Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley.

KEY MESSAGE: The hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm. The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

FLOURY ENDOSPERM 6 mutations enhance the sugary phenotype caused by the loss of ISOAMYLASE1 in barley.

KEY MESSAGE: Barley double mutants in two genes involved in starch granule morphology, HvFLO6 and HvISA1, had impaired starch accumulation and higher grain sugar levels than either single mutant. Starch is a biologically and commercially important glucose polymer synthesized by plants as semicrystalline starch granules (SGs). Because SG morphology affects starch properties, mutants with altered SG morphology may be useful in breeding crops with desirable starch properties, including potentially novel properties. In this study, we employed a simple screen for mutants with altered SG morphology in barley (Hordeum vulgare). We isolated mutants that formed compound SGs together with the normal simple SGs in the endosperm and found that they were allelic mutants of the starch biosynthesis genes ISOAMYLASE1 (HvISA1) and FLOURY ENDOSPERM 6 (HvFLO6), encoding starch debranching enzyme and CARBOHYDRATE-BINDING MODULE 48-containing protein, respectively. We generated the hvflo6 hvisa1 double mutant and showed that it had significantly reduced starch biosynthesis and developed shrunken grains. In contrast to starch, soluble α-glucan, phytoglycogen, and sugars accumulated to higher levels in the double mutant than in the single mutants. In addition, the double mutants showed defects in SG morphology in the endosperm and in the pollen. This novel genetic interaction suggests that hvflo6 acts as an enhancer of the sugary phenotype caused by hvisa1 mutation.

Regulation of germination by targeted mutagenesis of grain dormancy genes in barley.

High humidity during harvest season often causes pre-harvest sprouting in barley (Hordeum vulgare). Prolonged grain dormancy prevents pre-harvest sprouting; however, extended dormancy can interfere with malt production and uniform germination upon sowing. In this study, we used Cas9-induced targeted mutagenesis to create single and double mutants in QTL FOR SEED DORMANCY 1 (Qsd1) and Qsd2 in the same genetic background. We performed germination assays in independent qsd1 and qsd2 single mutants, as well as in two double mutants, which revealed a strong repression of germination in the mutants. These results demonstrated that normal early grain germination requires both Qsd1 and Qsd2 function. However, germination of qsd1 was promoted by treatment with 3% hydrogen peroxide, supporting the notion that the mutants exhibit delayed germination. Likewise, exposure to cold temperatures largely alleviated the block of germination in the single and double mutants. Notably, qsd1 mutants partially suppress the long dormancy phenotype of qsd2, while qsd2 mutant grains failed to germinate in the light, but not in the dark. Consistent with the delay in germination, abscisic acid accumulated in all mutants relative to the wild type, but abscisic acid levels cannot maintain long-term dormancy and only delay germination. Elucidation of mutant allele interactions, such as those shown in this study, are important for fine-tuning traits that will lead to the design of grain dormancy through combinations of mutant alleles. Thus, these mutants will provide the necessary germplasm to study grain dormancy and germination in barley.

A crucial role for a node-localized transporter, HvSPDT, in loading phosphorus into barley grains.

Grains are the major sink of phosphorus (P) in cereal crops, accounting for 60-85% of total plant P, but the mechanisms underlying P loading into the grains are poorly understood. We functionally characterized a transporter gene required for the distribution of P to the grains in barley (Hordeum vulgare), HvSPDT (SULTR-like phosphorus distribution transporter). HvSPDT encoded a plasma membrane-localized Pi/H+ cotransporter. It was mainly expressed in the nodes at both the vegetative and reproductive stages. Furthermore, its expression was induced by inorganic phosphate (Pi) deficiency. In the nodes, HvSPDT was expressed in both the xylem and phloem region of enlarged and diffuse vascular bundles. Knockout of HvSPDT decreased the distribution of P to new leaves, but increased the distribution to old leaves at the vegetative growth stage under low P supply. However, knockout of HvSPDT did not alter the redistribution of P from old to young organs. At the reproductive stage, knockout of HvSPDT significantly decreased P allocation to the grains, resulting in a considerable reduction in grain yield, especially under P-limited conditions. Our results indicate that node-based HvSPDT plays a crucial role in loading P into barley grains through preferentially distributing P from the xylem and further to the phloem.

Targeted genome modifications in cereal crops.

