Drought-tolerant transgenic wheat HB4®: a hope for the future.

Drought-tolerant transgenic [genetically modified (GM)] HB4® wheat carrying the drought-responsive sunflower gene Hahb4 was first developed in Argentina in 2019 and has already been approved for marketing and consumption as food/feed in at least ten countries. It has also been approved in Argentina and Brazil for commercial cultivation.

Genome-Wide Association Studies in Sunflower: Towards Sclerotinia sclerotiorum and Diaporthe/Phomopsis Resistance Breeding.

Diseases caused by necrotrophic fungi, such as the cosmopolitan Sclerotinia sclerotiorum and the Diaporthe/Phomopsis complex, are among the most destructive diseases of sunflower worldwide. The lack of complete resistance combined with the inefficiency of chemical control makes assisted breeding the best strategy for disease control. In this work, we present an integrated genome-wide association (GWA) study investigating the response of a diverse panel of sunflower inbred lines to both pathogens. Phenotypic data for Sclerotinia head rot (SHR) consisted of five disease descriptors (disease incidence, DI; disease severity, DS; area under the disease progress curve for DI, AUDPCI, and DS, AUDPCS; and incubation period, IP). Two disease descriptors (DI and DS) were evaluated for two manifestations of Diaporthe/Phomopsis: Phomopsis stem canker (PSC) and Phomopsis head rot (PHR). In addition, a principal component (PC) analysis was used to derive transformed phenotypes as inputs to a univariate GWA (PC-GWA). Genotypic data comprised a panel of 4269 single nucleotide polymorphisms (SNP), generated via genotyping-by-sequencing. The GWA analysis revealed 24 unique marker-trait associations for SHR, 19 unique marker-trait associations for Diaporthe/Phomopsis diseases, and 7 markers associated with PC1 and PC2. No common markers were found for the response to the two pathogens. Nevertheless, epistatic interactions were identified between markers significantly associated with the response to S. sclerotiorum and Diaporthe/Phomopsis. This suggests that, while the main determinants of resistance may differ for the two pathogens, there could be an underlying common genetic basis. The exploration of regions physically close to the associated markers yielded 364 genes, of which 19 were predicted as putative disease resistance genes. This work presents the first simultaneous evaluation of two manifestations of Diaporthe/Phomopsis in sunflower, and undertakes a comprehensive GWA study by integrating PSC, PHR, and SHR data. The multiple regions identified, and their exploration to identify candidate genes, contribute not only to the understanding of the genetic basis of resistance, but also to the development of tools for assisted breeding.

Rapid Evolution of Seed Dormancy During Sunflower De-Domestication.

Hybridization between crops and their wild relatives may promote the evolution of de-domesticated (feral) weeds. Wild sunflower (Helianthus annuus L.) is typically found in ruderal environments, but crop-wild hybridization may facilitate the evolution of weedy populations. Using 1 crop-specific mitochondrial marker (CMS-PET1) and 14 nuclear SSR markers, we studied the origin and genetic diversity of a recently discovered weedy population of sunflower (named BRW). Then, using a resurrection approach, we tested for rapid evolution of weedy traits (seed dormancy, herbicide resistance, and competitive ability) by sampling weedy and wild populations 10 years apart (2007 and 2017). All the weedy plants present the CMS-PET1 cytotype, confirming their feral origin. At the nuclear markers, BRW showed higher genetic diversity than the cultivated lines and low differentiation with one wild population, suggesting that wild hybridization increased their genetic diversity. We found support for rapid evolution towards higher seed dormancy, but not for higher competitive ability or herbicide resistance. Our results highlight the importance of seed dormancy during the earliest stages of adaptation and show that crop-wild hybrids can evolve quickly in agricultural environments.

Expressing the sunflower transcription factor HaHB11 in maize improves waterlogging and defoliation tolerance.

