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DNA extraction is a fundamental technique in molecular biology. For Chimonanthus praecox— a winter-flowering tree species — extensive and rapid DNA extraction is necessary to support genetic analyses. Currently, DNA extraction in C. praecox primarily relies on the traditional cetyltrimethylammonium bromide (CTAB) method, which is time-consuming and labor-intensive, hindering large-scale DNA extraction work. In this study, the different steps in the CTAB method are compared and evaluated to optimize the C. praecox leaf DNA extraction process. The water bath duration significantly impacts DNA extraction efficiency; the longer the water bath, the higher the DNA concentration. However, a 10-min water bath is sufficient to yield DNA of ideal concentration and purity. Additionally, a single extraction step is appropriate, with a 10-min precipitation at − 20 °C yielding high-quality DNA. Additionally, the pre-treatment step was modified by using a frozen pipette tip to crush samples directly in a centrifuge tube, reducing operational complexity and minimizing liquid nitrogen and sample consumption. Only 25 mg of sample is required, and high-quality DNA from C. praecox leaves can be extracted within 1 h. The amounts of required sample and liquid nitrogen were reduced by 75% and 90%, respectively. Moreover, the time required for the simplified extraction step was reduced by 77.14%. The applicability of the simplified scheme was evaluated using different C. praecox tissues, Calycanthaceae family members, and species of other families. The simplified scheme extracted DNA from the tepals and leaves of C. praecox with higher concentration purity. However, this protocol was biased toward the Chimonanthus family, Nicotiana tabacum , Populus tomentosa , and Lilium brownii . The proposed method enables the rapid and efficient extraction of high-quality DNA from 25 mg of plant leaves and is suitable for multiple species. This method reduces sample and liquid nitrogen consumption, lowering costs while significantly shortening the procedure time and enhancing extraction efficiency. This method is highly suitable for applications involving the extraction of large amounts of low-concentration DNA across various plant species.

Background Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce. Results The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.–vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p  < 0 . 005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p  < 0 . 005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods. Conclusion Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Extraction of high-quality RNA from oil palm tissues is challenging due to the presence of polysaccharides, polyphenols and other complexes that co-precipitate with RNA. Therefore, isolation of high-quality RNA from oil palm is challenging due to the presence of varying amounts of these constituents in diverse tissues. This communication describes a modified RNA extraction protocol based on the cetyltrimethylammonium bromide (CTAB) method which is useful for extracting high-quality RNA from different oil palm tissues. Total RNA isolation using a modified CTAB protocol was compared with two different methods, a conventional TRIzol method and the method for RNA isolation from palms (MRIP). Both methods were useful for isolating RNA from leaf tissues; however, they were not effective in isolating RNA from other tissues. The current protocol based on a modified CTAB method was efficient in isolating high-quality total RNA from oil palm fruit tissues, including mesocarp and endosperm, stem, root and flower tissues. This modified CTAB-based protocol gave approximately 20–30 μg of total RNA from 150 mg of tissue within 5–6 h.

The fungal genus Fusarium contains many toxigenic pathogens of maize with associated yield losses, reduction of grain quality, and accumulation of mycotoxins in harvested grains. To determine zearalenone (ZEN) concentration and identify the various Fusarium species in commercial maize grains, a survey of 75 maize samples, collected from 11 market centers in the five regions in northern Ghana was identified based on morphological characteristics, sequence analysis of the internal transcribed spacer region, and polymerase chain reaction using species-specific primers. ZEN levels were determined using HPLC. ZEN contamination was recorded in 33.3% of the maize samples, with concentrations ranging from 0.61 to 3.05 µg/kg. Based on VERT1/2 and TEF 1-α sequencing, F. verticillioides was the most prevalent species in the studied samples: 40.35% from the Upper East Region, 28.07% from the North East Region, 19.30% from the Upper West Region, 10.53% from the Savannah Region, and 1.75% for the Northern Region. Other fungal species found were F. equiseti and F. solani . A higher number of the Fusarium isolates were found in white maize (609 isolates from 27 samples) compared to yellow maize (225 isolates from 23 samples).

Premise Efficient protocols for extracting high‐molecular‐weight (HMW) DNA from ferns facilitate the long‐read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)‐based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time. Methods and Results We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8). Conclusions This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.

BackgroundThere is a growing interest in transcriptomics studies parallel to the advancement of transcriptome databases and bioinformatics, which provided the opportunity to study responses to growths, stimuli and stresses. There is an increase in demand for excellent RNA extraction techniques. General RNA extraction protocols can be used in RNA extraction, but the quality and quantity vary in different types of tissues from different organisms. Hence, a specific RNA extraction method for each organism’s tissue type is required to obtain the desired RNA quality and quantity.ResultsThe improved CTAB RNA extraction method is superior to the PCI method and MRIP method for thick waxy leaves that were applied for mature sago palm (Metroxylon sagu Rottb.) leaf tissue and produce total RNA extract with good purity (OD 260/280 ≥ 1.8, OD 260/230 ≥ 2.0) and integrity (RIN ~ 7). RNA sequencing was conducted with the extracted samples and showed good assembly results (Q20 ≥ 97, Q30 ≥ 91%, assembly mean length ≥ 700 bp).ConclusionThe improved CTAB RNA extraction method enables rapid, cost-effective, and relatively simple RNA extraction from waxy, fibrous and high-in-polyphenol sago palm (M. sagu Rottb.) leaf tissue with next-generation RNA sequencing recommended quality.

