Optimizing cannabis cultivation: an efficient in vitro system for flowering induction

doi:10.21203/rs.3.rs-4669256/v1

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Optimizing cannabis cultivation: an efficient in vitro system for flowering induction

https://doi.org/10.21203/rs.3.rs-4669256/v1

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published 12 Sep, 2024

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Background

Cannabis sativa is a versatile medicinal plant known for its therapeutic properties, derived from its diverse array of secondary metabolites synthesized primarily in female flower organs. Breeding cannabis is challenging due to its dioecious nature, strict regulatory requirements, and the need for photoperiod control to trigger flowering, coupled with highly dispersible pollen that can easily contaminate nearby female flowers. This study aimed to develop a protocol for in vitro flowering in cannabis, investigate factors affecting in vitro flower production, and generate viable in vitro seeds, potentially offering a method for producing sterile cannabinoids or advancing breeding techniques.

Results

We show that the life cycle of cannabis can be fully completed in tissue culture; plantlets readily produce inflorescences and viable seeds in vitro. Our findings highlight the superior performance of DKW medium with 2% sucrose in a filtered vessel and emphasize the need for low light intensity during flower induction to optimize production. The improved performance in filtered vessels suggests that plants conduct photosynthesis in vitro, highlighting the need for future investigations into the effects of forced ventilation to refine this system. All tested lines readily developed inflorescences upon induction, with a 100% occurrence rate, including male flowering. We revealed the non-dehiscent trait of in vitro anthers, which is advantageous as it allows for multiple crosses to be conducted in vitro without concerns about cross-contamination.

Conclusion

The current work developed and optimized an effective protocol for in vitro flowering and seed production in cannabis, potentially providing a platform for sterile cannabinoid production and an efficient tool for breeding programs. This system allows for the full and consistent control of plant growth conditions year-round, potentially offering the reliable production of sterile molecules suitable for pharmacological use. As a breeding strategy, this method overcomes the complex challenges of breeding cannabis, such as the need for large facilities, by enabling the production of hundreds of lines in a small facility. By offering precise control over factors such as plant growth regulators, light intensity, photoperiod, and temperature, this system also serves as a valuable tool for studying flowering aspects in cannabis.

in vitro breeding

in vitro seed production

Cannabis cultivars

in vitro photosynthetic capacity

in vitro anther development

Cannabis sativa L., a member of the Cannabaceae family, is classified as an annual dioecious plant species in which male and female flowers are borne on separate unisexual individuals.

Cannabis produces a wide array of specialized secondary metabolites, including more than 120 cannabinoids, terpenes, and flavonoids [1], which possess therapeutic properties and thus confer recognized medicinal value [2]. The global rise in the cannabis market has significantly boosted the growth of the cannabis industry. This includes intensive breeding efforts aimed at developing elite cannabis cultivars with enhanced cannabinoid profiles tailored to optimize therapeutic efficacy, meet consumer preferences, and improve yield [3]. Cannabinoids and terpenes are synthesized and accumulated in glandular trichomes [4], most densely concentrated on the bracts of female flowers, thus attributing commercial value to the plant. The female cannabis plant exhibits a bushy and robust structure that, upon a short photoperiod, develops complex inflorescences with numerous small flowers closely packed together with high biomass. These inflorescences are primarily located at the tips of the stem and branches, as well as on the leaf axils throughout the plant [5]. Consequently, the cannabinoid profile among inflorescences varies due to differential light exposure and variations in the source-sink relationship. This variability presents a significant challenge, prompting the adoption of various strategies to reduce these inconsistencies. One effective method is regular pruning to enhance light penetration and control plant density [6]. Another approach that has not yet become commercial, is the use of tissue culture systems, like cell suspensions, for the mass production of cannabinoids [7].

The breeding of cannabis is intricate and challenging for several reasons: 1. The induction of flowering requires controlled light regimes, which complicates agricultural practices [8]; 2. In most countries, cannabis is under strict regulation that mandates certain security measures, limits the growing space, and specifies the maximum number of plants; 3. The anemophilous nature of cannabis, relying on wind for pollination, introduces challenges in controlling cross-pollination [9]. A single male plant can produce a vast quantity of pollen grains. This requires strict isolation of male plants to prevent unwanted fertilization of nearby female plants, implementing firm measures that drastically limit the number of crosses feasible in a breeding program [10].

