Molecular diversity and phenotypic responses of two in vitro Solanum tuberosum varieties by physical mutagen

ABSTRACT

Induction of mutation in potato (Solanum tuberosum L.) plants by gamma radiation is considered an effective method for plant improvement. Solanum tuberosum L. is a vegetative plant, its tuber is starch-rich and it has worldwide economic importance. Two potato buds varieties (Valor and Spunta) were cultured in vitro on MS medium supplemented with 0.2 mg/l 6-Benzylaminopurine (BAP). The resulting plantlets were irradiated with gamma radiation doses of 10, 20, 30 and 40 Gy. Irradiated single node pieces were transferred onto fresh MS medium supplemented with BAP. Plantlet survival percentage and the other phenotypic criteria were calculated after eight weeks. Gamma radiation caused a moderate decrease in the survival percentage and the phenotypic criteria. Microtubers produced from irradiated plantlets changed in size compared to the control. The estimated proline content in irradiated plantlets was increased with increasing gamma radiation dose. The genomic DNA of the two varieties generated 150 bands. The maximum number of bands was 13, while the minimum number of bands was 5. Fifty-four polymorphic bands were amplified and the percentage of polymorphism ranged from 12.50 to 66.66% for two varieties. The highest genetic identity was 0.9672, between irradiated plantlets of the Valor variety with doses of 20 and 30 Gy. However, the highest genetic distance was 0.3995 observed between irradiated plantlets with a dose of 20 Gy in the Valor variety and 30 Gy in the Spunta variety. The dendrogram generated by cluster analysis distinguished the irradiated plantlets genetically. All of the mutants Spunta obtained via gamma irradiation were genetically very distant from the control.

KEYWORDS:

• Solanum tuberosum
• in vitro culture
• gamma radiation
• phenotypic criteria
• RAPD
• DNA polymorphism

1. Introduction

Potato is a crop of worldwide importance and it is an integral part of the diet of a large proportion of the world’s populations (Kane 2000; Drewnowski and Rehm 2013; Weaver and Marr 2013). Plant breeders suffer from the lack of availability or existence of required genotypes, therefore induced genetic diversity is the basic requirement in developing plant varieties (El-Fiki 1997). Mutation induction in combination with in vitro culture techniques may be effective methods for plant improvement (Novak 1991). The induction of genetic and phenotypic changes in plants using induced mutation has been found as the most efficient method. Induced mutation is widely used for changing genes which control important traits, and understanding the function and mode of action of such genes as well as developing improved crop varieties (Gaafar et al. 2016).

One of the most successful mutagenic agents is radiation, which induced mutation breeding in various crops and ornamental plants (Schum 2003). Radiation represented an excellent means of stimulating the expression of recessive genes, thus inducing new genetic variation (Song and Kang 2003). Gamma rays are ionizing radiation and interact with atoms or molecules inside the cells to produce free radicals. These radicals can damage or modify important components of plant cells and have been reported to affect differentially the morphology, anatomy, biochemistry and physiology of plants depending on the irradiation level. These effects include changes in the plant cellular structure, variation of some metabolic reactions, modulation of the antioxidative system and accumulation of phenolic compounds (Kovacs and Keresztes 2002; Kim et al. 2004; Wi et al. 2005).

Molecular genetic markers have become useful tools in providing a relatively unbiased estimation of genetic diversity and phylogeny in plants (Clegg 1990). Several different PCR techniques for DNA fingerprinting have been developed during the last decades, each one with specific advantages and disadvantages. Random amplified polymorphic DNA (RAPD) is the simplest and fastest DNA-based techniques in genetic similarity studies (Gwanama et al. 2002). A number of scientists have used RAPD markers to study polymorphism in various plants (Rout and Das 2002; Samal et al. 2003; Bered et al. 2005).

This work is an attempt to increase the genetic variability of two potato varieties (Valor and Spunta) using gamma radiation as physical mutagen and evaluate the phenotypic changes and forming molecular diversity analysis by using a RAPD marker.

