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The process of silique dehiscence is essential for the proper dispersal of seeds at the end of a dehiscent fruit plants lifecycle. Current research focuses on genetic manipulation to mitigate this process and enhance shatter tolerance in crop plants, which has significant economic implications. In this study, we have conducted a time-course analysis of cell patterning and development in valve tissues of Arabidopsis thaliana and closely related Triangle of U species (Brassica juncea, Brassica carinata, Brassica napus, Brassica rapa, and Brassica nigra) from Brassicaceae. The goal was to decipher the detailed temporal developmental patterns of the endocarp a and b cell layers of the valve, specifically their degradation and lignification respectively. Additionally, we propose a new classification system for the lignification of the endocarp a cell layer: L1 indicates the cell closest to the replum, with L2 and L3 representing the second and third cells, respectively, each numerical increment indicating lignified cells farther from the replum. Our findings provide a foundational framework absent in current literature, serving as an effective blueprint for future genomic work aimed at modifying valve structures to enhance agronomic traits, such as reducing fiber (lignin) or increasing shatter tolerance.

In plants, an increasing number of traits and new characteristics are being developed using gene editing. Simple traits represented by a single gene can be managed through backcross breeding, but this is typically not the case for more complex traits which may result from the function of a large number of genes. Here, we demonstrate two case studies of improving oleic oil content and developing pod shatter reduction in Brassica napus by using gene editing tools on an industrial scale. There are four BnaFAD2 genes involved in oleic oil content and eight BnaSHP genes involved in pod shatter tolerance. In order to develop these two traits, we delivered nuclease ribonucleoproteins with Gene Repair OligoNucleotides (GRONs) into protoplasts, with subsequent regeneration into plants on an industrial scale, which encompassed robust tissue culture protocols, efficient gene editing, robotics sampling, and molecular screening, vigorous plant regeneration, growth, and phenotyping. We can produce precise loss-of-function-edited plants with two improved agronomically important complex traits, high oleic oil or pod shatter reduction, in elite canola varieties within 1–3 years, depending on the trait complexity. In the edited plants carrying loss of function of four BnaFAD2 genes, the seed fatty acid oleic acid content reached 89% compared to 61% in the non-edited wildtype control. The plants carrying eight edited BnaSHP genes achieved 51% pod shatter reduction in multiple year field testing in the target environment compared to the wildtype control.

In plants, an increasing number of traits and new characteristics are being developed using gene editing. Simple traits represented by a single gene can be managed through backcross breeding, but this is typically not the case for more complex traits which may result from the function of a large number of genes. Here, we demonstrate two case studies of improving oleic oil content and developing pod shatter reduction in Brassica napus by using gene editing tools on an industrial scale. There are four BnaFAD2 genes involved in oleic oil content and eight BnaSHP genes involved in pod shatter tolerance. In order to develop these two traits, we delivered nuclease ribonucleoproteins with Gene Repair OligoNucleotides (GRONs) into protoplasts, with subsequent regeneration into plants on an industrial scale, which encompassed robust tissue culture protocols, efficient gene editing, robotics sampling, and molecular screening, vigorous plant regeneration, growth, and phenotyping. We can produce precise loss-of-function-edited plants with two improved agronomically important complex traits, high oleic oil or pod shatter reduction, in elite canola varieties within 1-3 years, depending on the trait complexity. In the edited plants carrying loss of function of four BnaFAD2 genes, the seed fatty acid oleic acid content reached 89% compared to 61% in the non-edited wildtype control. The plants carrying eight edited BnaSHP genes achieved 51% pod shatter reduction in multiple year field testing in the target environment compared to the wildtype control. [ABSTRACT FROM AUTHOR]

