Performance of Malaysian kenaf Hibiscus cannabinus callus biomass and exopolysaccharide production in a novel liquid culture

Figures & data

Figure 1. (a) Germinated kenaf seedlings. (b) Callus grown on kenaf stem. (c) Axenic kenaf callus. (d,e) Kenaf callus in cell suspension culture. (f) Kenaf callus biomass. (g) Kenaf extracellular polysaccharide.

Table 1. Experimental range and levels of independent variables.

Independent variables	Range and levels
	−1	0	1
Sucrose (g/L)	10.0	30.0	50.0
Agitation (rpm)	50.0	100.0	150.0
NAA (mg/L)	0.0	1.0	2.0
2,4-D (mg/L)	0.0	1.0	2.0

Figure 2. Growth curve of kenaf callus in cell suspension culture.

Table 2. RSM with CCD matrix and the outcomes of the KCB and EPS production.

Run	Experimental variables				Responses
	Sucrose concentration (g/L)	Agitation (rpm)	NAA (mg/L)	2,4-D (mg/L)	KCB (g/L)		EPS (g/L)
					Actual	Predicted	Actual	Predicted
1	50	150	2	2	9.87	9.32	1.70	1.77
2	30	150	1	1	7.15	6.87	0.95	0.76
3	50	150	0	0	4.22	4.65	0.75	0.85
4	30	100	1	1	9.07	8.10	0.56	0.65
5	30	100	0	1	7.26	7.43	0.18	0.00
6	50	50	2	0	4.80	4.92	0.38	0.41
7	10	50	0	2	4.80	4.90	0.11	0.04
8	30	100	1	0	10.95	10.68	1.06	0.70
9	50	50	2	2	2.24	2.35	0.28	0.26
10	50	150	2	0	13.41	13.26	1.59	1.63
11	50	50	0	0	0.79	0.19	0.34	0.47
12	30	100	1	1	8.06	8.10	0.66	0.65
13	10	50	2	2	1.58	1.08	0.05	0.03
14	50	50	0	2	1.97	1.65	0.22	0.21
15	50	150	0	2	4.68	4.75	0.88	0.88
16	30	100	1	2	9.60	10.31	0.59	0.74
17	30	100	1	1	8.76	8.10	0.45	0.65
18	30	100	1	1	8.43	8.10	0.68	0.65
19	10	50	0	0	1.23	1.71	0.08	0.09
20	50	100	1	1	4.15	5.04	1.86	1.50
21	30	100	2	1	9.56	9.83	0.33	0.34
22	30	100	1	1	7.78	8.10	0.55	0.65
23	10	150	0	2	3.1	2.92	0.19	0.24
24	30	50	1	1	2.97	3.69	0.08	0.05
25	10	100	1	1	3.07	2.62	0.82	0.96
26	30	100	1	1	7.82	8.10	0.39	0.65
27	10	50	2	0	2.04	1.92	0.02	0.00
28	10	150	0	0	1.24	1.09	0.01	0.00
29	10	150	2	0	4.91	5.17	0.64	0.73
30	10	150	2	2	2.41	2.97	1.23	1.07

Table 3. The results of analysis of variance (ANOVA) generated from the CCD quadratic model in RSM for KCB.

Source	Sum of Squares	df	Mean Square	F-value	p-Value
Model	335.48	14	23.96	61.17	<0.0001	Significant
A: Sucrose	26.28	1	26.28	67.09	<0.0001	Significant
B: Agitation	45.35	1	45.35	115.75	<0.0001	Significant
C: NAA	25.75	1	25.75	65.74	<0.0001	Significant
D: 2,4-D	0.6198	1	0.6198	1.58	0.2277
AB	25.93	1	25.93	66.20	<0.0001	Significant
AC	20.45	1	20.45	52.21	<0.0001	Significant
AD	3.00	1	3.00	7.66	0.0144	Significant
BC	15.00	1	15.00	38.28	<0.0001	Significant
BD	1.86	1	1.86	4.74	0.0459	Significant
CD	16.26	1	16.26	41.51	<0.0001	Significant
A^2	47.28	1	47.28	120.70	<0.0001	Significant
B^2	20.63	1	20.63	52.67	<0.0001	Significant
C^2	0.7225	1	0.7225	1.84	0.1945
D^2	14.84	1	14.84	37.88	<0.0001	Significant
Residual	5.88	15	0.3918
Lack of fit	4.50	10	0.4499	1.63	0.3065	Not significant
Pure error	1.38	5	0.2755
Cor total	341.36	29
Std. Dev	=0.6259		R2	=0.9828
Mean	=5.60		Adjusted R2	=0.9667
			Predicted R2	=0.9201

Figure 3. Three-dimensional (3D) response surface plot of KCB production of Hibiscus cannabinus showing the interaction between; (a) sucrose and agitation, (b) sucrose and NAA, (c) sucrose and 2,4-D, (d) agitation and NAA, (e) agitation and 2,4-D and (f) NAA and 2,4-D.

Table 4. The results of analysis of variance (ANOVA) generated from the CCD quadratic model in RSM for EPS.

