Transformation Of Wheat Research Articles

The transcription factor TaNF-YB4 overexpression in wheat increases plant vigor and yield

Ensuring food security is a priority in developing countries. This study aimed to improve wheat yield by overexpressing the TaNF-YB4 transcription factor, which is involved in carbon assimilation and stress tolerance. An expression cassette for TaNF-YB4 was developed in a modified wheat transformation vector (pSB219) and examined through transient expression in Nicotiana tabacum, followed by Agrobacterium-mediated transformation of wheat variety FSD-2008. T0 transgenic plants were propagated to obtain T3 generation PCR-positive plants. Transgene expression was assessed in PCR-verified T2 plants using RT-PCR and qRT-PCR at six weeks post-germination. qRT-PCR analysis using the ΔΔCT method indicated higher TaNF-YB4 expression in transgenic lines than in the wild-type control plants. Improved agronomic and phenotypic traits were observed with a 6-36% increase in 1000-grain weight in the selected transgenic lines. Root architecture assessments demonstrated enhanced root length, surface area, and projected area in transgenic lines compared with wild-type plants. Additionally, notable variances in total chlorophyll, protein, and sugar content levels were observed between the transgenic lines and control plants, demonstrating statistical significance with a p-value ≤ 0.05. This study indicates that low-level constitutive expression of TaNF-YB4 can enhance wheat yield, presenting a viable strategy for improving wheat productivity.

Setting of seeds during Agrobacterium-mediated winter wheat transformation by in planta method

Aim. To investigate the frequency of seeds setting and the formation of transgenic plants during Agrobacterium-mediated transformation of winter wheat by the in planta method using different methods of applying agrobacteria and the composition of inoculation media. Methods. Agrobacterium-mediated transformation by in planta method, molecular genetic analysis, methods of mathematical statistics. Results. A significant difference was found in the studied genotypes of winter wheat according to the indicator seed setting depending on the A. tumefaciens strain used and the inoculation medium. When comparing the effect of different strains of agrobacteria on this indicator, no significant differences between them were established, however, a tendency to increase the frequency of seed formation was observed when using the LBA4404 strain. When using the MS-22 inoculation medium, which additionally contained 12.5 mM MES, 4 mM NH4CI, 5.5 mM MgSO4, a certain increase in the number of formed seeds was also observed compared to the MS-21 medium supplemented with sodium thiosulfate, the frequency of seed setting increased in on average by 5 %. Conclusions. The genotypic dependence of the frequency of seed setting and the formation of transgenic plants with the use of different inoculation media and the method of the transformation procedure was established.

Engineering a One Health Super Wheat.

Wheat is the predominant crop worldwide, contributing approximately 20% of protein and calories to the human diet. However, the yield potential of wheat faces limitations due to pests, diseases, and abiotic stresses. Although conventional breeding has improved desirable traits, the use of modern transgenesis technologies has been limited in wheat in comparison to other crops such as maize and soybean. Recent advances in wheat gene cloning and transformation technology now enable the development of a super wheat consistent with the One Health goals of sustainability, food security, and environmental stewardship. This variety combines traits to enhance pest and disease resistance, elevate grain nutritional value, and improve resilience to climate change. In this review, we explore ways to leverage current technologies to combine and transform useful traits into wheat. We also address the requirements of breeders and legal considerations such as patents and regulatory issues.

Overexpression of Wheat Selenium-Binding Protein Gene TaSBP-A Enhances Plant Growth and Grain Selenium Accumulation under Spraying Sodium Selenite.

