红皮云杉体胚发生体系优化和超低温保存技术研究

doi:10.12302/j.issn.1000-2006.202007029

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3): 100-108.doi: 10.12302/j.issn.1000-2006.202007029

红皮云杉体胚发生体系优化和超低温保存技术研究

高芳¹^,²(), 陈士刚¹, 秦彩云¹, 才巨锋¹, 王聪慧³, 董环宇¹, 陶晶¹^,^*()

1.吉林省林业科学研究院林业生物技术研究所,吉林长春 130033
2.林木遗传育种国家重点实验室(东北林业大学),黑龙江哈尔滨 150040
3.吉林省林木种苗管理站,吉林长春 130022

收稿日期:2020-07-15 修回日期:2020-11-27 出版日期:2021-05-30 发布日期:2021-05-31
基金资助:
吉林省林业重大科技攻关计划项目(2015-002)

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

摘要/Abstract

摘要：

【目的】通过对红皮云杉(Picea koraiensis)体细胞胚胎发生条件的筛选和超低温保存技术研究, 建立其完整的体胚发生体系及超低温保存条件,为红皮云杉优良种质资源的大量繁殖及保存提供基础。【方法】以红皮云杉合子胚为外植体,以两种基本培养基、6种植物生长调节剂组合为培养条件,筛选胚性愈伤组织诱导的适宜条件。并将获得的胚性愈伤组织进行超低温保存和体胚发育与成熟萌发试验,以3种脱落酸(ABA)和Gelrite浓度以及4种基本培养基为培养条件,筛选出体胚成熟和萌发的适宜培养条件。【结果】①两种基本培养基相比较,改良RJW(诱导率为30.00%)优于1/2 LV(诱导率为22.50%)基本培养基。当萘乙酸(NAA)质量浓度为3.0 mg/L、6-苄氨基腺嘌呤(6-BA)质量浓度为0.5 mg/L时胚性愈伤组织诱导率最高为50.00%;② 胚性愈伤组织增殖培养3个月后进行超低温保存试验,经恢复培养10 d 左右可以清晰地观察到成活的胚性愈伤组织,3个细胞系的胚性愈伤组织均可成活;③ 体胚发育阶段,改良RJW培养基添加Gelrite 6.0 g/L和ABA 20 mg/L时,3个细胞系体胚数量均较高,但是畸形胚比例也偏高,特别是HY-1细胞系畸形胚比例高达77.14%;当Gelrite质量浓度增加到8.0 g/L时,3个细胞系畸形胚比例均降低, 其中HY-2细胞系体胚数量为396.00个/g,畸形胚比例为10.33%;④ 红皮云杉体胚成熟培养两个月进行体胚萌发,在1/2 LM上体胚萌发能力最弱,其次是LM基本培养基,改良RJW体胚萌发能力最佳。不同细胞系体胚萌发能力也不同,体胚萌发能力最高为65.00%。【结论】① 胚性愈伤组织诱导的适宜培养基为改良RJW基本培养基,适宜的激素组合为NAA 3.0 mg/L + 6-BA 0.5 mg/L;② 本试验进行超低温保存的细胞系均可成活;③ 体胚发育与成熟适宜的ABA质量浓度为20 mg/L,适宜Gelrite质量浓度为8.0 g/L;④ 体胚萌发的适宜基本培养基为改良RJW基本培养基。本研究建立了红皮云杉体胚发生体系并成功进行胚性愈伤组织的超低温保存条件,该技术体系可用于红皮云杉优良种质的快速繁殖。

关键词: 红皮云杉, 胚性愈伤组织, 体细胞胚, 超低温保存, 基本培养基, 再生植株

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

中图分类号:

S722.3

高芳,陈士刚,秦彩云,等. 红皮云杉体胚发生体系优化和超低温保存技术研究[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 100-108.

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 (Natural Science Edition), 2021, 45(3): 100-108.DOI: 10.12302/j.issn.1000-2006.202007029.

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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

红皮云杉体胚发生体系优化和超低温保存技术研究

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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