Establishment of Cell Suspension Culture of Carrot (Daucus carota)

Table of Contents

Carrot (Daucus carota) is a winter seasoned biennial plant that is grown for its edible taproot. It is one of the most consumed and produced vegetables because of its dietary variety in color, flavor, fiber, and vitamin A, as well as its consumption in both raw and cooked forms. More than 20 million tonnes of it are produced every year for human consumption.

Tissue culture practices have been very popular in the cloning of plants. The first study of carrot tissue culture was reported in 1939 by Gautheret and Nobecourt. However, Steward et al. and Reinert reported the first study of “carrot embryogenesis”; they tried to grow the roots using cell suspension and callus cultures.

Somatic embryogenesis is an efficient method to understand and investigate the biochemical, physiological, and genetic aspects of plant cell culture.

Many studies on the carrot tissue culture led to the development of a simple method of embryogenesis in carrots. According to the method, the growth of carrot cultures can be induced by just removing the hormone 2,4-D from cell suspension cultures. Now, the carrot is often used as an experimental model for the analysis of somatic embryogenesis in various labs worldwide.

Material and Equipment Required for the Establishment of the Culture

Sterilized Materials

• 5 Friable callus culture of carrot, uncontaminated, and maintained at 25 ℃.
• 5 Erlenmeyer flasks (250 ml), containing culture medium with 5 x 10-8 g/ml 2, 4-D.
• 5 measuring cylinders (100 ml) with foil caps.
• 2 spatulas.
• 25 sheets of aluminum foil (100 x 100 mm).
• 2 pieces of nylon, or stainless steel sieves with a porosity of 250 µm, which can be inserted into the opening of the measuring cylinders.
• 5 Petri-dishes.

Non-sterilized materials

• Waterproof marking pen
• Rotary shaking machine
• Bunsen burner
• 1 Erlenmeyer flask (150 ml) containing 95 % ethanol
• Parafilm

Procedure for Initiation and Establishment of the Culture

1. Take the tubes of callus culture, sterile the mouth of all 5 tubes and transfer the callus to the Petri dish separately (5 calli in 5 Petri dishes).
2. Take a 250 ml canonical flask containing the culture media and 2, 4-D.
3. Transfer all 5 calli from the Petri dish to 5 canonical flasks, separately.
4. Flame/sterile the mouth of the flasks and cover them with the cap. Secure the cap using parafilm.
5. Place all the flasks containing the culture on the rotary shaking machine in dark or low light intensity at 25 ℃.
6. After 7 days, transfer the flasks from the shaker to a sterile room.
7. Remove the parafilm and foil cap from each flask and flame/sterile the mouth of the flasks.
8. Transfer the suspension of each flask to a sterile 100 ml cylinder separately by passing it through a sieve.
9. Allow the content to settle for 10 minutes and then discard the supernatant.
10. Transfer the residues of cells left in the measuring cylinder to a 250 ml flask containing 60 ml of fresh medium.
11. Repeat step 10 for each culture flask.
12. Put all the five flasks on the shaker again.
13. After 7 days repeat the procedure you have done before (from step 6-10). However, this time only take 1/5th of the residual cells as the inoculum.
14. Repeat the procedure about 3-4 times. By doing so, only single cells or small aggregates will remain in the carrot suspension.
15. For carrot cell suspension, the number of appropriate cells ranges between 105 and 3 x 105 cells/ml or 100 and 300 mg (fresh weight) of inoculum in a volume of 60 ml of fresh medium containing media and 5x10-8 g/ml, 2,4-D.
16. A transfer period of 7 days is appropriate for the carrot to get single cells in the suspension culture.

Approximate Schedule of the Culture

Result

What should you observe while experimenting? Here are some points to remember while noting your results.

• Date and duration of the experiment.
• The number of cultures and treatments applied.
• Record morphology and number of infected cultures at an interval of three weeks.
• Determine PCV (packed cell volume) at the beginning and end of the transfer of the culture.
• Estimate the total cell number after 3 subcultures, at days 1, 2, 4, and 7, and plot a graph against time.
• Study the relationship between the density of inoculum and the pattern of growth.

The suspension culture provides a benefit over other cultures as it allows the movement of the tissue in the culture media that facilitates gaseous exchange. Furthermore, it removes any polarity of the tissue and nutrient gradient within and at the media.

Application of Tissue Culture for Carrot

1. To clone the taproots.
2. To study the mutant form of the carrots.
3. To study the physiological responses of the carrot that may facilitate an understanding of other plants as well.
4. To study the growth and development of the carrot from a single-cell.
5. To study the stress-response of the carrot that provides an insight into the responses of other plants as well.
6. To generate hundreds of transgenic plants from the suspension of single cells for any experimental purposes.

References

1. Reinert J. and Yeoman M. M. (1982). Plant Cell and Tissue culture: A laboratory manual. Springer-Verlag, Berlin Heidelberg, Newyork.
2. Hardegger M., Shakya R. (2004) Transformation of Carrot. In: Curtis I.S. (eds) Transgenic Crops of the World. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-2333-0_22.
3. Growth and division of carrot cells in suspension culture. (1970). Plant and Cell Physiology. DOI:10.1093/oxfordjournals.pcp.a074564.
4. https://www.atzlabs.com/prodcut-info/C1955-Carrot-...