不同抗褐化剂对青钱柳愈伤组织酶活性和生长的影响

doi:10.12302/j.issn.1000-2006.202203071

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南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (6): 167-174.doi: 10.12302/j.issn.1000-2006.202203071

不同抗褐化剂对青钱柳愈伤组织酶活性和生长的影响

王纪¹^,²(), 方升佐²^,^*()

1.南京晓庄学院,江苏南京 211171
2.南京林业大学林草学院,江苏南京 210037

收稿日期:2022-03-21 修回日期:2022-05-11 出版日期:2023-11-30 发布日期:2023-11-23
通讯作者: 方升佐
基金资助:
江苏省农业重点研发项目(BE2019388);国家自然科学基金项目(32071750)

Effects of different anti-browning agents on enzyme activity and growth in callus of Cyclocarya paliurus

WANG Ji¹^,²(), FANG Shengzuo²^,^*()

1. Nanjing Xiaozhuang University,Nanjing 211171, China
2. College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China

Received:2022-03-21 Revised:2022-05-11 Online:2023-11-30 Published:2023-11-23
Contact: FANG Shengzuo

摘要/Abstract

摘要：

【目的】青钱柳(Cyclocarya paliurus)在组织培养中的褐化问题一直是造成愈伤组织死亡和制约其规模化生产的重要原因,对褐化问题进行研究为青钱柳快速繁殖体系的建立和离体生产次生代谢物质奠定基础。【方法】以青钱柳无菌实生苗茎段、叶片和根系作外植体,研究维生素C(vitamin C, VC)、聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)和活性炭(active carbon, AC)3种抗褐化剂及其不同浓度对青钱柳愈伤组织褐化率、增长量及相关酶活性的影响。【结果】不同抗褐化剂与不同浓度处理对青钱柳愈伤组织褐化率、增长量、苯丙氨酸解氨酶(PAL)活性、多酚氧化酶(PPO)活性和过氧化物酶(POD)活性均具有显著影响(P < 0.05)。愈伤组织褐化率随PVP和AC浓度的增加而降低,随VC浓度的增加呈先降低后增加趋势。与VC和PVP相比,AC处理的愈伤组织褐化率最低,其中,以茎、根和叶为外植体的愈伤组织褐化率均以4.0 g/L AC处理最低,分别为16.3%、33.3%和26.7%。不同处理下,以叶片为外植体的愈伤组织褐化率高于茎段和根系的。愈伤组织累计增长量随PVP和AC质量浓度的增加而增加,随VC浓度的增加呈先增加后降低趋势。添加VC和PVP处理不同外植体愈伤组织累计增长量高于AC处理,以VC浓度为0.4 g/L时茎段愈伤组织累计增长量最高,为6.69 g。添加PVP和AC处理的茎段、叶片和根系愈伤组织PAL和POD活性略高于AC处理,而添加PVP处理的茎段、叶片和根系愈伤组织PPO活性高于添加AC和VC处理的。随着抗褐化剂浓度增加,愈伤组织PAL活性逐渐降低、PPO和POD活性呈单峰变化趋势。其中,以叶片为外植体的愈伤组织的PAL、POD和PPO活性均高于茎段和根系的。Pearson相关分析结果表明,青钱柳愈伤组织褐化率与PAL活性呈极显著正相关(P < 0.01),愈伤组织增长量与PPO活性、POD活性呈极显著正相关(P < 0.01)。【结论】抗褐化剂种类和浓度对青钱柳愈伤组织褐化及生长均有显著影响,以AC抗褐化效果最好,但高浓度的AC会一定程度抑制愈伤组织的生长。PAL活性在愈伤组织褐化过程中起关键作用,可以作为青钱柳组织培养过程中褐化控制的主要参考酶指标,同时外植体器官的类型选择也是控制褐化考虑的因素。

关键词: 青钱柳, 组织培养, 外植体, 愈伤组织, 褐化率, 酶活性

Abstract:

