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An in vitro grafting method to quantify mechanical forces of adhering tissues.

ABSTRACT: Grafting is an indispensable agricultural technology for propagating useful tree varieties and obtaining beneficial traits of two varieties/species-as stock and scion-at the same time. Recent studies of molecular events during grafting have revealed dynamic physiological and transcriptomic changes. Strategies focused on specific grafting steps are needed to further associate each physiological and molecular event with those steps. In this study, we developed a method to investigate the tissue adhesion event, an early grafting step, by improving an artificial in vitro grafting system in which two pieces of 1.5-mm thick Nicotiana benthamiana cut stem sections were combined and cultured on medium. We prepared a silicone sheet containing five special cutouts for adhesion of cut stem slices. We quantitatively measured the adhesive force at these grafting interfaces using a force gauge and found that graft adhesion started 2 days after grafting, with the adhesive force gradually increasing over time. After confirming the positive effect of auxin on grafting by this method, we tested the effect of cellulase treatment and observed significant enhancement of graft tissue adhesion. Compared with the addition of auxin or cellulase individually, the adhesive force was stronger when both auxin and cellulase were added simultaneously. The in vitro grafting method developed in this study is thus useful for examining the process of graft adhesion.

SUBMITTER: Kawakatsu Y

PROVIDER: S-EPMC8034679 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Blood flow and mechanical forces in the ventricle are implicated in cardiac development and trabeculation. However, the mechanisms of mechanotransduction remain elusive. This is due in part to the challenges associated with accurately quantifying mechanical forces in the developing heart. We present a novel computational framework to simulate cardiac hemodynamics in developing zebrafish embryos by coupling 4-D light sheet imaging with a stabilized finite element flow solver, and extract time-dependent mechanical stimuli data. We employ deformable image registration methods to segment the motion of the ventricle from high resolution 4-D light sheet image data. This results in a robust and efficient workflow, as segmentation need only be performed at one cardiac phase, while wall position in the other cardiac phases is found by image registration. Ventricular hemodynamics are then quantified by numerically solving the Navier-Stokes equations in the moving wall domain with our validated flow solver. We demonstrate the applicability of the workflow in wild type zebrafish and three treated fish types that disrupt trabeculation: (a) chemical treatment using AG1478, an ErbB2 signaling inhibitor that inhibits proliferation and differentiation of cardiac trabeculation; (b) injection of gata1a morpholino oligomer (gata1aMO) suppressing hematopoiesis and resulting in attenuated trabeculation; (c) weak-atriumm58 mutant (wea) with inhibited atrial contraction leading to a highly undeveloped ventricle and poor cardiac function. Our simulations reveal elevated wall shear stress (WSS) in wild type and AG1478 compared to gata1aMO and wea. High oscillatory shear index (OSI) in the grooves between trabeculae, compared to lower values on the ridges, in the wild type suggest oscillatory forces as a possible regulatory mechanism of cardiac trabeculation development. The framework has broad applicability for future cardiac developmental studies focused on quantitatively investigating the role of hemodynamic forces and mechanotransduction during morphogenesis.

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An in vitro grafting method to quantify mechanical forces of adhering tissues.

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