Source	Sum of Squares	df	Mean Square	F-value	p-Value
Model	6.68	14	0.4774	11.93	<0.0001	Significant
A: Sucrose	1.31	1	1.31	32.65	<0.0001	Significant
B: Agitation	2.26	1	2.26	56.50	<0.0001	Significant
C: NAA	0.6651	1	0.6651	16.62	0.0010	Significant
D: 2,4-D	0.0080	1	0.0080	0.2004	0.6608
AB	0.2233	1	0.2233	5.58	0.0321	Significant
AC	0.0028	1	0.0028	0.0689	0.7966
AD	0.0410	1	0.0410	1.02	0.3275
BC	0.7014	1	0.7014	17.52	0.0008	Significant
BD	0.0856	1	0.0856	2.14	0.1644
CD	0.0105	1	0.0105	0.2625	0.6159
A^2	0.8661	1	0.8661	21.64	0.0003	Significant
B^2	0.1579	1	0.1579	3.94	0.0656
C^2	0.6656	1	0.6656	16.63	0.0010	Significant
D^2	0.0103	1	0.0103	0.2582	0.6187
Residual	0.6004	15	0.0400
Lack of fit	0.5357	10	0.0536	4.14	0.0651	Not significant
Pure error	0.0647	5	0.0129
Cor total	7.28	29
Std. Dev	=0.2001		R2	=0.9176
Mean	=0.5877		Adjusted R2	=0.8406
			Predicted R2	=0.6833

Figure 4. Three-dimensional (3D) response surface plot of EPS production of Hibiscus cannabinus showing the interaction between; (a) sucrose and agitation, and (b) agitation and NAA.

Table 5. Optimized conditions for kenaf callus cell suspension culture.

To maximize	Experimental variables				Responses
	Sucrose (g/L)	Agitation (rpm)	NAA (mg/L)	2,4-D (mg/L)	KCB (g/L)	EPS (g/L)
KCB	40.03	129.59	1.87	0.04	11.97	-
EPS	49.99	134.25	1.34	1.983	-	0.73
KCB and EPS	50.00	147.02	2.00	0.00	11.87	0.91

Table 6. Kinetic parameters of kenaf callus in cell suspension culture.

Conditions	Inoculum size (% of the working volume)	Duration of cultivation (Days)	KCBmax (g/L)	KCBProductivity (g/L/day)	YieldKCB (gKCB/gsuc)	EPSmax (g/L)	EPSProductivity (g/L/day)	YieldEPS (gEPS/gsuc)	EPSSpecific production rate (g/g/day)
A	1	22	9.09 ± 1.2	0.41 ± 1.2	-	1.10 ± 0.02	0.05 ± 0.02	-	0.01 ± 0.02
B	1	22	11.97 ± 1.9	0.52 ± 1.9	0.30 ± 1.9	-	-	-	-
C	1	22	-	-	-	0.73 ± 0.04	0.03 ± 0.04	0.02 ± 0.04	-
D	1	22	11.87 ± 1.6	0.52 ± 1.6	0.25 ± 1.6	0.9 ± 0.02	0.04 ± 0.02	0.02 ± 0.02	>0.01 ± 0.02

Figure 5. FTIR spectra of EPS from kenaf callus in suspension culture.

Table 7. Comparison of the total biomass and extracellular polysaccharide produced with other literature.

Plant species	Callus used (% of working volume, g)	Sucrose (g/L)	Agitation (rpm)	NAA (mg/L)	2,4-D (mg/L)	Biomass produced (g/L)	EPS produced (g/L)	References
Hibiscus cannabinus*	1	30	100	0.2	0.2	9.09	1.1	Current study
Lycium schweinfurthii	2	30	120	2	0	NA	NA	[23]
Helicteres isora L.	7	30	50–60	0	0	19.2	NA	[36]
Betula pendula Roth	1	30	100	0	2.5	1.25	NA	[45]
Melissa officinalis L.	3	NA	110	NA	1	11.63	NA	[46]

*Based on the biomass and extracellular polysaccharide obtained in establishing kenaf growth curve. NA = not available.

Figure 6. Possible workflow for the mass-production of kenaf KCB and EPS.

Mamdouh D, Smetanska I. Optimization of callus and cell suspension cultures of lycium schweinfurthii for improved production of phenolics, flavonoids, and antioxidant activity. Horticulturae. 2022;8(5). doi: 10.3390/horticulturae8050394

 Web of Science ®Google Scholar

Shaikh S, Shriram V, Vinay Kumar TKA, et al. Establishment of callus and cell suspension cultures of Helicteres isora L. Res Plant Biol. 2018;8:01–07. doi: 10.25081/ripb.2018.v8.3366

 Google Scholar

Jafari Hajati R, Payamnoor V, Ghasemi Bezdi K, et al. Optimization of callus induction and cell suspension culture of Betula pendula Roth for improved production of betulin, betulinic acid, and antioxidant activity. Vitro Cell Deve Biol - Plant. 2016;52(4):400–407. doi: 10.1007/s11627-016-9773-6

 Web of Science ®Google Scholar

Meftahizade H, Lotfi M, Moradkhani H. Optimization of micropropagation and establishment of cell suspension culture in Melissa officinalis L. Afr J Biotechnol. 2010;9(28):4314–4321.

 Google Scholar

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.