Re: Dead Cells: The Bad Seed Free Download
Eden Alvardo
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The mechanism of inhibition of coffee (Coffea arabica cv. Rubi) seed germination by exogenous gibberellins (GAs) and the requirement of germination for endogenous GA were studied. Exogenous GA(4+7) inhibited coffee seed germination. The response to GA(4+7) showed two sensitivity thresholds: a lower one between 0 and 1 microM and a higher one between 10 and 100 microM. However, radicle protrusion in coffee seed depended on the de novo synthesis of GAs. Endogenous GAs were required for embryo cell elongation and endosperm cap weakening. Incubation of coffee seed in exogenous GA(4+7) led to loss of embryo viability and dead cells were observed by low temperature scanning microscopy only when the endosperm was surrounding the embryo. The results described here indicate that the inhibition of germination by exogenous GAs is caused by factors that are released from the endosperm during or after its weakening, causing cell death in the embryo and leading to inhibition of radicle protrusion.
Interpretive Summary: Bacterial spot is one of the most serious diseases of tomato. Contaminated and/or infected seed serve as a major inoculum source for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. To improve the seed certification process, a viability-qPCR assay in conjunction with a crude DNA extraction procedure was developed to selectively quantify viable Xanthomonads in tomato seed. The assay was shown to accurately quantify viable cells within cell mixtures. This assay provides a sensitive, specific, and cost-effective way to certify tomato seed free of the pathogens causing bacteria spot.
Technical Abstract: Bacterial spot is one of the most serious diseases of tomato. It is caused by four different species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed serve as a major inoculum source for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. However, current seed certification protocols rely mainly on culturing and PCR, and both methods have limited capability to accurately quantify viable pathogen cells. To improve the seed certification process, a viability-qPCR assay in conjunction with a crude DNA extraction procedure was developed to selectively quantify viable Xanthomonads in tomato seed. A DNA-binding dye, propidium monoazide (PMA), was used to treat seed extract where it selectively binds to the DNA of dead cells, thus preventing the DNA from dead cells from being amplified in downstream qPCR. The procedure was tested with tomato seed samples spiked with mixtures of viable and dead cells in different concentrations and proportions. The assay accurately quantified viable cells within mixtures of 1:10,000 (live:dead), and use of the crude DNA extract provided results similar as those obtained using purified DNA prepared from commercial kits. This assay will provide a sensitive, specific, and cost-effective way to certify tomato seed free of the pathogens causing bacteria spot.
You will learn that dead is not just dead. There are dead cells that have matured before dying, and cells that were aborted in their development. The relative abundance of each subtype on its own reveals important information that can even lead to a refinement of the term pollen viability.
I was impressed by all these field workers who were manually pollinating the flowers in the fields. Flower by flower, plant by plant, row by row. But I was also impressed by these plants, being exposed to that intense radiation and high temperature, and still producing green leaves, yellow flowers and red fruits. They must enjoy this daily sunbathing I thought for myself.
In Technology Implementation Projects we have a closer look at the pollen supply chain of customers. The pollen supply chain is a key process in breeding and seed production, and usually consists of various steps of pollen management, such as production, collection, preservation and pollination.
At the beginning of the projects we usually measure the pollen quality along the whole process, leading to the identification of bottlenecks and improvement of critical steps. Then we define quality checkpoints. These are specific points along the value chain in which standardized quality controls are implemented.
The supply chain of tomato pollen is comparably diverse and extensive, and thus usually has a great potential for optimization. The fact that pollen is collected, preserved and transported already opens up a broad field of applications for quality testing, because, you know, when things can go wrong, they will go wrong at some point. And another thing to bear in mind: The pollen quality can only decrease along the supply chain. It never increases. You have to start your process with the best material, otherwise the final quality may be insufficient to obtain a full seed set [1].
The samples number 900 to 1000 (on the right side of the plot) make 10 % of all the measurements, and they all contain less than 10 % viable pollen. I guess you can imagine what happens if you use such samples for pollination. The quality checkpoints in a well-organized supply chain monitoring program would not permit further processing or storing of such samples, unless they are used for dilutions.
