American Journal of Plant Sciences, 2013, 4, 2265-2270
Published Online November 2013 (
Open Access AJPS
Effect of Maltose Concentration on Plant Regeneration of
Anther Culture with Different Genotypes in Rice
(Oryza sativa L.)
Seul Gi Park, Mohammad Ubaidillah, Kyung-Min Kim*
Division of Plant Biosciences, School of Applied BioSciences, College of Agriculture and Life Science, Kyungpook National Uni-
versity, Daegu, Korea.
Email:,, *
Received September 14th, 2013; revised October 15th, 2013; accepted October 24th, 2013
Copyright © 2013 Seul Gi Park et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This study describes the impact of different concentrations of maltose on plant regeneration of anther culture for five
genotypes of rice (Oryza sativa). N6 medium was used for calli induction, while N6 medium supplemented with differ-
ent concentrations of maltose, 2.0 mg/L NAA and 0.5 mg/L kinase was used for plant regeneration. The result showed
that during the initial stages of calli induction the anther cultures had varying rates of calli formation among genotypes,
with the best frequency being observed for Dreami2/CaMsrB2-8-DH-1 with a calli frequency of 27.8%. Different
genotypes of rice cultured in regeneration media showed varying plantlet regeneration on media supplemented with
different concentrations of maltose, with low concentrations (0.04 g/L) leading to low frequency regeneration plantlet
but high green plant production. Indeed, when Dreami2/CaMsrB2-8-DH-2 and Dreami2/CaMsrB2-8-DH-5 were culti-
vated under these conditions, 100% green plants were observed. Another genotype also showed a small rate of albino
frequency in response to the lowest concentration of maltose, while increased maltose concentrations resulted in in-
creased rates of albino plants. Overall, the results of this study should facilitate establishment of an efficient plant re-
generation system from anther culture in rice.
Keywords: Anther Culture; Maltose; Oryza sativa; Plant Regeneration; Rice
1. Introduction
Anther culture has become the most popular method for
production of homozygous lines for rice cultivars world-
wide. Accordingly, rice anther culture has been inten-
sively investigated. However, despite significant im-
provements, its practical application remains limited [1].
Nevertheless, anther culture has been widely integrated
into rice breeding programs, allowing rapid production of
homozygous double haploid lines from F1 hybrids and
incorporation of new genes into breeding materials. The
production of double haploids from anthers or isolated
microspores culture in vitro is a rapid approach to ho-
mozygosity that shortens the time required for develop-
ment of new rice cultivars through conventional methods,
which require at least six generations. Haploids are also
valuable for detection and repair of desirable recessive
traits introduced through mutation [2] or hybridization
[3]. Double haploid techniques accelerate the breeding
cycle and allow the production fertile line. A large num-
ber of rice varieties have been developed through anther
culture and released for cultivation over several thousand
hectares because the number of green plants obtained
through anther culture did not meet the demands of prac-
tical exploitation [4]. Several factors influencing anther
culture have been studied, including genotype of the ex-
plants [5,6], growth condition of donor plants [7] and
culture methods [8]. Although different protocols have
been proposed to improve the efficiency of anther culture,
green plant regeneration frequencies have remained
rather low. To date, the application of anther culture to
rice breeding has been hampered by low calli induction
and plant regeneration frequency. Even good calli induc-
tion may result in predominant albino populations. A
number of factors have been examined to determine the
*Corresponding author.
Effect of Maltose Concentration on Plant Regeneration of Anther
Culture with Different Genotypes in Rice (Oryza sativa L.)
effects of various factors on calli regeneration frequency,
such as genotypes, hormonal composition, culture meth-
ods, and carbohydrate sources [9,10]. The carbohydrate
sources used for the induction and regeneration medium
can have a profound effect on anther culture response. It
has been known that some carbohydrate source, sucrose,
is most commonly used for all types of tissue cultures. In
comparison to sucrose, maltose has been found to be
superior for green shoot regeneration in barley and effec-
tive as a carbon source for androgenesis [11]. In this study,
we reported the effects of maltose as a carbohydrate
source on calli induction and regenerated plant frequency.
