Demonstration of different drought resistance mechanisms in Mediterranean shrub and tree species: offensive and defensive mechanisms. Demonstrator shows specific leaf morphology and anatomy by using Light Microscopy and Stereomicroscopy equipments.

NETCHEM Remote Access Laboratory Guide Demonstration of different drought resistance mechanisms in Mediterranean shrub and tree species: offensive and defensive mechanisms. In this exercise, you will: Observe plant structures by using Stereomicroscopy Observe plant structures by using LM microscopy Observe specific adaptations of Mediterranenan shrub species ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Background Plant species inhabiting Mediterranean areas of the World are faced with a long arid period and relatively high temperatures in their environments, at the same time. For this reason, many species have to develop mechanisms that allow them to keep water resources in their tissues. We consider such structures to be special adaptations that are featured through the reduction of the surface of the leaf, the formation of thicker cuticle, the improvement of the tissue for the transport of water, the forming of dense indumentums on their parts, or the taking of the role of photosynthesis by non-leaf structures. Specific evergreen shrublands in the Mediterranean climate zone in Europe is called macchia (Italian) or maquis (French). In such vegetation, the dominant life form is represented by drought-resistant, sclerophyll (in Greek sklēros - hard, and phyllon - leaf), mainly evergreen broadleaf trees and shrub species. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Stereo microscope The stereo or stereoscopic microscope is an optical microscope designed for low magnification observation of a samples, typically using light reflected from the surface of an object rather than transmitted through it. The instrument uses two separate optical paths with two objectives and eyepieces to provide slightly different viewing angles to the left and right eyes. This arrangement produces a three-dimensional visualization of the sample being examined. The stereo microscope is often used to study the surfaces of solid dry or fresh plant parts or to carry out close work such as plants specific details, that are not easily visible. Stereo microscopes are essential tools in botany as well as other fields of biology. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Stereomicroscope Leica MZ-16A (Leica Microsystems©, Wetzlar, Germany) ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Light microscope Leica DM 1000 and camera Leica DFC 290 (Leica Microsystems©, Wetzlar, Germany). ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Material For this laboratory exercise, you will need the following material : Herbarized plant material of Salvia officinalis L. (common sage), fam. Lamiaceae; Permanent microscopic preparations of Nerium oleander (oleander) leaf, fam. Apocynaceae; ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Procedure I (stereo microscope ): Prepare herbarium specimens for stereomicroscopic observations. Start your stereo microscope and additional hardware equipment. Put the preparation at the observation table. Turn on the light of the stereo microscope and adjust the intensity. After you get an image on the screen you can choose between different magnifications by using magnification button. Take the image of the specific structure by using “Acquire” button. Save the image in the destination folder you created. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Herbarium specimen of Salvia officinalis ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Result I: Based on stereo microscope observation, please note the folowing adaptations of the leaf surface to the environment: Dense, whitish to silverish indumentum on both leaf sides. Surface of the leaves as well as leaf margines are wrinkled. Nerves on the leaves are thick and prominent. Simple, long, interwoven, curly trichomes.

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Magnified image of plant leaf preparation on the monitor. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Procedure II (light microscope ): Prepare permanent cuts of plant tissues for light microscopic (LM) observations. Start your light microscope and additional hardware equipment. Put the plant preparation at the observation table. Turn on the light of the light microscope and adjust the intensity. After you get an image on the screen at minimal magnification, you can choose between magnifications by using different object glasses. Take the image of the specific tissue structure by using “Acquire” button. Save the image in the destination folder you created. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Magnified image of leaf cross section of Nerium oleander obtained by light microscope ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Closer view of the leaf cut on the monitor. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Result II: Based on light microscope observation, please note the folowing adaptations of the plant tissues to the environment: Thick cuticle Three-layered epidermis Palisade parenchyma Spongy parenchyma Intercellular space in spongy parenchyma Small vascular bundle Strongly developed central nerve Epidermal crypts consisting stomata and trichomes

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Remote Access Connection Instructions What makes these labs different and unique from other classroom experiments is that we have incorporated a section in each activity to remotely characterize your samples from your classroom. Request a remote lab session specifying information such as: the day, the time, and the instrument you are interested in using by visiting our web site: http://netchem.ac.rs/remote-access You will see the list of partners with the instruments provided to chose from. You will be contacted by a Remote Access staff member to set up a test run to ensure you are set up properly and have the required infrastructure. Send samples or verify the in-house sample you would like us to prepare and load for characterization. Send your samples to the Remote Access center that you chose on your request. There are two communications soft-ware packages, that will allow us to communicate instructions and answer questions during the session. - TeamViewer: You can obtain a free download at: https://www.teamviewer.com/en/index.aspx - Skype ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Remote Access Connection Instructions You will need: Computer with administrator access to install plug-ins and software An internet connection Speakers Microphone Projector connected to the same computer Web browser (Firefox preferred) During the test run you can refer to this guide to perform the following steps, but it’s very important that you only proceed with these steps during your scheduled times. You may interfere with other remote sessions and potentially damage equipment if you log in at other times. Open and logon to your Zoom/Team-viewer account. You will be given the access code to enter at the time of your test and then again during the remote session. If you are using the Zoom software, Remote Access staff will give you the access code. If you are using the Team-viewer software, Remote Access staff will give you the ID & password. You should soon see the Remote Access desktop and at this point you can interact with the icons on the screen as if it were your desktop. Switch to full screen mode by selecting the maximize screen option in the top right corner of the screen. Upon completion of the session, move your mouse to the top right corner of the screen, and click on the X to disconnect the remote session. It will ask if you want to end the remote session. Click Yes. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Author, Editor and Referee References This remote access laboratory was created thanks to work done primarily at University of Niš. Contributors to this material were: Bojan Zlatković Refereeing of this material was done by: _____________________ Editing into NETCHEM Format and onto NETCHEM platform was completed by: Milan Milošević ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

References and Supplemental Material The NETCHEM platform was established at the University of Nis in 2016-2019 through the Erasmus Programme. Please contact a NETCHEM representatives at your institution or visit our website for an expanded contact list. The work included had been led by the NETCHEM staff at your institution. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.