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RAPD ANALYSIS OF ASPLENIUM ADULTERINUM MILDE FROM IN VITRO CULTURE

. Asplenium adulterinum Milde represents one of the natural heritages which have to be protected due to their scientific importance. In the actual context of global warming with myriad effects on biodiversity, the ex-situ conservation of A. adulterinum should be a priority. The possibility to obtain genetically stable individuals through in vitro techniques is a requirement of ex situ conservation. The genetic similarity of A. adulterinum gametophytes maintained 8 years in vitro culture was assessed. Explants represented by gametophyte fragments were analyzed for genetic stability through RAPD. Close similarity (88.88%) was found between gametophytes maintained in the same conditions (medium, illumination, temperature, passages number).

So far, no studies concerning the genetic similarity of A. adulterinum regenerated through in vitro culture and maintained in medium-term conservation were identified. Thus, in the present study, the genetic similarity of A. adulterinum gametophytes maintained in vitro culture was rated with a view to certify that the medium-term conservation represents a proper conservation strategy for this species.
Asplenium adulterinum gametophytes were obtained through in vitro spore germination (Aldea et al. 2016). The spores were collected from one single plant. The medium variant was represented by the agarised MS (Murashige & Skoog 1962) added with 3% sucrose, IAA (1.8 mg/l), Kn (0.02 mg/l), and activated charcoal (1g/l), at 24 ± 2°C and 16 hrs light/8 hrs dark with the light intensity of 27μmol m -2 sec -1 . The in vitro cultures were maintained in the same conditions (medium variant, temperature, light) for 8 years with subculture passages at 3 months.
Eleven samples (10 -A. adulterinum and 1 -A. trichomanes) were used for total genomic DNA extraction to analyze the genetic stability. A. trichomanes was used as an outlier because the interest species is considered a natural hybrid between A. trichomanes and A. viride (Lovis 1955).
DNA was extracted from 0.5-1g/ sample using the DNA extraction method of Doyle and Doyle (1987). DNA concentration and quality were evaluated by NanoDrop 2000.
The amplification was realized with Kit A Roth RAPD primer. Amplification was done with an Eppendorf Mastercycler following the program: initial denaturation 95 o C 5 min, followed by 35 repeats: 95 o C 30s, 40 o C 30s, 72 o C 1 min, and final elongation at 72 o C for 5 min.
A total of 7 random primers were utilized for RAPD analysis, out of which 5 random decamer primers were selected based on the clarity of banding patterns. The primer sequences are presented in Table 1.
The PCR products were loaded on 1.5% agarose gel buffered with 1X TBE at a constant voltage of 4V/cm for 2 hrs. The size of the amplicons was determined by comparison with O'GeneRuler 1kb DNA Ladder (Thermo Scientific). PCR products were visualized under GENi Gel Documentation System from SynGene.
The presence/absence data matrix was used to generate a pairwise genetic distance matrix for binary data (using the GenAlEx 6.501 software) which was visualized using a cluster analysis (unweighted pair-group method with arithmetic averages, UPGMA) and illustrated in a dendrogram using MEGAX.
Despite its ecological role in biodiversity maintenance and the conservation interest underlined by its characteristics being a glacial relict (Holderegger 1994), a serpentine fern (Tutin et al. 1980), and the most threatened plant species in the Romanian flora (Bartók and Irimia 2015), few studies regarding A. adulterinum ex situ conservation were done. In this context, a study concerning the distribution, population rate, and conservation plant framework for this species was published by Zo£nierz et al. (2008), in Poland. Further studies highlight the usefulness of in vitro cultures method to create prothallia "gene bank" for serpentine fern species (Marszał-Jagacka and Kromer 2011, Aldea et al. 2016. After any type of ex situ conservation protocol is mandatory to verify the genetic stability of in vitro conserved plant germplasm. The samples used were maintained on the MS added with plant growth factors for 8 years. In the present investigation, the amplification profiles of 11 samples from medium-term culture were from 150 to 980 bp that could be scored. Of these bands, 16 were specific to the species A. adulterinum compared with A. trichomanes. For 10 samples of A. adulterinum induced through spores' germination of the same individual, a total of 27 bands were scored (Table 1). Cluster analysis was done based on the genetic distance matrix including the outlier (Fig. 1).  The similarity indices ranged from 59. 25 to 100% among gametophytes maintained for 8 years in the same in vitro conditions. All the samples (1-8) display a minimum 88.88% similarity (have 24 out of 27 matching loci). The 3 subsets of samples display total identity (1, 2, 7, 8), (3, 4), and (5, 6). However, two of the samples (9 and 10) display significant differences compared to the rest of the samples (up to 11 out of 27 mismatching loci) and amongst each other (5 mismatching loci).
The RAPD technique, being a random method is prone to errors. In the present study the 3 different loci could be authentic or an artifact due to different DNA concentrations in the samples or the randomized annealing of the primers. Recent genetic studies showed that in vitro culture induced different mutation types in all species studied (Chandra and Thoyajaksha 2018). Variations may be attributed to in vitro stress induced by biochemicals (Devarumath et al. 2002) or subcultures number. In the case of long-term micropropagated Olea europaea shoots, different level of genetic variability was identified through RAPD after the seventh subculture on the same medium variant (Peyvandi et al. 2009). Taking into account that the cause of genetic variability cannot be accurately identified, more analyses or elimination of individuals with variability from in vitro culture are required. There is no technique that can completely guarantee the genetic fidelity of the micropropagated plants (Mallón et al. 2010). Due to its attributes, the RAPD method is often used to verify genetic stability in vitro cultures.
Using the RAPD method an 88.88% similarity among the A. adulterinum gametophytes maintained in the same physical and chemical conditions for 8 years was observed. For a better analysis, the further molecular analysis should be performed.