The sunflower (Helianthus annuus) transcription factor HaHB11 (H. annuus  Homeobox 11) belongs to the homeodomain-leucine zipper family and confers improved yield to maize (Zea mays) hybrids (HiII × B73) and lines. Here we report that transgenic maize lines expressing HaHB11 exhibited better performance under waterlogging, both in greenhouse and field trials carried out during three growth cycles. Transgenic plants had increased chlorophyll content, wider stems, more nodal roots, greater total aerial biomass, a higher harvest index, and increased plant grain yield. Under severe defoliation caused by a windstorm during flowering, transgenic genotypes were able to set more grains than controls. This response was confirmed in controlled defoliation assays. Hybrids generated by crossing B73 HaHB11 lines with the contrasting Mo17 lines were also tested in the field and exhibited the same beneficial traits as the parental lines, compared with their respective controls. Moreover, they were less penalized by stress than commercial hybrids. Waterlogging tolerance increased via improvement of the root system, including more xylem vessels, reduced tissue damage, less superoxide accumulation, and altered carbohydrate metabolism. Multivariate analyses corroborated the robustness of the differential traits observed. Furthermore, canopy spectral reflectance data, computing 29 vegetation indices associated with biomass, chlorophyll, and abiotic stress, helped to distinguish genotypes as well as their growing conditions. Altogether the results reported here indicate that this sunflower gene constitutes a suitable tool to improve maize plants for environments prone to waterlogging and/or wind defoliation.

Genome-wide and comparative phylogenetic analysis of senescence-associated NAC transcription factors in sunflower (Helianthus annuus).

BACKGROUND: Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. RESULTS: To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. CONCLUSIONS: This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.

On-field phenotypic evaluation of sunflower populations for broad-spectrum resistance to Verticillium leaf mottle and wilt.

Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.

The "evil tribe" spreads across the land: A dated molecular phylogeny provides insight into dispersal, expansion, and biogeographic relationships within one of the largest tribes of the sunflower family (Vernonieae: Compositae).

PREMISE: With over 1500 species, the globally distributed Vernonieae is one of the most successful members of the largest family of flowering plants, the Compositae. However, due to its morphological complexity and limited geographic representation in previous studies, subtribal and biogeographic relationships are unclear. Here, new DNA sequence data spanning the geographic range of the tribe provides a taxonomically robust time-calibrated phylogeny, estimates migration pathways and timing of important biogeographic events, and allows inference of environmental factors that have contributed to the success of the Vernonieae worldwide. METHODS: Phylogenetic relationships were estimated for 368 taxa representing all Vernonieae subtribes. Molecular clock and ancestral range estimation analyses provide a framework for inference of the biogeographic history of the tribe. RESULTS: Relationships among the subtribes were established and correct placement determined for problematic taxa, along with the first model-based assessment of the biogeographic history of the tribe. The Vernonieae were estimated to have evolved ~50 mya. Africa was the first center of diversity, from which a single dispersal event established the monophyletic New World lineage. Long-distance dispersal from Africa and Brazil established the tribe on five continents and Oceania. CONCLUSIONS: The New World lineage is monophyletic, but Old World taxa are not. New subtribal taxonomies are needed. Moquinieae are nested in Vernonieae. Long-distance dispersal from Africa beginning 45 mya was key to establishing the tribe's near-global distribution. Migration corridors created by volcanic mountain chains and iron-rich soils in Africa and the Americas promoted radiation and range expansion.

Maternal control of early life history traits affects overwinter survival and seedling phenotypes in sunflower (Helianthus annuus L.).

When cultivated and wild plants hybridize, hybrids often show intermediate phenotypic traits relative to their parents, which makes them unfit in natural environments. However, maternal genetic effects may affect the outcome of hybridization by controlling expression of the earliest life history traits. Here, using wild, cultivated and reciprocal crop-wild sunflower (Helianthus annuus L.) hybrids, we evaluated the maternal effects on emergence timing and seedling establishment in the field and on seedling traits under controlled conditions. In the field, we evaluated reciprocal crop-wild hybrids between two wild populations with contrasting dormancy (the high dormant BAR and the low dormant DIA) and one cultivar (CROP) with low dormancy. Under controlled conditions, we evaluated reciprocal crop-wild hybrids between two wild populations (BAR and RCU) and one CROP under three contrasting temperature treatments. In the field, BAR overwintered as dormant seeds whereas DIA and CROP showed high autumn emergence (~50% of planted seeds), resulting in differential overwinter survival and seedling establishment in the spring. Reciprocal crop-wild hybrids resembled their female parents in emergence timing and success of seedling establishment. Under controlled conditions, we observed large maternal effects on most seedling traits across temperatures. Cotyledon size explained most of the variation in seedling traits, suggesting that the maternal effects on seed size have cascading effects on seedling traits. Maternal effects on early life history traits affect early plant survival and phenotypic variation of crop-wild hybrids, thus, they should be addressed in hybridization studies, especially those involving highly divergent parents, such as cultivated species and their wild ancestors.

