In vitro propagation, lactucin quantification, and antibacterial activity of Indian lettuce (Lactuca indica L.)

Lactuca indica L. (Asteraceae), a wild lettuce, is used as a vegetable and in traditional medicine. This study aims to establish in vitro propagation protocol and evaluate lactucin and antibacterial property from in vitro and natural plant tissues. Leaf blades and petioles were cultured in vitro on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP) and 1.2 mg L–1 indoleacetic acid (IAA). Leaf petioles and a lower BAP concentration (0.5 mg L–1) were optimal for direct shoot induction, while the leaf blade and a higher BAP (4 mg L–1) concentration performed best for callus induction. When the callus was subcultured, 98.7% of samples regenerated plants on MS medium supplemented with 1.5 mg L–1 BAP and 0.5 mg L–1 IAA. MS medium containing 1 mg L–1 IAA was best for in vitro rooting. A high-performance liquid chromatography analysis of the in vitro samples revealed a higher amount of lactucin (sesquiterpene lactones) in the root than the callus and the leaf, whereas in naturally grown plants, higher lactucin amounts were obtained from the juvenile root followed by the root of the flowering plant and juvenile leaf as the lowest concentration. All tissue extracts showed antibacterial activity against Pseudomonas fuscovaginae (a rice pathogen) and Escherichia coli, which was directly proportional to amount of lactucin produced. This in vitro regeneration and phytochemical investigation will facilitate the further exploitation of this useful wild plant.


Introduction
Lactuca indica L., popularly known as Indian lettuce, is a lactiferous medicinal herb in the Asteraceae. It is widely distributed across Asian countries, including the Republic of Korea, Indonesia, China, Japan, India, and Malaysia (Jeffrey 1966;Oliya et al. 2018). The leaves of this plant have long been used in salads, soups, and fermented foods (called "Kimchi" in Korea), as well as to wrap rice or meat. Traditionally, it is used in a tonic to heal fever, coughs, diabetes, and stomach disorders. The plant has antiinflammatory, antidiarrhea, cytotoxicity, antibacterial, antidiabetic, antioxidant, α-glucosidase-inhibiting, and immune-enhancing activities (Hou et al. 2003;Wang et al. 2003;Kim et al. 2007Kim et al. , 2008Kim et al. , 2010Harikrishnan et al. 2011;Lüthje et al. 2011;Park et al. 2014;Choi et al. 2016;Ha et al. 2017;Oliya et al. 2018). Like other Lactuca species, its leaves and stems release a milky latex upon wounding, which becomes sticky when exposed to air, helping to defend against herbivorous insects (Agrawal and Konno 2009). The latex is rich in various bioactive compounds, such as phenolics and sesquiterpene lactones (Abarca et al. 2019;Ilgün et al. 2020;Sessa et al. 2000). Sesquiterpene lactones are terpenoids whose structure is based on a guaiane skeleton bearing a lactone ring (Graziani et al. 2015). Various sesquiterpene lactones including lactucin, 8-desoyllactucin, and lactucopricin have been identified in the leaf and root of this plant (Nishimura et al. 1986;Michalska et al. 2009;Ha et al. 1 3 2017). Methanol extracts of L. indica exhibit antibacterial, anticarcinogenic, antidiabetic, antioxidant, and anticholesterolemic activity (Hou et al. 2003;Wang et al. 2003;Ghantous et al. 2010;Ha et al. 2017). Lactucin isolated from Lactuca species showed analgesic, sedative, and antimalarial activity (Bischoff et al. 2004;Wesołowska et al. 2006). In addition to its medicinal and therapeutic properties, Lactuca indica has a strong potential for breeding as a crop in its own right or as a possible candidate for improving the traits of cultivated lettuce (Lactuca sativa L.) (Kim et al. 2012;van Treuren et al. 2013;Lebeda et al. 2014;Ha et al. 2017;Oliya et al. 2018).
