Unit 6: Protoplast Isolation - Practice Quiz

1 A protoplast is a plant cell that has had which structure removed?

2 Which enzyme is commonly used to degrade the cellulose component of the plant cell wall during protoplast isolation?

3 Which enzyme is used to digest the pectin that holds plant cells together during protoplast isolation?

4 In the enzymatic method of protoplast isolation, what protects the freed protoplasts from bursting?

5 Which of the following is commonly used as an osmoticum during protoplast isolation?

6 The mechanical method of protoplast isolation was first demonstrated by which scientist?

7 Who pioneered the enzymatic method of protoplast isolation in 1960?

8 In the mechanical method, tissue is first treated to induce which condition before cutting?

9 Which is a major disadvantage of the mechanical method of protoplast isolation?

10 Which plant part is most commonly used as a source for isolating protoplasts?

11 Before enzyme treatment, leaves are often surface sterilised and the epidermis is removed to expose which layer?

12 When cellulase and pectinase are applied together at the same time, the method is called:

13 Which technique is commonly used to separate protoplasts from cell debris after enzyme digestion?

14 A freshly isolated protoplast typically has which overall shape?

15 Which stain is commonly used to test the viability of isolated protoplasts?

16 Compared with the mechanical method, the enzymatic method generally provides:

17 The fusion of two protoplasts from different species to form a hybrid is known as:

18 Enzymatic digestion during protoplast isolation is usually carried out at approximately which temperature?

19 Which enzyme mixture component specifically loosens the hemicellulose of the cell wall?

20 The outermost boundary of an isolated protoplast is formed by the:

21 A researcher wants to isolate protoplasts without using any enzymes. Which technique would be most appropriate?

22 Why is the mechanical method of protoplast isolation rarely used compared to the enzymatic method?

23 Which combination of enzymes is typically used to digest the plant cell wall during enzymatic protoplast isolation?

24 During enzymatic isolation, an osmoticum such as mannitol or sorbitol (0.3–0.7 M) is added to the enzyme solution. What is its primary role?

25 A student obtains a mixture containing protoplasts, undigested cells, and cell debris. Which method is most suitable for purifying the intact protoplasts?

26 Why is the two-step (sequential) enzymatic method sometimes preferred over the one-step method?

27 Leaf tissue is often used for protoplast isolation. Which pretreatment step improves enzyme access to mesophyll cells?

28 Which viability test relies on an enzyme within living protoplasts cleaving a non-fluorescent compound into a fluorescent product?

29 Evans blue is used during protoplast isolation to identify which cells?

30 The pH of the enzyme solution during protoplast isolation is typically maintained around 4.7–6.0. What is the main reason?

31 A protoplast preparation shows low yield because many cells remain clumped together. Which enzyme should be increased to improve cell separation?

32 Which of the following best explains why freshly isolated protoplasts appear spherical under the microscope?

33 If an isolated protoplast is placed in distilled water instead of an osmoticum, what will most likely happen?

34 Why are enzyme incubation times kept controlled (usually a few hours) during protoplast isolation?

35 Which source tissue is generally considered the most convenient starting material for protoplast isolation in dicots?

36 During density gradient purification, intact protoplasts collect as a distinct band because they:

37 What is the main advantage of the enzymatic method over the mechanical method of protoplast isolation?

38 Sterilisation of leaf material before enzyme treatment is important primarily to:

39 A laboratory switches from mannitol to a slightly higher concentration of sorbitol as osmoticum and observes fewer burst protoplasts. This is because the higher osmoticum:

40 Which observation best confirms that cell wall digestion is complete during enzymatic isolation?

41 During enzymatic protoplast isolation, plasmolysis is deliberately induced before adding the enzyme mixture. What is the primary mechanistic rationale for this pre-plasmolysis step?

42 A researcher obtains high protoplast yield but very low viability after enzymatic digestion. Increasing which single parameter is most likely to improve viability without drastically reducing yield?

43 Which combination of enzymes is required to fully degrade the plant cell wall (both primary wall polysaccharides and middle lamella) to release free protoplasts?

44 In the mechanical method of protoplast isolation, cells are first plasmolysed and then the tissue is sliced. Why is this method rarely used compared to the enzymatic method?

45 The two-step (sequential) enzymatic method differs from the one-step method primarily because it:

46 Protoplasts are purified from cell debris using a sucrose gradient during flotation centrifugation. Where do intact viable protoplasts accumulate and why?

47 Fluorescein diacetate (FDA) staining is used to assess protoplast viability. A viable protoplast fluoresces green because:

48 Osmotica used in protoplast isolation are classified as ionic (e.g., ) or non-ionic (e.g., mannitol, sorbitol). Why are non-ionic sugar alcohols generally preferred as the main osmoticum?

49 If leaf tissue is used, the lower epidermis is often peeled or the leaf is sliced before enzyme treatment. The main purpose of this pretreatment is to:

50 A protoplast preparation shows many burst protoplasts immediately upon dilution with distilled water. What does this observation confirm about protoplasts?

51 Prolonged enzyme incubation increases yield up to a point, after which yield and viability both decline. The decline is best explained by:

52 Calcium chloride () is frequently added to protoplast isolation and washing media even though mannitol provides the main osmotic support. Its key role is to:

53 Protoplast yield is often expressed as protoplasts per gram fresh weight. If protoplasts are counted from of leaf tissue using a haemocytometer, the yield is:

54 Why must protoplasts be washed several times (by gentle centrifugation and resuspension) after enzymatic digestion before culture?

55 During centrifugation of protoplasts, only low speeds (e.g., –) for short times are used. The main reason is that protoplasts:

56 Two protoplast batches are isolated from the same plant: one from young leaves and one from old senescing leaves. The young-leaf batch typically gives better protoplasts mainly because:

57 A student sterilizes plant material and prepares enzyme solution but observes microbial contamination in the isolation medium. The most appropriate corrective measure is to:

58 Which observation would best confirm that isolated protoplasts are truly wall-free rather than merely spherical single cells with a thin wall?

59 The osmotic potential of the isolation medium must roughly match that of the protoplast cytoplasm. If the medium is made slightly hypertonic relative to optimum, the expected effect on freshly released protoplasts is:

60 In comparing mechanical and enzymatic isolation, which statement correctly contrasts the quality/type of protoplasts obtained?