A Dube

E P Koshy

R Shukla

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Save Nature to Survive
9(3): 1077-1080, 2014
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1077
PRODUCTION OF HIGH QUALITY EMBRYOGENIC CALLUS OF RICE
R. SHUKLA1*, A. DUBE2  AND  E. P. KOSHY1
1Department of Tissue Engineering, Jacob School of Biotechnology and Bioengineering,
Sam Higginbottom  Institute of Agriculture Technology and Sciences, Allahabad 211007, U.P., INDIA
2Department of Biochemistry, College of Agriculture,
N.D. University of Agriculture and Technology, Kumarganj, Faizabad - 224 229, U.P., INDIA
e-mail: rahulbio07@hotmail.com
INTRODUCTION
Rice (Oryza sativa L.) is one of the most versatile cereal food
crop and it is the primary source of food and calories for
about half of the world population (Vega et al., 2009). India is
among the largest rice growing countries accounting for about
one third of the world acreage under the crop.During past few
decades, biotechnological techniques such as somaclonal
variation, in vitro selection, production of doubled haploid
lines from anther culture and genetic transformation are being
employed in ricedevelopment for the creation of novel rice
varieties. A mature rice seed, compared with immature tissues,
which contain a large number of actively dividing cells, is
suitable to induce embryogenic calli , it has distinct advantages
in practical experiments because it is readily available
throughout the year (Carsono and Yoshida, 2006). Maeda et
al. (2002) reported that white and green patches are often
seen on the surface stratum of callus with high regeneration
ability. Lee et al. (2002) found that the number, colour, size,
shape and appearance of the embryogenic calluses varied
among the rice genotypes depending upon the basal medium,
indicating that induction of high-quality rice callus influenced
by genotype, medium, and the kind of explants as well as
their interactions. Callus induction and regeneration is still a
challenging task in most rice varieties. Keeping the above facts
in view, the present study was investigated  to develop a
reproducible and an efficient procedure for callus induction
and plant regeneration of embryogenic calli from mature seeds
of SwarnaSub1 for future genetic transformation studies.
MATERIALS AND METHODS
The nuclear seeds of Oryza sativa L. cultivar Swarna Sub1
were collected from Department of Seed Technology, Narendra
Dev University of Agriculture and Technology ,Kumarganj,
Faizabad, Uttar Pradesh. The rice husks were removed from
the seeds and were washed for 20 minutes in running tap
water for removal of dust followed by disinfecting with
laboratory detergent (Domex , 15%) solution for 15-20 minutes
and also with indophil (0.1% fungicide) for five minutes. The
mature seeds were disinfected with 0.1% HgC1
2
 for five
minutes and finally rinsed with sterilized distilled water.
Murashige and Skoog (MS, 1962), Gamborg et al. (B
5
, 1968)
and Nitsch and Nitsch (N and N, 1969) media with varying
concentrations of plant growth regulator 2,4-D (2,4-
Dichlorophenoxyacetic acid) were used for callus induction
whereas MS medium supplemented with BAP, NAA and KIN
at various concentrations was used for plantlets regeneration.
RESULTS AND DISCUSSION
Callus induction and growth
Media for the present study was initially standardized by
inoculating seeds in MS, B
5
 and N and N media fortified with
2, 4-D (2 mg/L) for checking their callus induction efficiency
(Table 1). As MS media gave better results than B
5
 and N and
N media, MS media was selected for further studies. The rice
callus can be induced and grown on both MS and N and N
media (Rashid et al., 2004) but the low response in terms of
callusing as well as supporting regeneration from explants of
N and N and B
5 
media may be due to their lower N
2
 content
(Jubair et al., 2008). Pandey et al. (1994) reported that the
ABSTRACT
The study was undertaken to standardize an efficient and effective protocol for callus Induction, subsequent
growth and regeneration in rice variety Swarna Sub1.MS, B5 and N&N media were used for callus
induction.Overall,MS medium was found better for callus induction as compared to N&N medium. Callus
induction percentage was highest in SS29 (80.00) followed by SS4 which gave 76.67%, SS32 and SS44 gave
66.67%.  The growth Regulator 2, 4-D with varying concentrations were tested for callus induction. Production
of embryogenic calli increased as the concentration of 2, 4-D was increased from 0.5 mg/L to 1.5 mg/L.
