ACTA ZOOLOGICA BULGARICA Research Article Acta zool. bulg., Suppl. 11, 2018: 155-162 Ex situ conservation using medium-term cultures in Moehringia jankae Griseb. ex Janka (Caryophyllales: Caryophyllaceae) and genetic stability assessment using ISSR Irina M. Holobiuc1, Rodica D. Catanã1, Carmen R. Maximilian1, Victoria Cristea2 & Monica E. Mitoi1 1 Institute of Biology, Romanian Academy, Spl. Independentei St. No 296, Bucharest, Romania; iriholo@yahoo.com, monica. carasan@ibiol.ro; carmen.voichita@ibiol.ro, catanarodica@yahoo.com 2 Univ. Babeș Bolyai, Botanical Garden Al. Borza, Cluj-Napoca, Republicii St. No.42, Romania; crisvica@yahoo.co.uk Abstract: Moehringia jankae Griseb. ex Janka is a Balkan endemic plant which is spread in Romania and Bulgaria. Besides in situ conservation, a complementary ex situ conservation approach involves the elaboration of short and medium-term preservation protocols. For this purpose, several experimental variants consisted in the nutrients reduction or the supplementation of culture media with different chemical compounds as clormerquat, flurprymidol, abscisic acid and mannitol at different concentrations were tested and their efficiency for medium-term cultures establishment was evaluated. The results concerning growth reduction, regeneration and survival rate showed that this taxon can be efficiently medium-term preserved using 32 and 48 µM flurprymidol, mannitol at 0.16 M and clormerquat at 2.5 mM, this approach being a reliable and cheap method for ex situ preservation. From minimal cultures induced using factors with positive effect, after seven months well developed plants were obtained and evaluated concerning their genetic stability, using ISSR markers. Cultures established and maintained in the presence of abscisic acid, flurprymidol, mannitol or clormerquat did not affect significantly the stability of plants regenerated after seven months. Key words: medium-term, conservation, flurprymidol, clormerquat. Introduction Moehringia jankae Griseb. ex Janka (Syn. Arenaria jankae (Griseb. ex Janka) Fernald) is a Balkan endemic plant which grows on rocky, dry places in SE of Romania (in Dobroudja-Tulcea and Constanta county) and in NE of Bulgaria (in Shumen district) with relatively limited and diffused populations. This taxon is considered vulnerable in the Red Book of Vascular Plants from Romania (Dihoru & Negrean 2009) and endangered according to the Red Data Book of Republic of Bulgaria (Peev 2015). Biotechnological methods can help in situ measures providing an alternative for ex situ plant conservation (Engelmann 2011, Reed et al. 2011, Cruz et al. 2013, Pathak & Abido 2014; Rajasekharan & Sahiran 2015). Minimal cultures involving different methods of growth retardation has been widely used in the case of cultivated plants available for international exchanges (Cha-um & Kirdmanee 2007). To establish medium-term cultures, besides physical methods (low temperature storage, reduced illumination, small culture vessels and short photoperiod), different chemical methods were also applied alone or in combination. For inducing minimal in vitro cultures, different class of compounds were restricted or supplemented in the culture media as: the reduction of salts in the minimal media (Malik et al. 2005; Renau-Morata et 155 Holobiuc I. M., R. D. Catanã, C. R. Maximilian, V. Cristea & M. E. Mitoi al. 2006; Garcia-Jimenez et al. 2006); the addition of ethylene inhibitors or gibberellic acid inhibitors as flurprymidol, paclobutrazol or ancymidol (Sharkar et al. 2001, Madubanya et al. 2006) or abscisic acid (Jarret & Gawell 1991; Malik et al. 2005; Renau-Morata et al. 2006). Also, were used osmotic agents as: polyethylene glycol (Holobiuc et al. 2014); sugar and sugar alcohols (Hao & Deng 2003; Charoensub et al. 2004; Divakaran et al. 2006) In M. jankae, owing the positive in vitro response and the fast growth rate in the short-term culture, the elaboration of a protocol for growth reduction was necessary, this approach being a viable and