摘要
为了准确观测龙牙百合鳞茎生长点的生长分化过程,以龙牙百合冷藏期间的鳞茎为试验材料,在传统石蜡制片法的基础上,对脱水、透明等步骤进行优化并筛选了最佳的甲苯胺蓝染色方法。试验获得的百合鳞茎生长点石蜡切片制作改良方法为:将百合鳞茎剥开鳞片至可观察到茎尖生长点,修整为5mm大小的立方体投入FAA固定液中,保存一周;用85%乙醇(2 h)、95%乙醇(2h)、100%乙醇(3h)进行脱水处理,再用二甲苯与乙醇等比例溶液(2h)、纯二甲苯(3h)进行透明处理;将透明好的材料放入石蜡+二甲苯溶液(体积比2∶3)中,在37℃的恒温箱中浸蜡2d,期间多次少量加入石蜡;之后在60℃恒温箱更换纯石蜡2次,每次4 h,浸蜡后的材料在60℃的操作台上包埋,再切成厚10μm的薄片,待蜡片完全伸展,将蜡片粘于均匀涂抹了赫伯特粘片剂的载玻片上,37℃烤片5h,脱蜡复水后用1%甲苯胺蓝染液稀释150倍染色3min。
To accurately observe the growth and differentiation process of the growing point of Lilium brownii var.viridulum Baker bulb,this study employed the conventional method to prepare the paraffin sections of the bulbs stored in the refrigerator.The dehydration and transparentizing steps were optimized and the toluidine blue staining method was selected.The conditions for preparing the paraffin sections were improved as follows.First,the scales of a lily bulb were peeled off to expose the growth point,which was then trimmed into a cube of 5 mm×5 mm×5 mm.The cube was fixed in FAA solution for one week,dehydrated with 85%ethanol for 2 h,95%ethanol for 2 h,and 100%ethanol for 3 h,and then transparentized with the mixture of xylene and ethanol(1:1)for 2 h and pure xylene for 3 h.The transparent cube was then immersed in a mixture of paraffin and xylene(volume ratio of 2:3)at 37°C for 2 days,during which a small amount of paraffin was supplemented multiple times.Afterwards,the mixture medium was replaced with pure paraffin in a thermostat incubator at 60°C twice,each time for 4 h.After that,the material was embedded at 60°C.The wax block was sliced into pieces with a thickness of 10μm.After full extension,each slice was put onto a glass slide evenly coated with Herbert gelatin adhesive,and the slide was then heated at 37°C for 5 h.After dewaxing and rehydration,the slide was stained with 1%toluidine blue staining solution diluted by 150 times for 3 min.
作者
杨林澔
陈淼
李庠
马杰
李玉帆
YANG Lin-hao;CHEN Miao;LI Xiang;MA Jie;LI Yu-fan(College of Horticulture,Hunan Agricultural University,Changsha 410128,PRC;Hunan Engineering Research Center for the Breeding and Utilization of High-Quality Flowers and Trees in the Mid-Subtropical Zone,Changsha 410128,PRC;Hunan Cotton Science Institute,Changde 415000,PRC)
出处
《湖南农业科学》
2024年第6期10-13,20,共5页
Hunan Agricultural Sciences
基金
湖南省科学技术厅种业创新项目(2021NK1005)
湖南省自然基金项目(2023JJ30292)。
关键词
龙牙百合
鳞茎生长点
石蜡切片
技术改良
Lilium brownii var.viridulum Baker
growing point of bulb
paraffin section
technical improvement
