Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant.

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.

Alterations in Herbage Yield, Antioxidant Activities, Phytochemical Contents, and Bioactive Compounds of Sabah Snake Grass (Clinacanthus Nutans L.) with Regards to Harvesting Age and Harvesting Frequency.

Sabah snake grass or Clinacanthus nutans has drawn public interest having significant economic benefits attributable to the presence of phytochemicals and several interesting bioactive constituents that may differ according to harvesting age and harvesting frequency. The current study was aimed to evaluate the effect of harvesting age and harvesting frequency towards herbal yield, antioxidant activities, phytochemicals synthesis, and bioactive compounds of C. nutans. A factorial randomized completely block design with five replications was used to illustrate the relationship between herbal yield, DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ferric reducing antioxidant power (FRAP) assays, total phenolic and flavonoid content affected by harvesting age (week 8, 12, and 16 after transplanting), and harvesting frequency (harvest 1, 2, and 3). The bioactive compounds by HPLC were also determined to describe the interaction effect between both harvesting age and harvesting frequency. The yield, antioxidant activities, and phytochemical contents were gradually increased as the plant grew, with the highest recorded during week 16. However, the synthesis and activities of phytochemicals were reduced in subsequent harvests despite the increment of the herbal yield. All bioactive compounds were found to be influenced insignificantly and significantly by harvesting age and harvesting frequency, respectively, specifically to shaftoside, iso-orientin, and orientin. Among all constituents, shaftoside was the main compound at various harvesting ages and harvesting frequencies. These results indicated that harvesting at week 16 with 1st harvest frequency might enhance the yield while sustaining the high synthesis of polyphenols and antioxidant activities of C. nutans.

Unravelling the dynamics of genotype and environment interactions on chilli (Capsicum annuum L.) yield-related attributes in soilless planting systems.

Evaluation of genotypes to identify high-yielding and stable varieties is crucial for chilli production sustainability and food security. These analyses are essential, particularly when the breeding program aims to select lines with great adaptability and stability. Thirty chilli genotypes were evaluated for yield stability under four soilless planting systems viz; fertigation, HydroStock (commercial hydrogel), BioHydrogel (biodegradable hydrogel), and hydroponic to study the influence of genotype by environment interaction. The research used a split-plot randomized complete block design (RCBD) with two cropping cycles and five replications. The GGE biplot analysis was employed to assess the mean versus stability perspective in explaining the variation in genotypic and genotype-by-environment effects on the yield-related attributes for yield per plant, fruit number, fruit length, and width. Stability analysis denoted genotypes G26 and G30 as the most stable for yield per plant, while G16, G22, and G30 were stable for the number of fruits per plant. Among the four planting systems evaluated, HydroStock and BioHydrogel outperformed the others in yield per plant, demonstrating the highest level of informativeness or discrimination. These findings offer critical insights for future crop breeding programs and the optimization of agricultural practices.

Plant Growth Regulator- and Elicitor-Mediated Enhancement of Biomass and Andrographolide Production of Shoot Tip-Culture-Derived Plantlets of Andrographis paniculata (Burm.f.) Wall. (Hempedu Bumi).

Andrographis paniculata (Burm.f.) Wall. (Acanthaceae) is revered for its medicinal properties. In vitro culture of medicinal plants has assisted in improving both the quantity and quality of their yield. The current study investigated the effects of different surface sterilization treatments, plant growth regulators (PGRs), and elicitors on culture establishment and axillary shoot multiplication of A. paniculata. Subsequently, the production of andrographolide in the in vitro plantlets was evaluated using high-performance liquid chromatography (HPLC) analysis. The shoot-tip explant was successfully sterilized using 60% commercial bleach for 5 min of immersion with a 90% survival rate and 96.67% aseptic culture. The optimal PGR for shoot growth was 6-benzylaminopurine (BAP) at 17.76 µM, supplemented into Murashige and Skoog (MS) media, producing 23.57 ± 0.48 leaves, 7.33 ± 0.10 shoots, and a 3.06 ± 0.02 cm length of shoots. Subsequently, MS medium supplemented with 5 mg/L chitosan produced 26.07 ± 0.14 leaves, 8.33 ± 0.07 shoots, and a 3.63 ± 0.02 cm length of shoots. The highest andrographolide content was obtained using the plantlets harvested from 5 mg/L chitosan with 2463.03 ± 0.398 µg/mL compared to the control (without elicitation) with 256.73 ± 0.341 µg/mL (859.39% increase). The results imply that the protocol for the shoot-tip culture of A. paniculata was developed, and that elicitation enhanced the herbage yield and the production of andrographolide.

