Measuring Wheat Pollen Viability - A Delicate Procedure?!

Learn why it is important to measure wheat pollen viability and discover the best methods for pollen extraction.

Introduction

Georg Roell

Georg Roell

My name is Georg Roell, and I am part of the Amphasys support team. I have been working on wheat for more than 15 years. This includes working on a farm, in the breeding and plant protection areas and during my PhD studies. At all these different levels the main goal was to maximize yield by optimizing the management and/or the genetic material. Phenotyping of pollen characteristics can deliver a new tool to identify traits in plant breeding.

What this Article is About

Wheat crosspollination for breeding purposes is the only “natural” way to transfer genes between different lines. Flowering and pollen production are, on the one hand, fundamental processes implicated in the creation of new genetic combinations and, on the other hand, indispensable to secure the final crop yield. In the agricultural production system, little attention is devoted to pollen fertility. Nevertheless, it is well known from research that chemical treatments and stress conditions, like drought and heat, can have a strong impact on pollen viability.

The aim of this blog post is to offer practical guidance in collecting wheat pollen in the field in order to analyze its quality and to show you the background of some of our recommendations. I will cover aspects such as sample size, sample preparation methods and why pollen quality measurements should be done as quick as possible.

All measurements shown in this post were carried out with our Ampha Z32 instrument and our measurement buffer. If you want to learn more about our technology, please have a look here.

First, let’s learn something about wheat pollination and the reason for pollen viability measurements.

Wheat spike, anthers and pollen under the magnifying glass

Why Pollen Analysis Matters

Pollination in Wheat

Wheat is a strictly self-pollinated species and cross-pollination amounts to only about one percent [1]. Pollination normally occurs before the glume opens and is therefore called cleistogamic. The amount of pollen that is released after anther extrusion depends on the genetics and is considered as a genetic trait in breeding [2]. Based on literature, wheat pollen dies immediately after shedding [3]. This has to be taken into consideration when collecting and handling pollen. For manual pollination, ripe wheat anthers are normally harvested from the pollen donor (father plant) and immediately used for pollination of the pollen recipient (female plant).

Wheat spike with anthers

Hybrid Seed Production in Wheat

Wheat spike with grains

When it comes to hybrid seed production, the F1 generation (which the ordinary farmer will be using) must be repeatedly reproduced by the seed production companies. This means that the pollen must be transferred from the donor, via wind, to the recipient. To avoid self-pollination of the recipient line, male sterility must be introduced in the female inbred. The examination of the efficacy of this male sterility makes the characterization of pollen production mandatory.

For hybrid seed production, the male and female inbred lines are grown normally in stripes and only the female lines are harvested. To optimize seed production, a high female to male ratio is aspired. This requires the selection of an inbred male donor with a large pollen production and high viability to secure the fertilization.

 

Other Reasons for Pollen Analysis

Environmental conditions like drought and heat and the application of agrochemicals can have an impact on how much pollen is produced and how viable it is at release. Different genotypes respond differently to these environmental factors, thus the analysis of pollen can give more insights and improve your breeding process.

To support our customers and to develop our products even further, we performed a series of field trials in winter wheat cultivars with our Ampha Z32. Here you can see our “field lab” setup in the trunk of my car.

Poor wheat yield due to external impacts

Now the question is: How can we extract the pollen from the spikes to generate robust and valuable information?

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Wheat Pollen Extraction Methods

Extracting a representative sample for pollen viability determination is a critical step. Harvesting too early will result in low cell concentration and the collection of immature pollen. When harvesting too late the sample will likely contain also dead pollen and therefore your results will be distorted. The right pollen harvesting timepoint is when anthers are shining yellow and a few are already extruded in the central part of the spike.   

If you have found the right spike, you are ready to sample. Already extruded anthers have to be removed before pollen collection.

Now the question is: How can pollen be extracted to get representative viability data?

In our field trials we evaluated four extraction methods to quantify pollen viability. The selection criteria of these methods were how fast pollen can be extracted and how robust these methods are.

We named the methods we tested:

The right pollen harvesting timepoint. Wheat spike with anthers.

How to Extract Wheat Pollen - 3 Methods

Method 1: Anther picking method

The anther picking method is universally used in the breeding area for the emasculation of wheat spikes. You can easily do this by opening the glume and lemma, and picking out the anthers with a forceps, as shown in the picture. For our viability measurements, we experienced six to twelve anthers (shining are necessary to obtain enough pollen for a representative sample. If you run in the issue of a low cell concentration, the number of anthers can be increased further.