Wild species field cress (Lepidium campestre) has favorable agronomic traits, making it a good candidate for future development as an oil and catch crop. However, the species is very prone to pod shatter, resulting in severe yield losses. This is one of the important agronomic traits that needs to be improved in order to make this species economically viable. In this study, we cloned the L. campestre INDEHISCENT (LcIND) gene and prepared two LcIND-RNAi constructs with the IND promoter (long 400 bp and short 200 bp) from Arabidopsis. A number of stable transgenic lines were developed and evaluated in terms of pod shatter resistance. The majority of the transgenic lines showed increased resistance to pod shatter compared to the wild type, and this resistance was maintained in four subsequent generations. The downregulation of the LcIND gene by RNAi in the transgenic lines was confirmed by qRT-PCR analysis on T3 lines. Southern blot analysis showed that most of the analyzed lines had a single-copy integration of the transgene, which is desirable for further use. Our results show that it is possible to generate stable transgenic lines with desirable pod shatter resistance by downregulating the LcIND gene using RNAi in field cress, and thus speeding up the domestication process of this wild species. [ABSTRACT FROM AUTHOR]

Pod shattering causes severe yield loss in rapeseed (Brassica napus L.) under modern agricultural practice. Identification of highly shatter-resistant germplasm is desirable for the development of rapeseed cultivars for mechanical harvesting. In the present study, an elite line OR88 with strong shatter resistance and a lignified-layer bridge (LLB) structure was identified. The LLB structure was unique to OR88 and co-segregated with high pod-shatter resistance. The LLB structure is differentiated at stage 12 of gynoecium development without any gynoecium defects. Genetic analysis showed that LLB is controlled by a single recessive gene. By BSA-Seq and map-based cloning, the resistance gene location was delimited to a 0.688 Mb region on chromosome C09. Transcriptome analysis suggested BnTCP8.C09 as the gene responsible for LLB. The expression of BnTCP.C09 was strongly downregulated in OR88, suppressing cell proliferation in the pod valve margin. KASP markers linked to the candidate gene were developed. This pod shatter-resistant line could be used in rapeseed breeding programs by direct transfer of the gene with the assistance of the DNA markers.

The process of silique dehiscence is essential for the proper dispersal of seeds at the end of a dehiscent fruit plants lifecycle. Current research focuses on genetic manipulation to mitigate this process and enhance shatter tolerance in crop plants, which has significant economic implications. In this study, we have conducted a time-course analysis of cell patterning and development in valve tissues of Arabidopsis thaliana and closely related Triangle of U species ( Brassica juncea, Brassica carinata , Brassica napus, Brassica rapa , and Brassica nigra ) from Brassicaceae. The goal was to decipher the detailed temporal developmental patterns of the endocarp a and b cell layers of the valve, specifically their degradation and lignification respectively. Additionally, we propose a new classification system for the lignification of the endocarp a cell layer: L1 indicates the cell closest to the replum, with L2 and L3 representing the second and third cells, respectively, each numerical increment indicating lignified cells farther from the replum. Our findings provide a foundational framework absent in current literature, serving as an effective blueprint for future genomic work aimed at modifying valve structures to enhance agronomic traits, such as reducing fiber (lignin) or increasing shatter tolerance.

The genome of Brassica napus L. is the result of several polyploidization events that occurred during the history of B. napus. Due to its relatively short domestication history, diversity is relatively limited. An increasing number of loci in this crop's genome have been gene-edited using various technologies and reagent delivery methods for basic research as well as for trait development. New alleles have been developed as edits in single, 2, 4, or more homologous loci in this important oilseed crop. This comprehensive review will summarize new alleles that have been developed as they relate to weed control, flowering, self-incompatibility, plant hormone biology, disease resistance, grain composition, and pod shatter reduction. These new alleles have significantly augmented our understanding of both plant growth and development for basic research as well as for their potential commercial impacts. [ABSTRACT FROM AUTHOR]

