Ultrasound-assisted extraction of bioactive compounds from goji berries: Optimization, bioactivity, and intestinal permeability assessment.

Lycium barbarum L. berries have a remarkable chemical composition and extensive biological activities, being a valuable component of health and nutraceutical practices. Nevertheless, a deep insight on the intestinal permeation of the pro-healthy bioactive compounds is urgently needed to predict the real effects on human body. This study attempted, for the first time, to optimize the Ultrasound-Assisted Extraction (UAE) of goji berries using a Response Surface Methodology approach and establish the intestinal permeation of the principal pro-healthy compounds. The optimal extraction conditions were a solid:liquid ratio of 8.75 % for 56.21 min, using an intensity of 59.05 W/m2. The optimal extract displayed a remarkable antioxidant capacity, with LC/DAD-ESI-MS analysis unveiled a diverse phytochemical profile, encompassing different compounds (e.g. glu-lycibarbarspermidine F, 2-glu-kukoamine, rutin, 3,5-dicaffeoylquinic acid). The intestinal co-culture model demonstrated that glu-lycibarbarspermidine F (isomer 2) (73.70 %), 3,5-dicaffeoylquinic acid (52.66 %), and isorhamnetin-3-O-rutinoside (49.31 %) traversed the intestinal cell layer, exerting beneficial health-promoting effects.

Correction: In-season internal and external training load quantification of an elite European soccer team.

[This corrects the article DOI: 10.1371/journal.pone.0209393.].

Insights into the Bioactive Composition, Antioxidant Properties and In Vitro Cell Effects of Disphyma crassifolium.

Disphyma crassifolium, commonly known as sea fingers, is a halophyte plant recently introduced in gourmet cuisine. The present study aims to extract the bioactive compounds of D. crassifolium using ultrasound-assisted extraction and employing green solvents (water and ethanol). The antioxidant/antiradical activities, scavenging capacity against reactive species, phenolic profile, and intestinal effects were evaluated. The highest total phenolic (53.13 mg of gallic acid equivalent (GAE)/g on dry weight (dw)) and flavonoid contents (18.98 mg of catechin equivalent (CE)/g dw) as well as antioxidant (149.69 µmol of ferrous sulphate equivalent (FSE)/g dw) and antiradical capacities (9.37 mg of ascorbic acid equivalent (AAE)/g dw) were achieved for the alcoholic extract. Moreover, the alcoholic extract exhibited an efficient uptake of HOCl (IC50 = 1.97 µg/mL) and ROO• (0.34 µmol of Trolox equivalent (TE)/mg dw). A total of 34 phenolic compounds were identified in the extracts, with flavonols (isorhamnetin-3-O-rutinoside, quercetin-3-O-galactoside, and myricetin), flavanols (catechin), and phenolic acids (gallic and ellagic acids) being the principal classes. The intestinal cell viability assays attested that the alcoholic extract presented the lowest IC50 values (289.82 and 35.77 µg/mL for HT29-MTX and Caco-2), showing probable anticancer activity. These results emphasize the potential of D. crassifolium as a nutraceutical ingredient.

Identifying the ideal weekly training load for in-game performance in an elite Brazilian soccer team.

Introduction: The purpose of this study was to investigate the ideal training load to be applied during periods of fixture congestion to ensure an adequate dose-response effect for performance maintenance. Methods: Match performance data and corresponding pre-match training load sessions (both N = 498 match performance cases and training-block session cases) were collected (with the catapult system, VECTOR7) from 36 male professional soccer players (23.5 ± 5.2 years; 178 ± 4 cm; 75.5 ± 6.0 kg) belonging to the Brazilian First Division team during the 2022 season. The following data were collected in match and training sessions: jump, acceleration, deceleration, and change of direction (COD); running distance producing metabolic power at different intensities (>20, >20-35, >35-45, >45-55, and >55 W kg-1), total distance (m), relative distance (m/min), running distance at different speeds (>20, >25, and >30 km/h), number of sprints (running >25 km/h), and maximum speed (km/h). Mixed linear model (MLM), decision tree regression (DTR), and cluster K means model (SPSS v.26) approach were performed to identify the most critical variables (and their respective load) in the training sessions that could explain the athlete's match performance. Results: MLM and DTR regression show that training load significantly affects game performance in a specific way. According to the present data, an interference phenomenon can occur when a high load of two different skills (running in a straight line vs COD, deceleration, and jumping) is applied in the same training block of the week. The cluster approach, followed by a chi-squared test, identified significant associations between training load and athlete match performance in a dose-dependent manner. Discussion: The high load values described here have a beneficial effect on match performance, despite the interference between stimuli discussed above. We present a positive training load from a congested season from the Brazilian First Division team. The study suggests that an interference effect occurs when high physical training loads are applied to different specific physical skills throughout the season.