2. Materials and methods

2.1. Plant materials

Buds of two potatoes varieties (Spunta and Valor) were obtained from Agriculture Research Centre, Giza, Egypt. They were excised and surface sterilized by dipping in clorox (30%) for 10 min. followed by three rinses in sterile distilled water. The buds were cultured on solid MS medium (Murashige and Skoog 1962) without any hormone. Micropropagation began after 6–8 weeks when the plantlets were about 10–12 cm high. The culture was cut into single nodes and transferred onto fresh MS medium supplemented with 0.2 mg/l BAP. The pH of the culture medium was adjusted to 5.7 and it was incubated in a growth chamber at 25°C ± 2 under a 16 h photoperiod at 3000 lux.

2.2. Gamma irradiation

Irradiation was carried out using ⁶⁰Co as a source of gamma radiation at the dose rate of 10 Gy/23 min 34 s. at National Centre for Radiation Research and Technology, Cairo, Egypt. Mass cultures of in vitro grown plantlets derived from single nodes were treated with different doses of gamma rays (10, 20, 30, 40 Gy) and some nodes were not exposed to gamma rays and used as control.

2.3. Tuberization

The irradiated and control plantlets were transferred to liquid MS medium containing 8% sucrose, 2.0 mg/l BAP to enhance the formation of microtubers. The pH of the culture medium was adjusted to 5.7 before autoclaving. The cultures were incubated in a growth chamber at 20°C under a photoperiod of 8 h at 400 lux for 3–4 months. The resulting microtubers were cultured in green house for microtuber production.

2.4. Acclimatization

Irradiated and control plantlets were cultivated in sterile jars containing peat moss and sand with ratio 1:1 and covered with puncture plastic sheets. After one week the plastic cover sheets were removed and the plantlets were left in growth chamber one week before transfer to the green house.

2.5. Phenotypic changes analysis

Morphological criteria were recorded for 6–8-week-old plantlets. The measured criteria were number of growing plantlets, number of nodes, shoots and leaves and shoot length (cm). Survival percentage (%) and fresh and dry weight were also determined (fresh weights of the plants was determined directly after taking the samples, while dry weights were determined after drying the samples for 48 h at 80°C. Data were statistically analysed using ANOVA to compare the difference between irradiated and control plantlets using Costat software (http://www.cohort.com/costat.html).

2.6. Proline content estimation

The proline content was estimated according to Beatles et al. (1973) in irradiated and control plantlets of both varieties. Briefly, 0.5 g plant materials were extracted by 3% aqueous sulfosalicylic acid. The proline extracts were reacted with 2 ml acidninhydrin, 2 ml of glacial acetic acid and 4 ml toluene, and then the reaction was terminated in an ice bath. The absorbance of samples was read at 520 nm using a Jasco V530 spectrophotometer (JASCO Corporation,  Tokyo, JAPAN).

2.7. Genomic DNA isolation

Total genomic DNA was isolated according to the protocol described by Doyle and Doyle (1987) in control and irradiated potato plantlets of Valor and Spunta varieties.

2.8. Random amplified polymorphic DNA-polymerase chain reaction amplification

Fifteen primers were chosen arbitrarily. The 10-mer primers used were synthesized by Metabion International AG (Steinkirchen, Germany). RAPD amplification was performed according to Williams et al. (1990). Primer sequences (5′–3′) were as shown in Table 1. Amplification reactions were performed in a 50 μl volume, containing: 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 200 μM each of dNTPs, 1 μM primer, 30 ng of genomic DNA, and 1.5 U of Taq DNA polymerase. The reaction mixture was overlaid with two drops of mineral oil, incubated for 3 min at 94°C for initial denaturation, and then amplified for 35 cycles consisting of 1 min at 94°C, 30 s at 32.3°C and 1.30 min at 72°C, followed by 7 min incubation at 72°C. Amplification products were separated by gel electrophoresis on 1.0% agarose and visualized under UV transilluminator after staining with ethidium bromide and photographed.

