Sterile Mutant Research Articles

Comparison of Metabolic Profiling of Arabidopsis Inflorescences Between Landsberg erecta and Columbia, and Meiosis-Defective Mutants by 1H-NMR Spectroscopy

The online version contains supplementary material available at 10.1007/s43657-021-00012-3.

Germ granule dysfunction is a hallmark and mirror of Piwi mutant sterility

In several species, Piwi/piRNA genome silencing defects cause immediate sterility that correlates with transposon expression and transposon-induced genomic instability. In C. elegans, mutations in the Piwi-related gene (prg-1) and other piRNA deficient mutants cause a transgenerational decline in fertility over a period of several generations. Here we show that the sterility of late generation piRNA mutants correlates poorly with increases in DNA damage signaling. Instead, sterile individuals consistently exhibit altered perinuclear germ granules. We show that disruption of germ granules does not activate transposon expression but induces multiple phenotypes found in sterile prg-1 pathway mutants. Furthermore, loss of the germ granule component pgl-1 enhances prg-1 mutant infertility. Environmental restoration of germ granule function for sterile pgl-1 mutants restores their fertility. We propose that Piwi mutant sterility is a reproductive arrest phenotype that is characterized by perturbed germ granule structure and is phenocopied by germ granule dysfunction, independent of genomic instability.

Effects of Carbon Ion Beam Irradiation on Phenotypic Variations and Biochemical Parameters in Early Generations of Soybean Plants

Understanding the characteristics of carbon ion beam irradiation-induced mutation is essential to its potential application in plant breeding. A carbon ion beam-mutagenized soybean population was generated from the newly released soybean variety Dongsheng 28, with irradiation dosages of 100 Gy, 120 Gy and 140 Gy. Many phenotypic variations and novel mutants with heritable tendencies including plant height mutants, sterile mutants, early mature mutants, rolled leaves and short petioles mutants, yield-related mutants and lodging-resistant mutants were identified. Diverse variations in seed size, seed protein and oil concentration were found. Increasing irradiation dosage from 100 Gy to 140 Gy increased leaf chlorophyll concentration in M1 generation, but this effect was significantly reduced in M2 generation. The activities of superoxide dismutase (SOD), peroxidase (POD) and malondialdehyde (MDA) concentration all showed wider variation in M1 and M2 generation, the only exception being that the MDA concentration was similar to the control in the M2 generation. Overall, we suggest that treating soybean seeds with carbon ion beam irradiation at a dosage of 120 Gy (80 Mev/u) could be effective in soybean mutation breeding.

Progress on genic male sterility gene in soybean.

Male sterility refers to the phenomenon that stamens cannot grow normally and produce viable pollen grains in plants. Hybrid seed production by taking advantage of the trait of male sterility is an effective and quick strategy to increase crop yield. Up to date, the yield of rice (Oryza sativa L.), maize (Zea mays L.), wheat (Triticum aestivum L.) and other crops has been greatly increased based on hybrid vigor utilization. Soybean (Glycine max (L.) Merr.) is a self-pollination species, artificial emasculation is not only time-consuming, but also labor-intensive and economically impracticable. So far, large scale hybrid breeding has not been performed in soybean due to the shortage of male sterile lines suitable for hybrid production. Therefore, it is urgent to identify a stable male sterile system for the rapid utilization of heterosis in soybean. In this review, we summarize the progress on the discovery of soybean genic male sterility (GMS) mutants and GMS genes. Combining with the investigation of GMS genes in Arabidopsis, rice and maize, we provide important insights into the identification and potential utilization of GMS genes in soybean in the perspective of reverse genetics.

CLK-2/TEL2 is a conserved component of the nonsense-mediated mRNA decay pathway.

