3 Biotech (2017) 7:16

DOI 10.1007/s13205-016-0579-3

ORIGINAL ARTICLE

Plant regeneration from cell suspension culture in Saccharum

officinarum L. and ascertaining of genetic fidelity through RAPD
and ISSR markers
Avinash S. Thorat1,2 • Nishant A. Sonone1 • Vrushali V. Choudhari1 •

Rachayya M. Devarumath1 • K. Harinath Babu1

Received: 8 June 2016 / Accepted: 1 December 2016 / Published online: 8 April 2017
Ó The Author(s) 2017. This article is an open access publication

Abstract The aim of this study was to produce sugarcane in vitro regenerated plants was assessed by using RAPD and
plantlets from cell suspension culture and study its genetic ISSR markers. Analysis of the ten RAPD markers indicated
fidelity using molecular markers. The study was carried out that 90.48 and 86.95% true-to-type regenerated plantlets in Co
using sugarcane varieties Co 86032 and Q117. Callus cultures 86032 and Q117, respectively. However, in the ISSR markers,
of both the varieties were optimized using six different callus Co 86032 did not show any polymorphism and in the Q117,
induction media. After screening the growth response of cal- 92.18% true-to-type plantlets were found. These results con-
lus on six different callus induction media, it was observed that firmed that somaclonal variation occurs during the process of
medium no. VI supplemented with 500 mg l-1 of each PVP, indirect organogenesis and RAPD and ISSR marker based
Casein hydrolysate and MES buffer showed high amounts of molecular analysis is a suitable method for an early detection
callus in Co 86032 (79.66 ± 0.44%) and Q117 of variation in sugarcane.
(82.83 ± 1.69%). Addition of PEG 8000 at 2.5% to this
medium had a profound impact on inducing somatic Keywords Sugarcane  Callus  Cell suspension culture 
embryogenesis in Co 86032 (54.66 ± 1.76%) and Q117 RAPD  ISSR  Somaclonal variation  Genetic fidelity
(66.66 ± 2.60%) as compare to control (24.33 ± 1.76%) and
(27.33 ± 2.73%), respectively. Cell suspension cultures were
established by culturing embryogenic calli in liquid medium Introduction
showed well established suspension cultures with fever cell
aggregates. There was negligible cell division during initial Sugarcane (Saccharum officinarum L.), is an important cash
2 days of incubation and cell count increased rapidly between crop which contributes to around 70–80% of sugar produc-
2 and 8 days. Further incubation beyond 8 days resulted in a tion globally. It is cultivated in the tropical and sub-tropical
decrease in cell viability. Enhanced callus proliferation in regions and ranks amongst the top ten cultivated crops in the
Q117 while enhanced shoot regeneration in Co 86032 was world (Suprasanna et al. 2011). In India, sugarcane has
observed from cell suspension culture. The clonal fidelity of secured a distinct position after cotton as an agro-industrial
crop, due to it being a prominent source of vital product
(sugar) as well as by-products (baggasse, molasses, press-
Electronic supplementary material The online version of this mud, etc.) which plays a major role in the economic progress
article (doi:10.1007/s13205-016-0579-3) contains supplementary
material, which is available to authorized users.
of small and large scale industrial sectors. Sugarcane has
been recognized as the most competent crop which converts
& K. Harinath Babu solar energy into harvestable chemical energy in the form of
[email protected] sucrose and biomass (Joyce et al. 2010).
1 Currently, the development of somatic embryogenesis
Molecular Biology and Genetic Engineering Section,
Vasantdada Sugar Institute, Manjari (Bk), Pune, Maharashtra, through callus and suspension culture has great potential
India for propagation at a rapid rate (Dewanti et al. 2016). It was
2
Department of Botany, Shivaji University, Kolhapur, reported that all the regenerated plants from the tissue
Maharashtra, India culture are not true-to-type like the parent. Phenotypic