cheaper alternative to ex situ preserve during prolonged time without important additional costs which can be applied to other related species. This study had the aim to elaborate an efficient medium-term conservation protocol and to evaluate the genetic stability of the regenerants of the vulnerable taxon Moehringia jankae using ISSR markers. Material and methods Several experimental variants were tested for medium-term cultures establishment. After seven months of preservation in medium term culture, regenerated plants were evaluated using ISSR markers concerning their genetic stability. Medium-term cultures induction: from primary cultures previously obtained consisted in welldeveloped plants, grown in large Phytatray vessels, double node explants (1 cm height) were excised and were cultured on 13 variants of solid MS basal medium (Murashige & Skoog 1962) gelified with 8 g/l (w/v) agar and supplemented with different chemical compounds to induce growth reduction. Five explants/culture vessel in five repetitions were cultured for each experimental variant. The cultures were made in glass jars of 55 mm diameter and 85 mm high, containing 30 ml of medium and grown in a cultivation chamber at 25 ±1°C with 16 hrs of cool and white fluorescent light at 60 μM m-2 s-1 per day. As growth limitation factors were used: abscisic acid, clormerquat, flurprymidol and mannitol. Variant V1, consisted in MS medium added with 20g/l sucrose, used as control. V2 contains MS macro salts reduced at ½, V3- MS + sucrose (su) reduced at 10 g/l. The other media variants contain 20 g/l sucrose. All media had pH adjusted at 5.8 before autoclaving under 118 kPa and 120°C for 20min. V4-V13 variants contain retardation factors at different levels as follows: V4- MS+ 18.9 µM abscisic acid (ABA), V5- MS + 37.8 µM ABA, V6-MS+ 156 1.26 mM clormerquat (Cycocel), V7- MS+ 2.5 mM clormerquat, V8-MS+16µM flurprymidol, V9-MS+ 32 µM flurprymidol, V10- MS+ 48 µM flurprymidol, V11- MS+ 64 µM flurprymidol, V12- MS+ 0.16 M mannitol, V13- MS+ 0.32 M mannitol. The in vitro response of M. jankae during medium-term culture was evaluated after 1, 2, 3 and 6 months using several parameters: the maximum length (cm) of the best developed shoot/initial explant, the mean number of shoots/initial explants, the rooting rate (%) registered after different interval of time, the survival rate (%) registered after 6 months. After seven months of cultures, shoots induced in the presence of different growth retardants belonging to different initial clones were cultured on MS medium to obtain well-developed plants. Statistical analysis: all data were analyzed using One-Way ANOVA test (at p ≤ 0.05). The percent values were converted into arcsine x, prior statistical analysis. The significance of differences between experimental variants was assessed using a post-hoc test Bonferroni-Holmes at 95% confidence interval (Daniel’s XL Toolbox version 6.60). The results were expressed as the mean ± standard deviation (SD). Molecular analysis using ISSR- markers Plants derived from two clones maintained in medium-term cultures in the presence of the best variants reduced -growth as 37.8 µM ABA (V5), 2.5 mM clormerquat (V7), 32 µM (V9) and 48 µM (V10) flurprymidol and 0.16 M mannitol (V12), were analyzed concerning their genetic stability. For each sample, total DNA was extracted from 100 mg of shoots. Two clones of M. jankae were analyzed: -clone 1 consisted in 30 samples: ten from control (1.1-1.10), ten derived from medium V12 (2.12.10) and ten derived from variant V5 (3.1-3.10). The clone 2 included 40 samples: ten from control (4.1-4.10), ten from variant V7 (5.1-5.10), ten derived from variant V9 (6.1-6.10) and ten derived from variant V10 (7.1-7.10). Genomic DNA was extracted using NucleoSpin Plant II (MacherereyNagel) Kit and DNA concentration was determined using BioDrop Duo. Seven ISSR primers like UBC (The University of British Columbia Biotechnology Laboratory, Canada) were tested, having between 16-18 bases provided by Dexter