In vitro culturing and assessment of somaclonal variation of

Türk Biyokimya Dergisi [Turkish Journal of Biochemistry–Turk J Biochem] 2011; 36 (4) ; 296–302.
Research Article [Araştırma Makalesi]
Yayın tarihi 30 Aralık, 2011 © TurkJBiochem.com
[Published online 30 December, 2011]
In vitro culturing and assessment of somaclonal variation
of Solanum tuberosum var. desiree
[In vitro kültürde büyütülen Solanum tuberosum var. desiree’deki somaklonal
varyasyonun değerlendirilmesi*]
Faiza Munir1,
Syed Muhammad Saqlan Naqvi2,
Tariq Mahmood3
Departments of 1Biotechnology and 3Plant Sciences,
Faculty of Biological Sciences, Quaid-i-Azam
University, Islamabad-46320, 2Department of
Biochemistry, University of Arid Agriculture
Rawalpindi, Pakistan
Yazışma Adresi
[Correspondence Address]
Dr. Tariq Mahmood
Department of Plant Sciences, Quaid-i-Azam University, Islamabad-46320, Pakistan.
Tel: +92-51-90643144
Fax: +92-51-2601059
E-mail: [email protected]
* Translated by [Çeviri] Ebru Karabal.
Registered: 11 May 2011; Accepted: 19 September 2011
[Kayıt Tarihi : 11 Mayıs 2011; Kabul Tarihi : 19 Eylül 2011
http://www.TurkJBiochem.com
ABSTRACT
Aim: Genetic variability frequently occurs in micropropagated plants. In the present study,
random amplification of polymorphic DNA technique is used to assess somaclonal variation
in Solanum tuberosum var. desiree. Detection of somaclonal variants at an initial phase of
growth can be valuable in establishing better tissue culture and transformation system in
potato by quality control.
Materials and Methods: Callus culturing conditions optimized at different hormonal concentrations. Three growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and zeatin were used in different concentrations and combinations. Maximum callus regeneration (90%) was observed at 2.5 mg/L 2,4-D while minimum growth
was monitored at 1.5 mg/L Zeatin and 1.0 mg/L BAP combination. Potato plant was also
regenerated from callus cells with best results on Murashige and Skoog basal media supplemented with both 1.0 mg/L BAP and 1.5 mg/L Indole acetic acid. Genetic variability in
in vitro cultured callus at different hormonal concentrations was assessed by using random
amplification of polymorphic DNA technique.
Results: Ten different decamer oligonucleotide primers generated 111 reproducible amplified products. The similarity coefficient values calculated through Simqual subprogram of numerical taxonomy system of multivariate software ranged from 0.419-0.838. Cluster analysis
divided five samples into two groups, an in group and an out group. Maximum polymorphic
bands were revealed by using hormonal combination Zeatin 1.5 mg/L and BAP 1.0 mg/L in
calli cultures. Conclusion: Results indicate that random amplification of polymorphic DNA
is an effective technique for the assessment of genetic variability in in vitro cultured calli.
Conflict of Interest: The authors have no conflict of interest.
Key Words: Solanum tuberosum, somaclonal variation, in vitro culturing, RAPD.
ÖZET
Amaç: Mikropropagasyon uygulanan bitkilerde çoğunlukla genetik varyasyon oluşur. Çalışmamızda, Solanum tuberosum var. desiree’de rastgele çoğaltılmış polimorfik DNA (random
amplification of polymorphic DNA – RAPD) yöntemi ile somaklonal varyasyon değerlendirilmiştir. Somaklonal varyantların büyüme başlangıcında belirlenmesi, kalite kontrolü sayesinde patateste daha iyi doku kültürü ve transformasyon sistemi oluşturulmasında önemli
rol oynayabilir.
Gereç ve Yöntemler: Kallus kültür koşulları farklı hormonal konsantrasyonlarda optimize
edilmişlerdir. Üç büyüme regülatörü, 2,4-diklorofenoksiasetik asit (2,4-D), 6-benzilaminopurin (BAP) ve zeatin farklı konsantrasyonlarda ve bileşimlerde kullanılmıştır. En yüksek kallus
rejenerasyonu (%90) 2.5 mg/L 2,4-D konsantrasyonunda, en düşük büyüme ise 1.5 mg/L zeatin ve 1.0 mg/L BAP bileşiminde gözlenmiştir. Kallus hücrelerinden patates bitkisinin rejenere edilmesinde en iyi sonuç 1.0 mg/L BAP ve 1.5 mg/L indol asetik asit eklenen Murashige ve
Skoog temel besiyerinde elde edilmiştir. İn vitro kültürde farklı hormonal konsantrasyonlarda
büyütülen kalluslardaki genetik varyasyon, RAPD yöntemi ile değerlendirilmiştir.
