Enemies atpeace:Recentprogressin Agrobacterium-mediated cereal transformation

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

Development of powdery mildew resistant derivatives of wheat variety Fielder for use in genetic transformation

Genetic transformation is widely used to improve target traits and to study gene function in wheat.However,transformation efficiency depends on the physiological status of the recipient genotype and that is affected by several factors including powdery mildew(PM)infection.The widely used recipient variety Fielder is very susceptible to PM.Therefore,it would be beneficial to develop PM resistant derivatives with high regeneration ability for use in genetic transformation.In the present study PM resistant lines CB037 and Pm97033 carrying genes Pm21 and PmV,respectively,were backcrossed to Fielder with selection for PM resistance.Five lines,NT89,NT90,NT154,and WT48 with Pm21 and line FL347 with PmV were developed,identified by molecular markers and genomic in situ hybridization(GISH)or fluorescent in situ hybridization(FISH),and further subjected to detailed assessment of agronomic traits and regeneration ability following genetic transformation capacity.Lines FL347,WT48,NT89 and NT154 assessed as being equal to,or superior,to Fielder in regeneration and transformation ability are recommended as suitable materials for the replacement of Fielder for wheat gene transfer and genome editing study.

Wheat genome editing expedited by efficient transformation techniques:Progress and perspectives

Genome editing is one of the most promising biotechnologies to improve crop performance.Common wheat is a staple food for mankind. In the past few decades both basic and applied research on common wheat has lagged behind other crop species due to its complex,polyploid genome and difficulties in genetic transformation. Recent breakthroughs in wheat transformation permit a revolution in wheat biotechnology. In this review, we summarize recent progress in wheat genetic transformation and its potential for wheat improvement. We then review recent progress in plant genome editing, which is now readily available in wheat. We also discuss measures to further increase transformation efficiency and potential applications of genome editing in wheat. We propose that, together with a high quality reference genome, the time for efficient genetic engineering and functionality studies in common wheat has arrived.

Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation

Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon(-1000 to-1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules(CCRMs), CCRM1(-300 to-101), CCRM2(-650 to-400), and CCRM3(-950 to-750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qP CR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter(-300 to-1), spanning CCRM1 and core region(-100 to-1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering(DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2(-300 to-209) containing Complete HMW Enhancer(-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1(-208 to-101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.

Development and genetic analysis of wheat double substitution lines carrying Hordeum vulgare 2H and Thinopyrum intermedium 2Ai#2 chromosomes

Thinopyrum intermedium and barley are two close relatives of wheat and carry many genes that are potentially valuable for the improvement of various wheat traits. In this study we created wheat double substitution lines by hybridizing different wheat–Th. intermedium and wheat–barley disomic alien substitution lines, with the aim of using genes in Th. intermedium and barley for wheat breeding and investigating the genetic behavior of alien chromosomes and their wheat homoeologs. As expected, we obtained two types of wheat double substitution lines,2D2Ai#2(2B)2H( A) and 2A2 Ai#2(2B)2H(2D), in which different group 2 wheat chromosomes were replaced by barley chromosome 2 H and Th. intermedium chromosome 2Ai#2. The new materials were characterized using molecular markers, genomic in situ hybridization(GISH), and fluorescent in situ hybridization(FISH). GISH and FISH experiments revealed that the double substitution lines harbor 42 chromosomes including 38 wheat chromosomes, a pair of barley chromosomes, and a pair of Th. intermedium chromosomes. Analysis using specific DNA markers showed that two pairs of wheat homoeologous group 2 chromosomes in the new lines were substituted by a pair of 2H and a pair of 2Ai#2 chromosomes. Chromosome 2H showed a higher transmission rate than 2Ai#2, and both chromosomes were preferentially transmitted between generations via female gametes. Evaluation of botanic and agronomic traits demonstrated that,compared with their parents, the new lines showed similar growth habits and plant type but differences in plant height, flowering date, and self-fertility. Cytological observations using different probes suggested that the double substitution lines showed nearly normal genetic behavior before and during meiosis. The novel substitution lines can potentially be used in wheat meiosis research and breeding programs.