com, Romania (Table 1). The polymerase chain reaction (PCR) amplifications were performed in a 25 μl volume containing template DNA at concentration 50 ng/μl diluted with nuclease free water (Promega, USA), 2.5 μl of primer at concentration 10 μM and 12.5 μl Ex situ conservation using medium-term cultures in Moehringia jankae Griseb. ex Janka and genetic stability... Table 1. The primers tested and the results for ISSR amplifications. Range of No of scoNo of Total no. Polymorphic amplification rable bands polymorphic of ampliregenerats (pb) per primer bands fied bands Primer cod Secquence (5’-3’) Tm (°C) MC1 ACA CAC ACA CAC ACA CG 53 250-2000 6(clone1) - - - MC2 ATG ATG ATG ATG ATG ATG 43 350-2000 8 (clone1) 6 (clone2) 4 3 179 231 1.1, 1.2, 3.2 4.3, 4.9,4.10, 5.1,6.2,7.9 MC3 GAG AGA GAG AGAGAG AYG 49 250-1650 6 (clone1) 7 (clone2) 0 0 180 280 0 0 5 2 179 304 1.1,1.2,1.3, 1.7,1.8,2.6, 2.8 7.1-7.10 MC4 GAC AGA CAG ACA GAC A 47 250-1000 9 (clone1) 8 (clone2) MC5 GGA GAG GAG AGG AGA 48 250-2000 8 (clone1) 1 237 2.4, 2.10, 3.6 MC6 GAG AGA GAG AGA GAG AT 45 400-2000 6(clone1) - - - MC7 VHV GTG TGT GTG TGT GT 52 450-1000 7(clone1) Go taq Green Master Mix (Promega). The amplifications were made in an Eppendorf Mastercycler gradient (Germany). The PCR was optimized by modifying the annealing temperature to melting point (Tm) for individual primers. The cycling conditions were: initial denaturation step 2 min at 94°C, followed by 30 cycles of 30 s at 94°C (denaturation), 30 s at specific annealing temperature (Table 1), 40 s at 72°C (extension), and 1 cycle for 5 min final extension step at 72°C. Electrophoretic separation was performed with 7 μl of amplified products on 1,5% agarose gel. DNA amplification bands were stained with ethidium bromide at 0.02µg/ml gel concentration. The weight molecular marker (Mg) used was 1Kb Plus DNA Ladder (Invitrogen). The PCR amplification for each ISSR primer was made in two repetitions, only clear and completely reproducible bands were included in data evaluation. The bands were scored as presence (1) and absence (0) for each sample and were transformed into a binary character matrix. Genetic distances were determined using GenALex 6.5 software. Results In M. jankae, short-term culture was efficiently made through multiple axillary shooting (Holobiuc et al. 2017, in press), minimal culture or slowgrowth being the next level of ex situ preservation using in vitro methods. The behavior of the explants concerning the growth and regeneration depends on the factors modulated and the time interval recorded (Table 2). The use of minimal media with macro salts reduced at half (V2) or sucrose reduced at 10 g/l did not influence the growth or lateral shoots during two months. In the third month, only regeneration on V3 variant with reduced level of sucrose was lower. In the case of V1-V3 variants having similar growth rate to the control, the cultures could not be maintained more than 2-3 months. After two months of culture, the growth retardation effect was obtained applying different factors (Fig. 1 A-D). The use of ABA at the 37.8 µM level reduced the growth and lateral shooting was limited at 3-5 shoots/initial explant (Fig.1A). The application of clormerquat at both levels (1.26 and 2.5 mM) also determined a growth reduction, correlated with a good regeneration rate (Table 2, Fig. 1B), even after six months of culture (Fig. 1E). The origin of the new formed shoots are the lateral meristems of the double nodes stem explants. Testing of several concentrations of flurprymidol showed that 32 to 64 µM levels induced significant growth retardation- (Table 2), this behavior being maintained over six months with an acceptable viability rate of the cultures (Table 3). The level of 48 µM flurprymidol allowed a good response of regeneration even after six months, 18 shoots/initial explants being recorded (Fig. 1F). Mannitol at 0.16 M reduced the growth and also allowed a positive rate of regeneration (Fig.1D, 1G). Rooting process occurred easily on all