Optimization of Phenotyping Assays for the Model Monocot Setaria viridis

Abstract

Setaria viridis (green foxtail) is an important model plant for the study of C4 photosynthesis in panicoid grasses, and is fast emerging as a system of choice for the study of plant development, domestication, abiotic stress responses and evolution. Basic research findings in Setaria are expected to advance research not only in this species and its close relative S. italica (foxtail millet), but also in other panicoid grasses, many of which are important food or bioenergy crops. Here we report on the standardization of multiple growth and development assays for S. viridis under controlled conditions, and in response to several phytohormones and abiotic stresses. We optimized these assays at three different stages of the plant's life: seed germination and post-germination growth using agar plate-based assays, early seedling growth and development using germination pouch-based assays, and adult plant growth and development under environmentally controlled growth chambers and greenhouses. These assays will be useful for the community to perform large scale phenotyping analyses, mutant screens, comparative physiological analysis, and functional characterization of novel genes of Setaria or other related agricultural crops. Precise description of various growth conditions, effective treatment conditions and description of the resultant phenotypes will help expand the use of S. viridis as an effective model system.

Keywords: Setaria viridis; abiotic stress; phenotyping; phytohormones; seed germination; seedling growth and development.

FIGURE 1

Seeds used for germination and growth assays. (A) Diagrammatic representation of a Setaria floret. Upper anthecium which consists of hard upper lemma and palea attached to the caryopsis (fruit) was used in germination and growth assays. (B) Dark and pale seeds isolated form the same panicle. Dark seeds were used for all experiments. Scale bar is 250 μm.

FIGURE 2

Standardization of seed germination conditions for Setaria using synthetic media in plate-based assays. (A) Germination percentage of freshly harvested Setaria seeds was assessed in the presence of varying concentrations of exogenously added gibberellic acid (GA₃). (B) Seeds were treated with or without liquid smoke before sterilization and germination percentage was quantified from day 1 to day 4. (C) Percentage of seed germination of Setaria was counted from day 1 to day 4 on Phytagel- or Agar-based 0.5X MS media. The number of secondary roots at day 7 was also quantified (inset panel). (D) Effect of varying concentrations of sucrose on seed germination of Setaria was quantified for up to 4 days post-stratification. (E) The primary root length of Setaria seedlings were measured at 7 day post-stratification in the presence of varying concentrations of sucrose. (F) Secondary root number of Setaria seedlings was quantified at 7 day post-stratification. In all experiments, seeds sown on 0.5X MS media plates were stratified at 4 °C in the dark for 2 days followed by transfer to the growth chamber with light-12 h (31°C)/dark-12 h (22°C) cycle. Plates were placed vertically to measure the root length and secondary root number. Minimum 3 months old, dark-colored seeds were used in the experiments (C–F). Data were averaged from three biological replicates and at least 60–70 seeds were used in each experiment. For (A–D), error bars represent standard deviation (±SD). For (E,F) Error bars represent standard error (±SE). Asterisks represent P-values less than 0.05 as calculated using Student’s t-test.

FIGURE 3

Effect of exogenous abscisic acid (ABA), NaCl and glucose on Setaria seed germination in plate-based assays. Sterilized Setaria seeds were plated on varying concentrations of (A) ABA, (B) NaCl, and (C) glucose. Seed germination (n = 60 per treatment) was counted for up to 7 days and compared with germination on control media. Minimum 3 months old, dark-colored seeds were used. In all experiments, seeds were stratified at 4°C in the dark for 2 days followed by transfer to the growth chamber with light-12 h (31°C)/dark-12 h (22°C) cycle. Three biological replicates of each treatment were used for data analysis. Error bars represent standard deviation (±SD).

FIGURE 4

Effect of different plant hormones on post-germination growth of Setaria seedlings using plate-based assays. Sterilized Setaria seeds were plated on varying concentrations of (A) indole-3 acetic acid (IAA), (B) brassinolide (BL), (C) 1-aminocyclopropane-1 carboxylic acid (ACC), and (D) methyl jasmonate (JA). The plates were placed vertically. Root and coleoptile lengths were measured after 5 days of growth. For IAA treatment, seedlings were grown in light-12 h (31°C)/dark-12 h (22°C) cycle. The upper and lower panels in the picture show the entire seedlings and terminal root phenotypes (20 X magnification), respectively. For BL, ACC, and JA treatments the seedlings were grown in the dark. The pictures on the right show the phenotypes of 5 day old seedlings in the presence of specific hormone treatments. At least 3 months old, dark-colored seeds were used in the experiment. Data were averaged from three biological replicates. At least 20–30 seeds were used in each experiment. Error bars represent standard error (±SE). For comparative analysis, appropriate controls were included for each treatment. Asterisks represent P-values less than 0.05 as calculated using Student’s t-test. Scale bars are 5 mm for all figures except for the lower panel of IAA treated roots where it is 1 mm.

