Optimization of somatic embryogenesis system and cryopreservation of Picea koraiensis

doi:10.12302/j.issn.1000-2006.202007029

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (3): 100-108.doi: 10.12302/j.issn.1000-2006.202007029

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Optimization of somatic embryogenesis system and cryopreservation of Picea koraiensis

GAO Fang¹^,²(), CHEN Shigang¹, QIN Caiyun¹, CAI Jufeng¹, WANG Conghui³, DONG Huanyu¹, TAO Jing¹^,^*()

1. Jilin Provincial Academy of Forestry Sciences, Institute of Biotechnology, Changchun 130033, China
2. State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), Harbin 150040, China
3. Jilin Forest Tree and Seeding Management Station, Changchun 130022, China

Received:2020-07-15 Revised:2020-11-27 Online:2021-05-30 Published:2021-05-31
Contact: TAO Jing E-mail:1054293357@qq.com;taojing8116@126.com

Abstract

Abstract:

【Objective】This study aimed to establish a complete somatic embryogenesis system and optimal cryopreservation conditions to provide a basis for the breeding and preservation of Picea koraiensis (Korean spruce) planting resources. We screened somatic embryogenesis conditions and the cryopreservation of Korean spruce. 【Method】We explanted zygote embryos in two basal culture media with six plant growth regulators to define the optimal culture conditions in which the embryogenic mass was introduced. The embryogenic mass was cryopreserved, and the somatic embryo maturation and germination were assessed. We screened three abscisic acid (ABA) and Gelrite concentrations and four basic culture media to assess the optimal conditions for the somatic embryo maturation and germination. 【Result】①The two basal culture media were compared, and the modified induction ratio in RJW medium was 30.00% which was greater than that of 1/2 LV (induction ratio, 22.50%) basal culture medium. The highest induction ratio was 50.00% in the proliferation medium containing 3.0 mg/L of 1-naphthalacetic acid (NAA) and 0.5 mg/L of N6-benzyladenine (6-BA). ②The embryogenic mass was cultured for 3 months, then cryopreservation was assessed. The cryopreserved embryogenic mass from three cell lines survived after 10 days of restoration. ③The numbers of somatic embryos in the three cell lines were higher, but the ratio of abnormal HY-1 somatic embryos was 77.14% in the maturation medium containing Gelrite (6.0 g/L) and ABA (20 mg/L). Increasing the Gelrite concentration to 8.0 g/L decreased the ratio of abnormal somatic embryos in all three cell lines; the number of HY-2 somatic embryos was 396.00 per gram, and the ratio of abnormal somatic embryos was 10.33%. ④The somatic embryos of Korean spruce matured for two months in the germination medium. The amount of somatic embryo germination was the lowest on 1/2 LM, followed by on LM medium and was the highest on RJW medium. The germination ability of the cell lines differed, with a somatic germination ratio of 65.00%.【Conclusion】①The optimal basal culture medium for the embryogenic mass induction was modified RJW, and the appropriate plant growth regulator concentration was NAA 3.0 mg/L + 6-BA 0.5 mg/L. ②The embryogenic mass obtained survived cryopreservation. ③The optimal ABA and Gelrite concentrations for somatic embryo maturation were 20 mg/L and 8.0 g/L, respectively. ④Also, the optimal basal culture medium for somatic embryo germination was modified RJW. We established the ideal conditions for the regeneration, cryopreservation, and somatic embryogenesis of Korean spruce. This technique can be applied to propagate the best planting resources and to establish multi-varietal forestry by using Korean spruce.

Key words: Picea koraiensis(Korean spruce), embryonic mass, somatic embryo, cryopreservation, basic culture medium, regeneration plant

CLC Number:

S722.3

GAO Fang, CHEN Shigang, QIN Caiyun, CAI Jufeng, WANG Conghui, DONG Huanyu, TAO Jing. Optimization of somatic embryogenesis system and cryopreservation of Picea koraiensis[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(3): 100-108.

