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One of the common problems in strawberry (Fragaria × ananassa) micropropagation is the vitrification phenomenon (succulent plantlets, brittle stems, yellow leaves, etc.) leading to the reduction of plantlets quality and low survival rate in the greenhouse. In this study, the effects of silver nanoparticles (AgNPs) on explant disinfection, in vitro growth (shoot multiplication, and root formation), runner formation as well as ethylene accumulation during micropropagation of strawberry were investigated. The results showed that leaf explants treated with 200 mg/L AgNPs solution for 20 min was more effective in explant disinfection and shoot regeneration than using 1 g/L HgCl2. In addition, AgNPs stimulated the growth of shoot and plantlet and as well shortened the duration of root formation (4 days) as compared to those in control without AgNPs during micropropagation. Besides, AgNPs reduced the ethylene gas accumulation in the culture’s vessels of shoots (0.66 ppm) and plants (0.06 ppm) compared to controls (1.77 ppm; 0.15 ppm; respectively). Moreover, AgNPs combination with culture period (5; 10 or 15 days) effect root formation stage and acclimatization in the greenhouse. The plantlets that cultured on MS medium supplemethed with 0.5 mg/L AgNPs during 10 days showed higher survival rate (93.33%) after 15 days as well as runner formation per plant (8.00 runners) after 60 days in greenhouse than those in control.Key messageAgNPs improved explant disinfection and in vitro growth. AgNPs improved runner formation in the greenhouse. AgNPs limited ethylene accumulation during micropropagation.

Micropropagation has proven to be an effective method for large-scale plant production in a short time and a useful tool for plant breeding. Microbial contamination is one of the most difficult micropropagation challenges, resulting in reduced plant quality and loss of valuable stocks. Therefore, sterilization of culture media is a critical step in plant micropropagation. However, sterilized media might reduce the activity of plant growth regulators and nutritional components of culture media. The sterilization effects of silver nanoparticles (AgNP) on the growth of expiants and culture media were examined. The treatment with 250 ppm AgNP for 15 to 20 min of 4-wk-old ex vitro leaves proved optimal for controlling the contamination. Furthermore, the Murashige and Skoog medium containing 4 ppm AgNP resulted in 100% medium disinfection (no contamination) after 4 wk of culture. The plantlets obtained from non-sterilized MS medium (NoM) containing 4 ppm AgNP and 4 g L⁻¹ agar gave similar results as the control medium with 8 g L⁻¹ agar and the absence of AgNP. Large scale culture systems using NoM in large plastic containers of two different sizes (NoM1 and NoM2) could produce quality plantlets. Chrysanthemum plantlets in the NoM1 system showed higher antioxidant enzyme activities of ascorbate peroxidase and Superoxide dismutase than plantlets in the autoclaved medium. Furthermore, the plantlets from NoM were better acclimatized under greenhouse conditions than those from the autoclaved medium (AuM) system. The developmental stages (flower buds and blooming time) of NoM1 and NoM2 plantlets, were 1 wk earlier than those from the AuM system. The successful use of AgNP as a sterilizer and as a component of culture media would reduce the cost of micropropagation and improve plants' quality.

