsuhara, 1985; Hoque and Mansfi eld, 2004). but also by the composition of the culture medium
The type-III callus is dark and necrotic. In general, including plant growth regulators, and by the culture
immature embryos and meristematic tissues, having conditions. However, in particular, genotype and type
undifferentiated cells, are suitable for callus induction of explant are important factors for the successful
and plant regeneration than mature tissues (Morrish embryogenic callus induction and regeneration of
et al., 1987). However, such explants are available only rice plants (Rueb et al., 1994). In this study, genotypic
in a restricted period of the growth cycle in the rice differences among 15 Indonesian rice genotypes in
plant (Hoque and Mansfi eld, 2004), and to obtain callus induction potential was examined, combined
such explants all year round, we have to grow the with two types of explants (embryos of mature seed
plants in a greenhouse. However, embryos of mature and root segment) and two culture media: MS
seeds are available throughout the year, and are more (Murashige and Skoog, 1962) and CI (Potrykus et
suitable for rice callus culture. The embryogenic al., 1979). The success using MS medium (Seraj et
calluses induced by culture of mature seeds are al., 1997; Khanna and Raina, 1998; Lee et al., 2002),
effective used for genetic transformation by particle as well as CI medium (Jiang et al., 2000) for callus
bombardment (Jiang et al., 2000) or Agrobacterium- induction has already been reported.
mediated transformation (Kumria et al., 2001). The ǽThe objective of the present study was to identify
aseptic culture of root explants is also useful since it is callus induction potential of 15 Indonesian rice
relatively easy to provide in any season. In the in vitro genotypes in vitro. The genotypes identified to have
culture of rice explants, a signifi cant difference in a good induction potential could then be utilized in

Received 8 March 2005. Accepted 18 August 2005. Corresponding author : T. Yoshida ([email protected], fax
+81-28-649-5401).
 66 Plant Production Science Vol.9, 2006

Tableǽ1.ǽGenotypes used in this experiment.

No. Genotypes Parents

1. Fatmawati BP68C-MR-4-3-2/Maros
2. Gilirang B6672/Memberamo
3. Ciapus Memberamo//IR-66154-221-2-2/Memberamo
4. Cimelati Memberamo//IR-66160/Memberamo
5. IR-64 IR-5657/IR-2061
6. BP-23 IR-64/IRBB7//IR64
7. BP-140 IR-66738-18-1-2//Barumun/IRBB7
8. BP-360-2 B10182/Memberamo///IR-66160/Memberamo//Memberamo
9. BP-360-3 B10182/Memberamo///IR-66160/Memberamo//Memberamo
10. BP-205D-KN-78-1-8 Dacava line 85/Memberamo
11. BP-135 IR-65598-27-3-1/Suban//Barumun
12. BP-138 Muncul//IR-64/TB154E-TB-1
13. BP-143 Memberamo/IR-65598//Memberamo
14. B10597F IR-BB7/Cincklonik
15. BP-355E Memberamo//IR-665600/Memberamo///IR-65598/Cibodas
16. Nipponbare (check) Yamabiko/Sachikaze