KEYWORDS
Rice
SwanaSub1
embryogenic calli
2, 4-D and tissue culture
Received on :
20.12.2013
Accepted on :
04.07.2014
*Corresponding
author
1078
R. SHUKLA et al.,
Table 1: Selection of media for callus induction and growth in Swarna Sub1
Media Hormone Response
2,4-D (mg/l) Colour Type Appearance Callus induction
MS 2.00 Light Yellow Highly compact Smooth, oily Profuse
B
5
2.00 Yellow Friable Rough dry Scarce
N&N 2.00 Brown Friable Rough dry Scarce
- = no callusing; + = meager callusing; ++ = moderate callusing; +++ = high callusing; ++++ = profuse callusing; Values are means of 3 replicates.  Mean values followed by
the same letters are not significantly different at p ³ 0.05 DMRT.
Table 2: Effect of different plant hormones on rice callus induction, formation rate, appearance, colour and type
Media Code Hormone Concentration Induction % Formation rate Appearance Callus Colour Callus Type
SS1 M.S. Basal 0.00 ±0.00k - - - -
SS2 0.5mg/L 2,4D 43.33 ±4.71efg ++ Dry Light Yellow Compact
SS3 1.0 mg/L 2,4D 56.67 ±4.71cde +++ Oily Brownish Yellow Compact
SS4 1.5 mg/L 2,4D 76.67 ±4.71ab ++++ Oily Light Yellow Compact
SS5 2.0 mg/L 2,4D 63.33 ±4.71bcd ++++ Oily Light Yellow Compact
SS6 2.5 mg/L 2,4D 56.67 ±4.71cde +++ Oily Light Yellow Compact
SS7 3.0 mg/L 2,4D 56.67 ±4.71cde +++ Oily Dark Yellow Compact
SS8 3.5 mg/L 2,4D 46.67 ±4.71ef ++ Dry Yellow Compact
SS9 4.0 mg/L 2,4D 43.33 ±4.71efg ++ Dry Yellow Compact
SS10 4.5 mg/L 2,4D 43.33 ±4.71efg ++ Oily Blackish Brown Compact
SS11 5.0 mg/L 2,4D 30.00 ±8.16ghi + Oily Black Compact
SS12 0.5 mg/L 2,4-D + 0.5mg/L  NAA 16.67 ±9.47ij + Dry Light Yellow Compact
SS13 1.0 mg/L 2,4-D +0.5mg/L   NAA 26.67 ±4.71hij + Oily Brownish Yellow Compact
SS14 1.5 mg/L 2,4-D +0.5mg/L   NAA 30.00 ±8.16ghi + Dry Brownish Yellow Friable
SS15 2.0 mg/L 2,4-D +0.5mg/L   NAA 30.00 ±8.16ghi + Dry Brownish Yellow Friable
SS16 2.5 mg/L 2,4-D +0.5mg/L   NAA 26.67 ±4.71hij + Dry Black Compact
SS17 0.5 mg/L 2,4-D +1.0mg/L   NAA 43.33 ±4.71efg ++ Dry Brownish Yellow Compact
SS18 1.0 mg/L 2,4-D +  1.0mg/L NAA 63.33 ±4.71bcd ++++ Oily Light Yellow Compact
SS19 1.5 mg/L 2,4-D + 1.0mg/L  NAA 46.67 ±4.71ef +++ Oily Light Yellow Compact
SS20 2.0 mg/L 2,4-D + 1.0mg/L  NAA 13.33 ±9.43jk + Oily Brownish Yellow Friable
SS21 2.5 mg/L 2,4-D + 1.0mg/L  NAA 56.67 ±4.71cde +++ Oily Light Yellow Compact
SS22 0.5 mg/L 2,4-D +  1.5mg/L  NAA 30.00 ±8.16ghi + Oily Deadly Brown Compact
SS23 1.0 mg/L 2,4-D + 1.5mg/L   NAA 30.00 ±8.16ghi + Oily Brownish Yellow Compact
SS24 1.5 mg/L 2,4-D +1.5mg/L   NAA 26.67 ±4.71hij + Dry Light Brown Friable
SS25 2.0 mg/L 2,4-D + 1.5mg/L   NAA 16.67 ±9.47ij + Dry Brownish Yellow Friable
