Background:

Cauliflower (brassica oleracea) is a member of the mustard family and a long-domesticated food crop. The vegetable is grown for its massive, edible flower structures. Cauliflower is rich in several vitamins and nutrients including Vitamins C and K. They are an annual, cool weather crop that thrive best in temperatures of around 60 degrees Fahrenheit. The plants can reach 1.5 feet tall and have large, round leaves. White flowers are the most commonly grow commercially, but purple, orange, green, and brown cultivars exist as well.  

Cauliflower is also an easy plant to reproduce through tissue culture. Tissue culture is defined by the growth of cells derived from living tissue in an artificial medium. The technique has been used to produce vaccines, viral inhibitors, and most importantly, the commercial production of plants.  

Cauliflower is an ideal plant to use to demonstrate tissue culture for several reasons. First, its rapid growth allows the entire tissue culture growth process to be completed in 6 weeks. Second, growth requirements for cauliflower are minimal. Third, cauliflower is cheap and easy to obtain from supermarket. Additionally, its capable of withstanding a thorough sterilization treatment, reducing the loss of explant material to contamination during the preparation process. The cauliflower used should be as fresh as possible and contain no marks, wounds, or discoloration.  

 

Materials:

• Cauliflower flower sections.  
• Prepared Murashige & Skoog (MS) Medium. 
• Sterile petri plates containing around 25-30 milliliters of culture medium.  
• 1 sterile petri plate to use as a stage for cutting tissue.  
• Sterile boxes for rinsing and containing clean tissue. 
• Sterile water for rinse.  
• Sterile Tools: 
  • 1 scalpel.  
  • 1 tweezers (forceps). 
  • Tool rack.
• Disinfectants: 
  • 95% alcohol.  
  • 10% Clorox solution.  
  • Antimicrobial cleaner.  
• Bacticinerator.  
• Parafilm to wrap finished plates.

 

Sanitation Tips and Requirements:

The sanitation of the cauliflower, tools, and growth environment cannot be stressed enough. The sterility of the environment is the ultimate deciding factor in the success of the tissue culture explants. All tools should be sterilized in either an autoclave or a small machine called a Bacticinerator. A Bacticinerator is a small machine that sterilizes small tool such as scalpels, tweezers, and forceps by heating them up to high temperatures.  

Example of a Bacticinerator:

https://www.carolina.com/microbiology-supplies/bacti-cinerator-iv/FAM_703420.pr

In addition, the growth environment (countertops, tables, etc…) should be disinfected before the experiment. When preparing the explant, preparation should be done in a laminar flow hood. All jewelry and watches should be removed, long hair tied back, and hands washed up to the elbows. The flow hood generates a stream of air that flows outwards away from the hood reducing the risk of air-borne pathogens contaminating the explants or media.  

Details on how explants must be sterilized can be found in the “Preparation of Explant” section below.  

 

Media:

The most widely used medium is called the Murashige & Skoog (MS) basal medium. The MS medium should be prepared so that it has enough time to gel before performing the tissue culture propagation. It generally consists of a mixture of common macro- and micro- nutrients, sucrose, vitamins, hormones including Benzyladenine (BA), and Naphthalene acetic acid (NAA), and contains an agar base. The MS medium often comes premixed which saves both a tremendous amount of time as well as keeping track of an extensive inventory of media components. Directions are included with the purchase. The medium’s pH should be adjusted to 5.7 and autoclaved for 15 minutes before being poured into petri plates and allowed to gel.  

Murashige & Skoog Media:

https://phytotechlab.com/murashige-skoog-modified-basal-medium-m401.html

 

Preparation of Explant:

1) Selection:  

Select enough cauliflower flower pieces to make 3 petri dishes worth. Choose pieces that are healthy and not diseased or bruised.  

2) Disinfection: 

Wash all pieces in a solution containing water and several drops of soap. Drain the soapy water from the container. Replace soap solution with a solution made of 675 mL of distilled water and 75 mL 10% Clorox bleach. Take cauliflower pieces in disinfectant solution down to Laminar Hood room. Leave cauliflower tissues in solution for 15 minutes. (Be careful. Don’t leave tissues in disinfectant solution for too long or too short. Set a timer on phone or watch.)  

3) Personal Cleanliness: 

In the room with the laminar flow hoods:  

• Remove all jewelry and watches.  
• Wash your hands with soap and water up to your elbows. Rinse and dry.  

 

Transferring Explant to Media:

1) Move to laminar flow hood: 

Wash the work area with disinfectant.  

• Pour sterile water into the 3 sterile rectangular boxes with lids to rinse your tissue. Keep in mind that the outside of the boxes isn’t sterile.  
• Sterile forceps and scalpel in the bacticinerator and place on rack.  
• Use forceps to remove your leaves from the bleach and rinse in the sterile water.  
• Move through the 3 rinses in turn from 1 to 3.  
• Place your tissue in an empty sterile petri dish which you’ll use as a work stage. Replace the cover carefully to keep the tissue clean until you’re ready to use it.  

2) Wash the work area again with disinfectant.  

3) Sterilize your forceps and scalpel in the bacticinerator again.  

4) Transferring Explant to Petri Dishes: 

https://cedars.inverclyde.sch.uk/senior-school-blog-2014-15-archive/cell-and-tissue-culture

• Open the petri dish with your tissue and set the cover to the side. Do not let the bottom part of the cover touch the work surface.  
• Use your scalpel to remove all damage tissue and cut the cauliflower tissues into smaller pieces. 
• Place each tissue piece on the surface of the medium, evenly spread out from each other.  
• Use your forceps to gently press the pieces down onto the surface of the media to ensure adequate contact. Do not press too hard.  
• Wrap the petri dishes with parafilm to seal the edge between the lid and the base of the plate and label it with your initials and rate.  
• Place the finished plates into the incubator on the rack underneath lights.  

 

What to Expect and Care:

After approx. 1 week, the effectiveness of your sterility techniques will reflect in the results. If good, there will be no visible contamination on the cauliflower tissues or media surface. The plant materials should start to develop with roots and/or shoots visible in 4-6 weeks.  

Some examples of results:

https://www.saps.org.uk/secondary/teaching-resources/706-cauliflower-cloning-tissue-culture-and-micropropagation

If a few pieces in a petri dish are contaminated and a few are not, you can salvage the ones that aren’t contaminated by taking them and placing them into sterile petri dishes with fresh media.  

Once plantlets have established themselves, you must acclimate them to conditions outside the media and incubator. Carefully remove the cauliflower tissue from the medium and transfer the plantlets into a new environment with lots of humidity and moisture such as a greenhouse. To acclimate the new plantlets to a variety of conditions outside the greenhouse, you can gradually lower the humidity in the greenhouse.  

 

Works Cited:

Enders, J. F., Rogers, K., & Singh, S. (2015, July 7). Processing of Cultured Cells and Tissues. Retrieved October 22, 2019, from https://www.britannica.com/science/tissue-culture/Processing-of-cultured-cells-and-tissues. 

Haldeman, J. H., & Ellis, J. P. (1988). Using Cauliflower to Demonstrate Plant Tissue Culture. The American Biology Teacher, 50(3), 154–159. doi: 10.2307/4448679 

Petruzzello, M. (2019, February 25). Cauliflower. Retrieved October 22, 2019, from https://www.britannica.com/plant/cauliflower.