Key message: Elucidation of key regulators in Arabidopsis fruit patterning has facilitated knowledge-translation into crop species to address yield loss caused by premature seed dispersal (pod shatter). In the 1980s, plant scientists descended on a small weed Arabidopsis thaliana (thale cress) and developed it into a powerful model system to study plant biology. The massive advances in genetics and genomics since then have allowed us to obtain incredibly detailed knowledge on specific biological processes of Arabidopsis growth and development, its genome sequence and the function of many of the individual genes. This wealth of information provides immense potential for translation into crops to improve their performance and address issues of global importance such as food security. Here, we describe how fundamental insight into the genetic mechanism by which seed dispersal occurs in members of the Brassicaceae family can be exploited to reduce seed loss in oilseed rape (Brassica napus). We demonstrate that by exploiting data on gene function in model species, it is possible to adjust the pod-opening process in oilseed rape, thereby significantly increasing yield. Specifically, we identified mutations in multiple paralogues of the INDEHISCENT and GA4 genes in B. napus and have overcome genetic redundancy by combining mutant alleles. Finally, we present novel software for the analysis of pod shatter data that is applicable to any crop for which seed dispersal is a serious problem. These findings highlight the tremendous potential of fundamental research in guiding strategies for crop improvement. [ABSTRACT FROM AUTHOR]

The article offers information on seed dispersal in the plant Arabidopsis thaliana relative, Cardamine hirsuta. Topics include cellular innovations for the storage and rapid release of energy underpinning the evolution of trait, need of understanding the origin and evolution of complex traits in biology and botany, and variety of seed dispersal mechanisms in flowering plants.

Canola ( Brassica napus L.) often experiences high seed losses prior to and during harvest operations. Pod drop and pod shatter are responsible for pre-harvest seed losses in canola although relatively little is known about the significance of pod drop to total pre-harvest seed losses in canola. This research describes a method to measure the predisposition for pod drop among canola genotypes that was developed and tested across different sites from 2013 to 2015. This method, referred to as pod-retention resistance, consists of the use of a force gauge to quantify the force required to break the pod pedicle at the pedicle-rachis junction. Two sets of field experiments were used, the first developed and modeled the factors that influence pod-retention resistance, pod drop and seed shatter. A second set of experiments was used to validate these findings on a broader range of genotypes and determine whether a relationship between pod retention resistance and pod drop could be determined. Results showed that the pod-retention resistance was significantly greater for pods on the proximal half of the rachis compared to the distal half of the rachis. Several factors contributed strongly to variation in pod retention resistance (rachis position, genotype and site) and pod drop (site, rachis position and genotype). The relative importance of individual factors varied between pod retention resistance and pod drop suggesting no relationship between the two. However, weight-adjusted pod retention resistance, i.e., average pod retention resistance corrected for the average weight of dropped pods, was strongly correlated with pod drop, particularly in the validation experiments. This method could be used by canola breeders to select for and develop genotypes with reduced predisposition for seed losses via pod drop. [ABSTRACT FROM AUTHOR]

Asynchronousflowering ofBrassica napus(canola) leads to seeds and siliques at varying stages of maturity as harvest approaches. This range of maturation can result in premature silique dehiscence (pod shattering), resulting in yield losses, which may be worsened by environmental stresses. Therefore, a goal for canola crop improvement is to reduce shattering in order to maximize yield. We performed a comprehensive transcriptome analysis on the dehiscence zone (DZ) and valve ofArabidopsisandBrassicasiliques in shatter resistant and sensitive genotypes at several developmental stages. Among knownArabidopsisdehiscence genes, we confirmed that homologs ofSHP1/2,FUL,ADPG1,NST1/3andINDwere associated with shattering inB. junceaandB. napus. We noted a correlation between reduced pectin degradation genes and shatter-resistance. Tension between lignified and non-lignified cells in the silique DZ plays a major role in dehiscence. Light microscopy revealed a smaller non-lignified separation layer in relatively shatter-resistantB. juncearelative toB. napusand this corresponded to increased expression of peroxidases involved in monolignol polymerization. Sustained repression of auxin biosynthesis, transport and signaling inB. juncearelative toB. napusmay cause differences in dehiscence zone structure and cell wall constituents. Tension on the dehiscence zone is a consequence of shrinkage and loss of flexibility in the valves, which is caused by senescence and desiccation. Reduced shattering was generally associated with upregulation of ABA signaling and down-regulation of ethylene and jasmonate signaling, corresponding to more pronounced stress responses and reduced senescence and photosynthesis. Overall, we identified 124 cell wall related genes and 103 transcription factors potentially involved in silique dehiscence. [ABSTRACT FROM AUTHOR]