SS26 2.5 mg/L 2,4-D + 1.5mg/L   NAA 16.67 ±9.47ij + Dry Brownish Yellow Friable
SS27 0.5 mg/L 2,4-D +  0.5mg/L   BAP 43.33 ±4.71efg ++ Oily Brownish Yellow Compact
SS28 1.0 mg/L 2,4-D + 0.5mg/L   BAP 50.00 ±8.16def ++ Oily Brownish Yellow Friable
SS29 1.5 mg/L 2,4-D + 0.5mg/L   BAP 80.00 ±0.00a ++++ Oily Light Yellow Compact
SS30 2.0 mg/L 2,4-D + 0.5mg/L   BAP 56.67 ±4.71cde +++ Oily Brownish Yellow Friable
SS31 2.5 mg/L 2,4-D,    0.5mg/L   BAP 53.33 ±4.71cde +++ Oily Deadly Brown Friable
SS32 0.5 mg/L 2,4-D + 1.0mg/L   BAP 50.00 ±8.16def ++ Dry Dark Yellow Friable
SS33 1.0 mg/L 2,4-D + 1.0mg/L   BAP 66.67 ±4.71abc ++++ Oily Light Yellow Compact
SS34 1.5 mg/L 2,4-D +  1.0mg/L  BAP 36.67 ±8.47fgh ++ Oily Deadly Brown Friable
SS35 2.0 mg/L 2,4-D + 1.0mg/L   BAP 26.67 ±4.71hij + Oily Brownish Yellow Friable
SS36 2.5 mg/L 2,4-D + 1.0mg/L   BAP 26.67 ±4.71hij + Oily Brownish Yellow Friable
SS37 0.5 mg/L 2,4-D + 1.5mg/L   BAP 43.33 ±4.71efg ++ Oily Dark Yellow Compact
SS38 1.0 mg/L 2,4-D + 1.5mg/L   BAP 63.33 ±4.71bcd ++++ Oily Light Yellow Compact
SS39 1.5 mg/L 2,4-D + 1.5mg/L   BAP 46.67 ±4.71ef ++ Dry Deadly Brown Compact
SS40 2.0 mg/L 2,4-D + 1.5mg/L   BAP 43.33 ±4.71efg ++ Dry Light Yellow Friable
SS41 2.5 mg/L 2,4-D +  1.5mg/L  BAP 43.33 ±4.71efg ++ Dry Light Yellow Friable
SS42 0.5 mg/L 2,4-D +  0.5mg/L  Kin 53.33 ±9.47cde +++ Oily Light Yellow Compact
SS43 1.0 mg/L 2,4-D +  0.5mg/L  Kin 56.67 ±4.71cde +++ Dry Dark Yellow Friable
SS44 1.5 mg/L 2,4-D + 0.5mg/L   Kin 66.67 ±9.43abc ++++ Oily Light Yellow Compact
SS45 2.0 mg/L 2,4-D + 0.5mg/L   Kin 53.33 ±4.71cde +++ Oily Brownish Yellow Compact
SS46 2.5 mg/L 2,4-D +  0.5mg/L  Kin 43.33 ±4.71efg ++ Dry Brownish Yellow Friable
SS47 0.5 mg/L 2,4-D +  1.0mg/L  Kin 13.33 ±9.43jk + Oily Light Brown Compact
SS48 1.0 mg/L 2,4-D + 1.0mg/L   Kin 16.67 ±9.47ij + Dry Light Yellow Friable
SS49 1.5 mg/L 2,4-D + 1.0mg/L   Kin 36.67 ±4.71fgh ++ Oily Brownish Yellow Compact
SS50 2.0 mg/L 2,4-D + 1.0mg/L   Kin 56.00 ±4.71cde +++ Oily Deadly Brown Compact
SS51 2.5 mg/L 2,4-D + 1.0mg/L   Kin 26.67 ±9.43hij + Oily Brownish Yellow Compact
SS52 0.5 mg/L 2,4-D + 1.5mg/L   Kin 23.33 ±4.71hij + Oily Brownish Yellow Compact
SS53 1.0 mg/L 2,4-D + 1.5mg/L   Kin 46.67 ±4.71ef + Oily Deadly Brown Compact
SS54 1.5 mg/L 2,4-D + 1.5mg/L   Kin 56.00 ±4.71cde +++ Oily Brownish Yellow Compact
SS55 2.0 mg/L 2,4-D + 1.5mg/L   Kin 50.00 ±8.16def ++ Oily Brownish Yellow Compact
SS56 2.5 mg/L 2,4-D + 1.5mg/L   Kin 63.33 ±4.71bcd ++++ Oily Light Yellow Compact
1079
PRODUCTION OF HIGH QUALITY EMBRYOGENIC CALLUS OF RICE
success of in vitro culture largely depends on the nutritional
media, growth regulators, genotypes and the interaction of
genotype with the medium.
