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PLANT TISSUE CULTURE
Histo-morphological analysis of rice callus cultures reveals
differential regeneration response with varying media combinations
Bushra Ijaz
1
&Cristina Sudiro
2
&Muhammad Zeeshan Hyder
1
&Saad Imran Malik
3
&Sumaira Farrakh
1
&
Fiorella Lo Schiavo
2
&Tayyaba Yasmin
1
Received: 1 November 2017 /Accepted: 8 March 2019 / Editor: Yong Eun Choi
#The Society for In Vitro Biology 2019
Abstract
Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low
regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and
can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake
histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen
Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supple-
mented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L
Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by exam-
ining toulidine blue stained 5–8μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features.
Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli.
However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all
rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and
KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological
observations where characteristic embryogenic units (EU) were noticed in thesegenotypes. These meristematic regions displayed
high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent
nuclei and were located on the callus surface or inside surrounded by parenchymal cells.
Keywords Indica rice .In vitro regeneration .Oryza sativa .Histological analysis
Introduction
Rice (Oryza sativa) is the second most important cereal grain
being consumed by a large part of the world’s population
(Kumar et al. 2013). Breeding efforts in the past have been
able to tailor rice genotypes with high yield, better quality, and
tolerant/resistant to environmental stresses. However, im-
provement of rice varieties is comparatively slow due to the
non-availability of adequate genetic resources and lack of re-
sistant genes in the globally available rice germ plasm to be
used in conventional breeding by making compatible crosses.
To overcome these crossing barriers, genetic transformation
offers an opportunity to incorporate a gene of interest from a
remote source into an existing high yielding cultivar.
Nevertheless, genetic transformation exclusively relies on
the efficiency and resilience of tissue culture procedures,
which are least dependent on the genotype and reproducible
(Raina 1989;Dorosieve1996; Sikder et al. 2006). Producing
embryogenic calli and obtaining a good number of regenerat-
ed plants for further selection has always been regarded as a
key step towards rice improvement through genetic transfor-
mation (Cho et al. 2004; Wanichananan et al. 2010).
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11627-019-09974-6) contains supplementary
material, which is available to authorized users.
*Tayyaba Yasmin
tayyaba_yasmin@comsats.edu.pk
1
Department of Biosciences, COMSATS University Islamabad,
Islamabad, Pakistan
2
Department of Biology, University of Padua, Padua, Italy
3
Department of Plant Breeding and Genetics, PMAS Arid Agriculture
University, Rawalpindi, Pakistan
In Vitro Cellular & Developmental Biology - Plant
https://doi.org/10.1007/s11627-019-09974-6
Published data suggest that generally, it is not easy to
culture and regenerate monocot plants (Tan et al. 2011).
Although many tissue culture and plant regeneration pro-
cedures have been reported in rice varieties (Peng and
Hodges 1989; Aldemita and Hodges 1996;Rashidet al.
2001; Khanna and Raina 1998; Khan and Maliga 1999;
Bano et al. 2005; Toki et al. 2006; Vennapusa et al.
2015), indica rice is still not considered a good choice for
inducing embryogenic calli. In general, the japonica rice
cultivars are considered as easier to regenerate in vitro
compared to indica cultivars (Ge et al. 2006). There are
some reports of successful plant regeneration in indica rice
(Sahoo et al. 2011;Priyaet al. 2011;Waniet al. 2011)but
limited data is available for embryogenic callus of indica
rice as it easily turns brown during sub-culturing phases
reducing culture utilization for transformation studies.
Callus induction (CI) phase is crucial in producing succes-
sive somatic embryos and regeneration of whole plants. It
is well established that quality and morphology of calli is
essentially dependent on factors like genotype, explants,
induction medium, and its components apart from the con-
centration of exogenous hormones (Ozawa et al. 2003;
Naqvi et al. 2006;Nooret al. 2011;Pravinet al. 2011;
Joyia and Khan 2012). Calli developed from mature rice
seeds have been classified morphologically by Visarada
et al. (2002) into seven types which differ in their charac-
teristic and potential for plant regeneration. These calli
could be slick and compact or soft, friable, and loose in
texture, depending on the type of media used, hormones,
and culture conditions (Afrasiab and Jafar 2011; Rafique
et al. 2011; Joyia and Khan 2012).