Inducing flowering in tissue culture systems holds promise as a tool to address both issues: reliably producing cannabinoids in a highly controlled environment and enhancing breeding programs by allowing numerous crosses to be conducted in a small facility. In addition, it offers precise control over factors such as plant growth regulators, light intensity, photoperiod, and temperature, making it a valuable tool for studying the elements affecting flower initiation and floral organ development [11].

Plant tissue culture involves cultivating plant cells, tissues, or organs on defined nutrient media under aseptic conditions and controlled environments, occasionally supplemented with growth

regulators to induce specific cellular responses [12].

In this study, we developed an effective protocol for inducing flowering in cannabis in vitro. We optimized the media and growing conditions and demonstrated the ability to produce viable seeds. Therefore, this method can serve as a rapid and straightforward approach to breeding programs, a potential method for the production of cannabinoids, and a valuable tool for studying the flowering aspects of cannabis.

Experimental setup for in vitro flower induction of cannabis

In vitro flowering can serve as a valuable tool for studying specific aspects of flowering in cannabis and for producing important flower components, such as the calyx, to harvest cannabinoids under aseptic conditions. To establish our experimental system, we first followed a standard sterilization protocol to introduce explants into tissue culture. We selected single-node explants by cutting the stem of cannabis plants grown in a growth room under an 18/6 h photoperiod (Fig. 1a). Approximately 100 explants were sterilized in a 3L beaker using 2% sodium hypochlorite solution, followed by three rinses with sterile water. Subsequently, we placed these explants in MS or DKW media (see Materials and Methods) ensuring the proper orientation, with five stem sections per vessel.

The sterilization processes can impose stress on the explants, requiring a recovery period. At this stage, the stem sections are leafless, and a few days are required before leaves become apparent. To evaluate the requirement of initial vegetative growth for successful flowering, we subjected six vessels to an 18/6 h photoperiod for two weeks before transitioning to a 12/12 h photoperiod to induce flowering. We then compared this regime with direct placement under the flowering induction photoperiod (12/12 h cycle). The cannabis flower usually has two stigmas (Fig. 1f). To track the number of flowers and their rate of development, we followed the stigma development and counted the number of flowers every few days (Fig. 1g-i). Although the number of flowers developed within 38 days was similar between the two regimes, the plants that were first grown under a vegetative photoperiod appeared more developed. Therefore, we established this regime of two weeks under an 18/6 h cycle before transitioning to flower induction as part of our protocol.

Media Optimization for Enhanced In Vitro Flower Induction

Cannabis is known to exhibit a high rate of hyperhydricity in tissue culture systems, a phenomenon that varies depending on the genotype [13]. Several methods can be employed, to address this issue, including the incorporation of activated charcoal into the growth medium [14]. To determine the best media for inducing flowering in vitro in cannabis and to assess the tissue's response to activated charcoal, we tested two media commonly used for stem explants: MS and DKW-based media (see Materials and Methods), with or without activated charcoal. Adding activated charcoal to the DKW medium significantly reduced the number of flowers compared to the DKW medium alone but had no effect on the MS medium (Fig. 2a). These findings indicate that activated charcoal does not benefit cannabis flowering in tissue culture. Activated charcoal is known to absorb plant growth regulators and other organic supplements [15]. It might be that the presence of activated charcoal reduced the availability of important substances to the plant, thereby reducing flowering.

Statistical tests comparing the performance of plants in the two media without activated charcoal showed that plants in the DKW medium exhibited higher flower production than those in the MS medium at multiple time points (Fig. 2a, marked by an asterisk (*)).

Next, we tested how different concentrations of sucrose in the media affect flowering by comparing various sucrose concentrations in the MS medium (Fig. 2b) and DKW medium (Fig. 2c). In tissue culture systems, sugar serves two primary roles: as an energy source [16] and as a signal for flower induction [17]. However, there were no significant differences in flowering across the concentrations, except between the 2% and 1% sucrose concentrations in the MS medium at three-time points, where a decrease was observed at 1%.