Together we ran the measurement again. Same result. Then we had another look through the microscope. Indeed, there were lots of long pollen tubes all across the field of view. But also lots of small amorphous particles. According to the germination protocol of our partner such small particles are cells that should be ignored. This immediately caught my attention, as the pollen analyzer can measure virtually any kind of particle above a certain size. I was curious whether those were detected in the previous measurement.
Now the key question is: How do we treat those aberrant cells? The AmphaSoft software (software that comes with the pollen analyzer) includes a useful feature to mark and exclude unwanted populations from further analysis. This exclusion would have led to a perfect match with the results of the germination tests.
The effects of low pollen viabilities can be considerable. This was also one of the main findings of the collaborative research project done with Rijk Zwaan. In a large-scale research project, we found a considerable reduction in seed yield for low pollen qualities. And that was confirmed with all 12 crosses under investigation [1].
If aberrant cells are important factors leading to a reduced pollen viability, and a reduced pollen viability is associated with a lower seed yield, then those cells should receive the necessary attention also from an economic point of view.
One of our main goals at Amphasys is to provide a tool that can be used to pinpoint problems in the pollen supply chain. But finding the problem is not the end of the story. We also want to provide solutions.
When we first collaborated with a customer on this topic, our proven recipes for pollen supply chain optimization did not apply anymore, and we had to have a closer look to the very beginning of the chain. To the breeding and growing part.
The Ampha P20 is the first fully portable and autonomous pollen analyzer for on-site measurements: in the field and in the greenhouse. It is the optimal device for systematical screening for phenotyping and routine quality control.
Aberrant cells originate in the Grow part of the supply chain. The reason is linked to the beginning of this blog post, when I was standing in the middle of that tomato field in India.
There are numerous hints in literature that associate empty, sterile, degenerated, injured or abnormal pollen grains with plant stress, mostly heat [4,5]. The current hypothesis is that under stress conditions the microspore development can be arrested and the microspore is eventually aborted. As microspores grow in size during maturation, the aborted cell is nonviable and small in size.
I strongly believe that aberrant cells are linked to arrested or aborted pollen development. The abortion can be caused by abiotic factors such as heat or drought. We also made the observation that the fraction of aberrant cells shows some variation over time, but the overall abundance is line-specific. In addition, we saw large amounts of aberrant cells in pollen samples from buds, opening flowers and fully open flowers, indicating that they are not artefacts from pollen processing and storage.
Well, basically you need to remove the stressor. In case of heat stress in high-end greenhouses, cooling systems or ventilation could remove excess heat. In tomato hybrid seed production I have seen many simpler net house systems though. Here temperature regulation is usually not feasible. Under such circumstances the production window for a heat-sensitive line could be narrowed to cooler months of the year. During these months male plants could be grown on a larger surface to still produce enough pollen. Tomato production is particularly suitable for this approach, because pollen can be stored easily for months. There are even reports about storage over years. For such long-term storage make sure you optimize pollen dehydration to the right moisture content and choose a suitable storage temperature.
If you are working in breeding, I could imagine that heat- and drought-resistance are traits that are on your radar. Do you really pay enough attention to pollen development, the maybe most sensitive step in the whole plant reproduction cycle? How would a male line with impaired pollen maturation perform in hybrid seed production?
Clinical studies have identified that an excessive exposure of UV light can cause oxidative stress (OS) and tyrosinase enzyme over-expression, which are associated with multiple diseases including atherosclerosis, cancers, diabetics, rheumatoid arthritis. In this study, we investigated the impact of grape seed proanthocyanidin (GSPE) on regulating OS and tyrosinase activity in human epidermal melanocytes. This study revealed that GSPE did not affect cell viability and protected cells from UV induced damage in a dose-dependent manner. 5-(-6)-Carboxy-2,7-di-chlorodihydro-fluorescein diacetate staining (i.e., a fluorescence staining for intracellular (OS)) indicated that GSPE reduced OS level caused by UV exposure. A similar trend was also confirmed by flow cytometry analysis, where GSPE down-regulated OS level. Tyrosinase analysis showed that GSPE treatment decreased tyrosinase activity. Taken all data together, GSPE may restore the cellular damage caused by excessive UV-exposure and promote skin health by reducing tyrosine generation. Clinically, GSPE could be potentially utilized for improving skin health against excessive UV exposure.
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