Search Results (161)

Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu²⁺ ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu²⁺ and Zn²⁺ that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu²⁺ or Zn²⁺ presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu²⁺ and those treated with Zn²⁺ formed distinct groups based on DNA sequence and methylation markers. Zn²⁺ treated and control samples formed separate groups. Also, Cu²⁺ discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu²⁺ or Zn²⁺ ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye. Full article

Polyploidy plays a significant role in loquat breeding, particularly in triploid breeding for seedless fruit production. Currently, loquat polyploid breeding primarily relies on natural seedling selection and sexual hybridization approaches. In this study, unfertilized ovules from four loquat varieties were in vitro cultured. Gynogenesis and embryoid regeneration were achieved in ‘Xingning 1’ and ‘Huabai 1’, with ‘Xingning 1’ demonstrating the highest gynogenesis efficiency (21.63%). Flow cytometry and chromosome counting revealed that the obtained embryoid lines included haploid, diploid, tetraploid, hexaploid, and chimeric ploidy types. Further characterization of ‘Xingning 1’-derived embryoid lines through SSR markers and whole-genome resequencing confirmed that the haploid, diploid, tetraploid, and hexaploidy embryoid originated from haploid–somatic chimeras, diploid, doubled diploid and tripled diploid, respectively. Metabolic analysis showed a positive correlation between ploidy level and the content of both soluble sugars and organic acids. This study explored a novel platform for polyploid induction in loquat and may provide methodological insights for improvement of other perennial fruit trees. Full article

Cannabis sativa L. is a dioecious species known to produce over 1600 chemical constituents, including more than 180 cannabinoids classified into 11 structural groups. These bioactive compounds are predominantly synthesised in the glandular trichomes of female inflorescences. However, sex determination in C. sativa is influenced by both genetic and environmental factors, often leading to the development of male flowers on female plants. This unintended fertilisation reduces cannabinoid yield and increases genetic heterogeneity and challenges in medical cannabis production. Haploid and doubled haploid (DH) technologies offer a promising solution by rapidly generating homozygous lines from gametophytic (e.g., unpollinated ovaries and ovules) or sporophytic tissues (e.g., anthers and microspores) via in vitro culture or chromosome reduction during hybridisation. In land plants, the life cycle alternates between a diploid sporophyte and a haploid gametophyte generation, both capable of mitotic division to form multicellular bodies. A single genome regulates this phase transition and encodes the molecular, genetic, and epigenetic mechanisms that precisely control the developmental processes unique to each generation. While the application of haploid technology in C. sativa remains limited, through recent progress in haploid induction (HI) and CRISPR-based genome editing, the direct modification of haploid gametes or embryos enables the creation of null homozygous lines following chromosome doubling, improving genetic uniformity. Understanding the molecular mechanisms of spontaneous chromosome doubling may further facilitate the development of elite cannabis genotypes. Ultimately, enhancing the efficiency of DH production and optimising genome editing approaches could significantly increase the speed of genetic improvement and cultivar development in Cannabis sativa. Full article

Blackleg disease poses a major threat to global canola production. The resistance gene Rlm1, corresponding to the avirulence gene AvrLm1 in the pathogen Leptosphaeria maculans, has been widely used to mitigate the impact of the disease. To investigate the biochemical basis of Rlm1-mediated resistance against blackleg, we conducted an LC-MS–based analysis of a susceptible Topas double haploid (DH) line and its isogenic Rlm1-carrying resistant counterpart for metabolomic profiles during the infection process. Samples were labeled with ¹²C- and ¹³C for LC-MS analyses to enhance both chemical and physical properties of metabolites for improved quantification and detection sensitivity. Resistant plants showed early and sustained accumulation of several defense metabolites, notably pipecolic acid (PA, up to 326-fold), salicylic acid (SA), and gentisic acid (GA) in L. maculans-inoculated Topas–Rlm1 plants compared to mock-inoculated Topas–Rlm1 controls (adjusted p < 0.05), indicating activation of lysine degradation and hormonal defense pathways. Elevated glucosinolates (GLS), γ-aminobutyric acid (GABA), and melatonin precursors may further contribute to antimicrobial defense and cell-wall reinforcement. In contrast, flavonoid and phenylpropanoid pathways were down-regulated, suggesting metabolic reallocation during resistance. Exogenous application of PA, SA, GA, ferulic acid, and piperonylic acid (a known inhibitor of the phenylpropanoid pathway in plants) significantly reduced infection in susceptible canola varieties, validating their defense roles against blackleg. These results offer new insights into Rlm1-mediated resistance and support metabolic targets for breeding durable blackleg resistance in canola. Full article