Owing to the problem of calli browning during suc-
cessive subcultures in indica rice, histological studies of
primary calli can provide substantial information on their
regeneration potential and can be used for early grading
of calli expected to develop plantlets on regeneration
media. Nevertheless, the regeneration potential of indica
rice varieties has not been associated with histological
data so far. This study was aimed to assess mature seeds
of five economically elite rice cultivars viz. Shaheen
Basmati, Super Basmati, KSK-133, KS-282, and
DilRosh, for their embryogenic callus induction and sub-
sequent plant regeneration by characterizing their
morpho-histological features on three different media
combinations. Histochemical staining and microscopic
examination of primary calli produced by each line was
performed in order to establish if their structural mor-
phology could be an indicator of their regeneration fre-
quency (RF). Furthermore, we hypothesized if there is
any connection between the presence and absence of em-
bryogenic units in the selected cultivars that could pre-
dict their regeneration ability in vitro.
Materials and Methods
Explants preparation Certified seeds of five indica rice cultivars
viz.KSK-133, KS-282, Shaheen Basmati, Super Basmati, and
DilRosh were obtained from the National Agricultural
Research Council, (NARC) Islamabad, Pakistan. All contamina-
tion sensitive procedures were performed under laminar air-flow
cabinet and sterile conditions. The mature de-husked seeds were
surface sterilized with 70% ethanol for 1 min and in 2% NaOCl
for 15 min. The seeds were washed 5 times with sterile distilled
water before transfer to callus induction media.
Table 1. Callus induction
frequency on different media
combinations
Source DF Sum of squares Mean squares FPr>F
Model 14 25,888.245 1849.160 398.182 < 0.0001
Error 30 139.320 4.644
Corrected total 44 26,027.565
ANOVA for callus induction frequency showing sum of squares and Pvalues for each treatment. Computed
against model Y = Mean (Y)
Table 2. Regeneration frequency
of type II calli on regeneration
media
Source DF Sum of squares Mean squares FPr>F
Model 14 34,130.400 2437.886 129.992 < 0.0001
Error 30 562.625 18.754
Corrected total 44 34,693.025
ANOVA for regeneration frequency showing sum of squares and Pvalues for each treatment. Computed against
model Y = Mean(Y)
IJAZ ET AL.
Table 3. Callus browning of type
II calli on regeneration media Source DF Sum of squares Mean squares FPr>F
Model 14 34,130.399 2437.885 129.991 < 0.0001
Error 30 562.624 18.754
Corrected total 44 34,693.024
ANOVA for regeneration frequency showing sum of squares and Pvalues for each treatment. Computed against
model Y = Mean(Y)
Fig. 1. Effect of media on the
callus induction frequency (A)
and plant regeneration frequency
(B). KSK-133, KS-282, and
DilRosh showed high callus
induction media compared to
Shaheen Basmati and Super
Basmati. Plant regeneration
frequency was highest in KSK-
133 and KS-282 followed by
DilRosh. Calli of all cultivars in-
duced on CIM II showed an
overall high-regeneration fre-
quency as compared to other two
media combinations. Error bars
show standard error.
HISTO-MORPHOLOGICAL ANALYSIS OF RICE CALLUS CULTURES REVEALS DIFFERENTIAL REGENERATION RESPONSE WITH...
Culture media Three types of callus induction media (CIM)
were prepared using 3% sucrose, 0.8% plant agar (Sigma-
Aldrich St. Louis, MO), and 2 mg/L 2, 4-D as common ingre-
dients. CIM I and CIM III were N6 (Chu et al. 1975)andMS
(Murashige and Skoog 1962), respectively. CIM II was N6 sup-
plemented with additional 2 mg/L BAP along with 2 mg/L 2,4-
Dichlorophenoxyacetic acid. The pH of all media was adjusted
to 5.8 before autoclaving. The media were poured in sterile
plastic petri plates (60 mm × 15 mm). Surface sterilized seeds
of all varieties were cultured on these media combinations.
Cultured seeds were placed in dark in a plant growth chamber
set at 25 ± 1°C day-night temperature and 70% humidity.
Callogenesis Twenty-five mature seeds per variety per repli-
cate per media combination were used for callus induction.
Callus initiated from scutella of mature embryos and the callus
induction frequency (CIF) was calculated 7 d after culturing
mature seeds by the following relation.