Betulinic Acid for Glioblastoma Treatment: Reality, Challenges and Perspectives.

Betulinic acid is a naturally occurring compound that can be obtained through methanolic or ethanolic extraction from plant sources, as well as through chemical synthesis or microbial biotransformation. Betulinic acid has been investigated for its potential therapeutic properties, and exhibits anti-inflammatory, antiviral, antimalarial, and antioxidant activities. Notably, its ability to cross the blood-brain barrier addresses a significant challenge in treating neurological pathologies. This review aims to compile information about the impact of betulinic acid as an antitumor agent, particularly in the context of glioblastoma. Importantly, betulinic acid demonstrates selective antitumor activity against glioblastoma cells by inhibiting proliferation and inducing apoptosis, consistent with observations in other cancer types. Compelling evidence published highlights the acid's therapeutic action in suppressing the Akt/NFκB-p65 signaling cascade and enhancing the cytotoxic effects of the chemotherapeutic agent temozolomide. Interesting findings with betulinic acid also suggest a focus on researching the reduction of glioblastoma's invasiveness and aggressiveness profile. This involves modulation of extracellular matrix components, remodeling of the cytoskeleton, and secretion of proteolytic proteins. Drawing from a comprehensive review, we conclude that betulinic acid formulations as nanoparticles and/or ionic liquids are promising drug delivery approaches with the potential for translation into clinical applications for the treatment and management of glioblastoma.

Comparison of force variables and dynamic strength index between age groups in elite young Brazilian football goalkeepers.

Introduction: The application of muscle force is a determinant of football success as it is inherent to the motor control and sport. The aims of this study are: (1) to describe force variables Isometric Maximal Force (IMF), Concentric Peak Force (CPPF), and Dynamic Strength Index (DSI) in football goalkeepers from different age groups; (2) to compare these variables' behavior between those groups. Methods: The sample was formed by 19 youth players (15.97 ± 1.55 years old) from a first-division Brazilian football team. The CPPF and IMF variables were obtained through the Countermovement jump and isometric squat tests, respectively. For data collection, a force plate (Cefise, Brazil) was used with an acquisition frequency of 600 Hz and mono-axial. The DSI was calculated using the ratio between CPPF and IMF. For data analysis, the sample was separated into clusters by age. After the grouping, a descriptive analysis of the data and a comparison between the groups with p < 0.05. Results: The sample was grouped into three groups (GA, GB, and GC) and one of the individuals did not enter the group, totaling 18 individuals in the analyzed sample. The comparison between the ages of the groups showed a significant difference and small and moderate effect size (ES), validating the cluster strategy. The CPPF and IMF variables showed increased values according to chronological age. CPPF showed a significant difference between GA-GB, (ES = very large) GA-GC (ES = very large), and GB-GC (ES = moderate). The IMF variable had significant differences between GA-GB (ES = moderate) and GA-GC (ES = very large). However, DSI showed significant differences only between GA GB (ES = small) and GB-GC (ES = very large). Conclusions: The CPPF and IMF variables had constant increases and distinct values with an increase according to age, and this did not occur for DSI. The difference between CPPF and IMF compared to DSI bring to light the variability in dynamics and proportionality between muscular force in the concentric phase and maximal force in the isometric regime during the developmental process over chronological age in soccer goalkeepers.

Effects of applying a multivariate training program on physical fitness and tactical performance in a team sport taught during physical education classes.

