Development of callus initiation and regeneration system of different indigenous indica rice varieties

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Research Paper
Development of Callus Initiation and Regeneration
System of Different Indigenous indica Rice Varieties
Kazi Muhammad Ahasanul Hoque*, Z.A. Azdi and Shamsul H. Prodhan
Depart. of Genetic Engineering and Biotechnology, Shahjalal University of Sci. and Tech., Sylhet 3114, Bangladesh
*E-Mail: shaown9009@gmail.com
Abstract
The aim of the present study was to develop an efficient protocol for best callus induction and complete plant regeneration
for varieties of rice (Oryza sativa) i.e. Pakhi Biroin, Hati Baromashi, Kacha Biroin, Badal Boro, Porichok Amon, Khoiya
Boro, Joria Aman and BRRI Dhan53. Pakhi Biroin, HatiBaromashi, BadalBoro, Porichok Amon, KhoiyaBoro and Joria
Aman cultivate mainly in Sylhet & Sunamgonj district of Bangladesh. For callus induction from mature dehusked rice
seeds MS (Murashige & Skoog, 1962) media supplemented with different concentrations i.e. (1.5, 2.0, 2.5, 3.0 mg/L of 2,4-
D (2,4 dichlorophenoxyacetic acid)) were used. Khoiya Boro and Joria Aman did not show any efficiency in callus
induction. Maximum percentage of callus induction efficiency that is 100% was noted for Pakhi Biroin, Hati Baromashi
and BRRI Dhan53, but 75% was the highest percentage of callus for Kacha Biroin. For plantlet regeneration of Pakhi
Biroin, Hati Baromashi and Kacha Biroin, MS media with 0.5 mg/L NAA (1-Napthaleneacetic acid), 0.5 mg/L Kinetin and
various concentrations of BA (6-benzyladenine) (1.0, 2.0, 3.0, 4.0 mg/L) were employed. On the other hand, MS media
with 1.0 mg/L NAA, 1.0 mg/L Kinetin and various concentrations of BA (1.0, 2.0, 3.0, 4.0 mg/L) were used for BRRI
Dhan53 regeneration. Different result was recorded for different varieties at various hormone concentrations.
Keywords: Mature Embryos, Genotypic Variability, Embryogenic Callus, Plant Regeneration, Indica Rice
1. Introduction
Rice (Oryza sativa L) is one of the most versatile and
important cereal crops of Poaceae family cultivated for
more than 10,000 years (Sasaki, 2001). Currently this crop
supports more than 50% of the world population (Christou,
1997). In Asia it covers half of the arable land used for
agriculture in many countries (Cantrell & Hettel, 2004).
But the rate of growth in rice production has slowed down.
A considerable improvement has already been made by
exploiting the natural variation through conventional
breeding. Recent advancement in biotechn-ology, such as
transformation and In situ and In vitro hybridization has
enhanced the introgression of new genes from different
sources to the cultivated species (Sikder et al, 2006).
Efficient plant regeneration from cultured cells and tissues
requires successful application of biotechnology in crop
improvement. Therefore, the success of cell and tissue
culture research depends upon reliable callus cult-ure and
plant regeneration procedures. The frequencies of callus
induction and plant regeneration in tissue cultu-re of rice
are influenced by many factors: culture medi-um
composition explants source, genotype and environ-ment
(Torbert et al, 1998). Among them the genotype and
nutrient composition are regarded to be the major sources
of variation in in vitro culture (Khanna & Raina, 1998).
Pakhi Biroin, Hati Bromashi, Kacha Biroin, Badal Boro,
Porichok Aman, Khoiya Boro and Joria Aman are pure
varieties of Oryza sativa cultured mainly in Sylhet and
Sunamgonj, district of Bangladesh. BRRI Dhan53 are
hybrid rice variety from BRRI. It has moderate salt tolera-
nce.
Objective of the present research was to study the

Haque et al / Journal of Biology (2013), Vol. 01, Issue 02, pp. 46-51
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47
potentiality of the varieties in tissue culture as well as to
determine the most suitable concentration and combinat-
ion of growth regulators for excellent callus induction and
regeneration which is of great impotence for gene
transformation to create high yielding varieties.
