Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of psophocarpus tetragonolobus (L.) DC

Figures & data

Figure 1. Direct shoot regeneration from epicotyls and intermodal explants on medium containing TDZ. (A) Incubation of epicotyls explants on shoot induction medium containing AgNO₃ to stop tissue browning; (B) Shoot induction from epicotyls explants; (C) Shoot induction from intermodal explants.

Table 1. Direct shoot regeneration from epicotyls and internodal segments of P. tetragonolobus. Effect of TDZ concentrations in shoot-induction media on regeneration efficiency

	Epicotyl segments			Internodal segments
TDZ concentration (mgL^−1)	Explants survived (%)	Explants producing shoots (%)	Number of shoots per explant	Explants survived (%)	Explants producing shoots (%)	Number of shoots per explant
1.0	100 ± 0.0	0.0	0.0	100 ± 0.0	0.0	0.0
1.5	100 ± 0.0	0.0	0.0	100 ± 0.0	0.0	0.0
2.0	97 ± 2.3	0.0	0.0	91 ± 3.02	0.0	0.0
2.5	95 ± 3.43	62.15 ± 3.6	1.99 ± 3.13	90 ± 3.43	72.0 ± 4.08	2.06 ± 36
3.0	90 ± 2.11	89.2 ± 3.28	13.4 ± 2.04	88 ± 3.12	85.3 ± 4.16	6.5 ± 23
3.5	76 ± 4.22	68.2 ± 3.28	6.2 ± 3.14	66 ± 3.22	63.66 ± 2.57	1.2 ± 33
4.0	12.3 ± 3.58	0.0	0.0	22.1 ± 3.76	0.0	0.0
5.0	0.0	0.0	0.0	0.0	0.0	0.0

Data obtained from 3 independent experiments was calculated as average. Explants which did not produce any shoots, finally died upon further sub-culturings on to fresh media.

Table 2. Callus mediated shoot bud induction from cotyledon, leaf and hypocotyl explants of P. tetragonolobus, effect of BAP in combination with IAA or NAA concentrations on regeneration response

Growth regulator (mgL^−1)			Cotyledon		Leaf		Hypocotyl
BAP	NAA	IAA	% of Explants responded	Number of shoots per explant	% of Explants responded	Number of shoots per explant	% of Explants responded	Number of shoots per explant
1.0	0.1		21.3 ± 1.5	0.0	43.3 ± 4.21	0.0	32.3 ± 2.22	0.0
1.0		0.1	62.3 ± 2.5	0.0	42.3 ± 3.82	0.0	72.3 ± 3.35	1.22 ± 4.09
1.5	0.15		70 ± 3.55	5.3 ± 2.87	70.43 ± 3.34	0.0	66.75 ± 3.04	2.26 ± 3.53
1.5		0.15	72.0 ± 6.08	3.08 ± 1.98	76.5 ± 6.78	0.0	70 ± 3.66	5.76 ± 2.43
2.0	0.2		73.3 ± 4.16	6.0 ± 4.91	88.1 ± 1.3	0.0	86.13 ± 1.2	8.4 ± 2.91
2.0		0.2	99.66 ± 0.57	18.6 ± 3.45	99.3 ± 1.64	0.0	100 ± 0.0	23.2 ± 2.66
2.5	0.25		86.61 ± 3.82	2.83 ± 3.42	87.3 ± 4.13	0.0	73.3 ± 4.16	4.0 ± 5.11
2.5		0.25	93 ± 2.64	8.3 ± 2.16	99.66 ± 0.55	0.0	86.66 ± 4.47	6.48 ± 3.23
3.0	0.3		49 ± 1.0	0.0	79.67 ± 1.57	0.0	29 ± 2.05	1.23 ± 4.31
3.0		0.3	62 ± 2.08	2.65 ± 4.13	82.6 ± 2.99	0.0	49 ± 2.05	2.3 ± 3.13
4.0	0.4		22.3 ± 2.25	0.0	29.3 ± 1.88	0.0	27.3 ± 3.38	0.0
4.0		0.4	31.3 ± 2.05	0.0	29.3 ± 1.88	0.0	38.4 ± 2.84	0.0

Data was calculated as average obtained from 3 independent experiments. Number of explants produced callus against various PGR concentrations in the media, were considered to calculate% of explants responded. Callus obtained from explants on all concentrations of PGR s in the media were sub-cultured on to the fresh media containing similar combination of PGRs.

