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槲树不同发育时期胚珠石蜡切片制备体系的优化
祁永梁1, 李亚鹏1, 李世安,等2
1.北京林业大学 生物科学与技术学院,林木遗传育种全国重点实验室,国家林业和草原局刺槐工程技术研究中心,林木育种与生态修复国家工程研究中心;2.北京市绿地养护管理事务中心
摘要:
【目的】建立并优化适宜槲树子房或胚珠石蜡切片的制作体系,为后续槲树胚胎发育学研究提供技术支撑。【方法】以不同发育时期的槲树雌花为试验材料,将材料以60 d为界分为60 d前和60 d后两部分,对材料处理方式(授粉60 d前后的材料分别进行仅保留子房与胚珠处理)、浸蜡时间(18,24,36 h)与浸蜡条件(60 ℃烘箱内抽真空处理0,6,8,12 h)、切片厚度(6,8,10 μm)进行优化;在此基础上,以授粉70 d样品切片为材料,对染色方法(染色液分别为5 g/L苏木精、1 g/L甲苯胺蓝和10 g/L番红 固绿)及染色时间(1,2,5,10,15 min)进行优化,建立适合槲树胚珠的石蜡切片制备体系。【结果】授粉60 d前的样品仅保留子房部分,授粉60 d后的样品仅保留胚珠,两者在50 ℃烘箱经甘油乙醇浸泡48 h的软化处理后,所得切片效果最佳,可避免蜡带破裂等不良现象出现。将浸蜡时间延长至36 h,并于第1次浸纯蜡过程中在60 ℃烘箱内先进行12 h抽真空处理,可保证发育后期体积较大材料的充分浸蜡;脱蜡前将玻片进行3 h抽真空处理,可明显降低脱片率;授粉60 d前样品的切片厚度以8 μm为宜,授粉60 d及以后的样品以10 μm最佳。槲树胚株石蜡切片制备时,采用5 g/L苏木精染色5 min时的制片效果最好。【结论】建立并优化了槲树不同发育时期子房或胚珠石蜡切片的制备体系,采用该优化体系可获得蜡带连续、结构完整、染色均匀、背景清晰的槲树子房或胚珠切片。
关键词:  槲树胚珠;胚胎发育  石蜡切片;制备体系
DOI:
分类号:
基金项目:国家重点研发计划项目(2021YFD2200302-02);北京林业大学发展规划处专项(2018FGC03,2021YL06)
Optimizing preparation system of ovules paraffin sections of Quercus dentata Thumb. in different development stages
QI Yongliang,LI Yapeng,LI Shian,et al
Abstract:
【Objective】A system suitable for preparing paraffin sections of ovary or ovules of Quercus dentata Thumb. was established and optimized to provide support for subsequent studies on its embryo development.【Method】Female flowers of Quercus dentata Thumb. at different development stages were divided into two parts of within and after 60 d of pollination.Different material treatment methods (retaining ovary or ovule),wax dipping times (18,24 and 36 h),wax dipping conditions (vacuum treatments in a 60 ℃ oven for 0,6,8 and 12 h ) and section thicknesses (6,8 and 10 μm ) were analyzed and optimized.Using sections 70 d after pollination,staining methods (5 g/L haematoxylin,1 g/L toluidine blue and 10 g/L safranine solid green) and staining times (1,2,5,10 and 15 min) were also optimized.【Result】The ovary retained within 60 d after pollination and ovules retained more than 60 d after pollination had the best sections after being softened by soaking in a glycerol-ethanol softener in an oven at 50 ℃ for 48 h.The sections also avoided undesirable conditions such as the rupture of wax bands.When wax dipping time was extended to 36 h with 12 h evacuation in oven at 60 ℃ during the first dipping of pure wax,materials with large volumes at later development stages could be adequately dipped in wax.Vacuum treatment for 3 hours on glass slides before dewaxing significantly reduced the detachment rate.Section thickness of 8 μm was preferred for samples within 60 d after pollination,while 10 μm was best for samples 60 d after pollination.The best staining effect was achieved using 5 g/L hematoxylin for 5 min.【Conclusion】An preparation system was developed and optimized for paraffin sections preparation of ovary or ovules of Quercus dentata Thumb. at different development stages.The system obtained sections with continuous wax bands,structural integrity,uniform staining and clear background.
Key words:  Ovules of Quercus dentata Thumb.  embryonic development  paraffin section  preparation system