Besides sucrose, hormones can also play a role in increasing the number of flowers. Cytokinins, for instance, can influence meristem size, which in turn may enhance flower production [18]. Additionally, cytokinins have been shown to facilitate the transition from vegetative to floral meristems in tissue culture [19]. The 6-Benzylaminopurine (BA) cytokinin is particularly recognized as a highly effective inducer of floral induction in vitro [20, 21]. To test the effect of BA on the in vitro flowering of cannabis, we added either 2 mg/L of BA solely during the vegetative phase or 1 mg/L and 5 mg/L of BA during the flowering phase. Surprisingly, BA did not enhance flower production; instead, it resulted in a significant decrease (Fig. 2d). This effect might be attributed to our basic DKW medium, which contains adenine hemisulfate (see Materials and Methods), a precursor of cytokinins resulting in excessively high concentrations of cytokinin.

Other elements that have the potential to affect flower numbers, such as phosphate in the media or lower temperature during vegetative growth to increase meristem size, didn’t show better performance (Fig. S1 and S2).

To summarize, our analysis indicates that DKW media without activated charcoal and BA is most effective. Since there are no significant differences between 2% and 3% sucrose, and 2% is more economical and potentially reduces contamination, we will continue using 2% sucrose.

Evaluating photosynthesis and light response in tissue-cultured cannabis

Our results, showing no significant difference between 2% and 3% sucrose concentrations, raise the question of whether the plants in the vessel conduct photosynthesis. To address this, we first examined the presence of stomata, which is essential for gas exchange. Scanning electron microscopy analysis of abaxial fan leaves from plants growing in tissue culture and growth room reveals that leaves in tissue culture develop stomata similar to those in growth room plants (Fig. 3a). To compare stomatal density, we employed the nail polish method and found that leaves grown in tissue culture possess significantly fewer stomata (average of 35 per 0.15mm²) compared to those from growth room plants (average of 45 per 0.15mm²) (Fig. 3b). However, the normal appearance of the stomata of the tissue culture leaves suggests that the plants are potentially capable of photosynthesis. Subsequently, we tested the plants' capacity for photosynthesis by cultivating stem nodes in a medium devoid of carbon energy sources. Since photosynthesis requires gas exchange, we used two types of vessels: one hermetically sealed and the other equipped with a gas-permeable filter. Both were placed under 71±14 µmol· m⁻²⋅s⁻¹light in an 18/6 h photoperiod.

Plants cultured in media without sucrose appeared pale and less developed than those grown with 2% sucrose, although the ones in the filtered vessel exhibited better growth (Fig. 3d-e, Fig. S3). To quantify this, we measured the chlorophyll content and found that plants grown with 2% sucrose exhibited higher chlorophyll content in both vessels compared to those grown without a carbon source (Fig. 3f). However, plants cultivated in a filtered vessel, with or without sucrose, exhibited significantly higher chlorophyll content compared to their equivalents in a sealed box, implying that gas exchange allows in vitro plants to carry out photosynthesis. As a result, we have incorporated the use of a filtered box into our protocol.

The light intensity in ex-vitro-grown plants directly affects photosynthesis, with increased light levels enhancing photosynthetic activity until reaching a saturation point [22]. Light also impacts morphogenesis and the induction of flowering [23]. To test the effect of increasing light intensity on in vitro cannabis flowering, we cultured plants under an 18/6 h cycle for three weeks at two different light intensities measured at plant height: low (71±14 µmol· m⁻²⋅s⁻¹) and high (173±20 µmol· m⁻²⋅s⁻¹). We then transferred the plants to a flowering photoperiod (12/12 h) and redistributed them into the two light intensities, resulting in four treatment groups based on changes in light intensity: Low to Low, Low to High, High to Low, and High to High. Plants exposed to low light intensity during the flowering-promoting photoperiod produced significantly more flowers (Fig. 4b-d) with a final average of 9.3 (Low to Low) and 9.9 (High to Low) flowers per plant and appeared to have longer stigmas compared to those exposed to high intensity (Fig. 4e). Plants under high light intensity during the 12/12 h cycle had a final average of 7.3 flowers per plant, regardless of the light intensity during the vegetative phase. It is important to note that the selected light intensity was significantly lower than what is typically used in growth rooms (Fig. 4a), demonstrating that tissue-cultured plants are more susceptible to damage from high-intensity light.

