Salt tolerance in mungbean is associated with controlling Na and Cl transport across roots, regulating Na and Cl accumulation in chloroplasts and maintaining high K in root and leaf mesophyll cells.

Salinity tolerance requires coordinated responses encompassing salt exclusion in roots and tissue/cellular compartmentation of salt in leaves. We investigated the possible control points for salt ions transport in roots and tissue tolerance to Na+ and Cl- in leaves of two contrasting mungbean genotypes, salt-tolerant Jade AU and salt-sensitive BARI Mung-6, grown in nonsaline and saline (75 mM NaCl) soil. Cryo-SEM X-ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg, P, and S in various cell types in roots related to the development of apoplastic barriers, and in leaves related to photosynthetic performance. Jade AU exhibited superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis, and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell vacuoles compared to BARI Mung-6. Jade AU maintained higher [K] in root cells than BARI Mung-6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts and preferentially accumulated [K] in mesophyll cells than BARI Mung-6, resulting in higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in chloroplasts, and maintenance of high K in root and leaf mesophyll cells.

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.).

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L-1 hygromycin, 100 mg L-1 kanamycin and 200 mg L-1 cefotaxime. Additionally, 200 mg L-1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.

RNA-sequencing based gene expression landscape of guava cv. Allahabad Safeda and comparative analysis to colored cultivars.

BACKGROUND: Guava (Psidium guajava L.) is an important fruit crop of tropical and subtropical areas of the world. Genomics resources in guava are scanty. RNA-Seq based tissue specific expressed genomic information, de novo transcriptome assembly, functional annotation and differential expression among contrasting genotypes has a potential to set the stage for the functional genomics for traits of commerce like colored flesh and apple color peel. RESULTS: Development of fruit from flower involves orchestration of myriad molecular switches. We did comparative transcriptome sequencing on leaf, flower and fruit tissues of cv. Allahabad Safeda to understand important genes and pathways controlling fruit development. Tissue specific RNA sequencing and de novo transcriptome assembly using Trinity pipeline provided us the first reference transcriptome for guava consisting of 84,206 genes comprising 279,792 total transcripts with a N50 of 3603 bp. Blast2GO assigned annotation to 116,629 transcripts and PFam based HMM profile annotated 140,061 transcripts with protein domains. Differential expression with EdgeR identified 3033 genes in Allahabad Safeda tissues. Mapping the differentially expressed transcripts over molecular pathways indicate significant Ethylene and Abscisic acid hormonal changes and secondary metabolites, carbohydrate metabolism and fruit softening related gene transcripts during fruit development, maturation and ripening. Differential expression analysis among colored tissue comparisons in 3 cultivars Allahabad Safeda, Punjab Pink and Apple Color identified 68 candidate genes that might be controlling color development in guava fruit. Comparisons of red vs green peel in Apple Color, white pulp vs red pulp in Punjab Pink and fruit maturation vs ripening in non-colored Allahabad Safeda indicates up-regulation of ethylene biosynthesis accompanied to secondary metabolism like phenylpropanoid and monolignol pathways. CONCLUSIONS: Benchmarking Universal Single-Copy Orthologs analysis of de novo transcriptome of guava with eudicots identified 93.7% complete BUSCO genes. In silico differential gene expression among tissue types of Allahabad Safeda and validation of candidate genes with qRT-PCR in contrasting color genotypes promises the utility of this first guava transcriptome for its potential of tapping the genetic elements from germplasm collections for enhancing fruit traits.

Phenotypic characterisation and linkage mapping of domestication syndrome traits in yellow lupin (Lupinus luteus L.).