Determination of experimental domain factors of polyphenols, phenolic acids and flavonoids of lemon (Citrus limon) peel using two-level factorial design.

This study aimed to evaluate the significant extraction factors in achieving higher recovery yield of total polyphenols, phenolic acids and flavonoids content from Citrus limon peel using two-level factorial design. The effect of five independent factors including drying temperature (40-60 °C), methanol concentration (20-60%), extraction temperature (28-60 °C), extraction time (30-60 min) and storage duration (0-14 days) were evaluated. Among all the examined factors, results showed that drying temperature, storage duration and extraction temperature were the most significant and contributing factors affecting the total polyphenols, phenolic acids and flavonoids content of lemon peel at P < 0.05. On the contrary, methanol concentration and extraction time exhibited the least significant and contribution at P greater than 0.05. In conclusion, the experimental domain factors were successfully obtained from this experiment, Therefore, further study on optimization of the obtained factors will be conducted in the future study using response surface methodology.

The Efficient and Easy Micropropagation Protocol of Phyllanthus niruri.

Phyllanthus niruri (P. niruri) or Dukung Anak is a herbal plant in the Phyllanthaceae family that has been used traditionally to treat various ailments such as diabetes, jaundice, flu and cough. P. niruri contains numerous medicinal benefits such as anti-tumor and anti-carcinogenic properties and a remedy for hepatitis B viral infection. Due to its beneficial properties, P. niruri is overharvested and wild plants become scarce. This study was conducted to develop an appropriate in vitro culture protocol for the mass production of P. niruri. An aseptic culture of P. niruri was established followed by multiplication of explants using different types of basal medium and its strength and plant growth regulators manipulation. This study also established the induction of in vitro rooting utilizing various types and concentrations of auxin. Treatment of Clorox® with 30% concentration showed the lowest percentage (%) of contamination, 4.44% in P. niruri culture. Nodal segments of P. niruri were successfully induced in full-strength of Murashige and Skoog (MS) basal media with 2.33 number of shoots, 3.11 cm length of shoot and 27.91 number of leaves. In addition, explants in full-strength MS media without any additional cytokinin were recorded as the optimum results for all parameters including the number of shoots (5.0 shoots), the length of shoots (3.68 cm) and the number of leaves (27.33 leaves). Treatment of 2.5 µM indole-3-butyric acid (IBA) showed the highest number of roots (17.92 roots) and root length (1.29 cm). Rooted explants were transferred for acclimatization, and the plantlet showed over 80% of survival rate. In conclusion, plantlets of P. niruri were successfully induced and multiplied via in vitro culture, which could be a step closer to its commercialization.

Micropropagation of Ginger (Zingiber officinale Roscoe) 'Bentong' and Evaluation of Its Secondary Metabolites and Antioxidant Activities Compared with the Conventionally Propagated Plant.

'Bentong' ginger is the most popular variety of Zingiber officinale in Malaysia. It is vegetatively propagated and requires a high proportion of rhizomes as starting planting materials. Besides, ginger vegetative propagation using its rhizomes is accompanied by several types of soil-borne diseases. Plant tissue culture techniques have been applied in many plant species to produce their disease-free planting materials. As 'Bentong' ginger is less known for its micropropagation, this study was conducted to investigate the effects of Clorox (5.25% sodium hypochlorite (NaOCl)) on explant surface sterilization, effects of plant growth regulators, and basal media on shoots' multiplication and rooting. The secondary metabolites and antioxidant activities of the micropropagated plants were evaluated in comparison with conventionally propagated plants. Rhizome sprouted buds were effectively sterilized in 70% Clorox for 30 min by obtaining 75% contamination-free explants. Murashige and Skoog (MS) supplemented with 10 µM of zeatin was the suitable medium for shoot multiplication, which resulted in the highest number of shoots per explant (4.28). MS medium supplemented with 7.5 µM 1-naphthaleneacetic acid (NAA) resulted in the highest number of roots per plantlet. The in vitro-rooted plantlets were successfully acclimatized with a 95% survival rate in the ex vitro conditions. The phytochemical analysis showed that total phenolic acid and total flavonoid content and antioxidant activities of the micropropagated plants were not significantly different from the conventionally propagated plants of 'Bentong' ginger. In conclusion, the present study's outcome can be adopted for large-scale propagation of disease-free planting materials of 'Bentong' ginger.

The effects of genotypes and media composition on callogenesis, regeneration and cell suspension culture of chamomile (Matricaria chamomilla L.).