media without retardation factors (V1-V3). In the case of growth-limitation factors, only 0.16 M mannitol sustained some roots formation beginning the first month, while at the 0.32 M level the rooting rate was recovered after three months (Table 3). Mannitol ensured rooting with acceptable rates (80%) just in the third month of preservation. ABA inhibited root 157 Holobiuc I. M., R. D. Catanã, C. R. Maximilian, V. Cristea & M. E. Mitoi Fig 1. Growth retardation induced with different factors after 2 months of culture compared to the control (A - ABA 37.8 µM, B- clormerquat 1.26 mM, C- flurprymidol 48 µM, D - mannitol 0.16 M.); E. Details of medium-term regenerative cultures maintained during six months in the presence of clormerquat 2.5 mM, flurprymidol 48 µM (F), mannitol 0.16 M (G) (bar-1cm). formation. In the presence of clormerquat, rooting was induced beginning the second month of culture. Flurprymidol application initial inhibited roots development. Rooting occurred with reduced rates only at 16 and 32 µM level after two months, roots developed slowly, but 64 µM inhibited rooting. To obtain well rooted plants, shoots were cultured further on medium without retardants. The assessment of the genetic stability of regenerants from medium-term cultures In vitro culture per se and the addition of different compounds can induce somaclonal variation 158 consisted in different type of DNA alterations (Karp 1994). For this reason, for conservative purpose it is important to evaluate the genetic stability/variability of preserved plant material. In our study, from seven tested ISSR primers, only four provided clear and scorable bands with satisfactory intensity for clone 1 and 3 primers for clone 2. The analysis of patterns amplification in clone 1 (Fig. 2 A-D) showed a total of 775 bands generated for 30 regenerants derived from medium-term cultures. The four tested primers generated 31 loci from which 10 polymorphic loci, the total percentage of polymorphism in this Ex situ conservation using medium-term cultures in Moehringia jankae Griseb. ex Janka and genetic stability... Discussion Fig. 2. ISSR amplification profiles of M. jankae regenerants belonging to clone 1 (1.1-1.10-control, 2.1-2.10- derived from variant 0.16 M mannitol, 3.1-3.10-derived from variant 37.8 µM ABA) using MC2 (A), MC3 (B), MC4 (C) and MC5 (D) primers and clone 2 (4.1-4.10- control, 5.1-5.10-derived from variant 2.5 mM clormerquat, 6.16.10- from 32µM flurprymidol, 7.1-7.10- from 48 µM flurprymidol) using the MC3 (E) and MC4 (F) primers. clone was approximately 14%, mostly due to the 3 individuals in the control group that showed polymorphism by amplification with 2 from 4 primers. However, the number of samples that showed polymorphism is small (Table 1), and the genetic diversity among regenerants in the same experimental variant as well as per total for clone 1 have minimal values. In case of the second clone (Fig. 2 E, F), the amplification profiles for all three primers that produced reproducible bands, MC2, MC3 and MC4 showed the presence of 21 loci from which only 5 were polymorphic. The percentage of loci per clone is approximately 13% comparable to clone 1. Genetic diversity is very low for regenerants obtained from 2.5 mM clormerquat-supplemented medium and 32 µM flurprimydol treated regenerants. For regenerants obtained from cultures on higher level of flurprymidol (48 µM), genetic diversity was slightly increased than control (Table 4). Despite of the advantages of long-term preservation, minimal-slow growth is also considered to be useful for the conservation of plant crop genetic resources (Sarkar et al. 2001) and also for wild threatened plants (Reed et al. 2011). Among the factors tested to establish medium-term cultures in M. jankae, mannitol, is a sugar alcohol which was successfully used in medium-term cultures alone or in combination with other factors, at concentrations between 15-90 g/l (Hao & Deng 2003; Holobiuc et al. 2010, 2014). Usually, in vitro sugars and sugar