Table 1. Primer names and sequences.

No	Name	Sequence	No.	Name	Sequence
1	OP-A02	TGC CGA GCT G	9	OP-C08	TGG ACC GGT G
2	OP-A12	TCG GCG ATA G	10	OP-F06	AGG TGC GTC C
3	OP-B-01	CTG TCG TCG T	11	OP-L12	GGG CGG TAC T
4	OP-B07	AGG TGA CCG T	12	OP-L13	ACC GCC TGC T
5	OP-B10	CTG CTG GGAC	13	OP-L16	AGG TTG CAG G
6	OP-B11	CAG CAC TGC T	14	OP-L20	TGG TGG ACC A
7	OP-B12	CCT TGA CGC A	15	OP-Z03	CAG CAC CCC A
8	OP-C02	GTG AGG CGTC

2.9. Data analysis

The sizes of RAPD fragments were estimated by comparing the induced bands with the Gene ruler^TM (100bp) DNA Ladder (Fermentas #SM0328). RAPD fingerprints were recorded in the binary form (1 = presence of a band and 0 = absence of a band). The percentage of polymorphic fragments between control potato and their treatment were used to evaluate the genetic diversity in the two varieties. The similarity matrix between them and the UPGMA algorithm was used to perform hierarchical cluster analysis and to construct the dendrogram by using POPGENE package Version 3.5 (Yeh et al. 1999).

3. Results and discussion

Potatoes were propagated by using microcutting stem techniques on MS medium. The Valor variety was sensitive to gamma radiation doses; the resulting microtubers were decreased in size with increasing gamma radiation doses of as compared with control. In contrast, the Spunta variety was able to tolerate different gamma radiation doses (Figure 1). The acclimatization process in irradiated plantlets of both Spunta and Valor varieties as well as control plant as can be seen in Figure 2.

Figure 1. Formation of microtubers by irradiated (a) Spunta and (b) Valor varieties.

Figure 2. Acclimatization stages of irradiated Spunta and Valor plantlets.

All doses of gamma rays induced a moderate effect on phenotypic criteria as compared with control (Table 2). Shoot length ranged from 7.1 to 1.03 and 8 to 1.5 cm in Spunta and Valor varieties respectively. This observation could be correlated with the results obtained by Khalil et al. (1986) and Gaafar et al. (2017), who explained the decrease in shoot and root lengths at higher doses of gamma rays on the basis of reduction in the mitotic activity of plant tissues.

Table 2. Effect of different gamma radiation doses on several phenotypic criteria in Spunta and Valor varieties.

	No. of growing plantlets		Mean of shoots		Mean of nodes		Mean of leaves		Mean of shoot length (cm)
Radiation doses	Spunta cv.	Valor cv.	Spunta cv.	Valor cv.	Spunta cv.	Valor cv.	Spunta cv.	Valor cv.	Spunta cv.	Valor cv.
Control	100^a ± 0.48	101^a ± 0.45	2^a ± 0.0	2^a ± 0.0	5^a ± 0.0	5^a ± 0.0	8^a ± 0.0	8^a ± 0.0	7.5^a ± 0.45	8.26^a ± 0.49
10Gy	96^b ± 0.50	98^b ± 0.51	2^a ± 0.0	2^a ± 0.0	5^a ± 0.0	5^a ± 0.0	8^a ± 0.0	8^a ± 0.0	7.1^a ± 0.19	8^a ± 0.59
20Gy	86^c ± 0.45	92^c ± 0.35	1.46^a ± 0.50	2^a ± 0.0	5^a ± 0.0	5^a ± 0.0	8^a ± 0.0	8^a ± 0.0	6.4^b ± 0.72	6.4^b ± 0.39
30Gy	72^d ± 0.41	83^d ± 0.51	1.36^a ± 0.47	2^a ± 0.0	5^a ± 0.0	3.5^b ± 0.51	2.6^b ± 0.50	3.5^d ± 0.51	5.8^b ± 0.20	5.4^b ± 0.84
40Gy	30^e ± 0.65	32^e ± 0.51	0.6^b ± 0.31	0.73^c ± 0.17	2^b ± 0.63	2.2^c ± 0.41	1.8^c ± 0.56	2.3^e ± 1.04	1.03^c ± 0.29	1.5^c ± 0.51

Values (mean±sd) followed by different letters are significantly difference at 0.05 level.