Abstract: Adverse hot and dry environmental conditions prevailing during the period of oilseed rape ripening can be blamed for considerable yield losses during the crop''s mechanized harvesting in the Mediterranean area. In this paper, the effect of chemical dessication of oilseed rape with glyphosate applied 35 days after anthesis for reducing seed yield loss was studied. The experiment was conducted in four fields at two locations in central Greece and for two growth periods following a completely randomized plot design with 8 treatments in three replications. Treatments include all possible combinations of two crop varieties and four levels of glyphosate (as dessicant) application. A significant positive effect of dessicant application level on crop yield for both studied varieties was found. The application dose of 200g a.i. of glyphosate per hectare had the best results. The highest dry weight was found among the plants sprayed with 200g. a.i. of glyphosate per hectare. The highest seed yield and oil yield were found among the plants sprayed with 200g a.i. per hectare, which makes clear the positive effect of this spraying minimizing pod shattering and yield losses. The greatest 1000-seed weight was found among the plants sprayed with 200g a.i.ha−1, due to the greater size of seeds that remained on the plants until harvesting. [Copyright &y& Elsevier]

Plant genomic research now faces the ultimate challenge to develop applications in crop plants which implies the translation of gene functions from a model to a crop which is the field of ‘Plant translational genomics’. In this paper we discuss the perspectives of the candidate gene approach (CGA) as a tool for translational genomics in the ‘whole genome’ era. Factors to be considered for a successful application of the CGA in crops such as the type of crop, the complexity of the trait and the type of genes involved are discussed. Several crop traits that require improvement such as tolerance to stress, pod shatter in Brassicaceae and Fusarium resistance, are evaluated with regard to the potential of a CGA as a tool for crop improvement [ABSTRACT FROM AUTHOR]

Arabidopsis has proven to be extremely useful as a reference organism for studies in plant biology, and huge efforts have been employed to unravel various mechanisms of Arabidopsis growth. A major challenge now is to demonstrate that this wealth of knowledge can be used for global agricultural and environmental improvement. Brassica species are closely related to Arabidopsis and represent ideal candidates for model-to-crop approaches as they include important crop plants, such as canola. Brassica plants normally disperse their seeds by a pod-shattering mechanism. Although this mechanism is an advantage in nature, unsynchronized pod shatter constitutes one of the biggest problems for canola farmers. Here, we show that ectopic expression of the Arabidopsis FRUITFULL gene in Brassica juncea is sufficient to produce pod shatter-resistant Brassica fruit and that the genetic pathway leading to valve margin specification is conserved between Arabidopsis and Brassica. These studies demonstrate a genetic strategy for the control of seed dispersal that should be generally applicable to diverse Brassica crop species to reduce seed loss. [ABSTRACT FROM AUTHOR]

Floral induction is a key developmental switch in plants that leads to the production of flowers, fruits and seeds, which are of paramount importance for human life. To meet the demands of several crop harvests per year, or the growth of crop plants in regions with short vegetation times and for the production of ornamental plants, the timing of the floral transition is very important. The discovery of genes that are involved in flowering time control in model plants should allow the modulation of this developmental switch also in plants with economic value. By using a transgenic approach, we showed that a single MADS box gene accelerated flowering and seed ripening in summer rape plants. TheMADSBtransgene also partially substituted for the strict temperature requirements for flowering in winter rape plants. Transgenic winter rape plants expressing theMADSBtransgene also produced more rigid siliques than wild type winter rape plants, and this prevented precocious seed dispersal. [ABSTRACT FROM AUTHOR]

