Production of oilseed rape with increased seed shattering resistance

Rapeseed (Brassica napus) production is limited by the crop’s natural propagation mechanism which involves growing siliques that dry out upon maturity and break easily. The resulting pre-harvest yield loss makes shatter resistance an important breeding goal. Studies on Arabidopsis thaliana revealed a set of transcription factors controlling dehiscence zone establishment. INDEHISCENT (IND) and ALCATRAZ (ALC) are major regulators of tissue differentiation in the critical parts of the silique. While ALC is required for the development of a partially degraded separation layer, IND also regulates the essential lignification of neighboring cells, probably through induction of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 and 3 (NST1/3) expression. This study aimed at producing rapeseed lines with robust siliques through the use of Bnalc, Bnind, and Bnnst1 mutations. CRISPR/Cas9-mediated gene editing of the two BnALC homoeologs of cultivar ‘Haydn’ efficiently yielded four mutant alleles in a single transgenic T1 plant which were stably inherited. A tensile force test suggested the increased shatter resistance of T2 double mutants. However, the effect was masked by the innate silique robustness of ‘Haydn’. The Bnalc phenotype was therefore confirmed with EMS-induced mutant alleles in the shatter-prone cultivar ‘Express’. Bnind mutations derived from the same ‘Express’ mutant population were utilized for detailed analyses of shatter mechanics. Three phenotyping tests consistently identified a double mutant with especially robust siliques. No lignification defects were observed. Instead, shatter resistance was accounted to a broader replum-valve joint area in combination with smaller cells therein. CRISPR/Cas9-induced mutagenesis of four BnNST1 homoeologs yielded a chimeric T1 plant with multiple mutant alleles per gene copy which now have to be fixed in the progeny. Altogether, the developed mutant material provides novel variation for shatter resistance breeding

Evaluating canola genotypes and harvest methods to reduce seedbank addition and longevity

Seed loss in canola (Brassica napus, Brassica rapa and Brassica juncea) leads to considerable loss of revenue and dispersal of canola seeds into the soil seedbank. Once canola seeds enter the soil seedbank a portion can become secondarily dormant and persist for many years creating volunteer weed problems in following crops. Reducing canola seed loss and seedbank persistence can be an important strategy to reduce the incidence of volunteer weeds. The primary hypothesis of this research was that canola seedbank addition and dormancy induction in the seed are affected by genotype and harvest method. To test this hypothesis, three studies were conducted with the following objectives: i) to determine canola seedbank addition from windrowing and direct-harvesting operations on commercial farms in western Canada, ii) to determine agronomic- and harvest-related factors that may increase seed loss in canola, iii) to determine the effect of stage of crop maturity at harvest on potential to develop seed dormancy in canola, iv) to evaluate canola genotypes and harvest methods to reduce canola seedbank addition. A total of 66 canola fields were surveyed across Saskatchewan in 2010 and 2011. Shattered seeds from these fields were sampled within 3 weeks of harvest by using a vacuum cleaner. Agronomic- and harvest-related data were collected for each field using questionnaires. In a separate small plot study the effects of harvest methods (windrowing and direct-harvesting) and pod sealant products (Pod-Stik® and Pod Ceal DC®) on seed loss in five canola genotypes (InVigor5440, RR45H26, InVigor5020, RR4362, and CL8571) were evaluated in 2010 and 2011. In both years, 6 harvest samples were collected weekly from InVigor5440 and InVigor5020 genotypes starting at early stage of crop maturity until harvest to assess the effect of seed maturity on dormancy induction. On commercial farms, the average seedbank addition was 5,821 viable seeds m-2, which was equivalent to 7.3% of the total seed yield. There was no difference in the reported yield and seedbank addition between windrowed and direct-harvested canola on commercial farms. But in the small plot study, windrowing resulted in higher seedbank addition. Higher seedbank addition was observed when the yield of canola was higher and when producers had a larger area seeded to canola. The observed seedbank addition was also higher in Roundup Ready genotypes and when a conventional combine harvester was used to harvest canola. Little primary dormancy and low potential to secondary dormancy induction was observed in InVigor5440 and InVigor5020 seeds at an early stage of crop maturity. But at full maturity seeds of both genotypes had no primary dormancy but showed high potential for secondary dormancy induction. This indicates that windrowing the evaluated genotypes at early stage of crop maturity lowered the potential for secondary dormancy induction. There were appreciable differences in seedbank addition among the evaluated canola genotypes but pod sealant products did not affect seed yield and seed shatter in canola. The results of this study suggest that canola seedbank addition can be minimized by growing genotypes having reduced seed loss and with the adoption of direct-harvesting operations

The influence of agroecological and agrotechnological factors on the generative development of oilseed radish (Raphanus sativus var. oleifera Metzg.)

