Assessment of genetic stability on in vitro and ex vitro plants of Ficus carica var. black jack using ISSR and DAMD markers

Clonal propagation is one of the attributes of plant tissue culture. Therefore, analysis of genetic stability among the in vitro cultured plants is a crucial step. It helps to signify the clonal propagation of the micropropagated plants. Regenerated Ficus carica var. Black Jack plantlets were established using woody plant medium supplemented with 20 μM 6-Benzylaminopurine and 8 μM Indole-3-acetic acid under different light treatments such as normal fluorescent white light (60 μmol m−2 s−1), and four different LED spectra, white (400–700 nm), blue (440 nm), red (660 nm) and blue + red (440 nm + 660 nm). Genetic stability analysis was performed on the in vitro and ex vitro plants of Ficus carica var. Black Jack. Ten primers of each, ISSR and DAMD molecular markers, were used to assess the genetic stability of the eight samples of Ficus carica var. Black Jack. ISSR markers showed 97.87% of monomorphism whereas DAMD markers showed 100% monomorphism. Polymorphism of 2.13% was observed for the UBC840 ISSR–DNA primer which was negated under the genetic similarity index analysis for the eight samples. The findings of this study revealed that ISSR and DAMD markers are efficient in determining the polymorphism and monomorphism percentage among the in vitro and ex vitro samples of Ficus carica var. Black Jack. Monomorphism of 100% obtained using DAMD markers and more than 95% of monomorphism obtained using ISSR markers indicate that the regenerated plants are significantly genetically stable. These molecular markers can be used to test the genetic stability of in vitro regenerated plants. It is recommended that genetic stability analysis should be performed for long-term maintenance of such micropropagated plants.


Introduction
Micropropagation is a technique widely used for the commercialization of many woody plant species. Producing a whole plant using an explant is termed micropropagation. Varieties of plants will produce different responses under different growth conditions. In vitro regeneration of plants involves the use of various plant growth regulators (PGRs) [1][2][3] and growth conditions such as the number of subcultures [4,5] and the light intensities [6], to achieve optimum growing conditions for plants [7,8].
Micropropagation in plants solely depends on the phenomenon of totipotency among plants. The application of micropropagation includes growing any plant species on a large scale commercially, clonal propagation of endangered species of plants, somaclonal variations, producing transgenic plant cell lines and sterile hybrids with enhanced properties, and acquiring essential secondary metabolites.
The micropropagation system has a huge advantage in the commercial market of plant tissue culture, owing to its excellent multiplication techniques and viability. Using highly dividing meristematic tissues of the apical buds, leaves, or roots are used as explants to achieve plant regeneration through various techniques such as organogenesis or somatic embryogenesis [9][10][11].
The technique of regeneration of plants generally includes steps like acquiring the explant from a suitable mother plant, using PGRs in the culture media, and culturing in vitro plants on a suitable media [12]. Many factors affect the in vitro growing plant cultures. Using different PGRs is a major contributing factor to the change in plant genetics. The growth conditions such as the use of different light spectra or even the number of times the culture is subculture also affect the plant genetics [13,14]. Somaclonal variations in plants can also occur due to the difference in the in vitro growth conditions such as different light spectra and the photoperiod. Hence, evaluation of the genetic stability of the in vitro regenerated plants is a very crucial step that helps in concluding a successful micropropagation technique. The regeneration capabilities of the totipotent plants from a single cell into a whole new plant may introduce genetic changes once the tissues are dissected and obligated to a pathway of cell division using specific growth regulators [15,16].
The alterations caused on the plants due to the somaclonal variations can be in the form of change in plant morphology, variation in chromosome number, expression of genes, protein profile, and DNA sequences. Overall the assessment of the genetic stability among the regenerated plantlets is based on various analyses such as biochemical, cytological, and molecular analyses [17,18]. The studies on morphological and genetic variations in 40-five different varieties of figs (Ficus carica L.) were reported by Oukabli et al. [19]. They have concluded that regional constraint has an impact on the clonal variations among the different cultivars based on the environmental factors of the particular regions. Reports have also proven that overall genetic stabilities were maintained in the regenerated plants such as wasabi bananas Henckelia incana [7], Rauwolfia tetraphylla [8], Musa acuminata [20], Gladiolus cultivars [21], and Melonia cultivars [22].
Recently, polymerase chain reaction (PCR) based techniques have been known to be efficient for genetic studies among plants. PCR based single primer amplification reaction (SPAR) techniques using microsatellites, simple sequence repeats (SSR), inter simple sequence repeats (ISSR), random amplified polymorphic DNA (RAPD), and directed amplification of minisatellite DNA (DAMD) primers to analyse and detect the genetic stability among the in vitro generated plants are the most common practices in plant tissue culture experiments. Techniques which utilise single primers to amplify the DNA in PCR reactions are generally applicable universally [8,14,20,23]. The use of ISSR and DAMD markers has proven to be unbiased and cost-effective as compared with the RAPD markers [21,[24][25][26]. Consequently, the present study uses ISSR and DAMD markers to evaluate the genetic stability of the regenerated multiple shoots, mother plant and ex vitro acclimatised plants of Ficus carica var. Black Jack.
Prior to the current study, a full-strength woody plant medium (WPM) [27] supplemented with 20 μM 6-Benzylaminopurine (BAP) was used to acquire multiple shoots on the axenic apical buds of Ficus carica var. Black Jack. The apical buds were grown under five different light treatments namely, normal fluorescent white light (60 μmol m −2 s −1 ), and four different LED spectra, white (400-700 nm), blue (440 nm), red (660 nm) and blue + red (440 nm + 660 nm). The multiple shoots were rooted using WPM supplemented with 20 μM BAP and 8 μM indole-3-acetic acid (IAA). The rooted plantlets were used for ex vitro acclimatization using Biochar soil. The current study was intended to assess the genetic stability of the regenerated plantlets compared with the mother plant and ex vitro acclimatized plants of Ficus carica var. Black Jack using ISSR and DAMD markers.

