Higher Protein Yield Research Articles

Advancements in Escherichia coli secretion systems for enhanced recombinant protein production.

Escherichia coli is inarguably one of the most studied microorganisms across the spectrum of microbiology. It is very widely used in recombinant protein production owing to its rapid growth, ease of genetic manipulation, and relatively high protein yields. Despite all of its advantages, its inability to efficiently secrete proteins naturally remains a drawback leading to protein aggregation as inclusion bodies in the cytoplasm and consequent low overall protein yield. Therefore, many approaches to mitigate this weakness and enhance extracellular secretion to increase protein yield have been devised. This review explores the natural and engineered secretion systems in E. coli, highlighting their potential for enhanced protein secretion for non-glycosylated proteins. Natural one-step (e.g., Type I and III Secretion Systems) and two-step systems (e.g., Sec and Tat pathways) are detailed alongside recent advancements in genetic engineering, mutagenesis, and synthetic biology approaches aimed at improving protein yield, folding, and secretion efficiency. Emerging technologies, such as the ESETEC® and BacSec® platforms, promise scalable and cost-effective solutions for higher protein production. Challenges, including limited cellular capabilities and protein aggregation, are addressed through innovative strategies like cell wall modification, co-expression of chaperones, and medium optimization. This review emphasizes E. coli's adaptability to industrial applications, and the promising future of recombinant protein technologies.

High-power ultrasound pretreatment for enhanced protein extraction from lupin flour: Impact on yield, anti-technological and anti-nutritional factors, and techno-functional properties.

High-power ultrasound pretreatment for enhanced protein extraction from lupin flour: Impact on yield, anti-technological and anti-nutritional factors, and techno-functional properties.

Genome-wide analysis of Q binding reveals a regulatory network that coordinates wheat grain yield and grain protein content.

Genome-wide analysis of Q binding reveals a regulatory network that coordinates wheat grain yield and grain protein content.

Structure-based design of an immunogenic, conformationally stabilized FimH antigen for a urinary tract infection vaccine.

Adhesion of E. coli to the urinary tract epithelium is a critical step in establishing urinary tract infections. FimH is an adhesin positioned on the fimbrial tip which binds to mannosylated proteins on the urinary tract epithelium via its lectin domain (FimHLD). FimH is of interest as a target of vaccines to prevent urinary tract infections (UTI). Previously, difficulties in obtaining purified recombinant FimH from E. coli along with the poor inherent immunogenicity of FimH have hindered the development of effective FimH vaccine candidates. To overcome these challenges, we have devised a novel production method using mammalian cells to produce high yields of homogeneous FimH protein with comparable biochemical and immunogenic properties to FimH produced in E. coli. Next, to optimize conformational stability and immunogenicity of FimH, we used a computational approach to design improved FimH mutants and evaluated their biophysical and biochemical properties, and murine immunogenicity using a bacterial adhesion inhibition assay. This approach identified an immunogenic FimH variant (FimH-donor-strand complemented with FimG peptide 'triple mutant', FimH-DSG TM) capable of blocking bacterial adhesion that is produced at high yields in mammalian cells. By x-ray crystallography, we confirmed that the stabilized structure of the FimHLD in FimH-DSG TM is similar to native FimH on the fimbrial tip. Characterization of monoclonal antibodies elicited by FimH-DSG TM that can block bacterial binding to mannosylated surfaces identified 4 non-overlapping binding sites whose epitopes were mapped via a combinatorial cryogenic electron microscopy approach. Novel inhibitory epitopes in the lectin binding FimH were identified, revealing diverse functional mechanisms of FimH-directed antibodies with relevance to FimH-targeted UTI vaccines.

A universal method for the purification of C2H2 zinc finger arrays.

Zinc fingers (ZFs) are compact, modular, sequence-specific polynucleotide-binding domains uniquely suited for use as DNA probes and for the targeted delivery of effector domains for purposes such as gene regulation and editing. Despite recent advances in both the design and application of ZF-containing proteins, there is still a lack of a general method for their expression and purification. Here we describe a simple method, involving two chromatographic steps, for the production of homogeneous, functional ZF proteins in high yield (one milligram per liter of bacterial culture), and we demonstrate the generality of this method by applying it to a diverse set of eight C2H2-type ZF proteins. By incorporating a surface-exposed terminal cysteine residue that enables site-specific conjugation with maleimide-activated fluorophores, we confirm the suitability of these probes for in situ labeling of specific DNA sequences in human cells.