Grain yield establishment is a complex progress and the genetic basis of one of the most important yield components, 100-kernel weight, remains largely unknown. Here, we employed a double haploid (DH) population containing 477 lines which was developed from a cross of two maize elite inbred lines, PHBA6 and Chang7-2, to identify quantitative trait loci (QTL) that related to 100-kernel weight. The phenotypes of the DH population were acquired over three years in two different locations, while the DH lines were genotyped by next-generation sequencing technology of massively parallel 3ʹ end RNA sequencing (MP3RNA-seq). Eventually, 28,874 SNPs from 436 DH lines were preserved after SNP calling and filtering and a genetic map with a length of 837 cM was constructed. Then, single environment QTL analysis was performed using the R/qtl program, and it was found that a total of 17 QTLs related to 100-kernel weight were identified and distributed across the whole genome except chromosomes 5 and 6. The total phenotypic variation explained by QTLs detected in three different environments (BJ2016, BJ2107, and HN2018) was 22.2%, 32.9%, and 51.38%, respectively. Among these QTLs, three of them were identified across different environments as environmentally stable QTLs and explained more than 10% of the phenotypic variance each. Together, the results provided in this study preliminarily revealed the genetic basis of 100-kernel weight and will enhance molecular breeding for key agronomic kernel-related traits in maize. Full article

The production of carrot (D. carota L.) doubled haploids (DH) for the acceleration of this important vegetable crop breeding requires genome doubling of haploid regenerants. If spontaneous doubling does not occur, artificial chromosome doubling can be complicated by the lack of efficient genome-doubling protocols. We tested an antimitotic agent treatment of carrot at the embryo stage. It allowed us to produce and treat a large number of clonal carrot embryos (at least 30 embryos per treatment condition) in small volumes with minimal reagent amounts. We showed that 0.01–1 g/L colchicine did not perturb carrot development. Trifluralin showed no signs of toxicity at 0.001 and 0.01 g/L concentrations, but 0.1 g/L trifluralin reduced survival by 40% and delayed plantlet regeneration. We showed via DNA content flow cytometry that 0.01–1 g/L colchicine and 0.001–0.1 g/L trifluralin could double the carrot genome. The highest diploid percent was observed at 1 g/L colchicine (34%) and 0.1 g/L trifluralin (28%). The highest percent of diploids together with mixoploids (partial diploids) was at 0.01 and 0.1 g/L trifluralin (over 70%), followed by 1 g/L colchicine (56%). To our knowledge, this is the first report on trifluralin application for genome doubling in Apiaceae. In our study, we determined colchicine and trifluralin toxicity and doubling efficiency at different concentrations that can be used for carrot DH-line production and further improvement of genome doubling methods. Full article

Assessing the nutritional quality of silage maize (Zea mays L.) hinges largely on its oil content, a complex quantitative trait influenced by multiple genes. Mining candidate genes within oil content-related quantitative trait loci (QTLs) can provide genetic resources and a theoretical foundation for cultivating high-oil silage maize varieties. This study employed 274 doubled haploid (DH) lines derived from the parental lines BY4944 and DNF34-2 to perform main gene plus polygene mixed genetic analysis and complex interval mapping (CIM), with the goal of pinpointing oil content-related QTLs and genes distributed across the Z. mays L. genome. Leveraging 5400 single nucleotide polymorphism (SNPs), a high-resolution silage maize genetic linkage map covering 3864.51 cM was constructed with an average interval between markers of 0.74 cM. Analysis of the map revealed 13 oil content-related QTLs. The most significant large-effect QTL (qOIL-1-1), located on chromosome 1 within the region spanning 240.93 Mb to 256.57 Mb, exhibited a logarithm of odds (LOD) score of 3.34 and explained 5.06% of oil content-related phenotypic variation. Within these QTLs, 617 genes were annotated. Through transcriptome analysis combined with quantitative real-time polymerase chain reaction (RT-qPCR), five candidate genes potentially associated with oil content were predicted and subsequently validated within these genetic loci. This research underscores the potential of identifying candidate genes to enhance breeding efforts aimed at augmenting oil content, thereby advancing animal husbandry practices. Full article