2. Material and Method
This research work was conducted at the plant genetic
engineering lab of Department of Genetic Engineering and
Biotechnology, Shahjalal University of Science &
Technology (SUST), Sylhet, Bangladesh.
Mature Dehusked rice seeds were taken as source of
explant. Dehusked seeds were first washed in distilled
water mixed with Tween 20 (one drop/30 ml of water) for
ten minutes, steeped in 70% (v/v) ethanol for two minutes
with gentle agitation followed by rinsing three times with
sterile distilled water. The seeds were surface sterilized in
0.1% (w/v) HgCl
2
for 15 minutes with gentle agitation and
rinsed five times with sterile distilled water. Surface
sterilized seeds were thoroughly washed six to ten times
with autoclaved sterile distilled water. The seeds were
finally placed on the sterilized petriplate having sterile
filter papers with the help of forceps to remove excess
water.
After removing the water from the seeds surface, these
seeds were inoculated into culture tubes containing MS
(Murashige & Skoog, 1962) basal media supplemented
with different concentrations of 2, 4-dichlorophenoxyac-
etic acid (2,4-D) culture tubes were transferred and
maintained in an environmentally controlled growth room
for 4 weeks for callus and subcultured in the same medium
for 3 weeks. Regeneration efficacy was observ-ed with MS
media supplemented with different combina-tion and
concentration of NAA, Kn (Kinetin) and BA. The pH of
the media was adjusted to 5-8 with acid and alkali. The
media was autoclaved at 15 pound square inch (psi) for 20
minutes at 121 °C. Inoculation was carried out under a
sterilized environment in a laminar air flow cabinet. All
cultures were incubated at 25±1 °C with a photoperiod of
12 hours at 2000 lux light intensity of cool white
fluorescent light.
The response of all varieties of rice was determined in
terms of callus induction, callus growth and regeneration
frequencies. The data of callus growth was subjected to
ANOVA (Analysis of Variance) testing and Standard error
(SE) for 5% of means was calculated by using standard
statistical MSTAT-C software.
3. Result
Results are shows in Figure 1-4 and Tables 1-3. Callus
induction of Dehusked rice seeds of eight varieties i.e.
Figure 1. Callus Induction A) Pakhi Biroin on MS Medium with 2.5
mg/l 2,4-D B) Hati Bromashi on MS Medium with 2 mg/l
2,4-D C) Kacha Biroin on MS Medium with 2 mg/l 2,4-D
D) BRRI dhan53 on MS Medium with 2 mg/l 2,4-D
Pakhi Biroin, Hati Bromashi, Kacha Biroin, Badal Boro,
Porichok Aman, Khoiya Boro, Joria Aman and BRRI
Dhan53, was carried out on MS medium forti-fied with
different concentrations of 2,4-D (for landraces varieties
1.5 mg/l, 2 mg/l, 2.5 mg/l, 3 mg/l and for BRRI Dhan53,
1mg/l, 2mg/l and 3mg/l) (Figure 1). Badal Boro, Porichok
Amon, Khoiya Boro and Joria Aman did not show any
callus after 4 weeks. Calli were developed within 10 days
of inoculation of Pakhi Biroin, Hati Bromashi, Kacha
Biroin and BRRI Dhan53. Both embry-ogenic and non-
embryogenic calli were initiated. After four weeks, large
calli were formed from the scutellum.
The frequency of callus initiation were calculated as Pakhi
Biroin 75%, Hati Bromashi 100%, Kacha Biroin 43.75%
Figure 2. Subculture Callus A) Pakhi Biroin on MS Medium with 2.5
mg/l 2,4-D B) Hati Bromashi on MS Medium with 2 mg/l
2,4-D C) Kacha Biroin on MS Medium with 2 mg/l 2,4-D
D) BRRI dhan53 on MS Medium with 2 mg/l 2,4-D

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and BRRI Dhan53 79.16%. The first subculture was, then,
carried out and calli were removed from seed endosperms
and transferred onto fresh media (Figure 2).
The response of explants to different concentrations of 2,
4-D in terms of callus induction, degree of callusing
including the callus growth rate is shown in Table 1 and
Table 2.