The recent advent of customizable endonucleases has led to remarkable advances in genetic engineering, as these molecular scissors allow for the targeted introduction of mutations or even precisely predefined genetic modifications into virtually any genomic target site of choice. Thanks to its unprecedented precision, efficiency, and functional versatility, this technology, commonly referred to as genome editing, has become an effective force not only in basic research devoted to the elucidation of gene function, but also for knowledge-based improvement of crop traits. Among the different platforms currently available for site-directed genome modifications, RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases have proven to be the most powerful. This review provides an application-oriented overview of the development of customizable endonucleases, current approaches to cereal crop breeding, and future opportunities in this field.

Agrobacterium tumefaciens Enhances Biosynthesis of Two Distinct Auxins in the Formation of Crown Galls.

The plant pathogen Agrobacterium tumefaciens infects plants and introduces the transferred-DNA (T-DNA) region of the Ti-plasmid into nuclear DNA of host plants to induce the formation of tumors (crown galls). The T-DNA region carries iaaM and iaaH genes for synthesis of the plant hormone auxin, indole-3-acetic acid (IAA). It has been demonstrated that the iaaM gene encodes a tryptophan 2-monooxygenase which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM), and the iaaH gene encodes an amidase for subsequent conversion of IAM to IAA. In this article, we demonstrate that A. tumefaciens enhances the production of both IAA and phenylacetic acid (PAA), another auxin which does not show polar transport characteristics, in the formation of crown galls. Using liquid chromatography-tandem mass spectroscopy, we found that the endogenous levels of phenylacetamide (PAM) and PAA metabolites, as well as IAM and IAA metabolites, are remarkably increased in crown galls formed on the stem of tomato plants, implying that two distinct auxins are simultaneously synthesized via the IaaM-IaaH pathway. Moreover, we found that the induction of the iaaM gene dramatically elevated the levels of PAM, PAA and its metabolites, along with IAM, IAA and its metabolites, in Arabidopsis and barley. From these results, we conclude that A. tumefaciens enhances biosynthesis of two distinct auxins in the formation of crown galls.

Simultaneous regulation of F5H in COMT-RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis.

Ferulate 5-hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O-methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT-down-regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down-regulation of F5H in COMT-RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5-OH G units. Conversely, overexpression of F5H in COMT-RNAi transgenic plants reduced G units and increased 5-OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down-regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up-regulation and down-regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

QTLs maintaining grain fertility under salt stress detected by exome QTL-seq and interval mapping in barley.

Enhancing salt stress tolerance is a key strategy for increasing global food production. We previously found that long-term salinity stress significantly reduced grain fertility in the salt-sensitive barley (Hordeum vulgare) accession, 'OUC613', but not in the salt-tolerant accession, 'OUE812', resulting in large differences in grain yield. Here, we examined the underlying causes of the difference in grain fertility between these accessions under long-term treatment with 150 or 200 mM NaCl from the seedling stage to harvest and identified quantitative trait loci (QTLs) for maintaining grain fertility. In an artificial pollination experiment of the two accessions, grain fertility was significantly reduced only in OUC613 plants produced using pollen from plants grown under NaCl stress, suggesting that the low grain fertility of OUC613 was mainly due to reduced pollen fertility. Using QTL-seq combined with exome-capture sequencing and composite interval mapping of recombinant inbred lines derived from a cross between OUE812 and OUC613, we identified a QTL (qRP-2Hb) for grain fertility on chromosome 2H. The QTL region includes two genes encoding an F-box protein and a TIFY protein that are associated with male sterility, highlighting the importance of this region for maintaining grain fertility under salt stress.

Exome QTL-seq maps monogenic locus and QTLs in barley.

BACKGROUND: QTL-seq, in combination with bulked segregant analysis and next-generation sequencing (NGS), is used to identify loci in small plant genomes, but is technically challenging to perform in species with large genomes, such as barley. A combination of exome sequencing and QTL-seq (exome QTL-seq) was used to map the mono-factorial Mendelian locus black lemma and pericarp (Blp) and QTLs for resistance to net blotch disease, a common disease of barley caused by the fungus Pyrenophora teres, which segregated in a population of 100 doubled haploid barley lines. METHODS: The provisional exome sequences were prepared by ordering the loci of expressed genes based on the genome information and concatenating genes with intervals of 200-bp spacer "N" for each chromosome. The QTL-seq pipeline was used to analyze short reads from the exome-captured library. RESULTS: In this study, short NGS reads of bulked total DNA samples from segregants with extreme trait values were subjected to exome capture, and the resulting exome sequences were aligned to the reference genome. SNP allele frequencies were compared to identify the locations of genes/QTLs responsible for the trait value differences between lines. For both objective traits examined, exome QTL-seq identified the monogenic Mendelian locus and associated QTLs. These findings were validated using conventional mapping approaches. CONCLUSIONS: Exome QTL-seq broadens the utility of NGS-based gene/QTL mapping in organisms with large genomes.

Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci.

KEY MESSAGE: The genetic substitution of transformation amenability alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars. Barley (Hordeum vulgare) cv. 'Golden Promise' is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from 'Haruna Nijo' × 'Golden Promise,' as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross 'Golden Promise' × 'Full Pint.' Based on SNP genotype, we selected lines having 'Golden Promise' alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to 'Golden Promise.' The results validate that the genetic substitution of TFA alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars.

Mitochondrial genome sequences from wild and cultivated barley (Hordeum vulgare).

BACKGROUND: Sequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. Barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of 5.1 Gbp. Wild barley (Hordeum vulgare ssp. spontaneum) and cultivated barley (H. vulgare ssp. vulgare) have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes. RESULTS: We determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley. Two independent circular maps of the 525,599 bp barley mitochondrial genome were constructed by de novo assembly of high-throughput sequencing reads of barley lines H602 and Haruna Nijo, with only three SNPs detected between haplotypes. These mitochondrial genomes contained 33 protein-coding genes, three ribosomal RNAs, 16 transfer RNAs, 188 new ORFs, six major repeat sequences and several types of transposable elements. Of the barley mitochondrial genome-encoded proteins, NAD6, NAD9 and RPS4 had unique structures among grass species. CONCLUSIONS: The mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was highly differentiated from that of other grass species. Mitochondrial genome sequencing is essential for annotating the barley nuclear genome; our mitochondrial sequencing identified a significant number of fragmented mitochondrial sequences in the reported nuclear genome sequences. Little polymorphism was detected in the barley mitochondrial genome sequences, which should be explored further to elucidate the evolution of barley.

A genomics approach to deciphering lignin biosynthesis in switchgrass.

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.

Prognostic Impact of the ABCC11/MRP8 Polymorphism in Adjuvant Oral Chemotherapy with S-1 for Non-Small Cell Lung Cancer.

BACKGROUND: Postoperative 1-year administration of S-1, an oral derivative of 5-fluorouracil (5-FU), was shown to be feasible in lung cancer. The 5-year survival rates of postoperative patients treated with S-1 adjuvant chemotherapy and the prognostic impact of clinicopathological factors were examined. METHODS: The data of 50 patients with curatively resected pathological stage IB to IIIA non-small cell lung cancer, who were treated with S-1 postoperatively, were analyzed. The prognostic impacts of 22 clinicopathological factors including expressions of the 5-FU pathway enzymes were evaluated. A single-nucleotide polymorphism (SNP), i.e. 538G>A (rs17822931), of ABCC11/MRP8, which encodes a 5-FU excretion enzyme that is known as an earwax type determinant, was also evaluated. RESULTS: The 5-year overall and relapse-free survival rates were 72.5 and 67.5%, respectively. A performance status ≥ 1, lymphatic vessel invasion, blood vessel invasion, and the A/A type of SNP538, which is responsible for the dry earwax type, were significantly associated with shorter relapse-free survivals. In 34 patients who showed a relative performance of 70% or more for chemotherapy, multivariate survival analysis indicated significant hazard ratios only for the A/A type of SNP538 (p = 0.007). CONCLUSIONS: S-1 has sufficient power as adjuvant chemotherapy. However, its effect might be small in the dry earwax type patient group in an adjuvant setting.

Targeted Modification of Grain Dormancy Genes in Barley.

Transgenesis technologies, such as overexpression or RNA interference-mediated suppression, have often been used to alter the activity of target genes. More recently developed targeted genome modification methods using customizable endonucleases allow for the regulation or knockout mutation of target genes without the necessity of integrating recombinant DNA. Such approaches make it possible to create novel alleles of target genes, thereby significantly contributing to crop improvement. Among these technologies, the Cas9 endonuclease-based method is widely applied to several crops, including barley (Hordeum vulgare). In this chapter, we describe an Agrobacterium-based approach to the targeted modification of grain dormancy genes in barley using RNA-guided Cas9 nuclease.

Genome Editing to Produce Knockout Mutations of Seed Dormancy Genes in Wheat.

Knockout mutants provide definitive information about the functions of genes related to agronomic traits, including seed dormancy. However, it takes many years to produce knockout mutants using conventional techniques in polyploid plants such as hexaploid wheat. Genome editing with sequence-specific nucleases is a promising approach for obtaining knockout mutations in all targeted homoeologs of wheat simultaneously. Here, we describe a procedure to produce a triple recessive mutant in wheat via genome editing. This protocol covers the evaluation of gRNA and Agrobacterium-mediated transformation to obtain edited wheat seedlings.

