An improved technique for clearing and staining plant tissues for detection of nematodes.
An improved technique for clearing and staining plant tissues for detection of nematodes.
复制标题
DOI：
--
复制
发表时间：
1983-01
影响因子：
1.3
通讯作者：
D. W. Bybd;T. Kirkpatrick;K. Barker
中科院分区：
文献类型：
--
作者：
D. W. Bybd;T. Kirkpatrick;K. Barker
许多程序已被用于染色和清除线虫感染的植物组织（7）。酸性品红染色和乳酚（4，7）脱色是最广泛使用的技术。更快速的清除和染色根的方法包括高压灭菌（1），暴露于饱和水合氯醛（6），染色后根浸软（3）。但是，这些方法需要非常小心。与菌根ftmgi一起工作的研究人员发现氢氧化钾（KOH），硝酸，过氧化氢（H2 O2）或次氯酸钠（NaOC 1）可以有效地清除根（2，5），这取决于要清除的组织类型。碱性H：O.，对色素根的漂白效果最好（5）。用NaOCl过度漂白通常导致组织染色不良。本文描述了利用NaOC 1作为线虫感染的植物组织的预染色剂的改良的酸性品红染色-脱色植物细胞。清洗根，切成1-2-cm的段，将其置于装有50 ml自来水的150-ml烧杯中。加入20毫升含氯漂白剂（5.25%/1.0%NaOC 1），得到1.5%NaOC 1，并使根组织片在该溶液中保留4小时，偶尔有a~+离子。在NaOCl处理后，将根段在流水中冲洗（30-45秒），并使其在自来水中浸泡15分钟以除去残留的NaOCl。然后将材料排干并转移到含有30 ml水的烧杯中，向其中加入1 ml染色剂（3.5 g酸性品红、250 ml乙酸和750 ml蒸馏水）。然后将该溶液加热至沸腾约30秒。冷却至室温后，通过在流水中冲洗来去除多余的污渍。然后将根材料置于20-30 ml用几滴5 N HCl酸化的甘油中，加热至沸腾，并
Numerous procedures have been used to stain and clear nematode-infected plant tissues (7). Staining with acid fuchsin and destaining with lactophenol (4,7) has been the most widely used technique. More rap id means, of clearing and staining roots include autoclaving (1), exposure to saturated chloral hydrate (6), and root macera t ion after staining (3). However, extreme care is required for these methods. Researchers working with mycorrhizal ftmgi found potassium hydroxide (KOH), nitric acid, hydrogen peroxide (H202), or sodium hypochlorite (NaOC1) to be effective in clearing roots (2,5), depending on the type of tissue to be cleared. Alkal ine H:O., was the best bleaching agent for pigmented roots (5). Excessive bleaching with NaOCI often resulted in poor staining of tissues. A modified acid-fuchsin staining-destaining wocedure utilizing NaOC1 as a prestaining t rea tment for nematode-infected plant tissues is described herein. Roots are washed, chopped into 1-2-cm segments, aml placed in a 150-ml beaker with 50 ml tap water. Twen t y milli l i ters of chlorine bleach (5.25,.o/.0 NaOC1) are added to give 1.5% NaOC1, and the root tissue pieces are allowed to remain in this solution for 4 ntiu with occasional a~ ta t ion . Following the NaOCI treatment, the root segments are rinsed in runn ing water (30-45 sec) and allowed to soak in tap water for 15 min to remove residual NaOC1. T h e material is then drained and t ransferred to a beaker containing 30 ml of water to which has been added 1 ml of stain (3.5 g acid fuchsin, 250 ml acetic acid, and 750 ml distilled water). Th i s solution is then heated to boil ing for about 30 sec. After cooling to room temperature , excess stain is removed by rinsing in runn ing water. T h e root, mater ia l is then placed in 20-30 ml of glycerin acidified with a few drops of 5N HC1, heated to boiling, and