Callus induction frequency %ðÞ
¼Number of seeds producing calli
Total number of seeds sown on media 100
Calli were classified based on physical appearance as
described by on Ikeuchi et al. (2013) and Noor et al.
(2005,2011). Compact, nodular, and pale-white calli
were marked as embryogenic (type I). Loose-textured,
watery, and friable calli were regarded as non-embryogenic
(type II).
Histological analyses Three-week-old mature embryo de-
rived callus, grown on different media combinations,
were morphologically examined. Paraplast embedded sec-
tions of calli samples were prepared for histological inves-
tigations using Leica DMR transmission light microscope.
Each callus was fixed in a mixture of formaldehyde, gla-
cial acetic acid, and absolute alcohol (1:1:18) for 48 h.
The fixed calli were dehydrated in a series of graded al-
cohol (50, 70, 85, and 95 to 100%), submerged in absolute
ethanol and xylene (1:1) for 4 h followed by dipping in
pure xylene for 2 h. The samples were embedded in
Paraplast tissue embedding wax (Paraplast Plus, Sigma-
Aldrich) overnight. Paraplast embedded calli were sec-
tioned into 5–8-μm slices using a rotatory microtome
(Leica RM 2235, Nussloch, Germany). The sections were
dried overnight on poly-lysinated glass-slides followed by
staining with 0.05% toluidine blue for 2 min. Excess dye
was washed and sections were mounted. Sections were
photographed and optimized using Leica model DM500
microscope with an ICC50 camera and Leica LAS EZ
software. Five calli per variety per replicate were used
for histological analysis.
Plantlet regeneration Three-week-old well-proliferated
calli with regeneration capability, produced from all five
rice cultivars were transferred onto regeneration media
containing MS with 2 mg/L BAP, 1 mg/L NAA, 3%
sucrose, and solidified with 0.8% plant agar. When ma-
ture embryos with well-developed cotyledons and roots
emerged from calli, they were transferred to MS +
0.5 mg/L indole-3-butyric acid (IBA) to check regener-
ation frequency (RF) and plantlet formation. Twenty
calli per variety per replicate were used for plantlet
regeneration.
Fig. 2. Mean of callus induction frequency (A) of both type of calli on the
three CIM and average plant regeneration frequency of type I
(embryogenic) calli in different rice cultivars. (A) KSK-133, KS-282, and
DilRosh showed high CIF compared to Shaheen Basmati and Super
Basmati on all media combinations. (B) Plant regeneration frequency
remained highest in KSK-133 and KS-282 while significantly lower in
the rest of the genotypes. Error bars show standard error.
IJAZ ET AL.
The developed plantlets were counted and regeneration
frequency was calculated as:
Regeneration frequency %ðÞ
¼Number of calli producing plantlets
Total number of calli placed on regeneration media 100
Statistical analysis Each experiment was completely random-
ized and repeated three times. Means for relative callus induc-
tion and regeneration frequency (RF) were calculated.
Significant differences (P< 0.05) were determined using a
two-way analysis of variance (ANOVA) followed by a
Bonferroni post hoc test.
Results
Differential response of cultivars towards callus induction rate
and quality The ability of cultivars to form calli in vitro dif-
fered significantly (P≤0.01) on all three callus induction me-
dia (CIM), as revealed by two-way ANOVA (Table 4).
Relative CIF, RF, and callus browning of genotypes was cal-
culated on three different media combinations separately
(Table 1; Table 2;Table3;Fig.1A)aswellascombined
(Fig. 2A). Mean relative CIFs of KSK-133 and DilRosh were
recorded as 96 and 95% respectively (Fig. 2A). These two
genotypes exhibited the highest CIF as compared to the rest
of the genotypes, while KS-282 also showed a relatively high
rate of callus induction in all media combinations (Table 4).
Shaheen Basmati, instead, exhibited least ability to induce
calli in vitro, while Super Basmati had the lowest callus in-
duction rate on all media combinations except CIM III where
it was 71.3% (Table 4). Notably, Shaheen Basmati and Super
Basmati were more inclined towards germination rather than
callus formation on CIMs.
DilRosh showed the highest CIF (99%) on CIM I, which was
relatively lower on CIM II and CIM III (Table 4;Fig.1A). KSK-
133 showed better callusing on CIM II and CIM III, while CIM I
was found superior for KS-282 (Table 4). Super Basmati and
Shaheen Basmati followed a similar trend with low CIF in
CIM I and II but relatively high on media III (Table.4).
