Effects of H2SO4, GA3, and cold stratification on the water content, coat composition, and dormancy release of Tilia miqueliana seeds.

Introduction: Tilia miqueliana is an endemic species whose population is declining. The permeability barrier and mechanical constraint of the pericarp (seed coat) are important causes of its seed dormancy. Although there has been considerable research on this subject, questions remain regarding how the permeability barrier and mechanical constraint of the seed coat are eliminated during dormancy release and how water enters the seed. Therefore, protecting the species by improving its germination/dormancy breaking in the laboratory is urgent. Methods: In this study, the changes in the cellular structure, mechanical properties, and components of the Tilia miqueliana seed coat after an H2SO4-gibberellic acid (GA3) treatment were analyzed during dormancy release. Various analyses (e.g., magnetic resonance imaging, scanning electron microscopy, and paraffin section detection) revealed the water gap and water channel. Results: The H2SO4 treatment eliminated the blockage at the micropyle and hilum of the seeds. Water entered the seeds through the water gap (micropyle) rather than through the hilum or seed coat, after which it dispersed along the radicle, hypocotyl, and cotyledon to the endosperm. During the cold stratification period, the cellular structure was damaged and an increasing number of holes appeared on the inner and outer surfaces of the seed coat. Vickers hardness tests showed that GA3 decreased the seed coat hardness. Additionally, the seed coat lignin and total phenol contents continuously decreased during the cold stratification period. Notably, the Liquid chromatography-mass spectrometry (LC-MS) analysis of the seed coat detected polyethylene glycol (osmoregulator), which may have destabilized the water potential balance inside and outside the seed and increased the water content to levels required for germination, ultimately accelerating seed dormancy release. Discussion: This sophisticated and multi-level study reveals how H2SO4 and GA3 eliminate the permeability barrier and mechanical constraints of the seed coat during dormancy release of Tilia miqueliana seeds. This will be beneficial to artificially assist the natural regeneration and population expansion of Tilia miqueliana.

Cryo-attenuated properties of Tilia miqueliana pericarps and seeds.

Introduction: Cryo treatment of dry seeds is known to attenuate the structure of fruit and seed coats, but little is known about the microstructural impacts of such treatment. The seeds of Tilia miqueliana are dispersed within a hard pericarp, the manual removal (hulling) of which is time-consuming and inefficient. Rapid hulling technology is urgently needed for sustainable production and convenience of edible nuts. Methods: We explored the mechanistic basis of liquid nitrogen (N)-treatment weakening of the pericarp of T. miqueliana fruits using a range of microscopical, biophysical and chemical approaches. Results: Liquid N treatment (40 s) resulted in lower pericarp contents of cellulose and hemicellulose, and increased amounts of lignin. Profound changes in cell structure and mechanical properties included the emergence of large holes and gaps between the mesocarp and endocarp cells. Also, the toughness of the pericarp decreased, whilst the hardness and brittleness increased, thereby changing the fracture type from ductile to brittle. Liquid N treatment of dry fruits followed by tapping with a hammer, reduced the number of damaged seeds three-fold and pericarp peeling time four-fold compared with manual hulling, whilst seed viability was not negatively affected. Discussion: Comparable findings for the efficient and economical removal of hard covering structures from dispersal units of five more species from three other families following liquid N treatment indicates the potential application of our findings to large-scale production of seeds and seedlings for breeding, forestry and conservation/restoration purposes. Furthermore, it introduces a novel concept for postharvest treatment and pre-treatment of deep processing in nuts.

The metagenomics of soil bacteria and fungi and the release of mechanical dormancy in hard seeds.

Persistence in the soil is a function of seed physiology, particularly non-germination and inherent lifespan. However, for seeds with mechanical dormancy, non-germination is also a function of the composition and activity of the soil microbiota. We attempted to screen out microorganisms in the soil that can specifically and rapidly decompose the hard fruit pericarps of Tilia miqueliana Maxim., a unique native tree species in China. Using the classical replica plating method, more than 100 different culturable microorganisms that could rapidly erode the pericarp were collected from the surface of pericarps under different culture conditions. At the same time, we successfully extended the concept of metagenomics and applied it to the identification of mixed artificial cultures. The decomposition process of the pericarps in soil was also simulated artificially. The physical and chemical data suggested a potential mechanism of microbial scarification and cracking in pericarp, whilst the embryos inside the eroded fruits retained good viability. Our discoveries could pave the way for the removal of physical and mechanical obstacles that prevent hard coat seeds from germinating. We anticipate that the use of this technology will improve the germination of other hard coat seeds. More research is needed to investigate the impacts on other seeds. The findings of this research can inform the design of experiments on the seed ecology of persistence.