Optimization of Cell Suspension Culture Conditions of Vaccinium dunalianum

doi:10.11983/CBB21069

Abstract

Abstract: To increase the biomass of suspension culture of Vaccinium dunalianum, we used callus induced from leaves as start material, and investigated the effects of sucrose concentration, pH value, culture medium volume, initial inoculation size, and shaking speed using single factor experiment method. We optimized the suspension culture conditions using Box-Behnken response surface methodology. Our results showed that the optimal conditions for cell suspension culture were: 40 g·L^-1sucrose, pH5.2, culture medium volume 45 mL, callus inoculation size of 2.64 g, and shaking speed of 149 r·min^-1 based on the improved WPM medium. Under these optimal conditions, the dried cell biomass was 0.184 4 g which was close to the theoretical prediction of 0.184 5 g, and the growth curve of cells showed ‘S’ type. These results provide a basis for further study on the regulation of the secondary metabolites of cell suspension culture in V. dunalianum.

Key words: Vaccinium dunalianum, callus, cell suspension culture, culture condition, response surface methodology

Churan Li, Ling Fu, Yun Liu, Xiaoqin Yang, Guolei Zhu, Sida Xie, Huancheng Ma, Ping Zhao. Optimization of Cell Suspension Culture Conditions of Vaccinium dunalianum[J]. Chinese Bulletin of Botany, 2022, 57(2): 227-235.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21069

https://www.chinbullbotany.com/EN/Y2022/V57/I2/227

Figures/Tables 6

References 34

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Code	Factors	Levels
Code	Factors	-1	0	1
A	Sucrose concentration (g·L^-1)	30	40	50
B	pH value	4.5	5	5.5
C	Cultivation medium volume (mL)	40	45	50
D	Initial inoculation size (g)	2.0	2.5	3.0
E	Shaking speed (r·min^-1)	120	150	180

Code	Factors	Levels
Code	Factors	-1	0	1
A	Sucrose concentration (g·L^-1)	30	40	50
B	pH value	4.5	5	5.5
C	Cultivation medium volume (mL)	40	45	50
D	Initial inoculation size (g)	2.0	2.5	3.0
E	Shaking speed (r·min^-1)	120	150	180

No.	Sucrose concentration (g·L^-1)	pH value	Cultivation medium volume (mL)	Initial inocul- ation size (g)	Shaking speed (r·min^-1)	Biomass (g)
1	30	4.5	45	2.5	150	0.0936
2	50	4.5	45	2.5	150	0.1319
3	30	5.5	45	2.5	150	0.0921
4	50	5.5	45	2.5	150	0.1487
5	40	5.0	40	2.0	150	0.1066
6	40	5.0	50	2.0	150	0.1109
7	40	5.0	40	3.0	150	0.1306
8	40	5.0	50	3.0	150	0.1752
9	40	4.5	45	2.5	120	0.1277
10	40	5.5	45	2.5	120	0.1240
11	40	4.5	45	2.5	180	0.0986
12	40	5.5	45	2.5	180	0.0999
13	30	5.0	40	2.5	150	0.1021
14	50	5.0	40	2.5	150	0.1106
15	30	5.0	50	2.5	150	0.1068
16	50	5.0	50	2.5	150	0.1806
17	40	5.0	45	2.0	120	0.0969
18	40	5.0	45	3.0	120	0.1289
19	40	5.0	45	2.0	180	0.0942
20	40	5.0	45	3.0	180	0.1171
21	40	4.5	40	2.5	150	0.1123
22	40	5.5	40	2.5	150	0.1362
23	40	4.5	50	2.5	150	0.1368
24	40	5.5	50	2.5	150	0.1304
25	30	5.0	45	2.0	150	0.1123
26	50	5.0	45	2.0	150	0.1085
27	30	5.0	45	3.0	150	0.0988
28	50	5.0	45	3.0	150	0.1223
29	40	5.0	40	2.5	120	0.1119
30	40	5.0	50	2.5	120	0.1080
31	40	5.0	40	2.5	180	0.1136
32	40	5.0	50	2.5	180	0.1099
33	30	5.0	45	2.5	120	0.0846
34	50	5.0	45	2.5	120	0.1359
35	30	5.0	45	2.5	180	0.0811
36	50	5.0	45	2.5	180	0.1203
37	40	4.5	45	2.0	150	0.1196
38	40	5.5	45	2.0	150	0.0999
39	40	4.5	45	3.0	150	0.1211
40	40	5.5	45	3.0	150	0.1263
41	40	5.0	45	2.5	150	0.1819
42	40	5.0	45	2.5	150	0.1809
43	40	5.0	45	2.5	150	0.1816
44	40	5.0	45	2.5	150	0.1820
45	40	5.0	45	2.5	150	0.1823
46	40	5.0	45	2.5	150	0.1806

