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

BACKGROUND: Cannabis sativa L. 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.

Biological control of the cucurbit powdery mildew pathogen Podosphaera xanthii by means of the epiphytic fungus Pseudozyma aphidis and parasitism as a mode of action.

Epiphytic yeasts, which colonize plant surfaces, may possess activity that can be harnessed to help plants defend themselves against various pathogens. Due to their unique characteristics, epiphytic yeasts belonging to the genus Pseudozyma hold great potential for use as biocontrol agents. We identified a unique, biologically active isolate of the epiphytic yeast Pseudozyma aphidis that is capable of inhibiting Botrytis cinerea via a dual mode of action, namely induced resistance and antibiosis. Here, we show that strain L12 of P. aphidis can reduce the severity of powdery mildew caused by Podosphaera xanthii on cucumber plants with an efficacy of 75%. Confocal and scanning electron microscopy analyses demonstrated P. aphidis proliferation on infected tissue and its production of long hyphae that parasitize the powdery mildew hyphae and spores as an ectoparasite. We also show that crude extract of P. aphidis metabolites can inhibit P. xanthii spore germination in planta. Our results suggest that in addition to its antibiosis as mode of action, P. aphidis may also act as an ectoparasite on P. xanthii. These results indicate that P. aphidis strain L12 has the potential to control powdery mildew.

Pseudozyma aphidis Induces Salicylic-Acid-Independent Resistance to Clavibacter michiganensis in Tomato Plants.

The ability of plant pathogens to rapidly develop resistance to commonly used pesticides challenges efforts to maximize crop production. Fungal biocontrol agents have become an important alternative to chemical fungicides as a result of environmental concerns regarding conventional pesticides, including resistance issues. The complex mode of action of biocontrol agents reduces the likelihood that pathogens will develop resistance to them. We recently isolated a unique, biologically active isolate of the epiphytic fungus Pseudozyma aphidis. We show that the extracellular metabolites secreted by our P. aphidis isolate can inhibit Xanthomonas campestris pv. vesicatoria, X. campestris pv. campestris, Pseudomonas syringae pv. tomato, Erwinia amylovora, Clavibacter michiganensis, and Agrobacterium tumefaciens in vitro. Moreover, application of Pseudozyma aphidis spores on tomato plants in the greenhouse significantly reduced (by 60%) the incidence of bacterial wilt and canker disease caused by C. michiganensis subsp. michiganensis on those plants as well as disease severity by 35%. Furthermore, infected plants treated with P. aphidis were 25% taller than control infected plants. We found that P. aphidis activates PR1a-and other pathogenesis-related genes in tomato plants-and can trigger an induced-resistance response against C. michiganensis that proceeds in a salicylic-acid-independent manner, as shown using NahG-transgenic tomato plants.

The transcription factor SlSHINE3 modulates defense responses in tomato plants.

The cuticle plays an important role in plant interactions with pathogens and with their surroundings. The cuticle acts as both a physical barrier against physical stresses and pathogens and a chemical deterrent and activator of the plant defense response. Cuticle production in tomato plants is regulated by several transcription factors, including SlSHINE3, an ortholog of the Arabidopsis WIN/SHN3. Here we used a SlSHINE3-overexpressing (SlSHN3-OE) and silenced (Slshn3-RNAi) lines and a mutant in SlCYP86A69 (Slcyp86A69)--a direct target of SlSHN3--to analyze the roles of the leaf cuticle and cutin content and composition in the tomato plant's defense response to the necrotrophic foliar pathogen Botrytis cinerea and the biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria. We showed that SlSHN3, which is predominantly expressed in tomato fruit epidermis, also affects tomato leaf cuticle, as morphological alterations in the SlSHN3-OE leaf tissue resulted in shiny, stunted and permeable leaves. SlSHN3-OE leaves accumulated 38% more cutin monomers than wild-type leaves, while Slshn3-RNAi and Slcyp86A69 plants showed a 40 and 70% decrease in leaf cutin monomers, respectively. Overexpression of SlSHN3 resulted in resistance to B. cinerea infection and to X. campestris pv. vesicatoria, correlated with cuticle permeability and elevated expression of pathogenesis-related genes PR1a and AOS. Further analysis revealed that B. cinerea-infected Slshn3-RNAi plants are more sensitive to B. cinerea and produce more hydrogen peroxide than wild-type plants. Cutin monomer content and composition differed between SlSHN3-OE, Slcyp86A69, Slshn3-RNAi and wild-type plants, and cutin monomer extracted from SlSHN3-OE plants altered the expression of pathogenesis-related genes in wild-type plants.

