CRISPR/Cas9-Induced Loss-of-Function Mutation in the Barley Mitogen-Activated Protein Kinase 6 Gene Causes Abnormal Embryo Development Leading to Severely Reduced Grain Germination and Seedling Shootless Phenotype

The diverse roles of mitogen-activated protein kinases (MAPKs, MPKs) in plant development could be efficiently revealed by reverse genetic studies. In Arabidopsis, mpk6 knockout mutants complete the life cycle; however, ~40% of their embryos show defects in the development leading to abnormal phenotypes of seeds and seedlings' roots. Contrary to the Arabidopsis MPK6, the rice MPK6 (OsMPK6) is an essential gene as transfer DNA (T-DNA) insertion and CRISPR/Cas9 induced loss-of-function mutations in the OsMPK6 cause early embryo arrest. In this study, we successfully developed a viable transgenic barley line with the CRISPR/Cas9-induced heterozygous single base pair cytosine-guanine (CG) deletion [wild type (WT)/−1C] in the third exon of the HvMPK6 gene, a barley ortholog of the Arabidopsis and rice MPK6. There were no obvious macroscopic phenotype differences between the WT/−1C plants and WT plants. All the grains collected from the WT/−1C plants were of similar size and appearance. However, seedling emergence percentage (SEP) from these grains was substantially decreased in the soil in the T2 and T3 generation. The mutation analysis of the 248 emerged T2 and T3 generation plants showed that none of them was a biallelic mutant in the HvMPK6 gene, suggesting lethality of the −1C/−1C homozygous knockout mutation. In the soil, the majority of the −1C/−1C grains did not germinate and the minority of them developed into abnormal seedlings with a shootless phenotype and a reduced root system. Some of the −1C/−1C seedlings also developed one or more small chlorotic leaf blade-like structure/structures. The −1C/−1C grains contained the late-stage developed abnormal embryos with the morphologically obvious scutellum and root part of the embryonic axis but with the missing or substantially reduced shoot part of the embryonic axis. The observed embryonic abnormalities correlated well with the shootless phenotype of the seedlings and suggested that the later-stage defect is predetermined already during the embryo development. In conclusion, our results indicate that barley MPK6 is essential for the embryologically predetermined shoot formation, but not for the most aspects of the embryo and early seedling development.

Genetic Variability of Mineral Content in Different Grain Structures of Bean Cultivars from Mesoamerican and Andean Gene Pools

Beans (Phaseolus vulgaris L.) are an important source of proteins, carbohydrates, and micronutrients in the diets of millions of people in Latin America and Africa. Studies related to genetic variability in the accumulation and distribution of nutrients are valuable for biofortification programs, as there is evidence that the seed coat and embryo differ in the bioavailability of essential nutrients. In this study, we sought to evaluate the genetic variability of total mineral content in the grain and its constituent parts (seed coat, cotyledon, and embryonic axis) of bean genotypes from Mesoamerican and Andean centers of origin. Grain samples of 10 bean cultivars were analyzed for the content of proteins and minerals (Mg, Ca, K, P, Mn, S, Cu, B, Fe, and Zn) in the whole grains and seed coat, cotyledons, and embryonic axis tissues. Genetic variability was observed among the cultivars for protein content and all evaluated minerals. Moreover, differential accumulation of minerals was observed in the seed coat, cotyledons, and embryonic axis. Except for Ca, which accumulated predominantly in the seed coat, higher percentages of minerals were detected in the cotyledons. Furthermore, 100-grain mass values showed negative correlations with the contents of Ca, Mg, P, Zn, Fe, and Mn in whole grains or in the different grain tissues. In general, the Mesoamerican cultivars showed a higher concentration of minerals in the grains, whereas Andean cultivars showed higher concentrations of protein.

Optimization of Different Factors for Initiation of Somatic Embryogenesis in Suspension Cultures in Sandalwood (Santalum album L.)

Santalum album (L.) is a prized tropical tree species of high therapeutic and industrial importance. The wood of these naturally grown plants is extensively harvested to acquire therapeutically important metabolite santalol and be used for additional functions such as in wood statuette industries. Due to high demand, it is crucial to maintain a sufficient plant population. An easy protocol for establishing cell suspension culture initiated from the loose embryogenic callus mass of sandalwood was realized by shifting 6–8-week-old morphogenic calli acquired from the mature embryonic axis and cotyledon explant cultures in fluid media. The asynchronous embryogenic cultures were sloughed with clumps of flourishing cell clumps and embryos of various progressive phases along with diffident non-embryogenic tissues. The frequency of embryo proliferation was evidenced to determinethe expansion pace of embryogenic masses under diverse conditions. The intonation of initiation and creation of cell suspension was under the directive of the influence of exogenous plant growth regulators amended in the nutrient medium at different concentrations and combinations. Maximum relative growth rate (386%) and clumps/embryoids in elevated integers (321.44) were accomplished on MS nutrient medium fortified with 2.0 mg L−1 2,4-D in association with 0.5 mg L−1 BA and 30.0 g L−1 sucrose raised from mature embryonic axis-derived calli. Plantlet regeneration in higher frequency (84.43%) was evidenced on MS medium amended with 1.0 mg L−1 each of TDZ and GA3 in conjunction with 0.5 mg L−1 NAA and 20.0 g L−1 sucrose. Mature embryonic axis-derived calli were found to be constantly better than mature cotyledon-derived calli for raising profitable and reproducible cell suspension cultures. Regenerants displayed normal growth and morphology and were founded successfully in the external environment after hardening.