Increasing demand, unregulated harvesting, and the lack of a proper strategy for replenishing medicinal plants such as Lactuca indica have led to a decline in these natural resources in their natural habitat (Anis and Ahmad 2016). In nature, Lactuca indica propagates through seed production, but as with other lettuces, the seed has low viability and dormancy problems when the light requirement for seed germination is offset by low temperature (Eenink 1977;Sakpere et al. 2011;Obembe et al. 2017). Vegetative propagation is also possible, but limited. These are the factors limiting the sustainability and large-scale cultivation of this species; however, these obstacles could be solved through the use of a tissue-culture technique, which is used for mass propagation, disease-free plant production, genetic improvement, and analyzing secondary metabolites for pharmaceutical applications (Pink 1992;Ayan and KevseroĞlu 2007;Georgiev et al. 2011;Al Khateeb et al. 2012;Salgotra and Gupta, 2015). Also, this method provides a continuous supply of potential resources without any seasonal and environmental limitations, which ensures their practical application on a commercial scale (Kumari et al. 2016). Most of the in vitro regeneration research in lettuce has focused on the cultivated lettuce (Lactuca sativa) (Koevaxy et al. 1978;Alconero 1983;Webb et al. 1984;Pink 1992;Teng et al. 1992;Ampomah-Dwamena et al. 1997;Park and Lim 1997;Pink and Carter 1987;Mohebodini et al. 2011;Latif et al. 2014;Ahsan et al. 2015;Armas et al. 2017), with very few focusing on wild lettuce (Koevaxy et al., 1978;Obembe et al. 2017).
Investigation of the in vitro cultivation of Lactuca indica is still lacking; therefore, the objectives of this study are (1) to develop a protocol for in vitro propagation via direct and indirect organogenesis using leaf-blade and leaf-petiole explants; (2) to quantify the lactucin concentrations of in vitro-developed leaves, callus, and roots, and compare them with the quantities found in the roots, leaves, and stems of field-grown plants at the juvenile and flowering stages using high-performance liquid chromatography (HPLC); and (3) to compare the antibacterial performance of in vitro-grown tissue extracts with that of field-grown plant tissues collected at the juvenile and flowering stages.

Materials and methods
In vitro propagation -Plant materials, explant preparation, media, and culture conditions The plant material used in this study was obtained from the National Gene Bank of Korea (http:// www. seedb ank. re. kr). The seeds were grown in a soil bed in the Crop Genomics Lab, College of Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea. After 15 d, young aerial tissues (leaf blade and petiole) were collected for in vitro propagation. The tissues were washed under running tap water for 20 to 30 min, after which they were treated with liquid detergent (20 mL L -1 Tween-20 solution; Sigma-Aldrich, St. Louis, MO) for 30 min and washed five times with sterile distilled water. The pretreated tissues were then transferred into a laminar flow cabinet (Peak Scientific Instruments, Seoul, Republic of Korea) and surface-sterilized with 70% ethanol (Oci Company, Seoul, Republic of Korea) for 3 min before being treated with 1.5% sodium hypochlorite solution (v/v) (Oci Company, Seoul, Republic of Korea) for 8 min then washed three times (3 min per rinse) with sterile distilled water. After sterilization, the explants were left on sterile filter paper in a sterile Petri plate to absorb excess moisture. A sterile tissue-culture blade was used to prepare leaf-petiole and leaf-blade explants 0.5 cm in diameter, which were cultured in an in vitro culture establishment (ICE) medium.
The basal culture medium used in this study was Murashige and Skoog (MS) (Murashige and Skoog 1962) medium, containing the vitamins described by Gamborg et al. (1968) (Sigma-Aldrich) and 30 g L -1 sucrose, and with a pH of 5.8. Plant growth hormones such as cytokinins, 6-benzyl amino purine (BAP) (Sigma-Aldrich), and auxins indole acetic acid (IAA) (Sigma-Aldrich) and indolebutyric acid (IBA) (Sigma-Aldrich) were added in various concentrations, as outlined below. The pH of the media was adjusted with either 0.1 N NaOH or HCl, then supplemented with 8% agar (Sigma-Aldrich). A 50-mL aliquot of medium was poured into a tissue-culture glass bottle sealed with a transparent lid and autoclaved at 121°C for 20 min at 15 kg m −1 s −2 of pressure.