2. Materials and Methods
2.1. Preparation of Explants
The experiment was conducted in the field using the fol-
lowing eight genotypes of rice, japonica type,
Dreami2/CaMsrB2-8-DH-1, Dreami2/CaMsrB2-8-DH-2,
Dreami2/CaMsrB2-8-DH-3, Dreami2/CaMsrB2-8-DH-4,
Dreami2/CaMsrB2-23-DH-7, and Dreami2/CaMsrB2-
23-DH-9. Pre germinated seeds from each genotype were
sown under normal conditions during summer with com-
plete nutrition.
2.2. Anther Collection and Treatment
Anthers were collected at the early flowering stage, when
young panicles were still enclosed within the leaf sheath.
Selection was based on a maximum distance between the
auricle and the next subtending leaf of 5 - 10 cm. These
findings coincide with the mid-uninucleate stage, which
is most responsive to anther culture. Panicles were col-
lected from plants between 9:00 and 10:00 AM and then
washed with tap water. After clipping the flag leaves,
panicles were sprayed with 70% ethyl alcohol and then
incubated in polyethylene bags in the dark at 8˚C - 12˚C
for 14 days.
2.3. Calli Induction and Plant Regeneration
The panicles of eight rice were carefully sprayed with
70% ethanol (v/v) and catch with pinset and burn on the
fire just a few second, repeat this step 3 times. The pani-
cles enclosed in the leaves were then opened, after which
the disinfected capitula was plated on dry sterile filter
papers. Flowerlets of the outer ring on the capitula were
excised and torn open with two pairs of fine forceps, af-
ter which all five anthers were taken out from each flow-
erlet and immediately inoculated onto media for calli
induction and incubation at 25˚C in the dark for 30 days.
About 40 anthers were inoculated on calli induction me-
dium except those for comparison. Media for callies con-
sisted of N6-medium, 2.0 mg/L NAA, 0.5 mg/L kinase
and 4.0 g/L maltose. The pH was adjusted to 5.8 with 1N
HCl or 1N NaOH before the addition of Gelrite, after
which the media was autoclaved at 121˚C for 20 min.
Calli about 4 - 5 mm in size was transferred from the
calli induction media onto regeneration media (approxi-
mately 10 per petri dish). Samples were then incubated
under a 12-h light/12-h dark photoperiod with a light
intensity of about 20 lmol·m2·s1 at 25˚C ± 1˚C for 30
days. Regeneration consisted of N6-Y1 medium (Duchefa)
supplemented with various concentrations of maltose
(0.04 g/L, 0.4 g/L and 4.0 g/L), 2 mg/L NAA, and 0.5
mg/L Kinase. The pH was adjusted to 5.8 with 1N HCl
or 1N NaOH before the addition of gelrite and the media
was then autoclaved at 121˚C for 20 min.
The appearance and proliferation of calli were ob-
served and recorded every five days. In media induction
calli and after 30 days, the regenerated calli in regenera-
tion media was also observed every week, 8 dishes were
randomly chosen for analysis data. The total number of
calli formed and plantlet regeneration frequency (albino
and green) were recorded for analysis.
3. Results
3.1. Calli Induction from Anther Culture
Eight rice variety were tested for assessment of anther
calli induced response, anther from each genotype was
cultured separately on same N6 induction media. Of these
different genotype, total number of survived callidan
brown calli (death calli) were observed (Figure 1). The
result showed varian number of inoculated anther from
different genotype resulted different total number of calli
can be induced in this media. Among the eight respond-
ing genotype rice, almost all anther from ech genotype
can be induced to calli in this media, but the some of calli
showed not progress to grow up and marked with brown
color (Figure 1). The comparison total number brown
calli and white calli on different was showed in Figure
2(Y). In the response genotype on white calli, Dreami
2/CaMsrB2-8-DH-7 showed best response in N6 induc-
tion medium, where the frequency of calli induction was
27.9%, followed by Dreami2/CaMsrB2-8-DH-1 occur-
ring ata frequency of 26.8%. The lowest frequency of
white calli was 9.9% and 3.5% for Dreami2/CaMsrB2-
23-DH-5 at frequency and Dreami2/CaMsrB2-23-DH-2
in Figure 2(Z).