【Objective】The browning in the callus culture process of Cyclocarya paliurus is an important problem for the death of callus, which obviously restricts its large-scale production. Reducing the browning rate will lay the foundation for the establishment of a rapid propagation system and in vitro production of secondary metabolites. 【Method】The stem segments, leave and roots of C. paliurus seedlings were used as explants to study the effects of three different anti-browning agents, including vitamin C (VC), polyvinylpyrrolidone (PVP) and active carbon (AC) with different concentrations on the browning rate, increment as well as enzyme activities of C. paliurus callus. 【Result】Different anti-browning agents and their concentrations had significant effects on the browning rate, growth and activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) activity in C. paliurus callus (P < 0.05). The callus browning rate decreased with increasing PVP and AC concentrations, whereas the rate first decreased and then increased with increasing VC concentrations. Among them, the browning rate in callus of stem segments, roots and leaves was the lowest when the AC concentration of 4.0 g/L was added, reaching 16.3%, 33.3% and 26.7%, respectively. The growth of calli in the explants treated with VC and PVP was higher than that in the AC treatment. However, when the 0.4 g/L of VC was applied, the cumulative growth of calli in stem segments was the highest (6.69 g). With increasing concentrations of anti-browning agents, PAL activity in callus gradually decreased, whereas the activities of PPO and POD showed a unimodal trend. Moreover, the enzymatic activity of callus explants from leaves was higher than that of stem segments and roots. Pearson correlation analysis showed that callus browning rate was positively correlated with PAL activity(P < 0.01), whereas callus growth was positively correlated with PPO and POD activities(P < 0.01). 【Conclusion】The types and concentrations of anti-browning agents have a significant effect on the browning and growth of C. paliurus callus. AC has the best anti-browning effect, but somewhat inhibits callus growth. PAL enzyme activity plays a key role in preventing callus browning, and the selection of explant organs is crucial during tissue culture of C. paliurus.

Key words: Cyclocarya paliurus, tissue culture, explant, callus, browning rate, enzyme activity

中图分类号:

S682.29

王纪,方升佐. 不同抗褐化剂对青钱柳愈伤组织酶活性和生长的影响[J]. 南京林业大学学报（自然科学版）, 2023, 47(6): 167-174.

WANG Ji, FANG Shengzuo. Effects of different anti-browning agents on enzyme activity and growth in callus of Cyclocarya paliurus[J].Journal of Nanjing Forestry University (Natural Science Edition), 2023, 47(6): 167-174.DOI: 10.12302/j.issn.1000-2006.202203071.

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编号 code	处理treatment		茎段 stem segment	叶片 leaf	根系 root
编号 code	抗褐化剂 anti- browning agent	质量浓度/ (g·L^-1) meas concentration	茎段 stem segment	叶片 leaf	根系 root
z1	VC	0.1	50.0±10.00 a	66.7±5.77 a	43.30±5.77 abc
z2	VC	0.2	46.7±5.77 ab	56.7±11.55 ab	40.00±10.00 abc
z3	VC	0.4	43.3±5.77 ab	53.3±15.28 abc	36.70±11.55 abc
z4	VC	1.0	53.3±5.77 a	56.7±5.77 abc	53.30±5.77 a
z5	PVP	1.0	53.3±11.55 a	56.7±5.77 ab	53.30±20.82 a
z6	PVP	3.0	40.0±10.00 abc	50.0±10.00 abc	50.00±10.00 ab
z7	PVP	5.0	33.3±5.77 bcd	53.3±11.55 abc	43.30±5.77 abc
z8	AC	1.0	26.7±5.77 cde	43.3±15.28 bc	30.00±10.00 bc
z9	AC	2.0	23.3±5.77 de	36.7±11.55 bc	26.70±5.77 c
z10	AC	4.0	16.7±5.77 e	33.3±5.77 c	26.70±15.28 c

