ASSESSMENT OF THE EFFECT OF PLANT GROWTH REGULATORS ON IN VITRO MICROPROPAGATION AND METABOLIC PROFILES OF MELISSA OFFICINALIS L. (LEMON BALM)

In this study the effectiveness of plant growth regulators on micropropagation, total phenol content and metabolic profiles of Melissa officinalis L., important medicinal herb was assessed. The stem segments derived from one-month old in vitro germinated seedlings were used for initial explants. Of the eight different nutrient media tested the most favorable for micropropagation were found to be MP2 (MS medium enriched with 1mg/L BAP and 0.1 mg/L IBA) and MP3 (MS supplemented with 1.5 mg/L BAP and 0.5 mg/L NAA), which yielded 3.25- 4.0 shoots per explant within 4 weeks of culture. In vitro rooting (100%) was achieved on half strength MS medium contained 2% sucrose. Rooted plantlets were adapted successfully (98%) in a mixture of soil, peat, perlite, and sand in ratio 2:1:1:1 (v/v/v/v). Twenty metabolites were identified by GC/MS analysis of shoots grown on the MS media including different types of growth regulators. Analysis revealed the presence of phenolic, organic and fatty acids, sterols, triterpenes, fatty alcohols, saccharides and polyols. The studied samples have the same metabolic profile with quantitative differences in the individual metabolites. The maximum content of total phenols (13.92 mg/g extract) was obtained in shoots grown on MP2 medium and the lowest was recorded in the microplants cultured on MP1 control medium (8.45 mg/g extract). The obtained results indicated that it is possible to achieve the accumulation of desired metabolites by selection of plant growth regulators added in nutrient media.

. The cultivation of lemon balm through conventional methods is not difficult, but the main problem is to obtain a homozygous population producing valuable biologically active substances with constant quality . Through in vitro techniques it is possible to achieve both stable quantity and increase of the content of desired metabolites in plant culture (García-Pérez et al., 2011). Micropropagation allows true-to-type multiplication of a selected genotype (Debergh and Read, 1991). The differentiated organ cultures like shoots and roots cultured in vitro can present a metabolite profile similar to that of initial plants ( Kolewe et al., 2008), maintaining the same genetic characteristics of the highest productive clones (Chaturvedi et al., 2007). The various parts of lemon balm plants (shoots tips, cotyeldonary nodes, hypocotyeldon axes, leaf, petiole, stem, axillary buds) derived from seedlings or field grown plants were used as explants to initiate in vitro cultures ( The objective of this work was to study the effect of different types of plant growth regulators on micropropagation, total phenol contents and metabolic profiles of in vitro propagated M. officinalis shoots.

Sterilization
The seeds were collected from еx situ collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences. Seeds were surface washed with tap water and a detergent for 20 min followed by soaking in 70% ethanol for 2 min. Then two sterilizing agents were tested: 1) treatment with 50% commercial bleach (ACE) containing 4.85% sodium hypochlorite with a drop of Tween 20 or 2) 0.1% HgCl2 (Merck, Germany) each applied for 10 min. The sterilized seeds were washed three times with sterile distilled water for 5, 10 and 15 min.
In this study the effectiveness of plant growth regulators on micropropagation, total phenol content and metabolic profiles of Melissa officinalis L., important medicinal herb was assessed. The stem segments derived from one-month old in vitro germinated seedlings were used for initial explants. Of the eight different nutrient media tested the most favorable for micropropagation were found to be MP2 (MS medium enriched with 1mg/L BAP and 0.1 mg/L IBA) and MP3 (MS supplemented with 1.5 mg/L BAP and 0.5 mg/L NAA), which yielded 3.25-4.0 shoots per explant within 4 weeks of culture. In vitro rooting (100%) was achieved on half strength MS medium contained 2% sucrose. Rooted plantlets were adapted successfully (98%) in a mixture of soil, peat, perlite, and sand in ratio 2:1:1:1 (v/v/v/v). Twenty metabolites were identified by GC/MS analysis of shoots grown on the MS media including different types of growth regulators. Analysis revealed the presence of phenolic, organic and fatty acids, sterols, triterpenes, fatty alcohols, saccharides and polyols. The studied samples have the same metabolic profile with quantitative differences in the individual metabolites. The maximum content of total phenols (13.92 mg/g extract) was obtained in shoots grown on MP2 medium and the lowest was recorded in the microplants cultured on MP1 control medium (8.45 mg/g extract). The obtained results indicated that it is possible to achieve the accumulation of desired metabolites by selection of plant growth regulators added in nutrient media.

