Abstract
Quantification of the tissue and cellular structure of plant material is essential for the study of a variety of plant sciences applications. Currently, many methods for sectioning plant material are either low throughput or involve free-hand sectioning which requires a significant amount of practice. Here, we present an updated method to provide rapid and high-quality cross sections, primarily of root tissue but which can also be readily applied to other tissues such as leaves or stems. To increase the throughput of traditional agarose embedding and sectioning, custom designed 3D printed molds were utilized to embed 5–15 roots in a block for sectioning in a single cut. A single fluorescent stain in combination with laser scanning confocal microscopy was used to obtain high quality images of thick sections. The provided CAD files allow production of the embedding molds described here from a number of online 3D printing services. Although originally developed for roots, this method provides rapid, high quality cross sections of many plant tissue types, making it suitable for use in forward genetic screens for differences in specific cell structures or developmental changes. To demonstrate the utility of the technique, the two parent lines of the wheat (Triticum aestivum) Chinese Spring × Paragon doubled haploid mapping population were phenotyped for root anatomical differences. Significant differences in adventitious cross section area, stele area, xylem, phloem, metaxylem, and cortical cell file count were found.
Highlights
Anatomical plant traits represent an important, yet relatively unexploited route for crop improvement, in unfavorable or low-input conditions
Chinese Spring and Savannah were grown in 2 l pots filled with potting compost (John Innes number 2) in a glasshouse; rice (Oryza sativa) was grown in hydroponic solution in a controlled environment (CE) room at 28◦C with a 12 h photoperiod and a light intensity at plant height of 400 μmol m−2 s−1; Arabidopsis thaliana seedlings were grown on agar plates containing half-strength Murashige and Skoog (MS) growth medium pH adjusted to 5.7 with KOH, in a CE room with 12 h photoperiod and a light intensity at plant height of 150 μmol m−2 s−1; Thinopyrum ponticum was grown in 2 l pots filled with potting compost (John Innes number 2) in a glasshouse
Rice adventitious and seminal roots were collected from 60 day old plants, 10 cm from the root tip and Arabidopsis primary roots were collected from 7 day old plants, 5 cm from the root tip
Summary
Anatomical plant traits represent an important, yet relatively unexploited route for crop improvement, in unfavorable or low-input conditions. An increased number of root cortical aerenchyma (RCA) in maize has been shown to increase nitrogen acquisition in low nitrogen soils (Saengwilai et al, 2014). In rice (Oryza sativa L.) leaf tissue, decreased photosynthesis and hydraulic conductance under drought has been lined to decreasing major vein thickness, rather than changes in leaf vein density (Tabassum et al, 2016). These traits are only quantifiable through histological sectioning techniques, which are often time consuming and difficult to conduct, limiting their use in forward genetic screens
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