Enzyme-Assisted Extraction of Proteins from Cauliflower and Broccoli Waste Leaves

This research presents a novel approach to protein extraction from cauliflower (CL) and broccoli (BL) waste leaves by using enzymatic pretreatment, demonstrating its effectiveness on protein yield. Enzymatic pretreatment (pH 4.5, 10 h, t = 35 °C), was performed using commercial enzyme preparations, Viscozyme® L and Vinozyme®, in three different enzyme-to-substrate ratios E/S (0.2%, 2.5%, and 4.8%) of each enzyme. Leaf proteins (LPs) were obtained with alkaline extraction at pH 10–11 and their isoelectric precipitation at pH 4, following the control sample (extraction without enzymes). Protein yield (%), which was used as a parameter to monitor enzymatic efficiency, demonstrated a direct correlation with the enzyme-to-substrate (E/S) ratio. The highest protein yields were obtained at an enzyme concentration of 4.8% for both cellulolytic and pectolytic enzyme preparations, yielding 14.90 ± 0.12% for CL and 29.88 ± 0.86% for BL. The obtained proteins were characterized by FTIR spectroscopy and SDS-PAGE electrophoresis, and these methods confirmed the enzymatic efficiency of protein isolation. Isolated LPs showed high protein content for CLP 4.8% (77.27 ± 0.14%) and BPL 4.8% (84.66 ± 0.51%), and an increase in total amino acids, while the content of essential amino acids was over 40%. Protein solubility was assessed, revealing significant improvements (p < 0.05) in LPs derived from CL and BL at the highest E/S ratio of 4.8%, compared to the control sample C0%. Specifically, the solubility of CLP reached 29.4 mg/mL at pH 11, while BLP achieved 36.4 mg/mL at pH 10. As a result, these leaf proteins not only meet nutritional demands but also open innovative avenues of research in food science and biotechnology.

Comparative evaluation of quantitative, and qualitative attributes of oat (Avena sativa L.) varieties grown in semi-arid India

Oat is a valuable cereal of Poaceae family, which is progressively used as an elite healthy diet, as a source of protein, minerals and dietary fiber. The agro-morphological characteristics and variation in quality attributes were studied to understand the extent of variability in these traits. Morphological study revealed significant variations among the tested varieties. The yields of oat oils were varied from 2.49 to 5.91%. The Kent (5.02%), JHO-822 (5.64%), and JHO 99-2 (5.91%) were contains more than two-fold oil content as compared to JHO 99-1 (2.49%). The compositions of fatty acids such as PUFA (poly-unsaturated fatty acid) (36.5–40.08%), MUFA (mono-unsaturated fatty acid) (33.06–37.58%), and SFA (saturated fatty acid) (17.27–25.42%) were showed significant variations. The oats oil was constituted about 70% of unsaturated fatty acids (UFA) with good amounts of PUFA including linoleic acid (36.0–39.35%) and linolenic acid (0.42–0.73%). The varieties JHO-2004 (12.62%), JHO-2012-2 (12.53%) and JHO-851 (11.21%), contains higher amount of proteins. The oat varieties were contained high amounts of UFA (⁓ 70%) with good amounts of protein, which made it an ideal cereal for nutraceutical applications. Among these varieties, the JHO 99-2 and JHO-851 were superior due to high oil yield (5.91, 4.02%), PUFA (38.47, 40.08%) content, and good percentage of protein (10.24, 11.21%). The data on quantitative and qualitative attributes suggested that the JHO-851 and JHO-99-2 can be used as dual purpose and also recommended as super food.

