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

doi:10.12302/j.issn.1000-2006.202203002

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2023, Vol. 47 ›› Issue (6): 175-182.doi: 10.12302/j.issn.1000-2006.202203002

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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

Abstract

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

CLC Number:

S763

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, 2023, 47(6): 175-182.

Figures/Tables 5

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Table 1

Table 2

Table 3

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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

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

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