alcohols provide the carbon source, but higher levels induce osmotic stress and decrease leaf water potential and also determine the growth reduction. In our experiment, mannitol induced a growth retardation lower than flurprymidol, but more than clormerquat, this effect being detected after two months. The regeneration through axillary shooting occurred at good rate in the presence of 0.16 M mannitol, in M. jankae, mannitol being tolerated at lower level than in related species D. trifasciculatus Kit ssp. parviflorus (Holobiuc et al. 2014). For medium-term preservation, owing to the concern that osmotic compounds may affect the genetic stability through hypermethylation (Harding 1991) or because of abnormal physiological effects which can be inherited (Sarkar et al. 1999), growth retardants were also widely used (Sarkar et al. 2001). Abscisic acid (ABA) is a plant hormone (PGR) which besides its roles in leaves abscission, stress and plant pathogenesis, inhibits the cell division in the vascular cambium, stopping plant growth (Renau-Morata et al. 2006). The use of 37.8 µM ABA reduced the growth level, but few lateral shoots formed, without rooting. The survival rate was acceptable after seven months, while in Dianthus sp. ABA at similar level totally inhibited lateral meristems development from the explants (Holobiuc et al. 2010). Previously, ABA (0.1 mg/l) combined with ancymidol at 5 mg/l were also successfully used for minimal cultures induction in Glycyrrhiza glabra L. (Srivastava et al. 2013). Clormerquat usually is applied to promote flowering or inhibit ornamental plant elongation, being relatively safe and short-lasting (being metabolized). In our study, this compound had also a positive effect on growth limitation and promoted good regeneration rate through axillar shooting even after six months of maintenance. Flurprymidol reduces internode elongation through the inhibition of gibberellic acid (GA) biosynthesis. In M. jankae, flurprymidol at 35-48 µM levels proved to 159 Holobiuc I. M., R. D. Catanã, C. R. Maximilian, V. Cristea & M. E. Mitoi Variant Table 2 Parameters used to evaluate medium-term culture in M. jankae after different time intervals (values marked with the same letter are not significantly different at p≤0.05) Maximum growth of the shoots (cm) / initial explants Interval of culture (months) Number of shoots/ initial explant Interval of culture (months) 1 2 3 6 1 V1 6.02± 6.452a 9.82± 0.38 a 8.67± 0.54a - 2.76±1.50a V2 8.2± 6.56a 9.48± 0.25 a 10.2± 0.34a - 2.84±1.62a V3 5.32± 6.472a 7.084± 0.80 a 2 3 6 4.8±2.17a 4.8±1.11a - 5.16±3.06a 6.72±2.76a - 5± 0.76b - 2.68±0.94a 3.4±1.19a 4.36±2.76a - 2.07± 0.38cd 0.56± 0.18c 1.68± 0.47b 2±0.64b 1.96±1.62b 5.4±2.67a 1.76± 0.42 bd 1.90± 0.41cd V4 2.32 ± 0.452ab 2.016± 0.44b V5 0.58± 0.82b 0.60± 0.10c 1.64±0.48 b 1.92± 0.57b 2.16±0.68b 3±2.67c V6 0.86± 1.044b 2.28± 0.16b 2.88± 0.27c 3.16± 0.50a 2.04±0.61a 2.12±0.72b 4.1±2.04a 6.25±1.68a V7 1.62± 1.236b 2.37± 0.53b 2.33± 0.30c 2.06± 0.33b 1.72±0.97b 3.84±2.13a 5.68±2.42a 6.68±2.83a V8 1.98± 1.652b 4.57± 1.25c 5± 0.55b 4.71± 0.89a 1.36±0.63b 1.84±1.14b 2.56±1.32b 3.08±1.07c V9 0.56± 0.436b 0.75± 0.11d 0.98± 0.30d 0.55± 0.25c 1.36±0.48b 2.52±1.53a 4.24±2.35a 4.6±3.87b V10 0.36± 0.532b 0.65± 0.16d 0.81± 0.19d 0.32± 0.14c 1.24±0.43b 1.6±0.5b 2.4±1.11b 7.08±4.99a V11 0.42± 0.432b 0.58± 0.17d 0.66± 0.18d 0.52± 0.15c 1.4±0.57b 2.24±0.83b 2.56±1.11b 2.88±1.73c V12 0.42± 0.452b 1.06± 0.28bd 1.11± 0.24cd 1.17± 0.30bc 1.8±0.57b 3.88±1.83a 5.68±3.59a 5.64±3.60a V13 0.32± 0.348b 0.46± 0.11d 0.45± 0.15d 1.32±0.47b 2±0.64b 2.2±0.91b 3.96±3.52b 0.59± 0.13c Table 3. Rooting and survival rate registered after different time intervals. Rooting rate (%) interval of culture (months) Survival rate (%) after 6 months Variant Composition V1 MS 1 100 a 2 100a 3 100a 6 - V2 MS ½ 100a 100 a 100a - V3 MS+ 10 g 100 a 100a 100 a - V4 MS+ABA 18.9 µM 0 0 0 92±0.12a V5 MS+ABA 37.8 µM 0 0 0 88±0.12a V6 MS+C 1.26 mM 0 64±0.28b 92±0.12a 96±0.09a V7 MS+C 2.5 mM 0 28±2.47c 72±0.15b 96±0.09a