BACKGROUND: Chamomile is an important herb being used widely for medicinal purposes. Its multitherapeutic, cosmetic, and nutritional values have been established through years of traditional and scientific use and research. Increased use of medicinal plants necessitates rational use as well as sustainable production of such genetic resources. Plant in vitro micro-propagation poses unique opportunities for sustainable production of medicinal herbs, their regrowth and conservation. The present study aimed to investigate the effects of different explants, plant growth regulators (PGRs) combinations and media type on callogenesis, in vitro regeneration and cell suspension of six chamomile genotypes to enhance its sustainable production. METHODS: The shoot, lateral sprout, and leaf derived explants of six chamomile genotypes including Isfahan, Shiraz, Kazeron, Goral, Sharokashari and Presso were used for direct and indirect regeneration. For indirect regeneration various doses of NAA and kinetin were used to induce calli which were cultured on MS media containing PGRs for direct and indirect regeneration. Later, cell suspension was established and morphological characterization of CrO3 stained cells was carried out using microscopy. RESULTS AND DISCUSSION: Our findings revealed that the highest callus percentage and callus volume were observed from lateral sprouts and shoots of genotype Isfahan on MS medium containing 1 mg/L NAA and 1 mg/L kinetin. The in vitro regeneration was found to be genotype dependent while 77% and 77.5% was the highest percentage for indirect and direct regeneration, respectively. Additionally, the maximum shoot number (two shoots/explant) and shoot length (2.22 cm) were also observed in Isfahan genotype. Cell suspension culture showed the highest fresh weight (18.59 g) and dry weight (1.707 g) with 0.75 g inoculum of the callus derived from lateral sprouts cultured on MS medium. Microscopy of CrO3 stained cells was carried on each 3rd day for 27 days that revealed larger and spongier cells in the early days as compared to final days when the cell number was greater but cell size was smaller. CONCLUSION: The callogenesis, organogenesis, and cell suspension culture of chamomile may be genotype dependent. Hence, optimization of media ingredients and culture conditions is of utmost importance for devising tissue culture based conservation strategy of any chamomile genotype and secondary metabolite production.

In Vitro Responses of Plant Growth Factors on Growth, Yield, Phenolics Content and Antioxidant Activities of Clinacanthus nutans (Sabah Snake Grass).

Clinacanthus nutans, commonly known as Sabah snake grass, is one of the more important medicinal plants in Malaysia's herbal industry. C. nutans has gained the attention of medical practitioners due to its wide range of bioactive compounds responsible for various biological activities, such as anti-cancer, anti-venom and anti-viral activities. Due to its high pharmacological properties, the species has been overexploited to meet the demands of the pharmaceutical industry. The present study was conducted to establish a suitable in vitro culture procedure for the mass propagation of C. nutans. Murashige and Skoog (MS) basal medium, supplemented with different types of cytokinins, auxins, basal medium strength and sucrose concentrations, were tested. Based on the results, a full-strength MS basal medium supplemented with 12 µM 6-benzylaminopurine (BAP) and 30 g/L sucrose was recorded as the best outcome for all the parameters measured including the regeneration percentage, number of shoots, length of shoots, number of leaves and fresh weight of leaves. In the analysis of the phenolics content and antioxidant activities, tissue-cultured leaf extracts assayed at 100 °C exhibited the highest phenolic content and antioxidant activities. The propagation of C. nutans via a plant tissue culture technique was recorded to be able to produce high phenolic contents as well as exhibit high antioxidant activities.

Draft genome sequencing data of a pathogenic Pantoea stewartii subspecies stewartii strain SQT1 causing bronzing disease of jackfruit in Malaysia.

A Gram-negative bacterium, Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii) has been recognized as the causative agent for jackfruit bronzing disease in Malaysia. Here, we report the whole genome sequencing dataset of P. stewartii subsp. stewartii strain SQT1 isolated from local infected jackfruit. The paired-end libraries with an insert size of 350 bp was subjected to the Illumina Hiseq 4000, generating a genome size of 4,783,993 bp with a G+C content of 53.7%. A total protein of 4,671 was identified including virulence factors, resistance factors and secretion systems. Pantoea stewartii subsp. stewartii strain DC283 (NCBI accession no. CP017581.1) was used as a reference genome, where the query hit 72% coverage and average sequencing depth of 68. In total, 28,717 nucleotide polymorphisms, 520 small insertion/deletions and 142 structure variants were identified. The complete genome was deposited at the European Nucleotide Archive under the sample accession number ERP119356 and study accession number PRJEB36196.

