Mitosis Counts in Onion Root Tips and a Colchicine Slide
Class 10 students grew onion roots in water, cut 5 mm tips, fixed them in acetic-alcohol, hydrolysed them in 1 M HCl at 60 °C, stained with acetocarmine and squashed the tips under a cover slip. Under 400× magnification each group counted 200 cells in randomly chosen fields and recorded the stage of each.
The pooled class results were: interphase 160, prophase 14, metaphase 9, anaphase 6, telophase 11. The teacher stated that one complete cell cycle in these root-tip cells takes about 20 hours. A second batch of roots was treated with 0.05% colchicine for 4 hours before fixation; in that slide 46 of 200 cells were in metaphase and only 3 in anaphase or telophase. A third group used tips from garlic cloves stored in the dark for two months and obtained a mitotic index of 6%. One student wrote in her notebook: "Interphase is the resting phase, so most of these cells are resting."
1. Calculate the mitotic index and the time spent in each mitotic phase. State one assumption the calculation depends on.
Read the worked answer
Mitotic index = cells in mitosis ÷ total cells = (14 + 9 + 6 + 11) ÷ 200 = 40 ÷ 200 = 0.20, i.e. 20%.
Because a random sample of an asynchronous population spends time in each stage in proportion to that stage's duration, time in mitosis = 0.20 × 20 h = 4 h, and interphase = 16 h. Each phase is then scaled by its share of the 200 cells:
- Prophase: 14/200 × 20 h = 1.4 h
- Metaphase: 9/200 × 20 h = 0.9 h
- Anaphase: 6/200 × 20 h = 0.6 h
- Telophase: 11/200 × 20 h = 1.1 h
These add to 4.0 h, which checks the arithmetic.
The assumption is that all meristem cells cycle at the same steady rate and that the sample is genuinely random. If many cells are temporarily out of cycle, or if the squash is thick in some fields, the phase proportions and hence the times will be biased.
2. Explain the colchicine result: why are so many cells in metaphase, and why do those cells contain double the normal chromosome number?
Read the worked answer
Colchicine binds tubulin and prevents microtubules from assembling into the spindle. Chromosomes condense and, because DNA has already replicated in S phase, each is a pair of sister chromatids, but without a spindle they cannot be pulled to the poles. The spindle-assembly checkpoint keeps the cell arrested in metaphase, so metaphase cells accumulate: 46/200 = 23% against 4.5% in the untreated slide. Anaphase and telophase almost disappear (3 cells) because segregation cannot start.
Each arrested cell has twice the normal DNA content and twice the chromosome number of a gamete-producing cell - the cell is polyploid, not a normal diploid. If such cells are returned to a drug-free medium, the chromosomes may decondense into one nucleus, giving a polyploid plant. This is used deliberately in plant breeding to produce larger, thicker-leaved polyploids, and in the laboratory colchicine is used to hold cells in metaphase so that chromosomes can be counted and karyotyped.
3. Evaluate the student's notebook statement that interphase is a resting phase. Then explain whether the garlic result (6%) proves that garlic cells divide more slowly than onion cells.
Read the worked answer
The statement is wrong. Interphase occupies 160/200 = 80% of the cycle here, about 16 hours, but it is the busiest part: G1 is growth and synthesis of RNA and proteins, S is DNA replication, and G2 is further growth and preparation for mitosis. A cell that was truly resting would have left the cycle and would not be scored as cycling at all. Interphase looks quiet only because chromosomes are not condensed and therefore not visible as separate bodies.
The garlic figure cannot be used to compare species. The mitotic index depends on which part of the tip was taken: cells behind the meristem elongate and stop dividing, so a slightly older tip gives a low index. Storage for two months, temperature, staining quality and how fields were chosen all change the count. A fair comparison would need the same 1–2 mm meristem zone, the same season and temperature, and several roots per plant, with the index reported as a mean with a range.