V8 MS+F 16 µM 0 36±2.01c 56±2.21c 100 a V9 MS+F32 µM 0 36±2.02c 76±0.34b 88±0.12a V10 MS+F48 µM 0 0 0 88±0.22a V11 MS+F 64 µM 0 0 0 76±0.12ab V12 MS+Man 0.16 M 32±1.96b 69±1.26b 81±1.33b 88±0.12a V13 MS+Man 0.32 M 0 24±1.83c 80±0.18b 52±0.22b Table 4 Genetic distances and genetic identity between regenerants from each experimental variants with growth retardants and control (clone 1 - 37.8 µM ABA(V5), and 0.16 M mannitol (V12), respectively clone 2 - 2.5 mM clormerquat(V7), 32 µM (V9) and 48 µM (V10) flurprymidol). Experimental variants Clone 1 Clone 2 160 Genetic distance Genetic identity V12 0,033 0,967 V5 0,040 0,961 V7 0,015 0,985 V9 0,015 0,985 V10 0,087 0,917 Ex situ conservation using medium-term cultures in Moehringia jankae Griseb. ex Janka and genetic stability... be more efficient concerning growth limitation of shoots. The regeneration at 35-48 µM had a positive response concerning regeneration and survival even after six months of maintenance in restricted growth conditions. For the genetic evaluation of regenerants derived from medium-term cultures, we used ISSR markers which are considered relevant for their genetic stability/variability assessment. Somaclonal variation can be easily and rapid detected using ISSR primers consisted in microsatellites sequences being a dominant marker (Butiuc-Keul 2006) ensuring a rapid evaluation of repetitive sequences susceptible to mutations. In a related taxon of M. jankae, D. giganteus D’ Urv ssp. banaticus (Heuff.) Tutin, Jarda et al. (2014) performing the genetic analysis of donor plants and regenerants concluded that ISSR markers detected higher polymorphism than SSR ones In M. jankae, because in the case of the majority of the tested primers, polymorphic bands absented in the DNA pattern of regenerants compared to the control, a low genetic variability was detected. In other studies, based on similar analyses, the absence of genetic variations was also reported: in potato (Sarkar et al. 2001), in almond regenerants (Martin et al. 2004), in Swertia chirayita (Roxb. ex Fleming) H. Karst. (Joshi & Dhawan 2007). Soni & Kaur (2014) also recorded a very low level of polymorphism between in vitro regenerated clones in Viola pilosa Blume. The evaluation of medium-term cultures in M. jankae as alternative preservation strategy after 7 months of maintenance in the presence of different factors with growth limitation effect, showed that clormerquat, flurprymidol and mannitol are suitable for minimal cultures, being compatible with good plant regeneration and cultures survival. The results of genetic assessment of regenerated clones in M. jankae proved that the methods of growth retardation using factors as ABA, clormerquat, flurprymidol and mannitol did not affect the genetic stability of regenerated plants from medium term culture. Acknowledgements: This study was financial sustained by the project: “Innovative Biotechnologies for the preservation of endemic and/or endangered species of Caryophyllaceae from Romania for Natura 2000 Network consolidation “(TEHNAT) PN-II-PT-PCCA-2013-4-1764 Nr.71/2014, financed by ANCSI Romania. References Butiuc –Keul A. 2006. Markeri moleculari utilizaţi în genetica şi biotehnologia vegetală. Cluj-Napoca: Edit. Mega. 133 p. (In Romanian). Charoensub R. & Salak P. 2004. In vitro conservation of Rose colored leadwort: Effect of mannitol on growth of plantlets. Kasetsart Journal: Natural Science 38: 97-102. Cha-um S. & Kirdmanee C. 2007. Minimal growth in vitro culture for preservation of plant species. Fruit, Vegetable and Cereal Science and Biotechnology 1 (1): 13-25. Cruz-cruz C. A., González-Arna O. M. T. & Engelmann F. 2013. Biotechnology and conservation of plant biodiversity. Resources 2: 73- 95. Dihoru G. & Negrean G. 2009. Red book of vascular plants in Romania. Bucharest: Romanian Academy. 