Micropropagation
Stem segments (excised from one-month seedlings) were used as initial explants for multiplication. They were cultured on full-strength MS medium supplemented with one of the following cytokinins: 6-benzylaminopurine (BAP), kinetin (Kin), zeatin (Zea) or N 6 [2-isopentenyl]-adenine (2-iP), combined with the auxins: indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The media contained 3% sucrose and were solidified with 0.7% agar (w/v). The concentration and combinations of PGRs are shown in Table 2. Initiation of shoots and micropropagation experiments were carried out using glass tubes, 10 mL medium per tube. At the end of 4 weeks culture period, the frequency of shoot proliferation (evaluated as % of explants forming shoots), the multiplication rate (evaluated as mean number of shoots (>1 cm) per explant), and the mean shoot height were recorded. The hyperhydricity (%) and the rooting (%) observed in some variants were estimated too. Each treatment consisted of 20 explants distributed in 2 replications. Two subsequent sub-cultivations were done.

In vitro rooting and acclimatization of ex vitro obtained plants
For induction of in vitro rhizogenesis stem explants consisting of one node (1.5 -2 cm) were transferred to half strength MS medium containing 2 % sucrose. The in vitro obtained plants with well-developed roots were removed from culture vessels and washed free of agar. They were transferred to small pots containing soil, peat, perlite, and sand in ratio 2:1:1:1 (v/v/v/v). The pots were covered with clear plastic box to maintain high relative humidity and plants were watered daily. After two weeks the plastic boxes were removed. The survival percentage of plants was assessed after 4 weeks. Then the ex vitro adapted plantlets were transferred to the experimental field for further acclimation.

Culture conditions
All media were adjusted to pH 5.7 with 1N NaOH before autoclaving. The nutrient media were autoclaved at 120 ºC for 20 min at 1atm. The cultures were maintained in a growth chamber under a 16h day/8h dark photoperiod provided by cool-white fluorescent lamps, irradiance 40 μmol m -2 s -1 at temperature 21±2 °C.

Preparation of extracts and GC/MS analysis
Six samples were subjected to GC/MS analysis, each consisting of 100 mg dried in vitro shoots grown on MP1 control, MP2, MP4, MP5, MP7 and MP8 media. Samples were extracted with methanol by maceration for 24 hours at room temperature with added internal standard 3,4 dichloro-4-hydroxybenzoic acid (50 µg/mL) at the beginning of the extraction. The amounts of metabolites were estimated against this standard. For GC/MS analysis 300 µL of each extract was transferred to a vial and evaporated to dryness, then silylated with 50 µL of N, o-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) in 50 µL of pyridine for 2 h at 50 °C. The spectra were recorded on a Thermo Scientific Focus GC combined with a Termo Scientific DSQ mass detector as described previously (Nikolova et al.,  2019). The metabolites were identified as TMSi derivatives comparing their mass spectra and Kovats Indexes (RI) with those of an on-line available plant specific database. The measured mass spectra were deconvoluted by the Automated Mass Spectral Deconvolution and Identification System (AMDIS), before comparison with the databases. RI of the compounds was recorded with standard nhydrocarbon calibration mixture (C9-C36).

Total phenolic content
The total phenol content was determined spectrophotometrically using Folin-Ciocâlteu reagent and was expressed as mg per gram of the plant extract, in gallic acid equivalents (GAE) (Nikolova et al., 2013).

