Cannabis sativa L. photoautotrophic micropropagation: a powerful tool for industrial scale in vitro propagation.
Left to right: Adel Zarei, Behrang Behdarvandi, Elham Tavakouli Dinani and Jennifer Maccarone. Cannabis Micropropagation Laboratory, Safari Flower Co., Fort Erie, Ontario, Canada.
Global demands for an in vitro culture of cannabis have never been more sought after as countries shift their paradigm towards legalization. Cannabis conventional (photomixotrophic) micropropagation has not been suitable enough for large-scale propagation due to a high degree of plant hyperhydricity, low growth rate, poor rooting, and acclimation efficiency. In this study, cannabis photoautotrophic micropropagation method is introduced with the purpose of overcoming the difficulties that conventional micropropagation entails to meet the large scale micropropagation requirements. The roles of rockwool medium pH and moisture content, cutting length, basal wounding methods, light intensity, and culture vessel gas exchange capacity were assessed with the intention of increasing productivity of micropropagation method. This report provides an efficient in vitro propagation method that shortens the culture period, solving physiological disorders, offers high survival rate, and reduces costs using larger ventilated culture vessels and sugar-free medium. In our laboratory setup, up to 2260 rooted plantlets were produced per 10 m2 shelving area within a month suggesting this method can be utilized for commercial scale propagation of cannabis.
Adel Zarei, Behrang Behdarvandi, Elham Tavakouli Dinani and Jennifer Maccarone. Cannabis sativa L. photoautotrophic micropropagation: a powerful tool for industrial scale in vitro propagation. In Vitro Cellular & Developmental Biology-Plant 57, 932–941, 2021.
Detailed chromosome analysis of wild-type, immortalized fibroblasts with SV40T, E6E7, combinational introduction of cyclin dependent kinase 4, cyclin D1, telomerase reverse transcriptase
The cell immortalization enables us to expand the cultured cell infinitely. However, the process of immortalization sometimes changes the nature of the original cell with traditional virus derived oncogene method, such as SV40 large T antigen or human papilloma virus derived E6E7 oncogenes. In this study, we established immortalized embryonic fibroblasts with combinational expression of mutant cyclin dependent kinase 4 (CDK4), cyclin D1, telomerase reverse transcriptase (TERT) oncogenic SV40 and human papilla virus-derived E6E7, from identical primary wild type human embryonic fibroblasts (HE16). We compared the details of chromosome condition with the G-banding and Q-banding method. There is no example of detailed analysis so far about chromosome abnormalities, such as trisomy, ring chromosome, reciprocal translocation, and dicentric chromosomes. The detailed chromosome analysis revealed that immortalized cells with SV40 and E6E7 showed intensive chromosome abnormalities, such as gain or loss of the chromosomes all through the genome. However, we showed that the incidence of chromosome abnormities in the immortalized cell with the combinational introduction of R24C mutant of CDK4, cyclin D1, TERT is almost identical to that of wild type cell, which possibly explained intact function of p53 tumor suppressor gene. These results showed that cellular immortalization with CDK4, cyclin D1, and TERT is more advantageous in keeping the chromosome’s original condition than oncogenic immortalization methods.
Haruka Takada, Tomisato Miura, So Fujibayashi, Naomi Susaki, Kouhei Takahashi, Eriko Sugano, Hiroshi Tomita, Taku Okazi, Tohru Kiyono, Mitsuaki A. Yoshida, Tomokazu Fukuda. Detailed chromosome analysis of wild-type, immortalized fibroblasts with SV40T, E6E7, combinational introduction of cyclin dependent kinase 4, cyclin D1, telomerase reverse transcriptase. In Vitro Cellular & Developmental Biology-Animal, 57:998-1005, 2022.
Physical factors increased quantity and quality of micropropagated shoots of Cannabis sativa L. in a repeated harvest system with ex vitro rooting
Jeffrey Adelberg, Ryan Murphy
Cannabis sativa has been a plant of medicinal interest in recent years, mainly for its increased cannabinoid production in female inflorescences. As a dioecious plant, vegetative propagation of female cannabis plants is preferred, however, traditional stock plant maintenance for vegetative cuttings requires considerable production space and exposes propagules to pathogens. Recently, the cannabis industry has been plagued by pathogens, specifically Hops Latent Viroid, which reduces overall productivity and is easily transmissible. In vitro propagation methods, such as micropropagation, offer powerful tools for rapid, mass multiplication of pathogen-free, vegetative propagules of cannabis. A typical micropropagation system, which includes subculture and transfer of material into new vessels, is labor intensive and costly. This work was conducted on PGR-free DKW media and implemented a micropropagation technique called hedging, which focuses on the removal of the apical portion of the propagules, leaving the base of the propagule to remain in the original micropropagation vessel. The removal of the apical portion promotes lateral bud break, and subsequently additional apical shoots, increasing the multiplication potential. This process can be repeated over multiple cycles of hedging without having to subculture the propagule into a new vessel. The harvested unrooted, apical shoots were then introduced directly into a laboratory ex vitro propagation system for rooting, acclimatization, and hardening, without an intermediary step of subculturing propagules onto a rooting media in vitro, prior to ex vitro acclimatization. To produce good quality propagules for the laboratory ex vitro propagation system, it was found that pre-hardening the propagules in vitro was beneficial and was accomplished by using ventilated micropropagation vessels and a light intensity of approximately 100 μmol m−2 s−1 PPFD.
