抗松针褐斑病湿地松未成熟合子胚胚性愈伤组织的诱导

doi:10.12302/j.issn.1000-2006.202203002

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南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (6): 175-182.doi: 10.12302/j.issn.1000-2006.202203002

抗松针褐斑病湿地松未成熟合子胚胚性愈伤组织的诱导

程方¹^,²(), 孙婷玉¹(), 叶建仁¹^,^*()

1.南京林业大学林草学院,南方现代林业协同创新中心,江苏南京 210037
2.广东象头山国家级自然保护区管理局,广东惠州 516001

收稿日期:2022-03-01 修回日期:2023-07-04 出版日期:2023-11-30 发布日期:2023-11-23
通讯作者: *叶建仁(jrye@njfu.edu.cn),教授。
基金资助:
国家重点研发计划(2021YFD1400900)

Induction of embryogenic callus from immature zygotic embryos of Pinus elliottii resistant to brown spot needle blight (pathogen: Lecanosticta acicola)

CHENG Fang¹^,²(), SUN Tingyu¹(), YE Jianren¹^,^*()

1. Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry and Grassland, Nanjing Forest University, Nanjing 210037, China
2. Guangdong Xiangtoushan National Nature Reserve Administration, Huizhou 516001, China

Received:2022-03-01 Revised:2023-07-04 Online:2023-11-30 Published:2023-11-23

摘要/Abstract

摘要：

【目的】建立高效实用的抗松针褐斑病湿地松优良无性系的离体快繁体系,为湿地松优良种苗的商业化应用提供技术基础。【方法】以福建官庄林场采集的抗松针褐斑病家系湿地松未成熟合子胚为外植体,分析影响湿地松胚性愈伤组织诱导的主要因素:对外植体材料的预处理进行研究,利用单因素实验筛选适合湿地松未成熟球果的采集及冷藏时间;对培养基中影响胚性愈伤组织诱导的主要添加物中的碳源(麦芽糖、蔗糖和葡萄糖)、肌醇和抗坏血酸进行单因素实验,对α-酮戊二酸和丙酮酸进行双因素实验分析;最后对影响胚性愈伤组织诱导的主要激素,如生长素(2,4-二氯苯氧乙酸)和细胞分裂素(6-苄基氨基嘌呤和激动素)进行随机组合分析。【结果】①未成熟湿地松球果体胚最佳诱导时间为6月底至7月中上旬;②1 000 mg/L肌醇和30 g/L麦芽糖最适宜湿地松胚性愈伤组织的诱导;③抗坏血酸质量浓度为0、1、2、3 mg/L时,对湿地松胚性愈伤组织诱导的影响区别不大;但同时添加α-酮戊二酸和丙酮酸时明显促进了湿地松愈伤组织的诱导;④湿地松最佳诱导培养基与激素组合为基本培养基LP+2 mg/L 2,4-二氯苯氧乙酸(2,4-D)+2 mg/L 6-苄基氨基嘌呤(6-BA)+2.5 mg/L激动素(KT)。【结论】通过对湿地松球果的预处理,获得了适合湿地松愈伤组织诱导的材料,并且确定了适合胚性愈伤组织诱导的合子胚发育阶段,即2~4阶段,以及合适的采集期。另外,对诱导培养基中主要添加物和激素进行研究,筛选出了1种较优的湿地松愈伤组织诱导的培养基,确定了适合湿地松胚性愈伤组织诱导的主要条件,可为提高湿地松胚性愈伤组织诱导率、促进湿地松体胚发生提供参考。

关键词: 湿地松, 松针褐斑病, 合子胚, 体细胞胚胎发生, 胚性愈伤组织诱导

Abstract:

