坛紫菜叶状体的无菌化培养及其应用

对坛紫菜叶状体的无菌处理方法进行了优化,并利用紫菜外生菌对无菌处理后的紫菜叶片进行了人工感染。经0.7%KI和0.1%(w/v)氨苄青霉素对紫菜叶片进行预处理后,利用氨苄青霉素、硫酸链霉素、新霉素、庆大霉素及卡那霉素等5种抗生素及其不同组合对紫菜叶片进行处理,筛选出最佳抗生素组合、浓度和培养条件。结果表明:氨苄青霉素(终浓度300μg/mL)、卡那霉素(终浓度100μg/mL)与庆大霉素(终浓度100μg/mL)3种抗生素组合对坛紫菜叶状体进行无菌处理18 h,对紫菜细胞的毒害较小,并且3种抗生素合用对89.5%紫菜外生细菌的抑菌率达80%以上;外生菌感染结果研究表明:用105/mL真菌孢子液和细菌菌液回染紫菜,培养22 d后,实验组紫菜生长状况较对照组好,对照组紫菜出现褪色。用108/mL真菌孢子液分别回染健康紫菜、穿刺紫菜(用灭菌刀片在紫菜表面划1～2 mm的伤口),分别在18℃、28℃和35℃条件下培养。实验结果表明28℃和35℃高温和穿刺均会影响无菌紫菜健康生长,且加菌紫菜在高温和穿刺条件下,则会出现明显病斑,而18℃培养的加菌紫菜则生长良好。

Study on the axenic culture and application of Porphyra haitanensis thallus

The sterile processing of Porphyra haitanensis thallus was optimized. The artificial infection of thallus by epiphytic microorganisms from Porphyra was studied. The thalli of P. haitanensis were pretreated by 0. 7% KI and 0. 1% （w/v）ampicillin firstly to remove the outside organisms. Five antibiotics, such as ampicillin, streptomycin, neomycin, kanamycin and gentamicin, were applied independently or combinationally to remove the epiphytic microorganisms from the thallus of Porphyra. The appropriate combination, concentration and culture conditions were optimized. The cells of P. haitanensis thallus were minimum affected under the treatment of the combination of ampicillin （final concentration 300μg/mL）, kanamyein （final concentration 100μg/mL） and gentamicin （final concentration 100μg/mL） for 18 hours, and the inh!bitory rate of 89. 5% epiphytic microorganisms from Porphyra was more than 80%. The sterilized Porphyra thallus was infected by 105/mL fungal spores and bacteria suspensions. Tile results showed that the infected thallus grew well with the microorganisms, while the sterilized control thallus had eolour fading after they were cultured for 22 days. The sterilized healthy thallus were cut into 1 -2mm scratches on the surface and infected by the fungal spores suspensions under the concentration of 10^8/mL, then cultured at 18℃, 28℃ and 35℃, respectively. The cultured thalli were in bad condition and rotted obviously under both 28℃ and 35℃ and puncture treatments, while the P. haitanensis thallus cultured at 18℃ grew well even after being co-cultured with microorganisms.