A submerged culture system for rapid micropropagation of an ornamental aquarium plant ‘Amazon sword’

Members of Plant Biotech Lab, Post Graduate Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata 700118, India. From Left to Right (front row): Dr. SK Moquammel Haque (Post Doc. Student), Dr. Biswajit Ghosh (Associate Professor, Head of the Lab), From Left to Right (back row): Anupam Das, Tarun Halder, Md. Mabud Alam, Avijit Chakraborty & Indranil Santra (all are Ph.D. Students).

 

The production of aquarium plants via tissue culture has emerged as a rising industry all over the world. Amazon sword plant (Echinodorus ‘Indian Red’) is an underwater aquatic plant, very popular among aquarium hobbyists worldwide, and currently retails in the online market for $4.59–$14.95 per plant. An efficient method for in vitro propagation and plantlet acclimatization of this ornamental aquarium plant was developed. In vitro plants were cultured in submerged conditions in a solid-liquid bilayer medium, the culture-vessels containing plant implanted in the solid medium were further filled with water to maintain the submerged aquatic environments. Another important finding that the number of multiplied shoots increased with each consecutive regeneration cycle, from only 26.8 shoots per explant in the 1st cycle to 33.5 in 2nd and finally to 38.3 in 3rd regeneration cycle with the same medium composition. All earlier reports mentioned the tissue culture of aquatic plants used a solid culture medium, thereby providing terrestrial environments to those in vitro plants. As per our knowledge, the present study appears to be the first standardized to culture aquatic plants in their natural submerged aquatic environment. Along with the advantage of providing aquatic environments, this submerged system which also helps for easy detection of contamination in culture.  The regenerated plantlets were acclimatized within the waterbody of the aquarium with a 100% survival rate. Present submerged culture system is also suitable for the commercial propagation purpose of ‘Amazon sword’ and related aquatic plants those are widely used in aquarium-industries.

SK Moquammel Haque, Biswajit GhoshA submerged culture system for rapid micropropagation of the commercially important aquarium plant, ‘Amazon sword’ (Echinodorus ‘Indian Red’. In Vitro Cellular & Developmental Biology–Plant, 55: 81–87, 2019.


Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes

Members of Prof. Otoi’s laboratory at Tokushima University (Tokushima, Japan). From left: Ms. Shiho Nii, Ms. Quynh Anh Le, Ms. Nhien Thi Nguyen, Dr. Takeshige Otoi, Dr. Maki Hirata, and Dr. Fuminori Tanihara.

Pigs have gained considerable importance in biomedical research because they are similar to humans in several respects and genetically modified pigs can be used as highly favorable biomedical models for intractable human diseases. In a previous study, we established the GEEP (gene editing by electroporation of Cas9 protein) method, a simple method for CRISPR/Cas9 gene editing that requires the introduction of Cas9 protein and single-guide RNA (sgRNA) into in vitro fertilized zygotes by electroporation, to generate high-efficiency disruption of the targeted gene. However, mosaic offspring composed of more than 1 genetically distinct population of cells remains a challenge for genetically engineered animal production by gene editors. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine matured oocytes and putative zygotes. Our results demonstrate that the matured oocytes have greater sensitivity to the electrical pulse number than that of zygotes. Moreover, the type of eggs may influence the mutation rate in the resulting blastocysts. To ascertain the best model for porcine transgenesis, additional investigation of target mutations after electroporation with the CRISPR/Cas9 system is further required.

Maki Hirata, Fuminori Tanihara, Manita Wittayarat, Takayuki Hirano, Nhien Thi Nguyen, Quynh Anh Le, Zhao Namula, Masahiro Nii, Takeshige Otoi. Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes. In Vitro Cellular & Developmental Biology-Animal, 55:237-242, 2019.