tissue culture-based studies directed for the coming Tableǽ2.ǽComposition of media used in this study.
genetic transformation studies with the valuable genes
Components Medium MS* Medium CI*
of agricultural interest.
Inorganic salts
Materials and Methods
Macro elements
ǽFifteen indica rice genotypes from Indonesia and NH4NO3 ǽ1650 ǽ640
one japonica rice cv. Nipponbare as a check were used KNO3 ǽ1900 ǽ1212
in the experiment since this genotype has been widely CaCl2.2H2O ǽ440 ǽ588
used as model in tissue culture and plant improvement MgSO4.7H2O ǽ370 ǽ247
studies (Table 1). The fi rst fi ve genotypes listed in KH2PO4 ǽ170 ǽ136
the table are broadly cultivated in Indonesia and the Micro elements
following ten genotypes are promising ones bred by
KI ǽǽ0.83 ǽǽ0.83
Indonesia Institute for Rice Research (Sukamandi,
H3BO3 ǽǽ6.2 ǽǽ3.1
Subang, West Java).
MnSO4.4H2O ǽǽ22.3 ǽǽ11.15
ǽMature healthy seeds were dehusked manually and
ZnSO4.7H2O ǽǽ8.6 ǽǽ5.76
soaked in 70% ethanol for 3 min. with gentle agitation
followed by rinsing with sterile distilled water. The Na2MoO4. 2H2O ǽǽ0.25 ǽǽ0.24
seeds were then surface sterilized in 50% commercial CuSO4. 5H2O ǽǽ0.025 ǽǽ0.025
bleach (5% sodium hypochlorite) for 30 min. with CoCl2. 6H2O ǽǽ0.025 ǽǽ−
agitation and rinsed with sterile distilled water three CoSO4. 7H2O ǽǽ− ǽǽ0.028
times. The root explants were obtained from the FeSO4. 7H2O ǽǽ27.85 ǽǽ27.8
seedlings grown on MS-basal medium. The embryos Na2EDTA. 2H2O ǽǽ37.25 ǽǽ37.3
of mature seeds (seed explants) and root segment Organic supplement
(root explants, from 5- to 7-day-old seedling) were Nicotinic acid ǽǽ0.5 ǽǽ6.0
aseptically cultured on callus induction medium; Pyridoxine-HCl ǽǽ0.5 ǽǽ1.0
eight explants were placed on a Petri dish (9 cm Thiamine-HCl ǽǽ0.5 ǽǽ8.5
in diameter) containing 20 mL MS or CI medium Coconut water ǽǽ− ǽ100mlL-1
supplemented with 2 mg L-1 2,4 dichlorophenoxyacetic Glycine ǽǽ2.0 ǽǽ2.0
acid (2,4-D)(Table 2). Anticipate the contamination Carbon source
that would arise, we cultured additional explants of
Sucrose 30000 20000
each genotype on both media. The pH of medium was
Mannitol ǽǽ− 36430
adjusted to 5.8 before autoclaving, and the cultures
Phytohormone (2,4-D) ǽǽ2.0 ǽǽ2.0
were kept at 25 ±1ºC under dark condition. After
a 30-day culture, explants were examined for callus * : Concentration in mgL -1 . Both media did not contain
induction ability (CIA : total number of explants Myoinositol.
 Carsono and Yoshida źź Callus Induction of Indonesian Rice 67

Fig.ǽ1.ǽCalluses of the genotypes B10597F (A), BP-360-3 (B) and

Nipponbare (C), derived from seed explant (A, B, C followed by
subscript number 1 and 2) and root explant (with subscript number 3
and 4), cultured on MS (with subscript number 1 and 3) and CI (with
subscript number 2 and 4) media.

with calluses per total number of explants cultured × relatively hard, and sometimes dry, but those derived
100%), average diameter of callus (ADC in cm), % from root segment were highly mucilaginous, soft and
of white or cream or yellow calluses that belong to covered with a translucent sticky substance. In some
callus type I or II (WYC : number of white or cream or roots, calluses were produced on a few sites of the
yellow calluses with compact or friable structure per segment. Most of the root segments with hairs had a
number of explants cultured ×100%), and % of callus high ability to induce callus.
browning (CBW : number of callus browning per ǽA significant interaction was detected for genotype
number of explants cultured ×100%). A completely × medium × explant (p<0.01 for WYC and CBW),
randomized factorial design with three replications was medium × explant (p<0.01 for CIA, WYC and CBW),
employed in this study. Analysis of variance (ANOVA) genotype × explant (p<0.01 for CIA, WYC and p<0.05
and Dunnett’s pairwise multiple comparison t-test for for CBW), and genotype × medium (p<0.05 for WYC)
genotypes were performed by using SPSS 10 software. (Table 3). For WYC and similarly for CBW, except
for the genotype × medium interaction, main- and
Results
interaction effects were signifi cant. For CIA, the
ǽAll genotypes induced calluses, but CIA varied with medium × explant interaction accounted for the
the genotypes. Total number of explants with calluses greatest variation. In general, explant accounted for
was 76 for Nipponbare; on the average per dish was the greatest variation in ADC, WYC, and CBW, but not
6.3 (79.17%). Fig. 1 shows the induced callus of some in CIA. We found no significant interaction among
genotypes. B10597F had poor callus induction ability. the genotype × medium × explant for CIA and ADC.
Phenotypically, calluses derived from mature seed were Medium × explant and genotype × explant interaction
 68 Plant Production Science Vol.9, 2006

Tableǽ3.ǽAnalysis of variance for callus induction performance.