When the three media combinations were compared, CIM
I, II, and III proved to be significantly better for type I callus
formation in KSK-133 and KS-282(Table 4) with highest CIF
on CIM I. Shaheen Basmati, Super Basmati, and DilRosh,
however, induced type II callus on CIM I and II, while type
I Calli on CIM III (Table 4). In case of DilRosh and KS-282,
CIM I was marked as a relatively better combination (Table 4)
for callus induction. The overall callus induction response on
all three media combinations remained highest in KSK-133,
followed by KS-282 and DilRosh (Fig. 2A).
Table 4. Mean callus induction, regeneration, and callus browning response of five rice cultivars on different media combinations
Cultivars CIM Mean CIF (%) STDEV Mean RF (%) STDEV Calli type % callus browning STDEV
KSK-133 I 90.6
bcd
±4.11 81.0
a
± 6.02 I 19.00
a
±1.00
II 99.5
a
±3.24 81.9
a
± 6.11 I 19.39
a
±4.00
III 99.2
a
±6.79 63.8
b
± 7.09 I 36.12
c
±5.00
KS-282 I 97.1
ab
±3.98 71.5
ab
± 4.98 I 28.46
b
±1.37
II 89.9
cd
±4.01 76.0
ab
± 5.01 I 23.03
a
±3.00
III 85.6
d
±5.89 66.9
b
± 4.89 I 33.11
bc
± 11.00
Shaheen Basmati I 61.0
f
±7.82 14.2
e
±3.71 II 85.79
e
±1.06
II 54.3
f
±5.11 16.0
de
±1.29 II 83.97
e
±3.88
III 71.3
e
±6.79 10.8
e
± 4.45 I 89.23
e
±3.03
Super Basmati I 39.6
g
±7.91 12.2
e
±2.91 II 82.29
e
±1.04
II 17.0
h
±4.35 29.2
c
± 13.35 II 70.80
d
±7.01
III 72.0
e
±32.81 11.0
e
±1.87 I 88.9
e
±1.00
DilRosh I 99.3
a
±2.71 18.3
de
±3.46 II 70.56
d
±1.07
II 92.0
bcd
±1.99 33.8
c
±9.11 II 66.22
d
±2.04
III 93.1
abc
±3.45 40.7
c
±17.99 I 59.33
df
±5.03
Callus induction, regeneration, and callus browning response presented as relative frequencies calculated for calli in different media combinations. A
two-way ANOVA followed by a Bonferroni post hoc test (provided in the supplementary material) was used to rank both callus induction and
regeneration frequencies. Different lowercase letters represent significant difference between varieties. Treatment groups with at least one common
letter are not significantly different from each other at 5% significance level. ± STDEV = standard deviation
HISTO-MORPHOLOGICAL ANALYSIS OF RICE CALLUS CULTURES REVEALS DIFFERENTIAL REGENERATION RESPONSE WITH...
Morphological examination of calli produced by all culti-
vars, under a stereo-zoom microscope, revealed KSK-133 and
KS-282 producing more compact and nodular calli on all me-
dia combination classified as type I (embryogenic calli), while
Shaheen Basmati, Super Basmati, and DilRosh developed
masses of cells having loose and watery texture and whitish
in color on media I and II, designated as type II non-
embryogenic (Fig. 3).
Regeneration frequency (RF) Differential regeneration re-
sponse was observed for calli derived from different cultivars
which were previously induced on the three media
combinations. There was a significant effect of CIM com-
position on subsequent regeneration potential of calli de-
rived from all five cultivars, as revealed by analysis of
variance followed by Bonferroni post hoc test (Table 4).
KSK-133 showed the highest regeneration frequency from
calli derived from media I (81.0%) and II (81.9%).
However, the mean regeneration frequency from calli de-
rived from media III remained only 63.8% in this cultivar
(Table 4). For KS-282, callus induction media I and II
were similar and superior to media III towards better
RFs of 71.5%, 76.0%, and 66.9%, respectively
(Table 4). In contrast, DilRosh, Super Basmati, and
Fig. 3. Three-week-old primary
calli of rice cultivars on callus
induction media. Best callus
induction and proliferation was
seen in CIM III (MS + 2.0 mg/l
2,4-D). KSK-133 and KS-282
produced compact and nodular
calli (visible in white circle)
depicting their embryogenic po-
tential based on physical mor-
phology. Super Basmati, Shaheen
Basmati, and DilRosh produced
watery, friable, and loose-textured
non-embryogenic calli (shown by
arrow)onCIMIandIIwhile
somewhat nodular calli on CIM
III which are the characteristic
features of embryogenic callus
predicting regeneration potential
(scale bar = 2 mm).