Guidance by followers ensures long-range coordination of cell migration through α-Catenin mechanoperception

Morphogenesis, wound healing and some cancer metastases depend upon migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, extension of the embryonic axis is led by the mesendodermal polster that migrates towards the animal pole, followed by axial mesoderm that is undergoing convergence and extension. We here investigate how polster cells are guided towards the animal pole. Using a combination of precise laser ablations, advanced transplantations and functional as well as silico approaches, we establish that the directional information guiding polster cells is mechanical, and is provided by the anteriorward migration of the following cells. This information is detected by cell-cell contact through E-Cadherin/α-Catenin mechanotransduction and propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness.

Dormancia física de la semilla de Peltophorum pterocarpum (DC.) Backer ex Heyne: estudio morfoanatómico, germinación y emergencia de la plántula

Peltophorum pterocarpum (DC.) Backer ex Heyne es un árbol cuyo crecimiento rápido y adaptación a condiciones de estrés hídrico permiten su uso como ornamental en ciudades secas y calientes de los trópicos. Sin embargo, la propagación por semillas se dificulta debido a la dureza de la cubierta seminal, siendo necesaria la aplicación de tratamientos pregerminativos. El objetivo fue describir el proceso de germinación, emergencia y analizar las posibles causas morfoanatómicas de la dormancia en las semillas de P. pterocarpum. Para ello se estimaron los tiempos de germinación (T10, T50, T90), se estudió la fase plantular y se realizaron láminas semipermanentes. Las semillas son elípticas, homogéneas y opacas. Se evidenció la presencia del hilo, micrópilo, funículo, rafe y el lente o estrofíolo ubicados hacia el extremo hilar. El embrión es recto y el eje embrional está conformado por la radícula cónica, anfítropa, con dos cotiledones simples, elípticos, carnosos y endosperma escaso. La germinación fue epígea y fanerocotilar, con el primer par de prófilos bipinnados de ocho folíolos. Las características morfoanatómicas de las semillas proporcionan las estrategias para que la especie pueda propagarse a través del tiempo y el espacio debido a la presencia de la dormancia física, la cual se asocia anatómicamente con el tejido esclerenquimático presentado por la presencia de dos capas de células uniformemente engrosadas, lignificadas y compactas, dispuestas en empalizada, junto a una hilera de osteoesclereidas. Para facilitar los procedimientos a seguir por viveristas y productores durante la propagación sexual de la especie, se recomienda evaluar tratamientos pregerminativos. Palabra clave: Peltophorum pterocarpum, semilla, germinación, dormancia, morfoanatomía. Abstract Peltophorum pterocarpum (DC.) Backer ex Heyne is a tree whose rapid growth and adaptation to hydric stress conditions allow its use as an ornamental in hot, dry cities of the tropics. However, the propagation by seeds is difficult due to the hardness of the seminal cover, being necessary the application of pregerminative treatments. The objective was to describe the germination and emergence process and to analyze the possible morphoanatomic causes of dormancy in P. pterocarpum seeds. For this, the germination times (T10, T50, T90) were estimated, the seedling phase was studied and semi-permanent sheets were made. The seeds are elliptical, homogeneous, and opaque. The presence of the thread, micropyle, funiculus, raphe, and the lens or strophiolelocated towards the end of the line was evidenced. The embryo is straight and the embryonic axis is made up of the conical, amphitropic radicle, with two simple, elliptical, fleshy cotyledons and scant endosperm. Germination was epigeal and phanerocotylar, with the first pair of bipinnate prophiles with eight leaflets. The morphoanatomical characteristics of the seeds provide the strategies for the species to spread through time and space due to the presence of physical dormancy, which is anatomically associated with the sclerenchymal tissue presented by the presence of two layers of cells uniformly thickened, lignified and compact, arranged in a palisade, next to a row of osteosclereids. To facilitate the procedures to be followed by nurserymen and producers during the sexual propagation of the species, it is recommended to evaluate pregerminative treatments. Keywords: Peltophorum pterocarpum, seed, germination, dormancy, morphoanatomy.

The expansin EXP1 gene in the elongation zone is induced during soybean embryonic axis germination and differentially expressed in response to ABA and PEG treatments

During soybean seed germination, the expansive growth potential of the embryonic axes is driven by water uptake while cell wall loosening occurs in cells from the elongation zone (EZ). Expansins are regarded as primary promoters of cell wall remodelling in all plant expansion processes, with the expression profiles of the soybean expansins supporting their cell or tissue specificity. Therefore, we used embryonic axes isolated from whole seed and focused on the EZ to study seed germination. Using a suite of degenerate primers, we amplified an abundantly expressed expansin gene at the EZ during soybean embryonic axis germination, which was identified as EXP1 by in silico analyses. Expression studies showed that EXP1 was induced under germination conditions in distilled water and down-regulated by abscisic acid (ABA), which inhibits soybean germination by physiologically restraining expansion. Moreover, we also identified a time window of ABA responsiveness within the first 6 h of incubation in water, after which ABA lost control of both EXP1 expression and embryonic axis germination, thus confirming the early role of EXP1 in the EZ during this process. By contrast, EXP1 levels in the EZ increased even when germination was impaired by osmotically limiting the water availability required to develop the embryonic axes’ growth potential. We propose that these higher EXP1 levels are involved in the fast germination of soybean embryonic axes as soon as water availability is re-established. Taken together, our results show strong EXP1 expression in the EZ and postulate EXP1 as a target candidate for soybean seed germination control.