Selenium (Se) is an essential trace element for humans. Low concentrations of Se can promote plant growth and development. Enhancing grain yield and crop Se content is significant, as major food crops generally have low Se content. Studies have shown that Se biofortification can significantly increase Se content in plant tissues. In this study, the genetic transformation of wheat was conducted to evaluate the agronomic traits of non-transgenic control and transgenic wheat before and after Se application. Se content, speciation, and transfer coefficients in wheat grains were detected. Molecular docking simulations and transcriptome data were utilized to explore the effects of selenium-binding protein-A TaSBP-A on wheat growth and grain Se accumulation and transport. The results showed that TaSBP-A gene overexpression significantly increased plant height (by 18.50%), number of spikelets (by 11.74%), and number of grains in a spike (by 35.66%) in wheat. Under normal growth conditions, Se content in transgenic wheat grains did not change significantly, but after applying sodium selenite, Se content in transgenic wheat grains significantly increased. Analysis of Se speciation revealed that organic forms of selenomethionine (SeMet) and selenocysteine (SeCys) predominated in both W48 and transgenic wheat grains. Moreover, TaSBP-A significantly increased the transfer coefficients of Se from solution to roots and from flag leaves to grains. Additionally, it was found that with the increase in TaSBP-A gene overexpression levels in transgenic wheat, the transfer coefficient of Se from flag leaves to grains also increased.

Improved biolistic transformation and genome editing in wheat by using trehalose for high osmotic treatment.

The tissue culture process is usually involved in gene transfer and genome editing in plants. Like other species, there is enormous variation among wheat genotypes in tissue culture response. In the rapidly advancing system of CRISPR/Cas9 for genome editing, particle bombardment has received increasing attention as a delivery method for a large amount of nucleic acids and RNA-protein complexes. However, the efficiency of transformation by particle bombardment has been low in wheat, and only a limited number of varieties have been transformed. In this study, replacement of maltose with trehalose as an osmolyte for high osmotic treatment for the protection of tissues from physical impacts improved callus formation in immature wheat embryos and efficiency of transformation and genome editing in varieties that are relatively poor in tissue culture response. The range of varieties amenable to biolistic transformation and genome editing may be expanded by this modification.

In planta transformation in wheat: an improved protocol to develop wheat transformants.

Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Enhancing Wheat Crop Yield and Quality through Genetic Engineering Technologies: A Comprehensive Review

The increasing world population has created an alarming situation to meet the demand for food. Ultimately wheat yield is required to be increased proportionally to the world’s population. An estimated increase of 05 tonnes per hectare is direly needed from the current production of 3.3 tonnes by the end of 2050. To achieve this goal it is required to adopt new, emerging, and efficient breeding techniques which caused a recent breakthrough in wheat genome sequencing and provided sufficient information for the traits which are directly related to quality and yield of crops. This review provides a comprehensive overview of tools and techniques which are widely used for discovering the functionality of genes that results in specific phenotypes of Wheat. It gives a deep historical account of the development of wheat transformation techniques and provides an idea about in which way these techniques have been adapted to produce gain-of-function phenotypes through gene overexpression, loss-of-function phenotypes through the expression of antisense RNAs (RNA interference or RNAi). Recently, gene structure and expression manipulation using site-specific nucleases such as CRISPR/Cas9 for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to enhance yield, improve wheat's nutritional and health-promoting qualities, and boost the crop's resistance to various biotic and abiotic stresses. Keywords: Transformation, gene knock-Out, gene knock-In, RNAI, overexpression.

Modification of agricultural traits in cultivated varieties of barley and wheat

CRISPR/Cas technology makes it possible to induce mutations at defined positions. In breeding-oriented research, this opens up exciting opportunities for the targeted improvement of many agricultural crops. Wheat and barley are among the most important cereals in the world. However, the transformation poses a particular challenge for cereals and is strongly genotype dependent. This is because agrobacteria, which is mostly used for delivering the CRISPR/Cas system, have a limited compatibility with these non-host plants. Transformation of wheat is additionally difficult due to the large genome size and polyploidy.
 Besides obtaining improved genotypes, the object of the current study was to optimize the method of genomic editing based on the CRISPR/Cas system using particle bombardment for non-model varieties of barley and wheat. In barley, we targeted theNudgene that controls hulled/naked phenotype of the grain. Since the regeneration rate remains an issue for the cultivated cultivars, we used the JD633 vector that carries theGRF4-GIF1chimera to increase the efficiency of regeneration. We obtained five T0plants, carrying mutations. In wheat, targetingPpd-1genes that control photoperiod-dependent floral induction results in Cas9-induced mutations in 52 of 210 T0plants. The developed collection of wheat plants with different new alleles ofPpd-D1andPpd-B1genes is being studied for the expression under short day conditions and the effect on the vegetation period.
 Thus, we have obtained plants of the cultivated varieties of barley and wheat with edited agronomically important genes, using the improved protocols of biolistic transformation.
 This work was done within the framework of State Assignment Kurchatov Genomic Center of ICG SB RAS (No. 075-15-2019-1662).