Received: April 1st, 2022 ; Accepted: May 28th, 2022 ; Published: June 22nd, 2022 ; Correspondence: [email protected]; [email protected] the eight-year research period, we determined the peculiarities and regularities of morphological (length and diameter) and anatomical (stem thickness) features of oilseed radish pods considering their location within the generative part of plants for different types of spatial structure of the inflorescence generated in agrocenosises of different densities. We carried out the analysis and statistical grouping of morphological features of oil radish pod in the full range of possible technological options of pre-sowing construction of its agrocenosises, as well as within the selected three zones (tiers) of inflorescence by the nature of variation and variability of morphoparameters pod, namely, lower, middle and upper. We described in detail the stages of pod formation (microstages ВВСН 69-87) considering features of its linear and radial growth, peculiarities of formation of general internal anatomical structure with analysis of mathematical and statistical regularities of changes in these parameters in accordance as per order of its placement within an inflorescence (separately main axis and system of lateral branches). We determined the optimum technological intervals for the construction of oilseed radish agrocenosises, which ensure the combination of appropriate levels of morphometry formation of its fruit elements with the predicted level of reproductive effort and seed productivity. We made a general assessment of the peculiarities of formation of pod technological effectiveness in terms of ease of threshing and possible losses of seeds depending on the complex of factors under study

Characterization of the expression patterns of \u3ci\u3eCELLULOSE SYNTHASE-LIKE A (CSLA)\u3c/i\u3e genes in \u3ci\u3earabidopsis thaliana\u3c/i\u3e using reporter promoter-fusions and immunolocalization

Plant cell walls are the most abundant source of renewable biomass on Earth. Cell wall carbohydrates have many practical applications (e.g., forage, building materials, biofuels, textiles, paper, etc.), and within plants they contribute to structure, provide defense, and facilitate cellular communication. This study is focused on the CELLULOSE SYNTHASELIKE A (CSLA) family, members of which have been implicated in the synthesis of the backbone of mannan polysaccharides in plant cell walls. The Arabidopsis genome contains nine CSLA genes and we hypothesize that there is some degree of functional redundancy among these genes. A detailed investigation of transgenic Arabidopsis plants harboring promoter-GUS fusions at various phases of growth and development was conducted to examine the expression patterns of all nine CSLA genes. The expression patterns observed were largely overlapping, supporting the functional redundancy hypothesis. Some unique exceptions were also documented, providing insights into possible focal regions for future mutational studies

Functional analysis of Arabidopsis cold shock domain proteins

To understand the function of cold shock domain proteins in planta, I analyzed AtCSP3 (At2g17870), which is one of four A&barbelow;rabidopsis t&barbelow;haliana c&barbelow;old s&barbelow;hock domain p&barbelow;roteins (AtCSPs). Taq-Man probe quantitative RT-PCR (qRT-PCR) analysis confirmed that AtCSP3 transcripts were expressed dominantly in reproductive and meristematic tissues. The homozygous loss of function mutant atcsp3 displays a distinct phenotype with an overall reduced sized of seedlings, small sized orbicular rosette leaves, and curled leaf blades. Microscopic visualization of cleared leaves revealed a reduction in size and increased circular shape of palisade mesophyll cells in atcsp3 leaves. Image analysis of palisade cell layers indicated that the reduced size of the circular mesophyll cells is generated by a reduction of cell length and cell number along the leaf-length axis, resulting in an orbicular leaf shape. Also, I determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss of function mutant, but the leaf cell proliferation is not affected. Loss of function of AtCSP3 resulted in a dramatic reduction of LNG1 transcript involved in two-dimensional leaf polarity regulation. Subcellar localization of AtCSP3 in onion epidermal cells revealed nucleocytoplasmic localization. Collectively, these data suggest that AtCSP3 regulates leaf length specific polarity by affecting LNG1 transcript accumulation during leaf blade lateral expansion. I also discuss putative function of AtCSP3 as an RNA binding protein in relation to leaf development.;A&barbelow;rabidopsis t&barbelow;haliana C&barbelow;old S&barbelow;hock Domain P&barbelow;rotein 4 (AtCSP4; At2g21060) contains a well conserved cold shock domain (CSD) and glycine-rich motifs interspersed by two retroviral-like CCHC zinc fingers. GUS staining analysis in pAtCSP4:GUS transgenic Arabidopsis plants confirmed that AtCSP4 was expressed in all tissues but accumulates in reproductive tissues and those undergoing cell divisions. Overexpression of AtCSP4 resulted in a reduced length of siliques and embryo lethality. Interestingly, a T-DNA insertion atcsp4 mutant did not exhibit any phenotypes, implicating that the similar AtCSP2 gene is functionally redundant with AtCSP4. During silique development, overexpression of AtCSP4 induced early browning and shrunken seed formation beginning with the late heart embryo stage. A fifty percent segregation ratio of the defective seed phenotype was consistent with the phenotype of endosperm development gene mutants. Transcripts of FUS3 and LEC1 genes, which regulate early embryo formation, were not altered in the AtCSP4 overexpression lines. On the other hand, transcripts of MEA and FIS2 which are involved in endosperm development were affected by overexpression of AtCSP4 indicating that AtCSP4 may be a regulator of endosperm development via transcriptional or post-transcriptional regulation. Additionally, overexpression of AtCSP4 also affected the mRNA generation of several MADS box genes in stages of early silique development. Specifically, transcripts of AP, CAL, AG, and SHP2 were up-regulated. Collectively, these results indicate that AtCSP4 plays an important role during the late stages of silique development by affecting the expression of several development related genes.;Cold shock domain proteins (CSPs) have been reported to play an important role in tissue development and cold responses. Eukaryotic CSPs play a crucial role in cell differentiation and cell proliferation which result in regulating the timing of tissue development and cell division. Cold shock domain proteins have been identified in many plants, although functional analyses have been limited to Arabidopsis, wheat and rice and their in vivo functional roles remain unclear. Among the four Arabidopsis thaliana CSPs (AtCSPs) that I have characterized, AtCSP1 is highly similar to AtCSP3 in terms of its predicted amino acid sequence. Transcription of AtCSP1 in increased in the loss of function mutant atcsp3, implicating that AtCSP3 negatively affects AtCSP1 transcription. Using a gene trap line (GT606 defined as atcsp1), which has complete loss of full length AtCSP1 transcript, GUS gene expression was detected preferentially in tissue primordia and highly dividing tissues. The atcsp1 exhibited early germination after stratification but did not exhibit any further atypical phenotype in vegetative tissues. Germination of atcsp1 without stratification also occurred earlier than wild type but the germination time delayed 24 hours. Comparative analysis of GUS expression in seeds with or without stratification confirmed that AtCSP1 expression was affected by cold temperature during radicle emergence. In addition, ABA germination assays revealed a reduced sensitivity to ABA in atcsp1. Taken together, AtCSP1 may function to regulate germination timing which is in turn mediated by cold temperatures to promote embryo expansion. (Abstract shortened by UMI.)

