Maize Seedling Growth and Hormone Response Assays Using the Rolled Towel Method

Abstract

Root system architecture is a critical factor in maize health and stress resilience. Determining the genetic and environmental factors that shape maize root system architecture is an active research area. However, the ability to phenotype juvenile root systems is hindered by the use of field-grown and soil-based systems. An alternative to soil- and field-based growing conditions for maize seedlings is a controlled environment with a soil-free medium, which can facilitate root system phenotyping. Here, we describe how to grow maize under soil-free conditions for up to 12 days to facilitate root phenotyping. Maize seeds are sterilized and planted on specialized seed germination paper to minimize fungal contamination and ensure synchronized seedling growth, followed by imaging at the desired time point. The root images are then analyzed to quantify traits of interest, such as primary root length, lateral root density, seminal root length, and seminal root number. In addition, juvenile shoot traits can be quantified using manual annotation methods. We also outline the steps for performing rigorous hormone response assays for four classical phytohormones: auxin, brassinosteroid, cytokinin, and jasmonic acid. This protocol can be rapidly scaled up and is compatible with genetic screens and sample collection for downstream molecular analyses such as transcriptomics and proteomics. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maize seedling rolled towel assay and phenotyping Basic Protocol 2: Maize seedling hormone response assays using the rolled towel assay.

Keywords: auxin; brassinosteroid; cytokinin; jasmonic acid; maize.

Figure 1

Overview of the initial steps in Basic Protocol 1. First, maize kernels are surface‐sterilized in bleach and rinsed with water three times before placement on three sheets of labeled, fungicide‐treated Anchor seed germination paper. Towels are rolled up lengthwise, placed upright in a 2‐L Nalgene beaker filled with 600 ml of 0.5× LS, and put into a growth chamber.

Figure 2

Diagram of planted rolled towels. The rolled towels are oriented vertically in a 2‐L Nalgene beaker such that the kernels are at the top of the paper rolls. The beaker contains 600 ml of 0.5× LS, which does not come into contact with the kernels.

Figure 3

Brassinosteroid treatment assays. Brassinosteroid response assays in maize seedlings performed with brassinolide (BL), visualized with box and whisker plots. Seedlings were treated at 3 DAG for 2 days. (A) Primary root length of 5‐day‐old maize seedlings is inhibited with a 50 µM BL treatment (ANOVA, p‐value = 0.0108). Each dot is an individual seedling (biological replicate).

Figure 4

Cytokinin treatment assays. Cytokinin response assays in maize seedlings treated with benzylaminopurine (BAP). Seedlings were treated at 3 DAG for 2 days. (A) Representative 5‐day‐old (DO) maize seedlings grown with 0, 0.5, 2.5, and 5 µM cytokinin (as indicated next to the seedling) and imaged with a DSLR camera. Scale bar = 5.08 cm. (B) Primary root length is inhibited by 5 µM BAP (ANOVA < 0.0001). (C) Shoot length is increased under 0.5 µM (ANOVA = 0.0015) and 2.5 µM (ANOVA = 0.0043) BAP. (D) Lateral root number is significantly increased at all concentrations of BAP shown. (E) Lateral root density is significantly increased under 0.5 µM (ANOVA = 0.0037), 2.5 µM (ANOVA = 0.0008), and 5 µM (ANOVA = 0.0006) BAP. Each dot is an individual seedling (biological replicate).

Figure 5

Jasmonic acid response assays. Jasmonic acid response assays in maize seedlings treated with methyl jasmonate (JA). Seedlings were treated at 3 DAG for 2 days. (A) Representative 5‐day‐old (DO) maize seedlings grown with 0, 0.5, 50, and 100 µM JA (as indicated above each seedling) and imaged with a DSLR camera. Scale bar = 5.08 cm (B) Primary root growth is inhibited by all three concentrations of JA tested, ANOVA < 0.0001. Each dot is an individual seedling (biological replicate).

Figure 6

Indole‐3‐acetic acid response assays. Auxin response assays in maize seedlings treated with indole‐3‐acetic acid (IAA). Seedlings were treated with the corresponding hormone for 2 days at 3 DAG. (A) Representative 5‐day‐old (DO) maize seedlings grown with 0, 1, 10 and 100 µM IAA and imaged with a DSLR camera. Scale bar = 5.08 cm (B) Primary root length is significantly inhibited at all three concentrations of IAA that were tested (ANOVA ≤ 0.0001). (C) Shoot length is significantly inhibited under 100 µM IAA treatment (ANOVA= 0.0001). (D) Seminal root length is significantly inhibited under 100 µM IAA treatment (ANOVA ≤ 0.0001). (E) Lateral root density (the number of lateral roots divided by the primary root length) is significantly increased under all IAA treatments that were tested (ANOVA ≤ 0.0001). Each dot is an individual seedling (biological replicate).