IJAZ ET AL.
Shaheen Basmati showed a relatively low regeneration
capability from calli derived from all three media (Fig.
1B), recognizing the genotypic effect on plant regenera-
tion. Shaheen Basmati showed the lowest mean regener-
ation frequency among all cultivars tested (Fig. 1B).
After 2–3 wk on regeneration media, KSK-133, KS-
282, and DilRosh developed compact and nodular em-
bryogenic calli marked as type I. These calli developed
visible embryos with little callus browning (Fig. 4A–C).
It was noticed that type I calli exhibited a relatively fast
growth rate and late embryo stages emerged after 2 wk
on regeneration medium. Mature embryo differentiation
wasnoticedwithin2to3wkforKSK-133andKS-282
while in DilRosh, Shaheen Basmati, and Super Basmati,
the differentiations events were relatively delayed for up
to 5 wk along with obvious callus browning particularly
in Shaheen Basmati and Super Basmati (Fig. 4D,E).
Clumps (~ 4 cm) owning multiples embryos were excised
from the calli and transferred on new media to let them
complete their differentiation process (Fig. 5). Plantlets
were obtained in 6–8 wk. On the other hand, the friable
and loose-textured non-embryogenic type II calli showed
higher callus browning with relatively low callus prolif-
eration rate (Fig. 4E,F). Considering the combined ef-
fect of all three media combinations on plant regenera-
tion, KSK-133 and KS-282 showed the highest mean
regeneration frequencies which remained significantly
low in the rest of the cultivars (Fig. 2B).
Histological analyses of primary calli Compact embryogen-
ic calli obtained from mature seeds of all five cultivars after 3-
wk inoculation on CIM I, II, and III were separated from the
scutellum and subjected to histological examination.
Typical embryogenic structures showing compact and
globular shapes having tightly packed cells called em-
bryogenic units (EUs) were observed in calli of KS-282
Fig. 4. Calli forming plantlets on regeneration media. Six-week-old calli
derived from CIM I showing green embryo cotyledons spot formation in
(A) KSK-133, (B) KS-282, and (C) DilRosh. Six-week-old callus of
DilRosh (D) derived from CIM III media showing embryoid-like struc-
tures but no green embryo cotyledons spot formation after 3 wk on
regeneration media. Calli of low regeneration varieties (E) Shaheen
Basmati and (F) Super Basmati exhibiting callus browning and slow
growth which later had a low regeneration rate. Multiple shoots were
visible after 8 wk on regeneration media in (G)KSK-133,(H)KS-282,
and (I) DilRosh. Scale bar = 5 mm.
HISTO-MORPHOLOGICAL ANALYSIS OF RICE CALLUS CULTURES REVEALS DIFFERENTIAL REGENERATION RESPONSE WITH...
and KSK-133 obtained from CIM I, II, and III (Fig. 6)
and classified as type I. Such units were also observed in
calli obtained from DilRosh and induced on media II and
to some extent in calli obtained from media I, whereas,
these units were absent in the callus of DilRosh pro-
ducedonmediaIII(Fig.6). These embryogenic units
consisted of regularly organized small isodiametric,
densely stained and packed meristematic cells. In con-
trast, the callus of Super Basmati and Shaheen Basmati
induced on the callus induction media I, II, and III,
lacked such compact areas of active cell divisions within
EUs. Microtome sections of these two cultivars consisted
of unorganized, long tubular cells predicting their non-
embryogenic attributes (Fig. 6).Thenumberofembryo-
genic units per surface area in callus of KSK-133, KS-
282, and DilRosh induced on media II were more as
compared to the number of such units in histological
sections of calli from media I and III (Table 5)indicating
a probable complementary effect of BAP on the produc-
tion of type I calli having greater regeneration potential.
