Cryogenic preservation method of embryogenic callus of pine needle brown spot disease resistant Pine slash

A technology of embryogenic callus and cryopreservation, which is applied to the preservation of plants, gardening methods, botanical equipment and methods, etc., to achieve good growth status and improve the effect of cryopreservation

Active Publication Date: 2021-08-10
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1986, Ye Jianren and others established a disease-resistant slash pine seed garden in Fujian, but traditional breeding methods are difficult to meet the requirements of large-scale mass production in a short period of time (Ye Jianren et al., 1991)

Method used

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  • Cryogenic preservation method of embryogenic callus of pine needle brown spot disease resistant Pine slash
  • Cryogenic preservation method of embryogenic callus of pine needle brown spot disease resistant Pine slash
  • Cryogenic preservation method of embryogenic callus of pine needle brown spot disease resistant Pine slash

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Ultra-low temperature preservation of resistant slash pine embryogenic callus

[0027] 1 Pretreatment for cryopreservation

[0028] 10-12 days after subculture (the 24th time), take 0.3g pine embryogenic callus, place it on the hyperosmotic medium that has added 0.5mol / L sucrose and carry out pre-cultivation, set different culture time (12, 24, 36, 48h). Then, using different concentrations of DMSO (2.5, 5, 10, 15%), different concentrations of PEG4000 (0, 5, 10, 15%) and different concentrations of sucrose (0, 0.4, 0.5, 0.6mol / L) as Cryoprotectant pretreatment of callus. See Table 1 for details.

[0029] 2 cryopreservation

[0030] Place the pre-cultured embryogenic callus in a freezing tube, place the freezing tube with cryoprotectant in a programmed cooling device, set the cooling program, first drop from room temperature to 0°C, then -1°C / The temperature was lowered to -80°C at a rate of 1 min, and stored at -80°C for 30 minutes, and immediately put ...

Embodiment 2

[0052] Example 2 Determination of recovery of slash pine embryogenic callus after thawing

[0053] 1 Proliferation and maturation of callus after thawing

[0054] The washed callus was transferred to the LP proliferation medium, and the growth was resumed in the dark at a room temperature of 23±2°C, and the growth recovery of the callus was observed every 7 days. The recovered slash pine callus was transferred to the maturation medium, and the somatic embryo maturation of the callus was observed.

[0055] 2 Culture conditions and data analysis

[0056] Proliferation medium is LP+1.0mg / L NAA+0.3mg / L 6-BA+0.3mg / L 6-BA+15g / L maltose+6.5g / L agar, pH=5.8. The maturation medium is LP+10mg / L NAA+130g / L PEG8000+60g / L maltose+3g / L phytogel, pH=5.8. Cultivate in the dark at a temperature of 23±2°C.

[0057] 3 Recovery of callus tissue after thawing

[0058] As can be seen from Table 4, after the treated callus was frozen for 10 days, it was taken out and thawed out, and 14 of the 1...

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Abstract

The invention discloses a method for cryopreservation of embryogenic callus of pine needle brown spot disease-resistant slash pine. Appropriate pre-cultivation and the use of mixed cryoprotectants can help improve the effect of cryopreservation. After thawing at 32°C, the callus frozen for 90 days can resume regeneration after 1 month, and the regeneration rate of embryogenic callus is the highest. 100%, the regenerated callus grows well and still maintains the ability to differentiate into mature somatic embryos. This application preliminarily established a cryopreservation system for embryogenic callus of pine needle brown spot disease-resistant wetland pine embryogenic callus, providing technical reference for long-term maintenance of embryogenic callus of resistant wetland pine callus.

Description

technical field [0001] The invention relates to the preservation of plant callus, in particular to a method for ultra-low temperature preservation of pine needle brown spot disease-resistant slash pine embryogenic callus. Background technique [0002] Slash pine (Pinus elliottii) is a fast-growing evergreen tree of the genus Pinus in the family Pinaceae. It is native to the southeastern United States and is an important tree species for afforestation in southern my country. Since 1978, pine needle brown spot has occurred successively on slash pine introduced in southern my country, which severely limited the development of slash pine (Li Chuandao et al., 1987). Therefore, it is of great economic and ecological significance to carry out research on disease-resistant breeding of slash pine. In 1986, Ye Jianren and others established a disease-resistant slash pine seed garden in Fujian, but traditional breeding methods are difficult to meet the requirements of large-scale mass...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N3/00A01H4/00
CPCA01H4/00A01N3/00
Inventor 朱丽华杨帆夏馨蕊方天雨叶建仁
Owner NANJING FORESTRY UNIV
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