Genotyping-by-sequencing (GBS) reveals greater somatic mutations than simple sequence repeats (SSRs) in micropropagated cannabis plants
From left to right: Andrew Maxwell Phineas Jones, Kristian Adamek, and Davoud Torkamaneh
Micropropagation is a widely adopted method in horticulture aimed at producing large quantities of genetically identical and disease-free plants to ensure uniformity and high quality. However, this technique can lead to somaclonal variation, characterized by unusual phenotypic changes resulting from genetic mutations or epigenetic modifications. While some mutations are visually discernible through morphological or physiological abnormalities, many remain undetectable through observation alone. Although DNA markers have been utilized to assess genetic fidelity, their limited genome coverage poses challenges in accurately determining overall genetic fidelity. Genotyping-by-sequencing (GBS) has emerged as a cost-effective and widely used technique capable of sequencing and genotyping thousands of markers simultaneously. In this study, we compared the efficacy of GBS and simple sequence repeats (SSRs) in evaluating genetic fidelity in a clonal population of micropropagated cannabis plants. While SSR analysis revealed minimal genetic diversity, GBS identified thousands of genetic variants, uncovering genetic diversity within clonal lines across multiple subcultures. This study underscores the superior precision of sequencing-based technologies in detecting somaclonal mutations and underscores the limitations of traditional markers like SSRs in assessing genetic fidelity in micropropagation systems.
Kristian Adamek, Chris Grainger, Andrew Maxwell Phineas Jones, and Davoud Torkamaneh. Genotyping-by-sequencing (GBS) reveals greater somatic mutations than simple sequence repeats (SSRs) in micropropagated cannabis plants. In Vitro Cell. Dev. Biol.- Plant, 59, 757–766 (2023).
Cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) in tadpole cell lines from North American frogs
Stephanie DeWitte-Orr and Nguyen Vo
In the tributaries of Great Lakes, 3-trifluoromethyl-4-nitrophenol (TFM) is used as a lampricide to eradicate invasive sea lampreys in the Great Lakes. Toxic effects of TFM on non-target species including frogs have been widely reported. Current efforts are underway to develop next-generation lampricides that are more effective (in the elimination of lampreys) and safer (to protect the health of bystander aquatic animals). The focus of our research was to demonstrate that our newly established tadpole cell lines could be used as alternative toxicity models to study TFM toxicity and screen next-generation lampricides in high-throughput bioassays. The eight tadpole cell lines were derived from green frog (Lithobates clamitans), wood frog (Lithobates sylvaticus), and American toad (Anaxyrus americanus); all three species are native to the Great Lakes. Our research found that the tadpole cell lines were excellent tools that were comparable to whole-animal toxicity testing in evaluating TFM toxicity. The tadpole cell lines showed different sensitivity levels to TFM cytotoxicity, which would be useful in elucidating cellular mechanisms leading to toxicity or resistance in frogs. Among the eight tadpole cell lines tested, the WoodTad-HE1 cell line from wood frog and the GreenTad-gill cell lines from green frog would be useful alternative models in the toxicity screening of next-generation lampricides to ensure safety in frogs prior to their lamprey-eradicating use in the field.
Nguyen T. K. Vo and Stephanie DeWitte-Orr. Cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) in tadpole cell lines from North American frogs. In Vitro Cell. Dev. Biol.- Animal, 59, 790–795, (2024).
Establishment and characterization of a novel lung cell line derived from the common bottlenose dolphin
Left to right: Top: Kaede Tashiro, Kyosuke Hikobe and Takao Segawa; Bottom: Miwa Suzuki, Ken Maeda, and Takuya Itou
In a previous study published in this journal (DOI: https://doi.org/10.1007/s11626-023-00786-y), we reported on an infinite kidney cell line derived from bottlenose dolphin, and most of the long-term cultured cetacean cells reported are either skin- or kidney-derived. Additionally, cetaceans are often affected by respiratory diseases, which highlights the need for lung cell lines to study their unique respiratory physiology and pathogenesis of respiratory diseases. This paper introduced a novel lung cell line derived from the common bottlenose dolphin (Tursiops truncatus), named dLu. dLu is the first cetacean lung cell line that can be cultured for a long period, recording about 80 cell divisions by transfecting the simian virus 40 large T antigen. More importantly, this cell line showed altered cytokine gene expression upon immune stimulation with poly(I:C), making it a useful in vitro tool for studying immune responses in cetaceans. The results of this study may support the future expansion of cultured cetacean cells and develop cetacean research through the introduction of in vitro tools.
