Beyond the husk – shining light on seed quality through molecular imaging

Seed quality is absolutely key to the success of both agronomically relevant crops and forest tree species used for restoration strategies. Forest restoration activities, including reforestation and biodiversity conservation, are crucial to preserve forest ecosystems. Seeds are collected from trees in forest ecosystems, and transferred to nurseries to develop high-performing plants – thus, seed quality affects how well forest trees grow, how well they establish themselves and how well a long-term sustainable forest ecosystem can develop. Traditional methods to assess seed quality are often not precise enough to find hidden changes at the molecular or structural level that affect seed vigour or the methods are slow and laborious. This makes it harder for agriculture and forestry workers, as well as nurseries, to efficiently grow high-quality trees on a large scale. Moreover, the impact of climate change poses at risk this value chain which would benefit from the availability of highly resilient germplasm. 

Understanding the molecular basis of seed germination

Prof. Alma Balestrazzi, an expert of plant physiology and head of the Seed Molecular Physiology laboratory at the Department of Biology and Biotechnology - University of Pavia in Italy, and her team are trying to tackle this problem, in forest tree species whose seeds are quite sensitive to environmental changes. Their work focuses on what happens beyond the seed husk to improve our understanding of the physiological and molecular bases underlying the very early steps of seed germination. Molecular-level analysis provides a deeper understanding of the genetic, biochemical, physiological and structural properties that influence seed performance.

Based on these insights, they are trying to understand what predisposes seeds to respond well to certain vigourisation treatments that  improve seed germination. “The variability of the seed response is still a strong problem in seed biology” explains Alma, especially when it is only realized rather late that the expected reaction does not occur. “But if we have a molecular tool, an imaging tool, to screen the treatments in a fast way, then we will be able to identify any red flags very early.”

Having this vision in mind, Alma became very excited when the colleague Alessio Giovannelli from CNR-IRET (National Research Council-Research Institute on Terrestrial Ecosystems), PI together with Alma of a specific task in Spoke 4-National Biodiversity Future Centre told her about Euro-BioImaging’s DIMP Neuromed Node and the work of Nicola D’Ascenzo (the Node lead at that time), who – together with others –  has developed reliable methods to adapt the Positron Emission Tomography (PET) technology for the use with plants, also including a portable PET system to perform imaging on plants in the field. 

While positron emission tomography (PET) is widely recognized in medical research and clinical settings, adaptations to plants opens up exciting new possibilities for plant biology. This innovative technology allows researchers to explore and image various aspects of plant physiology, offering unprecedented insights into plant processes such as water and CO₂ metabolism. 

Innovative use of PET to measure seed quality

But up until now it has never been used to measure seed quality.  If this innovative approach of PET-technology could be pushed to investigate seed quality, this would not only allow to assess and compare changes in the seed pre-germinative metabolism in real-time but with the advent of portable PET, it would also enable scientists to directly operate on the fields or at national seed collection centers. 

This can open up new avenues to investigate and protect biodiversity. Protecting biodiversity is not just an environmental issue—it's considered to be a matter of national security in Italy. “The loss of so much biodiversity threatens our future. Within these ecosystems lie crucial genetic resources that hold the key to climate resilience and adaptation,” explains Alma.

Teaming up to combine the right expertise

Alma and her group had never used PET before. Thus, to push this idea forward, the teams of Alma and Nicola teamed up to combine the right expertise to investigate “if PET imaging can help us to better understand what happens in the seeds.” The main goal was to develop innovative protocols for a PET-based diagnosis of seed quality in model, crop, and native species (herbaceous and forest species). Anca Macovei, expert in seed molecular biology and head of the Plant Biotechnology Laboratory at the Department of Biology and Biotechnology-University of Pavia, joined the group and supported the challenging brainstorming on how to design an experimental model tailored for the PET-based investigation.

Initially, they focused on studying the water uptake in seeds and identifying when they are metabolically active. Therefore, several key parameters needed to be optimized, including seed size, resolution, ideal imaging time points, and the optimal concentration for tracer administration. “We were speaking different languages at the beginning,” Alma said. “But now we are aligned. It’s very rewarding to talk to colleagues from different disciplines.” 

In particular, Andrea Pagano, a post-doctoral researcher in Alma’s group, had the opportunity to broaden his expertise in this new and exciting field. He went to the DIMP Neuromed Node to provide the seeds and established the conditions for doing PET/CT on those seeds together with the team at Neuromed. 

Selecting the seed samples

Three maize seed lots were chosen for this purpose – high quality seeds, deteriorated seeds and viable seeds. Some seed samples were already sent to Neuromed before his visit, so that the PET experts at the Node could pre-optimize the equipment and setting. “It was a wonderful experience, with really excellent support,” Andrea said. “We are still analyzing the data, but it is getting more and more refined. Water uptake determines the reactivation of seed metabolism and the so-called seed repair response, associated with better seedling development and increased stress tolerance. Developing a model that can predict seed viability using PET/CT imaging provides the advantage to assess seed metabolism simultaneously with the morphological features in a non-invasive way that does not destroy the seed and allows for further analyses. Our aim is to link the PET/CT profiles with indicators of seed viability and/or deterioration. After optimization on large-sized maize seeds, I will try to implement it on the seeds of other crop and forestry species under different experimental conditions to test and expand the scope of PET/CT-based models. Additionally, conducting these PET studies directly in the fields is becoming increasingly vital, as it provides the most natural and dynamic context for such research.” 

Nicola D’Ascenzo and a team of international scientists as part of EU-funded projects PETAL and RI-Imagine Crops are optimizing the equipment for in field PET to make this wish come true. 

This article was written in collaboration with Euro-BioImaging’s Scientific Ambassador Judith Weber, Charité Berlin.