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variation is commonly observed amongst regenerated a rinse under tap water for 10–12 min. The outer mature
plants which are associated with genetic changes of an leaves of shoot tops were removed and discarded. The
organism. These changes could result from mutations, shoot tops were then surface sterilized using BavistinTM
epigenetic changes or a combination of both mechanisms. (0.1%) and streptomycin (0.1%) for 15 min followed by
Somaclonal variation is the genetic variability, which has HgCl2 (0.1%) for 30 min and then rinsed with sterile dis-
occurred during tissue culture or variants derived from any tilled water for 2–3 times.
form of cell or tissue cultures (Larkin and Scowcroft 1981).
The occurrence of somaclonal variation arising through Initiation of callus
cryptic gene defects can seriously limit the application of
micropropagation for clonal multiplication (Rani and Raina The immature leaf whorls from sterile cane tops were
2000). These variations in genomic DNA of regenerants sliced (about 1–2 mm thickness) and inoculated on six
restrict the utility of plant micropropagation techniques for different callus induction medium (Table 1) and incubated
large-scale multiplication. The inability for quick identifi- in dark at 28 ± 2 °C for 2 weeks. The medium showing
cation of these polymorphisms at cytological, biochemical, higher proliferation of callus from Co 86032 and Q117 was
phenotypic and molecular levels in micropropagated sug- selected for somatic embryogenesis and subculture was
arcane poses a challenge. The rapid detection is beneficial performed after 2 weeks of inoculation. The callus induc-
to check the homogeneity between mother and in vitro tion response (%) for each medium was recorded as:
grown plantlets (Devarumath et al. 2002). The PCR asso- Number of calli induced
ciated DNA molecular markers based analysis system such Callus induction ð%Þ ¼
Number of leaf whorls inoculated
as Random amplified polymorphic DNA (RAPD), Ampli-  100
fied fragment length polymorphism (AFLP), Simple
sequence Repeat (SSR), Inter-sequence simple repeats Effect of PEG on somatic embryogenesis
(ISSR) and microsatellite DNA/SSRs possess several
benefits over the traditional methods that have been used The proliferated calli of Co 86032 and Q117 were trans-
for detection of polymorphism and genotyping in plant ferred to medium with varying concentration of poly-
systems (Lal et al. 2008; Guasmi et al. 2012; Rajpal et al. ethylene glycol 8000 (PEG) (0, 2.5, 5.0, 7.0 and 10.0%)
2014). The RAPD and ISSR analysis has been widely used and incubated in dark at 28 ± 2 °C for 2 weeks. The effect
to determine polymorphism in genomic DNA in sugarcane of PEG on somatic embryogenesis was examined by
and several other plant systems due to numerous advan- measuring the number of somatic embryos formed after
tages like being a relatively fast, simple, cost-effective 2 weeks. The somatic embryogenesis (%) for each com-
technique which requires a small quantity of DNA sample bination was recorded as:
with no preliminary sequence information for primer
design (Suprasanna et al. 2006; Devarumath et al. 2007; Somatic embryogenesis ð%Þ
Lal et al. 2008; Rizvi et al. 2012; Dangi et al. 2014; Number of somatic embryos generated
¼  100
Kshirsagar et al. 2015). Number of calli initiated
The present work was performed with an objective to Development of cell suspension culture
establish sugarcane (Co 86032 and Q117) suspension cul-
ture and subsequent regeneration of plantlets and study Cell suspension culture was developed from 8 weeks old
their genetic variation occurred during the process of embryonic callus (2–2.5 g fresh weight) in liquid MS
regeneration using molecular markers (RAPD and ISSR). medium (Murashige and Skoog 1962) (20 ml) supple-
mented with coconut water (5% v/v), 2,4-D (3 mg l-1) and
casein hydrolysate (500 mg l-1) and pH was adjusted to
Materials and methods 5.8 ± 0.1 before autoclave. The culture bottles were con-
tinually agitated at 100 rpm and incubation in dark at
Plant materials and explants preparation 28 ± 2 °C. After 8–10 days, suspension culture was
transferred to fresh medium.
The sugarcane varieties Co 86032 and Q117 grown in
experimental farms of Vasantdada Sugar Institute, Manjari
(Bk.), Pune, Maharashtra, India were used in this investi- Growth kinetic studies of cell suspension culture
gation. The sugarcane tops were harvested from 4 to
6 months old field grown sugarcane varieties Co 86032 and In suspension culture growth of the cells was calculated by
Q117. The harvested shoot tops (25–30 cm) were cleaned measuring cell density using spectrophotometer (OD600)
with a surfactant TeepolTM (0.1%) for 4–5 min followed by (Mohler et al. 1996) and by haemocytometer (Aberkane