After picking the anthers, they have to be transferred to a 1.5 ml Eppendorf tube and stay there for 5 minutes to provoke the opening. To extract the pollen from the open anthers, the closed Eppendorf tube is moved over a lab rack quickly. Now the pollen is ready to be suspended in the measurement buffer and filtered for instant measurement.

This anther isolation method gives you the opportunity to extract pollen in a field where entire plants cannot be harvested. This is especially the case if you need the plants later, for example for seed production.

closeup of the wheat anther picking method

Method 2: Spike cutting method

Another option to collect fresh pollen is to cut the wheat spikelets horizontally near the end of the glumen tips with scissors. This method will provoke the extrusion of the anthers and the release of the pollen within five minutes. Please take a few seconds to look at the time-lapse recordings here. It is really fascinating to see how fast it goes (within 5 minutes)!

Picture of wheat anthers extruding from cut spikelets

To come up with the most robust pollen collection method for you, we used cut spikes in two different ways. Read on!

Using a paper funnel

The paper funnel method was a relatively simple approach. A paper funnel was created by an ordinary sheet of paper (download the funnel template here) and then a 150 µm filter was attached to the funnel outlet using tape. This composite was placed on an empty 1.5 ml Eppendorf tube. The freshly cut spikes (up to five) were put upside down in the funnel for five minutes until the anthers were extruded and the pollen was shed. Gentle finger flicking on the filter helped to release the pollen. After collecting a sufficient amount of pollen in the tube, measurement buffer was directly added to the Eppendorf tube. Slight shaking helped to suspend the pollen in the buffer. In the final step, the suspended pollen was filtered and transferred into a FACS tube for instant measuring.

In general, three to five spikes of the right developmental stage will always deliver you enough pollen for robust viability measurements.

Foto of wheat spikes in a paper funnel
Foto of a paper funnel with wheat anthers for pollen collection
Foto of a filter used to filter suspended pollen

Using an open falcon tube

Looking for an even more convenient method a test in open Falcon tubes was performed. The previously cut spikes were immediately transferred upside down into a 50 ml Falcon tube. The spikes remained there for five minutes until the pollen was shed. Gentle finger flicking on the Falcon tube helped to release the pollen.

After the pollen was shed, the spikes were removed and the Falcon tube was rinsed with 4 ml of our measurement buffer, and filtered (150 µm filter) into the FACS tube for instant measuring.

Foto of a hand in gloves holding a falcon tube with wheat anthers inside
A foto of wheat anthers in a Falcon tube

Method 3: Spike stripping falcon tube method

The third pollen extraction method was tested by stripping off the spikelets from the rachis with the bare hands onto a piece of paper. This method results in a partial ripping of the glume and lemma from the spikelets and leads to the release of the anthers. The individual spikelets were then transferred to a falcon tube and incubated for five minutes until the pollen was shed.

After the pollen was shed, we added 4 ml of measurement buffer, rinsed the Falcon tube and filtered the suspension with a 150 µm filter into the Facs tube. Then the sample was instantly measured.

Now the question is: Which method is the best for pollen viability measurements?

How to Extract Wheat Pollen - Which Method is the Best?

The overall results showed higher viability of the anther picking and the spike cutting (funnel) method compared to Falcon cutting and Falcon stripping. Even after looking at single cultivars there was the same trend.  The reason for lower viability of the Falcon methods is suspected in the creation of condensation droplets within the tube. This condensation can lead to instant dying (bursting) of the pollen grains. Therefore, I don’t recommend using the Falcon method for pollen viability measurements.

Focusing on the anther picking and spike cutting (funnel) method, we can see larger error bars in the anther picking compared to the spike cutting (funnel) method which tend to result from the picking bias. This bias is eliminated in the funnel method where all spikelets are cut and just the mature anthers extrude and shed pollen. This means if it is possible, I recommend harvesting the spikes and using the funnel method for your pollen viability measurements.

A bar plot showing a comparison of four different pollen extraction methods

Amphasys Method of Choice

For reliable results, harvest the spikes and use Method 2: Spike cutting, using a paper funnel.