Statistical analysis
Data were subjected to one-way ANOVA analysis of variance for comparison of means, and significant differences were calculated according to Fisher's least significance difference (LSD) test at the 5% significance level using a statistical software package (Statgraphics Plus, version 5.1 for Windows). Data were presented as means ± standard deviation.

Seed germination and establishment of in vitro culture
The surface sterilization with bleach was successful in ensuring contamination free seeds. Application of the mercuric chloride also resulted in 100% sterilized seeds, but they subsequently did not germinate on any of the tested nutrient media, indicating that the exposure time of this sterilizing agent was exceeded. Ninety percent of seeds germinated on MSG1 control medium free of plant growth regulators (Table1). The seeds cultured on MSG2 and MSG3 showed lower percentage of germinated seeds, 40% and 20%, respectively, which suggested a surplus of the gibberellic acid used.  The data are presented as means of 20 shoots per medium variant ± standard deviation. Different letters indicate significant differences assessed by Fisher LSD test (5%) after performing one way ANOVA analysis.

Influence of plant growth regulators on micropropagation
The effectiveness of micropropagation depended on the nutrient media composition ( Table 2). The explants cultured on control medium (MP1 free of PGRs) showed high shoot elongation, but only 15% of them produced 1-2 shoots per explants. The use of nutrient media supplemented with BAP and auxin (MP2, MP3 and MP4) resulted in maximum proliferation frequency (80 and 90%).
Concerning the quantity of the obtained shoots, the combination of BAP (1.5 mg/L) and NAA (0.5 mg/L) was optimum for lemon balm micropropagation and an average of 4.0 new shoots per explant within 4 weeks were formed ( Figure 1a); however, both MP3 and MP2 were commensurable because of the hyperhydricity (15%) observed in MP3. Increased concentration of BAP up to 2 mg/L (MP4) led to increase of multiplication rate (4.30±1.34), but most of the plants (60%) suffered from hyperhydricity. The nutrient media containing kinetin (MP5 and MP6) were appropriate for obtaining of vigorous shoots and the mean numbers of shoots per explant were 2.60±1.14 and 2.80±1.36, respectively. Media supplemented with Zea or 2-iP expressed less shoot proliferation and lower multiplication rate, compared with those containing BAP or Kin ( Table 2). The shoots produced on BAP or Kin containing media were characterized by very short internodes, small leaves and small shoot height in comparison with those grown on the media fortified with 2-iP or Zea, which were big, with longer internodes, and large leaf area.
In second subculture the multiplication rate was maintained and even increased in the optimal nutrient medium MP3 (4.50±1.44) (Figure 1b). However in subsequent subcultures there were noticed decrease of multiplication rate and regenerative capacity of M. officinalis. The shoots grew in height (8-12 cm) in short period of time (4 weeks) on all tested media and were cut into 3-4 nodal segments and again sub-cultured on fresh media. Thus, a large number of new shoots was obtained.

Rooting and acclimatization of in vitro obtained plants
Roots appeared during the first 5-6 days of culture on half strength MS medium. All in vitro shoots (100%) developed roots with an average number of 5-7 roots per shoot (Figure 1c). The 2:1:1:1 ratio of soil, peat, perlite and sand was found to be optimum for the hardening of the plants (the survival rate of 98%). This soil mixture had a beneficial effect on plant growth and development (Figure 1d). No phenotypic variations in the ex vitro adapted plants were observed.