DNA extraction procedure
Genomic DNA isolation was performed using the 'Genomic DNA Purification Kit' (Promega Wizard® Genomic DNA Purification Kit, Madison, WI 53711-5399 USA). DNA was extracted from the apical buds of the samples. The quality of DNA was checked by electrophoresis on 1.5% agarose gel. The concentration (ng/μL) and purity (A 260 /A 280 ) of the DNA extract was quantified using nanodrop spectrophotometer (ASP-2680, ACTGene Inc., New Jersey, USA).

Amplification of the genomic DNA using ISSR and DAMD markers
Genetic stability among the mother plant and regenerated plants (in vitro plants under normal incandescent light and six different LED spectra treatments) were analysed using ISSR and DAMD DNA markers. Chemicals and solutions used for the following study were purchased from the 1st Base Malaysia (exTEN 2X PCR Master Mix). The exTEN Master Mix contained 0.08 U/µl exTEN DNA Polymerase, 400 µM dNTP Mix, 3 mM MgCl 2 , reaction buffer, and a PCR enhancer.

PCR amplification using ISSR-DNA method
The molecular reagents in the PCR tubes consists of 12.5 µL of exTEN 2X PCR Master Mix, 2.5 µL of 1 µM Pimer, 3 µL Genomic DNA Template (< 250 ng), and 7 µL of Sterile de-ionised water. The total volume of the PCR tube is 25 µL. Table 1, shows the total ten ISSR primers [28] screened for the following study to assess the genetic stability of Ficus carica var. Black Jack. Overall, seven primers produced precise and reproducible bands. Thus, these seven primers were used in subsequent ISSR analysis. The ISSR primers were synthesised by 1 st Base Malaysia DNA. The PCR amplification was performed in the volume of 25 µL in 200 μL tube (Axygen Inc. California USA). The PCR profile was performed at an initial denaturation temperature of 95 °C for 4 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 65 °C (depending on the melting Temperature [Tm-5 °C]) for 30 s, extension at 72 °C for 1 min and then a final extension at 72 °C for 10 min (Table 2).

PCR amplification using DAMD-DNA method
A total of ten DAMD primers [28] were screened for the following study to assess the genetic stability of Ficus carica var. Black Jack (Table 3). A total of, seven primers produced precise and reproducible bands. Thus, these primers were used in subsequent DAMD analysis. The DAMD primers were synthesised by 1st Base Malaysia DNA. The PCR amplification was performed in the volume of 25 µL in a 200μL Eppendorf tube (Axygen Inc. California USA), and the composition of molecular reagents. The PCR profile was