The pasting properties of starch measured using the rapid visco analyzer (RVA) have important effects on the quality of wheat flour as well as flour-based foods. To identify quantitative trait loci (QTLs) for RVA parameters, a doubled-haploid population of 194 lines was used for linkage mapping in this study. A total of 39 QTLs with an LOD value ≥ 3.0 were detected across three years for six RVA parameters on 17 of the 21 chromosomes of common wheat (4A, 4B, 6B, and 7A were not considered). Among these QTLs, two QTLs for peak viscosity on 2A and 6A, two QTLs for trough viscosity on 2A and 6A, one QTL for breakdown on 5D, and two QTLs for setback on 5A and 7B were identified as the stable major QTLs detected in two or more environments, with phenotypic variation explanation exceeding 10%. Seven pleiotropic QTLs on 2A, 3D, 5A, 5B, 6A, 6D, and 7B were identified simultaneously with two or more RVA parameters. Molecular markers closely linked to the QTLs can be used to select the desired pasting property traits and provide assistance in breeding to improve wheat quality. Full article

In vitro anther culture is a technique used to produce haploid plants when regenerating varieties with specific traits. To generate haploid plants with preferred characteristics, an anther culture technique was established for Lilium longiflorum “Show Up”. Morphological characteristics were recorded, including the flower bud length and anther color corresponding to different stages of microspore development. The effects of different flower bud lengths, various concentrations of exogenous plant growth regulators (PGRs), low-temperature pretreatment at 4 °C, and incubation under dark conditions on the induction of callus formation were studied. When the flower buds were 2.2–2.4 cm in length and the microspores were in the mononuclear development phase, callus induction reached the highest rate (15.6%). Callus was not induced when the PGRs 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KT) were added separately to the growth medium, but the highest callus induction rate occurred when anthers were cultured on the medium containing 2,4-D (0.75–1.0 mg/L) and KT (4 mg/L). The low-temperature pretreatment significantly enhanced the induction rate of anthers, but prolonged low-temperature pretreatment reduced the induction rate. The optimal period of cultivation in darkness was 6 d. After 15 days of cultivation, the number of swollen anthers was recorded, and these were transferred onto the differentiation medium Murashige and Skoog (MS) + 1-naphthaleneacetic acid (NAA) (2.0 mg/L), sucrose (30 g/L), and agar (7 g/L) at pH 5.8, whereon 100% differentiation was recorded. Overall, 14 regenerated lines were obtained by in vitro anther culture. Chromosome ploidy was determined by counting chromosomes in the root tips of ten regenerated plants, and four were found to be haploids. This study lays the foundation for anther culture in lilies to shorten the breeding cycle, improve selection efficiency, facilitate efficient genetic transformation, and enable the effective production of both haploid and double-haploid plants. Full article