The effects of glucosinolates and their breakdown products on necrotrophic fungi.

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.

Pseudozyma aphidis induces ethylene-independent resistance in plants.

Species of the epiphytic fungus Pseudozyma are not pathogenic to plants and can be used as biocontrol agents against plant pathogens. Deciphering how they induce plant defense might contribute to their use for plant protection and expand our understanding of molecular plant-pathogen interactions. Here we show that Pseudozyma aphidis isolate L12, which is known to induce jasmonic acid- and salicylic acid-independent systemic resistance, can also activate local and systemic resistance in an ethylene-independent manner. We also show that P. aphidis localizes exclusively to the surface of the plant leaf and does not penetrate the mesophyll cells of treated leaves. We thus propose that P. aphidis acts via several mechanisms, and is an excellent candidate biocontrol agent.

Overexpression of AtSHN1/WIN1 provokes unique defense responses.

The plant cell cuticle serves as the first barrier protecting plants from mechanical injury and invading pathogens. The cuticle can be breached by cutinase-producing pathogens and the degradation products may activate pathogenesis signals in the invading pathogens. Cuticle degradation products may also trigger the plant's defense responses. Botrytis cinerea is an important plant pathogen, capable of attacking and causing disease in a wide range of plant species. Arabidopsis thaliana shn1-1D is a gain-of-function mutant, which has a modified cuticular lipid composition. We used this mutant to examine the effect of altering the whole-cuticle metabolic pathway on plant responses to B. cinerea attack. Following infection with B. cinerea, the shn1-1D mutant discolored more quickly, accumulated more H2O2, and showed accelerated cell death relative to wild-type (WT) plants. Whole transcriptome analysis of B. cinerea-inoculated shn1-1D vs. WT plants revealed marked upregulation of genes associated with senescence, oxidative stress and defense responses on the one hand, and genes involved in the magnitude of defense-response control on the other. We propose that altered cutin monomer content and composition of shn1-1D plants triggers excessive reactive oxygen species accumulation and release which leads to a strong, unique and uncontrollable defense response, resulting in plant sensitivity and death.

The epiphytic fungus Pseudozyma aphidis induces jasmonic acid- and salicylic acid/nonexpressor of PR1-independent local and systemic resistance.

Pseudozyma spp. are yeast-like fungi, classified in the Ustilaginales, which are mostly epiphytic or saprophytic and are not pathogenic to plants. Several Pseudozyma species have been reported to exhibit biological activity against powdery mildews. However, previous studies have reported that Pseudozyma aphidis, which can colonize plant surfaces, is not associated with the collapse of powdery mildew colonies. In this report, we describe a novel P. aphidis strain and study its interactions with its plant host and the plant pathogen Botrytis cinerea. This isolate was found to secrete extracellular metabolites that inhibit various fungal pathogens in vitro and significantly reduce B. cinerea infection in vivo. Moreover, P. aphidis sensitized Arabidopsis (Arabidopsis thaliana) plants' defense machinery via local and systemic induction of pathogenesis-related1 (PR1) and plant defensin1.2 (PDF1.2) expression. P. aphidis also reduced B. cinerea infection, locally and systemically, in Arabidopsis mutants impaired in jasmonic acid (JA) or salicylic acid (SA) signaling. Thus, in addition to direct inhibition, P. aphidis may inhibit B. cinerea infection via induced resistance in a manner independent of SA, JA, and Nonexpressor of PR1 (NPR1). P. aphidis primed the plant defense machinery and induced stronger activation of PDF1.2 after B. cinerea infection. Finally, P. aphidis fully or partially reconstituted PR1 and PDF1.2 expression in npr1-1 mutant and in plants with the SA hydroxylase NahG transgene, but not in a jasmonate resistant1-1 mutant, after B. cinerea infection, suggesting that P. aphidis can bypass the SA/NPR1, but not JA, pathway to activate PR genes. Thus, either partial gene activation is sufficient to induce resistance, or the resistance is not directed solely through PR1 and PDF1.2 but probably through other pathogen-resistance genes or pathways as well.

LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues.

BACKGROUND: Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium. FINDINGS: We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids. CONCLUSIONS: The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.

Identification and characterization of a novel miR159 target not related to MYB in tomato.

