Pradhan Laxmipriya, Singh Priya, Maurya Shiv Kumar, Sane Aniruddha P
Plant Gene Expression Lab, CSIR-National Botanical Research Institute, Lucknow, India.
Methods Mol Biol. 2025;2916:163-177. doi: 10.1007/978-1-0716-4470-6_15.
Isolation of petal abscission zones (AZs) for molecular and biochemical studies is challenging due to their small size and the difficulty in separating them cleanly from surrounding non-AZ tissues, followed by the rapid exposure of the excised tissue to wound and dehydration stresses, post-excision. Immediate freezing of all the excised tissue is essential but practically difficult. In this chapter, we present a refined protocol for isolating petal AZ tissues from rose petals by optimizing the dissection process to minimize contamination with non-AZ cells coupled with immediate freezing to prevent stress-induced artifacts. The protocol includes careful tissue handling, precise excision, and rapid freezing, enabling the acquisition of high-quality samples for subsequent molecular and biochemical analyses. We also provide a method that facilitates the study of promoters by GUS expression through agro-injection of rose flower buds.
由于花瓣脱落区（AZs）尺寸小，且难以将其与周围非脱落区组织清晰分离，再加上切除后离体组织会迅速受到伤口和脱水胁迫，因此分离花瓣脱落区用于分子和生化研究具有挑战性。立即冷冻所有切除的组织至关重要，但实际操作中很难做到。在本章中，我们提出了一种优化的方案，通过优化解剖过程以尽量减少非脱落区细胞的污染，并立即冷冻以防止应激诱导的假象，从而从玫瑰花瓣中分离花瓣脱落区组织。该方案包括小心处理组织、精确切除和快速冷冻，能够获取高质量的样本用于后续的分子和生化分析。我们还提供了一种通过农杆菌注射玫瑰花蕾来利用GUS表达促进启动子研究的方法。