Effects of feed nutrients on growth, development and the deposition of protein and fat in Tenebrio molitor larvae

Abstract An understanding of the impact of dietary nutritional composition on growth, development and composition of Tenebrio molitor larvae (mealworms) could help optimise production systems. Replicate containers of mealworms fed for 24 days on synthetic cellulose-based feeds containing different combinations of protein (casein, Cas), lipid (wheat germ oil, WGO), carbohydrate (glucose) and a mineral/vitamin premix (MV) were compared to a control diet of wheat bran (WB). Mealworms fed only cellulose (C) ceased to grow after 10 days, and did not pupate. Addition of casein alone did not restore growth but did induce pupation (much slower than in WB-fed worms). Mealworms fed C+Cas-based diets showed a marked increase in the relative proportion of body protein to fat, compared to those fed wheat bran. The addition of MV to the C+Cas diet restored growth and pupation, though not to the same level as WB. MV-fed worms maintained a significantly increased protein/fat composition. The further inclusion of WGO or WGO + glucose further increased growth, whilst WGO + glucose reduced pupation. Overall, the highest yield of protein (per container) was seen in the C+Cas+MV+WGO-fed mealworms, which was 50% higher than in the WB group (largely due to the decreased pupation rate). Furthermore, the lipid yield was 50% lower than in WB-fed mealworms. By contrast, the addition of glucose to this diet maintained a high protein yield but increased the lipid yield to similar levels as seen in WB-fed mealworms, with an associated significant increase in the relative proportion of the polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. The results indicate that sources of protein, lipid, minerals and multivitamins are all required to maximize protein whilst decreasing lipid deposition in mealworms. However, the addition of glucose (and presumably other digestible carbohydrates) increased lipid deposition and specifically the PUFA content.

ВЫБОР ЛЬНЯНОГО СЫРЬЯ ДЛЯ ПОЛУЧЕНИЯ ОСНОВЫ РАСТИТЕЛЬНОГО НАПИТКА

The purpose of the study is the choice of flax raw materials for obtaining the basis of a vegetable drink. Tasks: to obtain the basis of a milk-type drink from linseed raw materials; to analyze the physico-chemical parameters of the obtained model samples; choose linseed raw materials to obtain the basis of a drink such as milk. The studies were carried out on the basis of the laboratory for the processing of bast crops of the Federal Scientific Center for Bast Crops in Tver. Varietal flax seeds were used, as well as products of their processing in the form of flax cake and mucilage-free seeds. The basis of the vegetable drink was obtained in several stages: maximum grinding in a laboratory mill, additional grinding in a laboratory mortar, and mixing with an extractant using a blender. Extraction was carried out with a low-salt solution (0.6 % NaCl) with constant stirring for 1 h at 40 °C. The resulting extract was separated from the plant mass by centrifugation (3500 rpm, 30 min). Physicochemical parameters were determined by standard methods of analysis. It was revealed that flax seeds and linseed cake are high-quality raw materials for obtaining a vegetable drink such as milk. A sample of the base for a drink from seeds of the LM-98 variety was characterized by the highest protein yield (54.47 ± 2.72 %) and fairly high protein content in dry matter (33.77 ± 1.69 %).The base for the drink, obtained from linseed cake, was characterized by the maximum protein content of all the studied samples – 1.55 ± 0.08 % and the highest protein content in dry matter – 41.67 ± 2.08 %. It is shown that the yield of extractives, determined by the value of the dry residue of extracts, depends not only on the protein content in the raw material, but also on the polysaccharide complex of linseed mucus (its composition and quantity). The highest organoleptic indicators were characterrized by the base of the drink made from flax seeds of the Severny variety.

Breeding of winter chickpea genotypes with high photosynthetic productivity, adapted to mechanization, high yield and high protein content in the southern regions of Uzbekistan

Breeding of winter chickpea genotypes with high photosynthetic productivity, adapted to mechanization, high yield and high protein content in the southern regions of Uzbekistan

Characterizing the extractable proteins from tomato leaves - A proteomics study.

The ambition to utilize agricultural by-products has spotlighted tomato leaves as a promising source for plant-based proteins. High-yielding protein extractability is key for its industrial use, but previous studies reported decreased protein extractability at later stages of plant development. This study investigated the underlying factors in protein extractability through a comprehensive proteomics analysis across four plant developmental stages (vegetative, flowering, fruit-forming, mature-fruit). The findings linked reduced yields to a shift in leaf function, from anabolic to catabolic processes and (a)biotic stress responses. This functional shift is accompanied by decreased protein synthesis and increased protein degradation, leading to an overall decrease of the soluble protein fraction. Furthermore, incomplete extraction of soluble proteins from leaves of later developmental stages, suggested the presence of inhibitory molecules hindering the extraction process. These findings indicate that breeding strategies towards increased amounts of soluble proteins and reduced concentration of inhibitory molecules could enhance protein extraction yields.