The extraction of high-quality DNA is essential for molecular analyses in grapevine, yet differences among commonly used protocols remain underexplored. This study compared two cetyltrimethylammonium bromide (CTAB)-based methods, with and without polyvinylpyrrolidone (PVP), and three commercial kits (peqGOLD Plant DNA Mini Kit, Qiagen DNeasy Plant Mini Kit, and SPINeasy DNA Kit for Plant MP) using grapevine leaves and other tissues and further validated the CTAB protocol across 34 cultivars. DNA yield, purity, and integrity were assessed spectrophotometrically and by electrophoresis, while PCR suitability was confirmed for all methods. CTAB provided the highest yields and purity at low cost, with densitometry showing approximately 70–85% high-molecular-weight DNA (>20 kb). The Qiagen kit yielded reproducible results with moderate integrity (about 40–60% HMW fraction), making it suitable for high-throughput applications. The MP kit produced high concentrations but severe fragmentation (<10% HMW fraction) due to bead-beating, while the VWR kit performed worst in yield and purity. The addition of PVP improved DNA purity in polyphenol-rich tissues but reduced yield. All protocols generated DNA sufficient for PCR amplification. Overall, CTAB was robust and cost-effective across cultivars and tissues, Qiagen offered speed and reproducibility, and MP provided high concentration at the expense of integrity.

Apple (Malus pumila L) of the family Rosaceae, most cultivated fruit in temperate regions of the world and is used fresh or processed. The apple production is affected by several pathogens including fungi.  The present study was designed to identify disease-causing agents that reduce fruit production in the district Qilla Abdullah of Balochistan, Pakistan, which is the main apple production area of the province. Three varieties of apple: ‘Tur-Kulu’ (‘Red Delicious’), ‘Shin-Kulu’ (‘Golden Delicious’), and ‘Kaja’ were selected. Infected leaf samples were collected from eight different sites of tehsil Gulistan, district Qilla Abdullah. The cultures of fungal micro-flora were grown on two media, potato dextrose agar (PDA), and malt extract agar (MEA) followed by incubation for one week. The resulting colonies were observed under a microscope and identified based on morphological characters. Predominant fungal species was identified through ITS marker and PCR amplification. The isolated pathogens belonged to Zygomycota and Ascomycota divisions. The pathogens found were Aspergillus niger, A. oryzae, A. terrus, Colletotrichum gloeosporioides, Fusarium oxysporum, Mucor spp., Penicillium expansum, and one species of Absidia as well as Rhizopus. Colletotrichum gloeosporioides were predominantly found in all varieties. Morphological and phylogenetic analysis confirmed the identity of Colletotrichum gloeosporioides. As a result of this study, the predominant pathogen species Colletotrichum gloeosporioides is one of the causes of leaf infection in apple varieties.

Soy is considered an allergen under Regulation No. 1169/2011 of the European Parliament and of the Council, which mandates the labelling of soy allergen on food packaging. This study is focused on detecting soy in food products using two kinds of methods. The first method, enzyme-linked immunosorbent assay (ELISA), was performed using two commercial kits (Veratox for Soy Allergen; Neogen, USA). The second method, polymerase chain reaction (PCR), was carried out with two different deoxyribonucleic acid (DNA)-isolating kits and DNA isolation via the cetyltrimethylammonium bromide (CTAB) method. A total of 57 samples of food were tested, including 45 samples of animal origin and 12 samples of plant origin. The results were compared with information on the packaging. From the group of samples that contained soy according to the packaging (12 pieces), 9 samples were found to be positive by ELISA method, 10 samples by the CTAB method and 11 samples by GeneSpin. On the other hand, from the group of samples that should not contain soy according to the packaging (30 pieces), the presence of soy in 2 samples was detected by ELISA. No significant difference was found between the examined methods. Results show that the situation on the market is satisfactory and that only products declared as containing traces of soy appear to be problematic.

Glutathione is a tripeptide compound with many important physiological functions. A new, two-step reaction system has been developed to efficiently synthesize glutathione. In the first step, glutamate and cysteine are condensed to glutamyl-cysteine by endogenous yeast enzymes inside the yeast cell, while consuming ATP. In the second step, the yeast cell membrane is lysed by the permeabilizing agent CTAB (cetyltrimethylammonium bromide) to release the glutamyl-cysteine, upon which added glutathione synthetase converts the glutamyl-cysteine and added glycine into glutathione. The ATP needed for this conversion is supplied by the permeabilized yeast cells of glycolytic pathway. This method provided sufficient ATP, and reduced the feedback inhibition of glutathione for the first-step enzymatic reaction, thereby improving the catalytic efficiency of the enzyme reaction. In addition, the formation of suitable oxidative stress environment in the reaction system can further promote glutathione synthesis. By HPLC analysis of the glutathione, it was found that 2.1 g/L reduced glutathione is produced and 17.5 g/L oxidized glutathione. Therefore, the new reaction system not only increases the total glutathione, but also facilitates the subsequent separation and purification due to the larger proportion of oxidized glutathione in the reaction system.