Efficient culture protocol for plant regeneration from cotyledonary petiole explants of Jatropha curcas L.

Figures & data

Table 1. Effects of various concentrations of TDZ on the conventional regeneration of adventitious buds from in vitro cotyledonary petiole explants of J. curcas after 30 days.

TDZ concentration(mg/L)	Regeneration %	Number of buds per explant
0	0^d*	0^d
0.1	12.72 ± 2.85^c	2.27 ± 0.25^c
0.3	41.68 ± 3.01^a	4.31 ± 0.41^a
0.6	28.69 ± 5.73^b	3.38 ± 0.54^b

Note: Values represent means ± SD of 30 explants per treatment in three independent experiments.

*Data in the same column followed by different letters are significantly different (Duncan's test at p ≤ 5% level).

Figure 1. Induction of adventitious buds from cotyledonary petiole explants of J. curcas with conventional culture methods. In vitro petiole explants were horizontally placed on MS medium containing 0 mg/L (A), 0.1 mg/L (B), 0.3 mg/L (C) and 0.6 mg/L (D) TDZ after 30 days in culture.

Table 2. Effect of treating explants with TDZ solution of various concentrations on the regeneration of adventitious buds from in vitro cotyledonary petiole explants of J. curcas after 30 days.*

TDZ concentration(mg/L)	Regeneration %	Number of buds per explant
0	0^d**	0^d
10	52.57 ± 3.09^c	7.18 ± 0.48^c
20	88.42 ± 3.67^a	12.67 ± 0.42^a
30	72.19 ± 2.72^b	9.27 ± 0.52^b
60	56.08 ± 4.35^c	7.71 ± 0.81^c

Note: Values represent means ± SD of 30 explants per treatment in three independent experiments.

*All explants were treated with TDZ solution for 20 min.

**Data in the same column followed by different letters are significantly different (Duncan's test at p ≤ 5% level).

Figure 2. Direct induction of adventitious buds from cotyledonary petiole explants of J. curcas. Treatment of in vitro petiole explants with 10 mg/L (A), 20 mg/L (B), 30 mg/L (C) and 60 mg/L (D) TDZ solution for 20 min before transfer and horizontal placement on hormone-free MS medium after 30 days in culture (bar = 0.5 cm). Treatment of explants with 20 mg/L TDZ solution for 20 min before inoculation: in vitro petiole explants in vertical position (E), in vivo petiole in horizontal position (F), in vivo petiole in vertical position (G) on hormone-free MS medium after 30 days in culture (bars = 0.5 cm).

Table 3. Effect of the duration of treatment of explants with TDZ solution on the regeneration of adventitious buds from in vitro cotyledonary petiole explants of J. curcas after 30 days.*

Treatment duration (min)	Regeneration %	Number of buds per explant
0	0^d**	0^d
5	50.53 ± 6.37^c	6.62 ± 0.78^c
20	88.42 ± 3.67^a	12.67 ± 0.42^a
40	67.88 ± 4.96^b	8.35 ± 0.71^b
80	54.13 ± 5.22^c	6.74 ± 0.59^c

Note: Values represent means ± SD of 30 explants per treatment in three independent experiments.

*All explants were treated with 20 mg/L TDZ solution.

**Data in the same column followed by different letters are significantly different (Duncan's test at p ≤ 5% level).

Table 4. Effect of orientation (horizontal or vertical) and source of the explants (in vitro or in vivo) on the regeneration of adventitious buds from cotyledonary petiole explants of J. curcas after 30 days.*

Explant source	Explant orientation	Regeneration %	Number of buds per explant
In vitro	horizontal	88.42 ± 3.67^a**	12.67 ± 0.42^a
	vertical	50.92 ± 4.54^b	6.45 ± 0.48^b
In vivo	horizontal	68.46 ± 3.43^a	9.37 ± 0.72^a
	vertical	39.11 ± 3.36^b	4.76 ± 0.38^b

Note: Values represent means ± SD of 30 explants per treatment in three independent experiments.

*All explants were treated with 20 mg/L TDZ solution for 20 min.

**Data in the same column followed by different letters are significantly different (Duncan's test at p ≤ 5% level).

Table 5. Effects of various concentrations of L-arginine on elongation of the regenerated shoot buds from cotyledonary petiole explants of J. curcas after 15 days.

L-arginine (mg/L)	Length of shoot buds (cm)
0	1.27 ± 0.14^c*
7.5	1.81 ± 0.12^a
15	1.65 ± 0.09^ab
30	1.57 ± 0.04^b

Note: Values represent means ± SD of 30 explants per treatment in three independent experiments.

*Data followed by different letters are significantly different (Duncan's test at p ≤ 5% level).

Figure 3. Elongation of adventitious buds, rooting of elongated shoot buds and transplantation of the regenerated plants to the soil. (A) Regenerated adventitious buds were inoculated into elongation medium supplemented with 7.5 mg/L L-arginine for 15 days of culture; (B) close-up of an elongated shoot bud; (C) rooting of elongated shoot buds on half-strength MS medium containing 0.1 mg/L IBA after 30 days in culture; (D) a view from the bottom of the same culture; (E) 20-day-old acclimatized plant; (F) a regenerated plant growing in a pot after acclimatization (bars = 1 cm).

Table 6. Effects of various concentrations of IBA on the rooting of regenerated shoots from cotyledonary petiole explants of J. curcas.

Time (days)	IBA concentration (mg/L)	Rooting %	Number of roots per shoot*	Average root length (cm)
10	0	0^c**	0^c	0^c
	0.1	2.34 ± 2.42^a	1.83 ± 0.29^a	1.45 ± 0.08^a
	0.3	5.90 ± 5.24^b	0.67 ± 0.58^b	0.53 ± 0.46^b
	0.6	0^c	0^c	0^c
15	0	0^d	0^c	0^c
	0.1	21.94 ± 2.76^a	3.99 ± 0.65^a	2.33 ± 0.19^a
	0.3	18.28 ± 1.67^ab	1.33 ± 0.33^b	0.95 ± 0.08^b
	0.6	5.56 ± 4.81^c	0.67 ± 0.58^b	0.60 ± 0.52^b
20	0	0^d	0^d	0^d
	0.1	35.77 ± 3.77^a	5.37 ± 0.67^a	3.52 ± 0.51^a
	0.3	27.68 ± 2.91^b	4.66 ± 0.15^ab	2.47 ± 0.18^b
	0.6	15.97 ± 3.18^c	1.11 ± 0.19^c	0.84 ± 0.03^c
30	0	0^d	0^d	0^c
	0.1	42.83 ± 1.63^a	8.29 ± 0.93^a	4.20 ± 0.37^a
	0.3	29.60 ± 2.02^b	6.50 ± 1.32^b	3.62 ± 0.71^a
	0.6	21.35 ± 3.20^c	1.68 ± 0.59^c	2.50 ± 0.20^b
40	0	0^d	0^d	0^d
	0.1	43.39 ± 0.92^a	15.66 ± 0.97^a	6.24 ± 0.68^a
	0.3	31.31 ± 3.50^b	8.44 ± 1.35^b	3.74 ± 0.20^b
	0.6	23.04 ± 1.97^c	3.31 ± 0.62^c	2.67 ± 0.10^c

Note: Values represent means ± SD of 30 explants per treatment in three independent experiments.

*Number of roots per shoot = total number of roots/number of shoots that initiated at least one root.

**Data in the same column of the same culture time followed by different letters are significantly different (Duncan's test at p ≤ 5% level).