Introduction: A multivariate training program could be a pedagogical choice to improve physical and tactical performance in a team sport taught during physical education classes at different levels of education. Thus, the aim of this study was to verify the effects of applying a multivariate training program on physical fitness and tactical performance during the teaching of a basketball didactic unit in basic and secondary education. Methods: Seventy-five students from a Portuguese school, with an average age of 15.02 ± 1.31 years, included forty-two students from basic school and thirty-three students from secondary school. The FITescola® test battery was used to assess physical fitness (i.e., sit-ups, push-ups, horizontal impulse, shuttle test, 40 m sprint, agility 4 × 10 m). The Game Performance Assessment Instrument (GPAI) was used to assess students' tactical performance for each player's game performance during a 20-minute 3 vs. 3 match. The GPAI variables were decision making index (DMI), skill execution index (SEI), support actions index (SI), and adaptability index (AI). During a basketball didactic unit teaching, the students were randomly divided into two groups, a control group that will not carry out the training program and an experimental group that will carry out a strength training program, high intensity explosive exercises and activities based on small-sided games (SSG) for 6 weeks. The two groups were evaluated in two moments: before the application of the training program and after the application of the training program regarding changes in physical fitness and tactical performance. The independent samples t-test (samples from two groups) and paired sample Test (for the same group) were applied for pre and post-assessment comparisons. Results: All indexes present significant differences between basic and secondary students in the pre- and post-assessment tests with small effects (t = -6.54 to -4.82, Δ = -27.57 to -0.16, p<0.05-p< 0.001, d = 0.78-1.05). Discussion: The results allow to conclude that in a school environment, a well-structured multivariate training program can effectively improve students' tactical skills, increasing their physical conditioning levels.

Stalling the Course of Neurodegenerative Diseases: Could Cyanobacteria Constitute a New Approach toward Therapy?

Neurodegenerative diseases (NDs) are characterized by progressive and irreversible neuronal loss, accompanied by a range of pathological pathways, including aberrant protein aggregation, altered energy metabolism, excitotoxicity, inflammation, and oxidative stress. Some of the most common NDs include Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), and Huntington's Disease (HD). There are currently no available cures; there are only therapeutic approaches that ameliorate the progression of symptoms, which makes the search for new drugs and therapeutic targets a constant battle. Cyanobacteria are ancient prokaryotic oxygenic phototrophs whose long evolutionary history has resulted in the production of a plethora of biomedically relevant compounds with anti-inflammatory, antioxidant, immunomodulatory, and neuroprotective properties, that can be valuable in this field. This review summarizes the major NDs and their pathophysiology, with a focus on the anti-neurodegenerative properties of cyanobacterial compounds and their main effects.

The neuroprotective effect of traditional Chinese medicinal plants-A critical review.

Neurodegenerative and neuropsychiatric diseases are increasingly affecting individuals' quality of life, thus increasing their cost to social and health systems. These diseases have overlapping mechanisms, such as oxidative stress, protein aggregation, neuroinflammation, neurotransmission impairment, mitochondrial dysfunction, and excitotoxicity. Currently, there is no cure for neurodegenerative diseases, and the available therapies have adverse effects and low efficacy. For neuropsychiatric disorders, such as depression, the current therapies are not adequate to one-third of the patients, the so-called treatment-resistant patients. So, searching for new treatments is fundamental. Medicinal plants appear as a strong alternative and complement towards new treatment protocols, as they have been used for health purposes for thousands of years. Thus, the main goal of this review is to revisit the neuroprotective potential of some of the most predominant medicinal plants (and one fungus) used in traditional Chinese medicine (TCM), focusing on their proven mechanisms of action and their chemical compositions, to give clues on how they can be useful against neurodegeneration progression.

Perspectives on Player Performance during FIFA World Cup Qatar 2022: A Brief Report.

