Callus Induction and Secondary Metabolites Accumulation of Diploid Cyclocarya paliurus

doi:10.7525/j.issn.1673-5102.2025.04.006

Abstract

Abstract:

This study aimed to explore the effects of hormone combination on callus induction and proliferation of diploid Cyclocarya paliurus and the accumulation of main secondary metabolites during subculture. The effects of different hormone combinations on accumulation of secondary metabolites during callus induction and subproliferation were investigated using diploid C. paliurus leaves as explants by orthogonal experiment design. An optimal treatment for the maximum yields of total triterpenoids， total polyphenols， total flavonoid and crude polysaccharides were selected by entropy weight method. The highest callus induction rate was 100% in treatment 2（0.5 mg⋅L^-1 6-BA+1 mg⋅L^-1 2，4-D+0.2 mg⋅L^-1 NAA）， followed by treatment 9 （2.5 mg⋅L^-1 6-BA+1.5 mg⋅L^-1 2，4-D+0.2 mg⋅L^-1 NAA） and treatment 5（1.5 mg⋅L^-1 6-BA+1 mg⋅L^-1 2，4-D+0.4 mg⋅L^-1 NAA）， with an induction rate of 96.67% and 93.64%， respectively. The order of factors affecting callus induction rate was 2，4-D， NAA， 6-BA. An optimal concentration of 2，4-D and NAA was 1.0 mg⋅L^-1 and 0.2 mg⋅L^-1， with no significant effect of 6-BA. The colors of induced callus were light green and yellowish green； the morphology of the callus was characterized by loose texture. There were differences in the callus growth and accumulation of secondary metabolites among treatments when different combinations of hormones were used for subculture； the growth characteristics of calli in the same treatment changed greatly during continuous subculture. The first ranked formula was treatment 4 （0.2 mg⋅L^-1 KT+0.5 mg⋅L^-1 6-BA+0.75 mg⋅L^-1 NAA+0.4 mg⋅L^-1 2，4-D） with a score of 0.44，followed by treatment 2（0.75 mg⋅L^-1 6-BA+0.75 mg⋅L^-1 NAA+0.2 mg⋅L^-1 2，4-D） and treatment 6（0.2 mg⋅L^-1 KT+1.0 mg⋅L^-1 6-BA+0.5 mg⋅L^-1 NAA+0.2 mg⋅L^-1 2，4-D） with a score of 0.31 and 0.26， respectively. The yield of secondary metabolites in treatment 4 generally reached a high level from the 8th generation， with total triterpenoid yield of 28.91 mg⋅bottle^-1， total polyphenol yield of 6.89 mg⋅bottle^-1， total flavonoid yield of 1.77 mg⋅bottle^-1，and crude polysaccharide yield of 57.45 mg⋅bottle^-1. In summary， the optimal formula for callus induction of diploid C. paliurus was MS+0.5 mg⋅L^-1 6-BA+1.0 mg⋅L^-1 2，4-D+0.2 mg⋅L^-1 NAA. The subculture proliferation formula was MS+0.2 mg⋅L^-1 KT+0.5 mg⋅L^-1 6-BA+0.75 mg⋅L^-1 NAA+0.4 mg⋅L^-1 2，4-D. This study provided a reference for production of secondary metabolites by callus culture and further cell suspension culture.

Key words: Cyclocarya paliurus, callus induction, plant hormone, subculture proliferation, secondary metabolites

CLC Number:

Q813.1

Zhuosui LI, Yilin GAO, Han LIU, Xulan SHANG. Callus Induction and Secondary Metabolites Accumulation of Diploid Cyclocarya paliurus[J]. Bulletin of Botanical Research, 2025, 45(4): 533-545.

Figures/Tables 13

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水平Level	因素Factor
水平Level	KT/ （mg·L^-1）	6-BA/ （mg·L^-1）	NAA/ （mg·L^-1）	2，4-D/ （mg·L^-1）
1	0	0.50	0.50	0
2	0.2	0.75	0.75	0.2
3	0.4	1.00	1.00	0.4

试验号 No.	因素Factor			诱导率 Induction rate/%	愈伤组织颜色 Callus color	愈伤组织质地 Callus texture
试验号 No.	6-BA/（mg·L^-1）	2，4-D/（mg·L^-1）	NAA/（mg·L^-1）	诱导率 Induction rate/%	愈伤组织颜色 Callus color	愈伤组织质地 Callus texture
1	0.5	0.5	0	59.05±0.02^d	多数嫩绿色，少数黄绿色 Mostly light-green，a few yellow-green	疏松 Loose
2	0.5	1.0	0.2	100.00±0.00^a	嫩绿、黄绿各占半数 Half light-green，half yellow-green
3	0.5	1.5	0.4	77.75±0.06^bc	多数嫩绿色，少数黄绿色 Mostly light-green，a few yellow-green
4	1.5	0.5	0.2	74.44±0.08^bc	多数嫩绿色，少数黄绿色 Mostly light-green，a few yellow-green
5	1.5	1.0	0.4	93.64±0.06^a	嫩绿色 Light-green
6	1.5	1.5	0	67.94±0.11^cd	多数黄绿色，少数嫩绿色 Mostly yellow-green，a few light-green
7	2.5	0.5	0.4	71.59±0.12^cd	多数黄绿色，少数嫩绿色 Mostly yellow-green，a few light-green
8	2.5	1.0	0	86.90±0.07^ab	多数嫩绿色，少数黄绿色 Mostly light-green，a few yellow-green
9	2.5	1.5	0.2	96.67±0.06^a	嫩绿色 Light-green
k1	78.932	68.360	71.296
k2	78.672	93.514	90.370
k3	85.053	80.784	80.991
R	6.380	25.154	19.074

指标 Index	指标信息熵 Entropy	指标差异性系数 Difference coefficient	指标熵权 Entropy weight
总三萜产量 Total triterpenoid production	0.921	0.079	0.343
总多酚产量 Total polyphenol production	0.932	0.067	0.291
总黄酮产量 Total flavonoid production	0.957	0.043	0.186
粗多糖产量 Crude polysaccharide production	0.959	0.041	0.179

处理 No.	各指标得分 The index scores				综合得分 Comprehensive score	排名 Rank
处理 No.	总三萜产量 Total triterpenoid production	总多酚产量 Total polyphenol production	总黄酮产量 Total flavonoid production	粗多糖产量 Crude polysaccharide production	综合得分 Comprehensive score	排名 Rank
4	0.085	0.088	0.132	0.129	0.435	1
2	0.060	0.049	0.093	0.111	0.312	2
6	0.047	0.077	0.071	0.066	0.260	3
5	0.054	0.053	0.053	0.067	0.227	4
9	0.041	0.054	0.045	0.071	0.212	5