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Table 1 Media combination tested for callus induction

Component (l-1) I II III IV V VI

NH4NO3(g) 1.65 1.65 1.65 1.65 1.65 1.65

CaCl22H2O (mg) 440 440 440 440 440 440
MgSO47H2O (mg) 370 370 370 370 370 370
KH2PO4 (mg) 170 170 170 170 170 170
KNO3 (g) 1.9 1.9 1.9 1.9 1.9 1.9
Na2EDTA (mg) 37 37 37 37 37 37
FeSO4 (mg) 28 28 28 28 28 28
H3BO3 (mg) 6 6 6 6 6 6
CoCl26H2O (lg) 25 25 25 25 25 25
CuSO45H2O (lg) 25 25 25 25 25 25
MnSO47H2O (mg) 22 22 22 22 22 22
KI (mg) 1 1 1 1 1 1
Na2MoO42H2O (lg) 250 250 250 250 250 250
ZnSO47H2O (mg) 9 9 9 9 9 9
2,4-D (mg) 3 3 3 3 3 3
Sucrose (g) – 40 30 30 30 20
Maltose (g) 30 – – – – –
Coconut water (ml) – – 100 100 100 100
L-Glutamine (mg) – 100 – 30 – –
Kinetine (mg) 0.5 1 2 – – –
NAA (mg) – 0.5 0.5 – – –
PVP (mg) 100 100 100 100 100 500
Casein hydrolysate (mg) – – – 50 – 500
MES buffer (mg) – – – – – 500
Thiamine HCl (mg) – – – – – 1
Inositol (mg) – – – – – 20
Proline (mg) 500 – – 50 – 500

et al. 2002). Cell density was measured after every 24 h supplemented with PEG 8000 (2.5%) and incubated at
interval for nine subsequent days from initial culture. The 26 ± 2 °C under dark for the development of embryonic cal-
total number of cells/ml was calculated according to the lus. The regeneration capacity of callus was determined by
method explained by Dodds and Roberts (1986) and cell inoculating cell suspension and cell aggregates onto shoot
viability was determined by using trypan blue exclusion regeneration medium [MS basal medium fortified with ben-
test (Katsares et al. 2009) using the following formulas zylaminopurine (1 mg l-1), kinetin (0.5 mg l-1)] and incu-
Total cells counted bated at 26 ± 2 °C and 16/8 h (light/dark) photoperiod
Total number of cells=ml ¼ regimes.
Total squares counted
 Dilution factor  104
Total unstained cells counted Root induction and acclimatization
Number of viable cells=ml ¼
Total squares counted
 Dilution factor  104 Root induction was accomplished by incubating elongated
shoots (4–5 cm) from callus on to MS basal medium
augmented with naphthalene acetic acid (0.5 mg l-1) and
Plant regeneration from suspension culture
incubated at 26 ± 2 °C with the 16 h photoperiod. Plant-
lets with well-developed roots were washed with water and
Cells were harvested from suspension for callus induction by
transferred to plastic cups containing a mixture of soil,
filtering the suspension using whatmann filter paper no. 1. The
sand and coco peat (3:1:1) and incubated as above men-
filter paper containing harvested cells was carefully inoculated
tioned conditions. After a week of incubation, these
in petri plates containing callus induction medium