The architecture of the pod wall and dehiscence zone (DZ) was studied in populations of a resynthesized, shatter‐resistant, oilseed rape line, DK142, and the commercial cultivar Apex. The dimensions of the pod wall and its component tissues were significantly larger in DK142. However, the variation in the pod architecture of Apex, DK142 and F2 populations derived from crosses of DK142 and Apex was found to have little or no role in pod shatter. By contrast, variation in the dimensions of the DZ characters correlated strongly and positively with shatter resistance. The size of the main vascular bundle (MVBV) of DK142 as it exited the valve and joined the vascular tissue of the replum was, on average, 60% larger than in Apex, the DZ was 40% wider and there was a high preponderance of vascular tissue other than the MVBV. The variation in the size of the MVBV accounted for much of the variation in shatter resistance of all populations, including shatter‐susceptible Apex. The DZ width was also found to be important in explaining the limited range of shatter values in Apex, but in populations of DK142 and F2, where the amount of vascular intrusion into the DZ was much greater, the variation in DZ width was not important. The importance of the vascular tissue to shatter resistance was further highlighted by a novel microfracture test (MFT). By contrast, no significant difference between DK142 and Apex in the ease of separation of the thin‐walled DZ cells was detected using the MFT. [ABSTRACT FROM PUBLISHER]

The recessive mutation of the def gene of pea (Pisum sativum L.) leads to the loss of the hilum, the abscission zone between the seed and the pod. Thereby, it reduces the free dispersal of the seeds through pod shattering. As a prerequisite for a gene isolation via a map-based cloning approach, bulked segregant analysis followed by single plant analyses of over 200 homozygous individuals of a population of 476 F2 plants derived from a cross between 'DGV' (def wild-type) and 'PF' (def mutant), were used to detect markers closely linked to the def locus. The AFLP technique in combination with silver staining was used to maximize numbers of reproducible marker loci. Fifteen AFLP loci showed a genetic distance less than 5 and two of them less than 1 centiMorgans (cM) to the gene of interest. AFLPs were converted into sequence tagged sites (STSs) and into a newly refined AFLP-based single locus marker named the 'sequence specified AFLP' (ssAFLP). [ABSTRACT FROM AUTHOR]

Abstract Microscopic examination of cross sections of dorsal and ventral sutures of soybean pods (Glycine max cv. TGx1835-2E) at two different stages of maturity revealed that the dehiscence zone of soybean pods is functionally equivalent to the dehiscence zone known from crucifers. Enzymatic assays demonstrated the presence of endo-1,4-β -glucanases and endopolygalacturonases, the activity of which accumulated in the dehiscence zone and peaked during maturation. A single partial cDNA encoding an endopolygalacturonase was isolated by polymerase chain reaction and this clone was used to isolate the complete gene encoding the endopolygalacturonase in question. Approximately 1·2 kb of 5′ upstream sequence was cloned in the plant transformation vector pCAMBIA1301 in front of the uidA (GUS) gene and transformed into Arabidopsis thaliana . Expression analysis of the soybean endopolygalacturonase transcript revealed that the endopolygalacturonase is primarily found in dehiscence-related tissue and is presumably involved in the breakdown of the middle lamella prior to dehiscence. This result was corroborated by GUS stainings of the transgenic Arabidopsis lines. [ABSTRACT FROM AUTHOR]

Pod dehiscence in Arabidopsis thaliana is accompanied by an increase in the expression of a polygalacturonase (PG). The gene encoding this mRNA has been characterized and shown to have extensive homology to a similar PG gene from Brassica napus. The A. thaliana PG promoter was fused to β-glucuronidase (GUS) and the expression of this reporter gene analysed in transgenic B. napus plants. The GUS activity was detected throughout the dehiscence zone of pods from 35 d after anthesis and expression was restricted to those cells that undergo cell separation. Expression was also detectable at the junction between the seed and the funicular tissue and in mature anthers of flowers. Transgenic plants containing the PG promoter fused to barnase were sterile as a consequence of the anthers failing to undergo dehiscence. Fertilization of PG-barnase plants resulted in the development of pods that exhibited a reduced capacity to shatter. The role of PG during cell separation processes in plants is discussed. [ABSTRACT FROM AUTHOR]