The soft glumes of common wheat are sterile-lemmas as determined by the domestication gene Q

The Q gene in common wheat encodes an APETALA2(AP2) transcription factor that causes the free threshing attribute. Wheat spikelets bearing several florets are subtended by a pair of soft glumes that allow free liberation of seeds. In wild species, the glumes are tough and rigid,making threshing difficult. However, the nature of these "soft glumes", caused by the domestication allele Q is not clear. Here, we found that over expression of Q in common wheat leads to homeotic florets at glume positions. We provide phenotypic, microscopy, and marker genes evidence to demonstrate that the soft glumes of common wheat are in fact lemma-like organs, or so-called sterile-lemmas. By comparing the structures subtending spikelets in wheat and other crops such as rice and maize, we found that AP2 genes may play conserved functions in grasses by manipulating vestigial structures, such as floret-derived soft glumes in wheat and empty glumes in rice. Conversion of these seemingly vegetative organs to reproductive organs may be useful in yield improvement of crop species.

Development of a wheat material with improved bread-making quality by overexpressing HMW-GS 1S^(l)x2.3^(*)from Aegilops longissima

Wheat bread-making quality can be improved by use of high-molecular-weight glutenin subunits(HMW-GSs)from wild relatives.Aegilops longissima is a close relative of wheat that contains a number of HMW-GS-encoding genes including 1S^(l)x2.3^(*).In this study,transgenic wheat lines overexpressing 1S^(l)x2.3^(*)were obtained by Agrobacterium-mediated transformation and used to investigate the genetic contribution of 1S^(l)x2.3^(*)to wheat flour-processing quality.The 1S^(l)x2.3^(*)transgene was stably inherited and expressed over generations.Expression of 1S^(l)x2.3^(*)increased the relative expression of 1Dx2 and 1Dy12 and reduced that of 1By18 during grain development.In general,integration of 1S^(l)x2.3^(*)stimulated the accumulation of endogenous HMW-GSs and low-molecular-weight glutenin subunits in wheat kernels,greatly increasing the glutenin:gliadin ratio and resulting in faster formation of protein bodies in the endosperm during grain development.A wheat material with improved flour-making quality was developed in which 1S^(l)x2.3^(*)improved wheat bread-making quality.

The GATA transcription factor TaGATA1 recruits demethylase TaELF6-A1 and enhances seed dormancy in wheat by directly regulating TaABI5

Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting(PHS) under high-temperature and humid conditions. In this study,we report that the GATA transcription factor TaGATA1 is a positive regulator of seed dormancy by regulating TaABI5 expression in wheat.Our results demonstrate that TaGATA1 overexpression significantly enhances seed dormancy and increases resistance to PHS in wheat. Gene expression patterns, abscisic acid(ABA) response assay, and transcriptome analysis all indicate that TaGATA1 functions through the ABA signaling pathway. The transcript abundance of TaABI5, an essential regulator in the ABA signaling pathway,is significantly elevated in plants overexpressing TaGATA1. Chromatin immunoprecipitation assay(ChIP) and transient expression analysis showed that TaGATA1 binds to the GATA motifs at the promoter of TaABI5 and induces its expression.We also demonstrate that TaGATA1 physically interacts with the putative demethylase TaELF6-A1, the wheat orthologue of Arabidopsis ELF6.ChIP–qPCR analysis showed that H3K27me3 levels significantly decline at the TaABI5 promoter in the TaGATA1-overexpression wheat line and that transient expression of TaELF6-A1 reduces methylation levels at the TaABI5 promoter, increasing TaABI5 expression. These findings reveal a new transcription module, including TaGATA1–TaELF6-A1–TaABI5, which contributes to seed dormancy through the ABA signaling pathway and epigenetic reprogramming at the target site. TaGATA1 could be a candidate gene for improving PHS resistance.

Development of a haploid inducer by editing HvMTL in barley

The doubled-haploid technique mediated by parthenogenesis and androgenesis in plants can directly generate homozygous diploid lines after chromosome doubling with colchicine in one or two generations,and this method undoubtedly shortens the breeding process and improves breeding efficiency in crops.Previously,haploid plants were mainly induced by anther culture or microspore culture via androgenesis(Ohnoutkova et al.,2019).In wheat(Triticum aestivum),haploids can also be induced by maize(Zea mays)pollen and chromosome elimination via parthenogenesis(Liu et al.,2020b).However,the aforementioned induction methods are genotype-dependent,require complex manipulations,and are both time-consuming and inefficient.Therefore,it is necessary to develop new techniques or germplasm for simple and efficient haploid induction.

A fast technique for visual screening of wheat haploids generated from TaMTL-edited mutants carrying anthocyanin markers

Dear Editor,Breeding a new wheat variety using traditional methods typically takes at least 8 to 10 years,and the breeding period can be dramatically shortened via a doubled haploid strategy,which can yield homozygotes within one or two generations.In the past several decades,wheat haploids have been widely induced through anther or microspore culture and chromosome elimination via interspecific hybridization between wheat and maize.The first technique exhibits strong genotype dependency and a severe albino phenomenon,whereas the latter shows low induction efficiency(Sangam et al.,2015).In addition,haploid induction procedures using the aforementioned methods are complicated to perform and require specialized equipment and environmentally controlled conditions.Thus,the application of these technologies in wheat breeding has been limited(Sangam et al.,2015).