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plantlets were subsequently potted in soil and maintained using initial denaturation at 95 °C for 5 min followed by 40
under greenhouse conditions. cycles 1 min at 94 °C, 1 min at 52–54 °C (depending on the
primer), 2 min at 72 °C and final extension cycle of 10 min at
DNA extraction 72 °C. The PCR products were stored at 4 °C until further
analysis carried out. The PCR products were resolved on 1.5%
Total genomic DNA of young leaves of the mother plant and (w/v) agarose gel with 19 TBE buffer, stained with ethidium
in vitro raised plants were isolated using Aljanabi et al. bromide (EtBr) and documented under UV light. The frag-
(1999) method. The quality of genomic DNA was resolute on ment size was estimated using 1 kb DNA ladder.
0.8% agarose gel electrophoresis, while quantity by UV–Vis
spectrophotometry (UV-1700, Shimadzu Corporation, Statistical analysis
Japan). The final concentration of extracted genomic DNA
was made to 50 ng ll-1 and stored at -20 °C till further use. All experiments were performed in duplicate, with the three
independent replicates, as well as different DNA extraction
Genetic stability analysis using molecular markers of the same bulk sample to maintain the consistency of
(RAPD and ISSR) results. The data was analyzed using Microsoft Excel 2010
and SPSS for Windows (version 16). One way ANOVA was
Two sets of primers including arbitrary (RAPD) and semi applied to test mean differences of all treatments while a
arbitrary (ISSR) were used for analysis of genomic DNA statistical significant difference between mean values was
including mother plant and in vitro raised plants. Ten primers established at p B 0.05 while Duncan’s New Multiple
each from RAPD (OPH series, Operon Technologies, INC. Range Test was used. The results were expressed as
California, USA) (Table 2) and ISSR (UBC series, University mean ± SE. For RAPD and ISSR analysis, a band with the
of British Columbia, Vancouver, Canada) (Table 3) were same mobility were counted as an identical band while each
selected on the basis of preliminary screening. PCR analysis amplified product was scored as present (1) or absent (0),
was done by the method explained by Williams et al. (1990) bands of low intensity, which was difficult to be distin-
with slight modifications. For RAPD analysis, DNA amplifi- guished as present or absent were not considered.
cation was carried out with total reaction mixture volume of
20 ll consisting 50 ng template DNA (1 ll), 109 PCR buffer
with MgCl2 (2 ll), 250 lM dNTPs (2 ll), 0.25 lM primer Results and discussion
(2 ll), 1 U Taq DNA polymerase (0.2 ll) and sterile nucleus
free distilled water (12.8 ll). PCR was performed on Thermal Impact of culture medium on induction of callus
cycler (Applied Biosystem) at initial temperature of 94 °C
(5 min, 1 cycle), followed by 40 cycles of 1 min at 94 °C, Inoculated leaf whorl disks from both the varieties (Co
1 min at 37 °C, 2 min at 72 °C and final extension cycle of 86032 and Q117) shows variation in quality and rate of
10 min at 72 °C. During ISSR analysis, the reaction mixture callus generated in different media. During incubation
was made similar to RAPD. However, PCR was carried out browning of some disks due to secretion of diffusible

Table 2 Molecular polymorphism analyzed by 10 RAPD markers in sugarcane variety Co 86032 and Q117
Sr. No Primer code Primer sequence Co 86032 Q117 Size range (bp)
(50 –30 )
nSB nMB nPB nSB nMB nPB

1 OPH-01 GGTCGGAGAA 5 5 0 5 4 1 250–3000

2 OPH-02 TCGGACGTGA 5 5 0 6 6 0 250–3000
3 OPH-04 GGAAGTCGCC 5 5 0 6 5 1 250–2000
4 OPH-06 ACGCATCGCA 5 5 0 8 7 1 250–3000
5 OPH-09 TGTAGCTGGG 4 2 2 3 3 0 250–1000
6 OPH-11 CTTCCGCAGT 6 6 0 6 6 0 250–1000
7 OPH-13 GACGCCACAC 5 5 0 5 4 1 250–2000
8 OPH-16 TCTCAGCTGG 2 2 0 3 3 0 500–2000
9 OPH-17 CACTCTCCTC 2 2 0 2 2 0 500–1000
10 OPH-18 GAATCGGCCA 2 1 1 2 0 2 250–500
Total 42 38 4 46 40 6 –
Percentage (%) 90.48 9.52 – 86.95 13.05 –

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Table 3 Molecular polymorphism analyzed by 10 ISSR markers in sugarcane variety Co 86032 and Q117
Sr. No. Primer code Primer sequence Co 86032 Q117 Size range (bp)
(50 –30 )
nSB nMB nPB nSB nMB nPB