The doses of 10, 20 and 30 Gy had no effect on the number of shoots, nodes and leaves, while the 40 Gy dose has a drastic decreasing effect on them (Table 2). The decrease in morphological criteria of various crops has already reported by (Mejri et al. 2012; Gaafar et al. 2017), which supports the present investigation.

Gamma irradiation caused a weak gradual decrease in the survival percentage of both Spunta and Valor varieties. Only 40 Gy caused severe depression in the survival percentage of both varieties, reaching 28.5 and 30.4% in Spunta and Valor varieties respectively as shown in Figure 3. Also, gamma radiation doses had a moderate decrease in the fresh and dry weights of both varieties (Figure 4). The present results are in line with Kon et al. (2007) and Marcu et al. (2013).

Figure 3. Gamma radiation impact on potato survival percentage of Spunta and Valor varieties.

Figure 4. Gamma radiation impact on fresh and dry weight of Spunta and Valor plantlets.

The radiation sensitivity result based on survival percentage, shoot length and tuberization of irradiated and non-irradiated plantlets showed that there was a moderate significant reduction with increasing gamma dosage. These results were in accordance with radiation sensitivity test done by El-Fiki (1997) for potato; El-Fiki et al. (2005) for alfalfa, Norfadzrin et al. (2007) for tomato and okra and Kiong et al. (2008) for Orthosiphon stamineus El‐Fiki et al. (2015) and (El-Fiki et al. 2016) for tobacco.

Gamma rays are often used in developing varieties of some plants that are economically important and have high productivity potential (Jain et al. 1998). They are useful for mutations induction in breeding programs and in vitro mutagenesis in order to develop required features of plants and increase the genetic variability (Jain et al. 1998).

Proline content in irradiated potato plantlets with doses of 10, 20, 30 and 40 Gy and control plantlets was estimated. Gamma radiation doses had a positive impact on proline content in both varieties. The results showed that the proline content was increased with increasing gamma radiation dose (Figure 5). The accumulations of osmoprotectants, like proline, adapt the cell under stress towards the external environment factors (Huang et al. 2013). Therefore, accumulation of proline could be used as an important feature in selecting tolerant species or genotypes as previously reported by Ruffino et al. (2010). One of the most crucial functions of plant cell is to respond to gamma stress by developing defence mechanisms. This defence may be affected by alteration in the pattern of gene expression (Corthals et al. 2000), which may lead to modulation of certain metabolic and defensive pathways (Zolla et al. 2003).

Figure 5. Gamma radiation impact on proline content of Spunta and Valor plantlets.

Total genomic DNA from two radiated potato varieties Spunta and Valor were used as templates for RAPD finger printing. Of the 15 RAPD primers tested, 10 primers were selected based on the quality and repeatability of the amplified bands. The main changes observed in the RAPD profiles are appearance or disappearance of different bands with variation of their intensity (Figure 6); these effects might be connected with structural rearrangements in DNA caused by different types of DNA damages such as breaks, transpositions and deletion as clarified by Mejri et al. (2012). The appearance of new RAPD bands (Figure 6) could be due to the effect of DNA mutation enhanced by gamma radiation (Atienzar and Jha 2006). On the other hand, the disappearance of some RAPD bands could be explained as there was damage to DNA, such as presence of oxidized bases and/or modified bases or the presence of single or double-strand breaks. Point mutations, DNA–protein cross links and/or rearrangement of chromosomes can also be induced by gamma irradiation (Ginchner et al. 2008; Abhay et al. 2014). Moreover, free radicals induced by gamma radiation may interact with biomolecules like protein or DNA and remove electrons from them, which in turn can cause damage to both structure and activity of the DNA. Moreover, differences between DNA of two individuals DNA samples result in the loss or gain of some bands (Bardakci 2001).