Figures/Tables 9

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References 37

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指标 index	细胞系 cell lines	平方和 squares	自由度 df	平均值平方 MS	F检验 F-test	P
体胚数量 somatic embryo number	HY-1	335 646.923	2	167 823.462	76.877	0.000
	HY-2	397 716.667	2	198 858.333	71.091	0.000
	HY-3	41 116.667	2	20 558.333	49.340	0.000
畸形胚比例 abnormal somatic embryos percentage	HY-1	5 622.699	2	2 811.349	3.745	0.061
	HY-2	1 780.193	2	890.097	1.654	0.244
	HY-3	3 733.557	2	1 866.778	15.018	0.001

细胞系 cell lines	ABA质量浓度/ (mg·L^-1) ABA concentration	体胚数量/ (个·g^-1) somatic embryo number	畸形胚比例/% abnormal somatic embryos	细胞系 cell lines	Gelrite质量浓度/ (g·L^-1) gelrite concentration	体胚数量/ (个·g^-1) somatic embryo number	畸形胚比例/% abnormal somatic embryos
HY-1	10	122.50±21.75 a	31.79±4.31 b	HY-1	8	354.00±17.20 a	12.31±3.27 a
HY-2		157.50±22.50 b	48.69±10.57 a	HY-2		396.00±22.72 a	10.33±1.30 a
HY-3		57.50±6.29 b	36.90±8.50 a	HY-3		46.00±5.10 a	7.33±4.52 b
HY-1	20	382.00±28.18 a	77.14±4.53 a	HY-1	10	186.00±19.90 b	26.12±2.81 a
HY-2		460.00±38.94 a	68.75±4.62 a	HY-2		212.00±30.07 b	22.09±5.12 a
HY-3		142.50±16.52 a	37.91±4.58a	HY-3		24.00±5.10 b	25.00±11.18 a
HY-1	30	7.50±4.79 a	37.50±23.94 b	HY-1	12	12.00±5.83 c	23.33±14.53 a
HY-2		25.00±8.66 c	39.58±16.45 a	HY-2		4.00±2.45 c	20.00±20.00 a
HY-3		0.00±0.00 c	0.00±0.00 b	HY-3		0.00±0.00 c	0.00±0.00 b

指标 index	细胞系 cell lines	平方和 SS	自由度 df	平均值平方 mean squares	F检验 F-test	P
体胚数量 somatic embryo number	HY-1	292 440.000	2	146 220.000	120.843	0.000
	HY-2	384 640.000	2	192 320.000	80.920	0.000
	HY-3	5 293.333	2	2 646.667	30.538	0.000
畸形胚比例 abnormal somatic embryos percentage	HY-1	532.759	2	266.380	0.696	0.518
	HY-2	393.655	2	196.828	0.276	0.764
	HY-3	1651.481	2	825.741	3.406	0.067

处理 treatment	基本培养基 basic medium	不同细胞系体胚萌发百分比/% germination percentage
处理 treatment	基本培养基 basic medium	HY-1	HY-2	HY-3
1	改良RJW	60.00±5.77 a	65.00±6.71 a	56.67±4.94 a
2	1/2改良RJW	54.00±6.00 ab	52.00±6.70 ab	52.00±5.83 a
3	LM	40.00±7.07 b	42.00±4.90 b	44.00±8.12 a
4	1/2 LM	21.67±5.43 c	41.67±6.01 b	46.67±4.94 a

Optimization of somatic embryogenesis system and cryopreservation of Picea koraiensis

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质量浓度/(mg·L^-1) concentration		外植体总数 total explants	胚性愈伤组织诱导率/% embryogenic mass induction percentage
6-BA	NAA	外植体总数 total explants	胚性愈伤组织诱导率/% embryogenic mass induction percentage
0.5	2	40	10.00±5.77 b
0.5	3	40	50.00±5.77 a
0.5	5	40	15.00±9.57 b
1.0	2	40	25.00±5.00 b
1.0	3	40	25.00±9.57 b
1.0	5	40	5.00±5.00 b

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