Genetic diversity of Pantoea stewartii subspecies stewartii causing jackfruit-bronzing disease in Malaysia.

Jackfruit-bronzing is caused by bacteria Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii), showing symptoms of yellowish-orange to reddish discolouration and rusty specks on pulps and rags of jackfruit. Twenty-eight pure bacterial strains were collected from four different jackfruit outbreak collection areas in Peninsular Malaysia (Jenderam, Maran, Muadzam Shah and Ipoh). Positive P. stewartii subsp. stewartii verification obtained in the study was based on the phenotypic, hypersensitivity, pathogenicity and molecular tests. Multilocus sequence analysis (MLSA) was performed using four housekeeping genes (gyrB, rpoB, atpD and infB) on all 28 bacterial strains. Single gyrB, rpoB, atpD and infB phylogenetic trees analyses revealed the bootstrap value of 99-100% between our bacterial strains with P. stewartii subsp. stewartii reference strains and P. stewartii subsp. indologenes reference strains. On the other hand, phylogenetic tree of the concatenated sequences of the four housekeeping genes revealed that our 28 bacterial strains were more closely related to P. stewartii subsp. stewartii (99% similarities) compared to its close relative P. stewartii subsp. indologenes, although sequence similarity between these two subspecies were up to 100%. All the strains collected from the four collection areas clustered together, pointing to no variation among the bacterial strains. This study improves our understanding and provided new insight on the genetic diversity of P. stewartii subsp. stewartii associated with jackfruit-bronzing in Malaysia.

A comprehensive review on the determination of enzymatic assay and nonenzymatic antioxidant activities.

This review article presents a comprehensive review pertaining to antioxidants and various assays that determined enzymatic and nonenzymatic antioxidants. Antioxidants have gained attention at the global scale on its prominent beneficial roles that can fight against many chronic infirmities, including cancer and cardiovascular diseases. Many studies have investigated different types of samples, such as medicinal plants, fruits, and vegetables, by using various antioxidant assays. Antioxidants can be grouped into enzymatic and nonenzymatic antioxidants. To date, most studies had looked into nonenzymatic antioxidants due to lack of references on enzymatic antioxidant assays. Therefore, this review article depicts on seven assays of enzymatic antioxidants (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, ascorbate oxidase, guaiacol peroxidase, and glutathione reductase) and fifteen activities of nonenzymatic antioxidants (total polyphenol, total phenolic acids, total flavonoids, total ascorbic acid, anthocyanin content, DPPH scavenging activity, FRAP assay, hydrogen peroxide scavenging activity, nitric oxide scavenging activity, superoxide radical scavenging activity, hydroxyl radical scavenging activity, phosphomolybdate assay, reducing power, metal ion chelating activity, and ß-carotene), which are described in detail to ease further investigations on antioxidants in future.

Factors Affecting Cell Biomass and Flavonoid Production of Ficus deltoidea var. kunstleri in Cell Suspension Culture System.

A study was conducted to establish in vitro culture conditions for maximum production of biomass and flavonoid content for Ficus deltoidea var. kunstleri, locally named as Mas Cotek, known to have a wide variety of potential beneficial attributes for human health. Size of initial inoculum, cell aggregate and initial pH value have been suggested to influent content of biomass and flavonoid for cell suspension culture in several plant species. In the present study, leaf explants were cultured by cell suspension culture procedures in MSB5 basal medium supplemented with predetermined supplements of 30 g/L sucrose, 2.75 g/L gelrite, 2 mg/L picloram and 1 mg/L kinetin with continuous agitation of 120 rpm in a standard laboratory environment. Establishment of cell suspension culture was accomplished by culturing resulting callus in different initial fresh weight of cells (0.10, 0.25, 0.50, 1.0, and 2.0 g/25 mL of media) using similar basal medium. The results showed that the highest production of biomass (0.65 g/25 mL of media) was recorded from an initial inoculum size of 2.0 g/25 mL media, whereas the highest flavonoid (3.3 mg RE/g DW) was found in 0.5 g/25 mL of media. Cell suspension fractions classified according to their sizes (500-750 µm, 250-500 µm, and <250 µm). Large cell aggregate size (500-750 µm) cultured at pH 5.75 produced the highest cell biomass (0.28 g/25 mL media) and flavonoid content (3.3 mg RE/g DW). The study had established the optimum conditions for the production of total antioxidant and flavonoid content using DPPH and FRAP assays in cell suspension culture of F. deltoidea var. kunstleri.