Pod shatter in oilseed rape is associated with the degradation of the pectin-rich middle lamella at the site of dehiscence. It has been reported that, accompanying pod development, there is an increase in the activity of polygalacturonase (EC 3.2.1.15) and that this rise is restricted to the tissue undergoing cell separation. Using a PCR strategy a fragment of a polygalacturonase encoding an mRNA that is up-regulated specifically in the dehiscence zone tissue during pod development has been cloned. A full length clone (SAC66) complementary to this mRNA has been isolated from a dehiscence zone cDNA library and sequenced. The mRNA encoded by SAC66 shares significant amino acid homology with endopolygalacturonases from fruit of Actinidia deliciosa and Lycopersicon esculentum. The transcript size of the SAC66 mRNA is 1.7 kb. Northern analysis has revealed that expression of SAC66 mRNA increases at 30 d after anthesis (DAA), reaching a plateau at 45 DAA, and that the up-regulation is restricted to the site where dehiscence takes place. [ABSTRACT FROM PUBLISHER]

Seven distinct partial cDNAs, similar in sequence to previously described polygalacturonases (PGs), were amplified from cDNA derived from rape pod wall, dehiscence zone and leaves by the polymerase chain reaction. Northern analysis showed that one clone, PG35-8, was expressed at low levels in the dehiscence zone during the first five weeks after anthesis but was very abundantly expressed at week 6. In contrast, no PG35-8-related RNA was detected in the pod wall. Our data suggest that there are temporal and spatial correlations between the breakdown of the middle lamella, of the dehiscence zone cells and the pattern of synthesis of PG35-8 transcripts which may indicate a role for this particular PG in rape pod dehiscence. PG35-8 was used to isolate five cDNA clones from a rape dehiscence zone cDNA library. Restriction enzyme analysis and partial sequencing revealed that they were derived from four highly homologous transcripts which are probably allelic forms of a single gene. One full-length clone, RDPG1, was completely sequenced. The predicted protein of RDPG1 showed its highest identity with PG from apple fruit with an identity of 52%. [ABSTRACT FROM AUTHOR]

Bayer CropScience is a leader in the oilseed rape seeds business with a 2013 market share of 50% in Canada, based on the creation and use of a unique hybridization system enabling the development of high-yielding canola (B.napus) InVigor® hybrids. For the European markets, Bayer is developing non-transgenic hybrids that will be complemented with differentiating traits. To this end, a highly effective mutagenesis-based and omics-directed reverse genetics platform was established which enables the creation of novel productivity traits in canola.The reverse genetics process involves three major steps described in the following figure. The selection of relevant homoeologs is facilitated by Bayer's B.napus genome sequence and transcript atlas. The genome sequence allows the in silico identification of functional homoeologs and the transcript atlas enables to prioritize on homoeologs that are highly expressed in the right tissues. A new trait is created by stacking relevant mutant alleles in a single line. Bayer CropScience is using its canola reverse genetics platform to improve certain canola characteristics including pod shattering, grain yield and oil composition and to develop traits such as herbicide tolerance. Pod shatter reduction was the first trait developed with the platform. A first pod shatter-reduced InVigor hybrid, L140P, was commercially grown in Canada during the 2014 summer season. Bayer CropScience has established and is successfully using a this biotech-based platform for the improvement of the productivity of canola. The resulting traits do not require regulation and can be deployed in all continents. The major limitation of reverse genetics is that the scope of modification is limited to the crops’ own gene content and the expression levels of these genes. [ABSTRACT FROM AUTHOR]