For ICE, leaf-blade and petiole explants were inoculated onto solid MS media supplemented with one of four concentrations (0.5, 1.0, 2.0, or 4.0 mg L -1 ) of BAP with 1.2 mg L -1 of IAA. MS medium without growth hormones was used as the control. A sub-culture to the same hormonal combination was performed every 15 d. For the regeneration of multiple shoots, healthy calluses (green and compact but without shoot bud) developed in the ICE phase were cut to 1 cm 2 and transferred to MS media supplemented with one of four concentrations (0.5, 1.0, 1.5, or 2.0 mg L -1 ) of BAP combined with a fixed concentration (0.5 mg L -1 ) of IAA. In vitro-regenerated healthy shoots were excised and 1 3 transferred to full-strength MS medium supplemented with 1.0 or 2.0 mg L -1 of IAA or IBA. Each treatment was performed with ten replicates (five tissue-culture glass bottles each inoculated with two explants) and kept at 25 ± 2°C under an 8-/16-h dark/light photoperiod using cool white fluorescent light (with 4000 lux) at 23°C to 25°C during the day and 20°C at night under 60 to 70% relative humidity. For each culture, the responses were examined after 45 d of culture.
Hardening and acclimatization In vitro-developed plantlets (45 d old) were transferred to the acclimatization room 5 d before being transferred into the soil. The media attached to the root surface were removed from the root surface by careful washing, after which the plantlet was planted in a 500-mL plastic pot filled with a mixture of perlite, vermiculite, and soil (1:1:1). The plants were covered with a transparent polythene bag to maintain high environmental humidity and kept in the controlled room, providing the same environment in which it was cultured. The potted plants were watered with a half-strength MS basal medium devoid of sucrose at 3-d intervals for a period of 2 wk. After 7 d, the cover was removed 3 to 4 h daily to expose the plant to the air. When the plants were well adapted to the controlled room, they were transferred to the greenhouse, where the cover continued to be removed for 3 to 4 h daily. After a further month, the surviving plants were transferred into the field under natural conditions.

Lactucin quantification and antibacterial assay -Plant tissue selection, collection, and extraction
The in vitro-grown leaves, calluses (green color, hard texture), and roots were collected after 45 d of culture and stored at −20°C. The same accession used for tissue culture was sown in the greenhouse, and the 3-wk-old seedlings were transplanted into the field at Seoul National University experimental farm, Suwon, Republic of Korea (N 37° 16′ 12.094″, E 126° 59′ 20.756″). The in vivo sample was harvested at the juvenile stage (in July) and flowering stage (in September) and separated into leaf, stem, and root tissues. All collected samples (in vitro and naturally grown) were freeze-dried for 48 h after harvesting and stored at 4°C until required.
The freeze-dried samples were ground to a fine powder in the grinder. The lactucin extraction was performed using the method developed by Willeman et al. (2014) with some modifications. In brief, 0.1 g of tissue powder was dissolved in 1.5 mL of a solvent mixture of water:chloroform:methanol (30:30:40, v:v:v) and mixed by vortexing after which the sample was incubated in a water bath at 60°C for 1.5 h. During the incubation period, the samples were vortexed gently every 20 min. The tubes were agitated using a shaking incubator, SI-64 (Hanyang Science Lab Co., Seoul, Republic of Korea), for 24 h in the dark at room temperature and centrifuged at 12,000 rpm at 4°C for 10 min using Micro Refrigerated Centrifuge (Smart R17, Hanil Science Industrial, Incheon, Korea). The supernatant was filter-sterilized and stored at −20°C until used in the HPLC analysis and bioassays.
