Protocol

Authors

Lawas LMF, Oane R and Jagadish SVK

Author Affliations

International Rice Research Institute, DAPO BOX 7777, Metro Manila, Philippines

(Correspondence – k.jagadish@irri.org)

Overview

The protocol below is for (i) precise estimation of the impact of heat stress on spikelet fertility in rice and (ii) quantifying the influence of high temperature on the reproductive physiology, i.e. pollination success – pollen shedding and germination on the stigma and estimation of in vivo pollen tube growth in rice.

Background

Rice is extremely sensitive to heat stress at flowering; temperatures >38 ^∘C for even an hour can lead to increased spikelet sterility (Jagadish et al., 2007). Each rice panicle takes 5-7 days to finish flowering with a top-to-bottom approach. In addition, a delay in flowering among primary and secondary tillers makes precise quantification of the temperature effect more difficult. Hence, a spikelet marking approach has been devised to estimate the temperature effect on different flowering days and to avoid confounding results due to the quantitative effect of stress on different flowering days. The process of pollination and pollen germination leading to fertilization determines seed setting, and these processes are negatively affected by high-temperature stress coinciding with anthesis (Satake and Yoshida, 1978). Our protocol will provide details for estimating pollen germination and the events leading to fertilization (pollen tube reaching the ovary), using aniline blue staining (Jagadish et al., 2010). Aniline blue stains callose and helps track the rate of pollen tube growth in the stigma, style and until it reaches the ovary.

Materials/Equipment

• Acrylic paint (for estimating percent spikelet sterility)
• Vials
• Forceps
• Rigger brush (liner paint brush, size 1 or 2)
• Plastic Pasteur pipettes
• Microscope slide
• Glass cover slip
• Fixative (3:1 absolute ethanol:glacial acetic acid)
• Distilled water
• 8M NaOH
• 0.2% aniline blue dissolved in 0.1M K2HPO4
• Silicon glue
• Stereo microscope
• Fluorescent microscope with attached camera
• Light, temperature and humidity controlled growth chamber

Units, terms, definitions

pollination success – pollen shedding and germination on the stigma

Procedure

Quantifying the impact of heat stress on spikelet fertility

1. Tag the main tiller at the heading stage. Note: At least six replicate plants are needed to minimize experimental error.
2. On the predicted first day of anthesis, transfer plants from the greenhouse to a temperature-controlled chamber set at 38^∘C (threshold temperature for identifying true heat tolerance) and 75% RH. Note: (i) The chambers have to be programmed to follow a diurnal temperature pattern to reach 38^∘C from 0830 till 1430 with minimum light of 650 μmol m^-2 sec^-1; (ii) plants have to be transferred at 0700 well before the first spikelet starts to open to ensure that high-temperature treatment coincides with anthesis/flowering; and (iii) regular temperature and RH monitoring using data loggers is essential.
3. The plants have to be left inside the chamber for 6 hours (0830 to 1430).
4. Plants exposed to 6 hours of stress are moved out of the chamber to the greenhouse, and all the anthesed spikelets (with externally visible anthers) on the tagged tiller are marked using acrylic paint (Fig. 1).

Fig. 1. Marking individual spikelets with acrylic paint as a precise phenotyping protocol for quantifying heat tolerance in rice. Different colours mark spikelets that anthese on different days.

5. A couple of hours after marking the spikelets, carefully remove the anthers from anthesed spikelets to distinguish spikelets that flower the next day. Note: (i) If screening is done in a walk-in chamber, the marking can be done inside the chamber; (ii) avoid marking spikelets in the middle of the treatment period to ensure minimal manual interference. Data obtained with and without acrylic marks showed no change in spikelet fertility percentage.
6. Repeat marking and anther removal procedure on the second, third, and fourth day of anthesis (steps 1-5), using different-colored acrylic paint to estimate the quantitative effect of heat stress across different flowering days.
7. Maintain plants under non stress conditions until maturity and harvest the tagged panicles.
8. For each day of anthesis (identified by the different colors on the spikelets), record the total number of marked spikelets and the number of grains formed. Note: Plants used for estimating spikelet fertility should (if possible) not be used for sampling either leaves or spikelets for any other analysis.
9. Compute percent spikelet fertility as follows:

