DETECTION OF SOMACLONAL VARIATIONS IN POTATO USING RAPD MARKERS

otato (Solanum tuberosum L.) is an economically important vegetable crop in Egypt. It is the fourth most important crop by volume of production; it is high yielding, having a high nutritive value and gives high returns to farmers. Moreover, potato is considered a good source of antioxidants (Chen et al., 2007). It is vegetatively propagated, heterozygous and tetraploid, thus, traditional breeding of potato is very difficult (Solmon-Blackburn and Baker, 2001). Biotechnology could be contributed to solve this problem and realize great benefit to potato farmers. The regeneration of plants from cell and tissue culture represent an essential component of biotechnology and have the potential not only to improve the existing cultivars, but also for the generation of novel plants in a comparatively short time compared to conventional breeding. The success of plant biotechnology relies on several factors which include an efficient tissue culture system for regeneration of plants from cultured cells and tissues. During tissue culture, changes of interest to plant breeders may be heritable and result from changes in the plastid or nuclear genome. The introduction of variation may also be either problematic or useful for horticulturists and plant breeders and may occur in high frequency during adventitious plant regeneration or long-term callus culture (Kaeppler et al., 2000). Many researchers studied how to standardize the optimum concentrations of growth regulators for regeneration of potato and consequently great progress has been made in potato callus induction and plant regeneration (Ahloowalia, 1982; Dobranszki et al., 1999; Hansen et al., 1999; Ehsanpour and Jones, 2000; Fiegert et al., 2000; Yasmin et al., 2003; Shirin et al., 2007; Khadiga et al., 2009; Khalafalla et al., 2010; Shahabud-din et al., 2011).

Biotechnology could be contributed to solve this problem and realize great benefit to potato farmers.The regeneration of plants from cell and tissue culture represent an essential component of biotechnology and have the potential not only to improve the existing cultivars, but also for the generation of novel plants in a comparatively short time compared to conventional breeding.The success of plant biotechnology relies on several factors which include an efficient tissue culture system for regeneration of plants from cultured cells and tissues.During tissue culture, changes of interest to plant breeders may be heritable and result from changes in the plastid or nuclear genome.The introduction of variation may also be either problematic or useful for horticulturists and plant breeders and may occur in high frequency during adventitious plant regeneration or long-term callus culture (Kaeppler et al., 2000).Many researchers studied how to standardize the optimum concentrations of growth regulators for regeneration of potato and consequently great progress has been made in potato callus induction and plant regeneration (Ahloowalia, 1982;Dobranszki et al., 1999;Hansen et al., 1999;Ehsanpour and Jones, 2000;Fiegert et al., 2000;Yasmin et al., 2003;Shirin et al., 2007;Khadiga et al., 2009;Khalafalla et al., 2010;Shahabud-din et al., 2011).
DNA markers provided valuable tools in various analyses ranging from phylogenetic to the positional cloning of genes.Scoring of changes morphological and biochemical in plant can be useful in some studies, but there is limited diversity and trait may be affected by environmental influences.Molecular techniques such as Random Amplified Polymorphic DNA (RAPD) is often favored over traditional phenotypic, cytological and biochemical analysis, and generally assess even small variations in the genome.Detection of somaclonal variations using RAPD markers has several advantages, since RAPD P markers are technically simple, quick to perform with small amount of DNA and do not require previous information about genome or radioactive labeling (Michelmore et al., 1991).RAPDs are usually dominant and are inherited in a simple Mendelian fashion.Thus RAPD analysis is a useful tool in determining genetic relationships among regenerated potato and their original cultivars.The use of the PCR-based RAPD technique to detect somaclonal variations has been applied successfully to several plant species, such as Lolium (Wang et al., 1993), Allium sativum L (Al-Zahim et al., 1999) and Picea abies (Heinze and Schmidt, 1995).It has also been applied for tomato (Soniya et al., 2001) and potato (Khatab, 2000).The objectives of this study was to investigate the efficiency of callus induction and plant regeneration media for four potato cultivars and also to detect the somaclonal variations appeared after plant regeneration using RAPD markers.

Micropropagation
Three commercial potato cultivars; Desiree, Spunta, Silana and one exotic cultivar (Ijsselster) were used in this study.Sprouts were sterilized by immersing in 70% ethanol for 1 min, washed three times with distilled sterilized water to remove the traces of Ethanol, then immersed in 15% (v/v) Clorox and finally rinsed three times with distilled sterilized water.Disinfested sprouts were put on sterilized filter paper and cultured in cul-ture tubes containing MS medium (Murashige and Skoog, 1962).