The transformation of wild plants into domesticated crops usually modifies a common set of characters referred to as 'domestication syndrome' traits such as the loss of pod shattering/seed dehiscence, loss of seed dormancy, reduced anti-nutritional compounds and changes in growth habit, phenology, flower and seed colour. Understanding the genetic control of domestication syndrome traits facilitates the efficient transfer of useful traits from wild progenitors into crops through crossing and selection. Domesticated forms of yellow lupin (Lupinus luteus L.) possess many domestication syndrome traits, while their genetic control remains a mystery. This study aimed to reveal the genetic control of yellow lupin domestication traits. This involved phenotypic characterisation of those traits, defining the genomic regions controlling domestication traits on a linkage map and performing a comparative genomic analysis of yellow lupin with its better-understood relatives, narrow-leafed lupin (L. angustifolius L.) and white lupin (L. albus L.). We phenotyped an F9 recombinant inbred line (RIL) population of a wide cross between Wodjil (domesticated) × P28213 (wild). Vernalisation responsiveness, alkaloid content, flower and seed colour in yellow lupin were each found to be controlled by single loci on linkage groups YL-21, YL-06, YL-03 and YL-38, respectively. Aligning the genomes of yellow with narrow-leafed lupin and white lupin revealed well-conserved synteny between these sister species (76% and 71%, respectively). This genomic comparison revealed that one of the key domestication traits, vernalisation-responsive flowering, mapped to a region of conserved synteny with the vernalisation-responsive flowering time Ku locus of narrow-leafed lupin, which has previously been shown to be controlled by an FT homologue. In contrast, the loci controlling alkaloid content were each found at non-syntenic regions among the three species. This provides a first glimpse into the molecular control of flowering time in yellow lupin and demonstrates both the power and the limitation of synteny as a tool for gene discovery in lupins.

Prediction of pathogenicity genes involved in adaptation to a lupin host in the fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum via comparative genomics.

BACKGROUND: Narrow-leafed lupin is an emerging crop of significance in agriculture, livestock feed and human health food. However, its susceptibility to various diseases is a major obstacle towards increased adoption. Sclerotinia sclerotiorum and Botrytis cinerea - both necrotrophs with broad host-ranges - are reported among the top 10 lupin pathogens. Whole-genome sequencing and comparative genomics are useful tools to discover genes responsible for interactions between pathogens and their hosts. RESULTS: Genomes were assembled for one isolate of B. cinerea and two isolates of S. sclerotiorum, which were isolated from either narrow-leafed or pearl lupin species. Comparative genomics analysis between lupin-derived isolates and others isolated from alternate hosts was used to predict between 94 to 98 effector gene candidates from among their respective non-conserved gene contents. CONCLUSIONS: Detection of minor differences between relatively recently-diverged isolates, originating from distinct regions and with hosts, may highlight novel or recent gene mutations and losses resulting from host adaptation in broad host-range fungal pathogens.

Changes in gene expression during germination reveal pea genotypes with either "quiescence" or "escape" mechanisms of waterlogging tolerance.

Waterlogging causes germination failure in pea (Pisum sativum L.). Three genotypes (BARI Motorshuti-3, Natore local-2 [NL-2], and Kaspa) contrasting in ability to germinate in waterlogged soil were exposed to different durations of waterlogging. Whole genome RNAseq was employed to capture differentially expressing genes. The ability to germinate in waterlogged soil was associated with testa colour and testa membrane integrity as confirmed by electrical conductivity measurements. Genotypes Kaspa and NL-2 displayed different mechanisms of tolerance. In Kaspa, an energy conserving strategy was indicated by a strong upregulation of tyrosine protein kinsase and down regulation of linoleate 9S-lipoxygenase 5, a fat metabolism gene. In contrast, a faster energy utilization strategy was suggested in NL-2 by the marked upregulation of a subtilase family protein and peroxisomal adenine nucleotide carrier 2, a fat metabolizing gene. Waterlogging susceptibility in germinating seeds of genotype BARI Motorshuti-3 was linked to upregulation of a kunitz-type trypsin/protease inhibitor that blocks protein metabolism and may lead to excessive lipid metabolism and the membrane leakage associated with waterlogging damage. Pathway analyses based on gene ontologies showed seed storage protein metabolism as upregulated in tolerant genotypes and downregulated in the sensitive genotype. Understanding the tolerance mechanism provides a platform to breed for adaptation to waterlogging stress at germination in pea.