Biochemical and molecular approach of oxidative damage triggered by water stress and rewatering in sunflower seedlings of two inbred lines with different ability to tolerate water stress.

Water stress accelerates the generation of reactive oxygen species, which trigger a cascade of antioxidative defence mechanisms comprising enzymatic and nonenzymatic antioxidants. The aim of this study was to investigate the oxidative damage and the antioxidative defence systems in seedlings of the water stress-tolerant (B71) and the sensitive (B59) inbred lines of sunflower (Helianthus annuus L.) in response to water stress and rewatering. In addition, we characterised the transcriptomic profile associated with enzymatic antioxidative defence. An elevated electrolyte leakage in B59 indicated increased plasmatic membrane permeability, which correlated with greater sensitivity to water stress. In response to water stress, both lines showed an increase in malondialdehyde and H2O2 content but these increases were more noticeable in the sensitive line. In both lines, an increase in enzymatic activity (e.g. peroxidase and ascorbate peroxidase) was not sufficient to overcome the H2O2 accumulation triggered by water stress. Upon water stress, the overall expression level of genes associated with the enzymatic antioxidant system increased in B71 and decreased in B59, which showed downregulated levels of most genes in the shoots. The general profile of phenolic compounds was clearly different between organs and between inbred lines. The B59 line activated nonenzymatic antioxidant responses to counteract the oxidative stress caused by water stress. The tolerance of B71 to water stress could be associated with compensatory antioxidant mechanisms based on the expression of genes encoding enzyme components of the ascorbate-glutathione and redoxin cycles, which contributed to explaining, at least partly, the response of this line.

Successful field performance in warm and dry environments of soybean expressing the sunflower transcription factor HB4.

Soybean yield is limited primarily by abiotic constraints. No transgenic soybean with improved abiotic stress tolerance is commercially available. We transformed soybean plants with genetic constructs able to express the sunflower transcription factor HaHB4, which confers drought tolerance to Arabidopsis and wheat. One line (b10H) carrying the sunflower promoter was chosen among three independent lines because it exhibited the best performance in seed yield, and was evaluated in the greenhouse and in 27 field trials in different environments in Argentina. In greenhouse experiments, transgenic plants showed increased seed yield under stress conditions together with greater epicotyl diameter, larger xylem area, and increased water use efficiency compared with controls. They also exhibited enhanced seed yield in warm and dry field conditions. This response was accompanied by an increase in seed number that was not compensated by a decrease in individual seed weight. Transcriptome analysis of plants from a field trial with maximum difference in seed yield between genotypes indicated the induction of genes encoding redox and heat shock proteins in b10H. Collectively, our results indicate that soybeans transformed with HaHB4 are expected to have a reduced seed yield penalty when cultivated in warm and dry conditions, which constitute the best target environments for this technology.

Phylogeography and the Evolutionary History of Sunflower (Helianthus annuus L.): Wild Diversity and the Dynamics of Domestication.

Patterns of genetic variation in crops are the result of selection and demographic changes that occurred during their domestication and improvement. In many cases, we have an incomplete picture of the origin of crops in the context of their wild progenitors, particularly with regard to the processes producing observed levels of standing genetic variation. Here, we analyzed sequence diversity in cultivated sunflower (Helianthus annuus L.) and its wild progenitor (common sunflower, also H. annuus) to reconstruct phylogeographic relationships and population genetic/demographic patterns across sunflower. In common sunflower, south-north patterns in the distribution of nucleotide diversity and lineage splitting indicate a history of rapid postglacial range expansion from southern refugia. Cultivated sunflower accessions formed a clade, nested among wild populations from the Great Plains, confirming a single domestication event in central North America. Furthermore, cultivated accessions sorted by market type (i.e., oilseed vs. confectionery) rather than breeding pool, recapitulating the secondary development of oil-rich cultivars during its breeding history. Across sunflower, estimates of nucleotide diversity and effective population sizes suggest that cultivated sunflower underwent significant population bottlenecks following its establishment ~5000 years ago. The patterns inferred here corroborate those from previous studies of sunflower domestication, and provide a comprehensive overview of its evolutionary history.