FIGURE 5

Effect of different stresses on post-germination growth of Setaria using plate-based assays. Sterilized Setaria seeds sown on 0.5X MS media plates were stratified at 4°C in the darkness for 2 days followed by transfer to the growth chamber with light-12 h (31°C)/dark-12 h (22°C) cycle for another 2 days. The effect of ABA on post-germination growth was studied by measuring the primary root length at day 3 (A) and secondary root number at day 5 (B) after transfer to the media containing varying concentrations of ABA. The picture in (C) shows representative seedlings after growth in the presence of ABA. The effect of NaCl (D,E) and glucose (G,H) was analyzed by quantifying the primary root length and coleoptile length after transfer to the media containing varying concentrations of NaCl and glucose. Figures (F,I) show representative images of Setaria seedlings after growth in the presence of NaCl or glucose, respectively. For each experiment, 3 months old, dark-colored seeds were used. Three biological replicates of each treatment were performed and at least 20–30 seeds were used in each experiment. Appropriate controls were used for each treatment. Error bars represent standard errors (±SE). Asterisks represent P-values less than 0.05 as calculated using Student’s t-test. Scale bars are 5 mm.

FIGURE 6

Growth of Setaria in germination pouches and effect of different stresses on seedling growth. (A) Representative image of 2-week-old Setaria plants growing in germination pouch. At least 3 months old, dark-colored Setaria seeds were grown under control conditions by placing them on pre-wet pouches. (B) For different abiotic stress treatments, seeds were germinated on pre-wet germination pouches for 2 days. The media in the germination pouch was replaced with media supplemented with ABA (5 and 10 μM), NaCl (50 and 100 mM) and PEG (5% and 10%, equivalent to –0.05 and –0.15 MPa osmotic stress). (C) The media in germination pouches was replaced with different concentrations of IAA and (D) with either minus nitrogen or minus phosphate MS media. In all experiments, the primary root lengths were quantified after 2 weeks of growth. Appropriate controls were used in each experiment (B–D). Three biological replicates of each treatment were performed and at least 20 seeds were used for each experiment. Error bars represent standard errors (±SE). Asterisks represent P-values less than 0.05 as calculated using Student’s t-test.

FIGURE 7

Comparative analysis of vegetative and reproductive growth parameters of Setaria grown in controlled environment growth chambers and in greenhouses. (A) Representative images of 7-week-old Setaria plants grown in growth chamber (GC, left) and greenhouse (GH, right). (B) Plant height at 7 weeks, (C) Leaf number at 4 weeks (D) panicle number at 7 weeks and (E) length of mature panicle at 7 weeks were measured. (F) Total seed weight was quantified from the plants grown under in growth chambers versus greenhouse. Three biological replicates of each treatment and four plants in each experiment were used for data analysis. Error bars represent standard errors (±SE). Asterisks represent P-values less than 0.05 as calculated using Student’s t-test. Scale bar is 5 cm.

FIGURE 8

Effect of water and nitrogen deficit on vegetative and reproductive growth parameters of Setaria. (A) Representative image of 7 weeks old Setaria plants grown in the greenhouse under four different conditions: W = well-watered +15 mM nitrogen supplemented in the media, W-N = well-watered, no nitrogen supplement, L = low watered (50% compared to the well-watered control) +15 mM nitrogen, and L-N = low-water and no nitrogen supplement in the media. Different growth parameters such as (B) plant height and (C) leaf number were measured from seven and 4 weeks old Setaria plants, respectively. (D) Representative image of main panicle from 7 weeks old Setaria plants. (E) Panicles number per plant was counted from 7 weeks old plants. (F) Seed weight/plant was calculated after harvesting. Three biological replicates of each treatment and four plants in each experiment were used for data analysis. Error bars represent standard errors (±SE). Asterisks represent P-values less than 0.05 as calculated using Student’s t-test. Scale bars are 5 cm for (A) and 5 mm for (D).

FIGURE 9

Comparison of Setaria accessions A10.1 and ME034V grown in controlled environment growth chambers versus in greenhouses. (A) Representative images of 5 weeks old Setaria plants A10.1 (left) and ME034V (right) grown in growth chamber (GC) and greenhouse (GH). (B) Representative images of A10.1 (left) and ME034V (right) panicles grown in growth chamber (GC) and greenhouse (GH). The plants were growth conditions under similar conditions in growth chamber and greenhouse under conditions listed in Supplementary Table S1. Scale bars are 5 cm for (A) and 5 mm for (B).