What this Article is About
This article is dedicated to a topic that has always been important to humans and will become even more important in the future. It’s about one of the fundamental steps involved in the production of food. Without this process, life on this planet would look very differently. I would for sure not be sitting here writing that post. Maybe I would be an alga, tiny bacterium or a sponge, and my life would not have turned out that exciting.
Luckily lifeforms have evolved. And while writing I can enjoy a sip of espresso prepared from the finest coffee beans grown on a lush hill in Colombia. Coffee beans are seeds. And those seeds have been produced after successful pollination.
Pollination is a fascinating process. A pollen grain is adsorbing on the sticky stigmatic surface of the female flower, then a pollen tube starts protruding, and after growing at a tremendous speed through the style it eventually reaches its target – the ovule. If everything works out, fertilization occurs and a new seed starts developing.
Germinating and non-germinating tomato pollen
If you work in seed production, you most likely want to maximize the yield and quality of your seed. This requires:
- Optimum pollen quality. Dead pollen = no seed
- Optimum pollen quantity. Neither stinginess nor excessive generosity are rewarded
- Good timing. The stigma must be receptive
- Compatibility. I guess it’s like with humans. Sometimes there is good ‘chemistry’ and sometimes not
In the following sections, I will discuss those topics and present my view on how to characterize the pollination process.
The examples and illustrations used here are based on various observations in Solanaceae, but the principles are also applicable to other crops, particularly in the Cucurbitaceae family.
Let’s get started.
In my previous blog post about aberrant cells and the pollen supply chain I focused on the male part – the pollen. When we talk about pollination, however, pollen is only half of the story. The success of pollination depends not only on the pollen quality and quantity, but also on the environment in which the process takes place, the timing and interaction between pollen and the stigmatic surface.
In this post we also pay attention to those other aspects.
Emasculated tomato flower
When we start investigating a process, we usually come up with experimental designs in which we try to keep all variables constant except the one we want to characterize. Tomato pollen is usually available in quantities large enough for many experiments, and the powdery substance can be homogenized easily. That means that we have a possibility to keep the male part constant and focus on the female part for our first experiment.
Experiment 1: Finding the right conditions for pollination
- Characterize the female receptivity by pollinating under different conditions (time of the day, humidity, timepoint after emasculation…) and quantify the seed set obtained under those conditions.
- Use statistical models (e.g. multiple regression) to determine which variables affect the seed set.
- Use one pollen sample for all these experiments to keep this variable constant. If you don’t have a sample that is large enough, pool multiple samples and homogenize well. Also make sure you have a reproducible pollination routine, i.e. try to apply a similar amount of pollen each time.
- Characterize multiple lines and find the best conditions for each one.
Been there, done that? Then let’s look closer at the pollen.
Have you ever wondered how much pollen should be used for pollination?
- If you add too little, not all ovules will be fertilized, and you won’t get a good seed set.
- If you add just the right amount, you will get the highest seed set
- If you add too much you waste pollen and maybe the overload is even leading to a reduced seed set due to competition for water and traffic jams in the style (hypotheses).
In the end it’s the number of viable or germinable cells on the stigma that counts. That’s why it’s a matter of quality and quantity. In the following experiment we’ll have a look at those parameters by quantifying the number of viable pollen grains on the pollinated stigma.
Tomato flower in greenhouse
Experiment 2: Characterizing the reproducibility of the pollination process
The goal of this experiment is to see how much pollen is typically consumed for pollination, and how reproducible the pollination process is in terms of the pollen amount dispensed. The cool thing about these measurements is that you can have a look at the very last step of the pollen supply chain: Pollen on the stigma. That’s when it matters.
- Do your pollinations under the optimum conditions previously identified (Experiment 1) and collect the pollinated stigmas right afterwards (before pollen germination).
- Quantify the number of pollen grains on each stigma using the Ampha P20 Pollen Analyzer
- Compare the pollen load on individual stigmas. Determine the variation.
- In case the pollen has been diluted with a diluent (e.g. lycopodium, starch…), determine the diluent fraction of each sample to see whether the pollen-diluent mixture was homogenous.
Large Variation of Pollen Quantity on Stigma
In the graph below you see the results of a proof-of-concept experiment. The number of viable pollen grains on several freshly pollinated tomato stigmas was counted. Each dot in the boxplot corresponds to the number of pollen grains recovered from one stigma.