Lactucin quantification using HPLC Lactucin was characterized using a Thermo Dionex ultimate 300 HPLC (Thermo Fisher Scientific, Seoul, Republic of Korea) equipped with a UV-visible diode array detector (190 to 400 nm scanning) and monitored with an Inno-C-18 column (250 mm × 4.6 mm i.d., 5 μm particles; YoungJin Biochrom, Seongnam-si, Republic of Korea). A mobile phase of buffer A (0.1% trifluoroacetic acid in distilled water) and buffer B (acetonitrile) was used in the following solution gradient: 5% buffer B at 0 min, 5% B at 1 min, 50% B at 30 min, 90% B at 31 min, 90% B at 36 min, 5% B at 37 min, and 5% B at 40 min. The flow rate was 0.8 mL min -1 . Thermo Dionex Chromeleon 7.2 software (Thermo Fisher Scientific, Seoul, Republic of Korea) was used to calculate the lactucin content. Lactucin (CS30062-69727; Extrasynthese, Genay, France) was used as a standard compound. All the reagents and compounds used were of HPLC grade.

Antibacterial activity
The Lactuca indica extracts were tested for their potential antimicrobial activity in an agar disk diffusion assay using the Gram-negative bacteria Pseudomonas fuscovaginae and Escherichia coli. P. fuscovaginae is a soil bacterium that is pathogenic to rice (Oryza sativa L.), causing brown sheath rot, while E. coli colonizes in the intestines of mammals and can be pathogenic. The bacteria (5 × 10 5 CFUmL -1 ) were independently inoculated into molten Mueller-Hinton agar plates. A sterile filter paper disk (diameter 6 mm) was saturated with 40 μL of the tissue extract (10 μL was dispensed at a time and allowed to dry for 10 min before the next 10 μL was dispensed) and gently pressed onto the surface of the bacteria-seeded agar plate. For each bacterial strain, a positive control (10 μL of 1 mg L -1 ampicillin) and a negative control (sterile disk saturated with methanol) were used. The plates were incubated overnight at 28°C for P. fuscovaginae and 37°C for E. coli. The diameter of the zone of any growth inhibition was measured on each plate, and the area was calculated and compared with the zone of growth inhibition achieved by the positive and negative controls. Three test disks were used on each Petri dish, and three replicates were performed.
Statistical analysis Each treatment for the in vitro study comprises two explants contained in a tissue-culture glass bottle, and all treatments were replicated five times, totaling N = 10 explants per treatment group. For the lactucin quantification and antibacterial assays, three replications per treatment were used. All the data were analyzed using one-way analysis of variance (ANOVA) and the mean values for each parameter were compared for a significant difference using Duncan's multiple range test (DMRT) at P ≤ 0.05. The results were expressed as means ± standard error for each experiment. All statistical analyses were performed using the SPSS computer software (version 23; IBM, Armonk, New York).

Results
In vitro culture initiation The petiole explants were able to produce both calluses and direct shoots upon ICE. Depending on the medium used, it took 5 to 7 d to induce callus cultures and 4 to 12 d for direct shooting. Among the various concentrations of BAP used in the MS media, in combination with a constant concentration (1.2 mg L -1 ) of IAA, 4.0 mg L -1 BAP was significantly more able to generate calluses on 72.0% explants of the total petiole cultured, followed by 2.0 mg L -1 BAP (55.0%), 1.0 mg L -1 BAP (25.0%), and 0.5 mg L -1 BAP (10.7%), respectively. By contrast, the lowest BAP concentration (0.5 mg L -1 ) was significantly better for direct shooting, under which 87.3% of explants produced shoots; 1.0, 2.0, and 4.0 mg L -1 BAP induced shoots in 69.3%, 12.0%, and 0.3% of samples, respectively (Table 1, Fig. 1A-D). The leaf-blade explants took 9 to 12 d to induce a callus. There was no significant difference between the time taken for callus and shoot induction from the leaf-blade and leaf-petiole explants. The callus (greenish and compact) covered the leaf blade under the different media treatments after 3 wk of culture. Among the media used, 4.0 mg L -1 BAP induced calluses in significantly more explants (94.7%) than the other concentrations (2.0, 1.0, and 0.5 mg L -1 BAP induced calluses in 84.7%, 74.0%, and 72.7% of samples, respectively; Table 1, Fig. 1E-H).

