In vitro propagation from nodal segments of Lippia origanoides (chemotype A)

: This research described an efficient micropropagation protocol for Lippia origanoides (Verbenaceae). Sterile seeds were used to obtain germinated seedlings in Murashige and Skoog medium (MS) supplemented with sucrose and agar. The nodal segments obtained from seedlings were grown on MS medium supplemented with different concentrations of gibberellic acid (GA), benzylaminopurine (BAP) and 1-naphthalenacetic acid (NAA) with BAP. The callus induction, shoots length, shoots number and root length, were analyzed. The treatments showed high percentage of callus formation at 0.5 to 1.5 mg L -1 of BAP alone or in combination with NAA (0.1 mg L -1 ). The highest value of shoot number per nodal segments was obtained at 1.5 mg L -1 of BAP (4.3 ± 0.8). The obtained plantlets were better rooted in vitro in the absence of plant growth regulators (PGRs) and they showed acclimatization rate of 90%. We reported a protocol for in vitro propagation and acclimatization of L. origanoides for A chemotypes from Colombia.

Moreno et al. STASHENKO et al. 2013), a. A natural compound with pharmacological properties, including ischemic stroke, neurodegenerative diseases, and atherosclerosis (LAN et al. 2017). It is present only in the extract of Lippia origanoides chemotype A, which is exclusive of Colombia (STASHENKO et al. 2013). In addition, chemotype A also has the highest profitability in the extraction process of its products (ARIAS et al. 2020).
Attempts to propagate L. origanoides by conventional methods as "sticks and cuttings" and seeds have shown a lower rooting capacity and a delayed development (HERRERA-MORENO et al. 2013;SILVA et al. 2015). Therefore, it is essential to use alternative techniques to produce large amounts of biomass in a short time that allows supplying the industry demand. An option is plant propagation by tissue culture, a useful tool to optimize plant production of superior qualities, free from microorganisms and with high multiplication rates (LONE et al. 2020). However, a major limitation in large scale application of this technology is high mortality experienced by micropropagated plants during or following laboratory to land transfer (CHANDRA et al. 2010).
Although, the in vitro micropropagation technique has been efficient and described for some species of the genus Lippia (CASTELLANOS-HERNÁNDEZ et al., 2013;GUPTA et al. 2001;JOSÉ et al. 2019;JULIANI et al. 1999;PEIXOTO et al. 2006;RESENDE et al. 2015), currently, there is only a single report on the effects of different PGRs (benzylaminopurine, indole acetic acid and kinetin) on in vitro development of L. origanoides (CASTILHO et al. 2019). It is important to point out that this approach does not describe a useful methodology for ex vitro acclimatization. Considering that plant's response to in vitro culture varies between samples collected from different geographic locations (HAQUE & GOSH 2018;MANVI & PARASHARAMI 2019), this work established a protocol for in vitro propagation and ex vitro acclimatization of L. origanoides for A chemotype exclusive from Colombia.
The inflorescences of L. origanoides plants (A chemotype), were collected from experimental gardens at "Centro Nacional de Investigaciones para la Agro industrialización de Especies Vegetales Aromáticas Medicinales Tropicales (CENIVAM)", located at Universidad Industrial de Santander, (Bucaramanga, Colombia). These inflorescences were dried in the oven for 24 h at 34 °C until their use.
For seed germination, we used the MS medium (Murashige and Skoog, 1962) prepared at ½ strength and supplemented with 1.5% w/v sucrose and 0.7 % w/v agar (Caisson) at pH 5.8, as it was indicated by CASTELLANOS-HERNÁNDEZ et al., (2013). The seeds were immersed in 70% ethanol (1 min), followed by immersion in 4% sodium hypochlorite (12 min) and then were washed three times with sterile and distilled water. The seeds were germinated in 60 × 70 mm flask containing 20 mL of MS medium. The containers were maintained at 25 ± 2 °C with a 16 h photoperiod, provided by white and cold fluorescent tubes with an intensity of 50 μmol m -1 s -1 (Sylvania Toledo Glass Tube 9w TB G13). For determination of germination and contamination rates, five replications of 10 seeds were used and germination was scored by visual observation of radicle emergence posterior to 20 days. After six weeks, the nodal segments of the seedlings (with average length of 4.1 cm) were used as a source of explants for the multiplication stage.
The culture media (Table 1) was the MS medium with 3% w/v sucrose supplemented with NAA at 0.1 mg L -1 and different concentrations of GA and BAP, as follows: GA (0.5, 1, 1.5 mg L -1 ), BAP (0.5, 1, 1.5 mg L -1 ). Nodal segments (approximately 1 cm) were obtained from 6 weeks old in vitro seedlings and were inoculated into different media. Each treatment was composed by one explant per flask with four replicates and three repetitions. After the sixth week, we estimated the callus percentage, the number of shoots, and the shoot's length and roots per explants.