Nonsense-mediated mRNA decay (NMD) controls eukaryotic mRNA quality, inducing the degradation of faulty transcripts. Key players in the NMD pathway were originally identified, through genetics, in Caenorhabditis elegans as smg (suppressor with morphological effect on genitalia) genes. Using forward genetics and fluorescence-based NMD reporters, we reexamined the genetic landscape underlying NMD. Employing a novel strategy for mapping sterile mutations, Het-Map, we identified clk-2, a conserved gene previously implicated in DNA damage signaling, as a player in the nematode NMD. We find that CLK-2 is expressed predominantly in the germline, highlighting the importance of auxiliary factors in tissue-specific mRNA decay. Importantly, the human counterpart of CLK-2/TEL2, TELO2, has been also implicated in the NMD, suggesting a conserved role of CLK-2/TEL2 proteins in mRNA surveillance. Recently, variants of TELO2 have been linked to an intellectual disability disorder, the You-Hoover-Fong syndrome, which could be related to its function in the NMD.

Identification and genetic analysis of two maize CMS-T mutants obtained from out-space-flighted seeds

Male sterility is widely utilized for hybrid seed production. In this study, two new found male sterile mutants SauS4 and SauS5 were obtained from space flighted seeds of maize inbred line RP125. Then, genetic analysis, molecular markers identification, and cytological observation were conducted to confirm their male sterile types. For genetic analysis, the above two male sterile mutants were continuously backcrossed with two maize inbred line 18Hong and RP125, and four stable male sterile lines SauS4(18Hong), SauS5(18Hong), SauS4(RP125), SauS5(RP125) were generated by six-generation backcross. Restoring and maintaining relationship analysis showed that both Hui313 and Zifeng1 didn’t rescue the male sterility SauS4(18Hong) and SauS5(18Hong). Using CMS mitochondria-specific primers for PCR detection suggested that only a 440 bp band unique to CMS-T type was amplified in SauS4(18Hong), SauS5(18Hong), SauS4(RP125), and SauS5(RP125). Sequencing results showed that these bands sequences were identical in DNA level which compared with T-urf13. Cytological observations showed that the main abortion stages of SauS4 and SauS5 were at the middle stage of uninucleate microspores under the two nuclear backgrounds of 18Hong and RP125, exhibiting the characteristics of sporophyte sterility. All the above results pointed out the two male sterile mutants SauS4 and SauS5 belonged to the CMS-T type. Interestingly, some mitochondrial genome difference between SauS4(RP125) and SauS5(RP125) were revealed by AFLP analysis.

Phenotype Analysis and Fine Mapping of the Male Sterile Mutant ms10 in Rice (Oryza sativa L.)

There is a positive correlation between fertility and yield, and the decrease of fertility is bound to a greatly reduced crop yield. Male sterile mutants can be used in hybrid rice. Therefore, rice male sterility has an impo... | Find, read and cite all the research you need on Tech Science Press

Understanding Ustilago maydis Infection of Multiple Maize Organs.

Ustilago maydis is a smut fungus that infects all aerial maize organs, namely, seedling leaves, tassels, and ears. In all organs, tumors are formed by inducing hypertrophy and hyperplasia in actively dividing cells; however, the vast differences in cell types and developmental stages for different parts of the plant requires that U. maydis have both general and organ-specific strategies for infecting maize. In this review, we summarize how the maize–U. maydis interaction can be studied using mutant U. maydis strains to better understand how individual effectors contribute to this interaction, either through general or specific expression in a cell type, tissue, or organ. We also examine how male sterile maize mutants that do not support tumor formation can be used to explore key features of the maize anthers that are required for successful infection. Finally, we discuss key unanswered questions about the maize–U. maydis interaction and how new technologies can potentially be used to answer them.

Advances in Research and Application of Male Sterility in Brassica oleracea

Brassica oleracea is an important vegetable species which belongs to the genus Brassica and the mustard family Brassicaceae Burnett. Strong heterosis in B. oleracea is displayed in yield, quality, disease resistance, and stress tolerance. Heterosis breeding is the main way to improve B. oleracea varieties. Male sterile mutants play an important role in the utilization of heterosis and the study of development and regulation in plant reproduction. In this paper, advances in the research and application of male sterility in B. oleracea were reviewed, including aspects of the genetics, cytological characteristics, discovery of genes related to male sterility, and application of male sterility in B. oleracea. Moreover, the main existing problems and prospect of male sterility application in B. oleracea were addressed and a new hybrids’ production strategy with recessive genic male sterility is introduced.