Adapting legume crops to climate change using genomic approaches.

Our agricultural system and hence food security is threatened by combination of events, such as increasing population, the impacts of climate change, and the need to a more sustainable development. Evolutionary adaptation may help some species to overcome environmental changes through new selection pressures driven by climate change. However, success of evolutionary adaptation is dependent on various factors, one of which is the extent of genetic variation available within species. Genomic approaches provide an exceptional opportunity to identify genetic variation that can be employed in crop improvement programs. In this review, we illustrate some of the routinely used genomics-based methods as well as recent breakthroughs, which facilitate assessment of genetic variation and discovery of adaptive genes in legumes. Although additional information is needed, the current utility of selection tools indicate a robust ability to utilize existing variation among legumes to address the challenges of climate uncertainty.

The first genetic map for yellow lupin enables genetic dissection of adaptation traits in an orphan grain legume crop.

BACKGROUND: Yellow lupin (Lupinus luteus L.) is a promising grain legume for productive and sustainable crop rotations. It has the advantages of high tolerance to soil acidity and excellent seed quality, but its current yield potential is poor, especially in low rainfall environments. Key adaptation traits such as phenology and enhanced stress tolerance are often complex and controlled by several genes. Genomic-enabled technologies may help to improve our basic understanding of these traits and to provide selective markers in breeding. However, in yellow lupin there are very limited genomic resources to support research and no published information is available on the genetic control of adaptation traits. RESULTS: We aimed to address these deficiencies by developing the first linkage map for yellow lupin and conducting quantitative trait locus (QTL) analysis of yield under well-watered (WW) and water-deficit (WT) conditions. Two next-generation sequencing marker approaches - genotyping-by-sequencing (GBS) and Diversity Array Technology (DArT) sequencing - were employed to genotype a recombinant inbred line (RIL) population developed from a bi-parental cross between wild and domesticated parents. A total of 2,458 filtered single nucleotide polymorphism (SNP) and presence / absence variation (PAV) markers were used to develop a genetic map comprising 40 linkage groups, the first reported for this species. A number of significant QTLs controlling total biomass and 100-seed weight under two water (WW and WD) regimes were found on linkage groups YL-03, YL-09 and YL-26 that together explained 9 and 28% of total phenotypic variability. QTLs associated with length of the reproductive phase and time to flower were found on YL-01, YL-21, YL-35 and YL-40 that together explained a total of 12 and 44% of total phenotypic variation. CONCLUSION: These genomic resources and the QTL information offer significant potential for use in marker-assisted selection in yellow lupin.

Comparative Reaction of Camelina sativa to Sclerotinia sclerotiorum and Leptosphaeria maculans.

Sclerotinia sclerotiorum and Leptosphaeria maculans are two of the most important pathogens of many cruciferous crops. The reaction of 30 genotypes of Camelina sativa (false flax) was determined against both pathogens. C. sativa genotypes were inoculated at seedling and adult stages with two pathotypes of S. sclerotiorum, highly virulent MBRS-1 and less virulent WW-1. There were significant differences (P < 0.001) among genotypes, between pathotypes, and a significant interaction between genotypes and pathotypes in relation to percent cotyledon disease index (% CDI) and stem lesion length. Genotypes 370 (% CDI 20.5, stem lesion length 1.8 cm) and 253 (% CDI 24.8, stem lesion length 1.4 cm) not only consistently exhibited cotyledon and stem resistance, in contrast to susceptible genotype 2305 (% CDI 37.7, stem lesion length 7.2 cm), but their resistance was independent to S. sclerotiorum pathotype. A F5-recombinant inbred line population was developed from genotypes 370 × 2305 and responses characterized. Low broad-sense heritability indicated a complex pattern of inheritance of resistance to S. sclerotiorum. Six isolates of L. maculans, covering combinations of five different avirulent loci (i.e., five different races), were tested on C. sativa cotyledons across two experiments. There was a high level of resistance, with % CDI < 17, and including development of a hypersensitive reaction. This is the first report of variable reaction of C. sativa to different races of L. maculans and the first demonstrating comparative reactions of C. sativa to S. sclerotiorum and L. maculans. This study not only provides new understanding of these comparative resistances in C. sativa, but highlights their potential as new sources of resistance, both for crucifer disease-resistance breeding in general and to enable broader adoption of C. sativa as a more sustainable oilseed crop in its own right.