Callus induction and plant regeneration
Stem segments were cultured on MS medium supplemented with different concentrations of plant growth regulators for callus induction (Table 1).The explants were cultured on callus induction media for four weeks at 25 ± 2C in complete dark.For further proliferation, the produced calli were transferred to the fresh callus induction media every 21 days interval.Callus induction percentages, callus fresh weight and morphological appearance were determined.Well developed calli were cultured on two different regeneration media (Table 1) for shoot regeneration and kept at 25 ± 2C with photoperiod of 16 h of light using Phillips cool white florescent tubes (1500 Lux) and plantlets regeneration percentage was determined.

Acclimatization
In vitro rooted plants were removed from rooting medium, then washed to remove adhering gel and transplanted to plastic pots (10 cm) containing autoclaved garden soil and beat-moss at 3:1 ratio and covered with plastic pages.Plants were kept under culture room conditions for 15 days then transferred to green house.

Molecular analysis
Regenerated plantlets produced from plant regeneration medium (PRM2, which gave the highest numbers of plant-lets) were acclimatized and subjected to RAPD-PCR analysis.DNA was extracted from fresh leaves of the regenerated plants and their original cultivars by Cetyltrimethyl Ammonium Bromide (CTAB) according to Doyle and Doyle (1990).RAPD was performed using 10 random decamer primers (Table 2).Polymerase Chain Reaction (PCR) was carried out in presence of 1X Taq DNA polymerase buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 ), 100 µM dNTPs, 5 picomole single random primer, 25 ng template DNA, 0.5 unit of Taq DNA polymerase in a total volume of 25 µl.PCR amplification was performed in automated thermal cycler (MJ-Mini, Bio Rad) programmed as follow, 95C for 4 min followed by 40 cycles of 1 min for denaturation at 94C, 30 sec for annealing at 37C and 1.30 min for polymerization at 72C, followed by a final extension step at 72C for 7 min.The amplification products were resolved by electrophoresis in 1.5 % agarose gels in 0.5 X TBE buffer and documented on Gel Documentation UVITEC, UK.Produced bands were scored as present (+) and absent (-).

Callus induction
The highest percentage of callus induction and proliferation was observed on CIM1 medium.Based on morphological appearance, callus induction media (CIM1 and CIM2) produced three types of calli; type A, creamy white, compact and nodular for Desiree on CIM1, type B, yellow globular, non-compact and soft for Silana, Ijsselster, and Spunta on CIM1, type C, yellow dark, compact was observed on CIM2 for all studied cultivars (Fig. 1A, B, C, D and Table 3).Callus induction percentages and callus fresh weight on CIM1 were varied among the studied cultivars and ranged between 100% and 660 mg to 73.3% and 285.3 mg for Desiree and Ijsselster cultivars, respectively.On the other hand, the percentages of callus induction and callus fresh weight on CIM2 ranged between 12.3% and 19 mg to 33.3% and 36.3 mg for Ijsselster and Spunta, respectively.

Plant Regeneration
Regeneration started with the appearance of green spots on callus within four weeks then, turned to normal plantlets (Fig. 2A, B and Table 4).Plant regeneration medium (PRM2) produced the highest percentage of plant regeneration for all tested cultivars.Desiree cultivar yielded the maximum percentage of plantlets regeneration (46%) with average of seven plantlets per callus while, the lowest percentage of plantlets regeneration was recorded for Spunta (8%) with average of one plantlet per callus.

RAPD analysis for somaclones
Somaclonal variations among 14 regenerated plants and their original cultivars were tested by RAPD analysis.Out of 10 random primers used, only five successfully produced scoreable RAPD bands (200 bp to 3.05 kb) for all the tested genotypes.Four primers (OPV02, OPA12, OPQ14 and OPE02) produced polymor-phic RAPD profiles and only one primer (OPA05) gave monomorphic bands (Table 5).All used RAPD primers produced 61 bands in all the tested genotypes, out of them, 23 bands (37.71%) were common in the parental genotypes and the regenerated plants while, the rest were polymorphic (62.29%).Among the primers used, OPE02 produced the highest number of bands ( 14) while primers OPA05 produced the lowest number ( 7), (Table 5).The highest number (11) and the lowest number (1) of polymorphic bands were observed in Desiree and its regenerants using primers OPE02 (78.5%) and OPV02 (14.3%), respectively.Some new additional bands/loci were present in some regenerantes and absent in their originals, for example the band with size of 200 bp which amplified by OPQ14 for Ijsselster regenerants, also, bands with size of 320 bp by OPA12, 240 bp and 1100 bp by OPV02 for Silana regenrants (Table 6).Moreover, primers OPA12, OPQ14 and OE02 gave some additional bands in some regenrtants of Desiree.Contrary, some bands were present in the original cultivars and absent in one or more of their respective regenrants, for example, bands with size 350, 400, 480 and 500 bp by OPE02 for Desiree (Table 6).

