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Method for ultralow-temperature preservation of European spruce embryonic calluses

An embryogenic callus, ultra-low temperature preservation technology, applied in horticultural methods, plant preservation, botanical equipment and methods, etc., can solve the potential risks of large differences between genotypes, large differences, and recovery of frozen materials increase, etc.

Pending Publication Date: 2021-04-23
CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the reported literature related to the cryopreservation of embryogenic callus of European spruce, the dehydrating agent and protective agent used are not single enough and need to be used in combination, which brings certain uncertainties to the cryopreservation technology. Increased potential risk of material regrowing
[0006] In addition, it has been reported that the effect of cryopreservation on embryogenic callus of European spruce has great differences among genotypes (Dedicova B, Nilsson O, Egertsdotter U. Effect of cryopreservation on growth of different elite embryogenic cell lines of Norway spruce(Piceaabies(L.)Karst.)[J].Acta horticulturae,2011,908:203-206.; Hu Jiwen et al., Advances in Cryopreservation Technology of Picea Abies Embryogenetic Tissue. Temperate Forestry Research, Vol.2, No.1, 2019.03)
For example Norgaard et al. (Jens V N, Valerie D, Cystein J, et al. Variations in cryotolerance of embryogenic Picea abies cell lines and the association to genetic, morphological, and physiological factors [J]. Canadian Journal of Forest Research, 1993, 23 (12) : 2560-2567.) tested the cryopreservation of 81 different genotypes of Picea spruce embryogenic tissues, 95% of ETs could be refolded successfully, but only 63% of the genotypes were successfully restored to growth, and the differences between genotypes were large

Method used

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  • Method for ultralow-temperature preservation of European spruce embryonic calluses
  • Method for ultralow-temperature preservation of European spruce embryonic calluses
  • Method for ultralow-temperature preservation of European spruce embryonic calluses

Examples

Experimental program
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Effect test

Embodiment 1

[0036] In this example, the European spruce 2-4-17 cell line is used as the callus material, and the orthogonal test method is used to evaluate the suspension concentration treatment, pre-culture treatment, protective agent treatment and other conditions, and then screen the ideal cryopreservation The method specifically includes the following steps:

[0037] Step 1, preparation of embryogenic callus suspension:

[0038] The embryogenic callus of the Picea spruce 2-4-17 cell line was suspended in 10 ml of liquid medium to prepare a suspension.

[0039] Wherein the liquid culture medium formula used is: 950mg L -1 KNO 3 , 825mg·L -1 NH 4 NO 3 , 170mg·L - 1 K H 2 PO 4 , 925mg·L -1 mgSO 4 ·7H 2 O, 11 mg L -1 mgCl 2 ·6H 2 O, 10000mg·L -1 Sucrose, 15mg·L - 1 FeSO 4 ·7H 2 O, 18 mg·L -1 EDTA-Na 2 , 0.1mg·L -1 Thiamine HCl, 0.1mg L -1 Pyridoxine HCl, 0.5mg·L -1 Nicotinic acid, 15.5mg L -1 h 3 BO 3 , 10.5mg·L -1 MnSO 4 ·H 2 O, 21.5mg·L - 1 ZnSO 4 ·7H ...

Embodiment 2

[0061] Test the optimal cryopreservation method (treatment 4) obtained by the orthogonal experiment screening in Example 1, and its cryopreservation effect on other cell lines of Picea spruce.

[0062] The test materials are European spruce 2-4-4 cell line, European spruce 2-4-17 cell line, 2-4-45 cell line, 2-11-24 cell line, 2-11-29 cell line embryo Sexual callus material.

[0063] The cryopreservation method adopts the method described in Example 1 to process 4, specifically:

[0064] Step 1, preparation of embryogenic callus suspension:

[0065] The European spruce 2-4-4 cell line, European spruce 2-4-17 cell line, 2-4-45 cell line, 2-11-24 cell line, 2-11-29 cell line embryogenic healing Wounded tissues were suspended in 10ml of liquid medium, and suspensions with concentrations of 0.05, 0.1, and 0.2g / ml were made respectively (2-4-17 was only prepared with two concentrations of 0.05 and 0.2g / ml).

[0066] Wherein the liquid culture medium formula used is with embodime...

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PUM

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Abstract

The invention relates to a method for ultralow-temperature preservation of European spruce embryonic calluses. The method comprises the following steps of: preparing European spruce embryonic callus suspension; carrying out ultralow-temperature preservation pretreatment on the embryonic callus suspension by using a dehydrating agent and a cryoprotectant to prepare a frozen material; performing programmed cooling freezing on the frozen material; after freezing and cooling, storing the frozen material into a liquid nitrogen tank; and unfreezing the frozen material from a liquid nitrogen environment to a room temperature environment, recovering growth, and detecting the renaturation rate. According to the ultralow-temperature preservation method, the formula of the treating fluid is simple, the renaturation growth rate of a specific European spruce genotype strain is high, and growth is good.

Description

technical field [0001] The invention belongs to the technical field of low-temperature preservation of plant cells, and in particular relates to a method for ultra-low temperature preservation of European spruce embryogenic callus. Background technique [0002] Picea spp. is a large evergreen tree species with strong adaptability, strong stress resistance, and rapid growth. It is an important economic and timber tree species. European spruce (Picea abies) is widely distributed in central and northern Europe, and is vertically distributed at an altitude of about 2000m. European spruce likes a cool and humid climate, and is also resistant to atmospheric drought. It has a wide range of adaptability to temperature and soil. It has great potential for expansion and introduction in my country. It is currently the best performing tree species of foreign spruce plants in domestic suitable habitats. European spruce is an important tree species for pulpwood and construction timber. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N3/00A01H4/00
CPCA01N3/00A01H4/001A01H4/005
Inventor 朱天擎胡继文张一凡凌娟娟王军辉马建伟胡勐鸿
Owner CHINESE ACAD OF FORESTRY
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