Hemp (Cannabis sativa L. < 0.3% w/w total tetrahydrocannabinol [THC]) has been an increasingly important economic crop in the United States since the 2018 Farm Bill [1,2]. Hemp is naturally a diploid (2n = 2x = 20) dioecious species whose plant sex is determined by sex chromosomes, with heteromorphic (XY) signaling a male plant and homogametic (XX) signaling a female plant [3]. When grown for cannabinoids, only female plants are selected due to the synthesis of high concentrations of cannabinoids stored in glandular trichomes [4]. Male plants produce comparatively low amounts of cannabinoids [3,5]. Furthermore, pollination can drastically decrease the cannabinoid contents of female flowers by as much as 56% [6]. Initially, producers sourced high-cannabinoid female hemp cultivars from asexually propagated clones, and, in 2021, the total value of the production of hemp clones was $23.8 million, with a total of 20.2 million clones produced in the United States [7]. On average, the cost of asexually produced female floral hemp cultivars ranges from $5.00–10.00 per clonal propagule [8]. Female plants pollinated by genetically female pollen allow for the production of female seeds (“feminized seeds”) [9]. Much of the industry has shifted to developing or using feminized seed, allowing growers to reduce input costs, reduce plant disease risk, and increase growth vigor from the plant juvenile phase. The average cost of feminized seed from select hybrid lines is $0.50–1.00 per seed, depending on the amount ordered, source, and cultivar selected [8]. Therefore, producing feminized seeds is crucial to the floral hemp industry since only females are used in commercial production [9]. To produce feminized seeds, artificially stimulating male flowers and pollen production on genetically female plants becomes a critical step. Pollen that is genetically female does not carry the Y chromosome and can only result in female seeds.

Exogenous application of plant growth regulators (PGRs) and their inhibitors have been used to regulate and alter flower sex expression in many crops, including Cucumis sativus [10], Silene latifolia [11], and Coccinia grandis [12]. Specific PGR applications include gibberellins, which have been shown to favor male flower expression, while ethylene, cytokinin, and auxins all stimulate the formation of female flowers [9]. In hemp, exogenous application of plant growth regulators has been commonly used to produce male flowers from female plants for breeding purposes or to produce feminized seeds [9,13,14,15,16,17,18,19]. Two silver-based ethylene inhibitors, silver thiosulfate (STS) and colloidal silver and gibberellic acid (GA) spray, have been commonly applied for triggering male flowers on female Cannabis plants [9]. The STS method has shown the best efficiency in stimulating male flower counts [9] and has been commonly used in commercial feminized seed production and breeding processes [20].

Ethylene inhibitors are used in the horticultural industry to regulate flower senescence, increase fruit and flower shelf life, and alter plant habits [17]. Ethylene inhibitors operate through two primary mechanisms: inhibition of ethylene precursor biosynthesis or disruption of ethylene action via ethylene receptor binding [17]. The STS is one of the most commonly used ethylene inhibitors. The STS foliar spay is the product of silver nitrate and sodium thiosulfate. It has been used to initiate male flowers by inhibiting ethylene signals by breaking down the hormone into inactive compounds [21]. Ethylene receptors in the plant require copper co-factors for correct binding. When this binding fails, it allows physical interaction with CONSTITUTIVE TRIPLE RESPONSE (CTR1), a Raf kinase, to repress the positive feedback regulator ETHYLENE INSENSITIVE2 (EIN2), which produces ethylene [15,16,19]. Silver thiosulfate inhibits ethylene production by outcompeting copper ion cofactors in the ethylene receptors, allowing CTR1 to be active and signaling a slowed ethylene response, which initiates male flower production [19]. Through this mechanism, STS inhibits ethylene signals and stimulates male flower initiation.

The effectiveness of PGR application in hemp can be measured by the total number of male flowers and the quality of the resultant pollen. Male flowers are a direct comparative index of the PGR application effectiveness; however, a universally accepted standard method to estimate hemp quality is not yet available. Pollen germination and pollen staining methods are frequently used to investigate pollen qualities, and each method has its advantages and drawbacks. Pollen in vitro germination provides strong evidence of pollen viability. However, optimal methods might only be available for some plant species, but not yet for hemp. In hemp, the most updated pollen in vitro germination method showed only 30–50% germination of fresh pollen, and the rate significantly depended on the mother plant genotypes [21]. Another study compares the pollen viability of different genotypes through in vitro pollen germination rates ranging from 0 to 19% [22]. On the other hand, pollen staining methods that test the pollen non-abortion rate can be used to compare the qualities of pollen. Still, it might only partially indicate the real pollen viability (or fertility). Fluorescein diacetate (FDA) and acetocarmine staining are the most commonly used methods to investigate pollen quality (non-abortion rate). Fluorescein diacetate stains intact semi-permeable membranes, and intracellular esterases can hydrolyze FDA in the cytoplasm of the pollen cell, showing bright green inflorescence [23]. If the plasmalemma is not intact, the fluorescein does not accumulate, indicating lower-quality malformed pollen granules [24,25,26]. Acetocarmine stains chromatin located within the pollen nuclei, indicating pollen that is fully developed or has a complete structure [26]. In hemp, all three methods have been used in pollen quality investigations [21,22,27,28]; some are specifically used to compare pollen quality from different male flower stimulation methods [9].

There are several drawbacks to the traditional STS method when used in commercial agriculture environments. Traditional STS lacks shelf stability, requiring a freshly prepared stock solution to induce male flowers for every spray event [15]. The lack of shelf stability could increase the labor required because the buffer must be freshly made for each of the three applications [18,20]. The unstable silver nitrate in the STS can precipitate before foliar spraying, resulting in potentially inconsistent male flower stimulation. Chrysal ALESCO^® Anti-Ethylene Treatment (ALESCO^®) is a commercially available product for use as a potted plant conditioner. This product was originally used to avoid stress-induced premature shrinking or dropping of buds or blooms on ethylene-sensitive ornamental crops and to extend the shelf-life of ornamental products [29]. Since both traditional STS Spay and ALESCO^® target the blocking of ethylene-sensitive molecules by the same active ingredient, silver thiosulfate, using the shelf-stable commercial product, ALESCO^®, to replace traditional STS could be a worthwhile strategy.

This study is to evaluate the potential and efficiency of using ALESCO^® to replace the traditional STS foliar spray to stimulate male flower stimulation from female plants. In the current study, C. sativa plants were treated with the two traditional STS and ALESCO^® separately, and the effectiveness of male flower initiation (flower count per plant) and the quality of pollen were investigated and compared.