Callus regeneration
The callus regeneration began after 2 wk of culture. The medium supplemented with 1.5 mg L -1 BAP and 0.5 mg L -1 IAA regenerated significantly more callus cultures (98.7%) into shoots than the other media, producing an average of 8.5 shoots with a mean length of 4.3 cm. MS media supplemented with 1.0 or 2.0 mg L -1 BAP and 0.5 mg L -1 IAA led to the regeneration of shoots from 84% and 77% of calluses, respectively. The 1.0 mg L -1 BAP medium regenerated an average of 4.6 shoots per callus, with a mean shoot length of 3.8 cm, while the 0.5 mg L -1 BAP medium produced an average of 2.1 shoots per callus, with a mean shoot length of 2.1 cm (Table 2, Fig. 1I, J).
In vitro rooting, hardening off, and acclimatization The induction of roots on the microshoots was detected 15 d after their transplant onto rooting media. Among the auxin concentrations used, all microshoots produced roots in the media supplemented with 1.0 mg L -1 IAA, which was followed by 99.5% of microshoots on 1.0 mg L -1 IBA, 96.4% of microshoots on 0.5 mg L -1 IAA, and 92.5% of microshoots on 0.5 mg L -1 IBA. There was no significant difference between the overall root induction success on 1.0 mg L -1 of IAA or IBA; however, 1.0 mg L -1 IAA induced significantly more and longer roots (an average of 7.5 roots per callus, 3.9 cm in length; Table 3, Fig. 1K). Similarly, 0.5 mg L -1 IAA and IBA performed second and third best for the percentage of root induction and root lengths, with no significant difference between them (Table 3). The in vitro-rooted plants acclimatized to the pots of soil in the greenhouse, displaying a 100% survival rate in both the controlled room and the greenhouse (Fig. 1L). These surviving plants were successfully acclimatized in the field.
Lactucin quantification using a HPLC analysis Using a HPLC analysis, lactucin (standard 1-Lactucin-17.087) was detected at a retention time of 17 min in all tissue extracts. Its concentration ranged from 0.2 to 2.2 μg g -1 , with an average of 0.9 μg g -1 . Among the in vitro samples, the highest concentration of lactucin was detected in the root (0.9 μg g -1 ) followed by the callus (0.7 μg g -1 ) and the leaf (0.2 μg g -1 ).
In the naturally grown plants, the highest concentration of lactucin was detected in the root at the juvenile stage (2.2 μg g -1 ), followed by the root during flowering (1.3 μg g -1 ), the stem during flowering (0.8 μg g -1 ), the stem at the juvenile stage (0.7 μg g -1 ), the leaf during flowering (0.3 μg g -1 ), and the leaf at the juvenile stage (0.2 μg g -1 ) ( Fig. 2A, B). The DMRT indicated that the lactucin content in the juvenile root was significantly higher than in other samples. There was no significant difference between the lactucin amounts obtained from the tissue-cultured roots, calluses, and stems, as well as the flowering stems of the naturally grown plants.
Similarly, the lactucin contents of the tissue-cultured leaves and the naturally grown leaves of the juvenile and flowering  plants were significantly lower than those detected in the other tissues ( Fig. 2A).