Micropropagation of Lang Bian ginseng (Panax vietnamensis var. langbianensis), an endemic medicinal plant, via somatic embryogenesis (SE) and rhizome formation were investigated. Rhizome explants disinfected with 0.15% AgNPs for 30 min resulted in the most effective surface disinfection (contamination, necrosis and survival with callus induction of 36.00%, 14.70% and 49.30%, respectively). Moreover, shoot regeneration (45.33%) and the number of shoots (5.2 shoots) from 0.15% AgNPs disinfected- rhizome were significantly higher than those in others after 12 weeks of culture. The leaf was cut transverse thin cell layer (L-tTCL: 1 mm × 5 mm) and petiole cut longitudinally TCL (P-lTCL: 0.5 mm × 10 mm). L-tTCL explant cultured on MS medium supplemented with 7 mg/L NAA gave 100% SE and 32.80 embryos; meanwhile, P-lTCL explants also obtained 100% SE and 51.80 embryos in 1 mg/L 2,4-D treatment. Adventitious root formation and callus induction were also observed in the auxin-supplemented treatments. In addition, 12-week-old secondary somatic embryos, mainly cotyledonary stage, were obtained from the primary somatic embryo cultured on 1/2 MS medium supplemented with 1 mg/L 2,4-D. Plantlets derived from secondary embryos grew well with 63.34% rhizome formation, 1.27 cm in rhizome length and 0.65 cm in rhizome diameter. Meanwhile, plantlets derived from adventitious shoots only induced adventitious roots and callogenesis after 20 weeks of culture. The analysis of saponins by HPLC method showed that twenty-week-old in vitro rhizomes had Rg1, Rd and Rb1.Key message(1) Silver nanoparticles were effective in rhizome surface disinfection. (2) Primary and secondary somatic embryogenesis in Lang Bian ginseng. (3) Rhizome formation with ginsenoside accumulation.

This study investigates the influence of various parameters on the efficiency of cultivating and preserving Lemna aequinoctialis duckweed samples in vitro , with the aim of minimizing time, labor, and storage expenses. The method addresses the issue of limited space and the risk of sample loss by storing multiple duplicates (more than one tube or flask per sample) in a space-efficient and cost-effective container. Due to the practical implications and capacity to decrease operating expenses, our results are helpful for large-scale implementation. Using a single frond resulted in a longer preservation effect compared to sample densities of three and five fronds. Initial samples can be cultured in nutrient solution with or without sugar, stored in tap water or nutrient solution, kept at a stable temperature of 25 ± 2°C or room temperature. The test tubes with duckweed were not covered, or covered by black paper up to the solution surface or higher. The outcomes demonstrated that all three duckweed samples (HNP_005, HNP_026, and HNP_031), whether grown in SSM or SFM media, were best preserved if the tubes were covered by black paper higher than the solution surface (CH) and can be kept at room temperature for at least 9 mo.

This study investigates the endogenous hormone variation in the callus and adventitious root formation of Phyllanthus amarus using the thin cell layer (TCL) culture system. By employing longitudinal thin cell layer (lTCL) internode explants, this study examined the changes in key plant hormones, including auxin (AUX), cytokinins (CKs), gibberellin A3 (GA3), abscisic acid (ABA), and melatonin (MEL), using ultra-high-performance liquid chromatography (UHPLC). This experiment highlighted that the position of the internode had a significant impact on both callus and adventitious root formation. Internode explants closer to the root base (3rd internode) favored adventitious root formation, attributed to higher endogenous AUX levels, while the 2nd internode exhibited superior callus formation efficiency, associated with a more balanced AUX/CKs ratio. TCL explants demonstrated faster morphogenesis and higher efficiency compared to internode explants due to better nutrient and plant growth regulator absorption. The lTCL explants facilitated a more rapid and extensive formation of callus and adventitious roots. Hormonal analysis revealed dynamic changes in endogenous hormone levels throughout the culture period, with notable peaks of AUX and CKs at critical stages of callus and adventitious root development. The ratios between AUX and CKs, as well as interactions with GA3, ABA, and MEL, played a pivotal role in regulating morphogenesis. The findings provide valuable insights into the intricate hormonal mechanisms controlling in vitro morphogenesis in P. amarus and underline the potential of TCL culture systems for improving tissue culture strategies in medicinal plants.Key messageInternode position significantly influenced callus and adventitious root formation on Phyllanthus amarus. Thin cell layer culture positively impacted morphogenesis. Endogenous hormone alterations correlated with morphogenesis in Phyllanthus amarus.

