Question

The viability of seeds is tested through a chemical called 2,3,5-triphenyl tetrazolium chloride (TZ). The steps of the TZ tests are listed below. Find out the group with the most appropriate order of the steps-
(A). Dissection of the seeds to expose the embryo
(B). Placing the seeds in a 1.0% or 0.1% solution of 2,3,5-triphenyl tetrazolium chloride
(C). Soak the seeds in water overnight
(D). Examination of the treated seeds for coloration of the embryo
Choose the correct answer from the options given below:

• A. (C), (B), (A), (D).
• B. (D), (B), (C), (A).
• C. (B), (A), (D), (C).
• D. (C), (A), (B), (D).

Hint:

The biochemical basis of the TZ test:
Colorless Tetrazolium \( \xrightarrow{\text{Dehydrogenase enzymes (Living cells)}} \) Insoluble Red Formazan.
Hydration (C) and exposure of the embryo (A) must always precede exposure to the chemical staining solution (B).

Answer 1

The Correct Option is D

Step 1: Understanding the Concept:
The tetrazolium (TZ) test is a rapid, biochemical method used to estimate seed viability.
It relies on the activity of dehydrogenase enzymes within living, respiring plant cells.
These enzymes transfer hydrogen ions to the colorless tetrazolium salt (2,3,5-triphenyl tetrazolium chloride) absorbed by the tissues, reducing it into an insoluble red compound called triphenylformazan.
Living tissues stain bright red, while dead tissues remain uncolored.

Step 2: Detailed Explanation:
Let us arrange the steps of a standard tetrazolium viability test in chronological order:
1.

Step 1: Pre-conditioning/Soaking (C): The test begins by soaking the dry seeds in water overnight.
This hydration activates the embryonic dehydrogenase enzymes and softens the seed tissues, making them easier to cut and permitting even absorption of the staining solution.
2. Seed Dissection (A): After soaking, the seeds are dissected or cut (e.g., longitudinally through the embryo) to expose the internal embryonic structures (radicle, plumule, and cotyledons) to the chemical solution.
3.

Step 2: Staining/Chemical Treatment (B): The prepared seed halves are placed in a container with a \( 0.1% \text{ to } 1.0% \) aqueous solution of 2,3,5-triphenyl tetrazolium chloride.
The seeds are incubated in the dark at a warm temperature (typically \( 30^\circ\text{C} \text{ to } 35^\circ\text{C} \)) for several hours to allow the enzymatic reduction reaction to take place.
4.

Step 3: Evaluation/Examination (D): After incubation, the staining solution is decanted, the seeds are washed, and the embryo staining patterns are evaluated under a magnifying lens.
Seeds with fully stained, bright red embryos are classified as viable, while unstained or partially stained embryos are classified as non-viable.
Thus, the correct chronological sequence is (C) \( \rightarrow \) (A) \( \rightarrow \) (B) \( \rightarrow \) (D).

Step 3: Final Answer:
The correct sequence of the TZ test steps is (C), (A), (B), (D), corresponding to option (D).