Variability and response of cannabis cultivars to in vitro flowering

Cannabis cultivars exhibit significant variability in inflorescence traits, including flowering time, architecture, number of flowers, flower size, and compaction [5, 24-26]. To assess the response of different cultivars to in vitro flowering, we selected three cultivars that exhibit distinct inflorescence phenotypes when grown in a growth room (Fig. 5b) and introduced them to tissue culture. Following flower induction, we identified three distinct phenotypic differences: number of flowers, stigma length, and stigma browning (Fig. 5, Fig. S4, and Fig. S5)). In all cultivars, flowers began to appear on day 9. The Sky 1 cultivar produced significantly more flowers than the Magic 9 and TA5 cultivars (Fig. 5a), displaying a notable difference from its growth room equivalent (Fig. 5b). The TA5 stigmas appeared to be shorter than those of Sky 1 and Magic 9 and changed their color to brown before those of the other cultivars (Fig. 5e), suggesting that TA5's receptivity declines more quickly. This is consistent with the TA5 stigma observed in the growth room plants.

This experiment demonstrates that different cultivars behave differently in tissue culture, and their inflorescence characteristics do not always match their growth room phenotype, suggesting an interaction between genetic background and in vitro growth conditions.

In vitro pollination and seed development in cannabis

To assess the potential of in vitro flowering as a method for rapid breeding, we examined the ability of male plants to produce viable pollen and female plants to develop viable seeds. To this end, we cultured male cannabis plants (Bt cultivar) and female plants (cultivar Sky 1) for two weeks under an 18/6 h cycle before transferring them to a 12/12 h flowering photoperiod.

After 18 days, all plants produced flowers and were ready for pollination (Fig. 6a-d). Since the anther did not dehisce and release the pollen, we manually pinched the anther with a needle and tapped it over vessels containing female plants. All female inflorescent (25 plants) developed seeds in vitro (Fig. 6e) with an average of 4.41 ± 0.36 seeds per plant. However, only 2.44 ± 0.17 seeds per plant germinated (mean ± SE calculated per vessel, with five plants per vessel) (Table S1). In vitro hybrid plants were grown from seeds to fully developed plants (Fig. 6f-h).

Altogether, eight weeks were needed from the introduction of the stem segment to tissue culture to the production of viable seeds, highlighting the in vitro flowering system as an excellent method for fast and practical breeding.

Plants in tissue cultures are grown under controlled conditions that differ significantly from the natural environment. These optimized conditions enhance growth but can also induce stress due to the lack of fluctuations throughout the day and the lack of natural cues, leading to altered physiological responses. However, this platform allows us to fine-tune factors such as nutrient composition, light intensity, and temperature, thereby enhancing growth and development.

In this study, we focused on determining the optimal conditions for the in vitro flowering of cannabis. This approach holds immense potential for the sterile production of secondary metabolites free from pathogens like Botrytis cinerea, with high consistency and the likely capability to control the cannabinoid content. Given the complexity of breeding in cannabis, it also provides a valuable strategy for breeding programs, especially when resources are limited.

Using our protocol, we cultivated five plants per vessel and arranged the cultures on a shelf measuring 45 cm in width and 1 meter in length, accommodating a total of 32 vessels in a 4 by 8 configuration (Fig. S6). This setup allowed for the growth of 160 plants per shelf. By stacking four shelves vertically in a standard growth room, we demonstrated the capacity to grow a total of 640 plants on a 1-meter length floor area, highlighting the substantial potential of this method.

When we introduced the stem segments to tissue culture, the flowering time and the number of flowers produced were comparable, regardless of whether the plants were immediately subjected to a flowering-promoting photoperiod or first experienced a vegetative phase. We propose that since the day length cue is perceived in leaves to initiate the cascade that activates the flowering machinery, a stem node without leaves cannot sense day length and thus cannot induce the transition of the meristem to an inflorescence meristem. Therefore, a few days are needed to produce leaves, even under a short-day photoperiod, before flower meristems can form. This resulted in similar timing and numbers of flowers between the two regimes.

