关键词: 马铃薯, 花粉原生质体, 分离

Abstract: Mature pollen protoplasts of Solanum tuberosum were isolated by means of "cold pretreatment- germination-enzy- matic treatment". The buds were first incubated at 6℃, then mature pollens were germinated. When most of the pollens grains had just produced short pollen tubes, they were transferred to an enzymatic solution. The process of pollen protoplasts isolation was as follows: firstly, plasm membrance seperated from the cell wall, then the protoplasts inside pollen wall were partially extruded through the germinal hole after the action of enzyme solution, and finally the spherical protoplasts were detached from the exine of pollens. Pollen protoplasts yield was greatly influenced by the germination duration time and mannitol concentrations in enzymatic solution, and 30 min germination and 1 mol·L-1 mannitol were optimal. The enzymatic solution consists of 1% cellulase (R-10), 0.5% hemicellulase (Sigma), 1% pectinlase (Serva), 0.5% potassium dextran sulfate, 1% PVP, 0.01% CH and K3 medium. The highest pollen protoplasts yield was 19.40%. Wall degraded pollens were identified with 0.1% calcoflower white test and its vitality was tested by fluorochromatic reaction with fluorescein diacetate (FDA).

Key words: Solanum tuberosum L, Pollen protoplasts, Isolation