The western Mediterranean region provided the founder population of domesticated narrow-leafed lupin.

KEY MESSAGE: This study revealed that the western Mediterranean provided the founder population for domesticated narrow-leafed lupin and that genetic diversity decreased significantly during narrow-leafed lupin domestication. The evolutionary history of plants during domestication profoundly shaped the genome structure and genetic diversity of today's crops. Advances in next-generation sequencing technologies allow unprecedented opportunities to understand genome evolution in minor crops, which constitute the majority of plant domestications. A diverse set of 231 wild and domesticated narrow-leafed lupin (Lupinus angustifolius L.) accessions were subjected to genotyping-by-sequencing using diversity arrays technology. Phylogenetic, genome-wide divergence and linkage disequilibrium analyses were applied to identify the founder population of domesticated narrow-leafed lupin and the genome-wide effect of domestication on its genome. We found wild western Mediterranean population as the founder of domesticated narrow-leafed lupin. Domestication was associated with an almost threefold reduction in genome diversity in domesticated accessions compared to their wild relatives. Selective sweep analysis identified no significant footprints of selection around domestication loci. A genome-wide association study identified single nucleotide polymorphism markers associated with pod dehiscence. This new understanding of the genomic consequences of narrow-leafed lupin domestication along with molecular marker tools developed here will assist plant breeders more effectively access wild genetic diversity for crop improvement.

Exploring the genetic and adaptive diversity of a pan-Mediterranean crop wild relative: narrow-leafed lupin.

KEY MESSAGE: This first pan-Mediterranean analysis of genetic diversity in wild narrow-leafed lupin revealed strong East-West genetic differentiation of populations, an historic eastward migration, and signatures of genetic adaptation to climatic variables. Most grain crops suffer from a narrow genetic base, which limits their potential for adapting to new challenges such as increased stresses associated with climate change. Plant breeders are returning to the wild ancestors of crops and their close relatives to broaden the genetic base of their crops. Understanding the genetic adaptation of these wild relatives will help plant breeders most effectively use available wild diversity. Here, we took narrow-leafed lupin (Lupinus angustifolius L.) as a model to understand adaptation in a wild crop ancestor. A set of 142 wild accessions of narrow-leafed lupin from across the Mediterranean basin were subjected to genotyping-by-sequencing using Diversity Arrays Technology. Phylogenetic, linkage disequilibrium and demographic analyses were employed to explore the history of narrow-leafed lupin within the Mediterranean region. We found strong genetic differentiation between accessions from the western and eastern Mediterranean, evidence of an historic West to East migration, and that eastern Mediterranean narrow-leafed lupin experienced a severe and recent genetic bottleneck. We showed that these two populations differ for flowering time as a result of local adaptation, with the West flowering late while the East flowers early. A genome-wide association study identified single nucleotide polymorphism markers associated with climatic adaptation. Resolving the origin of wild narrow-leafed lupin and how its migration has induced adaptation to specific regions of the Mediterranean serves as a useful resource not only for developing narrow-leafed lupin cultivars with greater resilience to a changing climate, but also as a model which can be applied to other legumes.

An advanced reference genome of Trifolium subterraneum L. reveals genes related to agronomic performance.

Subterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano-based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super-scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4-fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re-annotated using transcriptome atlas data to contain 31 272 protein-coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium-specific and 6325 subterranean-clover-specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro-morphological traits, were fine-mapped on the high-density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker-assisted breeding, comparative mapping and identification of tissue-specific gene promoters for biotechnological improvement of forage legumes.

The loss of vernalization requirement in narrow-leafed lupin is associated with a deletion in the promoter and de-repressed expression of a Flowering Locus T (FT) homologue.

Adaptation of Lupinus angustifolius (narrow-leafed lupin) to cropping in southern Australian and northern Europe was transformed by a dominant mutation (Ku) that removed vernalization requirement for flowering. The Ku mutation is now widely used in lupin breeding to confer early flowering and maturity. We report here the identity of the Ku mutation. We used a range of genetic, genomic and gene expression approaches to determine whether Flowering Locus T (FT) homologues are associated with the Ku locus. One of four FT homologues present in the narrow-leafed lupin genome, LanFTc1, perfectly co-segregated with the Ku locus in a reference mapping population. Expression of LanFTc1 in the ku (late-flowering) parent was strongly induced by vernalization, in contrast to the Ku (early-flowering) parent, which showed constitutively high LanFTc1 expression. Co-segregation of this expression phenotype with the LanFTc1 genotype indicated that the Ku mutation impairs cis-regulation of LanFTc1. Sequencing of LanFTc1 revealed a 1.4-kb deletion in the promoter region, which was perfectly predictive of vernalization response in 216 wild and domesticated accessions. Linkage disequilibrium rapidly decayed around LanFTc1, suggesting that this deletion caused the loss of vernalization response. This is the first time a legume FTc subclade gene has been implicated in the vernalization response.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Camalexin Production in Camelina sativa is Independent of Cotyledon Resistance to Sclerotinia sclerotiorum.

Camelina sativa (L.) Crantz. has been proposed as a novel source of oilseed resistance to Sclerotinia rot (SR; causal agent Sclerotinia sclerotiorum (Lib.) de Bary). To assess factors likely important in determining the level of resistance to this pathogen, 30 diverse C. sativa genotypes were evaluated using a cotyledon test under controlled environmental conditions. Confirmed cotyledon SR-resistant (CS370) and SR-susceptible (CS2305) genotypes were assessed for camalexin production across time following inoculation at the 1-month vegetative stage of growth. There were significant differences among C. sativa genotypes in response to inoculation with S. sclerotiorum in terms of percent cotyledon disease index (%CDI), with the mean %CDI ranging from 30.9 to 69.4% across germplasm and confirmation screening, respectively. Genotype CS370 consistently showed low %CDI indicating high level of resistance to S. sclerotiorum, whereas CS2305 showed the highest %CDI value. These findings highlight the potential to develop highly SR-resistant cultivars of C. sativa by selection. Furthermore, liquid chromatographic analysis of leaves for both SR-resistant and SR-susceptible genotypes demonstrated that camalexin was produced when inoculated with S. sclerotiorum. However, camalexin production was not linked with disease severity in either genotype, indicating that SR resistance in C. sativa is independent of the level of camalexin production.

Genetic diversity and candidate genes for transient waterlogging tolerance in mungbean at the germination and seedling stages.