3.2. Plant Regeneration of Calli
In this study, we used regeneration media containing
different concentrations of maltose. After 4 - 6 weeks,
the calli diameter was about 1 cm, and embryogenic callies
Open Access AJPS
Effect of Maltose Concentration on Plant Regeneration of Anther
Culture with Different Genotypes in Rice (Oryza sativa L.)
Open Access AJPS
Figure 1. Calli formation at four weeks from anther culture during medium induction. A: Brown calli formation
(None-embryogenic), B: White calli formation (Embryogenic).
Figure 2. (Y) Total number of anthers inoculated and total number of brown callies induced by different genotype rice; (Z)
Percent of white calli induction by diffe rent genotype of rice. A: Dreami2/CaMsrB2-8-DH- 1, B: Dreami2/CaMsrB2-8-DH-2,
C: Dreami2/CaMsrB2-8-DH-3, D: Dreami2/CaMsrB2-8-DH-4, E: Dreami2/CaMsrB2-23-DH-2, F:
Dreami2/CaMsrB2-23-DH-5, G: Dreami2/CaMsrB2-23-DH-7, H: Dreami2/CaMsrB2-23-DH-9.
were generated upon transfer into regeneration media.
Approximately 4 weeks later, green spots appeared on
the surface of callies grown in regeneration media (Fig-
ure 3). The number of plantlets was then used as a pa-
rameter to determine the effects of different maltose
concentrations on regeneration of plantlets from callies.
Our data showed that different maltose concentrations
can influence the total number of plantlets and the albino
effect on plantlets. The different concentrations of mal-
tose had different effects on plant regeneration among
rice genotypes. On the concentration maltose 0.04 g/L
and 0.4 g/L has no major impact on the regeneration plant,
all genotype rice can generated in this media. The con-
centration maltose 4.0 g/L had indicated major impact on
the regeneration plantlet. This concentration was great ef-
fect on the some of genotype rice; however, in the contrast,
Effect of Maltose Concentration on Plant Regeneration of Anther
Culture with Different Genotypes in Rice (Oryza sativa L.)
Figure 3. Plant regeneration from anther derived callies. (A) Calli induction from anther culture at 20 days; (B) Embryogenic
callies with green spots after culture in regeneration medium; (C) Shoot regeneration from embryogenic callies approxi-
mately 28 days after culture on regeneration medium; (D) Plantlets from regenerated anthers, some of which showed green
color and others that showed no color (albino).
this effect was not great in Dreami2/CaMsrB2-23-DH-5
and Dreami2/CaMsrB2-23-DH-7, all callies could not be
generated in this concentration (Table 1). Moreover,
maltose effect on regeneration planlet was also deter-
mined by genotype of rice.
We also observed the ratio of albino plants to green
plant in response to different maltose concentrations (Ta-
ble 2). The results showed that the lowest albino plant
frequency occurred with the lowest concentration of mal-
tose 0.04 g/L. Additionally, Dreami2/CaMsrB2-8-DH-2
and Dreami2/CaMsrB2-8-DH-5 produced almost no al-
bino plants in this media, this maltose concentration in-
dicating that it could be used for plantlet generation. In
the concentration maltose 0.4 g/L, The frequency of the
total albino plants was great in some genotypes of rice
Dreami2/CaMsrB2-23-DH-1 and
Dreami2/CaMsrB2-23-DH-2. And the concentration 4.0
g/L, showed the frequency of albino plants was about
two folds greater than that of green plant in all genotype
of rice. The increased of maltose concentration have af-
fected on the total number of albino plantlet.
4. Discussion
The results showed that maltose as a carbon source used
for rice anther culture can have an effect on genetic re-
sponse. Maltose induced callies and green shoot regen-
eration in a variety of rice genotypes. Maltose could be
used to regenerate plantlets from callies, and the lowest
concentration of maltose was shown to be the best treat-
ment to obtain green plants. Increased maltose concen-
tration was associated with increased albino plants, but
the frequency of albinos varied by genotype. Dreami2/
CaMsrB2-8-DH-1, Dreami2/CaMsrB2-8-DH-2, and
Dreami2/CaMsrB2-8-DH-3 could all be regenerated on
all media investigated at different maltose concentrations.
