Induction and characterization of induced tetraploid, mixoploid and control diploid teak seedlings (Tectona grandis L.f.) from shoot cultures treated with oryzalin*

Figures & data

Table 1. Summary of the effects of clones and oryzalin doses on the plant recovery, identification of suspect polyploid plants, and identification of plant ploidy using flow cytometry, and their limited confirmation with chromosome counting.

Clones	Doses of oryzalin (µM)	Number of plants produced after oryzalin treatment	Number of suspects polyploid plants^a	Total number of plants analysed with flow cytometer	Plant ploidy determination with Flow cytometer analysis^b			Confirmation of ploidy with chromosome counting^c
					2X	Mixo	4X	total	2X	Mixo	4X
MK 44	0	20	–	–	–	–	–	–	–	–	–
	5	58	14	6	5	0	1	1	–	1 (2X)	–
	7.5	217	24	9	8	0	1	3	2	1 (2X)^4	–
	30	95	4	0	–	–	–	–	–	–	–
	60	68	5	0	–	–	–	–	–	–	–
MK 61	0	134	–	–	–	–	–	–	–	–	–
	2.5	34	15	14	14	0	0	–	–	–	–
	7.5	66	7	7	6	1	0	2	–	2(1 mixo, 1 2X)^5	–
	30	137	30	23	8	15	0	13	1	12 (mixo)	–
MK 17	0	1	–	–		0	0	–	–	–	–
	7.5	61	8	3	1	0	2	3		1	2
MK 71	0	44	–	–	–	–	–	–	–	–	–
	2.5	19	17	17	17	0	0	–	–	–	–
	7.5	38	21	21	21	0	0	2	1	1	–
MK 10	0	4	–	–	–	–	–	–	–	–	–
	1.25	22	21	20	20	0	0	–	–	–	–
	5.00	41	39	38	38	0	0	5	5	–	–
Total	–	1059	205	158	138	16	4	29	9	18	2

Note: ^aplants with suspected polyploid traits had thicker, stiffer, greener, wider and longer leaf size, with more serrated leaf edges than that of the control diploid.

b after ploidy identification using flow cytometer.

c Chromosome counting was only conducted from some of the plants with ploidy determined with flow cytometer.

d The notation of 1 (2X) under column of mixoploid indicates that chromosome counting found there is 1 mixoploid plant, but it was previously determined as diploid using flow cytometer.

e The notation of 2 (1 mixo, 1 2X) under column of mixoploid indicates 2 mixoploid plants were resulted from chromosome counting, 1 from a mixoploid flow cytometer determined plants and 1 from diploid flow cytometer determined plant.

Figure 1. Flow cytometry analysis of preselected plants from oryzalin treatment compared to the untreated control: (A) a diploid clone of KSP in the untreated control produced a peak at 198.48 with a coefficient of variation (CV) of 4.97%, (B) a mixoploid of MK61 produced two peaks at 181.35 and 369.10 with CVs of 7.78% and 5.31%, respectively, and (C) a tetraploid of clone MK17 peaked at channel 391.79 with a CV of 5.15% (CV).

Figure 2. Mitotic phases of root tip cells of teak plants: (A) prophase, (B) metaphase, (C) anaphase, (D) telophase, and (E) interphase, bar = 10 µm.

Table 2. The mitotic activities in root tips of teak plants showed as percentage of cells underwent different stages according to time of fixation from 05:45–11:30 AM.

Time of root tip fixation (AM)	Plant with root tip mitotic metaphase (%)^a	Number of cells per observation field^b	Percentage of cells (%)
			Prophase	Metaphase	Anaphase	Telophase	Interphase	Total
05:30–06:00	90.38	19.09	7.55	8.15	5.44	6.46	72.41	100
06:01–06:30	68.00	23.91	6.83	7.22	4.23	6.27	75.46	100
06:31–07:00	72.22	26.70	3.55	5.77	3.73	5.23	81.72	100
07:01–07:30	68.18	26.03	4.79	6.35	4.57	4.95	79.33	100
07:31–08:00	47.37	33.57	4.67	4.83	4.24	5.38	80.87	100
08:01–08:30	46.67	36.05	6.62	3.63	3.06	3.64	83.04	100
08:31–09:00	57.14	18.73	6.25	7.45	7.45	6.20	72.65	100
09:01–09:30	57.14	21.31	4.76	7.25	7.29	6.11	74.59	100
09:31–10:00	58.33	16.29	–	7.40	7.97	7.83	76.79	100
10:01–10:30	60.00	23.46	–	4.52	6.25	5.62	83.61	100
10:31–11:00	25.00	26.29	–	4.35	6.25	6.16	83.24	100
11:01–11:30	33.33	17.67	–	5.30	7.63	5.52	81.54	100
Average	–	24.07	–	–	–	–	–

Note: ^aPercentage of plants underwent mitotic metaphase = (number of plants with its root-tip showed mitotic metaphase/number of plants with root-tip fixed) x 100%.

^b observation field had area of 140 µm x 100 µm and an average of 24.07 cell number were observed in that field.

Figure 3. Metaphase chromosome number of root tips of teak plants from polyploidy experiment, (A) control diploid MK44 (2n = 2x = 36), (B) tetraploid of MK17clone (2n = 4x = 72), (C) mixoploid of MK61 clone (2n = 2x + 4x; tetraploid cell 4x = 72), (D) chimeric tissue with a diploid cell (chromosome number of ≈36) and a tetraploid cell (chromosome number of ≈72) of accession MK61-30-101.

Figure 4. The comparison of root tip cell size among tetraploid, mixoploid and diploid plants. (A) histogram of average root tip cell length, and the comparison of root cell size in an induced tetraploid (B), induced mixoploid (C), and a control diploid teak plant (D), bar = 20 µm. The same letters on figure (A) do not differ significantly according to the duncan multiple range test at a 5% significance level, where the p-value (pr > F) <0.001.

Figure 5. The comparison of the size and density of stomata from control diploids, and induced tetraploid and mixoploid teak. (A) stomata density which was the number of stomata/microscope observation field (350 µm x 250 µm), (B) length of stomata guard cell, (C) width of stomata guard cell. Bars in histogram indicates standard error. The same letters on the same parameter variable do not differ significantly according to the duncan test at a 5% significance level, where the p-value (pr > f) is <0.001 for stomata density, length and width of stomata guard cells.

Figure 6. The comparison of the size and density of stomata from induced tetraploid, mixoploid and control diploids under magnification of 400x and observation field of 350x250 µm² (A) tetraploid, (B) mixoploid, and (C) diploid. Bar = 50 µm, arrows mark the stomata.

Figure 7. The shoot and root morphology of the tetraploid and diploid plants. (A) a tetraploid plant (left) and a diploid plant (right); (B) Root of a tetraploid plant; (C) root of a diploid plant. Bar in the root = 50 mm.