Ryan Murphy, Jeffrey Adelberg. Physical factors increased quantity and quality of micropropagated shoots of Cannabis sativa L. in a repeated harvest system with ex vitro rooting. In Vitro Cellular & Developmental Biology-Plant, 57:923-931, 2021.
Shining light on anther culture, a poorly understood regeneration route in passion fruit (Passiflora gibertii N. E. BROWN): Histological, hormonal, and phytochemical aspects
Left to right: Top: Wellington Santos Soares*, Claudio Horst Bruckner*, Ana Claudia Ferreira Cruz* & Diego Ismael Rocha*; Bottom: Elyabe Monteiro Matos**, Andréa Dias Koehler*, Lyderson Facio Viccini** & Wagner Campos Otoni*. (Federal University of Viçosa* and Federal University of Juiz de Fora**, Brazil).
In vitro regeneration systems have been established to assist production of plant material and passion fruit breeding programs. Given the scarcity of studies that address the potential of passion fruit anthers in inducing morphogenetic pathways, we described the histological changes involved in the formation of P. gibertii embryogenic calluses. Moreover, we provided a histochemical, phytochemical, and hormonal characterization to assess the potential of passion fruit anthers for somatic embryogenesis induction and assess the ploidy of embryogenic calluses to determine the purpose of this regeneration system. In summary, our work provided a morpho-physiological description of P. gibertii somatic embryogenesis from anther culture. We believe that our results will contribute to a better understanding of this poorly characterized regeneration route in passion fruit. Moreover, the present findings can support further studies aimed at inducing androgenesis and haploid recovery, as well as contribute to micropropagation and genetic manipulation of passion fruit.
Wellington Santos Soares, Claudio Horst Bruckner, Ana Claudia Ferreira Cruz, Diego Ismael Rocha, Elyabe Monteiro Matos, Andréa Dias Koehler, Lyderson Facio Viccini, and Wagner Campos Otoni. Shining light on anther culture, a poorly understood regeneration route in passion fruit (Passiflora gibertii N. E. Brown): histological, hormonal, and phytochemical aspects. In Vitro Cellular & Developmental Biology-Plant, 57: 998–1008, 2021. https://doi.org/10.1007/s11627-021-10179-z
Integrin and growth factor receptor signaling cooperate to modulate the cellular radioresponse of fibroblasts
Left to right: Anne Vehlow and Nils Cordes, both National Center for Radiation Research in Oncology – OncoRay (Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Germany).
Upon interaction with the extracellular matrix, membrane bound integrin adhesion receptors regulate many important cellular functions, such as the survival of cells and their response to genotoxic stress. In focal adhesions, integrins coalesce with a plethora of regulatory macromolecules including growth factor receptors. While a cooperation of integrin and growth factor receptor signaling seems plausible, the functional consequences on cellular radiosensitivity are unclear. The presented study evaluates the influence of β1 integrin and epidermal growth factor receptor (EGFR) signaling on clonogenic survival and associated cell cycling of fibroblasts. An analysis of clonogenic survival without and in combination with X-ray irradiation reveals a dependency of basal survival on growth factors but not β1 integrins. In contrast, radiation enhanced G2/M arrest and, consequently clonogenic radiation survival, requires a convergence of signaling cascades downstream of both β1 integrins and EGFR. Particularly, the effect of EGFR signaling on the radiogenic G2/M arrest requires an activation of β1 integrin but not PI3K signaling. These data strongly suggest a pro-survival function of the tight association of integrin and growth factor receptor signaling upon irradiation. Further insight and understanding of the underlying complex mechanisms may lead to the identification of molecular targeted therapies for radiation oncology.
Anne Vehlow and Nils Cordes. Growth factor receptor and β1 integrin signaling differentially regulate basal clonogenicity and radiation survival of fibroblasts via a modulation of cell cycling. In Vitro Cellular & Developmental Biology-Animal, 58 (2):169-178. 2022.
Development of a Robust Transient Expression Screening System in Protoplasts of Cannabis
From Left to Right: Noel Cogan, Lennon Matchett-Oates, German Spangenberg. Inset: Ehab Mohamaden
Through factorial designs, the optimisation of protoplast isolation and transformation in Cannabis has been achieved in multiple cultivars. Range analyses were conducted to determine significant variables involved in isolation and transformation, with calculated optimised variable combinations performed. Protoplast yields as high as 5.7 x106 with a transformation efficiency of 23.2% was achieved, demonstrating the effectiveness of the developed protocol across multiple cultivars and for future genome editing attempts using protoplasts.
L. Matchett-Oates, Ehab Mohamaden, G.C. Spangenberg, N.O.I Cogan. Development of a Robust Transient Expression Screening System in Protoplasts of Cannabis. In Vitro Cellular & Developmental Biology-Plant, 57:1040-1050, 2021.