Kaede Tashiro, Kyosuke Hikobe, Takao Segawa, Miwa Suzuki, Ken Maeda, and Takuya Itou. Establishment and characterization of a novel lung cell line derived from the common bottlenose dolphin. In Vitro Cell. Dev. Biol.- Animal, 60, 98–105, (2024).
Development of an improved and simple shoot tip cryoconservation protocol for cryobanking of Bacopa monnieri (L.) Wettst. germplasm
Top from left : R. Gowthami, Neelam Sharma, Ramesh Chandra
Bottom from left: Jinsa Sara Kurian, Era Vaidya Malhotra and Anuradha Agrawal
In the world of herbal remedies, Bacopa monnieri is highly valued for its memory-enhancing properties, making it a valuable asset in traditional medicine. However, its popularity has led to over-exploitation, putting its genetic diversity at risk. To safeguard its future, we resorted to cutting-edge cryoconservation techniques. Researchers have been delving into the optimal methods for cryoconservation, aiming for high post-thaw regrowth that can be replicated across different genetic strains. In a series of experiments, we explored the impact of shoot tip size and cryoconservation techniques, including vitrification (V) and droplet-vitrification (DV), on the regrowth of Bacopa monnieri. We found that the best results were achieved with small-sized shoot tips, carefully treated with a special plant vitrification solution and subjected to precise dehydration processes. By employing the DV technique an impressive 80% recovery of growth was achieved. Crucially, genetic integrity of the regenerated plants remained intact, as revealed by meticulous analysis using genetic markers. The devised protocol was successfully tested on additional genetic strains of Bacopa monnieri. Overall, a four-fold increase in post-thaw regrowth, coupled with a significant reduction in preparation time was achieved. By effectively cryobanking its genetic material, we can preserve its medicinal properties for future generations while relieving the pressure on wild populations.
R. Gowthami, Neelam Sharma, Ramesh Chandra, Jinsa Sara Kurian, Era Vaidya Malhotra and Anuradha Agrawal. Development of an improved and simple shoot tip cryoconservation protocol for cryobanking of Bacopa monnieri (L.) Wettst. germplasm. In Vitro Cell. Dev. Biol.- Plant, 59, 744–756 (2023).
Identification of highly active compounds from insecticidal plant Oroxylum indicum L. (Vent.) and the induction of apoptosis by lapachol on Sf9 cells.
Top from left: Yujing Zhao, Jingjie An, and Zhihong Dang
Bottom from left: Jianglong Guo, Zhanlin Gao, Shujie Ma, and Yaofa Li
The frequent use of chemical pesticides has led to many environmental problems. Therefore, screening of green and safe insecticidal active ingredients from the abundant natural metabolites of insecticidal plants has become an important supplementary measure for IPM. Cell-based high-content screening (HCS) has been used for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Some effective compounds with high insecticidal activity can be detected at the cellular level by vitro cytotoxicity screening. To explore a high-efficiency and viable method for the screening of plant compounds with insecticidal activity, we screened for 34 active ingredients in the insecticidal plant, Oroxylum indicum L. Vent, using Sf9 cells. Among the main compounds identified, lapachol, chrysin, and baicalein had good proliferation inhibitory effects on Sf9 cells. Apoptosis assay further showed that lapachol promoted the production of caspase-3 and led to DNA fragmentation in Sf9 cells. Meanwhile, lapachol showed high biological activity against Aphis gossypii, Sitobion avenae, and Semiaphis heraclei. Our results showed that high-throughput screening of active ingredients in the insecticidal plant O. indicum using Sf9 cells is feasible, and the identifcation of lapachol as the main aphidicidal active substance is valuable for further study.
Yujing Zhao, Jingjie An, Zhihong Dang, Jianglong Guo, Zhanlin Gao, Shujie Ma, and Yaofa Li. Identification of highly active compounds from insecticidal plant Oroxylum indicum L. (Vent.) and the induction of apoptosis by lapachol on Sf9 cells. In Vitro Cell. Dev. Biol.- Animal, 59, 674 – 683(2023).
