摘要
【目的】对海金沙孢子无菌萌发与原叶体生长条件进行优化,为海金沙快繁体系建立及规模化种植提供理论依据。【方法】以海金沙孢子为试验材料,设不同孢子成熟度、NaClO溶液浓度与消毒时间组合、培养基无机盐含量、培养基琼脂浓度、培养基pH、接种孢子混悬液浓度、培养温度、暗处理时间、赤霉素(GA3)处理浓度,测定孢子萌发率和原叶体面积,筛选最适培养条件。【结果】同一孢子叶上的孢子成熟度不同,越早成熟散落的孢子萌发率越高,1~3 d散落的孢子培养15 d后最终萌发率达38.4%,显著高于其他时间段散落的孢子(P<0.05,下同),最佳的海金沙繁殖材料为孢子叶采收后1~3 d内自然散落的成熟孢子。不同NaClO溶液浓度与消毒时间处理的孢子在培养15 d内均未出现污染,但3%和5%的高浓度NaClO溶液消毒会抑制孢子萌发和原叶体生长,最适消毒方式为1%NaClO溶液消毒4 min。无机盐含量较低的Knop’s培养基有利于孢子萌发和原叶体生长;培养基琼脂浓度为7.5 g/L时,培养15 d后孢子最终萌发率和原叶体面积均显著高于8.5和9.5 g/L琼脂浓度;培养基为p H 5.8时孢子最终萌发率和原叶体面积均显著高于pH 5.4及pH 6.2;最适培养基配方为Knop’s培养基添加7.5 g/L琼脂和20.0 g/L蔗糖并调节pH为5.8。最佳的孢子接种步骤为采用1.0 mg/mL浓度孢子混悬液接种0.5 mL。最适培养温度为30℃恒温,培养3 d即启动萌发,培养15 d的最终萌发率可达52.5%,高于25℃恒温和20℃/30℃变温培养,原叶体面积也最大。暗处理15 d的孢子最终萌发率显著高于未经暗处理(0 d),但可能不符合大规模繁殖的效率要求。添加0.01 mg/L浓度GA3处理的孢子最终萌发率显著高于未经GA3处理(0 mg/L),但GA3浓度达100.00 mg/L时孢子最终萌发率显著低于未经GA3处理。【结论】筛选获得了海金沙孢子无菌萌发的最适培养条件,该培养条件也有利于原叶体的早期生长。
【Objective】The objectives of this study were to optimize the aseptic spore germination and the prothallus growth conditions of Lygodium japonicum,which could provide theoretical basis for the establishment of the rapid propa‐gation system and large-scale planting of Lygodium japonicum.【Method】Using Lygodium japonicum spores as experimen‐tal materials,the effects of different spore maturity levels,NaClO solution concentrations and sterilization durations,me‐dium inorganic salt content,agar concentration,pH value of medium,inoculation concentration of spore suspension,cul‐ture temperature,dark treatment duration,and gibberellin(GA3)concentrations were tested.Spore germination percen tage and prothallus area were measured to identify optimal culture conditions.【Result】Spores from the same sporophyll exhibited varying maturity levels,with earlier mature and dispersed spores having higher germination percentage.Spores dispersed within 1-3 d achieved a final germination percentage of 38.4%after 15 d of culture,significantly higher than those dispersed later(P<0.05,the same below).The best reproductive material was mature spores naturally dispersed within 1-3 d after sporophyll harvest.The spores treated with different NaClO solution concentrations and disinfection durations were not contaminated within 15 d of culture,but 3%and 5%high-concentration NaClO inhibited spore germi‐nation and prothallus growth.The optimal sterilization method was 1%NaClO solution soaking for 4 min.Knop’s me‐dium with lower inorganic salt content promoted spore germination and prothallus growth.The final germination percentage and prothallus area after 15 d cultured at 7.5 g/L agar concentration were significantly higher than those at 8.5 and 9.5 g/L.The final germination percentage and prothallus area spore in the medium with pH 5.8 were significantly higher than those in the media with pH 5.4 and pH 6.2.The optimal medium formula was Knop’s medium with 7.5 g/L agar,20.0 g/L sucrose,and pH value adjusted to 5.8.The optimal spore inoculation procedure was to inoculate 0.5 mL of spore suspen‐sion at a concentration of 1.0 mg/mL.The optimal culture temperature was 30℃constant temperature,initiating germina‐tion on 3 d of culture and achieving a final germination percentage of 52.5%on 15 d of culture,which was higher than constant 25℃and 20℃/30℃alternating temperatures,prothallus area was also the largest.The final germination per‐centage of spores under dark treatment for 15 d was significantly higher than that without dark treatment(0 d),but it might not meet the efficiency requirements for large-scale reproduction.The final germination percentage of spores treated with GA3 concentration of 0.01 mg/L was significantly higher than that without GA3 treatment(0 mg/L),but when the GA3 concentration reached 100.00 mg/L,the final germination percentage of spores was significantly lower than that without GA3 treatment.【Conclusion】Optimal aseptic germination conditions for Lygodium japonicum spores are selected,which also favor early prothallus growth.
作者
黄满霖
刘薇
童家赟
詹若挺
刘赛
HUANG Man-lin;LIU Wei;TONG Jia-yun;ZHAN Ruo-ting;LIU Sai(School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou,Guangdong 510006,China;Dongguan Institute of Guangzhou University of Chinese Medicine,Dongguan,Guangdong 523808,China;Key Laboratory of Chinese Medicinal Resource from Lingnan,Ministry of Education(Guangzhou University of Chinese Medicine),Guangzhou,Guangdong 510006,China;Dongguan Asia Pharmaceutical Technology Co.,Ltd.,Dongguan,Guangdong 523160,China)
出处
《南方农业学报》
北大核心
2025年第4期1224-1234,共11页
Journal of Southern Agriculture
基金
国家自然科学基金项目(32170400)
广东省乡村振兴战略专项资金种业振兴项目(2022-NJS-00-002)
东莞市科技特派员项目(2022441920000014)。
关键词
海金沙
孢子萌发
原叶体生长
无菌培养
Lygodium japonicum
spore germination
prothallus growth
aseptic culture