352 p. (in Romanian). Divakaran M., Babu K. N. & Peter K. V. 2006. Conservation of vanilla species in vitro. Scientia Horticulturae 110: 175-180. Engelmann F. 2011. Use of biotechnologies for the conservation of plant biodiversity. In vitro Cellular Developmental Biology Plant 47: 5-16. Hao Y. J. & Deng X. X. 2003. Genetically stable regeneration of apple plants from slow growth. Plant Cell, Tissue and Organ Culture 72: 253-260. Harding K. 1991. Molecular stability of the ribosomal RNA genes in Solanum tuberosum plants recovered from slow growth and cryopreservation. Euphytica 55: 141- 146. Holobiuc I., Mitoi M., Blîndu R. & Helepciuc F. 2010. The establishment of an in vitro gene bank in Dianthus spiculifolius Schur. and D. glacialis ssp. gelidus (Schott Nym. et Kotschy) Tutin: II. Medium-term cultures characterization in minimal growth conditions. Romanian Biotechnological Letter 15(2): 5111-5119. Holobiuc I., Voichiţă C., Catană R., Mitoi M. & Helepciuc F. 2014. Medium-term preservation of Dianthus trifasciculatus kit ssp. parviflorus through minimal cultures. Muzeul Olteniei Craiova. Oltenia. Studii şi comunicări. Ştiinţele Naturii 30 (1): 57-66. Holobiuc I., Catană R., Cogălniceanu G. & Cristea V. 2017. Biotechnological approach for ex situ conservation of the vulnerable species Moehringia jankae. Romanian Biotechnological Letters 22(3) DOI: 10.26327/RBL2017.40. Jarda L., Butiuc-Keul A., Höhn M., Pedryc A. & Cristea V. 2014. Ex situ conservation of Dianthus giganteus d’Urv. subsp. banaticus (Heuff.) Tutin by in vitro culture and assessment of somaclonal variability by molecular markers. Turkish Journal Biology 38: 21-30. Jarret R. L. & Gawel N. 1991. Abscisic acid-induced growth inhibition of sweet potato (Ipomoea batatas L.) in vitro. Plant Cell Tissue and Organ Culture 24: 13-18. Joshi P. & Dhawan V. 2007. Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biologia Plantarum 51: 22-26. Karp A. 1994. Origins, causes and the uses of variations in plant tissue culture. In Plant Cell and Tissue Culture. Basil K.I. and Thorpe T.A. (Eds). Dordrecht Springer Acad. Publisher, pp. 139- 151. Malik S. K., Chaudhury R. & Kalia R. K. 2005. Rapid in vitro multiplication and conservation of Garcinia indica: a tropical medicinal tree species, Scientia Horticulturae 106: 539-553. 161 Holobiuc I. M., R. D. Catanã, C. R. Maximilian, V. Cristea & M. E. Mitoi Martin M., Sarmento D. & Oliveira M. M. 2004. Genetic stability of micropropagated almond plantlets, assessed by RAPD and ISSR markers. Plant Cell Reports 23: 492-496. Murashige T. & Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15: 473- 497. Pathak M. R. & Abido M. S. 2014. The role of biotechnology in the conservation of biodiversity. Journal of Experimental Biology and Agricultural Sciences 2 (4). DOI: 10.18006. Rajasekharan P. E. & Sahijram L. 2015. In vitro conservation of plant germplasm. In: Bir Bahadur, Manchikatla Venkat Rajam, Leela Sahijram, K. V. Krishnamurthy (Ed.): Plant Biology and Biotechnology (Vol II: Plant Genomics and Biotechnology). New York: Springer Verlag, pp. 417-443. Peev D. (Ed.) 2015. Red Data Book of the Republic of Bulgaria. Vol. 1. Plants and Fungi. Sofia: BAS & MOEW. 881 p. Reed B. M., Sarasan V., Kane M., Bunn E. & Pence V. C. 2011. Biodiversity conservation and conservation biotechnology 162 tools. In Vitro Cellular & Developmental Biology - Plant 47 (1): 1-4. Renau-Morata B., Arrillaga I. & Segura J. 2006. In vitro storage of cedar shoot cultures under minimal growth conditions. Plant Cell Reports 25: 636-642. Sarkar D., Kaushik S. K. & Naik P. S. 1999. Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro. Plant Cell Reports 18: 897-903. Sarkar D., Swarup K. & Naik P. S. 2001. Slow-growth conservation of potato microplants: efficacy of ancymidol for long-term-storage in vitro. Euphytica 117: 133-142. Soni M. & Kaur R. 2014. Rapid in vitro propagation, conservation and analysis of genetic stability of Viola pilosa. Physiology and Molecular Biology of Plants 20 (1): 95-101. Srivastava M., Purshottam D. K., Srivastava A. & Misra P. 2013. In vitro conservation of Glycyrrhiza glabra by slow growth culture. International Journal of Plant Biotechnology & Research 3 (1): 49-58.