No.	Sucrose concentration (g·L^-1)	pH value	Cultivation medium volume (mL)	Initial inocul- ation size (g)	Shaking speed (r·min^-1)	Biomass (g)
1	30	4.5	45	2.5	150	0.0936
2	50	4.5	45	2.5	150	0.1319
3	30	5.5	45	2.5	150	0.0921
4	50	5.5	45	2.5	150	0.1487
5	40	5.0	40	2.0	150	0.1066
6	40	5.0	50	2.0	150	0.1109
7	40	5.0	40	3.0	150	0.1306
8	40	5.0	50	3.0	150	0.1752
9	40	4.5	45	2.5	120	0.1277
10	40	5.5	45	2.5	120	0.1240
11	40	4.5	45	2.5	180	0.0986
12	40	5.5	45	2.5	180	0.0999
13	30	5.0	40	2.5	150	0.1021
14	50	5.0	40	2.5	150	0.1106
15	30	5.0	50	2.5	150	0.1068
16	50	5.0	50	2.5	150	0.1806
17	40	5.0	45	2.0	120	0.0969
18	40	5.0	45	3.0	120	0.1289
19	40	5.0	45	2.0	180	0.0942
20	40	5.0	45	3.0	180	0.1171
21	40	4.5	40	2.5	150	0.1123
22	40	5.5	40	2.5	150	0.1362
23	40	4.5	50	2.5	150	0.1368
24	40	5.5	50	2.5	150	0.1304
25	30	5.0	45	2.0	150	0.1123
26	50	5.0	45	2.0	150	0.1085
27	30	5.0	45	3.0	150	0.0988
28	50	5.0	45	3.0	150	0.1223
29	40	5.0	40	2.5	120	0.1119
30	40	5.0	50	2.5	120	0.1080
31	40	5.0	40	2.5	180	0.1136
32	40	5.0	50	2.5	180	0.1099
33	30	5.0	45	2.5	120	0.0846
34	50	5.0	45	2.5	120	0.1359
35	30	5.0	45	2.5	180	0.0811
36	50	5.0	45	2.5	180	0.1203
37	40	4.5	45	2.0	150	0.1196
38	40	5.5	45	2.0	150	0.0999
39	40	4.5	45	3.0	150	0.1211
40	40	5.5	45	3.0	150	0.1263
41	40	5.0	45	2.5	150	0.1819
42	40	5.0	45	2.5	150	0.1809
43	40	5.0	45	2.5	150	0.1816
44	40	5.0	45	2.5	150	0.1820
45	40	5.0	45	2.5	150	0.1823
46	40	5.0	45	2.5	150	0.1806

Source	Sum of squares	Degree of freedom	Mean square	F-value	P-value
Model	0.0351	20	0.0018	11.1033	<0.0001**
A	0.0052	1	0.0052	32.6506	<0.0001**
B	1.58E-05	1	1.58E-05	0.0999	0.7545
C	0.0011	1	0.0011	6.9608	0.0141*
D	0.0019	1	0.0019	11.8855	0.0020**
E	0.0004	1	0.0004	2.7363	0.1106
AB	8.37E-05	1	8.37E-05	0.5295	0.4736
AC	0.0011	1	0.0011	6.7422	0.0155*
AD	0.0002	1	0.0002	1.784	0.2880
AE	3.66E-05	1	3.66E-05	0.2315	0.6346
BC	0.0002	1	0.0002	1.4517	0.2395
BD	0.0001	1	0.0001	0.9803	0.3316
BE	6.25E-06	1	6.25E-06	0.0395	0.8440
CD	0.0004	1	0.0004	2.3193	0.1403
CE	1E-08	1	1E-08	6.32E-05	0.9937
DE	2.07E-05	1	2.07E-05	0.1309	0.7205
A²	0.0110	1	0.0110	69.6601	<0.0001**
B²	0.0077	1	0.0077	48.9624	<0.0001**
C²	0.0049	1	0.0049	28.3590	<0.0001**
D²	0.0089	1	0.0089	56.9051	<0.0001**
E²	0.1539	1	0.1539	97.3289	<0.0001**
Lack of fit	0.0039	20	0.0002	0.0714	0.7925
Pure error	2.215E-06	5	4.43E-07
Cor total	0.0390	45