In Arabidopsis thaliana, leaf perception of day length induces the expression of the FLOWERING LOCUS T (FT) gene, known as florigen, which is transported through the phloem to the shoot apical meristem to promote flowering [27, 28]. In short-day plants, the mechanism of flowering induction is still being elucidated, but the process appears to be conserved [29]. Genes homologous to FT were identified in many short-day plant species [30, 31], including CsFT in cannabis [32].

Our results, showing normal stomata development in tissue culture and high chlorophyll content with improved growth in filtered vessels with and without a carbon source, suggest that cannabis shoots are capable of performing photosynthesis in tissue culture. However, the moderate plant growth in a sugar-free medium indicates that the plant cannot be fully autotrophic under our growth conditions with passive ventilation, likely due to limited CO₂. This is supported by the CO₂ level measured within a filtered vessel containing plants, showing values between 150 and 250 mmol/mol during the photoperiod, depending on the number of filters, while the CO₂ concentration in the room was maintained at 350-400 mmol/mol [33]. It would be interesting to test the cannabis performance under a forced ventilation system in the future.

In tissue culture, sucrose in the media is used to provide the necessary carbon for energy and growth, with standard concentrations generally ranging from 2% to 3% (w/v). As we did not observe a significant difference between these concentrations, we selected the 2% for our protocol, which offers several potential advantages. Low levels of sucrose can promote photosynthetic activity, partially by increasing rubisco activity, thereby supporting mixotrophic growth that generally leads to more viable plants [34-36]. Low sucrose level also reduces the risk of microbial contamination [33] and minimizes the phenomenon of hyperhydricity in sensitive cultivars [37]. Excess sucrose can cause metabolic imbalances that affect the synthesis of secondary metabolites [38]. Furthermore, using the lowest effective concentration is cost-effective for large-scale or long-term experiments.

Light intensity can promote mixotrophic growth, which relies on sucrose in the media and photosynthesis. Our results, however, show that increasing the light intensity during the flowering-promoting photoperiod led to significantly fewer flowers. The appearance of yellow leaves when high-intensity light is applied (Fig. S7) suggests that the plants might be experiencing light-induced stress.

In outdoor plants, prolonged exposure to excessive light can lead to photoinhibition, reducing photosynthetic capacity and subsequently causing chlorophyll degradation. The light intensity applied in tissue culture is considerably lower than that of outdoor environments[39] because most of the energy comes from the sucrose in the media and because high light intensity can induce stress.

Plants in tissue culture are placed in a closed box without buffer elements like wind, shading, and light fluctuations throughout the day. This may intensify the effects of high light and might cause increased temperature and humidity. Additionally, tissue cultures often consist of young tissues in early growth stages, making them more susceptible to environmental stresses due to the lack of protective structures like a thick cuticle or well-developed cell walls [40].

It might be that the lack of a phyllosphere microbiome due to sterile conditions in tissue cultures reduces plant fitness and increases susceptibility to light-induced stress. Microorganisms can produce protective compounds, such as pigments and antioxidants [41], that absorb excess light, and they can also form a physical barrier that reduces the amount of light reaching the leaf surface [42], thereby preventing potential damage from excessive light exposure.

In our experiment, when low light intensity was applied during the 12/12 h cycle, the plants produced more flowers regardless of the intensity during the vegetative phase. We propose that during the initial period under the flowering induction regime, prior to the transition to inflorescence meristem, the plants have time to recover. During this phase, the newly developing leaves grow under low light and develop into robust leaves that can support future inflorescence meristem and flower development.

Microflowering for rapid breeding

The individual female and male flowers that developed in vitro exhibited phenotypes similar to those of growth room flowers. However, the anthers did not burst and release pollen. Typically, anthers open under low relative humidity (RH), whereas high RH can delay or prevent this process [43]. This suggests that the high humidity levels within the culture vessel likely kept the cannabis anthers closed. In addition, pollen grains are typically partially desiccated upon dispersal, and before they germinate, they must rehydrate by absorbing water from the stigma on which they land [44]. It might be that the process of losing and gaining back moisture is crucial for pollen grain germination. Therefore, RH in the flower's surrounding environment can decrease the viability of the pollen from these flowers. Nevertheless, in our setting, although the anther did not dehisce naturally, it released pollen with our assistance, which proved to be viable according to our germination test and the successful fertilization.

