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Outline
To establish an efficient protocol for isolating high-quality protoplasts from barley leaf epidermal tissue, thereby providing technical support for studies on stomatal development, gene functional validation, and molecular breeding for stress resistance, this study used leaves of barley (Hordeum vulgare ‘Golden Promise’) at the three-leaf stage as experimental material. An orthogonal experimental design was employed to systematically optimize key parameters, including the concentrations of Cellulase R-10 and Macerozyme R-10, digestion time, and osmotic pressure. The results showed that enzyme concentration and digestion time significantly affected protoplast yield and morphology. The low-concentration enzyme combination (1.5% Cellulase R-10+0.75% Macerozyme R-10) failed to effectively release protoplasts. Increased enzyme concentrations (2% Cellulase R-10+1% Macerozyme R-10) improved yield, but levels remained relatively low. When the Macerozyme R-10 concentration was increased to 1.5% combined with a 6 h digestion, cell wall degradation was most complete, leading to significantly enhanced yield and improved morphological integrity. Osmotic pressure optimization revealed that increasing the mannitol concentration from 54.7 g·kg-1 to 98.4 g·kg-1 (resulting in an enzyme solution osmolality slightly higher than the leaf tissue osmotic potential of 630 mmol·kg-1) increased protoplast yield from 4.8×105 cells·mL-1 to 5.8×105 cells·mL-1. Furthermore, protoplast morphology transitioned from irregular shapes with debris to intact, uniformly spherical forms. The optimal conditions were determined to be 2% Cellulase R-10, 1.5% Macerozyme R-10, a 6 h digestion time, and a mannitol concentration of 98.4 g·kg-1, under which the protoplast yield was highest and the protoplasts were intact and uniformly spherical. This system is not only applicable to the lower epidermis of barley leaves but is also highly efficient for mesophyll tissue, providing reliable technical support for related cell biology and molecular biology research.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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