In conclusion, the characteristics of the embryogenic calli
were highly preserved in the EUs of KSK-133 and KS-
282 irrespective of the callus induction media. In case of
DilRosh, cultured cells from media II showed greater
number of embryogenic units per callus as compared to
callus induced on media I and III. On the other hand,
non-embryogenic type II callus was mainly observed in callus
of DilRosh induced on callus induction media III and in Super
Basmati and Shaheen Basmati cell culture irrespective of the
media combination used for callus induction.
Discussion
Histological analysis of the calli obtained from all varieties
used in present study revealed a media-dependent differen-
tial response. Calli of KSK-133 and KS-282, induced on
all three induction media combinations, showed the pres-
ence of densely packed meristematic zones. Whereas, calli
of Super Basmati and Shaheen Basmati lacked such meri-
stematic regions and had unorganized masses of tubular
cells. Similar masses of tubular cells were also found in
the histological sections of DilRosh callus induced on me-
dia III. Comparatively, fewer meristematic regions were
also observed in the callus of DilRosh induced on media
I and II but were missing in callus obtained from media III.
Moreover, the addition of BAP in callus induction media
enhanced the number of embryogenic units supporting the
fact that the right amount of growth regulators is an impor-
tant determinant of regeneration ability of indica rice calli.
Meristematic zones of active cell division have also been
previously observed in embryogenic calli of Pereskia
grandifolia (Leng and Keng 2007), Phaseolus vulgaris L.
(Mohamed et al. 1993), mung bean (Mendoza et al. 1992),
and rice (Sangduen and Klamsomboon 2001;Bevitori
et al. 2014). The histological studies have been conducted
on rice varieties previously indicating the presence of
parenchymatic cells in the central part of 3–4wkold
rice calli and meristematic cells in the peripheral areas
(Alfonso-Rubi et al. 1999;Vegaet al. 2009;Narciso
and Hattori 2010), but these studies have not been
linked to subsequent regeneration potential, particularly
for indica varieties.
To correlate histological observations with regeneration ca-
pabilities of calli, 3-wk-old calli were transferred onto regen-
eration media and their regeneration potential was evaluated
in terms of callus browning, appearance of green embryo cot-
yledons, and emergence of plantlets. This study demonstrates
that the rate of green embryo cotyledons formation, plant-
let development, and callus browning depends significant-
ly on the genotype, and that the CIM is an important future
determinant of regeneration potential for the calli.
Morphologically, two types of calli were obtained, em-
bryogenic calli appearing as nodular, compact, and pale
while non-embryogenic calli were friable, translucent,
and moist. Callus obtained from KSK-133 and KS-282
was somewhat more compact, hard, nodular and pale and,
therefore, categorized as embryogenic (type I). The callus
Fig. 5. A comparison of plantlets regenerated on regeneration media.
Regenerated plantlets of all selected rice cultivars were observed for
shoot and root formation. (A) KSK-133 showed maximum regeneration
potential with multiple shoots and roots followed by (B)KS-282and(E)
DilRosh. (C) Shaheen Basmati and (D) Super Basmati, however,
displayed a very low regeneration potential with poorly developed plant-
lets and a significant callus browning on regeneration media.
IJAZ ET AL.
of Super Basmati, Shaheen Basmati, and DilRosh induced
on CIM I and II was friable, soft, granular, moist, and loose
in texture, hence classified as non-embryogenic (type II).
While, the calli of the same cultivars on CIM III had nod-
ular regions predicting presence of some EU’s. It is been
reported that type I calli produce more plantlets than type II
and somatic embryos develop from compact and nodular
embryogenic calli derived from the rice scutellum (Molina
et al. 2002; Quiroz-Figueroa et al. 2006; Rachmawati and
Anzai 2006). Likewise, the presence of embryogenic units
in primary calli of cultivars is an index for their ability to
regenerate plantlets (Molina et al. 2002). In agreement to
this, we were able to find a positive correlation between the
number of EU’s and RF in the selected cultivars (Fig. 7). We
also found variation in callus size. DilRosh and Shaheen
Basmatishowedrelativelylarge-sized(>0.4cmindiameter)
Fig. 6. Histological observations
of 3-wk-old well-proliferated calli
of five rice cultivars.
