No abstract

A simple, rapid clearing and staining method was developed using intact in vitro blackberry (Rubus spp. ‘Silvan’) and strawberry (Fragaria × ananassa Duch. ‘Totem’) plantlets and sections of greenhouse-grown ‘Silvan’ and ‘Totem’ leaves. The clearing method involved three steps: 1) autoclaving in 80% ethanol to remove the chlorophyll, which took 15 to 20 min for plantlets and 25 and 35 min for greenhouse-grown ‘Silvan’ and ‘Totem’, respectively; 2) dissolution of the protoplasm using 5% NaOH at 80°C, which took 20 min for plantlets and 35 and 40 min for greenhouse-grown ‘Silvan’ and ‘Totem’ respectively; 3) post-alkali treatment with 75% bleach (4.5% NaClO). For tissue-cultured plantlets this took 5 to 10 min at room temperature, but greenhouse-grown material required 35 to 40 min at 55°. Aqueous safranin (10 mg·liter−1) was used for staining. This method gave consistently good results and required a maximum of 2 hr for completion.