Enhancing wheat regeneration and genetic transformation through overexpression of TaLAX1

In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat varieties currently restricts the use of genetic transformation for gene functional analysis and improved crop production. This research explores overexpression of TaLAX PANICLE1 (TaLAX1), which markedly enhances regeneration efficiency, thereby boosting genetic transformation and genome editing in wheat. Particularly noteworthy is the substantial increase in regeneration efficiency of common wheat varieties previously regarded as recalcitrant to genetic transformation. Our study shows that increased expression of TaGROWTH-REGULATING FACTOR (TaGRF) genes, alongside that of their co-factor, TaGRF-INTERACTING FACTOR 1 (TaGIF1), enhances cytokinin accumulation and auxin response, which may play pivotal roles in the improved regeneration and transformation of TaLAX1-overexpressing wheat plants. Overexpression of TaLAX1 homologs also significantly increases the regeneration efficiency of maize and soybean, suggesting that both monocot and dicot crops can benefit from this enhancement. Our findings shed light on a gene that enhances wheat genetic transformation and elucidate molecular mechanisms that potentially underlie wheat regeneration.

Wheat Transformation with ScTPS1-TPS2 Bifunctional Enzyme for Trehalose Biosynthesis Protects Photosynthesis during Drought Stress

Wheat cultivation makes an important contribution to human nutrition. Trehalose synthesis plays a role in the tolerance to drought stress. A bifunctional TPS-TPP enzyme gene from yeast was used to obtain transgenic wheat plants to increase trehalose synthesis. Mature wheat embryos were transformed using pGreen rd29A::TPS1-TPS2 or pGreen 35S::TPS1-TPS2 constructs. The transgene presence in mature leaves of T3 plants was confirmed by sequencing a PCR fragment of the inserted transgene. Transgenic and NT plants were submitted to drought stress for eight days. Transformed wheat lines retained a higher relative water content than NT plants during drought stress, and the Rubisco activity was unaffected. Plants transformed with the 35S construct showed a lower photosynthetic rate and lower fructose 1–6-bisphosphatase (FBPase) activity during drought, suggesting that constitutive trehalose and sucrose synthesis caused a reduced ribulose 1,5-bisphosphate (RuBP) regeneration. Lines transformed with the rd29A promoter showed a higher photosynthetic rate after eight days of drought, as the RuBP regeneration was unaffected. Transgenic wheat plants had higher biomass and grain weight than NT plants after drought. These results suggest that trehalose synthesis improves photosynthesis during stress and induces changes in the activity of some Calvin-cycle enzymes, reflected in plant metabolism and growth.

Uncovering the transcriptional regulatory network involved in boosting wheat regeneration and transformation.

Genetic transformation is important for gene functional study and crop improvement. However, it is less effective in wheat. Here we employed a multi-omic analysis strategy to uncover the transcriptional regulatory network (TRN) responsible for wheat regeneration. RNA-seq, ATAC-seq and CUT&Tag techniques were utilized to profile the transcriptional and chromatin dynamics during early regeneration from the scutellum of immature embryos in the wheat variety Fielder. Our results demonstrate that the sequential expression of genes mediating cell fate transition during regeneration is induced by auxin, in coordination with changes in chromatin accessibility, H3K27me3 and H3K4me3 status. The built-up TRN driving wheat regeneration was found to be dominated by 446 key transcription factors (TFs). Further comparisons between wheat and Arabidopsis revealed distinct patterns of DNA binding with one finger (DOF) TFs in the two species. Experimental validations highlighted TaDOF5.6 (TraesCS6A02G274000) and TaDOF3.4 (TraesCS2B02G592600) as potential enhancers of transformation efficiency in different wheat varieties.