Bulgular: On farklı dekamer oligonükleotid primeri, 111 tekrarlanabilir amplifiye ürün oluşturmuştur. Multivaryasyonun numerik taksonomi sistemi yazılımındaki Simqual alt-programı
ile hesaplanan benzerlik katsayı değerleri 0.419 ile 0.838 arasında bulunmuştur. Küme analizi
sonucu 5 örnek, iç ve dış olmak üzere 2 gruba ayrılmıştır. Maksimum polimorfik bantlar,
kallus kültürlerinde 1.5 mg/L zeatin ve 1.0 mg/L BAP bileşimi kullanımında gözlenmiştir.
Sonuç: Bulgular, in vitro kültürde büyütülen kallluslardaki genetik varyasyonun değerlendirilmesinde, RAPD’nin etkili bir yöntem olduğunu göstermektedir.
Çıkar Çatışması: Yazarların çıkar çatışması bulunmamaktadır.
Anahtar Kelimeler: Solanum tuberosum, somaklonal varyasyon, in vitro kültür, RAPD.
296
ISSN 1303–829X (electronic) 0250–4685 (printed)
Introduction
Potato (Solanum tuberosum L.) of family Solanaceae is
one of the economically valuable vegetables worldwide
[1]. For research purpose, potato plantlets are in vitro
propagated to obtain multiple copies in limited time period. In vitro propagation of potato is beneficial in solving numerous difficulties linked with cultivation and
productivity. For genetic modification in potato, in vitro
regeneration is valuable method to produce multiple copies of the plant. Moreover, high yields of disease free
potato plantlets can be produced. Tissue culture procedure leading to great cellular reprogramming level may
be a consequence of increased somaclonal variations
[2]. Earlier, Labra et al. [3] reported various somaclonal variations in transgenic Arabidopsis thaliana plants
resulted from callus formation. Somaclonal variation
in potato calli can be utilized to find suitable variants
with desired characters, such as drought or salt stress
tolerance [4]. Assessment of somaclonal variation can be
helpful in potato breeding technique. Earlier, research
studies have been conducted in sweet potato for salinity
tolerance by evaluating the extent of somaclonal variation in regenerated plants [5]. It has been reported that
somaclonal variation can generate disease resistance in
potato. In this context, Rosenberg et al. [6] carried out
a study to identify variants with early and late blight
tolerance. The food value in terms of quality and quantity, defense strategy against infectious agents and
genetic makeup for desired traits in potato can be improved through biotechnological practices as tissue culture
and genetic transformation [7]. In potato transformation,
early identification of somaclonal variants can be beneficial in eliminating undesired variants that may affect
morphological characters and agronomic functions. The
propagation method in various plants results in repeated
occurrence of somaclonal genetic variation [8].
Genetic variations can be effectively revealed through
random amplification of polymorphic DNA (RAPD)
that has been confirmed as a highly valuable technique in this aspect [9]. According to Kaeppler et al. [10],
somaclonal genetic variation results from micropropagated plant cultures. Genetic likeness and contrasts in
plants propagated through tissue culture have been discovered by the productive application of RAPD [11-14].
In plant tissue culture system the rate of somaclonal variation enhances with the increase of subcultures in micropropagation protocols [15]. Biswas et al. [16] applied
random amplification of polymorphic DNA technique
for assessing the genetic fidelity in strawberry cultures
by investigating three successive subclones of in vitro
propagated strawberry.
Earlier, Vasconcelos et al. [17] indicated random amplification of polymorphic DNA as a practically accessible
method used for the identification of somaclonal variation in in vitro cultured maize plant and it was concluded that greater extend of somaclonal variation occurs
Turk J Biochem, 2011; 36 (4) ; 296–302.
during the callus development stage due to prolonged
culturing duration and growth regulators utilized for
callusing as compared to general plant regeneration.