Agarose gel electrophoresis (AGE) analysis
PCR amplified products were separated using electrophoresis in 1.5% (w/v) agarose. Agarose (0.6 gm) was dissolved in 40 mL 1 × Tris-Borate-EDTA (TBE), melted in a microwave oven. Once it was a little warm, 2 μL red-safe stain (Intron Biotechnology™) was added. The gel was casted in a Mini Gel Caster (Bio-Rad Laboratories, Inc., USA) and was allowed to solidify at room temperature for 20 min before transferring to the Wide Mini Sub-Cell® GT agarose gel electrophoresis System (Bio-Rad Laboratories, Inc., USA) base filled with 1X TBE buffer. Subsequently, 3 µL of Thermo Scientific GeneRuler and 100 bp plus DNA Ladder ready to use (Thermo Scientific, EU, Lithuania) were loaded into the well to function as the molecular markers. Ready to use PCR amplified products were then loaded into the following wells (5 µL) (Fig. 1). The electrophoresis system was attached to the PowerPac™ Basic Power Supply (Bio-Rad Laboratories, Inc., USA). The samples were electrophoresed at 70 V for 60 min.

Determination of polymorphism analysis
Genetic similarity index for the samples of Ficus carica var. Black Jack was assessed. The visible bands were manually scored as 1 or 0 for the presence or absence of the band. Similarity index was calculated according to Nei and Li [29], and Harirah and Khalid [30]: Similarity index (Si) = 2Nxy (Nx + Ny)  3  3  3  3  3  3  3  3  3  0  1.0  200-600  UBC818  3  3  3  3  3  3  3  3  3   where Nxy = number of monomorphic bands between the control and treatment groups; Nx = total number of bands in the control group; Ny = total number of bands in the treatment group.
The polymorphism percentage among the samples was calculated using the formula,

ISSR-DNA analysis
For the ISSR analysis, seven out of the selected ten primers produced precise and reproducible band outcomes ( Fig. 2; Table 3). In general, all 7 ISSR primers resulted in 24 bands from each of the treatments the mother plant sample were produced from each primer ranged from 1 (N8) to 7 (UBC864) bands (Table 3). The genetic stability between the mother plant and the in vitro regenerated plants from seven different treatments was compared. Banding profiles obtained from the ISSR-DNA primers showed a similar banding pattern for all the treatments. Thus, the amplicons produced were monomorphic. Results indicate that there was 2.12% polymorphism and 97.87% monomorphism (Tables 3 and 4). All primers, except one (UBC840), produced an SI of 1.0. Based on Table 4, the SI value of approximately 0.95 was calculated among the 8 samples of Ficus carica var. Black Jack for primer UBC840. Thus based on the SI partial polymorphism can be conferred in primer UBC840.

DAMD-DNA analysis
A total of ten DAMD primers were used to analyse the genomic DNA acquired from the samples of Ficus carica var. Black Jack. Seven DAMD primers produced well-separated and reproducible band outcomes ( Fig. 3; Table 4). Overall, 23 monomorphic bands were produced from samples collected from each treatment. The amplified products generated band sizes from 150 to 1000 bp. Scorable bands were produced from each primer ranged from 1 (URP2F) to 7 (URP25F) bands (Table 4).
Comparing banding profiles generated using the DAMD-DNA primer it can be determined that genetic stability between the mother plant, ex vitro, and the in vitro regenerated plants from four different LED treatments is maintained. All the bands acquired using DAMD primers were monomorphic. Thus, these results indicate that there is   (Tables 3 and  4). Thus the plant genetic stability of Ficus carica var. Black Jack is maintained throughout the regeneration experiments as indicated by the SI value of 1.0 as indicated in Table 4.