Rapid and highly efficient morphogenic gene-mediated hexaploid wheat transformation.

The successful employment of morphogenic regulator genes, Zm-Baby Boom (ZmBbm) and Zm-Wuschel2 (ZmWus2), for Agrobacterium-mediated transformation of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) has been reported to improve transformation by inducing rapid somatic embryo formation. Here, we report two morphogenic gene-mediated wheat transformation methods, either with or without morphogenic and marker gene excision. These methods yield independent-transformation efficiency up to 58% and 75%, respectively. In both cases, the tissue culture duration for generating transgenic plants was significantly reduced from 80 to nearly 50 days. In addition, the transformation process was significantly simplified to make the procedure less labor-intensive, higher-throughput, and more cost-effective by eliminating the requirement for embryonic axis excision, bypassing the necessity for prolonged dual-selection steps for callus formation, and obviating the prerequisite of cytokinin for shoot regeneration. Furthermore, we have demonstrated the flexibility of the methods and generated high-quality transgenic events across multiple genotypes using herbicide (phosphinothricin, ethametsulfuron)- and antibiotic (G418)-based selections.

A bifunctional selectable marker for wheat transformation contributes to the characterization of male-sterile phenotype induced by a synthetic Ms2 gene.

An engineered selectable marker combining herbicide resistance and yellow fluorescence contributes to the characterization of male-sterile phenotype in wheat, the severity of which correlates with expression levels of a synthetic Ms2 gene. Genetic transformation of wheat is conducted using selectable markers, such as herbicide and antibiotic resistance genes. Despite their proven effectiveness, they do not provide visual control of the transformation process and transgene status in progeny, which creates uncertainty and prolongs screening procedures. To overcome this limitation, this study developed a fusion protein by combining gene sequences encoding phosphinothricin acetyltransferase and mCitrine fluorescent protein. The fusion gene, introduced into wheat cells by particle bombardment, enabled herbicide selection, and visual identification of primary transformants along with their progeny. This marker was then used to select transgenic plants containing a synthetic Ms2 gene. Ms2 is a dominant gene whose activation in wheat anthers leads to male sterility, but the relationship between the expression levels and the male-sterile phenotype is unknown. The Ms2 gene was driven either by a truncated Ms2 promoter containing a TRIM element or a rice promoter OsLTP6. The expression of these synthetic genes resulted in complete male sterility or partial fertility, respectively. The low-fertility phenotype was characterized by smaller anthers than the wild type, many defective pollen grains, and low seed sets. The reduction in the size of anthers was observed at earlier and later stages of their development. Consistently, Ms2 transcripts were detected in these organs, but their levels were significantly lower than those in completely sterile Ms2TRIM::Ms2 plants. These results suggested that the severity of the male-sterile phenotype was modulated by Ms2 expression levels and that higher levels may be key to activating total male sterility.

CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.

CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.

Wheat leaf rust fungus effector Pt13024 is avirulent to TcLr30.