DISCUSSION
Somaclonal variations could be the result of mutation occurrence during the tissue culture process and in particular during plant regeneration from callus.PCR-based polymorphisms may be random or specific depending on the type of the primer used.RAPD amplification polymorphism is also a powerful technique for detection of somaclonal variations.Genotypes play a vital role in shoot regeneration as well in transformation efficiency among potato varieties (Sheerman and Bevan, 1988;Wenzeler et al., 1989;Phillip and Hampson, 1995;Khalafalla et al., 2010;Shahab-ud-din et al., 2011).In this study, it was observed that Desiree cultivars showed over all highly significant mean of callus induction percentage and fresh weight on CIM1 which contains 2,4-D and Kin.This result is in support of the results obtained by Fiegert et al. (2000), Jayasree et al. (2001) and Yasmin et al. (2003).Among all the growth regulators used 2,4-D was found to be the most effective growth regulator for potato callus induction either when used alone or in combination with cytokinins.Castillo et al. (1998) reported that 2,4-D by itself or in combination with cytokinins has been widely used to enhance callus induction and maintenance.Moreover, This result is in agreement with those of Shirin et al. (2007), who used 2,4-D for callus induction from internodal potato cultivars and found that 3.0 mg/l 2,4-D was found to be the most effective concentration.Produced calli cultured on MS medium supplemented with BAP and NAA gave plantlets more than using GA3 (Table 1).The necessity of cytokinins for shoot initiation is well documented (Beck and Coponetti, 1983;Evans et al., 1984;Khadiga et al., 2009).
In this study somaclonal variations were detected using 10 primers.Four pri-mers gave polymorphic bands.Some of these primers could reveal additional DNA bands, for example OPQ14, OPA12 and OPV02 were able to detect the extra DNA bands after PCR amplification, while OPE02 and OPA12 detected the missing DNA bands.Similar results in potato callus using RAPD-PCR have been reported by Bordallo et al. (2004), who observed somaclonal variations after different treatments with plant growth regulators based on additional or missing bands detected in the pattern of DNA using RAPD primers.In this study, additional or missing DNA bands were detected due to somaclonal variations.It has also been documented that if the callus phase is not long enough during plant regeneration, less somaclonal variation could be expected by Soniya et al. (2001).Since, even a single base change at the primer annealing site is manifested as appearance or disappearance of RAPD bands, it could be suggested that tissue culture conditions have induced varied amounts of genetic changes in different regenerated plants.Some of these changes appeared identical in different plants as represented by appearance of non-parental bands.Such commonness of genetic variation in these plants could be because they were all derived from the same callus.Somaclonal variation might be useful for selection of callus for desirable traits, such as biotic and /or a biotic stresses or secondary metabolites production.In this study an efficient callus induction and plant regeneration protocols were developed for some new potato cultivars recently grown in Egypt and not examined be-fore.Moreover, RAPD-PCR as a molecular marker was applied for detection of somaclonal variation in the studied cultivars.In conclusion, the system established in the present study for tissue culture of potato can get enough callus and plant regeneration efficiency to perform transgenic operation.Moreover, as the potentiality of shoot multiplication from callus continued for a long time, regenerates may be characterized by somaclonal variation and giving birth to traits of agronomic importance.

SUMMARY
Investigating of the efficiency of callus induction and plant regeneration for four potato cultivars (Desiree, Spunta, Silana and Ijsselster) as well as detection of somaclonal variations appeared after plant regeneration were studied.For callus induction and plant regeneration, in vitro stem explants were cultured on MS medium supplemented with different types of plant growth regulators.The highest percentage of callus induction was obtained using callus induction media (CIM1) that contained 3 mg/l 2,4-D and 0.5 mg/l Kin.Moreover, the medium containing 1 mg/l Kin, 0.5 mg/l NAA and 2 mg/l BA gave the highest percentage of plant regeneration.Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic variability of the hardened regenerated plants.Ten arbitrary decamer primers were used to amplify genomic DNA of the four original potato cultivars and 14 regenerated plants.Thirty eight bands out of 61 were polymorphic (62.29%).RAPD patterns generated by these primers suggested high percentage of polymorphic fragments, indicating high level of genetic variations among genotypes.Desiree cultivar showed the highest number of polymorphic fragments, while Silana cultivar showed the lowest percentage of somaclonal variations.This study established an efficient system for potato plant regeneration that could be used to perform transgenic operation.However, the obtained somaclonal variants can be used for selection of potato toward desirable traits.Yasmin, S., K. M. Nasiruddin, R. Begum and S. K. Talukder (2003).Regeneration and establishment of potato plantlets through callus formation with BAP and NAA.Asian J. Plant Sci., 2: 936-940.

Fig
Fig. (1): Callus induction efficiency of the four studied potato cultivars on two different callus induction media.

Table ( 3
): Callus induction means, fresh weight (mg) and color for the four potato cultivars.

Table ( 4
): Plant regeneration efficiency of the four potato cultivars under study on two different regeneration media.

Table ( 5
): Distribution of RAPD markers among the four potato cultivars and their fourteen selected somaclones.