Why Pollen Quality Matters?
The relevance and benefits of knowing the reproductive properties of your lines
Introduction
Dr. Jörg Schrickel
Hi there, I am Jörg, Head of Marketing at Amphasys. For more than four years I work with customers in the seed industry, explaining the importance and benefits of our technology for plant breeding and seed production. The link is not obvious: why should the use of our technology (measuring pollen viability) be related to an economic benefit? Having a closer look, it gets more obvious, and this is what the article is about.
What this Article is About
Would you phenotype 20 different crop lines by means of pollen germination to find out the best pollinator line? For sure not. Even if the knowledge about the reproductive properties of different lines is the most important for successful breeding and seed production.
This article is not about the different methods and technologies of pollen characterization. This article is about how to use the knowledge about pollen properties of lines to improve the results in breeding and seed production. And how easy it is to gain this knowledge by using Amphasys’ technology.
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Pollen quality matters!
If you are a follower of Amphasys, you will have heard a lot already about the importance of pollen analysis. This is what we at Amphasys talk about all the time.
Assuming you are a newcomer, why should pollen quality matter? Irrespective of whether you are a breeder or a seed producer – you are interested in the fruit and the yield and the properties of the crop. But pollen?! Of course, you are aware that pollen is needed to get the fruit or seed, but is there more to it?
In breeding, pollen is the carrier of half – the male – of the genetic information that will make up a new genotype. Any reason not to pay attention to it?
In seed production, pollen is one of the most important factors to obtain full seed or fruit set. Bad pollen quality means low fruit or seed set. You still think you should neglect pollen quality?
In fact, pollen analysis was not easy in the past and mostly then carried out when obvious pollination issues were observed. The analysis was mainly done by means of germination or staining assays, with visual analysis under the microscope. Both methods are tedious, not very fast, and relatively unprecise as only a small number of cells are counted visually (Figure 1). Other shortcomings are that for germination assays not always the most appropriate buffers are used, whereas staining does not work for all crops anyways. These assays resulted in the information if pollen was viable or dead – not less and not more. Due to the tedious work, pollen analysis was not performed routinely but only in specific cases.
What is now what Amphasys’ pollen analyzers can do? First of all: everything much faster and with higher accuracy. But most important: the content of information. Amphasys’ pollen analyzers routinely provide the information of viable, dead, or aberrant cells in quality and quantity. (Aberrant cells are cells which were aborted during the maturation of pollen cells and are smaller than “normal” dead cells. Often the appearance and amount of aberrant cells are an indicator for abiotic stress, e.g., heat stress). More important: the value of Amphasys’ pollen analyzers lies in the possibility to systemically screen large numbers of lines in breeding and seed production for their pollen properties. The comparison of the results of these measurements brings highly valuable information and knowledge about the pollen and the lines with a high impact in cost and efficiency of breeding and seed production (Figure 2).
Pollen of all crops can be measured and Amphasys has prepared already instructions for more than 250 different crop species. Due to the fast sample preparation and fast measurement, a high throughput of samples is possible: 30 to 40 samples within one hour including sample preparation are possible. The measurement is automated, and machine based, so it is not user biased and therefore standardizable. The measurement principle furthermore is not based on optics, which means it must not be calibrated and gives the same results all over the world. Finally, our Ampha P20 is portable, allowing in-field measurements which is beneficial for crops with short-lived pollen like wheat, corn or rice.
For the first time, a routine quality control of pollen is possible and that allows a systematic screening of lines which leads to relevant information.
What is now the information you can obtain from pollen measurements with one of our Amphasys Pollen Analyzers (Figure 3)?
The measurement results show, if viable, aberrant and dead cells are present, and in addition, the exact number of each, either absolute or as concentration in the sample. With that it is possible for example to phenotype lines where the number of pollen cells per flower is the important metric.
In addition to normal pollen cells, the developmental stages of microspores can be determined – very important for an efficient DH production process.
Finally, the ploidy of a cell can be determined by measuring pollen cells.
After knowing what the pollen analyzers do and what the outcome is, the question is: for what can you use this information, where is it helpful, which value does it provide? The value lies in the systematic screening and the information and knowledge about the lines you obtain with pollen quality measurements and that you use in your business processes.
DH production
An efficient DH production starts with the right starting material. If the microspores are not in the right developmental stage, DH production will be even less efficient. Amphasys’ technology is able to measure microspores and clearly identify the developmental stages they are in. With the microspores in the right developmental stage the induction and subsequent formation of embryos is much higher. This will result in a more efficient DH production.
My colleagues from R&D have worked extensively on microspores and the results you can find here: https://amphasys.com/doubled-haploid-plant-production/
Line selection
The following example (Figure 4) demonstrates the value of the information for line selection: from 30 broccoli lines, grown under the same conditions, the proportion of viable and aberrant cells were determined. These measurements can be performed within 2 hours only, whereas with conventional methods it can take 2 days – and you still would not get the same amount of information!