Upon transferring the calli to regeneration media, green
spots were visible on the calli within 7-10 (Figure 3) days
and after 4-5 weeks fully regenerated roots and shoots
were observed (Figure 4). At every treatment, regeneration
of roots, shoots, and the frequency were noted as shown in
Table 3.
4. Discussion
Mature Dehusked rice seeds were used as an explants
because calli initiated from scutellum of mature seeds of
all rice varieties have high embryogenic potential (Ge et al,
2006 and Khaleda & Al-Forkan, 2006) and was excellent
material for transformation of rice by Agrobacterium
(Rashid et al, 1996; Toki, 1997; Rashid et al, 2001; 2003;
Cho et al, 2004 and Ge et al, 2006). In this study, by
offering suitable growth regulators a great number of
embryogenic calli from mature seeds of four indica
varieties i.e. Pakhi Biroin, Hati Baromashi, Kacha Biroin
and BRRI Dhan53, were successfully induced.
MS medium was used as basal media as MS and N6
(Cho’s N6 basal media) were the most commonly used
basal media (Pandey et al, 1994; Rashid et al, 1996 and
Toki, 1997). Mostly 2,4-D has been used as the only
growth regulator in callus induction media (Katiyar et al,
1999 and Zhenyu et al, 1999). The present study showed
that the MS medium supplemented with 2,4-D alone
enables the production of calli from the seeds of Pakhi
Table 1. Effect of Callus Induction of Pakhi Biroin, Hati Bromashi and Kacha Biroin Varieties on MS Supplemented with Different
Concentrations of 2, 4-D
Concentration
of 2,4-D (mg/l) Varieties Frequency of Callus
Initiation (%) Degree of
Callus Callus Growth Rate on 1
st
Subculture
in Size ± SE (mm/week)
Pakhi Biroin 50 + 1.61±0.11
Hati Bromashi 100 + 1.61±0.35
1.5
Kacha Biroin 0 - 0
Pakhi Biroin 75 +++ 1.97±0.23
Hati Bromashi 100 ++++ 2.13±0.16
2
Kacha Biroin 62.5 ++++ 1.56±0.13
Pakhi Biroin 100 ++++ 2.18±0.13
Hati Bromashi 100 +++ 1.63±0.62
2.5
Kacha Biroin 62.5 ++ 1.50±0.83
Pakhi Biroin 75 ++ 1.83±0.35
Hati Bromashi 100 + 1.58±0.18
3
Kacha Biroin 52 + 1.41±0.14
Size of Callus = (width + length)/2
Table 2. Effect of Callus Induction of BRRI dhan53 Variety on MS Supplemented with Different Concentrations of 2,4-D
Concentration
of 2,4-D (mg/l) Frequency of Callus Initiation (%) Degree of Callus Callus Growth Rate on 1
st
Subculture in Size± SE (mm/week)
1 66.66 ++ 1.90±0.28
2 100 ++++ 2.36±0.12
3 100 +++ 2.14±0.26
4 50 + 2.13±0.28
Size of Callus = (width + length)/2

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Biroin, Hati Baromashi, Kacha Biroin & BRRI Dhan53.
Similar results were reported by Ge et al (2006) for the
varieties of Zhenshan 97, Minghui 63, and 93-11. These
findings are as per the reports of several researchers
(Jubair et al, 2008; Summart et al, 2008 and Tariq et al,
2008).
Figure 3. Plantlet Regenerate Initiation A) Pakhi Biroin on MS
Medium with 2 mg/l BA + 0.5 mg/l NAA + 0.5 mg/l Kn B)
Hati Bromashi on MS Medium with 3 mg/l BA + 0.5 mg/l
NAA + 0.5 mg/l Kn C) Kacha Biroin on MS Medium with
3 mg/l BA + 0.5 mg/l NAA + 0.5mg/l Kn D) BRRI dhan53
on MS Medium with 2 mg/l BA + 1 mg/l NAA + 1 mg/l Kn
We have found that the Hati Bromashi produced 100%
callus, BRRI Dhan53 produced 79.16% callus and Pakhi
Biroin produced 75% callus. Similar result was also report-
ed by Kabir et al (2008) for BRRI Dhan32 variety. They
also reported that BRRI Dhan29 produced 69.45% callus
which is relevant to Kacha Biroin variety produced
43.75% callus. Saharan et al (2004) observed callus induc-
tion frequency was 60.5% in cv. HKR-46, whereas in
HKR-126 it was 83.5 per cent. This finding was also
similar with our results.