Regulation of multiple developmental processes by AtFH1 and AtARP3 mediated actin cytoskeleton in Arabidopsis thaliana

The actin cytoskeleton plays a multifaceted role in plant biology. It is involved in several developmental processes and is needed to cope with both biotic and abiotic stresses. Actin is a highly conserved, the most abundant and multifunctional globular protein that can exist either as a globular sub-unit (G-actin) or filamentous (F-actin) form. F-actin is the microfilament part of the cytoskeleton polymerized from G-actin. Actin cytoskeleton polymerization is facilitated by several proteins like formins (polymerizing linear actin cytoskeletons) and the ARP2/3 complex (polymerizing branched actin cytoskeletons). AtFH1 and AtARP3 are important regulators of actin cytoskeleton in Arabidopsis thaliana and belong to the formins and ARP2/3 complex, respectively. The effect of AtFH1 and AtARP3 on actin cytoskeleton reorganization and its subsequent regulation on multiple developmental processes in Arabidopsis thaliana were studied using both single and double mutants of these genes. Simultaneous mutation of AtFH1 and AtARP3 appears to have a lethal effect. Although fh1-1 was not a true knockout mutant, the double mutant fh1-1/arp3-1 further recovered some expression of the AtFH1 gene compared to fh1-1 single mutant but a homozygous double mutant was not obtained. This double mutant showed several unique characteristics compared to the wild type and each single mutant, such as small plants with short, narrow and pale green leaves; short root, slow root growth rate; greater gravitropic response; altered lateral root locations etc. At the cellular level, the double mutant exhibited deformities in epidermal cell circularity; short root hairs; distinct trichome phenotype; small mesophyll cells with lower chloroplasts content; small pollen size, a number of which were structurally distorted. The double mutant produced tiny flowers with distinct floral organ structures that vastly affected the fertility resulted a short silique and a smaller number of seeds due to aborted ovule or embryo. Most of these characteristics were absent in the single mutants, fh1-1 and arp3- 1, and/or were not as severe as in the double mutant. The aberrant actin cytoskeleton organizations that were distinctive in each mutant were observed in epidermal pavement cells, trichome cells and mesophyll cells. So, AtFH1 and AtARP3 appears to regulate several biological processes in Arabidopsis thaliana by maintaining the proper organization of actin cytoskeleton

Plants in Space

Plants will play a critical role in the survival of human beings on long-duration space missions, probably beginning pretty soon with a mission to Mars. Plants can adapt to extreme environments on Earth, and model plants have been shown to grow and develop through a full life cycle in microgravity. In space, long-term human space exploration missions require a life support system in which higher plants play a vital role. Growing crops in space is as much about developing the humans’ technological capacity to provide plants with adequate growth conditions in the unique microgravity environment, as is about the symbiotic relationship between plants and space travelers. After several decades of research, we have learned a lot about the impediments to growing plants in microgravity, in outer space, and on other planets. As human space exploration advances, we should feel confident about our ability to grow plants on board spacecraft during long-term space missions, on the Moon, and on other planets. Plants will require specialized environments for growth and development in microgravity, but – at least on a small scale – we already know how to produce such growth chambers and greenhouses