The average number of female flowers produced was six, with an average of 4.4 seeds developing per inflorescence/plant. Low pollen grain viability might have led to incomplete fertilization, preventing some flowers from producing seeds. Another possibility is that the plant's small size could not support extensive seed development. Out of the collected seeds, only an average of 2.4 seeds per plant germinated (Table S1). However, achieving one successful succession per generation is sufficient for a breeding program aimed at hybrid seed production and generating numerous homozygous plants to test various combinations.

This study has successfully established a method for in vitro flowering induction in cannabis, providing a valuable tool for both research and practical applications. Our findings highlight the superior performance of DKW medium with 2% sucrose in a filtered vessel and emphasize the need for low light intensity during flower induction to optimize production. The improved performance in filtered vessels suggests that plants conduct photosynthesis in vitro, underscoring the need for future investigations into the effects of forced ventilation to refine this system. We show that this method is applicable to several cultivars and for male flowering as well. Additionally, we use this system to produce viable seeds in vitro. Altogether, this method offers a robust framework for cannabis research and cultivation, with potential applications in sterile cannabinoid production and efficient breeding strategies.

Plant material

Cannabis sativa L. plants from cultivars TA5, Sky 1, Magic 9 (female cultivars), and Bt (male plant) (our private lines) were used in this study. The plants were grown in a growth room in 1-liter pots with 'Bental 11' planting soil (Tuff Substrates), regularly irrigated with tap water, and fertilized with BOUNTY1 (N.P.K. 4:2:6, Zalmanson Dshanim). The plants were grown at 26 °C (measured at plants’ canopy) under Metal halide 600W light bulbs (UPLUX, USA) in a long-day photoperiod of 18/6 h light/dark cycle to sustain vegetative growth.

Tissue culture

TA5 vegetative plants were initially introduced to tissue culture by cutting them into single-node stem explants. Sterilization was done by submerging and stirring the explants in a 2% sodium hypochlorite solution containing 0.1% (w/w) Triton for eight minutes, followed by three subsequent rinses with sterilized water. Two basal media were used in this study: Murashige and Skoog (MS) [45] consist of 4.4 g/L MS including Nitsch vitamins (M0256, Duchefa), 3% sucrose (w/v), and 0.5 g/L MES and Driver and Kuniyaki Walnut (DKW) [46] containing 5.6 g/L DKW/JUGLANS basal salt mixture including vitamins (D0247, Duchefa), 3% sucrose (w/v), 80 mg/L adenine hemisulfate (A0908, Duchefa) and 0.5 g/L MES. The pH of the media was adjusted to 5.8 and 0.8% (w/v) plant agar (P1101, Duchefa) was used as the gelling agent. The media were sterilized by autoclaving at 121 °C for 20 minutes and poured into either a ventilated round polypropylene microbox (O118/80+OD118, SacO2) with a volume of 65 ml or into a closed 10x10x10 cm square polycarbonate box (Fig. 5d) with a volume of 84 ml. Five stem explants were cultured in each vessel for two weeks under an 18/6 h light/dark photoperiod to promote vegetative growth. The plants were then transferred to fresh media and placed under a 12/12 h light/dark cycle to induce flowering and re-cultured in fresh media every two weeks. Both growth conditions were set at a temperature of 25 °C. Flowers were counted using a stereomicroscope every several days.

Fertilization: To conduct an in vitro pollination procedure, male flowers were carefully removed from the plant and placed on a sterile Petri dish, followed by pinching with a sterile needle. In the laminar flow cabinet, the anthers were then rubbed onto the stigma using forcep

Media Optimization

For cytokinin effect, plants were cultured on DKW medium for two weeks in vegetative conditions (18/6 h) and then cultured under flowering-promoting photoperiod (12/12 h) in three media: control (DKW medium + 80 mg adenine), 1 mg/L 6-Benzylaminopurine (BA) (B001, Caisson) and 5 mg/L BA. In addition, one group was cultured on DKW with 2 mg/L BA medium during the vegetative phase and re-cultured to control DKW medium when transferred to flowering induction.

For the activated charcoal effect, 0.5 g/L activated charcoal (C9157, Sigma) was added to DKW or MS media during both the vegetative phase and flowering-promoting photoperiod.