Enzymatic Hydrolysis Systems Enhance the Efficiency and Biological Properties of Hydrolysates from Frozen Fish Processing Co-Products.

Co-products from the frozen fish processing industry often lead to financial losses. Therefore, it is essential to transform these co-products into profitable goods. This study explores the production of fish protein hydrolysates (FPH) from three co-products: the heads and bones of black scabbardfish (Aphanopus carbo), the carcasses of gilthead seabream (Sparus aurata), and the trimmings of Nile perch (Lates niloticus). Four enzymatic hydrolysis systems were tested: an endopeptidase (Alcalase, A), an exopeptidase (Protana, P), two-stage hydrolysis with an endopeptidase followed by an exopeptidase (A + P), and a single stage with endo- and exopeptidase (AP). The results show that combined enzymatic treatments, especially single-stage Alcalase and Protana (AP), achieved high protein yields (80%) and enhanced degrees of hydrolysis (34 to 49%), producing peptides with lower molecular weights. FPH exhibited significant antioxidant activity, in 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, with EC50 values below 5 mg/mL. Additionally, AP hydrolysates demonstrated over 60% angiotensin-converting enzyme (ACE) inhibition at 5 mg/mL, indicating potential antihypertensive applications. Antidiabetic and anti-Alzheimer activities were present, but at relatively low levels. AP hydrolysates, especially from gilthead seabream, proved to be the most promising. This study highlights the value of fish co-products as sources of functional peptides, contributing to waste reduction, and their potential applications in food, agriculture, and nutraceuticals.

Efficient production of non-immunogenic, long circular RNAs for high protein yield.

Efficient production of non-immunogenic, long circular RNAs for high protein yield.

Investigation of the Structural Characteristics of Seed Surfaces of Some Soybean Genotypes by Using Scanning Electron Microscopy (SEM)

Soybean (Glycine max) is a partly climbing, branched, annual plant in the Fabaceae family, native to East Asia. The plant is grown for its edible beans. Soybeans have many uses in agronomic practice. It plays a crucial role in East Asian cuisine and the animal feed industry as it is one of the plants with the highest protein yield per cultivated area and is rich in nutritional value. Therefore, the characterization of soya genotypes and seed surface structural properties is critical. SEM technique, one of the techniques that can be used for this purpose, provides high-resolution images of the surfaces and allows detailed examination of the microstructure of the materials. This study used SEM microscopy to determine seed surface characteristics such as surface roughness, reticulation, tubercles, and raised and grooved surface decorations in 12 soya genotypes. As a result of the study, soybean genotypes were divided into 3 groups according to their micro-morphological characteristics and their similarities and differences were revealed. In conclusion, it was once again demonstrated that this classification based on surface characteristics can be useful for various agricultural, botanical and research purposes by helping to identify and compare seeds.

Evaluation of Functional Properties and the Lipase Inhibitory Activity of Proteins from Amaranthus cruentus Seeds

Aims/Background: Obesity is characterized by excessive body fat and is a chronic and complex disease. The medications used to date for the treatment of obesity have exhibited various side effects. Thus, new substances must be sought from alternative sources as anti-obesity drugs. Recently, there has been increased interest in the study of bioactive peptides generated from plant proteins for functional food development. Plant proteins have exhibited pancreatic lipase inhibitory activity and can be potentially studied as sources of anti-obesity drugs for long-term use. Amaranthus cruentus seeds are very nutritious, with high protein con-tent and medicinal properties. Hence, this study focuses on pancreatic lipase inhibitory activity and functional properties of Amaranthus cruentus seed protein isolates. Materials and Methods: Proteins were isolated using conventional and thermal isoelectric pre-cipitation methods. The protein's ability to inhibit lipases was assessed using both synthetic and natural substrates. The functional properties, such as nitrogen solubility, foaming test, emulsi-fication test, and water and oil holding abilities, were evaluated. The protein isolates were char-acterized by SDS-PAGE and DSC. Cell line studies were performed on 3T3-L1 cell lines to determine the cytotoxicity and anti-adipogenic activity of protein isolates. Results: Proteins 1, 2, 3, and 4 were found to have high protein yield and protein content. SDS-PAGE characterization showed protein bands at different molecular weights (kDa). Protein iso-lates demonstrated good functional properties and antilipase activity as compared to flour. In the cell line experiments, protein 1 exhibited a dose-dependent reduction in the lipid content. Conclusion: A. cruentus seed protein could pave the way for the development of nutraceutical formulation in the treatment of obesity.