Mungbean [Vigna radiata var. radiata (L.) Wilczek] production in Asia is detrimentally affected by transient soil waterlogging caused by unseasonal and increasingly frequent extreme precipitation events. While mungbean exhibits sensitivity to waterlogging, there has been insufficient exploration of germplasm for waterlogging tolerance, as well as limited investigation into the genetic basis for tolerance to identify valuable loci. This research investigated the diversity of transient waterlogging tolerance in a mini-core germplasm collection of mungbean and identified candidate genes for adaptive traits of interest using genome-wide association studies (GWAS) at two critical stages of growth: germination and seedling stage (i.e., once the first trifoliate leaf had fully-expanded). In a temperature-controlled glasshouse, 292 genotypes were screened for tolerance after (i) 4 days of waterlogging followed by 7 days of recovery at the germination stage and (ii) 8 days of waterlogging followed by 7 days of recovery at the seedling stage. Tolerance was measured against drained controls. GWAS was conducted using 3,522 high-quality DArTseq-derived SNPs, revealing five significant associations with five phenotypic traits indicating improved tolerance. Waterlogging tolerance was positively correlated with the formation of adventitious roots and higher dry masses. FGGY carbohydrate kinase domain-containing protein was identified as a candidate gene for adventitious rooting and mRNA-uncharacterized LOC111241851, Caffeoyl-CoA O-methyltransferase At4g26220 and MORC family CW-type zinc finger protein 3 and zinc finger protein 2B genes for shoot, root, and total dry matter production. Moderate to high broad-sense heritability was exhibited for all phenotypic traits, including seed emergence (81%), adventitious rooting (56%), shoot dry mass (81%), root dry mass (79%) and SPAD chlorophyll content (70%). The heritability estimates, marker-trait associations, and identification of sources of waterlogging tolerant germplasm from this study demonstrate high potential for marker-assisted selection of tolerance traits to accelerate breeding of climate-resilient mungbean varieties.

Leaf formononetin content of Trifolium subterraneum increases in response to waterlogging but its proportion of total isoflavones is little changed.

The isoflavone formononetin (F) impacts livestock fertility and cultivars of the pasture legume Trifolium subterraneum L. (subclover) have been selected for F levels ≤0.2% of leaf dry weight. However, the impact of waterlogging (WL) on isoflavones is little studied. We investigated the response of isoflavones, biochanin A (BA), genistein (G) and F, to WL for: (1) Yarloop (high F) and eight low F cultivars each from subspecies subterraneum , brachycalycinum and yanninicum (Experiment 1); and (2) four cultivars and 12 ecotypes of ssp. yanninicum (Experiment 2). WL impacted F: estimated means increased from 0.19% (control) to 0.31% (WL) in Experiment 1 and from 0.61% to 0.97% in Experiment 2. Isoflavones under WL were highly heritable, particularly F (H 2 =95%). The proportions of BA, G and F were little changed by WL, with strong positive correlations between free-drained and waterlogged treatments. Isoflavone contents were not related to WL tolerance, as assessed by shoot relative growth rate. In conclusion, isoflavones varied among genotypes and increased with WL, but the proportion of individual isoflavones in each genotype was stable. High F under WL was unrelated with genotype tolerance to WL. Instead, it was a consequence of inherently high F for that particular genotype.

Productivity gains in vegetables from rice husk biochar application in nutrient-poor soils in Timor-Leste.

Response to fertilisation with biochar is greatest in field crops on acidic tropical soils, but limited information is available for vegetable crops. As a case-study using vegetable production in Timor-Leste, we assessed if biochar alleviates nutritional constraints to vegetables in low-nutrient soils. Field trials on vegetable crops were conducted with fertiliser combinations of rice husk biochar, phosphate and local fertiliser at three sites. A pot soil incubation trial of biochar was undertaken with soil from the acid site, where rice husk biochar had a larger effect on productivity than the other fertilisers in chili pepper, tomato and soybean with an average yield increase with biochar of 230% over control. Combining phosphate with biochar augmented the yield over biochar alone in chili pepper, tomato and soybean. At neutral and alkaline sites, fertilisation with biochar lifted mean yield over the control. Soil constraints alleviated by fertiliser were primarily from P and Zn deficiencies. Marked increases in vegetable yields, among the highest globally, were achieved with fertilisation with biochar individually and in combination with phosphate in low nutrient soil in Timor-Leste. Clearly, rice husk biochar is a promising avenue to fertilise the soil with P and Zn and increase crop productivity in Timor-Leste.

Tolerance of four grain legume species to waterlogging, hypoxia and anoxia at germination and recovery.