Callus Average Callus Callus

induction diameter of white/ browning
Source of variation ǽdf
ability (CIA) callus (ADC) yellow (CBW)
(WYC)
Genotype (g) ǽ15 ǽ2669.92** 0.04** ǽ2337.67** ǽ557.29**
Medium (m) ǽ1 ǽ8138.02** 0.16** ǽ2929.69** 3987.63**
Explants (e) ǽ1 14179.69** 1.87** 19300.13** 9847.00**
Genotype x Medium (g x m) ǽ15 ǽ276.91ns 0.01ns ǽ462.67* ǽ291.45ns
Genotype x Explants (g x e) ǽ15 ǽ617.19** 0.02ns ǽ1079.64** ǽ397.35*
Medium x Explants (m x e) ǽ1 17347.00** 0.02ns ǽ6018.88** 6302.08**
Genotype x Medium x Explants (g
ǽ15 ǽ319.23ns 0.01ns ǽ610.89** ǽ435.76**
x m x e)
Error 128 ǽ243.33 0.01 ǽ263.67 ǽ191.24
Total 192

Data show mean square value, ns : nonsignificant, * : significant at p = 0.05, ** : significant at p = 0.01, respectively.

for ADC, genotype × medium interaction for both CIA reported that white and green patches are often seen
and ADC were also not significant. The main effect on the surface stratum of callus with high regeneration
of each factor for these traits was significant (p<0.01, ability. Moreover, in sorghum, Kaeppler and Pedersen
Table 3). (1997) reported that the production of a high quality
ǽAlthough a signifi cant genotype × medium × callus depends mainly on the genotype. Lee et al.
explant interaction was obtained for WYC and CBW (2002) found that the number, colour, size, shape and
as previously mentioned, we mainly analyzed the appearance of the embryogenic calluses varied among
genotypic difference among the traits observed. In the rice genotypes depending on the type of basal
comparison with Nipponbare, Fatmawati, Ciapus, medium, indicating that induction of high-quality rice
BP-23, and BP-360-3 had similarly superior callus callus is infl uenced by genotype, medium, and the
induction-related traits irrespective of the medium kind of explant as well as by their interactions.
and explants used according to Dunnett’s pairwise ǽCallus browning is one of the major problems in in
multiple comparison t-test at 0.05 probability level vitro culture. Abe and Futsuhara (1991) and Ogawa et
(Table 4). On the other hand, BP-135 was the only al. (1999) argued that callus browning is genetically
genotype showing significantly inferior performance in controlled, which correspond to the present study. A
all traits as compared with Nipponbare. decrease in the rate of callus growth is occasionally
ǽIn the overall analysis, MS medium and seed explant related to the appearance of a brown colored area
were superior for CIA, ADC, and WYC, whereas CI (Maeda et al., 2002). A low 2,4-D concentration
medium and root explant were superior for CBW. (Mitsuoka et al., 1994) and high temperature (Hoque
and Mansfield, 2004) seem to cause callus browning or
Discussion
regeneration of albino plants. However, in the present
ǽIt is well known that the genotype (Abe and study, 2 mg L-1 2,4-D and 25oC were used for callus
Futsuhara, 1986), the interaction between genotype induction, which are supposed to be an optimum
and medium (Khanna and Rainna, 1998) and also the for callus induction. However, the result of callus
interaction between genotype and explant (Hoque browning in this study should be considered carefully,
and Mansfield, 2004) have significant effects on callus since explants of the genotype that could not produce
induction. In this study, the effect of genotype x the calluses were not examined. If we examined the
medium x explant interaction on WYC and CBW was browning of the culture including the browning
significant, but that on CIA and ADC was not (Table of explants, the result would be slightly different,
3). The WYC and CBW are believed to be related to particularly in the genotypes that had low ability in
the quality of callus. White/cream/yellow calluses as callus induction ability, i.e., Gilirang, BP-138, BP-143,
reported by Visarada et al. (2002) are embryogenic and B10597F (Table 4).
calluses and are also associated with at least two ǽIn the present study, we provided root explants
other types of calluses, i.e. 1) a soft, transparent, from 5- to 7-day-old seedlings; although Hoque and
unorganized callus, which is nonmorphogenic or may Mansfield (2004) found that the younger explants (3-
form only roots, and 2) soft mucilaginous callus, which to 5-day-old seedlings) of indica rice were most efficient
often form roots. Maeda (2000) and Maeda et al. (2002) in both callus induction and plant regeneration. This
 Carsono and Yoshida źź Callus Induction of Indonesian Rice 69

Tableǽ4.ǽComparison of callus induction-related traits of 15 genotypes with those of Nipponbare and overall
means for media and explants.