Antibacterial activity
The tissue extracts of all plant samples (except the leaves from the naturally grown juvenile parental plants) were effective against both bacteria tested (Fig. 2C,   D). The solvent mixture used as the negative control did not inhibit bacterial growth. The positive control, ampicillin, showed a larger bacterial inhibition area than test samples (465.6 mm 2 ) for E. coli; however, the area of inhibition of P. fuscovaginae achieved using the positive control (185.7 mm 2 ) was not significantly different from the juvenile root  extract (176.5 mm 2 ). Among the tissue-cultured samples, the root was able to kill both bacteria, achieving areas of bacterial inhibition for E. coli and P. fuscovaginae of 103.8 mm 2 and 131.0 mm 2 , respectively. Using the callus extract, bacterial inhibition areas of 74.5 mm 2 and 80.7 mm 2 were achieved against E. coli and P. fuscovaginae, respectively. The leaf extract was less effective at inhibiting these bacteria where bacterial inhibition areas against E. coli and P. fuscovaginae were 47.5 mm 2 and 49.1 mm 2 , respectively (Fig. 2C, D). Among the naturally grown samples, the juvenile root was able to kill both bacteria. The areas of E. coli inhibition achieved (in decreasing order) by the root of the juvenile plant; the root, stem, and leaf of the flowering plant; and the stem of the juvenile plant were 168.0, 125.9, 100.3, 85.8, and 61.0 mm 2 , respectively. These values were significantly different, as determined using the DMRT. For P. fuscovaginae, the inhibition areas obtained (in decreasing order) were 176.7, 112.1, 69.0, and 67.4 mm 2 for the root of the juvenile plant, and the root, stem, and leaf of the flowering plant, respectively. We did not identify any significant difference in the antibacterial activity of the juvenile and mature stem extracts for P. fuscovaginae; however, there was a significant difference in the antibacterial activities of the juvenile and mature root extract (Fig. 2C, D).
The E. coli inhibition levels achieved by the cultured tissue samples were less effective than the naturally grown juvenile and mature roots, but more effective than the stem extracts from the juvenile and flowering plants. The antibacterial activity of the callus was higher than the juvenile stem and lower than the other extracts. The tissue-cultured leaf extract was not significantly different from the juvenile stem, but significantly less effective than other extracts. Similarly, for P. fuscovaginae, the antibacterial activity of the tissue-cultured root was markedly lower than the juvenile root but considerably higher than the other extracts. The callus showed significantly lower antibacterial activity than the juvenile and mature root, but substantially higher activity than the other extracts. The antibacterial activity of the tissue-cultured leaf was lower than the root and leaf of the juvenile and flowering plants, but not significantly different to the stems of plants at either developmental stage (Fig. 2C).

Discussion
The in vitro propagation of a traditional medicinal plant such as Lactuca indica is essential for enabling various technological breakthroughs that will enhance the breeding, conservation, and research of this species (Anis and Ahmad 2016;Chavan et al. 2018;Oliya et al. 2021). Here, a protocol was developed for direct and indirect organogenesis using leaf and petiole explants. Three major factors can influence the regeneration progress: the selection of a cultivar with adequate regeneration efficiency, the optimization of the explant source material, and the adaptation of the medium (Ampomah-Dwamena et al. 1997). The rapid production of microshoots could be helpful for the production of pathogen-free clones, which can be used more efficiently for producing true-to-type plants in a short timeframe. In indirect organogenesis, the plant cells multiply in a disorganized manner to produce a callus, which can in turn regenerate multiple shoots, roots, and even full embryos with the proper supplementation of growth hormones, pH, and light. This indirect organogenesis is an essential prerequisite to new cultivar production, cryopreservation, genetic transformation, and large-scale high-value secondary metabolite production (Rout et al. 2006;Dunwell 2008).
Different tissues may contain different levels of endogenous hormones; therefore, the source material of the explants has a critical impact on callus induction and regeneration success (Das et al. 2013;Oliya et al. 2021). Different studies in lettuce tissue culture have focused on ranking different cultivars according to their callus formation or direct shoot induction capacities (Ampomah-Dwamena et al. 1997;Latif et al. 2014). Leaves and leaf petioles have been used to generate explants in several plants to facilitate direct and indirect embryogenesis (Soneji et al. 2002;Arumugam et al. 2009;Ahsan et al.2015). These reports showed that genotype, explant age, plant growth hormone concentrations, the amount of micro-and macronutrients, and the carbon source all play a vital role in regeneration. Callus and shoot induction using various explants have been performed in wild and cultivated lettuce (Park and Lim 1997;Mohebodini et al. 2011;Ahsan et al. 2015;Armas et al. 2017;Obembe et al. 2017) however, our study is the first to achieve the in vitro propagation of Lactuca indica.