The SAP function in pistil development was proved by two allelic mutations in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

BackgroundPistil development is a complicated process in plants, and female sterile mutants are ideal material for screening and cloning pistil development-related genes. Using the female sterile mutant (fsm1), BraA04g009730.3C was previously predicted as a candidate mutant gene encoding the STERILE APETALA (SAP) transcriptional regulator. In the current study, a parallel female sterile mutant (fsm2) was derived from EMS mutagenesis of a Chinese cabbage DH line ‘FT’ seeds.ResultsBoth fsm2 and fsm1 mutant phenotypes exhibited pistil abortion and smaller floral organs. Genetic analysis indicated that the phenotype of mutant fsm2 was also controlled by a single recessive nuclear gene. Allelism testing showed that the mutated fsm1 and fsm2 genes were allelic. A single-nucleotide mutation (G-to-A) in the first exon of BraA04g009730.3C caused a missense mutation from GAA (glutamic acid) to GGA (glycine) in mutant fsm2 plants. Both allelic mutations of BraA04g009730.3C in fsm1 and fsm2 conferred the similar pistil abortion phenotype, which verified the SAP function in pistil development. To probe the mechanism of SAP-induced pistil abortion, we compared the mutant fsm1 and wild-type ‘FT’ pistil transcriptomes. Among the 3855 differentially expressed genes obtained, 29 were related to ovule development and 16 were related to organ size.ConclusionOur study clarified the function of BraA04g009730.3C and revealed that it was responsible for ovule development and organ size. These results lay a foundation to elucidate the molecular mechanism of pistil development in Chinese cabbage.

Deficiencies in the formation and regulation of anther cuticle and tryphine contribute to male sterility in cotton PGMS line

BackgroundMale sterility is a simple and efficient pollination control system that is widely exploited in hybrid breeding. In upland cotton, CCRI9106, a photosensitive genetic male sterile (PGMS) mutant isolated from CCRI040029, was reported of great advantages to cotton heterosis. However, little information concerning the male sterility of CCRI9106 is known. Here, comparative transcriptome analysis of CCRI9106 (the mutant, MT) and CCRI040029 (the wild type, WT) anthers in Anyang (long-day, male sterile condition to CCRI9106) was performed to reveal the potential male sterile mechanism of CCRI9106.ResultsLight and electron microscopy revealed that the male sterility phenotype of MT was mainly attributed to irregularly exine, lacking tryphine and immature anther cuticle. Based on the cytological characteristics of MT anthers, anther RNA libraries (18 in total) of tetrad (TTP), late uninucleate (lUNP) and binucleate (BNP) stages in MT and WT were constructed for transcriptomic analysis, therefore revealing a total of 870,4 differentially expressed genes (DEGs). By performing gene expression pattern analysis and protein-protein interaction (PPI) networks construction, we found down-regulation of DEGs, which enriched by the lipid biosynthetic process and the synthesis pathways of several types of secondary metabolites such as terpenoids, flavonoids and steroids, may crucial to the male sterility phenotype of MT, and resulting in the defects of anther cuticle and tryphine, even the irregularly exine. Furthermore, several lipid-related genes together with ABA-related genes and MYB transcription factors were identified as hub genes via weighted gene co-expression network analysis (WGCNA). Additionally, the ABA content of MT anthers was reduced across all stages when compared with WT anthers. At last, genes related to the formation of anther cuticle and tryphine could activated in MT under short-day condition.ConclusionsWe propose that the down-regulation of genes related to the assembly of anther cuticle and tryphine may lead to the male sterile phenotype of MT, and MYB transcription factors together with ABA played key regulatory roles in these processes. The conversion of fertility in different photoperiods may closely relate to the functional expression of these genes. These findings contribute to elucidate the mechanism of male sterility in upland cotton.

