Analysis of DNA Methylation Related to Callus Differentiation and Rooting Induction of Paeonia ostii‘Fengdan’

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

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (8): 1735-1746.doi: 10.16420/j.issn.0513-353x.2021-0547

• Research Papers • Previous Articles Next Articles

Analysis of DNA Methylation Related to Callus Differentiation and Rooting Induction of Paeonia ostii‘Fengdan’

ZHANG Wanqing, ZHANG Hongxiao, LIAN Xiaofang, LI Yuying, GUO Lili, HOU Xiaogai^*()

College of Agriculture/College of Tree Peony,Henan University of Science and Technology,Luoyang,Henan 471023,China

Received:2022-05-19 Revised:2022-08-17 Online:2022-08-25 Published:2022-09-05
Contact: HOU Xiaogai E-mail:hkdhxg@haust.edu.cn

Abstract

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

CLC Number:

S685.11

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.

Figures/Tables 9

References 40

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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）

Analysis of DNA Methylation Related to Callus Differentiation and Rooting Induction of Paeonia ostii‘Fengdan’

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模式 Mode	占比/% Percentage	类型 Type	非胚性愈伤带型 Non-embryogenic callus type		胚性愈伤带型 Embryogenic callus type		条带数 Number of strip	%	甲基化变化 Methylation changes
模式 Mode	占比/% Percentage	类型 Type	H	M	H	M	条带数 Number of strip	%	甲基化变化 Methylation changes
无变化 No change	22.04	A1	1	1	1	1	139	42.90	非→非Un-→Un-
		A2	1	0	1	0	99	30.56	半→半Hemi-→Hemi-
		A3	0	1	0	1	86	26.54	全→全Full-→Full-
去甲基化 Demethylation	28.78	B1	1	0	1	1	69	16.31	半→非Hemi-→Un-
		B2	0	1	1	1	83	19.62	全→非Full-→Un-
		B3	0	0	1	1	61	14.42	超→非Hper-→Un-
		B4	0	0	1	0	87	20.57	超→半Hper-→Hemi-
		B5	0	0	0	1	123	29.08	超→全Hper-→Full-
甲基化 Methylation	40.48	C1	1	1	1	0	76	12.77	非→半Un-→Hemi-
		C2	1	1	0	1	81	13.61	非→全Un-→Full-
		C3	1	1	0	0	92	15.46	非→超Un-→Hper-
		C4	1	0	0	0	147	24.71	半→超Hemi-→Hper-
		C5	0	1	0	0	199	33.45	全→超Full-→Hper-

组培苗 Callus	条带数（%）
组培苗 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
生根Rooted	447（27.49）	378（23.25）	452（27.80）	349（21.46）
未生根Unrooted	347（21.34）	497（30.57）	355（21.83）	427（26.26）

模式 Mode	占比/% Percentage	类型 Types	未生根组培苗 Unrooted tissue culture seedlings		生根组培苗 Rooting tissue culture seedlings		条带数 Number of strips	%	甲基化变化 Methylation changes
模式 Mode	占比/% Percentage	类型 Types	H	M	H	M	条带数 Number of strips	%	甲基化变化 Methylation changes
无变化 No change	18.37	A1	1	1	1	1	133	44.63	非→非Un-→Un-
		A2	1	0	1	0	97	32.55	半→半Hemi-→Hemi-
		A3	0	1	0	1	68	22.82	全→全Full-→Full-
去甲基化 Demethy- lation	38.41	B1	1	0	1	1	103	16.53	半→非Hemi-→Un-
		B2	0	1	1	1	97	15.57	全→非Full-→Un-
		B3	0	0	1	1	114	18.30	超→非Hper-→Un-
		B4	0	0	1	0	122	19.58	超→半Hper-→Hemi-
		B5	0	0	0	1	187	30.02	超→全Hper-→Full-
甲基化 Methylation	30.15	C1	1	1	1	0	81	16.56	非→半Un-→Hemi-
		C2	1	1	0	1	63	12.88	非→全Un-→Full-
		C3	1	1	0	0	70	14.31	非→超Un-→Hper-
		C4	1	0	0	0	163	33.33	半→超Hemi-→Hper-
		C5	0	1	0	0	112	22.90	全→超Full-→Hper-

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