A lower BAP concentration significantly increased the shoot induction rate, while a higher concentration of BAP increased the callus induction rate, which is consistent with previous findings in cultivated lettuce and chicory (Cichorium intybus L.) (Park and Lim 1997;Mohebodini et al. 2011;Armas et al. 2017). The MS medium supplemented with 1.5 mg L -1 BAP and 0.5 mg L -1 IAA performed best for callus regeneration, consistent with an earlier report in Cichorium pumilum Jacq. (Al Khateeb et al. 2012). Similarly, 2.5 mg L -1 BAP combined with 0.5 mg L -1 NAA performed best for shoot induction in Launaea taraxacifolia (Willd.) Amin ex C. Jeffrey, an African wild lettuce, using single-node culture (Obembe et al. 2017). Ahsan et al. (2015) used leaf segments of lettuce (Lactuca sativa) variety (BARI Lettuce-1) for in vitro regeneration, revealing that MS medium supplemented with 1.5 mg L -1 BAP and 0.1 mg L -1 NAA was best for callus induction, while MS medium containing 2.5 mg L -1 BAP and 0.1 mg L -1 NAA was best for shoot induction. Callus quality and carbohydrate source were most critical for cell growth and differentiation in a suspension culture of lettuce (Teng et al. 1992). The highest frequency and speed of multiple shoot regeneration from cotyledon explants of L. sativa were recorded on MS regeneration medium supplemented with 0.5 mg L -1 activated charcoal, 3% sucrose, 0.5 mg L -1 BAP, and 0.5 mg L -1 NAA, which induced shoots through direct regeneration. In the present study, 1.0 mg L -1 IAA performed best for rooting over the other treatments, consistent with earlier studies in lettuce and chicory (Pink and Carter 1987;Yucesan et al. 2007;Dolinski and Olek 2013).
One of the challenges in plant tissue culture is the process of acclimatization. Under in vitro conditions, plants are raised in a controlled environment and with high humidity. These plants therefore have adequate moisture in their leaves and stems, resulting in their production of leaves containing large spaces between their palisade cells and few stomata. When the plant is exposed to the outer atmosphere, there is a high degree of transpiration and fungal contamination, leading to plant death. The success of acclimatization depends on the genotype, root growth, season, and handling. The periodic removal of the plastic cover to gradually lower the high atmospheric humidity, irrigation with half-strength MS basal medium devoid of sucrose for 3 wk at 3-d intervals, and raising plants in a controlled room for another 6 wk before transferring into the greenhouse or field often resulted in a 100% survival rate for Lactuca indica in our study. Previously, Conner et al. (2019) studied the ex vivo growth response of Cichorium intybus (cultivar 'Grasslands Puna') and Lactuca sativa (cultivar 'Cobham Green') plants regenerated from the nodal segment that had gone phage change to in vitro flowering (adult plant), and the cauline leaf culture of the adult plant (rejuvenated plants). The resulting rejuvenated plants for both species exhibit substantially improved performance in greenhouse conditions with increased frequency of plant survival, a doubling of the frequency of plants that flowered, and substantially increased seed production compared to adult plants. Similarly, De Souza et al. (2007) obtained 100% survival for in vitro raised plantlets of Lychnophora pinaster, a threatened endemic medicinal plant belonging to Asteraceae when planted in soil from the area of occurrence of the species, whereas 0% survived when planted in commercial substratum Plantmax.