Changing the date of the FIFA World Cup Qatar 2022 may represent a factor to consider for the expected performance of participating players. This was due to fixture congestion at the start of the season and expected weather conditions during the competition. Thus, the main purpose of this brief report was to critically analyze the potential impact of changing the competition date and weather conditions on players' performance. In addition, a brief description about the performance during the World Cup is also provided. For the research, the Web of Science, PubMed and SPORTDiscus databases were accessed using the primary keywords FIFA World Cup and World Soccer Cup associated with the secondary keywords match running performance, fixture congestion, fatigue and weather conditions. After applying inclusion and exclusion criteria, 52 articles were considered for analysis. The results seem to indicate that although changes were expected due to the modifications made (i.e., the competition date and scheduling congestion), the performance of the players seems not to have been affected in terms of the analyzed indicators. Furthermore, it seems possible to identify some patterns in the behavior of the teams that reached the most advanced stages of the competition.

Resultant equations for training load monitoring during a standard microcycle in sub-elite youth football: a principal components approach.

Applying data-reduction techniques to extract meaningful information from electronic performance and tracking systems (EPTS) has become a hot topic in football training load (TL) monitoring. The aim of this study was to reduce the dimensionality of the internal and external load measures, by a principal component approach, to describe and explain the resultant equations for TL monitoring during a standard in-season microcycle in sub-elite youth football. Additionally, it is intended to identify the most representative measure for each principal component. A principal component analysis (PCA) was conducted with a Monte Carlo parallel analysis and VariMax rotation to extract baseline characteristics, external TL, heart rate (HR)-based measures and perceived exertion. Training data were collected from sixty sub-elite young football players during a 6-week training period using 18 Hz global positioning system (GPS) with inertial sensors, 1 Hz short-range telemetry system, total quality recovery (TQR) and rating of perceived exertion (RPE). Five principal components accounted for 68.7% of the total variance explained in the training data. Resultant equations from PCA was subdivided into: (1) explosiveness, accelerations and impacts (27.4%); (2) high-speed running (16.2%); (3) HR-based measures (10.0%); (4) baseline characteristics (8.3%); and (5) average running velocity (6.7%). Considering the highest factor in each principal component, decelerations (PCA 1), sprint distance (PCA 2), average HR (PCA 3), chronological age (PCA 4) and maximal speed (PCA 5) are the conditional dimension to be considered in TL monitoring during a standard microcycle in sub-elite youth football players. Current research provides the first composite equations to extract the most representative components during a standard in-season microcycle in sub-elite youth football players. Futures research should expand the resultant equations within training days, by considering other well-being measures, technical-tactical skills and match-related contextual factors.

Concentric Isokinetic Strengthening Program's Impact on Knee Biomechanical Parameters, Physical Performance and Quality of Life in Overweight/Obese Women with Chronic Meniscal Lesions.

Meniscal injuries are one of the most common intra-articular knee injuries. Different treatments are presented depending on the symptoms and duration of meniscus tears, such as arthroscopic partial meniscectomy, physiotherapy, or even pharmacological treatment. The purpose was to investigate the effect of a concentric isokinetic knee muscle strengthening program on strength, joint range of motion, physical performance, quality of life and pain tolerance in overweight/obese women with chronic meniscal lesions. Twenty-four overweight/obese women were randomized into two groups. A control group (CG) performed a usual rehabilitation program plus isokinetic muscle strengthening (IMS) in the continuous passive motion mode and measured with an isokinetic dynamometer. An experimental group (EG) performed the same program in combination with IMS in the isokinetic active mode. The peak torque of the knee extensors (PTE) and flexors (PTF), sit-to-stand test, stair climb test, joint amplitude, heel-to-buttock distance, Thessaly test, KOOS questionnaire of pain and quality of life were measured pre- and post-protocol. After the intervention, the sit-stand number for both groups was significantly higher (p < 0.001) and the time to climb stairs was significantly reduced for the EG (p < 0.001). A significant improvement in joint range of motion, life quality (p < 0.001), relief in knee pain (p < 0.001) and in the post-program evaluation at the EG (p < 0.05) was noted. There was no significant difference in PTE and PTF between groups at 60°/s and 180°/s. The IMS in the active mode could be an effective therapeutic modality in managing middle-aged patients suffering from a degenerative meniscal tear.

A Deep Learning Neural Network to Classify Obesity Risk in Portuguese Adolescents Based on Physical Fitness Levels and Body Mass Index Percentiles: Insights for National Health Policies.

