抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究

doi:10.12302/j.issn.1000-2006.202312024

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南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (1): 87-94.doi: 10.12302/j.issn.1000-2006.202312024

抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究

柯欣, 费琪, 夏馨蕊, 叶建仁, 朱丽华^*()

南京林业大学,南方现代林业协同创新中心,南京林业大学林草学院,江苏南京 210037

收稿日期:2023-12-25 修回日期:2024-02-07 出版日期:2025-01-30 发布日期:2025-01-21
通讯作者: * 朱丽华(lhzhu@njfu.com.cn),教授。
作者简介:
柯欣(1176350062@qq.com)。
基金资助:
国家自然科学基金项目(31971659)

The factors influencing the embryogenic callus initiation and somatic embryo yield in Pinus elliottii resistant to pine needle brown spot disease

KE Xin, FEI Qi, XIA Xinrui, YE Jianren, ZHU Lihua^*()

Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China

Received:2023-12-25 Revised:2024-02-07 Online:2025-01-30 Published:2025-01-21

摘要/Abstract

摘要：

【目的】建立和优化抗性湿地松(Pinus elliottii)体胚发生体系,为湿地松优良抗性材料的大规模快速繁育奠定基础。【方法】以抗性湿地松4个家系的未成熟合子胚为材料,探究不同家系对胚性愈伤组织诱导的影响;选取增殖效果好、具有胚性胚柄细胞团(ESM)的胚性愈伤组织开展体胚成熟试验,从基因型、植物磺肽素(phytosulfokine,PSK)浓度及继代次数方面对体胚成熟条件进行研究。【结果】抗性湿地松4个家系之间胚性愈伤组织诱导率差异显著(P<0.05),其中,30号家系诱导率最高,为(29.8±5.6)%;32号家系诱导率最低,仅为(7.3±3.4)%,最终共获得37个可稳定增殖的胚性细胞系。35个供试细胞系中25.7%的细胞系可产生体胚,其中2007-3和2007-5成熟体胚产量显著高于其他细胞系,成熟子叶型胚数量平均分别为(91.3±7.0)和(89.8±8.4)个/mL,其余74.3%的细胞系未产生体胚。添加PSK对抗性湿地松体胚产量具有显著影响,其质量浓度为1 mg/L时,可显著提高体胚产量。随着继代次数的增加,成熟体胚数量呈下降趋势。其中细胞系2007-3成熟体胚数量在继代8次后逐渐下降,但第20代仍保持胚性;2007-5的成熟体胚数量随继代次数增多而下降,直至20代完全丧失胚性。【结论】家系对胚性愈伤组织的诱导影响显著。基因型和PSK含量可显著影响抗性湿地松体胚产量,同时胚性愈伤组织的胚性随继代次数的增加而降低。本研究优化了抗性湿地松体胚发生技术体系,可为湿地松优良基因型体胚繁育提供技术支撑。

关键词: 湿地松, 体胚发生, 基因型, 植物磺肽素(PSK), 体胚成熟, 抗病育种

Abstract:

【Objective】The somatic embryogenesis induction system of resistant Pinus elliottii is established and optimized to provide technical support for the large-scale rapid propagation of high-resistant materials of P. elliottii. 【Method】 The immature zygotic embryos from four families of P. elliottii are used to explore the effects of different families on the embryogenic callus initiation rate. The embryogenic callus with good proliferation rate and generating large quantity of embryo suspensor mass is selected to test somatic embryo maturation. The maturation conditions of somatic embryos are optimized based on genotype, phytosulfokine (PSK) concentration, and subculture times. 【Result】A significant difference is observed in the initiation frequencies of embryogenic callus among the four families (P < 0.05). Family No. 30 has the highest initiation rate (29.8 ± 5.6)%, while family No. 32 has the lowest (7.3 ± 3.4)%. A total of 37 embryogenic cell lines with stable proliferation are obtained. Of these, 25.7% of 35 cell lines produce matured somatic embryos, and the yields (number of somatic embryos) in 2007-3 and 2007-5 are significantly higher than that of other cell lines, with an average yield of (91.3 ± 7.0) and (89.8 ± 8.4) cotyledonary somatic embryos per mL, respectively, while the remaining 74.3% does not produce any somatic embryos. The addition of PSK significantly affects the yield of somatic embryos of resistant P. elliottii, and embryo production is considerably increased when the PSK concentration is 1 mg/L. The number of mature somatic embryos decreases with the increase in subcultures. The number of mature somatic embryos of cell lines 2007-3 gradually decreases after eight subcultures but maintains embryogenic competence in the 20^th subculture. For cell lines 2007-5, the number of mature somatic embryos reduces as subcultures rise, ultimately losing maturation capacity after 20 subcultures. 【Conclusion】The initiation of embryogenic callus is greatly influenced by the genotype. Genotype and PSK concentration significantly affect the somatic embryo yield of resistant P. elliottii. The yield of somatic embryos decreases with the increase in subcultures. The somatic embryogenesis technology system of resistant P. elliottii is optimized to provide technical support for the somatic embryogenesis of excellent genotypes of P. elliottii.