The addition of maltose to the plant regeneration medium
appeared to have a beneficial effect beyond that seen
with maltose in the medium. However, maltose does not
appear to be essential for green shoots as they are also
formed without any carbohydrate source. The effects of
maltose and alternative carbohydrates have been docu-
mented in several tissue culture systems, but the reason
for their superiority to sucrose is not known. Other re-
searchers have indicated that maltose improves the os-
motic stability of culture medium compared to sucrose
[11]. It has also been reported that maltose stimulates
embryogenesis at low concentrations [12], which is
analogous with the finding that increased maltose con-
centrations resulted in the generation of higher numbers
of plantlets in the present study. Albino plants, which are
frequently regenerated from pollen-derived callies, are
often limiting factors in rice anther culture [13]. Addi-
tionally, the albino plant generation is specific to anther
culture [14] and only a few albino plants are generated
from somatic cells [15]. t is well known that the I
Open Access AJPS
Effect of Maltose Concentration on Plant Regeneration of Anther
Culture with Different Genotypes in Rice (Oryza sativa L.)
Table 1. Total plantlet regeneration per 100 anthers from different genotypes to various maltose concentrations.
Total number of plantletsregenerated per 100 anthers
Genotypes of rice
0.04 g/L Maltose 0.4 g/L Maltose 4 g/L Maltose
Dreami2/CaMsrB2-8-DH-1 34 ± 2.5* 30 ± 2.1 66 ± 2.5
Dreami2/CaMsrB2-8 DH-2 27 ± 2.2 19 ± 2.5 97 ± 5.1
Dreami2/CaMsrB2-8-DH-3 18 ± 1.4 37 ± 4.9 64 ± 1.7
Dreami2/CaMsrB2-23-DH-5 7 ± 1.1 8 ± 1.7 0 ± 0.0
Dreami2/CaMsrB2-23-DH-7 25 ± 3.1 14 ± 1.3 0 ± 0.0
*Mean ± SD.
Table 2. Plantlet regeneration ratio (green and albino) from different genotypes of rice to various maltose concentration.
Plantlet regeneration ratio frequency (%)
0.04 g/L Maltose 0.4 g/L Maltose 4 g/L Maltose
Genotype of rice
Normal Albino Normal Albino Normal Albino
Dreami2/CaMsrB2-8-DH-1 88.5 ± 1.4 11.4 ± 1.4 43.3 ± 0.1 56.6 ± 1.1 30.3 ± 3.5 69.6 ± 2.1
Dreami2/CaMsrB2-8 DH-2 100.0 ± 3.1 0.0 ± 0.0 31.5 ± 3.1 68.4 ± 1.5 36.0 ± 1.4 64.0 ± 2.5
Dreami2/CaMsrB2-8-DH-3 94.4 ± 3.5 5.4 ± 3.1 64.8 ± 3.5 35.1 ± 3.1 34.3 ± 4.5 65.6 ± 5.1
Dreami2/CaMsrB2-23-DH-5 100.0 ± 4.6 0.0 ± 0.0 62.5 ± 13.0 37.5 ± 2.5 0.0 ± 0.0 0.0 ± 0.0
Dreami2/CaMsrB2-23-DH-7 84.0 ± 0.4 16.0 ± 3.5 85.7 ± 6.6 14.2 ± 2.2 0.0 ± 0.0 0.0 ± 0.0
Average 93.4 ± 2.6 6.6 ± 1.6 57.6 ± 5.3 42.4 ± 2.1 33.6 ± 1.9 66.4 ± 1.9
frequency of albino plants during anther cultures in rice
is controlled by nuclear genes [16]. The results of the
present study indicated a correlation between the maltose
concentration in media and frequency of albino plants.
Specifically, application of the lowest concentration of
maltose minimized the frequency of albino plants from
anthers, but they still occurred. Overall, the results of this
study can be used as a reference for development of ad-
ditional anther culture techniques.
5. Acknowledgements
This work was supported by a grant from the Next-Gen-
eration BioGreen 21 Program (No. PJ0080912013), Ru-
ral Development Administration, Republic of Korea.
[1] S. K. Raina, “Doubled Haploid Breeding in Cereals,” In:
J. Janick, Ed., Plant Breeding Reviews, Vol. 15, John
Wiley & Sons, Inc., Canada, 1997, pp. 141-186.