Germplasm improvement is essential for maize breeding. Currently, intra-heterotic-group crossing is the major method for germplasm improvement, while inter-heterotic-group crossing is also used in breeding but not in a systematic way. In this study, five inbred lines from four heterotic groups were used to develop a connected segregating population through inter-heterotic-group line crossing (CSPIC), which comprised 5 subpopulations with 535 doubled haploid (DH) lines and 15 related test-cross populations including 1568 hybrids. Significant genetic variation was observed in most subpopulations, with several DH populations exhibiting superior phenotypes regarding traits such as plant height (PH), ear height (EH), days to anthesis (DTA), and days to silking (DTS). Notably, 10.8% of hybrids in the population POP5/C229 surpassed the high-yielding hybrid ND678 (CK). To reduce field planting costs and quickly screen for the best inter-heterotic-group DH lines and test-cross hybrids, we assessed the accuracy of genomic selection (GS) for within- and between-population predictions in the DH populations and the test-cross populations. Within the DH or the hybrid population, the prediction accuracy varied across populations and traits, with an average hybrid yield prediction accuracy of 0.41, reaching 0.54 in POP5/Z58. In the cross DH population predictions, the prediction accuracy of the half-sib population exceeded that of the non-sib cross population prediction, with the highest accuracy observed when the non-shared parents were from the same heterotic group, and the average phenotypic prediction accuracies of POP3 predicting POP2 and POP2 predicting POP3 were 0.54 and 0.45, respectively. In the cross hybrid population predictions, the accuracy was highest when both the training and the test sets came from the same DH populations, with an average accuracy of 0.43. The proportion of shared polymorphisms with respect to SNPs between the training and the test sets (PSP) exhibited a significant and strong correlation with the prediction accuracy of cross population prediction. This study demonstrates the feasibility of creating new heterotic groups through inter-heterotic-group crossing in germplasm improvement, and some cross population prediction patterns exhibited excellent prediction accuracy. Full article

Cucumber mosaic virus (CMV) is one of the most dangerous viral diseases threatening Solanaceae crops, in particular Capsicum sp. This study aims to develop double haploid (DH) pepper lines from germplasm resistant to CMV in order to speed up the breeding process. For this purpose, six genotypes previously tested for CMV resistance were used. Two induction mediums (17-2 and 17-3) with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) for anther incubation and further plant regeneration were applied. L10 was the most responsive genotype, exhibiting the highest direct embryogenesis and the most plant regenerants on both mediums. Medium-specific response was observed in genotype L9 where regenerants were observed only on 17-2. Further, eight DH lines were evaluated with two CMV isolates (L-BG and PV-0418) and checked for local and systemic presence of the pathogen in leaves and fruits for a period of 60 days by DAS-ELISA. Of the tested DH lines, four (DH2, DH6, DH7 and DH9) were resistant to both strains, two (DH5 and DH14) were resistant to L-BG, and two (DH19 and DH21) were susceptible to both isolates. Field evaluation of DH7, DH9, and DH14 for some agronomic and morphological traits divided them into two groups according to the original genotypes. Full article

Brassicas are considered the third most important source of vegetable oil globally. With the escalating production of Brassica varieties, there is growing demand for high-yielding genotypes. Doubled haploid (DH) techniques have become very popular in various Brassica breeding programs. Such DH techniques can play a significant role in plant breeding by accelerating the production of homozygous lines and increasing selection efficiency. Among these methods, isolated microspore culture stands out as the most effective, facilitating the generation of a higher number of embryos compared to conventional methods of plant breeding. Different chemical compounds such as herbicides, brassinosteroids, and polyethylene glycol have an antimitotic effect and have been found to generate DH plants and improve microspore embryogenesis in Brassica species. Colchicine and trifluralin have proven to be efficient chromosome-doubling agents as well as important supplements that can increase the rate of embryogenesis. This review serves as a comprehensive summary and effectiveness evaluation of the latest research findings in the Brassica oil crops to help increase efficiency of the future research focusing on DH methods and application of antimitotic agents in the various oilseed species of the genus Brassica. Full article

In barley having adherent hulls, an irreversible connection between the pericarp with both palea and lemma is formed during grain maturation. A mutation in the NUDUM 1 (NUD1) gene prevents this connection and leads to the formation of barley with non-adherent hulls. A genetic model of two isogenic lines was used to elucidate the genetic mechanisms of hull adhesion: a doubled haploid line having adherent hulls and its derivative with non-adherent hulls obtained by targeted mutagenesis of the NUD1 gene. Comparative transcriptomics analysis of the grain coats was performed at two stages of development: the milk stage, when the hulls can still be easily detached from the pericarp, and the dough stage when the hull adhesion process occurs. It was shown that the main differences in the transcriptomes lie in the genes related to DNA replication and chromatin assembly, cell wall organization, and cuticle formation. Meanwhile, genes involved in lipid biosynthesis mostly show minor differences in expression between stages and genotypes and represent a limited set of active genes. Among the 3-ketoacyl-CoA synthase (KCS) genes active during grain development, candidates for key enzymes responsible for very long-chain fatty acid elongation were identified. Full article