1 UBC-811 (GA)8C 5 5 0 5 4 1 50–1500

2 UBC-828 (TG)8A 4 4 0 6 6 0 50–1500
3 UBC-835 (AG)8YC 7 7 0 5 3 2 50–3000
4 UBC-836 (AG)8YA 7 7 0 7 7 0 50–1000
5 UBC-844 (CT)8RC 6 6 0 8 8 0 50–1500
6 UBC-849 (GT)8YA 6 6 0 6 5 1 50–1000
7 UBC-855 (AC)8YT 5 5 0 6 6 0 50–1500
8 UBC-857 (AC)8YG 8 8 0 9 8 1 50–1500
9 UBC-864 (ATG)6 5 5 0 7 7 0 50–1500
10 UBC-868 (GAA)6 9 9 0 5 5 0 50–2000
Total 62 62 0 64 59 5 –
Percentage (%) – 100 0 – 92.18 7.81 –
nSB total number of scorable bands, nMB number of monomorphic bands, nPB number of polymorphic bands

polyphenolic compounds was noticed. Significantly physiological responses, enabling plants to bear the
superior callus induction of Co 86032 and Q117 variety undesirable effects of abiotic stress (Nounjan et al. 2012).
was noticed in callus induction medium number VI The medium fortified with MES and proline had a sig-
(79.66 ± 0.44 and 82.83 ± 1.69%), while least callus nificant impact on cell proliferation during growth of rice
induction was observed in medium number IV suspension culture (Kermanee 2004).
(57.50 ± 1.04 and 65.66 ± 1.45%) (Fig. 1). This might
be due to the fortification of an appropriate concentration Determination of callus type
of thiamine, casein hydrolysate, MES buffer, proline, PVP
and inositol along with other nutrients in callus induction The calli developed in the induction medium was pheno-
medium number VI (Table 1). These supplemented typically heterogeneous and hence differentiated visually
components in specified concentration and in combination based on morphology into two types i.e. embryogenic
with each other, while other media components are (compact, friable, creamy and embryogenic) and, non-
known to augment the callus induction. The excess embryogenic (loose, filamentous, whitish, watery and non-
amount of PVP (500 mg l-1) restricted the phenolics embryogenic) (Fig. 2). The embryogenic callus facilitated
secretion by leaf whorl callus into the culture medium. the development of somatic embryos. The generation of
This is due to the chelating ability of PVP which binds embryogenic callus along with a rate of callus induction
ions responsible for activation of polyphenol oxidative was more prominent in Q117 than Co 86032. In some
enzymes and these results are in agreement with previous plates embryogenic and non-embryogenic type of calli
reports of sorghum tissue culture by Liu et al. (2015). developed from the same explant and somatic embryos
Thiamine, which serves as a cofactor in numerous meta- formed on cut edges of leaves. In-vitro embryogenesis was
bolic pathways is essential for the growth of all cells in triggered by physiological and genetic factors. These
tissue (Goyer 2010). Inositol stimulates the cell growth findings have supported the results of Basnayake et al.
and has a vital role in cell division and hence added in a (2011) and Silveira et al. (2013). Additionally, plant
small quantity of the growth medium (Saad and Elshahed growth hormones (e.g. Abscisic acid, auxins like 2,4-D,
2012). Casein hydrolysate used in the culture medium is a etc.) along with explants source, carbohydrates, and
mixture of amino acids, which are the ambient source of nitrogen source plays a crucial role for initiation or inhi-
organic nitrogen. The medium supplemented with casein bition in embryogenic cells and tissues (Dewanti et al.
hydrolysate has growth promoting activity on different 2016).
genotypes of rice (Visarada et al. 2002) while the stim-
ulatory effect on embryo initiation and callus regeneration Effect of PEG on somatic embryogenesis
in sugarcane Nasir et al. (2011). MES is a buffering agent
added to stabilize the pH of growth medium (de Klerk The effect of PEG 8000 on development and growth of
et al. 2008) while proline as a compatible solute which somatic embryos was determined by incorporating PEG
plays a critical osmoprotective role in numerous 8000 at a different concentration in the callus induction

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Fig. 1 Callus induction

response of Co 86032 and Q117
to different media

Fig. 2 Type of callus: a compact, friable, creamish and embryogenic and b loose, filamentous, whitish, watery and non-embryogenic