Wheat leaf rust, caused by Puccinia triticina Eriks. (Pt), is a global wheat disease threatening wheat production. Dissecting how Pt effector proteins interact with wheat has great significance in understanding the pathogenicity mechanisms of Pt. In the study, the cDNA of Pt 13-5-72 interacting with susceptible cultivar Thatcher was used as template to amplify Pt13024 gene. The expression pattern and structure of Pt13024 were analyzed by qRT-PCR and online softwares. The secretion function of Pt13024 signal peptide was verified by the yeast system. Subcellular localization of Pt13024 was analyzed using transient expression on Nicotiana benthamiana. The verification that Pt13024 inhibited programmed cell death (PCD) was conducted on N. benthamiana and wheat. The deletion mutation of Pt13024 was used to identify the virulence function motif. The transient transformation of wheat mediated by the type III secretion system (TTSS) was used to analyze the activity of regulating the host defense response of Pt13024. Pt13024 gene silencing was performed by host-induced gene silencing (HIGS). The results showed that Pt13024 was identified as an effector and localized in the cytoplasm and nucleus on the N. benthamiana. It can inhibit PCD induced by the Bcl-2-associated X protein (BAX) from mice and infestans 1 (INF1) from Phytophthora infestans on N. benthamiana, and it can also inhibit PCD induced by DC3000 on wheat. The amino acids 22 to 41 at N-terminal of the Pt13024 are essential for the inhibition of programmed cell death (PCD) induced by BAX. The accumulation of reactive oxygen species and deposition of callose in near-isogenic line TcLr30, which is in Thatcher background with Lr30, induced by Pt13024 was higher than that in 41 wheat leaf rust-resistant near-isogenic lines (monogenic lines) with different resistance genes and Thatcher. Silencing of Pt13024 reduced the leaf rust resistance of Lr30 during the interaction between Pt and TcLr30. We can conclude that Pt13024 is avirulent to TcLr30 when Pt interacts with TcLr30. These findings lay the foundation for further investigations into the role of Pt effector proteins in pathogenesis and their regulatory mechanisms.

An established protocol for generating transgenic wheat for wheat functional genomics via particle bombardment.

Wheat is one of the most important food crops in the world and is considered one of the top targets in crop biotechnology. With the high-quality reference genomes of wheat and its relative species and the recent burst of genomic resources in Triticeae, demands to perform gene functional studies in wheat and genetic improvement have been rapidly increasing, requiring that production of transgenic wheat should become a routine technique. While established for more than 20 years, the particle bombardment-mediated wheat transformation has not become routine yet, with only a handful of labs being proficient in this technique. This could be due to, at least partly, the low transformation efficiency and the technical difficulties. Here, we describe the current version of this method through adaptation and optimization. We report the detailed protocol of producing transgenic wheat by the particle gun, including several critical steps, from the selection of appropriate explants (i.e., immature scutella), the preparation of DNA-coated gold particles, and several established strategies of tissue culture. More importantly, with over 20 years of experience in wheat transformation in our lab, we share the many technical details and recommendations and emphasize that the particle bombardment-mediated approach has fewer limitations in genotype dependency and vector construction when compared with the Agrobacterium-mediated methods. The particle bombardment-mediated method has been successful for over 30 wheat genotypes, from the tetraploid durum wheat to the hexaploid common wheat, from modern elite varieties to landraces. In conclusion, the particle bombardment-mediated wheat transformation has demonstrated its potential and wide applications, and the full set of protocol, experience, and successful reports in many wheat genotypes described here will further its impacts, making it a routine and robust technique in crop research labs worldwide.

Underlying Mechanisms for Low-Molecular-Weight Dissolved Organic Matter to Promote Translocation and Transformation of Chlorinated Polyfluoroalkyl Ether Sulfonate in Wheat.

Dissolved organic matter (DOM) such as fulvic acid (FA) and humic acid (HA) in soil considerably affects the fate of per- and polyfluoroalkyl substances (PFASs). However, the effect of DOM on their behavior in plants remains unclear. Herein, hydroponic experiments indicate that FA and HA reduce the accumulation of an emerging PFAS of high concern, 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA), in wheat roots by reducing its bioavailability in the solution. Nevertheless, FA with low molecular weight (MW) promotes its absorption and translocation from the roots to the shoots by stimulating the activity and the related genes of the plasma membrane H+-ATPase, whereas high-MW HA shows the opposite effect. Moreover, in vivo and in vitro experiments indicate that 6:2 Cl-PFESA undergoes reductive dechlorination, which is regulated mainly using nitrate reductase and glutathione transferase. HA and FA, particularly the latter, promote the dechlorination of 6:2 Cl-PFESA in wheat by enhancing electron transfer efficiency and superoxide production. Transcriptomic analysis indicates that FA also stimulates catalytic activity, cation binding, and oxidoreductase activity, facilitating 6:2 Cl-PFESA transformation in wheat.