Powdery mildew is a prevalent wheat disease that affects yield and quality. The characterization and fine mapping of genes associated with powdery mildew resistance can benefit marker-assisted breeding. In this study, quantitative trait loci (QTL) associated with powdery mildew were mapped using a high-density 35K DArT genetic linkage map developed from a population of double haploid lines (DHs) created by crossing “Jinmai 33 (a highly resistance line) with Yannong 19 (a highly susceptible line)”. Three stable QTLs for powdery mildew were identified on chromosomes 1B, 2B, and 6A combined with the composite interval graphing method and multiple interval mapping, explaining phenotypic variations (PVE) that range from 4.98% to 13.25%. Notably, Qpm.sxn-1B and Qpm.sxn-2B were identified across three environments, with the PVE ranging from 9.37% to 13.25% and from 4.98% to 5.23%, respectively. The synergistic effects of these QTLs were contributed by the parental line “Jinmai 33”. Qpm.sxn-1B was the major stable QTL, and Qpm.sxn-2B was close to Pm51. Furthermore, Qpm.sxn-6A was identified in two environments, accounting for PVE values of 7.13% and 7.65%, respectively, with the resistance effects originating from the male parent. Remarkably, this locus has not been reported previously, indicating that Qpm.sxn-6A represents a newly dis-covered QTL governing powdery mildew genes. Conclusions Five molecular markers available for mark-er-assisted selection were selected for tracking Qpm.sxn-1B and Qpm.sxn-2B in the program. The identification of this novel newly discovered QTL and markers reported in this study will be useful for marker-assisted selection of powdery mildew resistance. Full article

Maize (Zea mays L.) is one of the most widely cultivated grain crops globally. In sub-Saharan Africa (SSA), it plays an important role in ensuring both food and income security for smallholder farmers. This study was conducted to (i) assess the performances of testcross hybrids constituted from maize lethal necrosis (MLN) tolerant doubled haploid (DH) lines under various management conditions; (ii) estimate the combining ability effects and determine the nature of gene action in the DH lines; and (iii) identify DH lines and testcross hybrids for resistance to MLN, high grain yield, and other important traits. Eleven DH lines were crossed with 11 single-cross testers using the line-by-tester mating design, and 115 successful testcross hybrids were generated. These hybrids, along with five commercial check hybrids, were evaluated across four optimum management conditions, two MLN artificial inoculations, and one managed drought environment in Kenya. Under each management condition, the effects of genotypes, environments, and genotype-by-environment interactions were significant for grain yield (GY) and most other traits. Hybrids T1/L3, T10/L3, and T11/L3 exhibited higher grain yields under at least two management conditions. A combining ability analysis revealed that additive gene effects were more important than non-additive effects for GY and most other traits, except for leaf senescence (SEN) and MLN disease severity score. DH line L3 exhibited a desirable general combining ability (GCA) effect for GY, while L5 was the best general combiner for anthesis date (AD) and plant height (PH) across all management conditions. DH lines L2, L6, and L7 showed negative GCA effects for MLN disease severity. Single-cross testers T11 and T10 were good general combiners for GY under all management conditions. Hybrids T2/L11, T9/L10, and T2/L10 demonstrated high specific combining ability (SCA) effects for GY under all conditions. This study identified DH lines and testers with favorable GCA effects for grain yield, MLN resistance, and other agronomic traits that can be used in breeding programs to develop high-yielding and MLN-resistant maize varieties. Better-performing testcross hybrids identified in the current study could be verified through on-farm testing and released for commercial production to replace MLN-susceptible, low-yield hybrids grown in the target ecologies. Full article