Plantlets with well-developed roots (root length greater than 3 cm) were removed from the culture medium and washed with distilled and sterilized water to remove the adhering medium. They were transplanted in sealable plastic bags with different substrates such as river sand, black peat and vermicompost, and all were previously sterilized ( Figure 1). Fifteen plantlets were transplanted for each substrate. The bags were transferred to a greenhouse and were opened gradually until ten days and were irrigated with tap water every 3 days. After 6 weeks, plantlets were transferred to field conditions. All experiments were performed under a completely randomized design. The collected data were normalized (Log 10 ), and a factor variance analysis was performed at a significance level of P < 0.05, followed by a Tukey multiple range test. The RStudio 1.0.143 program for Windows was used.
In vitro germination of L. origanoides seeds collected from the field was performed to obtain axenic seedlings as sources of explants for plant propagation. After ten days the average germination rate of seedlings explants was 60%. Contamination was observed in less than 20% of seeds. Similar results were obtained with L. graveolens (CASTELLANOS-HERNÁNDEZ et al., 2013) since we used the same medium.
Nodal explants showed a high percentage of callus formation in treatments with BAP alone or in combination with NAA (Table 1)    Ciência Rural, v.52, n.7, 2022. Moreno et al. Culture of nodal explants on media supplemented with 1.5 mg L -1 of BAP resulted in significant differences in the number of shoots compared to control medium (Table 1). The highest values for shoot number per explants was obtained in the medium supplemented with BAP despite the fact that they did not have significant differences with other treatments. The addition of 0.1 mg L −1 NAA in combination with BAP showed no statistically significant difference from treatments with BAP alone in terms of number of shoots ( Figure 2; Table 1).
The beneficial effects of cytokinin during in vitro plant propagation, as report here, are in agreement with the results obtained in other species of Lippia, such as L. junneliana (JULIANI et al. 1999 (PEIXOTO et al. 2006), but in this case, the combination with NAA results in the highest multiple shoot response.
Nevertheless, in vitro propagation of L. origanoides from Brazil, the best result showed shoot regeneration was with a medium free of PGRs (CASTILHO et al. 2019), demonstrating that plant's response to in vitro culture varies between different geographic locations, regardless of being the same species.
Healthy elongated shoots were obtained on MS medium with 0.5 and 1.5 mg L -1 of GA and medium free of PGRs (4.7 ± 0.7). The effect of GA has not been reported in other Lippia species. This result is consistent with similar findings in L. dulcis (URREA et al. 2010) andL. junelliana (JULIANI et al. 1999), where the mean shoot length was higher in a medium free of PGRs. However, it is the opposite of CASTILHO et al.( 2019) reported since the medium without PGRs did not affect the shoot length. Once more, indicating the need to establish propagation protocols for plants of the same species with differences in their localities.
Conversely using 1 mg L -1 of BAP or BAP in combination with NAA showed a lower capacity of the development of the length of the shoot. In contrast, the use of NAA and BAP + NAA in in vitro cultures explants from L. filifolia (PEIXOTO et al. 2006) and L. rotundifolia (RESENDE et al. 2015); respectively, improved shoot length. Since the incorporation of BAP into the culture medium, it is used primarily to induce shoot proliferation (BHOJWANI et al. 2013); therefore, it is expected that it will not have a positive effect on stem elongation by itself. The highest value for shoot rooting was observed at 0.5 mg L -1 of GA (8.6 ± 3.1) and control treatment (6.5 ± 4.6). Increasing the GA concentration reduced the length of roots. Although, nodal explants have a positive response on MS medium with 0.5 mg L -1 of GA, our results suggested that it is unnecessary PGRs to obtain elongated and rooting shoots of L. origanoides since there was not significant differences with control. This was also reported for L. dulcis (URREA et al. 2010), where MS medium was the best treatment.
After hardening in sand and peat substrates for four weeks, regenerated L. origanoides plantlet survival was over 90% but only 60 % in vermicompost substrate (Figure 1). Despite the high mortality often observed upon transfer plants to ex vitro conditions (CHANDRA et al. 2010), plants obtained in vitro from L. origanoides do not present this difficulty, since it was only necessary to gradually decrease the humidity to get healthy plants. All the plants survival the transfer to the field.
The method described in the present study provided an efficient reproducible protocol for in vitro micropropagation of L. origanoides (A). This technique is an alternative to traditional propagation methods for this promising aromatic species. The application of the protocol will facilitate research into the any major volatile component that proved to have industrial value and it could stimulate their development on a commercial scale.

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL
The projects were also approved by the Ethical Committee (Record N o . 22) from UIS.