Thawaro and Te-chato [18], utilized random amplification of polymorphic DNA technique to confirm hybridization for different groupings of hybrid of oil palm half
seeds utilized in the investigation. Similarly, various
plants that are at the risk of becoming extinct as Anisodus tanguticus [19], Neolitsea sericea [20], Heptacodium miconioides [21] have been analyzed through random amplification of polymorphic DNA for evaluating
genetic level variations. The main objective of the present research was to develop an efficient callus induction and regeneration protocol for Solanum tuberosum
var. desiree and to investigate genetic variations from in
vitro propagated calli maintained at different hormonal
concentrations and combinations using RAPD markers.
Material and Methods
In vitro plant propagation
In vitro cultures of Solanum tuberosum var. desiree
were maintained by optimizing a regeneration protocol.
Nodal segments were cultured on MS basal media [22]
supplemented with 30 g/L sucrose and 1.5 mg/L gibberellic acid (GA 3) for plant regeneration. Inoculation was
done in test tubes with one node per tube. The multiplication cultures were maintained in a growth chamber
at 25°C under cool white fluorescent light (2000 LUX)
and a photoperiod regime of 16 hrs light and 8 hrs dark.
Shoot growth and multiplication rate were examined after every other day until the shoots reached 8-10 cm in
size. Full length shoots were regularly sub-cultured by
shifting on fresh regeneration media.
Callus induction and regeneration
Nodal segments from tissue cultured Solanum tuberosum var. desiree, used as explants were inoculated on
callus induction media by using 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (BAP) and zeatin in different combinations and concentrations (Table
1). The growth rate of callus was recorded. The healthy
calli were transferred on fresh media and sub-cultures
were established.
Callus DNA extraction
Genomic DNA was isolated from five samples of callus tissues growing on different combinations of growth
hormones. Cetyl Trimethyl Ammonium Bromide
(CTAB) method illustrated by Richards [23] was used
for DNA isolation. DNA was quantified by spectrophotometric analysis and DNA purity was calculated from
OD260/OD280 ratio that was 1.7. Then the DNA samples
were checked by running them on 1% agarose gel prepared in 0.5X Tris Acetate Ethylene Diamine Tetra Acetic
Acid (TAE) buffer.
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Plant regeneration from callus
Callus tissue was shifted to the regeneration media. MS
basal media supplemented with different concentrations
of growth regulators Indole acetic acid (IAA), BAP and
GA 3 (Table 2) were used.
RAPD markers
RAPD amplifications were carried out by using ten decamer RAPD primers from OPC series, OPC1-OPC10
(Table 3). PCR reaction was optimized by increasing
the annealing temperature in 0.5ºC increments in successive optimization runs. Polymerase chain reaction
(PCR) mixture of 25µl was prepared by using 25 ng/
µl of genomic DNA template (40 ng/µl determined by
spectrophotometry), 25 pmol primer, 12.5 µl 2x PCR
master mixture and 10.5 µl of PCR water (MBI Fermentas). Polymerase chain reaction conditions employed for
the amplification were initial denaturation at 94ºC for
1 minute followed by 44 cycles of denaturation at 94ºC
for 30 seconds, annealing at 35ºC, 35.5ºC, 36ºC, 36.5ºC,
37ºC, 37.5ºC, 38ºC, 38.5ºC, 39ºC, 39.5ºC and 40ºC for 1
minute and extension at 72ºC for 2 minutes with a final
extension for 7 minutes at 72ºC in a gradient MultiGene
Thermal Cycler (Labnet). Polymerase chain reaction generated amplimers were separated on 1.5% agarose gel
prepared in 0.5X TAE buffer. Gel was stained with ethidium bromide (0.6 mg/ml) solution and gel documentation was conducted by using Dolphin Doc Plus Gel Image
System (Wealtec).
Table 1. Callus texture and rate of development at different hormonal concentrations optimized.
Samples
Explants
Growth Hormone (mg/l)
Rate of Callusing (%)
Callus Texture
1
Nodes
2,4-D – 2.5
90
Friable, soft
2
Nodes
2,4-D – 2.0
80
Friable, soft
3
Nodes
2,4-D – 3.0
70
Soft, watery
4
Nodes
BAP – 1.0 + 2,4-D – 2.0
50
Friable
5
Nodes
Zeatin – 1.5 + BAP – 1.0
35
Hard
Table 2. Regeneration media containing different concentrations of IAA, GA3 and BAP for potato plant culturing from callus tissue.