Spikelet fertility = (Number of grains formed/Total number of marked spikelets) x100

In vivo pollen germination and pollen tube growth

Spikelet sampling

1. On the first day of anthesis, a careful “wait and watch” approach has to be followed and spikelets starting to open (the tip of the lemma moving away from the palea) are very delicately marked with a waterproof marker. Note: If a sufficient number of spikelets do not open at the same time, different colors can be used to distinguish batches of spikelets opening at different times to obtain the desired number of spikelets with the set target duration.
2. At 45 min or a maximum of 1 hour after the start of anthesis, marked spikelets are collected using a forceps and placed in a fixative containing absolute ethanol and glacial acetic acid in 3:1 proportions.
3. Samples are stored at 4 ^∘C for further microscopic observation.

Dissection and staining

1. Stored spikelets are transferred into a container with distilled water to dilute the fixative.
2. Using a stereo microscope, dissect each spikelet by carefully removing the lemma and palea to get the pistil. Remove filaments near the ovary using a sharp blade. Note: Use utmost care to avoid damaging the ovary.
3. Using a rigger brush (size 1 or 2), transfer the dissected pistil (stigma, style, and ovary) into a vial containing 8N NaOH. Leave the sample in solution for 24-36 hours at room temperature to clear the tissue. Note: Clearing time is genotype-dependent. It is recommended to optimize clearing duration before detailed experimentation.
4. The pistil samples are removed from the NaOH solution using a similar brush and placed on a microscope slide. Excess NaOH has to be removed using a Pasteur pipette.
5. Add 3-4 drops of 0.2% aniline blue on the sample. Stain for 10-15 min. Note: This step should be done in the dark since aniline blue is light-sensitive.
6. Using a pipette, remove the stain and wash the sample with distilled water to remove excess aniline blue. Finally, suspend stained pistil samples on the slide with distilled water.
7. Place a small amount of silicon glue on four corners of the cover slip, which will serve as a spacer to prevent crushing the ovary, and place the cover slip on top of the slide. Add water to fill the space between the slide and cover slip to avoid interference with air bubbles.

Microscopic observations

1. The slide containing the sample is observed under an epifluorescent microscope (Axioplan 2, Carl Zeiss, Germany) using 365nm excitation filter and 397nm barrier filter (LP, Filter #1). Note: The HBO lamp of the fluorescent microscope should be switched on at least 15min before observation.
2. Photomicrographs of the stigma and ovary at 50x magnification (to accommodate the entire pistil) are taken using a camera (Olympus DP70) attached to the microscope.
3. View the stigma at 100x magnification to count the total number of pollen grains deposited on the stigma, the number of germinated pollen, and the number of pollen tubes reaching the ovary (Fig. 2). A pollen grain is considered germinated if the pollen tube length is equal to or greater than the pollen diameter. Viable pollen and pollen tube would give a fluorescent green color as a result of the reaction of the dye with callose. Note: The sample has to be observed on different focal planes to view all pollen on the entire surface of the stigma.
4. Pollen tube length is measured with Image Pro Plus or other image analysis software after calibration. Pollen tube length is measured from the base of the germ pore to the tip of the germinating pollen tube.

Fig. 2. Rice stigma with germinated pollen grains under control temperatures (A) and ovary showing pollen tubes reaching the ovule (B).

Literature references

Jagadish SVK, Craufurd PQ, Wheeler TR (2007) High temperature stress and spikelet fertility in rice. Journal of Experimental Botany, 58(7), 1627-1635.

Jagadish SVK, Muthurajan R, Oane R, Wheeler TR, Heuer S, Bennett J, Craufurd PQ (2010) Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.). Journal of Experimental Botany, 61(1), 143-156.

Satake T, Yoshida S (1978) High temperature induced sterility in indica rice at flowering. Japanese Journal of Crop Science 47, 6-10.

Health, safety & hazardous waste disposal considerations

Wear suitable protective clothing and gloves when handling the stain. Avoid contact with skin and eyes. Separate brush and Pasteur pipettes used in staining. Dispose of excess stain according to local health and safety regulations.