Figure 2. Callus mediated shoot bud induction and shoot regeneration form hypocotyls and cotyledon explants on media containing BAP and NAA or IAA. (A) and (B). Callus induction from hypocotyl explants on media containing various concentrations of plant growth regulators, callus with regeneration potential was indicated with arrow markings; (C) Callus produced from cotyledon explants; (D) Callus induction from leaf discs incubated BAP and NAA containing medium. (E) Shoot buds and shoots produced from the callus which was obtained from hypocotyls explants; (F) Incubation of shoots for further proliferation on shoot induction medium.

Table 3. Effect of BAP, NAA and IAA concentrations on morphology of the callus associated with regeneration response. Observations were made when hypocotyl segments were used as explants

BAP (mgL^−1)	NAA (mgL^−1)	IAA (mgL^−1)	Callus color	Texture	Dry weight after 30 day inoculation of callus*(mg)	Explants responded (%)	Average no. of shoots per explant
1.0	0.1		Yellow green	Soft Loose	9.8 ± 0.15	32.3 ± 2.22	0.0
1.0		0.1	Yellow green	Soft	11.8 ± 0.28	72.3 ± 3.35	1.22 ± 4.09
2.0	0.1		Green	Soft	12.5 ± 0.5	39 ± 3.05	1.93 ± 4.31
2.0		0.1	Green	Soft Loose	12.7 ± 0.25	44 ± 2.6	2.43 ± 3.63
2.0	0.2		Light Green	Soft	15.3 ± 0.43	86.13 ± 1.2	8.4 ± 2.95
2.0		0.2	Green	Very Soft	15.7 ± 0.36	100 ± 0.0	23.2 ± 2.66
2.5	0.25		Yellow Green	Soft Loose	12.3 ± 0.30	73.3 ± 4.16	4.0 ± 5.15
2.5		0.25	Green	Hard	12.6 ± 0.15	86.66 ± 4.47	6.48 ± 3.23

* 200mg callus obtained from the media containing respective PGR combinations was used as initial inoculum Callus obtained after 30days of inoculation was wrapped in aluminum foil and dried at 75°C in hot air oven for 48 hours. The dry weight of the each callus was measured.

Figure 3. Transfer of plants obtained from in vitro regenerated shoots of; (A) Rooting of isolated shoot on rooting media; (B) Transfer and survival plants in green house conditions, well rooted plants were transferred to plastic pots and acclimatized before transferring to soil.

Table 4. Contents of secondary metabolites in different parts of P. tetragonolobus plants from green house and tissue culture raised plants

Plant source	Organ	Gallic acid (μg/g)	Protocatechuic Acid (μg/g)	Chlorogenic acid (μg/g)	Rutin (μg/g)	Ferulic acid (μg/g)	Quercetin (μg/g)	Caffeic acid (μg/g)	Kaempferol (μg/g)
Green house grown	Leaf	9.6 ± 2.3	0.52 ± 0.6	0.61±0.4	15.39 ± 3.4	5.32 ± 1.3	3.37 ± 1.2	7.67 ± 2.4	93.5 ± 11.0
	Stem	47.3 ± 4	2.38 ± 0.8	3.09 ± 1.2	14.50 ± 2.8	51.7±7.2	82.0 ± 3.2	14.20 ± 3.8	59.4 ± 7.4
	Root	23.2 ± 5.8	8.9 ± 1.9	20.1 ± 3.0	109.8 ± 11.9	12.5 ± 2.0	11.7 ± 1.6	11.66 ± 3.1	32.8 ± 4.1
Tissue culture raised	Leaf	4.01 ± 1.2	3.4 ± 0.9	4.62 ± 1.2	74.0 ± 5.0	20.5 ± 1.6	8.4 ± 1.7	5.4 ± 2.4	49.2 ± 5.6
	Stem	4.53 ± 1.0	9.6 ± 1.3	43.7 ± 4.6	66.27 ± 4.6	14.8 ± 2.3	70.3 ± 4.6	7.74 ± 1.5	42.5 ± 6.2
	Root	5.47±2.0	4.3 ± 1.0	14.0 ± 2.2	21.75 ± 3.8	0.49 ± 0.12	63.2 ± 5.2	0.4 ± 0.1	22.8 ± 3.2

Figure 4. Comparative analysis of polyphenolic compounds present in stem, leaf and roots of green house grown (labeled as green) and tissue culture raised (labeled as red) P. tetragonolobus plants; (A) Graph showing contents of poyphenolic compounds in Stems of both type of plants; (B) Graph showing contents of poyphenolic compounds in Leaves of both type of plants; (C) Graph showing contents of poyphenolic compounds in Roots of both type of plants. Graphs were plotted with concentrations of polyphenolic compounds (mg/g) on x-axis and various types of polyphenolic compounds on y-axis.