This study substituted sodium molybdate dehydrate (Na2MoO4.2H2O) in MS medium (Murashige and Skoog 1962) with molybdenum trioxide nanoparticles (MoO3NPs) to evaluate their impact on the morphogenesis, growth, absorption of metal-mineral elements and the activity of antioxidant enzymes of chrysanthemum. The results indicated that 100% callus formation was observed when 1-month-old leaf explants were cultured on medium supplemented with MoO3NPs and Na2MoO4.2H2O in basic MS medium, while leaf explants cultured on MS medium without Na2MoO4.2H2O did not induce callus induction. In addition, the treatment with 223.5 µL/L MoO3NPs resulted in the highest shoot regeneration (33.33%), with 1 shoot per explant, and shoot height (1.14 cm), and fresh weight (1.21 g) compared to those in others and control treatments. Meanwhile, 1-month-old stem node (1 cm) explants cultured on medium supplemented with 149 µL/L MoO3NPs recorded 100% shoot regeneration and the highest number of shoots larger than 2 cm (5 shoots/stem node), shoot height (3.23 cm) and fresh weight (1.87 g) of the shoot cluster after 30 days of culture. During the regeneration stage, the activity of antioxidant enzymes in 149 µg/L MoO3NPs treatment was better than the control (+) treatment (except for SOD) and all the other treatments. Similar results were also observed during the shoot multiplication phase, where either the lack or surplus of Mo in the culture medium also caused the decline of SOD, CAT, and APX enzymes activity. Besides, at the concentration of 6.4 µg/L MoO3NPs in the culture media, nutrients are absorbed more efficiently and rapidly by explants. These findings suggest that substituting ion salt in the culture medium with MoO3NPs led to enhanced absorption, providing a micro-mineral source for plants to support biosynthesis and essential functions. The chrysanthemum plantlets exhibited enhanced rooting and growth when treated with 149 µg/L MoO3NPs, particularly during the rooting stage after 15 days of culture.Key messageMolybdenum trioxide nanoparticles effected on the morphogenesis, growth, absorption of metal-mineral elements and the activity of antioxidant enzymes of chrysanthemum

This study investigated the changes in ethylene gas accumulation, antioxidant system activity, and secondary metabolite synthesis during the in vitro formation of adventitious roots in Phyllanthus amarus . The results indicated that longitudinal thin cell layer (lTCL) internode explants exhibited significantly higher ethylene accumulation and increased root weight during adventitious root formation. The results also showed enhanced antioxidant enzyme activities, including APX, CAT, SOD, DPPH, phenolic, and vitamin C in lTCL-internode explants. In addition, the levels of important secondary metabolites were enhanced in lTCL-internode explants compared to internode explants. Phyllanthin and hypophyllanthin contents in adventitious roots were significantly increased compared with the control. These findings suggest that the TCL culture technique can effectively promote adventitious root growth and optimize the production of bioactive compounds in P. amarus explants. This study provides valuable insights into the relationship between ethylene, antioxidant systems, and secondary metabolite synthesis, thus opening up possibilities for improving plant tissue culture and secondary metabolite production in this plant. Key message lTCL-internode explants in Phyllanthus amarus showed higher ethylene accumulation, antioxidant system activity, and secondary compound production, enhancing medicinal value compared to internode explants during adventitious root formation.

The use of silver nanoparticles (AgNPs) is a new direction that promises high economic efficiency in micropropagation of Limonium sinuatum (L.) Mill. ‘White’. In this study, the effect of AgNPs on the explant surface disinfection; in vitro growth, development and acclimatization of L. sinuatum were investigated. Especially, endogenous hormone alterations in shoot multiplication and rooting stages of plantlets derived from with or without AgNPs (control) treatments were recorded. The results showed that explants treated with 200 mg/L AgNPs for 20 min not only had a higher disinfection effect but also had better effects on shoot induction than the explants treated with 1000 mg/L HgCl2 for 5 min. The explant cultured on medium supplemented with 1.0 mg/L AgNPs showed higher shoot number (5.66 shoots/explant) than the control (2.66 shoots/explant) via reducing ethylene content (0.690 µg/g) and increasing zeatin content (0.947 µg/g). The rooting time of plantlets was shortened by 3 days in the medium supplemented with 0.4 mg/L AgNPs as compared to the control, which corresponds to low ethylene content and high content of IAA, GA3, and ABA. The results indicated that AgNPs stimulated shoot multiplication and rooting of L. sinuatum through altering endogenous hormones. Furthermore, 0.4 mg/L AgNPs-derived plantlets showed well acclimatization as compared to those of the control in greenhouse conditions.Key messageThe supplementation of AgNPs in the culture medium might alter endogenous hormone levels resulting in higher shoot multiplication, rooting, survival and growth rate of Limonium sinuatum.