Genetic Diversity, Population Structure and Linkage Disequilibrium Assessment among International Sunflower Breeding Collections.

Sunflower germplasm collections are valuable resources for broadening the genetic base of commercial hybrids and ameliorate the risk of climate events. Nowadays, the most studied worldwide sunflower pre-breeding collections belong to INTA (Argentina), INRA (France), and USDA-UBC (United States of America-Canada). In this work, we assess the amount and distribution of genetic diversity (GD) available within and between these collections to estimate the distribution pattern of global diversity. A mixed genotyping strategy was implemented, by combining proprietary genotyping-by-sequencing data with public whole-genome-sequencing data, to generate an integrative 11,834-common single nucleotide polymorphism matrix including the three breeding collections. In general, the GD estimates obtained were moderate. An analysis of molecular variance provided evidence of population structure between breeding collections. However, the optimal number of subpopulations, studied via discriminant analysis of principal components (K = 12), the bayesian STRUCTURE algorithm (K = 6) and distance-based methods (K = 9) remains unclear, since no single unifying characteristic is apparent for any of the inferred groups. Different overall patterns of linkage disequilibrium (LD) were observed across chromosomes, with Chr10, Chr17, Chr5, and Chr2 showing the highest LD. This work represents the largest and most comprehensive inter-breeding collection analysis of genomic diversity for cultivated sunflower conducted to date.

Exploring gene networks in two sunflower lines with contrasting leaf senescence phenotype using a system biology approach.

BACKGROUND: Leaf senescence is a complex process, controlled by multiple genetic and environmental variables. In sunflower, leaf senescence is triggered abruptly following anthesis thereby limiting the capacity of plants to keep their green leaf area during grain filling, which subsequently has a strong impact on crop yield. Recently, we performed a selection of contrasting sunflower inbred lines for the progress of leaf senescence through a physiological, cytological and molecular approach. Here we present a large scale transcriptomic analysis using RNA-seq and its integration with metabolic profiles for two contrasting sunflower inbred lines, R453 and B481-6 (early and delayed senescence respectively), with the aim of identifying metabolic pathways associated to leaf senescence. RESULTS: Gene expression profiles revealed a higher number of differentially expressed genes, as well as, higher expression levels in R453, providing evidence for early activation of the senescence program in this line. Metabolic pathways associated with sugars and nutrient recycling were differentially regulated between the lines. Additionally, we identified transcription factors acting as hubs in the co-expression networks; some previously reported as senescence-associated genes in model species but many are novel candidate genes. CONCLUSIONS: Understanding the onset and the progress of the senescence process in crops and the identification of these new candidate genes will likely prove highly useful for different management strategies to mitigate the impact of senescence on crop yield. Functional characterization of candidate genes will help to develop molecular tools for biotechnological applications in breeding crop yield.

Dynamic regulation of chromatin topology and transcription by inverted repeat-derived small RNAs in sunflower.

Transposable elements (TEs) are extremely abundant in complex plant genomes. siRNAs of 24 nucleotides in length control transposon activity in a process that involves de novo methylation of targeted loci. Usually, these epigenetic modifications trigger nucleosome condensation and a permanent silencing of the affected loci. Here, we show that a TE-derived inverted repeat (IR) element, inserted near the sunflower HaWRKY6 locus, dynamically regulates the expression of the gene by altering chromatin topology. The transcripts of this IR element are processed into 24-nt siRNAs, triggering DNA methylation on its locus. These epigenetic marks stabilize the formation of tissue-specific loops in the chromatin. In leaves, an intragenic loop is formed, blocking HaWRKY6 transcription. While in cotyledons (Cots), formation of an alternative loop, encompassing the whole HaWRKY6 gene, enhances transcription of the gene. The formation of this loop changes the promoter directionality, reducing IR transcription, and ultimately releasing the loop. Our results provide evidence that TEs can act as active and dynamic regulatory elements within coding loci in a mechanism that combines RNA silencing, epigenetic modification, and chromatin remodeling machineries.