Chlorophyll extraction and measurements

Chlorophyll content was assessed on plants cultured as follows: first, all stem explants were cultured on DKW medium with 2% sucrose in a ventilated micro box (four explants per micro box) for three weeks under a long-day photoperiod (18/6 h). Next, they were divided into four treatments: DKW medium with or without 2% sucrose in a ventilated micro box and DKW medium with or without 2% sucrose in a closed 10x10x10 cm square polycarbonate box. Plants were grown for four weeks under the same conditions as before without media renewal. For chlorophyll measurements, two fresh leaves (numbers 3 and 4 from the top) from each plant (a total of eight leaves per vessel) were taken for extraction with 25ml of 80% acetone. Chlorophyll was measured according to Arnon 1949 [47]. The leaves were weighed before extraction to determine their fresh weight, and spectrophotometric measurements at 645 and 663 nm were conducted using the Infinite M Plex (Tecan). The chlorophyll concentration was calculated using the formula: ((20.2 × X A₆₄₅) + (8.02 × A₆₆₃)) * V /(1000 × W), where V is the solution volume and W is the weight of the leaves.

Light intensity experiments

Explants were introduced to culture as described. During the vegetative phase (18/6 h), vessels were divided into two light-intensity treatments: Low (71±14 µmol·m^-2s^-1) and High (173±20 µmol·m^-2s^-1), measured at the plant height level. After three weeks, plants from each light treatment were re-cultured in fresh media and placed under a flowering-promoting photoperiod (12/12 h) at the two different light intensities, resulting in a total of four treatments: Low to Low, Low to High, High to Low, and High to High. Light intensity was measured using a LI-180 Spectrometer (LI-COR).

Stomata counting analysis

A silicone impression material (Elite HD+, Zhermack) was used to create molds of abaxial fan leaves. Next, patterns from those molds were taken using nail polish and checked under a light microscope. From each leaf, stomata were counted in three areas (0.15 mm² each) along the main vascular bundles (Fig. S8).

Pollen germination

Pollen germination was assessed following the protocol of Flajšman et al., 2021 [48]. The germination medium consisted of 170 g/l sucrose, 0.1 g/l H₃BO₃, 0.432 g/l Ca(NO₃)₂ ∗ 4H₂O. The pH was adjusted to 7.0, and 0.7% (w/v) plant agar was used as the gelling agent. The medium was sterilized by autoclaving before being poured into Petri dishes. Male flowers were gently tapped to distribute pollen across the plate. Subsequently, the Petri dishes were incubated in the dark at room temperature for 24 hours. Images were taken using a Nikon SMZ-18 stereo zoom microscope (Nikon).

Scanning electron microscopy

Scanning electron microscopy (SEM) was conducted following the method described previously [49]. Briefly, fresh fan leaves from plants grown in tissue culture or growth room were initially fixed in 100% methanol for 10 min. Subsequently, they were rinsed three times for 30 minutes in 100% ethanol, dried using a K850 critical point dryer, and then mounted on stubs. The specimens were further coated with a thin layer (2 nm) of gold-palladium using a Q150T ES (Quorum Technologies Ltd, Lewes, UK) for SEM imaging in a JSM-IT100 SEM (Jeol Ltd, Tokyo, Japan).

Experimental design and statistical analysis

The experiment utilized a completely randomized design, with a specified number of replicates, each comprising the average of five explants cultured within a single vessel. The data were analyzed using JMP software (SAS Institute, Cary, NC, USA). Means comparison was conducted using analysis of variance (ANOVA) with post-hoc Tukey–Kramer honest significant difference (HSD) test (for multiple comparisons) or Student’s t-test (for one comparison) at α = 0.05 significance level and the data are represented as mean ± SE.

Competing Interests

KB and LEW are listed as inventors on a PCT Patent Application PCT Patent Application No. PCT/IL2022/050007

Author Contribution

LEW, OL, and KB: Conceptualization; LEW and OL: design of the work; OL: conducted the experiments; LEW and OL: Writing Original Draft Preparation; LEW and OL: Writing Review and Editing. All authors read and approved the final manuscript

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Competing interest reported. KB and LEW are listed as inventors on a PCT Patent Application PCT Patent Application No. PCT/IL2022/050007

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Optimizing cannabis cultivation: an efficient in vitro system for flowering induction

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