An efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species. A quick transformation system is also crucial for the functional analysis of genes along with the study of proteins and their interactions in vivo. Presently, however, quick and effective transformation systems are still lacking for many plant species including pineapple. This has limited the full exploration of the genetic repository of pineapple as well as the study of its genes, protein localization and protein interactions. To address the above limitations, we have developed an efficient system for protoplast isolation and subcellular localization of desired proteins using pineapple plants derived from tissue culture. A cocktail of 1.5% (W/V) Cellulase R-10 and 0.5% (W/V) Macerozyme R-10 resulted in 51% viable protoplasts with 3 h digestion. Compared to previously reported protocols, our protoplast isolation method is markedly faster (saving 4.5 h), requires only a small quantity of tissue sample (1 g of leaves) and has high yield (6.5 × 105). The quality of the isolated protoplasts was verified using organelle localization in protoplasts with different organelle markers. Additionally, colocalization analysis of two pineapple Mg2+ transporter genes in pineapple protoplasts was consistent with the results in a tobacco transient expression system, confirming that the protoplast isolation method can be used to study subcellular localization. Further findings showed that the system is also suitable for protein–protein interaction studies. Based on our findings, the presently described method is an efficient and effective strategy for pineapple protoplast isolation and transformation; it is convenient and time saving and provides a greater platform for transformation studies.

Ralstonia solanacearum is a phytopathogen causing bacterial wilt diseases of tomato and affecting its productivity, which leads to prominent economic losses annually. As an alternative to conventional pesticides, green synthesized nanoparticles are believed to possess strong antibacterial activities besides being cheap and ecofriendly. Here, we present the synthesis of silver nanoparticles (Sn-AgNPs) from medicinally important aqueous plant extracts of Salvia nubicola. Characterization of biologically synthesized nanoparticles was performed through UV-vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy (TEM), and thermogravimetric analysis. The antibacterial activity of the biosynthesized silver nanoparticles was tested against the phytopathogen R. solanacearum through in vitro experiments. Preliminary phytochemical analysis of the plant extracts revealed the presence of substantial amounts of flavonoids (57.08 mg GAE/g), phenolics (42.30 mg GAE/g), tannins, and terpenoids. The HPLC phenolic profile indicated the presence of 25 possible bioactive compounds. Results regarding green synthesized silver nanoparticles revealed the conformation of different functional groups through FTIR analysis, which could be responsible for the bioreduction and capping of Ag ions into silver NPs. TEM results revealed the spherical, crystalline shape of nanoparticles with the size in the range of 23-63 nm, which validates SEM results. Different concentrations of Sn-AgNPs (T1 (500 μg/mL) to T7 (78.1 μg/mL)) with a combination of plant extracts (PE-Sn-AgNPs) and plant extracts alone exhibited an efficient inhibition of R. solanacearum. These findings could be used as an effective alternative preparation against the bacterial wilt of tomato.

Stress is one of the important factors that directly or indirectly affects the plant architecture, biochemical pathways, and growth and development. Melatonin (MEL) is an important stress hormone; however, the exogenous addition of melatonin to culture media stimulates the defense mechanism and releases higher quantities of secondary metabolites. In this study, submerged adventitious root cultures (SARCs) of diabetically important Stevia rebaudiana were exposed to variable concentrations (0.5-5.0 mg/L) of MEL in combination with 0.5 mg/L naphthalene acetic acid (NAA) to investigate the biomass accumulation during growth kinetics with 07 days intervals for a period of 56 days. The effects of exogenous MEL on the biosynthesis of stevioside (Stev.), total phenolics content (TPC), total flavonoids content (TFC), total phenolics production (TPP), total flavonoids production (TFP), total polyphenolics content (TPPC), fresh and dry weight (FW & DW), and antioxidant potential were also studied. Most of the SARCs displayed lag, exponential, stationary, and decline phases with variable biomass accumulation. The maximum fresh (236.54 g/L) and dry biomass (28.64 g/L) was observed in SARCs exposed to 3.0 mg/L MEL and 0.5 mg/L NAA. The same combination of MEL and NAA also enhanced the accumulation of TPC (18.96 mg/g-DW), TFC (6.33 mg/g-DW), TPP (271.51 mg/L), TFP (90.64 mg/L), and TPPC (25.29 mg/g-DW). Similarly, the highest stevioside biosynthesis (91.45 mg/g-DW) and antioxidant potential (86.15%) were observed in SARCs exposed to 3.0 mg/L MEL and NAA. Moreover, a strong correlation was observed between the biomass and the contents of phenolics, flavonoids, antioxidants, and stevioside. These results suggest that MEL is one of multidimensional stress hormones that modulate the biosynthetic pathways to release higher quantities of metabolites of interest for various industrial applications.

