研究目的
To develop a robust method for confocal laser scanning microscopy (CLSM) by which oomycetous and plant tissue could be clearly differentiated, using the example of the previously unstudied infection process of P. salviae-officinalis in association with its host, S. officinalis.
研究成果
The staining procedure involving decolourization with ethanol and KOH and staining with a 1:1 aniline blue–trypan blue mixture allows detailed visualization of downy mildew structures outside and inside the host tissue, as well as of callose deposition as an immune response of the plant. The method is also applicable to other downy mildew species, demonstrating its potential for studying the infection processes of downy mildews in general.
研究不足
The study acknowledges the challenges in fluorescence staining and imaging of oomycete–plant interactions due to the similarity in cell wall composition of both plant and microorganism. The method requires ample experience of microscopic observation for the distinction of intercellular hyphae or haustoria.
1:Experimental Design and Method Selection:
The study aimed to develop a staining method for CLSM to differentiate oomycetous and plant tissue clearly. A histological study of various infection events of P. salviae-officinalis on S. officinalis was conducted.
2:Sample Selection and Data Sources:
Sage plants at the 4-leaf-stage were spray-inoculated with a suspension of freshly harvested conidia of P. salviae-officinalis. Samples were also taken from Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum.
3:List of Experimental Equipment and Materials:
Confocal laser scanning microscope (TCS SP8; Leica), aniline blue, trypan blue, ethanol series, KOH, phosphate-buffered saline (PBS), glycerol.
4:Experimental Procedures and Operational Workflow:
Leaf samples were decolourized with ethanol and KOH, stained with a 1:1 mixture of aniline blue and trypan blue, and examined under CLSM.
5:Data Analysis Methods:
Images were captured using LAS X software for Leica microscopes, and Adobe Photoshop CS2 was used for image enhancement.
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