器官 organ	处理时间/d treatment time	增长量grewth
器官 organ	处理时间/d treatment time	z1	z2	z3	z4	z5	z6	z7	z8	z9	z10
茎段 stem segment	10	0.43±0.10 ab	0.68±0.20 a	0.70±0.21 a	0.38±0.07 ab	0.36±0.08 ab	0.39±0.08 ab	0.40±0.16 ab	0.13±0.03 b	0.16±0.05 b	0.14±0.02 b
	20	0.98±0.16 a	1.13±0.38 a	1.09±0.39 a	0.75±0.15 a	0.70±0.16 a	0.90±0.22 a	0.92±0.12 a	0.12±0.07 b	0.12±0.03 b	0.15±0.05 b
	30	1.11±0.44 a	1.72±0.96 a	1.57±0.61 a	1.37±0.42 a	0.96±0.1 a	1.28±0.27 a	1.64±0.55 a	0.01±0.05 c	0.03±0.11 c	0.15±0.06 b
	40	1.33±0.28 b	2.32±2.6 ab	3.33±1.06 a	2.60±0.39 ab	1.40±0.07 b	1.92±0.57 ab	2.18±1.07 ab	0.09±0.46 c	0.03±0.05 d	0.04±0.19 d
叶片 leaf	10	0.50±0.12 a	0.43±0.13 ab	0.4±0.15 ab	0.36±0.11 ab	0.52±0.15 a	0.35±0.11 a	0.51±0.14 ab	0.19±0.06 b	0.19±0.04 b	0.23±0.06 b
	20	0.82±0.23 ab	0.68±0.22 b	0.55±0.14 b	0.52±0.20 b	0.75±0.14 a	0.70±0.25 ab	1.02±0.23 ab	0.12±0.04 c	0.10±0.04 c	0.17±0.07 c
	30	1.28±0.37 ab	1.06±0.33 ab	1.06±0.18 ab	0.87±0.20 ab	1.07±0.14 a	1.17±0.48 ab	1.60±0.14 ab	-0.05±0.01 b	-0.02±0.01 c	0.07±0.03 d
	40	0.73±0.20 c	2.23±0.53 ab	2.16±0.43 ab	1.33±0.26 b	1.66±0.06 a	1.84±0.34 bc	3.31±0.75 ab	-0.02±0.00 d	0.00±0.00 e	0.07±0.02 e
根系 root	10	0.36±0.11 a	0.28±0.08 ab	0.31±0.07 a	0.29±0.10 a	0.24±0.06 a	0.40±0.09 a	0.44±0.06 a	0.13±0.04 c	0.18±0.05 bc	0.20±0.07 bc
	20	0.36±0.11 a	0.53±0.16 ab	0.54±0.16 ab	0.61±0.23 ab	0.66±0.22 ab	0.76±0.28 b	0.86±0.28 a	0.44±0.15 b	0.17±0.06 c	0.18±0.03 c
	30	0.61±0.22 a	0.88±0.33 ab	1.01±0.49 ab	0.94±0.31 ab	1.01±0.25 ab	1.43±0.57 b	1.56±0.36 a	-0.24±0.06 c	0.11±0.04 bc	0.18±0.03 b
	40	0.98±0.3 ab	0.93±0.16 ab	2.72±1.04 b	2.20±0.63 a	2.50±0.51 ab	2.20±0.62 b	2.70±0.21 ab	0.11±0.04 c	0.11±0.04 c	0.14±0.05 c

指标index	PAL活性 PAL activity	PPO活性 PPO activity	POD活性 POD activity
褐化率browning rate	0.516^**	0.359	0.347
愈伤组织增长量callus growth	0.317	0.608^**	0.631^**

不同抗褐化剂对青钱柳愈伤组织酶活性和生长的影响

Effects of different anti-browning agents on enzyme activity and growth in callus of Cyclocarya paliurus

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