Cytological and molecular characterizations of a novel 2A nullisomic line derived from a widely-grown wheat cultivar Zhoumai 18 conferring male sterility.

A dwarf, multi-pistil and male sterile dms mutant was previously reported by us. However, the genetic changes in this dms are unclear. To examine the genetic changes, single nucleotide polymorphism (SNP) association, chromosome counting, and high-resolution chromosome fluorescence in situ hybridization (FISH) techniques were employed. By comparing tall plants (T) with dwarf plants (D) in the offspring of dms mutant plants, SNP association analysis indicated that most SNPs were on chromosome 2A. There were three types in offspring of dms plants, with 42, 41 and 40 chromosomes respectively. High-resolution chromosome painting analysis demonstrated that T plants had all 42 wheat chromosomes; the medium plants (M) had 41 chromosomes, lacking one chromosome 2A; while D plants had 40 wheat chromosomes, and lacked both 2A chromosomes. These data demonstrated that dms resulted from a loss of chromosome 2A. We identified 23 genes on chromosome 2A which might be involved in the development of stamens or pollen grains. These results lay a solid foundation for further analysis of the molecular mechanisms of wheat male sterility. Because D plants can be used as a female parent to cross with other wheat genotypes, dms is a unique germplasm for any functional study of chromosome 2A and wheat breeding specifically targeting genes on 2A.

Genetic and cytological analysis of male sterile mutant <italic>msm2015-1</italic> in mungbean

<p indent=0mm>In China, the low yield per unit has always been a problem in the mungbean industry. The heterosis of mungbean is an effective way to increase yield. The male sterile line is a valuable resource to realize the utilization of hybrid. In this study,⁶⁰Co-γ radiation mutagenesis technology was used to induce male sterile mutants in mungbean, and we gained one mutant named <italic>male sterile mungbean 2015-1</italic> (<italic>msm2015-1</italic>) for the first time. There was no significant phenotype difference between <italic>msm2015-1</italic> and Sulyu 1 during the vegetative growth stage. The floral organ of <italic>msm2015-1</italic> was normal, but the anthers of<italic> msm2015-1</italic> were white and could not crack and disperse powder normally in the flowering stage. In the mature stage, hardly any normal pods grew up. Genetic analysis showed the separation ratio of fertility and sterility in the fertile segregation population was in line with 3:1, indicating that the sterility trait was controlled by a single recessive nuclear gene. Cytological analysis of pollen development revealed that there were few pollen grains of <italic>msm2015-1</italic> attached to mature stigmas. The pollen grains of <italic>msm2015-1 </italic>did not germinate <italic>in vitro</italic>. The cytoplasm of <italic>msm2015-1</italic>’s pollen could not be stained by Alexander dye. Three abortion types showed up when stained by I₂-KI dye: typical abortion, spherical abortion and stained abortion. DAPI staining showed the nuclei of <italic>msm2015-1</italic>’s pollen developed abnormally. Magenta acetate staining indicated that abortion occurred in the early stage of pollen development, while the asymmetry and more anomalous fission of meiosis during the tetrad stage were the main causes of pollen abortion.

The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

A rice GDSL esterase/lipase protein (GELP) is required for anther and pollen development

Male reproductive development is vital for crop seed production. However, the mechanism underlying remains largely unknown. Here, we isolated a male sterile mutant osgelp34-1 in rice. Cytological analysis indicated that the mutant exhibited delayed degradation of anther wall layers and abnormal pollen development, which resulted in the production of shrunk and non-viable pollen grains. MutMap and CRISPR/Cas9 analysis confirmed that a point mutation in OsGELP34 is responsible for the male sterile phenotype. OsGELP34 is highly expressed in reproductive tissues and encodes a putative GDSL lipase. OsGELP34 protein is located to the endoplasmic reticulum (ER) and conserved in land plants. Collectively, our findings elaborated that OsGELP34 plays a vital role in rice male reproduction and has potential applications in rice hybrid breeding.

