Plant Science

Based on the availability of oxygen, plant growth environment can be normoxic (normal environment), hypoxic (reduced oxygen, <21%), or anoxic (complete depletion of oxygen). Hypoxic/anoxic environment is created when a plant is exposed to stresses such as submergence, flooding, or pathogen attack. Survival of the plants following stress conditions is in part dependent on their ability to overcome the stress induced by anoxia/hypoxia conditions. This shows the need for the development of strategies for understanding the mechanisms involved in plant tolerance to anoxia. Previous studies have employed different methods for establishing an anerobic environment. Here, we describe a simple method for creating anoxic environment using an anaerobic atmosphere generation bag. Anoxic conditions can be maintained in a cylindrical jar, a rectangular box, or a vacuum sealer bag, enabling the screening of a large number of samples. This protocol is particularly useful to screen plant mutants that are tolerant to anoxia. The method is simple, easy, cost-efficient, reproducible, and does not require any sophisticated instruments.

Graphic abstract

Spores are the primary way of spread and reproduction for ferns, a clade of seed-free vascular plants. However, no detailed protocol for ferns spore cultivation has been reported yet. Here we provide a modified approach for axenic cultivation of fern Adiantum capillus-veneris L., based on Cao’s and Li’s method (Cao, et al., 2010; Li, et al., 2013).

Our approach can be briefly divided into four steps: 1) collect spores; 2) sterilize the spores with 5% sodium hypochlorite solution and wash twice; 3) incubate the spores in liquid Knop’s medium in the dark for five days; 4) cultivate the spores on Knop's plate medium. To increase the germination rate, we constrain the sterilization time under 25 min and add dark treatment step after spore sterilization. After these modifications, the germination rate raises from 2% to 25%.