Potential of Impedance Flow Cytometry to Assess the Viability and Quantity of Cannabis sativa L. Pollen

Abstract

Over the last decade, efforts to breed new Cannabis sativa L. cultivars with high Cannabidiol (CBD) and other non-psychoactive cannabinoids with low tetrahydrocannabinol (THC) levels have increased. In this context, the identification of the viability and quantity of pollen, which represents the fitness of male gametophytes, to accomplish successful pollination is of high importance. The present study aims to evaluate the potential of impedance flow cytometry (IFC) for the assessment of pollen viability (PV) and total number of pollen cells (TPC) in two phytocannabinoid-rich cannabis genotypes, KANADA (KAN) and A4 treated with two different chemical solutions, silver thiosulfate solution (STS) and gibberellic acid (GA3). Pollen was collected over a period of 8 to 24 days after flowering (DAF) in a greenhouse experiment. Impedance flow cytometry (IFC) technology was used with Cannabis sativa to assess the viability and quantity of pollen. The results showed that the number of flowers per plant was highest at 24 DAF for both genotypes, A4 (317.78) and KAN (189.74). TPC induced by STS was significantly higher compared to GA3 over the collection period of 8 to 24 DAF with the highest mean TPC of 1.54 × 105 at 14 DAF. STS showed significantly higher viability of pollen compared to GA3 in genotype KAN, with the highest PV of 78.18% 11 DAF. Genotype A4 also showed significantly higher PV with STS at 8 (45.66%), 14 (77.88%), 18 (79.37%), and 24 (51.92%) DAF compared to GA3. Furthermore, counting the numbers of flowers did not provide insights into the quality and quantity of pollen; the results showed that PV was highest at 18 DAF with A4; however, the number of flowers per plant was 150.33 at 18 DAF and was thus not the maximum of produced flowers within the experiment. IFC technology successfully estimated the TPC and differentiated between viable and non-viable cells over a period of 8 to 24 DAF in tested genotypes of Cannabis sativa. IFC seems to be an efficient and reliable method to estimate PV, opening new chances for plant breeding and plant production processes in cannabis.

Influence of Sucrose, Boric Acid and Calcium Chloride on in vitro Pollen Germination and Tube Elongation of Alfalfa

“…There are also studies (Heidmann et al, 2016) showing that impedance flow cytometry (IFC) determines the electrical properties of cells by using microfluidic chips and analyzes a high throughput of pollen cells for viability screening in a standardized method, which is widely used in many species such as wheat, corn, hazelnut, tomato, tobacco, etc. (Rafiq et al, 2021). At present, most scholars believe that in vitro pollen germination is the most common and simplest method and can be applied to study many basic and applied aspects of pollen biology (Gomez-Mena et al, 2022).…”