Influence of plant growth regulators on metabolite profiles and total phenolic content
The applied plant growth regulators in nutrient media influenced not only micropropagation but also the accumulation of metabolites in in vitro obtained shoots. The results of GC/MS analysis of the metabolic profiles of the shoots grown on media containing 0.1 mg/L IBA and 4 different cytokinins in concentration 1 mg/L, are presented in Table 3. Twenty metabolites were identified. The studied samples have the same metabolic profile with quantitative differences in the individual metabolites. The phenolic acidscaffeic and rosmarinic were found to be accumulated in the highest amount in shoots grown on MS media containing BAP (MP2 and MP4). The organic acids were established in the highest quantity in the shoots cultured on MP7 containing Zea. At the same cultural conditions increased accumulation of sterols, triterpenes and fatty acids, as well as glycerol was observed. Also the fatty acids were presented in high content in the shoots grown on MP8 supplemented with 2-iP. In these cultural conditions the myo-inositol was accumulated in the highest degree. The content of saccharides was found to increase in all tested nutrient media containing PGRs compared to the control medium. A reverse trend was noticed for the content of pyroglutamic acid, which was significantly higher in shoots from the control medium. The results of total phenolic determination are presented at Figure 2. It was found that shoots from variants with higher micropropagation efficiency such as BAP and Kin containing media, produced an increase amount of total phenols in comparison with control media MS free of PGRs. The maximum amount of total phenols (13.92 mg/g extract) was obtained in shoots grown on MP2 (MS medium enriched with 1 mg/L BAP and 0.1 mg/L IBA) and the lowest one was recorded in the microplants cultured on MS control medium (8.45 mg/g extract). In this study an optimized protocol for M. officinalis micropropagation as well as metabolitic profile and total phenol content of in vitro grown shoots were presented. Surface sterilization (100% efficiency) was attained by consecutive soaking the seeds in 70% ethanol for 2 min and in bleach solution for 10 min. The use of 2.5% sodium hypochlorite for 3 minutes was effective disinfectant for obtaining of lemon balm in vitro aseptic seeds (Kiani et al., 2017).
Other authors recommended applying of more toxic sterilizing agent HgCl2 (0.1%) for 15 minutes, but they did not mention the disinfection efficiency (Ülgen et al., 2017). In our study the sterilization procedure with 0.1% HgCl2 failed to induce germination of seeds on all nutrient media tested. The highest germination efficiency of seeds was achieved on MS nutrient medium free of PGRs (90%) and Studied samples lower was recorded on MS media supplemented with GA3. Although GA3 usually stimulated seed germination in some cases it is suppressed by applying inappropriate concentrations (Chetouani et al., 2017). In literature there are many trials to stimulate M. officinalis seed germination: using of magnetic field application (100 mT) for 1 hour resulted in 52% germinated seeds (Ülgen et al., 2017), pre-chilling treatment lead to 68% germination (Aghilian et al., 2014), incubation at 25 ºC temperature was optimum for 93% seeds germination (Vârban et al., 2010). Kiani et al. (2017) reported that the most appropriate medium for in vitro germination (81.33%) was half strength MS medium containing 1% sucrose; however in our study we achieved 90% germination on full strength MS medium with 2 % sucrose. The most favorable media among the tested eight variants were found to be those supplemented with 1.5 mg/L BAP and 0.5 mg/L NAA, or with 1 mg/L BAP and 0.1 mg/L IBA. Most authors recommended addition of BAP at concentration of 1 to 3 mg/L in combination with auxins NAA or IAA to nutrient medium MS for M. officinalis shoot multiplication using different type of initial explants (shoot tips, apical nodes, cotyledonary nodes) ( Tavares al., 2013). The nutrient media MS supplemented with BAP and IBA led to higher activity of antioxidant enzymes in micropropageted Hyssopus officinalis L. shoots in comparison with those cultured on nutrient media with TDZ or Zea (Zayova et al., 2018). It was found that plant growth regulators initiate or mediate many different physiological processes and interact with each other in various ways during these processes (Phillips and Garda, 2019). Future research should be done in order to clarify the role of specific growth regulators in the accumulation of corresponding metabolites.

CONCLUSION
The results obtained from this study pointed that the type and concentration of plant growth regulators affected not only micropropagation and growth of shoots in vitro, but also the quantity of total phenols and the individual metabolites of Melissa officinalis L. However, all samples showed the same metabolic profile assessed by GC/MS analysis. In addition an optimized protocol for micropropagation of M. officinalis was presented, allowing 98% ex vitro adapted plants. The results reveal that lemon balm in vitro plants may offer a promising source of desired substances of this important medicinal plant.