Callus Average Callus white/ Callus

induction diameter of yellow browning
No. Genotype
ability callus (%,WYC) (%,CBW)
(%,CIA) (cm,ADC)
1. Fatmawati 69.79ns 0.29ns 44.79ns 11.46ns
2. Gilirang 59.38* 0.19* 34.38* 19.79ns
3. Ciapus 80.21ns 0.26ns 50.00ns 14.58ns
4. Cimelati 67.71ns 0.24* 36.46* 20.83ns
5. IR-64 79.17ns 0.24* 57.29ns 8.33ns
ns ns ns
6. BP-23 86.46 0.30 65.63 6.25ns
7. BP-140 90.63ns 0.33ns 64.58 ns 25.00*
8. BP-360-2 67.71ns 0.20* 33.33* 25.00*
9. BP-360-3 82.29ns 0.33 ns 52.08ns 17.71ns
ns ns ns
10. BP-205D-KN-78-1-8 69.79 0.25 44.79 23.96*
11. BP-135 46.88* 0.19* 28.13* 22.92*
12. BP-138 57.29* 0.26 ns 31.25* 17.71ns
13. BP-143 61.46* 0.20* 34.38* 13.54ns
14. B10597F 33.33* 0.15* 15.63* 14.58ns
ns ns ns
15. BP-355E 70.83 0.29 45.83 27.08*
16. Nipponbare (check) 79.17 0.38 57.29 6.25

Seed explant 77.47 0.36 53.52 24.35

Root explant 60.29 0.16 33.46 10.03
MS medium 75.39 0.29 47.40 21.75
CI medium 62.37 0.23 39.58 12.63
ns : nonsignificant, * : significant compared to Nipponbare’s performance using Dunnett’s pairwise multiple
comparison t-test at 0.05 probability level with right-sided (>control) for CIA, ADC and WYC, and left-sided (<control)
for CBW. Values are means of combination of 2 media and 2 explants with 3 replicates. The difference between seed
explant and root explant or between MS medium and CI medium are significant (Table 3).

is because a large variation was found in root length cv. Memberamo (from crossing between B6555 and
in the 3-to 5-day-old seedlings, in some genotypes root IR-19661), and twelve of them are also derived from
length was only a few centimeters. the IR-series (breeding lines) developed by IRRI (Table
ǽMedium composition plays an important role in 1), thus all genotypes carry a genetic composition
callus induction (Khanna and Raina, 1998; Ogawa, (female/male parent) from IR-series.
1999). The nutrient, particularly the nitrogen source, ǽIn Table 3, we found a high contribution of the
affects the callus induction of somatic embryos, explant to the variation of traits observed. The seed
particularly in monocots (Leifert et al., 1995). MS explant was suitable for obtaining high CIA, ADC,
medium was suitable for obtaining high CIA, ADC and and WYC, and root explant for low CBW (Table 4).
WYC, while CI medium was suitable for obtaining low In addition, four genotypes i.e., Fatmawati, Ciapus,
CBW, although the difference was slight (Table 4). BP-23, and BP-360-3 have been identified as excellent
MS medium, compared with CI medium, has a higher genotypes for high CAI, ADC, WYC and low CBW
concentration of macro and micro elements, but compared with Nipponbare (Table 4). This shows that
lower content of organic supplement, only one carbon the seed explants of these superior genotypes have
source (i.e. sucrose) and no coconut water (Table 2). a high callus induction potential which depends on
Such differences might cause the differences in tissue the activity of genes that determine and maintain the
culture response. meristematic activity of the cells, level of hormones,
ǽAlthough genotype significantly contributed to the and sensitivity to hormones, as well as on the activity
variation of traits observed, the contribution was not of other genes that control different stages of plant
as large as we expected (Table 3). This may be due to morphogenesis (Ezhova, 2003). Fatmawati, released
the similar genetic background of the materials used. as a new cultivar in Indonesia in 2003, and BP-360-3
Eight of them are derived from the same parent, i.e., are new plant type (NPT) with low tillering capacity
 70 Plant Production Science Vol.9, 2006

and large panicles. Ciapus is also a new cultivar from transformed embryonic callus of an elite indica rice via
released in 2003 and BP-23 is a promising line, Agrobacterium. Plant Cell Tiss. Org. Cult. 67 : 63-71.
though not released yet. These genotypes should be Lee, K., Jeon, H. and Kim, M. 2002. Optimization of mature
further examined for their plant regeneration ability embryo-based in vitro culture system for high-frequency
or may be used as genetic transformation materials. somatic embryogenic callus induction and plant regeneration
from Japonica rice cultivars. Plant Cell Tiss. Org. Cult. 71 :
We believed that these genotypes with good callus
237-244.
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Leifert, C., Murphy, K.D. and Lumsden, P.J. 1995. Mineral and
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