Wheat Pollen Lifetime

Pollen Survival after Release

If you are digging in the literature, you will find ranges for wheat pollen survival between 30 minutes and three hours. This is really short compared to species like sunflower or tomato. Obviously, this time span is also dependent on the genotype and environment. It has a direct impact on planning pollen viability measurements in field trials, especially if you are planning batchwise sample preparation.

Therefore, we performed a wheat pollen “survival” experiment under different conditions to give you a recommendation.

Pollen Viability

Pollen viability can range considerably between different species. Read more on pollen viability of tomato pollen.

Wheat Pollen Deteriorates Quickly after Extraction

To test how fast pollen viability drops over time, we performed a pollen life-time experiment by keeping the pollen at room-temperature (~22°C) and in the fridge at (4°C). The pollen was collected from ten different spikes using the previously shown Method 2 – using a paper funnel. After collection, the pollen was aliquoted in Eppendorf tubes, the tubes were closed and kept either at room-temperature or in the fridge. In total, eight time points were evaluated.

If we look at the results we can see that pollen viability was partly preserved in the fridge compared to room-temperature.

The initial wheat pollen starting materials showed high viabilities between 80% and 90%. The pollen viability at room temperature dropped faster than the storage in the fridge.

At room temperature, the pollen viability dropped within the first 60 minutes by around 35 – 40 %. Similar pollen viability was observed when storing more than 180 minutes in the fridge. At room temperature, the collected pure pollen samples start deteriorating immediately after release and the viability drops very quickly, making pollen viability measurements only directly after extraction meaningful.

The fridge storage showed slower deterioration, but pollen viability also drops very fast. I highly recommend measuring pollen viability immediately after extraction.

Expert Tip

If it is not possible to measure within 30 minutes from pollen collection, we recommend storing whole spikes instead.

Harvesting Spikes Allows You to Store Wheat Pollen Longer

In case your wheat pollen viability measurements cannot be performed directly in the field, we tested another approach. This included the harvest of the whole plant and the transfer into a bucket of water at room temperature in the lab. After different timepoints pollen was collected from the spikes by using the spike cutting (funnel) method.

A graph showing wheat pollen lifetime from a harvested spike over time

The results showed no significant loss of pollen viability for 24 hours.

This indicated reliable results if spikes were kept in water at room temperature.

After having found the right sampling and extraction method for pollen viability, the question arose of how you can determine the other important variable: The quantification of the amount of pollen within a field. This question will be addressed in the next blog post, so stay tuned.

Take Home Message

Pollen viability can be either measured by anther picking, spike cutting (funnel) or spike stripping. All these methods differ in work intensity and accuracy. This blog post should give you an overview about the methods that we tested and what the main findings were. In the end it should fit in your production system and your routine. Nonetheless, if you can harvest your wheat spikes I highly recommend using the spike cutting (funnel) method. It gives you the chance reduce the variation found between different spikelets. If you cannot harvest the spikes, I recommend using the anther picking method.  Please make sure to collect the anthers at the right stage.

Pollen collection method

Work intensity

Accuracy/
Precision

Recommendation

medium

high

- Funnel

high

very high

- Falcon

high

low

X

In general, it is important to keep the time from the extraction to measurement as short as possible, because wheat pollen dies very fast. We observed some delaying of the dying process under cooling conditions, but I highly encourage you to measure as fast as possible. If pollen collection and measurement cannot be synchronized to measure as fast as possible, I recommend you to harvest the whole stem, put it into water and perform pollen extraction in the lab within the next 24 hours.

 

Now you only have to grab your spikes and you are ready to harvest.

If you want to know more about wheat sampling or if you run into any issue, please contact me.

Best Georg

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Sources

[1] Griffin, W. B. (2012). Outcrossing in New Zealand wheats measured by occurrence of purple grain Outcrossing in New Zealand wheats. 8233. https://doi.org/10.1080/00288233.1987.10421885

[2] Pickett AA (1993) Hybrid wheat results and problems. Paul Parey Scientific Publishers, Berlin Pinto RS, Reynolds MP, Mathews KL, McIntyre CL, Olivares-Villegas JJ, Chapman SC (2010) Heat and drought adaptive QTL in a wheat population designed to minimize confounding agronomic effects. Theor Appl Genet 121:1001–1021

[3] Fritz, S. E., & Lukaszewski, A. J. (1989). Pollen Longevity in Wheat, Rye and Triticale. Plant Breeding, 102(1), 31–34. https://doi.org/10.1111/j.1439-0523.1989.tb00311.