Figure 4. Plantlet Regenerated A) Pakhi Biroin on MS Medium with
2 mg/l BA + 0.5 mg/l NAA + 0.5 mg/l Kn B) Hati Bromashi
on MS Medium with 3 mg/l BA + 0.5 mg/l NAA + 0.5 mg/l
Kn C) Kacha Biroin on MS Medium with 3 mg/l BA + 0.5
mg/l NAA + 0.5 mg/l Kn D) BRRI dhan53 on MS Medium
with 2 mg/l BA + 1 mg/l NAA + 1 mg/l Kn
Three varieties i.e. Hati Bromashi, Kacha Biroin, BRRI
Table 3. Regeneration Frequency of Pakhi Biroin, Hati Bromashi, Kacha Biroin, and BRRI dhan53 Varieties on MS Supplemented with
Different Hormonal Combinations and Concentrations
Pakhi Biroin, Hati Bromashi and Kacha Biroin BRRI dhan53
Concentration of Growth
Regulator (BA+NAA+Kn)
(mg/l) Varieties Frequency of
Regeneration
(%)
Concentration of Growth
Regulator (BA+NAA+Kn)
(mg/l)
Frequency of
Regeneration
(%)
Pakhi Biroin 0
Hati Bromashi 0
1 + 0.5 + 0.5
Kacha Biroin 0
1 + 1 + 1 50
Pakhi Biroin 75
Hati Bromashi 50
2 + 0.5 + 0.5
Kacha Biroin 25
2 + 1 + 1 25
Pakhi Biroin 50
Hati Bromashi 75
3 + 0.5 + 0.5
Kacha Biroin 75
3 + 1 + 1 75
Pakhi Biroin 25
Hati Bromashi 25
4 + 0.5 + 0.5
Kacha Biroin 25
4 + 1 + 1 50

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Dhan53 showed better callus induction response on MS
medium supplemented with 2mg/l 2,4-D similar to Pandey
et al (1994). They founded that MS medium, supplemented
with 2.0 mg/l 2, 4-D, produced the most desired calli for
10 rice genotypes. Other researchers (Islam et al, 2005 and
Khalequzzaman et al, 2005) found better callusing freque-
ncy at a concentration of 2.5 mg/L 2,4-D. It coincides with
our findings as we found that Pakhi Biroin shows better
callusing frequency at a concentration of 2.5 mg/l 2,4-D
and embryogenic calli obtained from mature seed explant
have high regeneration capacity (Khalequzzaman et al,
2005). Combinations of auxin and cytokinin along with the
effect of basal salts played an important role for plant
regeneration (Prodhan et al, 2001 and Lee et al, 2002). In
this study we found Pakhibiroin shows best results at the
concentration of NAA 0.5 mg/l + BA 2 mg/l + Kinetin 0.5
mg/l, Hati Baromashi and Kacha Biroin shows best results
at NAA 0.5 mg/l + BA 3 mg/l + Kinetin 0.5 mg/l and
BRRI Dhan53 shows best regeneration frequency at, MS +
NAA 1 mg/l + BA 2 mg/l + Kinetin 3 mg/l which are simi-
lar to Jubair et al (2008) study, in which they found best
regeneration frequency for local variety Topa at MS +
NAA 0.5 mg/l + BA 3 mg/l + Kinetin 0.5 mg/l.
5. Conclusion
The investigation was conducted for establishment of
callus initiation and regeneration system for these local
rice varieties using mature seed embryo as explants. This
highly efficient, reproducible system will be used to devel-
op genetic transformation techniques for these important
rice cultivars.
Acknowledgement
The authors gratefully acknowledge to the Plant Genetic
Engineering Lab of Depart. Genetic Engineering and
Biotechnology, Shahjalal University of Sci. and Tech. who
gave all the lab facilities.
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