[2] Q. F. Chen, C. L. Wang, Y. M. Lu, M. Shen, R. Afza, M.
V. Duren and H. Brunner, “Anther Culture in Connection
with Induced Mutations for Rice Improvement,” Euphytica,
Vol. 120, No. 3, 2001, pp. 401-408.
[3] T. He, Y. Yang, S. B. Tu, M. Q. Yu and X. F. Li, “Selec-
tion of Interspecific Hybrids for Anther Culture of Indica
Rice,” Plan Cell Tissue and Organ Culture, Vol. 86, No.
2, 2006, pp. 271-277.
[4] D. Y. Zhu, X. H. Ding, J. H. Ying, N. Q. Jie and H. L.
Xiong, “Genetic Studies on Anther Culture Ability of In-
dica Rice,” In: H. Hu and L. H. Wang (Eds.), Plant Cell-
engineering and Crop Improvement, Beijing Industrial
University Press, Beijing, 1990, pp. 38-43.
[5] J. H. Shen, M. F. Li, Y. Q. Chen and Z. H. Zhang,
“Breeding by Anther Culture in Rice Varieties Improve-
ments,” Scientia Agricultura Sinica, Vol. 2, No. 2, 1982,
pp. 15-19.
[6] M. F. Li, “Anther Culture Breeding of Rice,” In: C. J.
Yan (Ed.), Tissue Culture of Field crops, Shanghai Scien-
tific and Technical Publishers, Shanghai, 1991, pp. 135-
[7] Y. Chen, “In Vitro Development of Plant from Micro-
spores of Rice,” In: H. Hu and Y. Chen (Eds.), Plant So-
matic Genetics and Crop Improvement, Beijing University
Press, Beijing, 1988, pp. 27-67.
[8] R. D. Qu and Y. Chen, “A Preliminary Research on the
Function of Enhancement of Calli Induction Frequency
by Cold Pretreatment in Rice Anther Culture,” Acta Phy-
tophysiologica Sinica, Vol. 9, No. 4, 1983, pp. 375-381.
[9] J. Asad, Q. M. Hassan, T. Fatima and T. Hasnain, “Tissue
Culture Response of Local Varieties of Rice (Oryza sativa
L.) of NWFP,” Journal of Biological Sciences, Vol. 1, No.
5, 2001, pp. 387-390.
[10] J. M. She, B. L. Zhang and W. C. Ni, “Factors Affecting
Plant Regeneration from Calli of Mature Embryo of
‘Yangdao 6’,” Jiangsu Journal of Agricultural Sciences,
Open Access AJPS
Effect of Maltose Concentration on Plant Regeneration of Anther
Culture with Different Genotypes in Rice (Oryza sativa L.)
Vol. 18, No. 4, 2002, pp. 199-202.
[11] U. Kuhlmann and B. Foroughi-Wehr, “Production of
Double d Haploid Line in Frequencies Sufficient of Bar-
ley Breeding Program,” Plant Cell Reports, Vol. 8, No. 2,
1989, pp. 78-81.
[12] S. D. Strickland, J. W. Nichol, C. M. McCall and D. A.
Stuart, “Effect of Carbohydrate Source on Alfalfa Somatic
Embryogenesis,” Plant Science, Vol. 48, No. 2, 1987, pp.
[13] M. Yamagishi, “Heterogeneous Plastid Genomes in An-
ther Culture-Derived Albinorice Plants,” Euphytica, Vol.
123, No. 1, 2002, pp. 67-74.
[14] H. Niizeki and K. Oono, “Induction of Haploid Rice Plant
from Anther Culture,” Proceedings of the Japan Academy,
Vol. 44, No. 6, 1968, pp. 554-557.
[15] M. Tsukahara, T. Hirosawa and H. Murayama, “Effect of
Culture Methods on the Regeneration of Albino Rice
(Oryza sativa L.) Plantlets,” Plant Cell Reports, Vol. 15,
No. 8, 1996, pp. 597-600.
[16] C. A. Quimio and F. J. Zapata, “Diallel Analysis of Calli
Induction and Green-Plant Regeneration in Rice Anther
Culture,” Crop Science, Vol. 30, No. 1, 1990, pp. 188-
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