Genetic Diversity and Population Structure of Wild Sunflower (Helianthus annuus L.) in Argentina: Reconstructing Its Invasion History.

Studying the levels and patterns of genetic diversity of invasive populations is important to understand the evolutionary and ecological factors promoting invasions and for better designing preventive and control strategies. Wild sunflower (Helianthus annuus L.) is native to North America and was introduced, and has become invasive, in several countries, including Argentina (ARG). Here, using classical population genetic analyses and approximate Bayesian computation (ABC) modeling, we studied the invasion history of wild sunflower in ARG. We analyzed 115 individuals belonging to 15 populations from ARG (invasive range) and United States (US, native range) at 14 nuclear and 3 chloroplast simple sequence repeat markers along with 23 phenotypic variables. Populations from ARG showed similar levels of nuclear genetic diversity to US populations and higher genetic diversity in the chloroplast genome, indicating no severe genetic bottlenecks during the invasion process. Bayesian clustering analysis, based on nuclear markers, suggests the presence of 3 genetic clusters, all present in both US and ARG. Discriminant analysis of principal components (DAPC) detected an overall low population structure between central US and ARG populations but separated 2 invasive populations from the rest. ABC modeling supports multiple introductions but also a southward dispersal within ARG. Genetic and phenotypic data support the central US as a source of introduction while the source of secondary introductions could not be resolved. Finally, using genetic markers from the chloroplast genome, we found lower population structure in ARG when compared with US populations, suggesting a role for seed-mediated gene flow in Argentina.

Arabidopsis and sunflower plants with increased xylem area show enhanced seed yield.

Plant architecture plasticity determines the efficiency at harvesting and plays a major role defining biomass and seed yield. We observed that several previously described transgenic genotypes exhibiting increased seed yield also show wider stems and more vascular bundles than wild-type plants. Here, the relationship between these characteristics and seed yield was investigated. Hanging weight on the main stem of Arabidopsis plants provoked significant stem widening. Such widening was accompanied by an increase in the number of vascular bundles and about 100% of yield increase. In parallel, lignin deposition diminished. Vascular bundle formation started in the upper internode and continued downstream. AUX/LAX carriers were essential for this response. The increase of vascular bundles was reverted 3 weeks after the treatment leading to an enlarged xylem area. Aux1, lax1, and lax3 mutant plants were also able to enlarge their stems after the treatment, whereas lax2 plants did not. However, none of these mutants exhibited more vascular bundles or seed yield compared with untreated plants. Weight-induced xylem area enhancement and increased seed yield were also observed in sunflower plants. Altogether these results showed a strong correlation between the number of vascular bundles and enhanced seed yield under a long-day photoperiod. Furthermore, changes in the levels of auxin carriers affected both these processes in the same manner, suggesting that there may be an underlying causality.

Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type.

HaHB4 is a sunflower transcription factor belonging to the homeodomain-leucine zipper I family whose ectopic expression in Arabidopsis triggers drought tolerance. The use of PCR to clone the HaHB4 coding sequence for wheat transformation caused unprogrammed mutations producing subtle differences in its activation ability in yeast. Transgenic wheat plants carrying a mutated version of HaHB4 were tested in 37 field experiments. A selected transgenic line yielded 6% more (P<0.001) and had 9.4% larger water use efficiency (P<0.02) than its control across the evaluated environments. Differences in grain yield between cultivars were explained by the 8% improvement in grain number per square meter (P<0.0001), and were more pronounced in stress (16% benefit) than in non-stress conditions (3% benefit), reaching a maximum of 97% in one of the driest environments. Increased grain number per square meter of transgenic plants was accompanied by positive trends in spikelet numbers per spike, tillers per plant, and fertile florets per plant. The gene transcripts associated with abiotic stress showed that HaHB4's action was not dependent on the response triggered either by RD19 or by DREB1a, traditional candidates related to water deficit responses. HaHB4 enabled wheat to show some of the benefits of a species highly adapted to water scarcity, especially in marginal regions characterized by frequent droughts.

