In vitro callus induction and evaluation of antioxidant activity of Rhinacanthus nasutus (L.) Kurz.

Rhinacanthus nasutus (L.) Kurz is used in Thai traditional medicine for the treatment of skin diseases, ringworm, and eczema. This research studied the effects of cytokinin and auxins on callus induction and evaluated antioxidant activity of R. nasutus. Nodes, young, and mature leaf explants were cultured on MS medium containing 0, 1, 2, 3, and 4 mg/l kinetin (6-furfurylaminopurine) and 0, 1 mg/l 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) for 6 weeks to induce callus. Calli derived from nodes, young and mature leaves, and other plant parts were ultrasonically extracted with methanol to determine total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity by ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhtdrazyl (DPPH), and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assays. Nodal explants on MS medium containing 1 mg/l kinetin combined with 1 mg/l 2,4-D were most efficient in callus production with the average fresh and dry weight per explant of 2.29 ± 0.14 and 0.18 ± 0.01 g, respectively. Addition of kinetin combined with NAA or 2,4-D had a positive effect on callus induction from young and mature leaf explants. The leaf extract showed the highest TPC, TFC, FRAP, and IC50 of DPPH and ABTS assays (ca 113 mg GAE/g extract, 45 mg QE/g extract, 121 mg TE/g extract, 53 µg/ml and 14 µg/ml, respectively), followed by callus derived from nodes. Overall, phenolic content was higher than flavonoid content. A strong positive correlation was found between FRAP assay, TPC (r = 0.973), and TFC (r = 0.798), indicating that phenolic and flavonoid compounds are responsible for antioxidant activity of R. nasutus.

The Optimization of Medium Conditions and Auxins in the Induction of Adventitious Roots of Pokeweed (Phytolacca americana L.) and Their Phytochemical Constituents.

Pokeweed, Phytolacca americana L., is considered a widely spreading invasive plant, while saponin contents accumulated in the roots have pharmaceutical uses, such as rheumatism treatments and anti-inflammation. Adventitious root cultures are an important source of diverse secondary metabolites, which have significant applications in various fields. This study focused on the optimization of parameters for root induction using different medium states and treatments with auxins on a pokeweed leaf. Semisolid and liquid MS (Murashige and Skoog, 1962) media were supplemented with indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) at 0.5, 1, 2, and 4 mg/L. Root growth parameters, e.g., induction percentage, root numbers, length, and weight, were measured to determine the adventitious root induction efficiency. Total phenolic content, total flavonoid content, total saponin content, and antioxidant activity were recorded. Results showed that adventitious roots induced in semisolid MS medium supplemented with 0.5 mg/L NAA exhibited a high density of lateral roots. Appropriate medium state and auxin for adventitious root induction in pokeweed were determined as semisolid medium supplemented with 2 mg/L NAA. Considering phytochemicals, adventitious roots induced in liquid medium containing 0.5-1 mg/L NAA had the highest yield extract percentage. Additionally, adventitious roots cultivated in a liquid medium enriched with 1 mg/L NAA exhibited the highest phenolic and saponin contents. A principal component analysis (PCA) biplot and hierarchical cluster analysis (HCA) heatmap demonstrated different response patterns between semisolid and liquid media applied with NAA. The results of the semisolid media were grouped together due to high expression levels of the root induction parameters, while elevated phytochemical values were observed in the liquid media treatments. The results suggested two different media that provide the highest adventitious root induction efficiency and the greatest phytochemical contents: semisolid medium with 2 mg/L NAA and liquid medium with 1 mg/L NAA, respectively. These culture media can be applied to optimize adventitious root culture of pokeweed and in vitro phytochemical production.

Optimal protocol for in vitro polyploid induction of Cymbidium aloifolium (L.) Sw.

BACKGROUND: Cymbidium aloifolium is a popular ornamental flower in Thailand with both economic and medical values. Polyploid induction techniques are used to improve plant quality. This study identified polyploidy levels of C. aloifolium induction by colchicine. Protocorms of C. aloifolium were treated on solid New Dogashima Medium (NDM) with various concentrations of colchicine (0, 0.01, 0.02, 0.03, 0.04 and 0.05% w/v) for 2, 4 and 8 weeks. RESULTS: Colchicine effectively induced plant polyploidy. Tetraploid plants were found after treatment with 0.03% and 0.04% colchicine for 8 weeks, while at increased concentration and duration, survival, response and growth performance decreased. Tetraploid plants showed the lowest growth performance but highest size of stomatal and epidermal cells. Growth performance and leaf surface anatomy data were analyzed by Pearson's correlation and PCA. Results showed that stomatal and epidermal cell sizes had strongly negative correlations with other variables, while HCA revealed that stomatal and epidermal cell sizes of tetraploid plantlets were larger but stomatal and epidermal cell densities decreased when compared with the diploids. CONCLUSION: Colchicine at suitable concentrations and duration produced polyploid plants with alteration of morphological and anatomical traits. This study provides potential information to support orchid quality production as ornamental plants and a source of pharmaceutical raw materials.

