Abstract
The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%; with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h; considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h; meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.
Highlights
IntroductionNagata and Takeble first reported tobacco mesophyll protoplast isolated, cultured and regenerated plants obtained in 1970 [5]
Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD), an allohexaploid species, is the principal food crop used for humans and livestock globally
With the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%; with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h; considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h; with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min
Summary
Nagata and Takeble first reported tobacco mesophyll protoplast isolated, cultured and regenerated plants obtained in 1970 [5]. Protoplasts removing the cell wall and including the protoplasm and plasma membrane are the living material of a plant or bacterial cell. Due to the absent of cell wall, plant protoplasts have been widely used for genetic transformation, cell fusion and somatic mutation to generate unique and novel plants, which result in the requiring of large quantities of high activity protoplasts [8] [9] [10] [11] [12]. This paper made a preliminary study on the preparing conditions of wheat protoplasts, which can lay the foundation for genetic programs and breeding of new varieties
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