【Objective】This study aimed to establish an efficient and practical in vitro rapid propagation system for excellent clones of slash pine (Pinus elliottii) resistant to brown spot needle blight (pathogen: Lecanosticta acicola), and provided a technical basis for the commercial application of excellent seedlings of P. elliottii. 【Method】Immature zygotic embryos of P. elliottii from the resistant families were used as explants to explore the main factors affecting embryogenic callus induction. First, the pre-treatment of the explant material was investigated, using single-factor tests to screen for the appropriate collection of P. elliottii immature cones and the time of refrigeration of pine cones. Furthermore, single-factor tests were performed on the carbon sources (maltose, sucrose and glucose), inositol, and ascorbic acid in the main additions to the medium affecting the induction of embryogenic callus, as well as two-factor tests for α-ketoglutaric acid and pyruvic acid. Finally, random combination probability analysis was performed for auxin 2,4-dichlorophenoxyacetic acid and cytokinins (6-benzylaminopurine and kinetin). 【Result】(1) The best time for embryogenic tissue induction for immature P. elliottii cones was in late June to early mid-July in Guanzhuang Forest Farm, Fujian Province; (2) the addition of 30 g/L maltose and 1 000 mg/L inositol were the most suitable for the induction of P. elliottii embryogenic callus; (3) ascorbic acid (0, 1, 2, 3 mg/L) had little effect on callus induction of P. elliottii; however, the addition of α-ketoglutarate and pyruvate added simultaneously obviously promoted callus induction in P. elliottii; (4) the best induction medium and hormone combination of P. elliottii were LP(lobbly pine) media supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/L 6-benzylaminopurine (6-BA) and 2.5 mg/L kinetin (KT).【Conclusion】We obtained a better material for P. elliottii embryogenic tissue induction by pretreatment of P. elliottii cones and identified suitable zygotic embryo developmental stages, i.e., stages 2-4, for embryonic tissue induction. To study the main additives and hormones in the induction medium, we obtained a better callus induction medium for P. elliottii and determined the main conditions suitable for the induction of embryonic calli, thus providing a basis for improving the embryogenic callus induction rate and promoting the development of P. elliottii somatic embryogenesis.

Key words: Pinus elliottii, brown spot needle blight(pathogen: Lecanosticta acicola), zygotic embryos, somatic embryogenesis, embryogenic callus initiation

中图分类号:

S763

程方,孙婷玉,叶建仁. 抗松针褐斑病湿地松未成熟合子胚胚性愈伤组织的诱导[J]. 南京林业大学学报（自然科学版）, 2023, 47(6): 175-182.

CHENG Fang, SUN Tingyu, YE Jianren. Induction of embryogenic callus from immature zygotic embryos of Pinus elliottii resistant to brown spot needle blight (pathogen: Lecanosticta acicola)[J].Journal of Nanjing Forestry University (Natural Science Edition), 2023, 47(6): 175-182.DOI: 10.12302/j.issn.1000-2006.202203002.

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处理 treatment		不同类型愈伤组织诱导率/% callus initiation rate					出愈率/% initiation rate
处理 treatment		Ⅰ、Ⅱ	Ⅲ	Ⅳ	Ⅴ	Ⅵ	出愈率/% initiation rate
采集时间(月-日) collection time n=50	06-15	0	31.7	2.8	38.9	26.6	34.5
	06-22	0	33.5	3.2	50.9	12.4	36.7
	06-28	0	58.4	6.8	32.5	2.3	65.2
	07-04	0	64.3	11.3	22.6	1.8	75.6
	07-13	0	76.8	12.1	9.8	1.3	88.9
	07-28	0	12.3	2.2	34.6	51.9	14.5
	08-03	0	13.6	0	32.5	53.9	13.6
家系号 family trees code n=35	9	0	68.5	12.1	14.1	5.3	80.6
	8	0	59.9	7.9	28.6	3.6	67.8
	34	0	65.6	15.6	15.9	2.9	81.2
	2	0	66.8	12.8	18.6	1.8	79.6
	23	0	72.4	13.4	14.2	0	85.8
	39	0	68.9	7.8	19.5	17.2	76.7
	11	0	70.5	10.0	14.6	4.9	80.5
	25	0	70.7	11.7	12.5	4.9	82.4
球果冷藏时间/d cone refrigeration time n=32	0	0	58.9	19.6	12.8	8.7	78.5
	5	0	55.8	24.8	13.6	5.8	80.6
	15	0	51.3	24.5	10.3	13.9	75.8
	30	0	53.8	16.6	16.9	12.7	70.4
	60	0	52.5	16.2	20.5	10.8	68.7