Embryogenic units darkly stained
with toluidine blue (0.05%) are
visible in calli of KSK-133 and
KS-282 induced on CIM I, II, and
III. Such organization of cells was
also observed in the calli of
DilRosh induced on media I and
II. Presence of EUs in 3-wk-old
callus demonstrates that the bud-
ding regions can instigate from
superficial cells. Cells in these
EUs were found to be smaller in
size and darkly stained. The em-
bryogenic cells were heavily cy-
toplasmic with prominent nuclei
located on the callus surface or
inside surrounded by parenchy-
mal cells (scale bar =100μm).
No such units were visible in
Shaheen Basmati and Super
Basmati calli from all media
combinations as well as in
DilRosh callus induced on media
III. These histological sections
consisted of mass of unorganized,
loosely packed cells, or non-
embryogenic units (NEU).
HISTO-MORPHOLOGICAL ANALYSIS OF RICE CALLUS CULTURES REVEALS DIFFERENTIAL REGENERATION RESPONSE WITH...
calli, whereas KSK-133, KS-282, and Super Basmati produced
small-sized (< 0.4 cm in diameter) calli. Previously, efficient
callus induction has been reported in Super Basmati on N6
media supplemented with 2 mg/L 2,4-D (Noor et al.
2011); however, the intricate interaction between geno-
types, media, and hormones remained uncovered.
After 2–3 wk, embryogenic calli developed visible
green cotyledons spots that later on developed into plant-
lets on regeneration media. Among the five varieties test-
ed KS-282 and KSK-133 showed highest regeneration
response that was also manifested by the prior histolog-
ical observations, whereas DilRosh, Super Basmati, and
Shaheen Basmati exhibited lower regeneration frequen-
cies in general. We also observed calli browning in
DilRosh, Super Basmati, and Shaheen Basmati, as re-
ported by previous studies for some other indica varieties
(Peng et al. 1992;Qianet al. 2004). However, KS-282
and KSK-133 are superior in this context and could be
exploited for genetic engineering or biotechnological ex-
periments as compared with other varieties.
Conclusions
Our data implies that regeneration potential of calli can
be envisaged by histological observations. Calli mani-
festing greater number of EUs, characterized by meriste-
matic zones, are more likely to return higher regenerat-
ed plantlets. The absence of EUs is an indicator of calli
expected to display low/null regeneration frequency.
DilRosh seems to be an exception, showing the pres-
ence of embryogenic units in the presence of BAP
while lacking such units in callus induced on basal me-
dia without BAP. However, DilRosh exhibited a low
regeneration potential and was able to develop embryos
and subsequent regeneration of plantlets.
Fig. 7. A correlation plot
representing the presence of
embryogenic units and its effect
on regeneration frequency of the
selected cultivars. A positive
correlation was found between
the mean number of EU’s/ mm
2
of calli and their mean
regeneration frequency (%) as
analyzed by Pearson correlation
analysis.
Table 5. Classification of embryogenic and non-embryogenic callus
based on the number of embryogenic units per mm
2
of paraplast embed-
ded sections
Cultivar CIM Type of callus Number of EU/mm
2
KSK-133 I Embryogenic 7.6 ± 2.41
II Embryogenic 4.8 ± 1.64
III Embryogenic 7.6 ± 3.05
KS-282 I Embryogenic 8.4 ± 2.19
II Embryogenic 7.6 ± 1.82
III Embryogenic 8.4 ± 3.85
Shaheen Basmati I Non-embryogenic 1.4 ± 1.14
II Non-embryogenic 1.6 ± 0.89
III Non-embryogenic 0.6 ± 0.89
Super Basmati I Non-embryogenic 1.6 ± 0.89
II Non-embryogenic 1 ± 1.22
III Non-embryogenic 0.4 ± 0.55
DilRosh I Embryogenic 6.2 ± 2.77
II Embryogenic 7 ± 3.67
III Non-embryogenic 1.8 ± 1.30
Number of embryogenic units per mm
2
of calli induced on three media
combinations. Mean ± standard deviation with n= 5 calli/variety per cal-
lus induction media combination
IJAZ ET AL.
Acknowledgments We are thankful to Prof. Barbara Baldan, Department
of Biology, University of Padua, Italy, for providing the microtome facil-
ity for histological analysis. We are also grateful to Dr. Tahir Abbas Shah,
Department of Biosciences, CUI, Islamabad, Pakistan for assistance in
the statistical analysis of data.
Funding information This work was supported by Erasmus Mundus
Mobility for Asia (2014) grant no. ID06884 awarded to Ms Bushra Ijaz.
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