Key words: Pinus elliottii, somatic embryogenesis, genotype, phytosulfokine, somatic embryo maturation, breeding for disease resistance

中图分类号:

Q943
S722

柯欣,费琪,夏馨蕊,等. 抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究[J]. 南京林业大学学报（自然科学版）, 2025, 49(1): 87-94.

KE Xin, FEI Qi, XIA Xinrui, YE Jianren, ZHU Lihua. The factors influencing the embryogenic callus initiation and somatic embryo yield in Pinus elliottii resistant to pine needle brown spot disease[J].Journal of Nanjing Forestry University (Natural Science Edition), 2025, 49(1): 87-94.DOI: 10.12302/j.issn.1000-2006.202312024.

图/表 5

图1

表1

抗性湿地松37个胚性细胞系的发育阶段及其体胚产量情况"

家系号 family No.	细胞系 cell line	显微结构 microstructure	成熟体胚数量/ (个·mL^-1) number of somatic embryos	体胚形态 the morphology of somatic embryos	家系号 family No.	细胞系 cell line	显微结构 microstructure	成熟体胚数量/ (个·mL^-1) number of somatic embryos	体胚形态 the morphology of somatic embryos
7	2007-1	PEM Ⅰ、Ⅱ	0	未产生体胚	27	2027-5	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-2	PEM Ⅱ、Ⅲ	0	未产生体胚		2027-6	PEM Ⅰ、Ⅱ	2.3±1.3 e	白色、胚轴膨大、子叶闭合
	2007-3	SE	91.3±7.0 a	白色、胚轴伸长、子叶展开		2027-7	PEM Ⅱ、Ⅲ	0	未产生体胚
	2007-4	PEM Ⅱ、Ⅲ	0	未产生体胚		2027-8	PEM Ⅱ、Ⅲ	0	未产生体胚
	2007-5	PEM Ⅰ、Ⅱ	89.8±8.4 a	白色、胚轴伸长、子叶展开		2027-9	PEM Ⅱ、Ⅲ	0	未产生体胚
	2007-8	PEM Ⅱ、Ⅲ	2.0±0.8 e	黄色、胚轴粗壮、子叶闭合		2027-10	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-9	PEM Ⅱ、Ⅲ	0	未产生体胚		2027-11	PEM Ⅰ、Ⅱ	8.0±2.5 d	黄色、胚轴粗壮、子叶闭合
	2007-10	SE	28.0±2.9 b	黄绿色、胚轴粗壮、子叶展开		2027-12	PEM Ⅱ、Ⅲ	9.0±1.6 d	白色、胚轴膨大、子叶展开
	2007-12	PEM Ⅱ、Ⅲ	0	未产生体胚	30	2030-2	PEM Ⅱ、Ⅲ	0	未产生体胚
	2007-14	PEM Ⅱ、Ⅲ	0	未产生体胚		2030-5	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-15	PEM Ⅰ、Ⅱ	0	未产生体胚		2030-7	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-17	SE	0	未产生体胚		2030-9	SE	0	未产生体胚
	2007-19	SE	0	未产生体胚		2030-11	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-21	PEM Ⅱ、Ⅲ	0	未产生体胚		2030-12	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-24	SE	21.3±2.6 c	黄绿色、胚轴膨大、子叶闭合		2030-13	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-26	PEM Ⅰ、Ⅱ	0	未产生体胚		2030-14	PEM Ⅰ、Ⅱ	0	未产生体胚
	2007-27	SE	20.7±3.1 c	黄色、胚轴伸长、子叶展开	32	2032-1	PEM Ⅰ、Ⅱ	—	—
27	2027-1	SE	0	未产生体胚		2032-2	PEM Ⅰ、Ⅱ	—	—
	2027-4	PEM Ⅱ、Ⅲ	0	未产生体胚

表1

图2

图3

图4

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抗松针褐斑病湿地松胚性愈伤组织诱导及体胚产量影响因素的研究

The factors influencing the embryogenic callus initiation and somatic embryo yield in Pinus elliottii resistant to pine needle brown spot disease

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