Unraveling the Amino Acid Synthesis in Maturity Sesame Seeds Based on the Integrative Analysis of Transcriptome and Metabolome

ABSTRACTSesame plays a vital role in food industry due to its high oil yield, antioxidant potential, and substantial protein content. Notably, there are significant differences in amino acid composition in sesame seeds at various developmental stages. However, the molecular basis and regulatory mechanism underlying amino acid production largely remain unexplored. To unravel these mechanisms, we analyzed the metabolome and transcriptome profiles of a sesame variety across four distinct growth stages (S1–S4). Our analysis identified a total of 17 amino acids, with glutamic acid (Glu), arginine (Arg), proline (Pro), and tyrosine (Tyr) exhibiting significantly higher abundances in mature stages. This increased abundance correlated with the elevated expression of genes involved in amino acid synthesis and regulatory genes. Using weighted gene co‐expression network analysis, we discovered modules associated with glutathione metabolism, arginine biosynthesis, proline, and tyrosine synthesis, along with candidate genes that regulate amino acid production and metabolism. Notably, the differential expression of genes within the amino acid pathways resulted in significant variations in the contents of Glu, Arg, Pro, and Tyr at the mature stage (28 days after flowering, S4) compared to other growth stages. Correlation analysis revealed strong association among the enzymes glutamine synthetase (GS), glutamate/aspartate–prephenate aminotransferase (PAT), and polyamine oxidase (PAO) with the 17 amino acids, suggesting their potential role in the amino acid synthesis. Our findings provide novel insights into the synthesis and accumulation of amino acids during the growth stages of sesame seeds, highlighting key regulatory genes and metabolic pathways involved in this process. Our study lays the groundwork for future studies aiming to enhance the nutritional quality and yield of sesame varieties.

Biochemical characterization of a recombinant laccase from Halalkalibacterium halodurans C-125 and its application in the biotransformation of organic compounds

ObjectivesThis study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application.ResultsThe rLac-HhC125 was partially purified by size exclusion chromatography and concentrated by ultrafiltration (10 kDa) with a yield of 57.6%. Oxidation reactions showed that adding 2 mM CuSO4 to the assay solution led to activating the laccase. To increase its initial activity, the rLac-HhC125 was treated at 50 °C for 20 min before the assays, improving its performance by fourfold using the syringaldazine as a substrate. When treated with EDTA, methanol, ethanol, and DMSO, the rLac-HhC125 maintained more than 80% of its original activity. Interestingly, the acetonitrile induced a twofold activity of the rLac-HhC125. The putative rLac-HhC125 demonstrated a capability of efficient transformation of different organic compounds at pH 6, known as dye precursors, into coloured molecules.ConclusionThe rLac-HhC125 was active at high temperatures and alkaline pH, exhibited tolerance to organic solvents, and efficiently transformed different hydroxy derivatives into coloured compounds, which indicates that it can be used in various biotechnological processes.

Controlled hydrolysis with preheating treatment and glutathione addition for enhanced foaming properties of soy protein isolate

Controlled hydrolysis with preheating treatment and glutathione addition for enhanced foaming properties of soy protein isolate

THE RECOMBINANT BACTERIA LACTOCOCCUS LACTIS EXPRESSING SARS-COV-2 SPIKE PROTEIN OR ITS RECEPTOR-BINDING DOMAIN ELICITS AN IMMUNE RESPONSE FOLLOWING ORAL IMMUNISATION OF RATS