MicroRNA 159 (miR159) is a highly conserved miRNA with roles in flowering under short days, anther development and seed germination via repression of GAMYB-like genes. In tomato, the function of miR159 (Sl-miR159) is currently unknown and target transcripts have not been experimentally validated. Here, we identified and characterized a new miR159 target gene (SGN-U567133) in Solanum lycopersicum (tomato) that is not related to MYB. SGN-U567133 is predominantly expressed in flowers and encodes a nuclear-localized protein that contains a unique NOZZLE-like domain at its N terminus. In tomato, SGN-U567133 represents a small gene family and orthologs have been identified in other plant species, all containing a conserved miR159 target site in their coding sequence. Accordingly, 5'-RACE cleavage assay supported miRNA-mediated cleavage of SGN-U567133 transcripts in vivo. Moreover, the SGN-U567133 transcript accumulated in P19-HA-expressing tomato leaves in which miRNA-mediated cleavage is inhibited. In addition, transgenic tomato plants expressing a miR159-resistant form of SGN-U567133 accumulated higher levels of the SGN-U567133 transcript and exhibited defects in leaf and flower development. Together, our results suggest that SGN-U567133 represents a novel class of miR159 targets in plants and raise the possibility that its post-transcriptional regulation by Sl-miR159 is essential for normal tomato development.

Transgenic expression of tomato bushy stunt virus silencing suppressor P19 via the pOp/LhG4 transactivation system induces viral-like symptoms in tomato.

During natural infection, the Tomato bushy stunt virus (TBSV) silencing suppressor protein P19 is expressed at high levels, which are required for optimum viral pathogenicity and silencing suppression. To date, expression of P19 in transgenic host plants has failed to achieve comparable expression levels and thus has provided only limited information on its in planta effects. To obtain high P19 expression and study its effects on host plant development in the absence of virus infection, we generated HA-tagged P19 (P19HA)-transgenic tomato reporter plants using the pOp/LhG4 transactivation system, which separates transformation from transgene expression. Upon reporter plant activation with a strong constitutive promoter, the transactivated F1 plants expressed high levels of a functional P19HA protein and displayed multiple abnormal phenotypes, some of which were highly reminiscent of the symptoms described previously for TBSV-infected tomato. Moreover, phenotype severity correlated with P19HA expression level, amount of bound miRNA/miRNA* duplexes, and accumulation of miRNA target transcripts. Together our results demonstrate that the tomato miRNA pathway is markedly compromised by P19, in particular when this protein is relatively abundant, as occurs during natural infection. We suggest that such interference with endogenous silencing may be responsible for at least some of the symptoms characteristic of TBSV-infected tomato.

Identification of trans-acting siRNAs in moss and an RNA-dependent RNA polymerase required for their biogenesis.

Trans-acting small interfering RNAs (tasiRNAs) are a class of higher-plant endogenous siRNAs that, like miRNAs, direct the cleavage of non-identical transcripts. tasiRNAs derive from non-coding transcripts (TAS) that are converted into dsRNA by a RNA-dependent RNA polymerase (RDR6), following their initial miRNA-guided cleavage. The dsRNA is then processed by a dicer-like enzyme 4 into phased 21-nucleotide siRNAs. To date, tasiRNAs have been identified only in Arabidopsis, and their identity and function in other land plants are unknown. Here, a set of endogenous small RNAs that correspond in a phased manner to a non-coding transcript (contig13502) were identified in the moss Pyscomitrella patens. Northern analysis suggests that contig13502-derived small RNAs are expressed in the juvenile gametophyte. In addition, miR390-guided cleavage of contig13502 at two sites flanking the small RNAs cluster was validated by 5' RACE. These cleavages are predicted to provide defined termini for the production of phased siRNAs. To elucidate the biogenesis of identified siRNAs, we cloned and generated knock-out mutants for an RDR6 moss homologue (PpRDR6). These mutants exhibited an accelerated transition from juvenile to mature gametophyte. In addition, RNA blots demonstrated that they lacked contig13502-derived siRNAs, suggesting that PpRDR6 is required for siRNA biogenesis. A target gene, which showed homology to an AP2/EREBP transcription factor, for one phased siRNA, was validated, corroborating its identity as a trans-acting siRNA. Taken together, our data indicate that contig13502 is a novel TAS locus and suggest a role for derived tasiRNAs in the regulation of gene expression in moss.