Effect of Heat Stress on Some Physiological and Anatomical Characteristics of Rice (Oryza sativa L.) cv. KDML105 Callus and Seedling.

Global warming is a serious problem, with significant negative impacts on agricultural productivity. To better understand plant anatomical adaptation mechanisms as responses to heat stress, improved basic knowledge is required. This research studied the physiological and anatomical responses of Khao Dawk Mali 105 (KDML105) to artificial heat stress. Dehusked seeds were sterilized and cultured on Murashige and Skoog (MS) medium, supplemented with 3 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) for callus induction. The cultures were maintained at 25 °C and 35 °C for 4 weeks, while the other culture was treated with heat shock at 42 °C for 1 week before further incubation at 25 °C for 3 weeks. Results revealed that elevated temperatures (35 °C and 42 °C) adversely impacted seedling growth. Plant height, root length, leaf number per plant, fresh and dry weight, chlorophyll a, chlorophyll b and total chlorophyll content decreased after heat stress treatment, while malondialdehyde (MDA) and electrolyte leakage percentage significantly increased, compared to the control. Heat stress induced ROS accumulation, leading to lipid peroxidation and membrane instability. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) results also confirmed negative correlations between MDA, electrolyte leakage and other parameters. MDA content and electrolyte leakage are effective indicators of heat stress in rice. Surface anatomical responses of rice seedlings to heat stress were studied but significant alterations were not observed, and heat stress had no significant negative effects on KDML105 calli. Size and mass of calli increased because heat stress stimulated gene expression that induced thermotolerance. Our results provide useful information for rice breeding and heat stress tolerance programs to benefit long-term global food security.

Salinity Stress Response of Rice (Oryza sativa L. cv. Luem Pua) Calli and Seedlings.

Soil salinity limits plant growth and production. This research investigated a suitable medium for callus induction and plantlet regeneration in the Luem Pua rice cultivar. The effect of salt stress on seedling growth was determined using in vitro culture and soil conditions. An efficient protocol for callus induction has been developed by culture sterilized seeds on the Murashige and Skoog (MS, 1962) medium containing 0.5 mg/l benzyladenine (BA) with 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) that resulted in a 100% callus induction. Plantlet regeneration percentage of 49% was recorded on the MS medium containing 4 mg/l BA with 0.5 mg/l 1-naphthaleneacetic acid (NAA) after 4 weeks. For salt stress investigation, the calli were treated on an induction medium containing various concentrations of NaCl (0, 50, 100, 150, and 200 mM), while two-week-old rice seedlings were planted in soil and treated with the same concentration of NaCl for 4 weeks. In vitro culture revealed that callus survival percentage decreased when NaCl concentration increased, similar to soil culture. Seedling growth under salinity treatment also decreased when NaCl concentration increased, while other physiological parameters such as total chlorophyll, chlorophyll a, chlorophyll b, green intensity, and chlorophyll fluorescence under light conditions increased under salinity stress. These changes define the growth and physiological salinity tolerance characteristics of Luem Pua rice calli and seedlings. They can be utilized as a baseline for demand-driven in vitro rice propagation, providing useful information that can be combined with other agronomic features in rice development or breeding programs to improve the flexibility of abiotic stress-tolerant cultivars.

Micropropagation of pokeweed (Phytolacca americana L.) and comparison of phenolic, flavonoid content, and antioxidant activity between pokeweed callus and other parts.

