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Edexcel GCE

Biology
Advanced Subsidiary

CORE PRACTICAL 06

Demonstration of totipotency using plant tissue culture techniques.

Amal Hulangamuwa (BSc)

Describe how totipotency can be demonstrated practically using plant tissue culture techniques. Tissue culture techniques for the vegetative propagation of plants (micropropagation) have a number of advantages over other methods. Growth is very rapid. Growth is independent of seasons. The technique can be applied to a number of species that are otherwise difficult to propagate. Apical meristems in infected plants often remain virus-free. Their use for tissue culture has, therefore, permitted the elimination of viruses from infected stocks of a range of species. Certain types of callus culture give rise to clones that have inheritable characteristics different from those of the parent plant. Improved varieties arising in this way can be propagated and used commercially. In the future, it is likely that new varieties and hybrids produced using other modern techniques will be commercially propagated by tissue culture methods.
Procedure

Collect all the apparatus that you will need and prepare any solutions that are required. Set up and sterilize your work-bench as suggested in the notes on aseptic handling techniques. 1. Select a clean floret from a fresh cauliflower head. Place it on a tile while holding it with forceps, Carefully trim off mini-fIorets from the curd to produce 20 cuboids of curd tissue, approximately 3 mm x 5mm x 5 mm. These will be your explants.The next stage of the procedure is to steriiize the surface of the explants with a chlorate (I) solution (Bleach). Caution: You will be using a fairly strong bleach solution so take extreme care. From now on you must use aseptic handling techniques so return your forceps to the ethanol beaker. 2. Quickly transfer your explants to a clean (preferably sterile) screw-top jar and add chlorate (l) solution to leave a small head space. Reseal the jar and shake the contents for five seconds. 3. Shake the jar for five seconds every minute for exactly 10 minutes. 4. After exactly 10 minutes pour off the hypochlorite solution into the 'waste beaker, using the Jar lid to trap the sterilized explants. 3 5. wash the explants four times as follows. Add approximately 100cm sterile water to the jar. Reseal it, shake for five seconds and pour off the liquid into the 'waste' beaker as in Step 4. The explants may be left in the last wash until they are required. 6. Using sterile forceps (cooled in the wash water) transfer six explants to each of the three petri dishes containing growth medium. The explants should be widely spaced and pressed gently onto the agar. Flame sterilize and cool your forceps when each dish is complete.

7. Seal each dish with Parafilm or insulating tape to reduce dehydration. 8. Label each dish clearly on its base and incubate them in the light at 20 0C to 280C. 9. Examine each culture weekly. record and sketch any changes you observe, If some of the explants show signs of contamination, the remainder should be aseptically transferred to fresh medium (Steps 68). Aseptic techniques the systematic precautions taken to keep cultures pure and free from contamination. All apparatus (Petri dishes, pipettes, flasks, etc) must be sterilised by autoclaving at 1210C for 15 minutes at 103 kPa Or by irradiating with ultraviolet rays (Wavelength - 254nm). Sterile the culture medium by autoclaving. Wipe the bench / work surfaces with 70% ethanol before and after work. Flame the necks of bottles to prevent air-borne contamination. Flame wire loops and forceps before and after use or sterilise by dipping in ethanol. Lids of containers should not left on benches. Petri dishes opened slightly during operation to avoid contamination. Cultures after study and contaminated equipment must be autoclaved / sterilised before disposal or reuse.

Q 01 Plant tissue culture is a method used to propagate plants. The flow diagram shows one method of plant tissue culture.

(a) Name the type of reproduction involved in plant tissue culture. (1 mark) (b) Give two advantages of producing plants using this method rather than from seeds. (2 marks) (c) Why is a viral infection more likely to destroy a complete batch of plants grown by plant tissue culture than a batch of plants grown from seeds? (1 mark) (d) Callus tissue develops into either shoots or roots depending on the relative concentration of the plant growth regulators used. Use your knowledge of genes to suggest how these plant growth regulators determine the type of plant tissue formed. (1 mark) Q 02 Take-all is a disease of wheat caused by a fungus. It can cause serious damage to the crop. There is no gene for resistance to this fungus in wheat. There is, however, a gene for resistance to this fungus present in oats. The diagram shows how this gene might be transferred to wheat.

(a)

(i) The wheat plant with the resistance gene contains recombinant DNA. What is recombinant DNA?(1) (ii) The plasmids act as vectors for the resistance gene. What is a vector? (1) (iii) Suggest how cells with the resistance gene might be selected. (2)

(b)

(i) Suggest which tissue would be most suitable for the explants. (1) (ii) Explain why explants containing large quantities of xylem do not usually grow into a callus. (2)

c) State what is meant by the term totipotency. (2) d) Distinguish between differentiation and dedifferentiation. (2) e) Suggest why adult animal cells usually do not develop into complete organisms, but plant tissues do.(2)