The increasing prevalence of overweight and obesity among adults is a risk factor for many chronic diseases and death. In addition, obesity among children and adolescents has reached unprecedented levels and studies show that obese children and adolescents are more likely to become obese adults. Therefore, both the prevention and treatment of obesity in adolescents are critical. This study aimed to develop an artificial intelligence (AI) neural network (NNET) model that identifies the risk of obesity in Portuguese adolescents based on their body mass index (BMI) percentiles and levels of physical fitness. Using datasets from the FITescola® project, 654 adolescents aged between 10-19 years old, male: 334 (51%), female: n = 320 (49%), age 13.8 ± 2 years old, were selected to participate in a cross-sectional observational study. Physical fitness variables, age, and sex were used to identify the risk of obesity. The NNET had good accuracy (75%) and performance validation through the Receiver Operating Characteristic using the Area Under the Curve (ROC AUC = 64%) in identifying the risk of obesity in Portuguese adolescents based on the BMI percentiles. Correlations of moderate effect size were perceived for aerobic fitness (AF), upper limbs strength (ULS), and sprint time (ST), showing that some physical fitness variables contributed to the obesity risk of the adolescents. Our NNET presented a good accuracy (75%) and was validated with the K-Folds Cross-Validation (K-Folds CV) with good accuracy (71%) and ROC AUC (66%). According to the NNET, there was an increased risk of obesity linked to low physical fitness in Portuguese teenagers.

Obesity Status and Physical Fitness Levels in Male and Female Portuguese Adolescents: A Two-Way Multivariate Analysis.

Obesity and decreasing fitness levels among the youth are growing concerns in Portugal, similar to other developed countries, with implications for health and psychomotor development. Understanding the influence of health determinants such as sex and age are crucial for developing effective public health strategies. This study aimed to analyze the association between sex and chronological age with obesity status and physical fitness in Portuguese adolescents. A total of 170 adolescents (85 males and 85 females) were evaluated for body mass index, abdominal adiposity, aerobic fitness, abdominal resistance, upper limb resistance, lower limb power, and maximal running speed in a 40 m sprint using the FITescola® physical fitness battery, a Portuguese government initiative. The general model, analyzed using Pillai's trace, showed a significant effect of age and sex on body mass index, abdominal circumference, aerobic fitness, abdominal resistance, upper limb resistance, lower limb power, and maximal running speed (V = 0.99, F (7) = 10,916.4, p < 0.001, partial η2, sex = 0.22; age = 0.43, sex and age interaction = 0.10). Boys had higher physical fitness levels than girls in most tests, but both sex groups had a significantly higher proportion of non-fit adolescents, with boys showing the highest number of participants classified as non-fit.

Physical Activity and Sedentary Behavior in Adults from Penafiel, Portugal: A Cross-Sectional Study.

The aim of this study was to compare the physical activity (PA) and sedentary behavior (SB) levels of young and middle-aged adults living in and around the municipality of Penafiel and to determine whether they meet PA recommendations. The researchers used the "International Physical Activity Questionnaire" (IPAQ) to measure moderate to vigorous PA and time spent on sedentary behavior (high vs. low). A prospective observational cross-sectional sample of 1105 adults aged 18-63 years, living in the municipality of Penafiel and its surroundings (45% women, 55% men), was used. The results indicated that more than half of the population was inactive (53.8%) and sedentary (54.0%). Men were more likely to be sedentary (59.2%) and inactive (55.6%) than women (inactive: 51.7%, high SB: 47.7%). Regarding daily PA and SB levels, women had higher levels of walks (3.8 ± 2.3; p = 0.034) and vigorous PA (2.2 ± 1.8 min; p = 0.005) per days/week, as well as vigorous PA per minutes/week (75.4 ± 82.1 min; p = 0.034). The time spent on vigorous PA per day was also higher in women (26.2 ± 22.8 min; p = 0.030). However, men had higher values in walking minutes per day (26.3 ± 17.1 min; p = 0.030), SB for weekdays (429.2 ± 141.2 min; p = 0.001), SB for weekends (324.7 ± 163.7 min; p = 0.033) and time spent on SB per minutes/week (2795.6 ± 882.0 min; p = 0.001). The results also showed that the older the adults, the lower the frequency and total time of vigorous PA per week. Young adults (18-28 years) had higher levels of vigorous PA (p = 0.005) than the other age groups (29-39; 40-50 and 51-63 years). Finally, the study found no significant correlation between individual level factors, such as number of children, marital status and monthly income, and PA or SB. Conversely, a significant and negative correlation between SB and levels of PA was found, indicating that the higher the level of PA practice, the lower the SB level. The authors suggest that promoting new PA habits and healthy lifestyles is an important future challenge for sustainability and improving the quality of life in public health.