BACKGROUND: Pokeweed (Phytolacca americana L.) is regarded as an invasive plant in many parts of the world but possesses therapeutic characteristics used for antitumor and rheumatism treatment. This study investigated the effects of auxins and four explants on pokeweed callus induction. The effects of cytokinins and combinations between cytokinins and NAA on shoot and root induction were also studied. TPC, TFC and antioxidant activity of calli were screened and compared with other pokeweed plant parts. METHODS: Four explants were used to induce callus using 2,4-D and IBA at 1, 2, 3 and 4 mg/l for each auxin. Direct shoot organogenesis from nodal explants was investigated using BAP, kinetin and TDZ (1, 2 and 4 mg/l for each cytokinin). Combined effects between cytokinins and NAA at 0.1, 0.2 and 0.3 mg/l were further simultaneously estimated with root induction. Calli derived from the leaves were compared with other plant parts for TPC, TFC and antioxidant activity using the Folin-Ciocalteu, AlCl3 colorimetric assay and DPPH assays, respectively. RESULTS: Results showed that MS medium containing 2 mg/l 2,4-D induced callus formation on leaf explants that provided highest fresh and dry weights. Three types of synthetic cytokinins as kinetin, TDZ and BAP were used for direct shoot organogenesis from pokeweed nodes. MS medium containing 2 mg/l kinetin was effective in stimulating normal shoots, with the largest number of shoots and leaves and the longest shoots. The combination between cytokinins and NAA showed no positive effect on shoot and root induction from pokeweed nodal explants. For TPC and TFC determination, pokeweed seeds and leaves possessed the highest phenolic and flavonoid contents, respectively. Highest phenolic content of pokeweed seeds led to lowest IC50 by DPPH assay. Phenolic content was higher than flavonoid content. CONCLUSION: Results suggested promising conditions for callus induction. Leaf explants cultured on MS medium with 2 mg/l 2,4-D and nodal explants cultured on MS medium with 2 mg/l kinetin provided the largest number of normal shoots and leaves. NAA did not show positive effects on shoot and root induction when combined with cytokinins. Chemical constituent screening indicated that seeds and leaves provided highest TPC and TFC, respectively, while pokeweed calli contained higher phenolic than flavonoid content. This is the first report describing chemical constituent screening and antioxidant activity of calli and other parts of the pokeweed plant. Results provided significant information to further enhance bioactive compound contents of pokeweed calli using elicitation methods.

Effect of stress on the leaf anatomy of sugarcane cultivars with different drought tolerance(Saccharum officinarum, Poaceae)

Introduction: Drought is an important stress factor for sugarcane production in many areas of the world. Water proportion and moisture indices are applicable information for agronomic planning to forecast water excess or deficit during the crop cycle. Objective: Leaf anatomical features of two different sugarcane Saccharum 'UT12' (drought susceptible cultivar) and Saccharum 'UT13' (drought tolerant cultivar) were compared under early drought stress situation between 30 and 90 days after planting. Methods: Forty leaf anatomical features were investigated using peeling and free hand sectioning technique. Results: Some anatomical characteristics showed response to drought stress. Saccharum 'UT12' demonstrated higher sensitivity toward anatomical characteristics than Saccharum 'UT13'. A total of 23 and 15 out of the 40 anatomical characteristics showed significance in Saccharum 'UT12' and Saccharum 'UT13', respectively. Some anatomical features such as cell wall and cuticle thickness, vascular bundle size, stomatal size and density can be used as important markers for drought stress assessment in sugarcane leaf. Conclusions: This is the first report describing comparative leaf anatomy of sugarcane Saccharum 'UT12' and Saccharum 'UT13' in Thailand under drought stress. Results will provide important information to improve adaptation mechanisms of tolerant sugarcane cultivars under initial drought stress situations.

Introducción: La sequía es un factor de estrés importante para la producción de caña de azúcar en muchas áreas del mundo. La proporción de agua y los índices de humedad son información aplicable en la planificación agronómica para pronosticar el exceso o el déficit de agua durante el ciclo del cultivo. Objetivo: Se compararon las características anatómicas de las hojas de dos cañas de azúcar diferentes Saccharum 'UT12' (cultivar susceptible a la sequía) y Saccharum 'UT13' (cultivar tolerante a la sequía) bajo una situación de estrés por sequía temprana entre 30 y 90 días después de la siembra. Métodos: Se investigaron las características anatómicas de cuarenta hojas utilizando la técnica de seccionamiento de pelado y manos libres. Resultados: Algunas características anatómicas mostraron respuesta a estrés por sequía. Saccharum "UT12" demostró una mayor sensibilidad hacia las características anatómicas que Saccharum 'UT13'. Un total de 23 y 15 de las 40 características anatómicas mostraron significancia en Saccharum 'UT12' y Saccharum 'UT13', respectivamente. Algunas características anatómicas como la pared celular y el grosor de la cutícula, el tamaño del haz vascular, el tamaño y la densidad de los estomas se pueden utilizar como marcadores importantes para evaluar el estrés por sequía en la hoja de caña de azúcar. Conclusiones: Este es el primer reporte que describe la anatomía comparada de la hoja de la caña de azúcar Saccharum 'UT12' y Saccharum 'UT13' en Tailandia bajo estrés por sequía. Los resultados proporcionarán información importante para mejorar los mecanismos de adaptación de cultivares tolerantes de caña de azúcar bajo situaciones iniciales de estrés por sequía.