处理 treatment			不同类型愈伤组织诱导率/% callus initiation rate												出愈率/% initiation rate
处理 treatment			Ⅰ		Ⅱ		Ⅲ		Ⅳ		Ⅴ		Ⅵ		出愈率/% initiation rate
糖种类及质量浓度/(g·L^-1) sugar type and concentration n=35	S1		0		0		65.4		5.1		22.5		7.0		70.5
	S2		0		0		70.2		8.4		19.8		1.2		78.6
	S3		0		0		55.4		8.2		25.8		10.6		63.6
	S4		0		0		48.6		11.1		30.5		9.8		59.7
	S5		0		0		48.4		7.6		25.5		18.5		56.0
	S6		0		0		47.8		9.5		22.7		10.0		57.3
肌醇质量浓度/(mg·L^-1) inositol concentration n=35	0		0		0		33.5		12.3		40.2		14.0		45.8
	100		0		0		32.7		14.0		44.6		8.7		46.7
	300		0		0		35.6		8.3		45.8		10.3		43.9
	500		0		0		38.9		9.7		45.7		5.7		48.6
	1 000		2.9		0		25.5		7.0		35.8		8.8		35.4
	2 000		0		0		30.5		11.8		38.7		19.0		42.3
抗坏血酸质量浓度/(mg·L^-1) ascorbic acid concentration n=35		0		0		0		58.5		18.4		18.6		4.5	76.9
		1		0		0		55.5		15.1		18.2		11.2	70.6
		2		0		0		52.0		20.5		17.5		10.0	72.5
		3		0		0		56.0		22.1		16.0		5.9	78.1
α-酮戊二酸和丙酮酸质量浓度/(mg·L^-1) alpha-ketoglutaric acid and pyruvate concentration n=40		A		0		0		45.5		25.3		20.6		8.6	70.8
		B		0		0		48.8		27.5		16.3		7.4	76.3
		C		0		0		53.3		22.6		14.8		9.3	75.9
		D		0		0		66.0		19.8		8.5		5.7	85.8

处理号 treatment code	激素质量浓度/(mg·L^-1) hormone concentration			不同类型愈伤组织诱导率/% callus initiation rate						出愈率/% initiation rate
处理号 treatment code	2,4-D	6-BA	KT	Ⅰ	Ⅱ	Ⅲ	Ⅳ	Ⅴ	Ⅵ	出愈率/% initiation rate
Ⅰ-26	2	2.0	2.5	0	2.9	52.3	23.7	15.4	5.7	78.9
Ⅰ-36	2	4.0	2.5	0	0	55.4	30.3	10.1	4.2	85.7
Ⅰ-40	4	0.5	1.5	0	0	51.6	32.2	12.6	3.6	83.8
Ⅰ-127	10	2.0	4.0	0	0	49.3	31.2	13.0	6.5	80.5
Ⅰ-128	10	3.0	0	1.8	0	56.9	31.7	7.5	2.1	90.4
Ⅰ-129	10	3.0	0.5	0	0	45.3	30.4	15.9	8.4	75.7
Ⅰ-130	10	3.0	1.5	0	0	48.6	29.8	16.0	5.6	78.4
Ⅰ-14	2	0.5	0.5	0	0	15.5	4.1	47.9	32.5	19.6
Ⅰ-16	2	0.5	2.5	0	0	23.6	9.0	43.1	24.3	32.6
Ⅰ-17	2	0.5	4.0	0	0	24.3	5.1	46.8	23.8	29.4
Ⅰ-18	2	1.0	0	0	0	19.2	8.3	40.4	32.1	27.5
Ⅰ-20	2	1.0	1.5	0	0	18.5	7.7	48.1	25.7	26.2
Ⅰ-38	4	0.5	0.5	0	0	15.7	4.7	47.1	32.5	20.4
Ⅰ-39	4	0.5	1.5	0	0	14.2	11.1	35.8	38.9	25.3

抗松针褐斑病湿地松未成熟合子胚胚性愈伤组织的诱导

Induction of embryogenic callus from immature zygotic embryos of Pinus elliottii resistant to brown spot needle blight (pathogen: Lecanosticta acicola)

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