Whereas in MS media, callus induction percentage was highest
in SS29 (80.00) followed by SS4 which gave 76.67%, SS32
and SS44 gave 66.67% (Table 2). Production of
embryogeniccalli increased as the concentration of 2, 4-D
was increased from 0.5 mg/L to 1.5 mg/L. Before now, it was
reported that among the auxins, 2,4-D increase the rate of cell
division and this attributes to increased amount of callus and
to initiate and sustain embryogenic callus growth in rice
(Wagiran et al., 2008; Revathi and Pillai, 2011), when applied
at low concentration (2 mg/L 2,4-D and or 1.5 and 2.5 mg/L
2,4-D) showed high callus induction percentage in all the rice
dehulled seeds (Tam and Lang, 2003). However, the
combination of 2, 4-D with kinetin was more effective in
producing embryogenic and or organogeniccalli (Wang et
al., 2004), when addition of NAA and or BAP could enhance
the quality of the initiated callus (Turhan and Baser, 2004).
While cytokinin may increase the growth rate of pre-
embryogenic masses (Kommamine et al., 1992). Results
obtained by Rashid et al. (2009) that increase in the
concentration of 2, 4-D and kinetin reduced the percentage of
embryogenic calli production. Moreover, difference in callus
proliferation rate between different auxins may be due to the
difference in the physiological activity of the auxins (Wagiran
et al., 2008) and differences in response of genotypes,
especially even when carried out after a short time of callus
maintenance (Muhammad et al., 2005; Htwe et al., 2011).
The embryogeniccalli were relatively smooth (oily), compact,
nodular in appearance and milky white (yellowish) in colour
(Rachmawati and Anzai, 2006; Mahmood et al., 2012). Yellow
or milky white colour of the 2, 4-D derived callus might be
due to the inhibitory effect of 2, 4-D on chlorophyll formation.
Our observations are  confirmed with Li et al. (2009) and
Narciso and Hattori, (2010) who reported that embryogenic
type of callus in indica rice varieties was characterized as
yellowish, compact, smooth, big in size and iso-diametric cells
termed as embryogenically active. Embryogeniccalli grew
about 5 to 10-fold in volume 30 days after transfer. However,
the non embryogeniccalli turned brown and died.
In the present study, a combination of BAP along with 2, 4-D
was found to be superior to kinetin + 2, 4-D in callus induction.
Embryogeniccalli being composed of individual compact and
spherical cells that were rather regular in size and tightly held
together and appeared to organize globular embryos in small
compact clusters. By comparison, non embryogeniccalli
showed elongated and loosely held cells on the surface.
Fazelienasab et al. (2004) reported that the compact type of
callus indicates the unorganized cell division of tissues with
highly viable cells for further growth of culture. These
observations are in close conformation with Haeyoung et al.
(2007), Singh et al. (2009) and Rashid et al. (2009).
Callus induction is the first step in rice regeneration to obtain
high quality of embryogeniccalli because of the efficiency of
regeneration of a plant is highly dependent on the quality of
the calli. Rachmawati et al. (2004) visually selected the
embryogenic rice calli on the basis of their appearance. The
embryogenic callus was relatively compact, oily and nodular
in appearance. On the basis of performance of callus on the
above characters, callus obtained from eleven treatments were
selected for further studies, which are SS4, SS5, SS18, SS19,
SS21, SS29, SS33, SS38, SS42, SS44 and SS56.
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R. SHUKLA et al.,


PRODUCTION OF HIGH QUALITY EMBRYOGENIC CALLUS OF RICE

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PRODUCTION OF HIGH QUALITY EMBRYOGENIC CALLUS OF RICE

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