medium. PEG 8000 had a considerable impact on the stress conditions developed by PEG 8000 due to binding of
development of somatic embryos in Co 86032 nutrients and free water required for cellular metabolism.
(54.66 ± 1.76%), Q117 (66.66 ± 2.60%) at 2.5% PEG. Moreover, PEG 8000 at higher concentrations developed
However, in the case of control (medium without PEG the high osmotic potential in the culture medium because
8000) only 24.33 ± 1.76 and 27.33 ± 2.73% of embryos of accumulation of minerals around it.
were observed respectively (Fig. 3). The sugarcane variety
Co 86032 performed an almost equal percentage of somatic Initiation and maintenance of cell suspension culture
embryogenesis at 2.5 and 5% concentrations of PEG 8000
in the medium. At lower concentration, PEG along with The cell suspension culture was initiated by inoculating
sucrose controls cellular necrosis enhancing somatic calli in liquid MS basal medium fortified with coconut
embryogenesis (Rizvi et al. 2012). At the above concen- water (500 ml l-1), 2,4-D (3 mg l-1) and casein hydro-
tration of 5%, the growth gradually reduced because of lysate (500 mg l-1) and cultures were maintained on

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the acclimatization of cells to the media components and

growth conditions. This was followed by vigorous cell
division (as OD600 and cell count increased rapidly) between
2 and 8 days. Further incubation beyond 8 days resulted in
the decrease in cell viability due to depletion of nutrients and
it comes to stationary phase. (Ho and Vasil 1983) also
reported the similar results and observed a maximum number
of cells on the 8th day of culture. Hence longstanding med-
ium needs to be replaced for maintaining a high live cell
count (Shah and Seth 2010).

Determination of cell type and size

Fig. 3 Effect of PEG on somatic embryogenesis in Co 86032 and
Q117
The cell suspension culture was composed of two basic
types of cells, i.e. embryogenic: spherical cells with
continuous shaking at 100 rpm. During incubation shaking
prominent and actively dividing nucleus, rich in cytoplasm
serves both, to aerate the culture and to disperse the eel in
and starch granules and non-embryogenic: cylindrical cells
medium (Shah and Seth 2010). The initiation of cell sus-
with large vacuoles (Supporting Figure 1). The cell size
pension culture requires comparatively a good quantity of
measured with ocular micrometry indicated it to be in
well-differentiated callus mass to serve as inoculums. Due
between 5 and 10 lm in the case of embryogenic cells and
to continuous agitation, the actively dividing and newly
10–45 lm in the case of non-embryogenic cells in both Co
formed cells gradually free themselves from the inoculated
86032 and Q117 tissues. The embryogenic and non-em-
fragile callus (Parekh et al. 2008). The well-established
bryogenic cells were developed in the same cultures,
suspension cultures with no cellular clumps were passage
however, non-embryogenic cells were comparatively
into fresh medium and incubated under constant agitation
higher in number than embryogenic cells. The number of
for maintaining viable cells in a free form.
embryogenic cells in Q117 was observed higher than Co
86032. The similar evidence of embryogenic and non-
Determination of growth in suspension culture
embryogenic cells was reported in sugarcane cell suspen-
sion culture in clone 68–1067 by Ho and Vasil (1983) and
The growth of sugarcane varieties Co 86032 and Q117 cell
Falco et al. (1996).
density in suspension culture was measured by the spec-
trophotometric method at OD600 and cell size by haemocy-
tometer, while cell viability was estimated using tryphan Plant regeneration from cell suspension culture
blue dye exclusion method. The growth curve plotted using
OD600 and cell count is shown in Fig. 4a, b and it was noticed Callus induction of Co 86032 and Q117 from suspension
that cell dividing rate is faster in Co 86032 as compared to culture achieved by inoculation of cells harvested from
Q117. There was negligible cell division (as OD600 and cell suspension culture into the callus induction medium VI
count was constant) during first 2 days of incubation due to supplemented with PEG 8000 (2.5%) is shown in Fig. 5a,

Fig. 4 Cell suspension culture growth of Co 86032 and Q117 by spectrophotometer (OD600) (a) and by haemocytometer (b)