Regeneration Media Combinations
Growth Regulators mg/l
IAA
GA3
BAP
R1
1.0
-
1.0
R2
1.5
-
1.0
R3
-
1.0
1.5
R4
1.0
2.0
-
Table 3. Primers used in RAPD analysis
Sr. No.
Name
Sequence
1
OPC1
5’-TTCGAGCCAG -3’
2
OPC2
5’-GTGAGGCGTC -3’
3
OPC3
5’-GGGGGTCTTT -3’
4
OPC4
5’-CCGCATCTAC -3’
5
OPC5
5’-GATGACCGCC -3’
6
OPC6
5’-GAACGGACTC -3’
7
OPC7
5’-GTCCCGACGA -3’
8
OPC8
5’-TGGACCGGTG -3’
9
OPC9
5’-CTCACCCTCC -3’
10
OPC10
5’-TGTCTGGGTG -3’
Turk J Biochem, 2011; 36 (4) ; 296–302.
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Munir et al
Results
Micropropagation
High quality potato explants were regenerated in good
number (Figure 1) with an efficient high frequency protocol optimized by using 1.5 mg/L GA 3. There was no
bacterial or fungal contamination observed and the cultures obtained were 100% contamination free. Potato
callus was produced by using different types of growth
regulators in variable concentrations. Callus growth was
affected by different explants used and various concentrations of growth hormones used. Callus development in
terms of mass and weight was high with nodal segments
used as explants while leaves as explants revealed low
callus growth. It was observed that 2.5 mg/L of 2,4-D revealed best results showing 90% callus production. Callus production was 80% and 70% by using 2.0 mg/L and
3.0 mg/L of 2,4-D, respectively. Combination of BAP 1.0
mg/L and 2,4-D 2.0 mg/L resulted in 50% callus growth
while Zeatin 1.5 mg/L and BAP 1.0 mg/L combination gave 35% callus development (Figure 2). Graphical
representation of percentage growth of callus at various
hormonal concentrations was analyzed (Figure 3). The
effects of different growth regulators on potato callus
production have been summarized in Table 1. Using
callus cells, whole potato plantlets were successfully regenerated (Figure 4). IAA, GA 3 and BAP were used in
different concentrations (Table 2) for potato plant culturing from callus tissue. On the basis of results obtained,
it was observed that MS basal media supplemented with
1.0 mg/L BAP and 1.5 mg/L IAA showed best regeneration potential from callus tissue utilized as explants.
Fig. 1. In vitro propagated S. tuberosum var. desiree on MS basal
media.
Fig. 2. Healthy potato callus developed from nodes through in vitro
propagation with different hormonal concentrations. A: 2.5 mg/L
of 2,4-D; B: 2.0 mg/L of 2,4-D; C: 3.0 mg/L of 2,4-D; D: 1.0 mg/L
BAP and 2.0 mg/L 2,4-D; E: 1.5 mg/L Zeatin and 1.0 mg/L BAP.
RAPD Analysis
RAPD primers were applied to evaluate somaclonal genetic variability in five different samples of potato calli
obtained through in vitro propagation at different hormonal combinations and concentrations (Table 1). There was
difference in the intensity and resolution of banding pattern at different annealing temperatures. At 35ºC, 35.5ºC
and 36ºC there were faint bands while sharp bands were
observed at 36.5ºC. A gradual decrease in band intensity
was observed with further rise of temperature at 37ºC,
37.5ºC, 38ºC, 38.5ºC, 39ºC and 39.5ºC, while at 40ºC there were no bands. All of the ten primers (Table 3) have
produced sharp DNA bands at annealing temperature of
36.5ºC. In total 111 reproducible bands were generated
with ten primers. The number of bands and percentage of
polymorphism generated have been given in Table 4. The
banding pattern of amplified DNA samples ranged from
200 bp to 1500 bp in size. Six primers OPC2, OPC3, OPC5,
OPC7, OPC8 and OPC 9 resulted in some polymorphism
while OPC1, OPC4, OPC6 and OPC10 primers gave monomorphic bands. Figure 5 is a representative profile of
RAPD analysis indicating polymorphic bands generated
with OPC5, OPC7 and OPC8 primers. Among ten primers,
OPC5 produced maximum number of bands (20) from five
Turk J Biochem, 2011; 36 (4) ; 296–302.
