Lab 01-carrot - bleach solution 5 Rinse the carrots three times with sterile water(100 – 150ml covering the sections and stirring for 1-2 min

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
Lab 1 Establishment and Maintenance of Carrot Callus Objective: Students will sterilize carrot tissue and establish it in tissue culture Protocol: 1. In the lab, rinse a carrot root under running tap water to remove any surface soil. Make sure the root is undamaged; particularly that it doesn’t have any large cracks. Cut off the top and bottom on the carrot and peel off the epidermis with a peeler. Trim the carrot into 6 - 10mm sections and place them into a beaker in the flow hood. – The carrot has already be sliced up for you. 2. You will be given carrot root slices that have been soaked in 2.5mg/L 2,4-D for 24 hr. Treat them as follows, the same as the untreated slices, but keep them separate. 3. Cover the carrot sections with about 150ml 70% EtOH and sitr for 5 minutes. Pour off the EtOH into a waste beaker. 4. Immediately cover the carrot sections with about 150ml of a 20% bleach solution + a drop of Tween 20 (a surfactant) for approximately 20 min, then decant off the
Background image of page 1
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: bleach solution. 5. Rinse the carrots three times with sterile water (100 – 150ml), covering the sections and stirring for 1-2 min each time. 6. Transfer a section into a sterile dish. Using scalpel and forceps, cut small (5mm square) pieces of root, making sure to include the cambium (the innermost ‘ring’ of the root). Sterilize Instruments often. 7. Place 4 explants in each of the provided Petri plates. You will be given two types of plates - containing MS medium and MS medium + 2,4-D (prepared on 1/5). Prepare one of each media type for each root treatment. 8. Place 4 of the slices into a Petri plate with sterile filter paper which has been moistened with sterile distilled water. 9. Seal each dish with parafilm, label and incubate in the dark. 10. After 4 weeks, subculture the callus onto the same medium, discarding necrotic tissue. 11. After another 4 weeks, transfer all tissue to hormone-free medium (MS media without 2,4-D)....
View Full Document

This note was uploaded on 07/23/2011 for the course HOS 6737c taught by Professor Moore during the Spring '09 term at University of Florida.

Ask a homework question - tutors are online