In this study, the lactucin concentrations of the in vitro-grown roots and calluses were higher than the leaves, lower than the roots, and not significantly different from the stems of the naturally grown mother plant. This result suggests that tissue-cultured materials are similarly biochemically potent to the naturally grown plants. In general, we found roots were the best tissue for the accumulation of lactucin; however, this compound can be extracted from the entire plant. The variation in the concentration of lactucin among the tissues may be due to transcriptomic difference between the tissue, and abiotic environmental factors (Badri et al. 2010;Sampaio et al. 2016;Hubbard et al. 2017;Soorni et al. 2021). Previously, Michalska et al. (2009) used ultraviolet-HPLC and thin-layer chromatography to identify six different types of sesquiterpene lactones (8-deoxylactucin, jacquinelin, crepidiaside B, lactucopicrin, glucozaluzanin C, and lactuside-A) in the roots and leaves of Lactuca indica; however, they did not detect lactucin in this plant. In another previous study, a small amount of lactucin, 8-desoyllactucin, and lactucopricin was detected in the leaves at the bolting stage, while lactucin was only observed in the flowering stage (Ha et al. 2017). Moreover, in the same study, lactucin concentrations ranging from 1.9 to 98.7 μg g -1 were reported in the leaves of flowering plants from 61 Lactuca indica accessions collected across the Republic of Korea. In the present study, using a HPLC analysis, we have quantified lactucin from the leaf, stem, and root in the flowering and juvenile periods of naturally grown plants. A higher amount of lactucin was obtained from the roots of plants in the juvenile period, followed by the leaves of flowering individuals. Comparing the work of Ha et al. (2017) with the present study, we conclude that lactucin biosynthesis in Lactuca indica depends on the plant tissue involved, phenological development, and the environment.
Sesquiterpene lactones, a diverse group of terpenoids isolated from Asteraceae species, exhibit a broad spectrum of biological activities. Several of them are already commercially available as a drug, such as artemisinin isolated from Artemisia annua is used as an antimalarial drug (Liu et al. 2019;Zhang et al. 2019;Moujir et al. 2020). Here, we explored the antibacterial activity of the different tissue extracts against P. fuscovaginae and E. coli, with both bacteria reacting similarly. The root extract, which contained a significantly higher lactucin concentration, also showed a significantly higher antibacterial activity. The leaf extract, which contained a low lactucin concentration, showed weaker or no antibacterial activity. Thus, the antibacterial activity of the tissue extracts could be attributed to the presence of lactucin; however, as reported by previous studies, diverse sesquiterpenoids, phenolic, flavonoids, and minerals are also present in the crude extract and could potentially have an antibacterial effect (Nishimura et al. 1986;Kim et al. 2008;Michalska et al. 2009;Kim and Yoon 2014;Padilla-Gonzalez et al. 2016;Ha et al. 2017;Abdalla et al. 2021). A previous study by Pavlović et al. (2011) showed that the extracts of Lactuca sativa grown in greenhouses showed high antibacterial activity against various bacterial strains, particularly against Staphylococcus aureus and Proteus mirabilis. Lactuca indica extracts have been shown to have various therapeutic activities, including antibacterial activity 1 3 against uropathogenic E. coli (Hou et al. 2003;Wang et al. 2003;Kim et al. 2007Kim et al. , 2010Lüthje et al. 2011;Park et al. 2014). Lactucin in Cichorium intybus showed antibacterial activity against various bacteria (Petrovic et al. 2004;Nandagopal and Kumari 2007;Liu et al. 2013). Together with its derivatives lactucopicrin and 11beta,13-dihydrolactucin, which are characteristic bitter sesquiterpene lactones of Lactuca virosa and Cichorium intybus, isolated from root and leaves, lactucin was evaluated for analgesic and sedative properties in mice (Wesołowska, et al. 2006). Lactucin and lactucopicrin also showed antimalarial activity (Bischoff et al. 2004). Though the therapeutic activities of plant extracts were reported, the present study is the first to identify the antibacterial effect of Lactuca indica against the plant pathogen P. fuscovaginae. This finding broadens the therapeutic horizon of Lactuca indica and gives insight into its potential applicability as a pesticide. Further research on bioassay-guided fractionation and purification is strongly recommended in this field.

Conclusion
This study established an efficient in vitro propagation protocol for direct and indirect organogenesis, which have many potential applications for medicinal and horticultural species such as Lactuca indica, including the true-to-type large-scale propagation of superior genotypes and genotypic improvement via mutagenesis and/or genetic engineering. This study validates the practical application of a newly established tissue-culture protocol for phytochemical and therapeutic research. In addition, this study found variation in the lactucin concentrations and antibacterial activities, which were affected by the developmental stage of the plant, the environment, and the tissue type sampled, which is beneficial for germplasm conservation and the economic exploitation of Lactuca indica. Our results could guide the further study of lettuce and other medicinal plants.