Fig. 3. Comparison of callus growth rate at different hormonal
concentrations and combinations.
Fig. 4. Whole potato plant regeneration from callus tissue.
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Fig. 5. Representative RAPD profile of genomic DNA isolated
from in vitro propagated potato callus at five different hormonal
concentrations with OPC5, OPC7 and OPC8 primers. 1: callus sample
1 (2,4-D 2.5 mg/L) 2: callus sample 2 (2,4-D 2.0 mg/L) 3: callus
sample 3 (2,4-D 3.0 mg/L) 4: callus sample 4 (BAP 1.0 mg/L and 2,4D 2.0 mg/L) 5: callus sample 5 (Zeatin 1.5 mg/L and BAP 1.0 mg/L).
DNA samples analyzed, while OPC1 resulted in minimum
number of amplified bands (5). Minimum polymorphic
bands were found in sample 2 (2,4-D 2.0 mg/L), while
maximum number of polymorphic bands were resulted
in sample 5 (Zeatin 1.5 mg/L and BAP 1.0 mg/L). Highly
reproducible banding patterns were resulted for PCR runs
operated over a period of one month. For good reproducibility in RAPD analysis, the experiments were repeated
three times. Further, 2x PCR master mix (MBI Fermentas) was used for amplification reactions instead of using
PCR mixture made by adding individual components separately to avoid errors that can affect reproducibility. The
reproducibility of RAPD pattern was calculated according
to the computation done by Daya et al. [24]. In our RAPD
analysis the percentage reproducibility was 97%.
The similarity coefficients as estimated using Simqual
subprogram of NTSYS-pc software indicated maximum similarity (0.838) between the sample 1 (2,4-D 2.5
mg/L) and sample 3 (2,4-D 2.0 mg/L). However, the minimum genetic similarity was observed (0.419) between
the sample 3 (2,4-D 3.0 mg/L) and sample 5 (Zeatin 1.5
1.0 mg/L and BAP 1.0 mg/L) (Table 5). The phylogenetic cluster analysis was conducted with Sequential Agglomerative Hierarchical and Nested clustering (SHAN)
from the NTSYS-pc statistical package (version 2.02).
The cladogram comprised of an in group and an out
group (Figure 6). In group showed two sub-groups, in
which sub-group 1 comprised of sample 1 and 3 with
83% genetic relationship, while sample 2 and 4 (Table1)
formed the sub-group 2 with 81% genetic similarity (Figure 6). The out group represented by the sample 5 that
appeared to form a separate group. In case of sample 1, 2,
3 and 4 having 2,4-D 2.5 mg/L, 2,4-D 2.0 mg/L, 2,4-D
3.0 mg/L and BAP 1.0 mg/L + 2,4-D 2.0 mg/L, respectively, forming an in group, 2,4-D was a common hormone. Sample 5 with Zeatin 1.5 mg/L and BAP 1.0 mg/L
separated from rest of the samples 2,4-D was not present,
so different types of growth regulators may be the cause of genetic variability. Sample 5 showed low rate of
callus growth (35%) and required maximum culturing
period. A number of factors as culturing time period and
culturing conditions may result in genetic instability.
Turk J Biochem, 2011; 36 (4) ; 296–302.
Fig. 6. Dendrogram generated by the amplified products of five callus
samples by 10 OPC primers.
Growth regulators are the main components of media
that can affect the in vitro culturing in terms of growth
rate, therefore the optimization of their concentration is
the key requirement. As shown by the present data, 5
mg/L GA3 revealed maximum micropropagation of potato plant while for potato callus 2.5 mg/L of 2,4-D showed
maximum callusing. The genetic instability or variations
in in vitro cultures can be analyzed through RAPD markers. It has been observed that the rate of polymorphism
increases with the increase of culturing time period. According to present results the sample 5 (Zeatin 1.5 mg/L
and BAP 1.0 mg/L) required maximum time period for
callus production and resulted in maximum polymorphic
bands that indicated somaclonal genetic variation was
high and occurred in calli cultures by the use of hormonal combination of Zeatin 1.5 mg/L and BAP 1.0 mg/L.