Figure 6. RAPD profiles of irradiated and control Spunta (S) and Valor (V) varieties. M, marker; 1, V control; 2, V 10 Gy; 3, V 20 Gy; 4, V 30 Gy; 5, V 40 Gy; 6, S control; 7, S 10 Gy; 8, S 20 Gy; 9, S 30 Gy; 10, S 40 Gy.

Figure 6. (Continued).

Yoko et al. (1996) reported that the large strands of DNA were broken into small strands at low irradiation dose of gamma rays. However, small and large DNA strands were broken at higher irradiation doses. Moreover, Kahrizi et al. (2012) showed that when gamma doses were increased, the nuclear DNA content was decreased in all the genotypes studied. Likewise, Hussein (2012) clarified that the appearance or disappearance of bands under gamma irradiation might be considered as molecular markers for radiation processes.

Altogether, 150 bands were amplified (Table 3). The maximum number of bands was 13, produced by primer OP-B07 with Valor samples. The lowest number of bands was 5, produced by primers OP-B11 with Spunta samples, OP-L13 with Valor samples and by OP-L16 and OP-Z03 with both varieties. All 10 primers were polymorphic and generated 54 polymorphic bands (Table 3). The polymorphism percentage ranged from 14.28 to 66.66% with an average of 38.94% in the Spunta variety, while the polymorphism percentage of the Valor variety ranged from 12.50 to 55.55% with an average of 31.62%. These polymorphism percentages displayed genetic diversity between control and treated samples in both varieties. This indicates that gamma ray irradiation can be considered as an effective tool of mutant induction (Pestanana et al. 2011; Taheri et al. 2014) and RAPD is of relatively high discriminative power for mutants or mutant loci (or the genetic diversity between them). The discovery that PCR with random primers can be used to amplify a set of randomly distributed loci in any genome facilitated the development of genetic markers for a variety of purposes (Williams et al. 1990; Welsh and McClelland 1994). The main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterization of the genome. The concept of polymorphism is used to determine the genetic variability in the population, which in recent decades has become the subject of many studies in various disciplines such as genetics, ecology, botany, and zoology (Chesnokov and Artemyeva 2015). Fingerprinting based on RAPD technique was used for identification and characterization of potato varieties in North America (Sosinski and Douches 1996), Australia (Ford and Taylor 1997) and India (Chakrabarti et al. 1998).

Table 3. Number and percentage of polymorphic loci obtained from gamma radiated Spunta and Valor varieties.

Primer No.	Spunta				Valor
	Gel polymorphism				Gel polymorphism
	Total band	Monomorphic bands	Polymorphic bands (unique + non-unique bands)	Polymorphism (%)	Total band	Monomorphic bands	Polymorphic bands (unique + non-unique bands)	Polymorphism (%)
OP-B01	9	6	3	33.33%	9	6	3	33.33%
OP-B07	12	4	8	66.66%	13	11	2	15.38%
OP-B11	5	4	1	20.00%	7	4	3	42.85%
OP-B12	9	7	2	22.22%	10	6	4	40.00%
OP-F06	8	3	5	62.50%	9	4	5	55.55%
OP-L12	7	6	1	14.28%	8	7	1	12.50%
OP-L13	7	3	4	57.14%	5	3	2	40.00%
OP-L16	5	3	2	40.00%	5	3	2	40.00%
OP-L20	6	4	2	33.33%	6	5	1	16.66%
OP-Z03	5	3	2	40.00%	5	4	1	20.00%
Total	73	43	30		77	53	24
Mean	7.3	4.3	3.0	38.94%	7.7	5.3	2.4	31.62%

The genetic identity and genetic distance between the 10 gamma radiated treatments of both potato varieties, Valor and Spunta, are presented in Table 4. The Nei’s genetic identity was the highest (0.9672) in treatment pairs 30 and 20 Gy in the Valor variety. The lowest genetic identity was (0.6707) in treatments pairs 30 Gy in the Spunta variety and 20 Gy in the Valor variety. Likewise, the highest Nei’s genetic distance was 0.3995 between the two treatments 20 Gy in the Valor variety and 30 Gy in the Spunta variety. The lowest Nei’s genetic distance was 0.0333 within irradiated Valor with doses 30 and 20 Gy.

