Comparing methods for controlled capture and quantification of pollen in Cannabis sativa

Abstract

Cannabis sativa L. (Cannabaceae) is a dioecious plant, producing male and female flowers on separate unisexual individuals (Sinoto, 1929; Valle et al., 1968). Although both male and female plants are capable of producing cannabinoids in equal concentrations (Valle et al., 1968), female plants produce greater floral biomass than male plants (Ohlsson et al., 1971) and thus are exclusively used in commercial marijuana production facilities. Moreover, after pollination, female plants alter their relative investment in phytochemicals by reducing the production of secondary metabolites like cannabinoids, flavonoids, and terpenoids (Pijlman et al., 2005). In the absence of pollen, stigmas on female plants continue to grow and thus produce more surface area on which cannabinoids can be produced (Small and Naraine, 2016). Because of this negative impact of pollination on cannabinoid yield, industrial growers rarely maintain male plants in production facilities, and instead propagate their stock of female plants by vegetative cloning (Flores-Sanchez and Verpoorte, 2008; Decorte, 2010). However, the "mother" plants used to produce clones eventually become non-regenerative and new mother plants are grown from seed, which necessitates pollination (Valle et al., 1968). Therefore, careful consideration must be given as to the most effective and efficient ways to collect pollen for controlled crosses while preventing pollen escape into production areas. Cannabis is anemophilous (wind-pollinated) (Small and Antle, 2003), and therefore relies on air movement for pollen transfer from male to female plants, sometimes across long distances (Small and Antle, 2003). Pollen dispersal mechanisms often reflect pollen ornamentation, as seen in C. sativa's smooth exine layer, triporate (i.e., three aperture) morphology, and low mass-features intended to maximize pollen dispersal distance and chance of successful ovule fertilization (Hesse et al., 2009). The aerodynamic morphology of C. sativa's pollen highlights the difficulty associated with controlling its movement, as any airflow following anther dehiscence can result in pollen movement, a frequent issue when studying dispersal in anemophilous species (Whitehead, 1969, 1983). It is therefore important to determine the most efficient method of capturing windborne pollen upon anthesis, in terms of both the number of pollen grains collected and the time spent collecting pollen. Procedures for controlled pollen capture are typically required in crop breeding programs to ensure precise knowledge of paternity so as to breed progeny with preferred traits (Richey, 1950; Briggs and Knowles, 1967; Allard, 1999). For example, standard methods for maize breeding were established in the early 1900s, with an abundance of literature outlining the procedure for controlled crosses (

No Correlation Between Pollen Fertility and Viability: Differential Measures of Male Gametophytic Fitness inCannabis sativaL.

“…14 (Zeiss Primo Star Upright Light Microscope, Carl Zeiss Canada Ltd., Toronto, Ont.). Similarly to previous work (Wizenberg et al, 2020), we kept a tally of the number of burst pollen grains on each slide, separate from total pollen grains; burst pollen grains represented <1% of all sampled pollen grains. Any pollen forming clump were excluded from all counts because we could not differentiate between viable and aborted pollen.…”

“…Pollen stored in wheat flour and liquid nitrogen maintained its ability to germinate in vitro for up to four months, though the germination rates remained low, even for fresh pollen (Gaudet et al, 2020). Noting the inconsistencies in methodologies and conclusions among previous publications on this topic, and building on our previous work (Wizenberg et al, 2020), we set out to investigate C. sativa pollen viability and fertility, under the broader goal of developing a framework for measuring male gametophytic fitness. Accordingly, our research asks:…”

“…We closely monitored the five selected male plants until the inflorescences began to swell, showing visible protrusion of mature pollen sacs, indicating that anther dehiscence would occur. On the first day of anther dehiscence, we hand collected pollen (Wizenberg et al, 2020) in 1.7 mL centrifuge tubes (LIFEGENE graduated micro-centrifuge tubes, Modiin, Israel). Pollen samples in centrifuge tubes were left unsealed to dry for 1h before sealing, following which we stored them under one of two experimental conditions; samples were labelled and stored at either 'room temperature' conditions of 22°C ± 0.95°C and ~43% relative humidity, or 'freezer' conditions of -4°C and ~100% relative humidity.…”

Bibliometric and Scientometric Analysis of Scopus-Indexed Literature on Hemp (Cannabis sativa) Breeding

“…The most commonly used index keywords were 'Cannabis', Cannabis sativa', 'Hemp' and 'Cultivar'. The sampled papers also reported on research themes such as genetic diversity [29][30][31][32][33], fibre quality [34,35], pollen quantification and sex genetics [36,37], cannabinoid content [38], seed oil [39] and pulp yield [40].…”

Methods for characterizing pollen fitness in Cannabis sativa L.

“…Pollen stored in wheat flour and liquid nitrogen maintained its ability to germinate in vitro for up to 4 months, though the germination rates remained low over-all, even for fresh pollen [ 57 ]. Noting the inconsistencies in methodologies and conclusions among previous publications on this topic, and building on our previous work [ 58 ], we set out to investigate C . sativa pollen viability and abortion rates, under the broader goal of developing a framework for measuring male gametophytic fitness.…”