Wow, quite a spread!
You can see that the number of pollen grains on the pollinated stigma varies greatly. This may be related to the operator performing the pollination, the stigmatic surface area and the pollination method. You can also see that the number of viable cells on the stigma is in the order of a few thousands. Compared to the number of seed typically obtained from a tomato fruit (in the range of 25 – 250), it is evident that there are many more viable pollen grains than ovules, and that many pollen grains therefore do not successfully fertilize an ovule. This can have many reasons, such as:
- No access to the stigmatic surface
- Pollen not germinable
- Failure in tube growth and finding ovary
- Target ovule already fertilized
- Failure in fertilization process
Those remarkably large differences between minimum and maximum pollen load immediately ring alarm bells and makes me think about process optimization:
Making the pollination process more reproducible (i.e. using roughly the same quantity of pollen) will:
- Prevent wasting precious pollen by not overloading the stigma
- Prevent insufficient pollination and therefore a reduction of seed yield
A higher reproducibility can be achieved on two levels: Standardization of the pollination method practiced by the operators and homogenization of the pollen sample.
Homogenization is particularly important if you use pollen diluents, such as dead pollen or lycopodium spores. Make sure that there are no micro-heterogeneities in the sample by mixing very well. Use the Pollen Analyzer to quantify the homogeneity of the sample by measuring several subsamples drawn from it and quantifying the variation.
Finding out How Much Pollen is Enough
Now let’s tackle the burning question of how much pollen is needed to achieve a full seed set. The question of “how much” is a question about quality (viability) and quantity (pollen load):
Viable cells = Viability x Pollen Load
The stigma is a sticky surface onto which pollen grains can adsorb. If there is only little pollen, the stigmatic surface will not be occupied much. The more pollen adsorbs onto the surface, the more difficult it gets for additional pollen grains to find a free spot. At some point the surface becomes saturated.
This simple description of the pollen-stigma system reminds me a lot of my first Biophysics and Biochemistry courses. In these fields, such phenomena are usually modelled using some sort of a saturation model. Applied to pollination, the model would look like this:
For small quantities of viable pollen, only little seed can be produced. Pollen is the limiting factor. If the pollen load is increased, more seed can be produced. At some point the number of ovules and the stigmatic surface area become limiting factors and adding more pollen will not increase the seed set anymore. This is called saturation.
If you want to maximize the seed yield, you have to operate close to or at the saturation.
So, what is the quantity of viable pollen needed to obtain a full seed set in tomato?
As always in Biology, there is no one-fits-all type of answer. It depends on the cultivation conditions and the compatibility between mother and father line.
Different crosses can show different saturation behaviors, as illustrated here:
I created three categories: Low compatibility, intermediate compatibility and high compatibility. The term compatibility here is related to the likelihood that a pollen grain is able to fertilize an ovule.
- For low compatibility crosses the maximum hypothetical seed set cannot be reached. The better your pollen quality, the better the seed set. However, the full potential cannot be exploited.
- For intermediate compatibility crosses the full seed set can be reached if high quality pollen has been harvested and the pollen supply chain is well under control to prevent substantial pollen quality decrease.
- For high compatibility crosses an intermediate pollen quality is already sufficient for a full seed set. For such crosses you have a good chance to reduce pollen consumption.
Knowing the behavior of each of your crosses will finally allow you to create specific pollination recipes and individually maximize the seed yield.
For The Geeks:
Experiment 3: Find cross-specific correlation pattern between pollen quality and seed set
Quantify the seed set obtained from a wide range of pollen qualities and fit a saturation model to your dataset. Be aware, this is a relatively large experiment. Invest some time in a sound experimental design.
If you include not only the pollen quality measurements in your experiment, but also quantify the average number of pollen grains on the stigma for each pollination round, you can extend the dataset by the quantity dimension.
Tip for Cucurbitaceae:
If you work in Cucurbitaceae seed production, including the pollen load into your experimental design is particularly important, as in such pollinations the variation in pollen number is usually substantially higher than the variation in pollen viability. Therefore, correlating the number of viable pollen grains (combination of viability and pollen number) with seed set is a more robust experimental setup than correlating pollen viability with seed set.
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This will give you a complete view of the pollination behavior of your cross at given optimized pollination conditions.
Why is that information useful?
It will allow you to create a specific pollination recipe and to make a yield forecast. How to get there will be the topic of my next post.
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