Assessment of the roles of SPO11-2 and SPO11-4 in meiosis in rice using CRISPR/Cas9 mutagenesis.

In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.

A Fragment Substitution in Promoter of MS92/PTC1 Causes Male Sterility in Rice

Persistent tapetal cell1 (PTC1) plays a curial role in pollen development, and is thought to function as a transcriptional activator in rice. However, the molecular mechanism of PTC1 in regulating pollen development and its cis-elements are not well understood. We identified a novel weak male sterility mutant (ms92) which exhibited expanded tapetum and shrink pollen grains. Map-based cloning and allelic analysis suggested that the male sterility of ms92 was caused by a DNA fragment substitution in the promoter of PTC1. The decreased expression of MS92/PTC1 in ms92 and cis-element analysis indicated that the substituted sequence contained several potential binding cis-element of negative feedback. MS92/PTC1 was specifically expressed in tapetum and microspores at the young microspore stage, and its protein was localized in nucleus. We further found that MS92/PTC1 functions as a transcription activator by recognizing H3K4me3. Transcriptomic analysis revealed that a number of genes involved in tapetum degeneration and pollen wall formation were down-regulated in ms92, which are the potential targets of MS92/PTC1. The substitution fragment in MS92/PTC1 promoter was essential for pollen development, and we provided a novel mutant for further identifying the cis-elements in promoter and the molecular network of MS92/PTC1.

Characterization and transcriptome analysis of a dominant genic male sterile cotton mutant

BackgroundMale sterility is an efficient trait for hybrid seed production and germplasm innovation. Until now, most studies on male sterility were on cytoplasmic and recessive genic sterility, with few on dominant genic male sterility, especially in cotton, due to lack of such mutant.ResultsWe discovered a natural male sterile (MS) Sea Island cotton (G. barbadense) mutant. Genetic analysis showed the mutation was caused by a dominant mutation in a single nuclear gene. Comparative cytological observation of anther sections from MS and wild-type (WT) uncovered cellular differences in anther at and after the tetrad stage of pollen mother cells (PMC). In the MS anthers, the outer wall of pollen grains was free of spinules, the tapetum was vacuolated and showed delayed degradation, consequently, no functional pollen grains. Comparison of transcriptomes from meiosis, tetrad, mononuclear and binuclear pollen, and pollen maturation stages identified 13,783 non-redundant differentially expressed genes (DEGs) between MS and WT. Based on the number of DEGs, analyses of enriched GO terms and KEGG pathways, it was evident that significant transcriptomic changes occurred at and after the tetrad stage, consistent with cytological observation, and that the major differences were on metabolism of starch, sucrose, ascorbate, aldarate, alanine, aspartate and glutamate, and biosynthesis of cutin, suberine and wax. WGCNA analysis identified five modules containing 920 genes highly related to anther development, especially the greenyellow module with 54 genes that was highly associated with PMC meiosis and tetrad formation. A NAC transcription factor (Gh_D11G2469) was identified as a hub gene for this module, which warrants further functional characterization.ConclusionsWe demonstrated that the MS trait was controlled by a single dominant nuclear gene and caused by delayed tapetum degradation at the tetrad stage. Comparative transcriptome analysis and gene network construction identified DEGs, enriched GO terms and metabolic pathways, and hub genes potentially associated with anther development and the MS trait. These results contribute to our understanding of dominant genic male sterility (DGMS) and provided source for innovation of cotton germplasm.

A detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata [L.] Walp.)

BackgroundThe legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement.ResultsAttempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3–4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes.ConclusionThe transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in genes involved in both vegetative and reproductive developmental programs. The method and tested editing constructs and components have potential application for a range of crop legumes.