Table 4. Genetic identity (above diagonal) and genetic distance (below diagonal) values among of 10 gamma radiation treatments in potato varieties Spunta and Valor.

ID	1	2	3	4	5	6	7	8	9	10
1	****	0.8800	0.8559	0.8559	0.8757	0.9167	0.7740	0.7986	0.7235	0.8389	1 = Cont. Valor
2	0.1278	****	0.7163	0.7650	0.7540	0.8984	0.8311	0.7461	0.8408	0.7542	2 = 10Gy Valor
3	0.1556	0.3336	****	0.9672	0.9006	0.8244	0.7067	0.7671	0.6707	0.8721	3 = 20Gy Valor
4	0.1556	0.2678	0.0333	****	0.8684	0.8389	0.7219	0.7671	0.7206	0.8209	4 = 30Gy Valor
5	0.1327	0.2824	0.1047	0.1411	****	0.8610	0.7912	0.7842	0.6985	0.8729	5 = 40Gy Valor
6	0.0869	0.1071	0.1931	0.1757	0.1496	****	0.8463	0.8542	0.7720	0.8346	6 = Cont. Spunta
7	0.2562	0.1850	0.3472	0.3259	0.2341	0.1669	****	0.8820	0.8877	0.8780	7 = 10Gy Spunta
8	0.2249	0.2929	0.2652	0.2652	0.2431	0.1576	0.1256	****	0.8034	0.8936	8 = 20Gy Spunta
9	0.3237	0.1734	0.3995	0.3277	0.3587	0.2588	0.1191	0.2189	****	0.7795	9 = 30Gy Spunta
10	0.1757	0.2821	0.1369	0.1974	0.1360	0.1809	0.1301	0.1125	0.2491	****	10 = 40Gy Spunta

A dendrogram based on Nei’s (1972) genetic distance using the unmeasured pair group method of arithmetic mean (UPGMA) was established with 10 gamma irradiation treatments in potato varieties Valor and Spunta (Figure 7). These treatments segregated into two main clusters at about 29. The first cluster contained control Spunta and Valor and all irradiated Valor. The second cluster includes the Spunta variety irradiated with different gamma radiation doses 10, 20, 30 and 40 Gy. The first cluster was divided into two subclusters at about 26. The first subcluster contained the two controls for Spunta and Valor and irradiated Valor with 10 Gy, while the second subcluster contained irradiated Valor with 20, 30 and 40 Gy. The second cluster was divided into two sub clusters at about 24. In the first sub cluster were the two irradiated Spunta with 10 and 20 Gy, while irradiated Spunta with 30 and 40 Gy were in the second subcluster. Upon evaluation of the dendrogram demonstrating genetic distances, irradiated Valor with 10 Gy appeared to be very close to the controls, while irradiated Spunta with 40 Gy were very distant. Generally, all of the Spunta mutants obtained via gamma irradiation were genetically very distant from the control plant. The high difference in gene diversity among accessions/varieties reveals the presence of strong difference in the genetic structure between them and thus significant differences exist in the genotypic among them (Ashraf et al. 2014).

Figure 7. Dendrogram of 10 gamma irradiated treatments of potato varieties Valor and Spunta based on genetic distances produced by RAPD.

4. Conclusion

It can be concluded that the usage of gamma radiation as a tool for genetic variations had a negative impact on growth rate, microtubers and size of potato plantlets. So, it can be concluded that gamma ray treatment was an effective method for mutation induction in potato varieties and the mutants were successfully identified by using RAPD analysis.

Disclosure statement

No potential conflict of interest was reported by the authors.