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Fig. 5 Different stages of plant

regeneration through cell
suspension culture in S.
officinarum (Co 86032 and
Q117). a suspension culture on
callus induction medium
[MS ? 3 mg l-1 2,4-D ? 20 g/
l sucrose ? 100 ml l-1 coconut
water ? 500 mg l-1
PVP ? 500 mg l-1 casein
hydrolysate ? 500 mg l-1
MES buffer ? 1 mg l-1
thiamine HCL ? 20 mg l-1
inositol ? 500 mg l-1
proline ? 2.5% PEG (8000)].
b Callus proliferation after
15 days of suspension culture,
c shoot regeneration
[MS ? 1 mg l-1 BAP,
0.5 mg l-1 kinetin],
d regenerated plantlets,
e plantlets on root induction
medium [MS ? 5 mg l-1
NAA], f fully developed
plantlets on shoot elongation
medium

b. It was observed that the number of Q117 calli induced (embryogenic) in Q117 callus as compared to Co 86032.
and proliferated were comparatively more, prominent and The callus induced from the harvested cells were inocu-
actively dividing in contrast with Co 86032 (Fig. 4a, b). lated onto regeneration medium viz. MS basal medium
The possible reason for this could be the presence of containing benzylaminopurine (3 mg l-1) and kinetin
potentially dividing cells with prominent nuclei (1 mg l-1) further incubated at 26 ± 2 °C with 16/8 h

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Fig. 7 RAPD profile of cell suspension culture regenerated plants of

sugarcane. a OPH-02. b OPH-06. Lane M: 1 kb marker, 1: parent of
Co 86032, 2–9: Co 86032 plants from cell suspension culture, 10:
parent of Q117 and 11–18: Q117 plants from cell suspension culture

whole plant regeneration, many authors (Ho and Vasil

1983; Ahloowalia and Maretzki 1983) did not mention
clearly the process of plant regeneration. In other hand not
many researchers have attempted to regenerate whole
plants due to the difficulties associated with regeneration
from suspension culture of sugarcane.

Assessment of genetic stability using RAPD

and ISSR
Fig. 6 Rooting profile of regenerated plants of sugarcane. a Co86032
and b Q117 The quality of total genomic DNA extracted from the
young leaves of field grown mother plant and in vitro
photoperiod regimes (Fig. 5c). It was noticed that the raised plants was determined by agarose gel electrophore-
proliferation of Q117 callus was much higher as compared sis using kHind III as molecular marker and quantity on
to Co 86032 but the shoot regeneration was significantly UV–Vis spectrophotometer by recording OD at 260 and
superior in Co 86032 (41.4 ± 4.95 per 20 calli) (Fig. 5d) 280 nm. Genetic fidelity analysis using genomic DNA of
with respect to Q117 (10.6 ± 1.99 per 20 calli). The pre- the parent and in vitro raised plants was carried out to
sent results of plant regeneration from cell suspension confirm genetic stability using RAPD and ISSR markers.
through the embryogenic cells supports the observations of The fingerprinting profiles of the parent and in vitro
Vasil and Vasil (1982) and Ho and Vasil (1983) and also regenerated plantlets through cell suspension culture (Co
support the principle approach of regeneration in tissue and 86032 and Q117) using the RAPD and ISSR markers
cell culture of the poaceae is passing through somatic produced distinct and reproducible amplified products
embryogenesis. The roots were induced by incubating (Figs. 7, 8) and scoring data are summarized (Tables 2, 3).
elongated shoots excised from plantlets developed from RAPD analysis resulted in a total of 42 and 46 reproducible
proliferating callus on MS basal medium supplemented bands of Co 86032 and Q117 in vitro grown plants
with naphthalene acetic acid (Fig. 5e, f). It was observed respectively (Table 2). The number of bands produced per
that roots induced in Co 86032 and Q117 were compara- RAPD primer was ranging from 2 to 6 in Co 86032 and
tively equal in number and lengths as well as they were 2–8 in Q117. The percentage of monomorphic bands in Co
morphologically similar (Fig. 6a, b). The variation in 86032 and Q117 were 90.48 and 86.96%, which poly-
proliferation, shoot and root regeneration response might morphic bands in Co 86032 and Q117 were 9.52 and
be due to the influence of growth regulators and hormones 13.04% respectively. The size of amplified DNA fragments
on growing and developing callus (Salehi et al. 2014), produced by using selected ten primers ranged from 250 to
genotypic and phenotypic characters of the mother plant 3000 bp for Co 86032 and Q117 (Table 2). It was also
(Benderradji et al. 2012). Due to the time necessity for observed that only three primers (OPH-02, OPH-09 and