Discovery and Genome Characterization of a Closterovirus from Wheat Plants with Yellowing Leaf Symptoms in Japan.

Many aphid-borne viruses are important pathogens that affect wheat crops worldwide. An aphid-transmitted closterovirus named wheat yellow leaf virus (WYLV) was found to have infected wheat plants in Japan in the 1970s; however, since then, its viral genome sequence and occurrence in the field have not been investigated. We observed yellowing leaves in the 2018/2019 winter wheat-growing season in an experimental field in Japan where WYLV was detected five decades ago. A virome analysis of those yellow leaf samples lead to the discovery of a closterovirus together with a luteovirus (barley yellow dwarf virus PAV variant IIIa). The complete genomic sequence of this closterovirus, named wheat closterovirus 1 isolate WL19a (WhCV1-WL19a), consisted of 15,452 nucleotides harboring nine open reading frames. Additionally, we identified another WhCV1 isolate, WL20, in a wheat sample from the winter wheat-growing season of 2019/2020. A transmission test indicated that WhCV1-WL20 was able to form typical filamentous particles and transmissible by oat bird-cherry aphid (Rhopalosiphum pad). Sequence and phylogenetic analyses showed that WhCV1 was distantly related to members of the genus Closterovirus (family Closteroviridae), suggesting that the virus represents a novel species in the genus. Furthermore, the characterization of WhCV1-WL19a-derived small RNAs using high-throughput sequencing revealed highly abundant 22-nt-class small RNAs potentially derived from the 3'-terminal end of the WhCV1 negative-strand genomic RNA, indicating that this terminal end of the WhCV1 genome is likely particularly targeted for the synthesis of viral small RNAs in wheat plants. Our results provide further knowledge on closterovirus diversity and pathogenicity and suggest that the impact of WhCV1 on wheat production warrants further investigations.

Optimizing genome editing efficiency in wheat: Effects of heat treatments and different promoters for single guide RNA expression.

Genome editing is a promising method for simultaneously mutagenizing homoeologs in the three subgenomes of wheat (Triticum aestivum L.). However, the mutation rate via genome editing must be improved in order to analyze gene function and to quickly modify agronomic traits in wheat. Here, we examined the Cas9-induced mutation rates in wheat plants using two promoters for single guide RNA (sgRNA) expression and applying heat treatment during Agrobacterium tumefaciens-mediated transformation. Using the TaU6 promoter instead of the OsU6 promoter from rice (Oryza sativa L.) to drive sgRNA expression greatly improved the Cas9-induced mutation rate. Moreover, a heat treatment of 30°C for 1 day during tissue culture increased the Cas9-induced mutation rate and the variety of mutations obtained compared to tissue culture at the normal temperature (25°C). The same heat treatment did not affect the regeneration rates of transgenic plants but tended to increase the number of transgene integration sites in each transgenic plant. These results lay the foundation for improving the Cas9-induced mutation rate in wheat to enhance research on gene function and crop improvement.

A bifurcated palea mutant infers functional differentiation of WOX3 genes in flower and leaf morphogenesis of barley.

Barley (Hordeum vulgare) is the fourth most highly produced cereal in the world after wheat, rice and maize and is mainly utilized as malts and for animal feed. Barley, a model crop of the tribe Triticeae, is important in comparative analyses of Poaceae. However, molecular understanding about the developmental processes is limited in barley. Our previous work characterized one of two WUSCHEL-RELATED HOMEOBOX 3 (WOX3) genes present in the barley genome: NARROW LEAFED DWARF1 (NLD1). We demonstrated that NLD1 plays a pivotal role in the development of lateral organs. In the present study, we describe a bifurcated palea (bip) mutant of barley focusing on flower and leaf phenotypes. The palea in the bip mutant was split into two and develop towards inside the lemma surrounding the carpels and anthers. The bip mutant is devoid of lodicules, which develop in a pair at the base of the stamen within the lemma in normal barley. bip also exhibited malformations in leaves, such as narrow leaf due to underdeveloped leaf-blade width, and reduced trichome density. Map-based cloning and expression analysis indicated that BIP is identical to another barley WOX3 gene, named HvWOX3. The bip nld1 double mutant presented a more severe reduction in leaf-blade width and number of trichomes. By comparing the phenotypes and gene expression patterns of various WOX3 mutants, we concluded that leaf bilateral outgrowth and trichome development are promoted by both NLD1 and HvWOX3, but that HvWOX3 serves unique and pivotal functions in barley development that differ from those of NLD1.

Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.