Mucosal delivery of antigens represents an effective strategy for the development of vaccines to control diseases with the upper respiratory and/or gastrointestinal tracts as the main routes of transmission. Lactic acid bacteria (LAB) are food-grade bacteria used for the expression of target antigens in the development of mucosal vaccines. Over the last two decades, research on the use of LAB has focused on the construction of genetically modified strains. Current study aimed to develop and examine the recombinant Lactococcus lactis strains, expressing SARS-CoV-2 spike (S) protein or the receptor-binding domain (RBD) of S protein as immunizing antigens.The spike gene from the component II of Sputnik V vaccine and two synthesized coding sequences of the RBD region of SARS-CoV-2 spike protein (319–529 residues named HA-spike and 318–590 residues named mini-spike) were subcloned into expression vector pNZ8121. The resulting plasmids were verified by sequencing and subsequently transformed into competent L. lactis NZ3900 cells. The expression of recombinant proteins in obtained strains was analyzed by western blot using SARS-COV-2 Spike RBD antibodies. The results showed that the spike gene could be efficiently expressed in the cells of recombinant L. lactis. The major 150 kDa band detected in the cell lysate of L. lactis pNZ::spike represented the full-length S protein. Protein bands of 35 kDa and 23 kDa from the lysates of L. lactis pNZ::mini-spike and L. lactis pNZ::HA-spike respectively were detected indicating that the recombinant RBD protein was also successfully expressed. The mini-spike was expressed more abundantly than the HA-spike variant. Based on the growth kinetics and protein expression profiles of recombinant strains, the highest protein yield was obtained under the following conditions: overnight culture was inoculated (1/25 inoculum) and grown until reaching an optical density of 0,9 at 600 nm, then gene expression was induced by adding nisin (1 ng/ml), cells were harvested after 3 h incubation with the inducer. To examine the immunogenicity of recombinant strains, 1 ml of cell suspension (1,5×109 CFU) of L. lactis pNZ::spike, pNZ::mini-spike or L. lactis pNZ::HA-spike was orally administered to Wistar rats. The same volume of peptone water was administered to the control group. Serum was collected from the animals at 28 days and IgG level was determined by ELISA for the quantitative determination of IgG antibodies to SARS-CoV-2 (COVID-19) Spike RBD protein in rat serum. As expected, the serum from control animals lacked antibodies to the RBD antigen. The serum of rats immunised once with cell suspension of the recombinant strains showed the following levels of specific IgG: 124,24±12,56 ng/ml in group pNZ::mini-spike, 165,91±59,13 ng/ml in group pNZ::spike, and 213,60±54,08 ng/ml in group pNZ::HA-spike. The highest IgG level (545,56±230,24 ng/ml) was detected in the serum of rats immunized with ultrasound-treated cell suspension of L. lactis pNZ::spike. No adverse effects were observed in vaccinated animals at regular check-ups throughout the study period.The current results have demonstrated that the RBD and full spike protein were successfully expressed in L. lactis. Oral administration of the recombinant L. lactis expressing S protein or RBD to rats appeared to generate the anti-SARS-CoV-2 antibodies in serum, positioning this bacteria as promising basis for creating oral vaccine.

Management of maize-legume conservation agriculture systems rather than varietal choice fosters human nutrition in Malawi

Malawi smallholder farmers are facing climate-induced challenges that have increased food and nutrition insecurity in the country, thus sustainable intensification practices has been widely recommended. The objective of this study was to assess the effects of cropping systems with improved varieties on total system productivity and nutrition under different environments. The study involved on-farm experiments in ten communities in Central and Southern Malawi, incrementally established from 2005/2006 to 2018/2019 cropping seasons. Each community had six demonstration plots with three main treatments: conventional ploughing (CP): sole maize grown on seasonally constructed ridges and furrows; no-tillage (NT): sole maize grown on retained ridges with minimum soil disturbance and residue retained; and Conservation agriculture (CA): maize intercropped either cowpea, pigeon pea or groundnut on retained ridges as in NT. Our results show that total system nutrition was higher in CA treatments than NT and CP. The yields of maize were at least 800 kg ha−1 higher in CA and NT than CP despite the variety that was grown. Legume yields were also higher under CA and NT than CP. High protein yield was observed in CA systems (at least 100 kg ha−1 higher than CP) where maize and legume intercrops were rotated with grain legumes. Our results show nutrients and energy gains in CA and NT systems that can be invested in practices that increases the resilience of smallholder farmers to climate change. Conservation agriculture and NT systems have more influence on productivity of smallholder farms, despite the genotypes used (hybrids or OPVs).