SummaryThe effects of iron nanoparticles (FeNPs) on in vitro rooting, chlorophyll content, antioxidant and hydrolysis enzyme activities, acclimatization and subsequent growth of Gerbera jamesonii var. Revolution Yellow plantlets under greenhouse conditions were examined in this study. The single shoots (2 cm in length) were cultured in different medium treatments, including: MS0 (MS medium - control), MS0 replacing 27.8 mg/L FeSO4.7H2O with FeNPs (ranging from 0 to 11.2 mg/L). The results showed that 2-cm shoots cultured in medium supplemented with 100 mM FeNPs induced in vitro rooting 2 days earlier than shoots cultured in MS0 medium. In addition, plantlet height (6.83 cm), number of main roots and lateral roots (13.00 roots and 4 roots), root length (5.27 cm), leaf length and width (2.07 cm and 1.83 cm), fresh and dry weights (1188.67 and 155 mg), dry matter ratio (13.04%), and chlorophyll a, b, a + b content (25.25, 18.35 and 43.6 mg/g, respectively) of the shoot culture in 5.6 mg/L FeNPs treatment were also recorded higher than other amounts of FeNPs and control (MS0) treatments after 4 weeks of culture. Furthermore, the enzyme activities of SOD (41.32 U/g), CAT (308.70 U/g), and APX (0.55 U/g) were higher in the 100 µM FeNPs treatment than those in the MS0 and MS-Fe treatments, and hydrolysis enzyme activities (cellulase and pectinase) showed opposite results after 4 weeks of culture. The plantlets derived from the 5.6 mg/L FeNPs treatment had the highest survival rate (93.00%) in comparison to those in MS0 medium (83.67%) and without FeNPs (73.33%) after 2 weeks under greenhouse conditions, and the early flower bud formation (10.17 weeks), increased flower diameter (7.87 cm), and peduncle length (22.17 cm) after 12 weeks.Key messageIron nanoparticles improved in vitro rooting, changed antioxidant and hydrolytic enzyme activities, increased acclimatization, growth and flowering of G. jamesonii var Revolution Yellow.

The stem elongation, somatic embryogenesis of Tuberous begonias (Begonia × tuberhybrida Voss) plantlet under different lighting (fluorescent lamps - FL, blue light-emitting diode - LED, red LED and blue to red LED ratio), and subsequent growth on medium containing cobalt nanoparticles were investigated. The results showed that shoots (1.5 cm in length) cultured under the red LED condition produced higher values of shoot length (6.13 cm), number of internodes per shoot (6.00 internodes), fresh and dry weights (640.34 mg and 78.25 mg, respectively) compared to those under the other lighting conditions after 60 days of culture. Meanwhile, the S-tTCL explants cultured under red LED achieved higher somatic embryogenesis (64.71%), number of somatic embryos (46.67 embryos) and percentage of somatic embryos with torpedo-shape (34.39%) and cotyledon (65.61%) as compared to those under the other lighting conditions after 60 days of culture. For plantlet formation and subsequent growth, somatic embryos (cotyledon shape) cultured on medium containing 0.0465 µg/L CoNPs enhanced plantlet growth, acclimatization and flowering of plantlets in the greenhouse.Key messageRed light-emitting diodes enhanced stem elongation and somatic embryogenesis of tuberous begoniaMedium containing cobalt nanoparticles enhanced plantlet growth and their acclimatization