Discussion
Potato plant cultures were maintained by using nodal/intermodal segments as explants. In earlier studies, Sarker
and Mustafa [25] and Haque et al. [26] reported that for
in vitro potato plant propagation nodal segments can show
multiple shoot production. In another report, Bordallo et
al. [27] analyzed somaclonal genetic variation by random
amplification of polymorphic DNA analysis in potato callus. The authors found that somaclonal variations were
induced by different growth regulators used for callus induction. Chakrabarti et al. [28] investigated the evaluation
of fingerprinting consistency in potato through RAPD by
using two potato varieties from greenhouse and four in vitro propagated cultivars. They reported more than 90% fingerprint likeness among various in vitro generated tissues
from a specific variety that suggested the usefulness of
random amplification of polymorphic DNA fingerprints
for the detection of potato cultivars with high accuracy.
In our results, 33 polymorphic bands were produced by
six OPC primers (OPC2, OPC3, OPC5, OPC7, OPC8
and OPC 9), while 73 monomorphic bands were generated by remaining four OPC primers (OPC1, OPC4,
OPC6 and OPC10). It was observed that the overall percentage of polymorphism is 30% (Table 4). There was a
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Table 4. Number of bands and percentage of polymorphism generated by using 10 OPC primers for RAPD analysis.
Total
Bands
Polymorphic
Monomorphic
Bands
Rare
Bands
Unique
Bands
Polymorphism
S.No.
Primers
1
OPC 1
5
5
0
0
0
0
2
OPC2
17
10
6
1
0
35
3
OPC3
10
5
5
0
0
50
4
OPC4
5
5
0
0
0
0
5
OPC5
20
10
8
2
0
40
6
OPC6
5
5
0
0
0
0
Bands
(%)
7
OPC7
15
5
8
2
0
53
8
OPC8
19
15
4
0
0
21
9
OPC9
10
8
2
0
0
20
10
OPC10
5
5
0
0
0
0
111
73
33
5
0
30
Total
Table 5. Similarity coefficient values for five samples analyzed through Simqual subprogram of NTSYS-pc software.
Sample No
1
2
3
1
1.0000000
2
0.7096774
1.0000000
3
0.8387097
0.8064516
4
0.6451613
0.8064516
0.7419355
1.0000000
5
0.4838710
0.5483871
0.4193548
0.4838710
5
1.0000000
difference in intensity of polymorphic bands generated
by different primers. The bands generated by OPC7 and
OPC8 primers were more intense as compared to those
resulted from OPC2, OPC3, OPC5 and OPC 9. Difference in band intensity occurs because each primer hybridizes in different extents to target DNA and the undefined
target DNA may exist in multiple copies per genome. Similar results have already been reported by Skroch and
Nienhuis [29] that RAPD bands amplified by one primer
vary in intensity from those amplified by another primer.
Earlier, random amplification of polymorphic DNA
analysis was conducted on the genetic resources of Plantago spp to access genetic variability [30]. It was reported that variation in growth regulators concentrations
and their proportion in culture media, culturing time period, nutrients [31], plant species, explants used and culturing conditions [32, 33] can be the factors responsible
for somaclonal genetic variation in micropropagated
plants. Detection of somaclonal variations by random
amplification of polymorphic DNA analysis has been
reported from other plants as well. One such example is
from in vitro propagated banana cultivar where genetic
stability was confirmed by monomorphic banding pattern shown by 50 RAPD primers [It was concluded from
our experimentation that various hormonal concentrations/combinations and culturing duration were the main
factors contributing the somaclonal genetic variability
in micropropagated potato calli. As our results showed
Turk J Biochem, 2011; 36 (4) ; 296–302.
4
1.0000000
that maximum culturing period in case of callus production by Zeatin (1.5 mg/L) and BAP (1.0 mg/L) resulted
in high genetic variability in terms of polymorphism.
Therefore, random amplification of polymorphic DNA
analysis can be a reliable tool for the assessment of somaclonal variation in Solanum tuberosum.
Acknowledgements
We are thankful to Pakistan Science Foundation, Islamabad, Pakistan for providing financial assistance for this
research work through the project No. C-QU/Bio (419).
Conflict of Interest
The authors have no conflict of interest.
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