Drought stress has been shown to have harmful effects on crop productivity worldwide, including in Pakistan, due to rapid climate change scenarios. Extensive work has been reported on the influential role of melatonin (MEL) in either foliar or seed-primed applications; however, its role in root application is seldom reported. We investigated plant biochemical responses, including anti-oxidants, plant pigments, leaf water characteristics, and maize crop production, with MEL treatment under mild and severe drought stress. Maize Cvar. Jalal was subjected to drought stress (60% and 80% of full irrigation) at the four-leaf stage, and MEL was applied as pretreatment with irrigation water at different doses (0, 100, and 200µM). The findings of the study revealed that the Chl a, b, and a + b contents and the carotenoid content significantly increased with MEL application during severe and mild drought stress. After applying 200 µM MEL, leaf water attributes, comprising relative water content (RWC), leaf water content (LWC), and relative saturation deficit (RSD), increased by 1.9%, 100%, and 71.2%, respectively, during mild drought and 17%, 133%, and 32% under severe drought. The anti-oxidant activities of POD, CAT, and APX were remarkably enhanced with MEL during drought stress. Our results showed that root application of 200 µM melatonin boosted seed yield and water productivity by 31% and 38%, and plant biomass increased by 32% and 29% under mild and severe drought stressors compared to plants with no MEL, leading to increased drought tolerance.

Understanding the genetic diversity of domestic chicken breeds under the impact of socio–cultural and ecological dynamics is vital for the conservation of natural resources. Mae Hong Son chicken is a local breed of North Thai domestic chicken widely distributed in Mae Hong Son Province, Thailand; however, its genetic characterization, origin, and diversity remain poorly understood. Here, we studied the socio–cultural, environmental, and genetic aspects of the Mae Hong Son chicken breed and investigated its diversity and allelic gene pool. We genotyped 28 microsatellite markers and analyzed mitochondrial D-loop sequencing data to evaluate genetic diversity and assessed spatial habitat suitability using maximum entropy modeling. Sequence diversity analysis revealed a total of 188 genotyped alleles, with overall nucleotide diversity of 0.014 ± 0.007, indicating that the Mae Hong Son chicken population is genetically highly diverse, with 35 (M1–M35) haplotypes clustered into haplogroups A, B, E, and F, mostly in the North ecotype. Allelic gene pool patterns showed a unique DNA fingerprint of the Mae Hong Son chicken, as compared to other breeds and red junglefowl. A genetic introgression of some parts of the gene pool of red junglefowl and other indigenous breeds was identified in the Mae Hong Son chicken, supporting the hypothesis of the origin of the Mae Hong Son chicken. During domestication in the past 200–300 years after the crossing of indigenous chickens and red junglefowl, the Mae Hong Son chicken has adapted to the highland environment and played a significant socio–cultural role in the Northern Thai community. The unique genetic fingerprint of the Mae Hong Son chicken, retaining a high level of genetic variability that includes a dynamic demographic and domestication history, as well as a range of ecological factors, might reshape the adaptation of this breed under selective pressure.