Over the years, the Root Development lab has developed different techniques and protocols to achieve his goals. We provide here some descriptions and links towards the publications describing these.
An efficient way to study the histology of the root is sectioning material previously embedded in a resin. It offers the possibility to precisely localize the expression of markers such as GUS, to perform different staining, and finally to image the sections with high resolution. If this method finds more and more competition with the use of confocal microscopes for the root of Arabidopsis thaliana, it remains the best choice to analyse the structure of the roots in others plant species containing a higher number of cell layers.
A first technique described in Beeckman et Viane (1999) allows the embedding of samples in a resin block with very precise orientation using a two steps method.
Construction of flat molds for embedding small plant material. A. Double sided tape (in grey) attached to both sides of a sheet of transparency are cut into strips. B. Strips 21 x 3.7 cm cut into 1.5 x 3.7 cm pieces. C. Enlarged side view of a strip with double-sided tape on each side of the transparency. D. Enlarged view of a small piece of transparency with a central cavity. E. Fenestrated pieces attached to another transparency strip resulting in a flat mold as shown in (F). G. Drop of embedding solution is placed in the central cavity, the specimen is added, and the whole covered by a piece of transparency. H. After polymerization, the tissue is embedded in a thin plate of resin in the central cavity of mold. I-J. A piece of embedded tissue may be cut out of the platelet for further analysis (I), or can be re-embedded in bigger molds in the desired orientation with other similar pieces (J).
Transverse Sectioning of Arabidopsis thaliana Leaves Using Resin Embedding
In collaboration with our laboratory, the laboratory of Gerrit T.S. Beemster has developed a protocol for transverse sectioning of Arabidopsis thaliana leaves using resin embedding. This protocol provides a reliable platform to yield high quality images of cross sections allowing study of development of various tissue layers across the transversal axis of the leaf. As this method is an adaptation of the protocol developed for the Arabidopsis root tip by Beeckman and Viane (1999) and De Smet et al. (2004), it can easily be modified to accommodate other organs and species.
For the purpose of GWAS, a phenotyping pipeline was created to visualize, trace and measure root system architecture traits.
A. Set-up of the growth system dedicated to the GWAS. B. Example of root growth 14 days after germination. C. A3 scanner with a small and a big petri dish. D. scheme illustrating image processing using software dedicated to the measurement of root system architecture.
Some steps of in-vitro growth and phenotyping protocols routinely run in the laboratory have been adapted to analyze plants at older stages of development, i.e. 14 days after germination. Plants are synchronously germinated in 12x12 cm petri dishes before being transferred to larger 24.5x24.5 cm NunclonTM square plates. Gelrite (Duchefa Biochemie) is used as a medium gelling agent, at the final concentration of 0.7%. Plants are grown in growth chambers at 21°C with a light cycle of 16 hours light and 8 hours dark. 14 days after germination, roots are imaged using an EPSON Expression 11000XL A3 scanner. Finally, the images are analyzed with different detection software, such as ImageJ to measure the number of lateral roots and the primary root length, and GiA Roots (Galkovskyi et al., 2012, DOI: 10.1186/1471-2229-12-116) to measure the width and depth, the total root length, the total root surface, the root convex area, the root length distribution and more complex parameters.
Small-Scale Aeroponic System for Non-Destructive Maize Root and Shoot Imaging
The laboratory has developed a small scale aeroponic system for the observation of root systems of young maize plants.
A. Small-scale aeroponic system placed in a maize dedicated plant growth chamber. B. cover of the system holding the plants. C. Defensor 505 spraying device. D. Roots 7 days after germination.
Plants are first synchronously germinated in paper rolls and later transferred to the system. Hoagland solution is sprayed every 160 seconds using a Defensor 505. Two units allow growing simultaneously 2 x 24 plants, up to 2 weeks after germination. This stage of growth corresponds to an optimal development of the embryonic primary and seminal roots as well as the formation of lateral roots on the embryonic roots and the formation of the first ring of nodal roots. This stage also corresponds to the emergence of the second leaf collar on the shoot. The system can easily be opened and allows the observation of the root systems at different stages of development.
The Roots lab acquired a large scale hydroponic system that allows various analyses of both root and shoot simultaneously in a more in vivo approach. The system consists of a main air pump aerating up to 8 hydroponics boxes simultaneously via a manifold, resulting in an equal saturation of oxygen and constant flow of liquid media in the hydroponic boxes. The hydroponic boxes are each equipped with a floating support that can hold 36 plants (of various species) while compatible with different volumes/types of liquid media. The systems flexibility allows prolonged growth up to the moment of flowering unlike most in vitro growth setups. Furthermore the system is less artificial than most, and corresponds more to breeding of crops in agriculture.
Principle of the hydroponics system. A. Scheme of hydroponics setup. B. Setup with 8 hydroponics boxes containing A.thaliana in flowering stage. C. Detail of submerged part of supports, easy access to roots of A.thaliana separated by the tube holders.
MVX 10 Macro Fluorescence Imaging System
The Roots lab has recently installed a tilted macroscope that allows fluorescence imaging of whole tissues as well as at the cellular level in a tilted enclosed setup. This setup consists of a focus drive onto which the MVX10 is mounted in conjunction with a software regulated stage adapted for holding square petridishes in a vertical manner. Automated filterwheels allow sequential fluorescence imaging of multiple fluorophores such as DAPI, GFP, YFP and RFP. As such several roots of A.thaliana seedlings can be observed as a whole for screening or in detail individually. When imaged in detail it is possible to do multi time-lapse imaging of several individual growing roots of A.thaliana in parallel for TL of 20h and more.
MVX 10 system. A. Horizontal encased MVX 10 setup. B. Imaging overview of several rosettes/roots of A.thaliana. C. Imaging root tip of several roots of A.thaliana.