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum,hexaploid triticale,and rye(Secale cereale L.)using their immature embryos.Transgenic plants were efficiently generated.During the transformation process,the Agrobacterium infection efficiency was assessed by histochemical staining forβ-glucuronidase(GUS).Finally,the transgenic nature of regenerated plants was verified by polymerase chain reaction(PCR)-based genotyping for the presence of the GUS and bialaphos resistance(bar)genes,histochemical staining for GUS protein,and the QuickStix strip assay for bar protein.The transformation efficiency of T.monococcum genotype PI428182 was 94.4%;the efficiencies of four hexaploid triticale genotypes Lin456,ZS3297,ZS1257,and ZS3224 were 52.1,41.2,19.4,and 16.0%,respectively;and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.Fluorescence in situ hybridization(FISH)and genomic in situ hybridization(GISH)analyses indicated that the GUS transgenes were integrated into the distal or near centromere(proximal)regions of the chromosomes in transgenic T.monococcum and hexaploid triticale plants.In the transgenic hexaploid triticale plants,the foreign DNA fragment was randomly integrated into the AABB and RR genomes.Furthermore,the transgene was almost stably inherited in the next generation by Mendel’s law.The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Wheat breeding history reveals synergistic selection of pleiotropic genomic sites for plant architecture and grain yield

Diversity surveys of crop germplasm are important for gaining insights into the genomic basis for plant architecture and grain yield improvement,which is still poorly understood in wheat.In this study,we exome sequenced 287 wheat accessions that were collected in the past 100 years.Population genetics analysis identified that 6.7%of the wheat genome falls within the selective sweeps between landraces and cultivars,which harbors the genes known for yield improvement.These regions were asymmetrically distributed on the A and B subgenomes with regulatory genes being favorably selected.Genome-wide association study(GWAS)identified genomic loci associated with traits for yield potential,and two underlying genes,TaARF12 encoding an auxin response factor and TaDEP1 encoding the G-proteinγ-subunit,were located and characterized to pleiotropically regulate both plant height and grain weight.Elite single-nucleotide haplotypes with increased allele frequency in cultivars relative to the landraces were identified and found to have accumulated over the course of breeding.Interestingly,we found that TaARF12 and TaDEP1 function in epistasis with the classical plant height Rht-1 locus,leading to propose a“Green Revolution”-based working model for historical wheat breeding.Collectively,our study identifies selection signatures that fine-tune the gibberellin pathway during modern wheat breeding and provides a wealth of genomic diversity resources for the wheat research community.

CRISPR/Cas9 editing of wheat TaQ genes alters spike morphogenesis and grain threshability

The Ta Q alleles as one of the AP2-like transcription factors in common wheat(Triticum aestivum) play an important role in the evolution of spike characteristics from wild and domesticated emmer to modern wheat cultivars. Its loss-of-function mutant not only changed threshability and spike architecture but also affected plant height, flowering time, and floret structure. However, the comprehensive functions of Ta AQ and Ta Dq genes in wheat have not been fully elucidated yet. Here, CRISPR/Sp Cas9 was used to edit wheat Ta AQ and Ta Dq. We obtained homozygous plants in the T1 generation with loss of function of only Ta AQ or Ta Dq and simultaneous loss of function of Ta AQ and Ta Dq to analyze the effect of these genes on wheat spikes and floret shapes. The results demonstrated that the Ta AQ-edited plants and the Ta AQ and Ta Dq simultaneously-edited plants were nearly similar in spike architecture, whereas the Ta Dq-edited plants were different from the wild-type ones only in plant height. Moreover, the Ta AQ-edited plants or the Ta AQ and Ta Dq simultaneously-edited plants were more brittle than the wild-type and the Ta Dqedited plants. Based on the expression profiling, we postulated that the VRN1, FUL2, SEP2, SEP5, and SEP6 genes might affect the number of spikelets and florets per spike in wheat by regulating the expression of Ta Q. Combining the results of this report and previous reports, we conceived a regulatory network of wheat traits, including plant height, spike shape, and floral organs, which were influenced by AP2-like family genes. The results achieved in this study will help us to understand the regulating mechanisms of Ta AQ and Ta Dq alleles on wheat floral organs and inflorescence development.