Discussion
ISSR and DAMD markers have been used for the present study to determine the genetic stability among the mother plant and all the in vitro plants grown under different light treatments.
Genetic stability or diversity among different plant species is studied using different molecular markers such as RAPD, ISSR, DAMD, SSR, and minisatellite DNA. Assessment of efficiency for such markers was performed using 25 varieties of Musa acuminata (Banana). Comparative analysis for testing the polymorphism among the banana varieties using RAPD, ISSR, and DAMD showed that ISSR markers were highly efficient in producing polymorphic bands showing 90.06% of polymorphism. It was concluded that ISSR and DAMD markers span only selected repeats on the DNA, which allows these markers to competently detect differences in the amplicons [20]. Genetic stability among Prickly pear cultivars (Opuntia ficus-indica L.) was also studied using RAPD markers. Out of 87 amplicons, only 82 bands were monomorphic produced, and overall 91% of genetic similarity among the plantlets [23].
Commercial application of micropropagation on woody plant species can be considered to be successful only when genetic stability is maintained among the plants. Many factors can contribute to the molecular level alterations among the tissue culture plants. The use of PGRs, culture conditions such as light and humidity can initiate genetic instability among the in vitro plants. Morphological, cytological, and biochemical changes occurring in the plants can be an indicator of genetic alterations. Thus the molecular assessment of the tissue cultured plants during their juvenile in field stages is crucial. The study of somaclonal variation also allows for the improvement in micropropagation techniques intending to produce genetically identical plants [18].
Morphogenetic processes among plants such as stem and root elongation, leaf expansion, biochemical pathways, and metabolisms are greatly affected by the availability of quantity and quality of light. Morphological effects on the in vitro growing plants can occur based on the availability of the photosynthetically active radiation (PAR) [32,33]. LED spectra are used for micropropagation of plants to enhance the cellular level properties of the plants. LED differs from the normal light in the sense that LEDs provide a directed flow of the required red and blue photons to the growing cultures. The application of red and blue LEDs has shown efficacy in the yields of many plants [34,35]. Changes in the photosynthetic parameters and yields of the plants can be attributed to the genetic level changes occurring among the plants [6]. Different LEDs were used for the study of growth and yield patterns in a wheat plant. Genes regulating the biosynthetic pathway, for the production of phenylpropanoids in wheat plants growing under white LED were identified using PCR technique. Short sequence primers and qRT-PCR were used to analyse the gene expression in the in vitro plants. It was reported that TaPAL3, TaPAL4, and TaDFR are potential genes that upregulate the production of phenylpropanoids [36]. As a result of somaclonal variations, the DNA undergoes alterations. Polymorphism is a term used to describe such changes in DNA sequences. A study of date palm genetic diversity using ISSR and DAMD markers found 85.45% polymorphism. Chrysanthemum hybrids, such as Lady Salmon and Lady Vitroflora, and melon species were also studied for polymorphism [22,37,38].
Monomorphism and polymorphism in the DNA fragments can be detected using the concept of similarity index. It uses a mathematical equation to find the relationship between the DNA fragments acquired from PCR and separated using agarose gel electrophoresis. Analysis of polymorphism observed in a single primer was performed using a correlation table. This table relates the similarity index of each individual from the selected population to each other and calculates the similarity index quotient [31]. Molecular analysis was performed on 194 varieties of figs (Ficus carica L.) to detect genetic diversity among different germplasm accessions. Genetic diversity among four types of figs such as Common, san Pedro, Smyrna, and Caprifigs was studied using microsatellite markers. Sixteen chosen microsatellite markers showed substantial polymorphism. The heterozygous alleles on different loci indicated exhibited genetic diversity among the cultivars [39].
Genetic similarity analysis performed using the range of bands acquired from the PCR analysis for both ISSR and DAMD primers shows that all plants are genetically similar to each other. This indicates that throughout the experiment, there is no occurrence of somaclonal variations. However, only primer UBC 840 produced a single polymorphic band with a similarity index of 0.95. Table 5 shows a correlation and analyses the genetic similarity coefficient for all the treatments. According to the analysis the similarity coefficient should be more than 0 and less than 1. In case of the primer UBC 840 the value of the similarity coefficient generated is 0.95, which indicates that all the treatments are genetically similar. The result is due to the presence of an extra polymorphic band in the samples. Such a kind of polymorphism is still considered negligible owing to the possibility of error dynamics in the PCR process.
For in vitro generated Opuntia ficus-indica L. cultivars, genetic stability assessment using RADP markers presented with a low percentage of polymorphism (2.79%) among the in vitro cultures of over 5 years. Thus it was reported that genotypes showing the similarity index percentage of more than 90% are genetically close to each other [23]. For the different treatments of Ficus carica var. Black Jack, polymorphism percentage obtained at 2.12% using ISSR marker. Therefore, a higher percentage of monomorphic bands (97.87%) proves genetic stability among the plants. Thus the plant genetic stability of Ficus carica var. Black Jack is maintained throughout the regeneration experiments.

Conclusions
Assessment of genetic stability among the regenerated plantlets and ex vitro plants of Ficus carica var. Black Jack was successfully performed using ISSR and DAMD molecular markers. Banding profile obtained using DAMD markers showed 100% monomorphism. The banding profile obtained from ISSR markers showed 97.87% of monomorphism with 2.13% of polymorphism. The average genetic similarity index was 0.94 among all the treatments of Ficus carica var. Black Jack. Thus the in vitro and ex vitro plants of Ficus carica var. Black Jack was found to be genetically stable.