The Association between Training Frequency, Symptoms of Overtraining and Injuries in Young Men Soccer Players.

Overtraining is a prevalent issue among young men soccer players, particularly those who are driven to enhance their skills. While an intense training volume and effort might contribute to athletic growth, it can also have negative implications, including injury. The current study aimed at examining the association between training frequency, symptoms of overtraining and injuries in young men soccer players. A path analysis approach was used to examine the causal relationships between variables. The sample consisted of 189 young men soccer players aged 13-17 years old (age = 14.81, SD = 1.37). Participants reported that they were training, on average, 5.77 days (SD = 1.53) per week. Athletes were competing at a regional (n = 100) or national (n = 89) level. Concerning injuries, participants indicated, on average, 2.03 (SD = 1.16) injuries since they started practicing soccer. The results displayed a significant association, as theoretically expected, namely: (i) training frequency was significantly associated with overtraining symptoms (ß = 0.15 [IC95% = 0.01, 0.29]); (ii) overtraining symptoms were significantly associated with the number of injuries (ß = 0.19 [IC95% = 0.02, 0.35]). An indirect effect between training frequency and injuries was also observed (ß = 0.15 [IC95% = 0.01, 0.29]). Thus, there is preliminary evidence that overtraining symptoms could play a mediating role. In conclusion, investigating the links between overtraining symptoms and injury in young men soccer players is critical, as it can assist in identifying overtraining warning signs, promote young players' health and safety, customize training regimens to individual needs, and contribute to a better understanding of sports-related injuries.

AGPs as molecular determinants of reproductive development.

BACKGROUND AND AIMS: Morphogenesis occurs through accurate interaction between essential players to generate highly specialized plant organs. Fruit structure and function are triggered by a neat transcriptional control involving distinct regulator genes encoding transcription factors (TFs) or signalling proteins, such as the C2H2/C2HC zinc-finger NO TRANSMITTING TRACT (NTT) or the MADS-box protein SEEDSTICK (STK), which are important in setting plant reproductive competence, feasibly by affecting cell wall polysaccharide and lipid distribution. Arabinogalactan proteins (AGPs) are major components of the cell wall and are thought to be involved in the reproductive process as important players in specific stages of development. The detection of AGPs epitopes in reproductive tissues of NTT and other fruit development-related TFs, such as MADS-box proteins including SHATTERPROOF1 (SHP1), SHP2 and STK, was the focus of this study. METHODS: We used fluorescence microscopy to perform immunolocalization analyses on stk and ntt single mutants, on the ntt stk double mutant and on the stk shp1 shp2 triple mutant using specific anti-AGP monoclonal antibodies. In these mutants, the expression levels of selected AGP genes were also measured by quantitative real-time PCR and compared with the respective expression in wild-type (WT) plants. KEY RESULTS: The present immunolocalization study collects information on the distribution patterns of specific AGPs in Arabidopsis female reproductive tissues, complemented by the quantification of AGP expression levels, comparing WT, stk and ntt single mutants, the ntt stk double mutant and the stk shp1 shp2 triple mutant. CONCLUSIONS: These findings reveal distinct AGP distribution patterns in different developmental mutants related to the female reproductive unit in Arabidopsis. The value of the immunofluorescence labelling technique is highlighted in this study as an invaluable tool to dissect the remodelling nature of the cell wall in developmental processes.

An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus.

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for ß-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.