Agrobacterium-mediated transformation – method of genetic modification of Triticum aestivum L. plants

Aim. Investigate the effect of Agrobacterium-mediated transformation in planta on seed tying and the frequency of transformation in winter wheat (Triticum aestivum L.). To analyze changes in the level of free L-proline (Pro) in transformed and control seedlings under normal / stress conditions and productivity indicators of biotechnological plants (T1) under normal growing conditions. Methods. Agrobacterium-mediated transformation in planta; PCR analysis, DNA electrophoresis; determination of seed tying frequency and transformation, Pro content, yield structure indicators. Results. Obtained transgenic wheat plants. The level of Pro in the tested variants under normal / stress conditions and indicators of T1 productivity of plants and their initial form under optimal water supply were studied. Conclusions. The susceptibility of the studied wheat genotypes to agrobacterial infection is shown. The frequency of seed tying after genetic transformation was 12.7 % and 5.4 % for plants of UK 106/19 and UK 171/19h, respectively. Transgenic seedlings had elevated levels of Pro. Complete incorporation of the vector construct was identified in 14 and 11 variants of genotypes UK 161/19 and UK 171/19h, respectively. Control and T1 biotechnological plants under normal growing conditions had similar yields.

Applications of In Vitro Tissue Culture Technologies in Breeding and Genetic Improvement of Wheat.

Sources of new genetic variability have been limited to existing germplasm in the past. Wheat has been studied extensively for various agronomic traits located throughout the genome. The large size of the chromosomes and the ability of its polyploid genome to tolerate the addition or loss of chromosomes facilitated rapid progress in the early study of wheat genetics using cytogenetic techniques. At the same time, its large genome size has limited the progress in genetic characterization studies focused on diploid species, with a small genome and genetic engineering procedures already developed. Today, the genetic transformation and gene editing procedures offer attractive alternatives to conventional techniques for breeding wheat because they allow one or more of the genes to be introduced or altered into an elite cultivar without affecting its genetic background. Recently, significant advances have been made in regenerating various plant tissues, providing the essential basis for regenerating transgenic plants. In addition, Agrobacterium-mediated, biolistic, and in planta particle bombardment (iPB) gene delivery procedures have been developed for wheat transformation and advanced transgenic wheat development. As a result, several useful genes are now available that have been transferred or would be helpful to be transferred to wheat in addition to the current traditional effort to improve trait values, such as resistance to abiotic and biotic factors, grain quality, and plant architecture. Furthermore, the in planta genome editing method will significantly contribute to the social implementation of genome-edited crops to innovate the breeding pipeline and leverage unique climate adaptations.

Research and Application of Factory Hydroponic Technology in Wheatgrass Greenhouse

Conversion of farmland to forest is one of the important policies of China’s western development strategy, which is of great significance to the protection of ecological environment in western China. In the context of this era, the traditional grazing-based breeding method that the western region has long adhered to needs to be replaced by the factory-based house feeding with large-scale construction of forage bases. On the basis of a self-developed multi-layer three-dimensional intelligent environmental control system, this study comprehensively optimized the temperature and humidity control system, automatic micro-spraying system, intelligent ventilation system and automatic light filling system, so as to develop an efficient wheat hydroponic planting system. Based on the above planting system, it only takes 7 days from sowing to harvesting. The roots, stems and leaves of wheat grains are all fodder, and 7 kilo-grams of green forage grass can be produced by per kilogram of wheat. In addition, through hydroponic transformation of wheat, protein starch, which is difficult or inefficient to be absorbed in the form of wheat seed, can be transformed into easily absorbed amino acids and glucose. Meanwhile, the vitamin content of green feed is significantly increased, which is of great significance to improve the feed biological conversion rate.