‘凤丹’牡丹愈伤组织分化和生根诱导中的DNA甲基化分析

doi:10.16420/j.issn.0513-353x.2021-0547

摘要/Abstract

摘要：

牡丹愈伤组织分化率低和组培苗生根困难一直是其再生体系建立的两大制约因素。利用甲基化敏感扩增多态性（MSAP）技术,对‘凤丹’非胚性与胚性愈伤组织、未生根与生根组培苗的DNA甲基化进行了比较分析。结果表明,非胚性和胚性愈伤组织的DNA甲基化水平均以全甲基化水平较高,二者的超甲基化位点数相差较大,与非胚性愈伤组织相比,胚性愈伤组织分化引起由全甲基化转变为超甲基化的占比多达33.45%;与未生根组培苗相比,组培苗生根过程是以去甲基化模式占比最多,为38.41%。因此推测,‘凤丹’胚性愈伤组织分化较少,可能与其超甲基化的形成有关;而组培苗生根可能与其去甲基化有关。

关键词: 牡丹, 愈伤组织, 组培苗, 胚性细胞, 生根苗, 甲基化

Abstract:

The low differentiation rate of callus and the difficulty of rooting in tissue culture have always been two restrictive factors for the establishment of regeneration system in peony. Methylation Sensitive Amplification Polymorphism（MSAP）was employed to compare the DNA methylation level between in vitro non-embryogenic and embryogenic callus,as well as unrooted seedlings and rooted plantlets in tissue culture of Paeonia ostii T. Hong et J. X. Zhang‘Fengdan’. The results showed that permethylation was the main methylation type in both non-embryogenic and embryogenic calli of ‘Fengdan’,while there existed significant differences in the number of hypermethylation sites between these two callus types. Compared with non-embryogenic callus,the transition from permethylation to hypermethylation caused by embryogenic callus differentiation accounted for as much as 33.45%. Compared with unrooted tissue culture seedlings,the demethylation was the main mode in the rooting process of seedlings,accounting for 38.41%. Therefore,it is speculated that the less differentiation of embryogenic callus of‘Fengdan’may be related to the formation of hypermethylation. The formation of rooting plantlets in tissue culture may be related to the demethylation.

Key words: Paeonia ostii, callus, tissue culture seedling, embryonic cell, rooting plantlet, methylation

中图分类号:

S685.11

张婉青, 张红晓, 廉小芳, 李昱莹, 郭丽丽, 侯小改. ‘凤丹’牡丹愈伤组织分化和生根诱导中的DNA甲基化分析[J]. 园艺学报, 2022, 49(8): 1735-1746.

ZHANG Wanqing, ZHANG Hongxiao, LIAN Xiaofang, LI Yuying, GUO Lili, HOU Xiaogai. Analysis of DNA Methylation Related to Callus Differentiation and Rooting Induction of Paeonia ostii‘Fengdan’[J]. Acta Horticulturae Sinica, 2022, 49(8): 1735-1746.

图/表 9

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接头和引物 Adapters and primers	EcoRⅠ（E）（5′-3′）	HpaⅡ/MspⅠ（H/M）（5′-3′）
Adapter‐Ⅰ	CTCGTAGACTGCGTACC	GACGATGAGTCCTGAG
Adapter‐Ⅱ	AATTGGTACGCAGTCTA	TACTCAGGACTCAT
预扩增Pre-amplification	GACTGCGTACCAATTC	GATGAGTCCTGAGCGG
选择性扩增 Selective amplification	E + AAC	HM + AGC
	E + AAT	HM + AGG
	E + ATG	HM + ATA
	E + AGG	HM + CAT
	E + ACG	HM + CTA
	E + CAA	HM + CTC
	E + CAT	HM + CTG
	E + GTG
	E + GTT
	E + TCA
	E + TCC
	E + TCG

接头和引物 Adapters and primers	EcoRⅠ（E）（5′-3′）	HpaⅡ/MspⅠ（H/M）（5′-3′）
Adapter‐Ⅰ	CTCGTAGACTGCGTACC	GACGATGAGTCCTGAG
Adapter‐Ⅱ	AATTGGTACGCAGTCTA	TACTCAGGACTCAT
预扩增Pre-amplification	GACTGCGTACCAATTC	GATGAGTCCTGAGCGG
选择性扩增 Selective amplification	E + AAC	HM + AGC
	E + AAT	HM + AGG
	E + ATG	HM + ATA
	E + AGG	HM + CAT
	E + ACG	HM + CTA
	E + CAA	HM + CTC
	E + CAT	HM + CTG
	E + GTG
	E + GTT
	E + TCA
	E + TCC
	E + TCG

培养基 Medium	浓度/（mg · L^-1）Concentration						愈伤增殖率/% Callus proliferation rate	胚性愈伤比率/% Embryogenic callus ratio
培养基 Medium	NAA	6-BA	TDZ	2,4-D	IBA	水解酪蛋白 Hydrolyzed casein	愈伤增殖率/% Callus proliferation rate	胚性愈伤比率/% Embryogenic callus ratio
WPM	0.05	0.5	0			0	87.22 ± 4.08 a	8.89 ± 5.88 ab
WPM	0.02	0			0.05	0	72.67 ± 2.61 c	5.00 ± 5.00 cd
MS	0	2.0			0.30	0	71.94 ± 2.96 c	0 d
WPM	0.05	0.5	0.3			0	83.61 ± 3.50 ab	0 d
MS	0.1	0.8	0.5			0	80.27 ± 3.06 abc	3.33 ± 3.33 cd
1/2MS	0.1	0.8	0.5			0	71.11 ± 3.38 c	6.95 ± 4.52 bc
MS	0.3	1.5		0.5		100	74.79 ± 6.75 bc	13.33 ± 4.02 a

培养基 Medium	浓度/（mg · L^-1）Concentration						愈伤增殖率/% Callus proliferation rate	胚性愈伤比率/% Embryogenic callus ratio
培养基 Medium	NAA	6-BA	TDZ	2,4-D	IBA	水解酪蛋白 Hydrolyzed casein	愈伤增殖率/% Callus proliferation rate	胚性愈伤比率/% Embryogenic callus ratio
WPM	0.05	0.5	0			0	87.22 ± 4.08 a	8.89 ± 5.88 ab
WPM	0.02	0			0.05	0	72.67 ± 2.61 c	5.00 ± 5.00 cd
MS	0	2.0			0.30	0	71.94 ± 2.96 c	0 d
WPM	0.05	0.5	0.3			0	83.61 ± 3.50 ab	0 d
MS	0.1	0.8	0.5			0	80.27 ± 3.06 abc	3.33 ± 3.33 cd
1/2MS	0.1	0.8	0.5			0	71.11 ± 3.38 c	6.95 ± 4.52 bc
MS	0.3	1.5		0.5		100	74.79 ± 6.75 bc	13.33 ± 4.02 a

愈伤组织 Callus	条带数（%）
愈伤组织 Callus	Ⅰ 非甲基化Un-methylation	Ⅱ 半甲基化Hemi-methylation	Ⅲ 全甲基化Full-methylation	Ⅳ 超甲基化Hyper-methylation
胚性Embryogenic	388（26.39）	383（26.05）	428（29.12）	271（18.44）
非胚性Non-embryogenic	352（23.94）	322（21.90）	358（24.35）	438（29.80）