Effect of Ahasl1-1 and Ahasl1-4 alleles on herbicide resistance and its associated dominance in sunflower.

BACKGROUND: Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1-1 and Ahasl1-4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole-plant levels, and to determine the dominance relationships for imazapyr and metsulfuron-methyl herbicides. RESULTS: Resistant near-isogenic lines showed significantly lower specific AHAS activity than susceptible near-isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose-response for six near-isolines carrying different combinations of Ahasl1-1 and Ahasl1-4 alleles and two herbicides (imazapyr and metsulfuron-methyl) were evaluated at whole-plant and enzymatic levels. Ahasl1-1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron-methyl. Conversely, Ahasl1-4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole-plant level showed a semi-dominant behavior among the alleles for both herbicides. CONCLUSION: Ahasl1-4 allele confers higher resistance levels than Ahasl1-1 when evaluated with imazapyr and metsulfuron-methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids. © 2018 Society of Chemical Industry.

Evaluating rumen fermentation kinetics and nutritive value of sunflower seed meal supplemented with acetonic extract of pomegranate peel using in vitro gas production technique

This study was conducted to investigate effects of supplementing different levels (0.0, 0.5 and 1.0% of buffered rumen fluid) of acetonic extract of pomegranate peel on rumen fermentation kinetics of sunflower seed meal (SFM), using in vitro gas production technique. The samples were incubated in syringes containing rumen liquor obtained from three cannulated Iranian Ghezel rams for 2, 4, 6, 8, 12, 24 and 36h. Results indicated that, addition of acetonic extract of pomegranate peel resulted in increase in gas production volume in all of incubation times (p < 0.0001). Amount of gas production, also increased by increasing dose of the extract. Amounts of a (the gas production from the immediately soluble fraction), b (the gas production from the insoluble fraction) and a + b (the potential gas production) for pomegranate peel extract supplemented sunflower seed meal were higher (p < 0.05) than that of control meal. Adding pomegranate peel extract resulted in increase estimated short chain fatty acids (SCFA) production as well as digestible organic matter (DOM), metabolizable energy (ME) and net energy for lactation (NEL) content of SFM. Production of SCFA as well as DOM, ME and NEL content of SFM increased (p < 0.05) by enhancing the level of the extract supplementation. In conclusion, it can be suggest that, supplementing acetonic extract of pomegranate peel may be lead to higher ruminal fermentation and better nutritive value of SFM in ruminants.(AU)

Evaluating rumen fermentation kinetics and nutritive value of sunflower seed meal supplemented with acetonic extract of pomegranate peel using in vitro gas production technique

This study was conducted to investigate effects of supplementing different levels (0.0, 0.5 and 1.0% of buffered rumen fluid) of acetonic extract of pomegranate peel on rumen fermentation kinetics of sunflower seed meal (SFM), using in vitro gas production technique. The samples were incubated in syringes containing rumen liquor obtained from three cannulated Iranian Ghezel rams for 2, 4, 6, 8, 12, 24 and 36h. Results indicated that, addition of acetonic extract of pomegranate peel resulted in increase in gas production volume in all of incubation times (p < 0.0001). Amount of gas production, also increased by increasing dose of the extract. Amounts of a (the gas production from the immediately soluble fraction), b (the gas production from the insoluble fraction) and a + b (the potential gas production) for pomegranate peel extract supplemented sunflower seed meal were higher (p < 0.05) than that of control meal. Adding pomegranate peel extract resulted in increase estimated short chain fatty acids (SCFA) production as well as digestible organic matter (DOM), metabolizable energy (ME) and net energy for lactation (NEL) content of SFM. Production of SCFA as well as DOM, ME and NEL content of SFM increased (p < 0.05) by enhancing the level of the extract supplementation. In conclusion, it can be suggest that, supplementing acetonic extract of pomegranate peel may be lead to higher ruminal fermentation and better nutritive value of SFM in ruminants.