Legume seeds, when relay sown following rice, may suffer from soil waterlogging and the associated hypoxia or even anoxia. This study evaluated the tolerance of grain legume species, grass pea (three genotypes), lentil (two genotypes), faba bean (two genotypes) and field pea (one genotype), to soil waterlogging in a glasshouse, to anoxia and hypoxia in temperature-controlled room at germination and seedling stages. Changes in oxygen in the surface layers of soil, with time after waterlogging, were measured by microelectrode profiling. The soil profiling showed that soil oxygen declined and then stabilized by the fourth day after waterlogging and oxygen was not detected at 8 mm below the soil surface. Germination of seeds under waterlogging for up to 12 days and seedling survival after the soil was drained for up to 36 days were measured in pot experiments. Seed germination and/or survival in anoxia (N2-flushed solutions) and hypoxia (1.0 and 2.5 kPa oxygen) were evaluated, and so were post-anoxia or post-hypoxia recoveries, all in comparison with aerated controls. Lentil had higher seedling emergence (55 %) than the other species during soil waterlogging. However, lentil had lower seedling survival (9 %) than grass pea (28 %) during recovery following soil drainage. Grass pea seeds were more tolerant of anoxia and of hypoxia than the seeds of the three other species. In conclusion, grass pea, with higher percent germination and seedling survival during recovery, is more tolerant to waterlogging and subsequent soil drainage than the three other grain legume species. Grass pea was also more tolerant of hypoxia and of anoxia at the seed germination stage. These findings demonstrate the superior waterlogging tolerance of grass pea in relay sowing, as compared with the other grain legumes.

Response of Mungbean (cvs. Celera II-AU and Jade-AU) and Blackgram (cv. Onyx-AU) to Transient Waterlogging.

Mungbean [Vigna radiata (L.) Wilczek] and blackgram [Vigna mungo (L.) Hepper] are important crops for smallholder farmers in tropical and subtropical regions. Production of both crops is affected by unexpected and increasingly frequent extreme precipitation events, which result in transient soil waterlogging. This study aimed to compare the waterlogging tolerance of mungbean and blackgram genotypes under the varying duration of waterlogging stress at germination and seedling stages. We evaluated the responses to different durations of transient waterlogging in a sandy clay loam under temperature-controlled glasshouse conditions. Waterlogging durations were 0, 1, 2, 3, 4, 5, 6, 7, and 8 days during germination and 0, 2, 4, 8, and 16 days during the seedling stage. We used two mungbean genotypes (green testa), Celera II-AU (small-seeded), and Jade-AU (large-seeded), contrasting in seed size and hypocotyl pigmentation, and a blackgram genotype (black testa), Onyx-AU. Waterlogging reduced soil redox potential, delayed or even prevented germination, decreased seedling establishment, and affected shoot and root development. In the seedlings waterlogged (WL) at 15 days after sowing (DAS), adventitious root formation and crown nodulation varied between the genotypes, and 16 days of waterlogging substantially reduced growth but did not result in plant death. Plants in soil with waterlogging for 8-16 days followed by drainage and sampling at 39 DAS had reduced shoot and root dry mass by 60-65% in mungbean and 40% in blackgram compared with continuously drained controls, due at least in part to fewer lateral roots. Soil plant analysis development (SPAD) chlorophyll content was also reduced. Onyx-AU, a blackgram genotype, was more tolerant to transient waterlogging than Jade-AU and Celera II-AU in both growth stages. Of the two mungbean genotypes, Celera II-AU had a greater seedling establishment than Jade-AU post waterlogging imposed at sowing. In contrast, Jade-AU had more plant biomass and greater recovery growth than Celera II-AU after waterlogging and recovery during the seedling stage. Both species were delayed in emergence in response to the shorter periods of transient waterlogging at germination, and with the longer waterlogging germination and emergence failed, whereas at the seedling stage both showed adaptation by the formation of adventitious roots.