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Martin et al. (2004) and Lakshmanan et al. (2007)

suggested that use of multiple markers amplifying various
regions of the genome, allowing high probability for the
successful identification of polymorphism. RAPD (arbi-
trary, dominant) and ISSR (semi-arbitrary, medium to
highly reproducible, dominant and more stringent) usually
based on the non-coding regions of DNA. These two
markers are simple, cost effective and proved efficient in
evaluating the genetic homogeneity, genetic diversity and
evolutionary studies (Harish et al. 2014; Rathore et al.
2014). RAPD and ISSR has been successfully applied to
determine genetic fidelity/stability in several micropropa-
gated plant systems viz. sugarcane (Suprasanna et al. 2006;
Devarumath et al. 2007; Lal et al. 2008), Chlorophytum
borivilianum (Rizvi et al. 2012), Terminalia beelerica
(Dangi et al. 2014), Aloe barbandesis (Sahoo and Rout
2014), Swertia lawii (Kshirsagar et al. 2015), Dendro-
Fig. 8 ISSR profile of cell suspension culture regenerated plants of calamus strictus (Goyal et al. 2015), Dendrocalamus
sugarcane. a UBC 811. b UBC 857. Lane M: 1 kb marker, 1: parent of hamiltonii (Singh et al. 2013) etc. These markers have been
Co 86032, 2–9: Co 86032 plants from cell suspension culture, 10:
successfully applied for the detection of polymorphisms
parent of Q117 and 11–18: Q117 plants from cell suspension culture
that are either induced or incorporated during in vitro
regeneration of several plant species (Asthana et al. 2011).
OPH-18) and five primers (OPH-01, OPH-04, OPH-06, Ahloowalia and Maretzki (1983), Ho and Vasil (1983), and
OPH-13 and OPH-18) were showing polymorphism in Co Falco et al. (1996) were developed plants from sugarcane
86032 and Q117 respectively. The monomorphic bands in cell suspension culture, however not mentioned about
RAPD represent the presence of the same allele at a par- variation in the regenerated plants.
ticular locus, while polymorphic bands indicate two or Based on the above observations, the plant regeneration
more alleles at a single locus. The uniformity in banding through cell suspension culture was developed for both the
pattern confirms the genetic fidelity of in vitro raised varieties of sugarcane. This protocol imparts successful
sugarcane plantlets from cell suspension culture. technique that can be utilized for developing somaclones
Ten ISSR primers were selected for analysis after a and genetic trans-formation for its further improvement.
preliminary screening of 10 RAPD primers. All the ten This study clearly indicates that the RAPD and ISSR
ISSR markers yielded 62 and 64 scorable and reproducible marker technique can be utilized for detection of genetic
bands in Co 86032 and Q117 respectively. Each primer variation in early stages. To the best of our knowledge this
produced a unique set of amplification products ranging in is the first report which clearly mentions that the plants
size from 50 to 2000 bp in both the variety and number of regenerated from sugarcane cell suspension culture and
bands produced per ISSR primer was ranging from 4 to 9 in detects the genetic variation by using molecular markers.
Co 86032 and 5–9 in Q117 (Table 3). The percentage of
Acknowledgements The authors are grateful to Shri. Shivajirao
monomorphic bands in Co 86032 and Q117 were 100 and Deshmukh, Director General, Vasantdada Sugar Institute, Manjari
92.18% and the rest of the 7.81% polymorphic in Q117. It (Bk.), Pune for his constant support, encouragement and providing all
was also observed that only four primers (UBC-811, UBC- the facilities. One of the Authors (AT) received research fellowship
835, UBC-849 and UBC-857) were showing polymor- (Late Madhubhau Chaudhari Fellowship) during research work.
phism in Q117. The uniformity in banding pattern confirms Compliance with ethical standards
the genetic fidelity of in vitro raised sugarcane plantlets
from cell suspension culture. The possible reasons for Conflict of interest The authors declare that they have no conflict of
polymorphism observed during RAPD and ISSR analysis interest in the publication.
that occurred in in vitro raised plantlets regenerated from
Open Access This article is distributed under the terms of the
mother plants using cell suspension culture due to source of Creative Commons Attribution 4.0 International License (http://
explants, mode of in vitro regeneration, duration of cell and creativecommons.org/licenses/by/4.0/), which permits unrestricted
tissue multiplication, accumulating mutations during the use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
process of indirect organogenesis, etc. (Rizvi et al. 2012;
link to the Creative Commons license, and indicate if changes were
Kshirsagar et al. 2015). made.

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