The purple bars in the diagram show the viability of each line, the orange bars represent aberrant pollen grains. As can be seen, there is a large variability between the lines. The lines within the green circle are good pollinators : high pollen viability and very low amount of aberrant cells. With these lines no problems with reproduction will arise. The lines in the red circle are bad pollinator lines: nearly the same or even higher amount of aberrant cells compared to viable cells. If you continue breeding with these lines, time-to-market will be much later and breeding will not be efficient. Similarly, in seed production these lines will always create problems and seed set can be expected to be low compared to other lines. This is where you will lose money, you will not earn.
Detection of abiotic stress
During the early phases of development, pollen is especially susceptible to abiotic stresses. The environment will influence pollen abortion during development, in a way that is also dependent on the genetics of the plant. Therefore, the presence of aberrant cells can be an indicator for abiotic stress if the level in non-stressed conditions is detected too. The knowledge about susceptibility of lines to abiotic stress and especially heat stress can be used in breeding but also in seed production to avoid losses caused by wrong line selection or wrong crop placement.
More about aberrant cells and abiotic stress: https://amphasys.com/unraveling-hidden-secrets-behind-dead-pollen/
Crops with short lived pollen
Many crops, especially wind pollinated ones, have pollen with short lifetime. Wheat, corn, and rice are examples of those crops. In addition, cells are very sensitive to water and burst after a short while. This made it so far impossible to determine pollen viability with means of traditional pollen analysis. Amphasys’ pollen analyzers only need a minute for sample preparation and less than a minute for the measurement which allows to determine pollen viability before the cell is damaged. This enables for the first time phenotyping and line characterization based on pollen for wheat, rice, corn and other short lived species. In addition, with the portable Ampha P20 it is even possible to run measurements directly in the field.
Sunflower pollen is not short-lived but pollen measurements in the sunflower field are more efficient than taking collected pollen to the lab. More information about measurements of short-lived wheat pollen you can access here: https://amphasys.com/measuring-wheat-pollen-viability/
Crop placement
Finding the right place and environment for seed production is crucial for commercial success (Figure 5). Even a good line will lead to a bad result if it is grown in the wrong area. The following example shows three different corn lines grown in the colder location 1 and the warmer location 2.
The bars represent the pollen viability of each line. In general, pollen viability of corn is high. Line 1 shows a strong impact from the warmer temperatures, exhibiting a strong reduction in viable pollen in the warmer area compared to the colder area. In comparison to that, Line 2 is tolerant to temperature whereas Line 3 also shows some temperature sensitivity. In the case of Line 1 the difference in the seed set was obvious so that seed production was moved to location 1, resulting in higher seed yield.
Production pattern
The female to male ratio (Figure 6) is also a factor that can make you lose a lot of money if you do not know the properties of your lines: how much pollen they produce, how viable it is, how long it is viable, under which conditions it can be stored (if applicable). If you plant too many male lines, you lose money because you spend resources on something which is not necessary. If you do not plant enough male lines, the fecundation of the females will be insufficient, and you lose as well, as your seed yield will be lower than possible.
The example above shows the effect of an improved female to male ratio: if you reduce the male surface by 0.5 ha, increasing the surface for females at the same time for the same amount, you will increase your seed production and your benefit accordingly.
Amphasys technology helps to determine the right female to male ratio and makes sure that you will gain the most out of it.
Storage protocols and shipping of pollen
Growing pollen in pollen farms and ship it to the locations where the females are grown is becoming more and more frequent practice (Figure 7). Shipping is always related to pollen storage. For pollen storage, the right protocol needs to be in place and during storage pollen viability always decreases. Therefore, it is important to have the right storage protocol in place but more important, to have a method for quality control in place. Amphasys’ technology helps you to define the conditions to preserve viability during cooling and storage.
At the receiving location where the females are grown, Amphasys’ technology enables a fast and accurate quality control that is not affected by local conditions, such as the user and does not need calibration. This ensures comparable results, e.g., if pollen is shipped to third party growers.
Take home message:
Amphasys offers a technology for the easy, fast, and accurate screening of lines for their pollen quality. These properties, and the comparison between lines, provides information which is relevant with respect to the reproductive properties of specific lines. This knowledge can be used in plant breeding and seed production and helps to improve processes and economic results.
Our technology has further developed in the last decade, and we provide even applications with automated data analysis to make measurements as easy and comfortable as possible. The fact, that all leading seed companies are our customers – among many others – and that the technology is used in supporting industries like supplementary pollination technologies, is a strong sign for becoming the standard technology for pollen quality measurements.
If you need more information, if you want to carry out a project with us, if you want a demo: don’t hesitate to contact us.
Best regards,
Jörg