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Method for ultralow-temperature preservation of embryonic tissues of larix principis-rupprechtii

A technology for cryopreservation and embryogenic tissue, which is applied in the field of cryopreservation of embryogenic callus of larch cell line and maintenance of plant callus

Pending Publication Date: 2020-07-31
BEIJING FORESTRY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for cryopreservation of larch embryonic tissue in North China in view of the deficiencies in the pretreatment method, cryoprotectant and thawing method after cryopreservation in the cryopreservation process of the existing plant callus, Improve pre-culture efficiency and restore growth efficiency by adjusting pre-treatment medium culture

Method used

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  • Method for ultralow-temperature preservation of embryonic tissues of larix principis-rupprechtii
  • Method for ultralow-temperature preservation of embryonic tissues of larix principis-rupprechtii
  • Method for ultralow-temperature preservation of embryonic tissues of larix principis-rupprechtii

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Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1 test material

[0066] The embryogenic suspension of the stable North China larch embryogenic cell line BL13-25a for 3-4 days was selected as the test material for ultra-low temperature cryopreservation research, as follows:

[0067] (1) Induction of embryogenic callus of Larix chinensis

[0068] Using immature zygotic embryos of Larix chinensis as explants, embryogenic callus was induced. The solid medium for embryogenic callus induction is: 1 / 2LV+2,4-D 1.5-2.5mg / L+6-BA 0.5-1.5mg / L+sucrose 30g / L+plant gel 3g / L+hydrolyzed casein 400mg / L+glutamine 500mg / L, preferably 1 / 2LV+2,4-D2mg / L+6-BA 1mg / L+sucrose 30g / L+plant gel 3g / L+hydrolyzed casein 400mg / L+glutamine 500mg / L, pH 5.8, cultured in the dark at 24±1°C for 6 weeks, induced embryogenic callus. (LV medium is: KNO 3 1900mg / L+NH 4 NO 3 1650mg / L+KH 2 PO 4 340mg / L+CaCl 2 .2H 2 O 22mg / L+MgSO 4 .7H 2 O 1850mg / L+H 3 BO 3 31mg / L+ZnSO 4 .7H 2 O 43mg / L+MnSO 4 .H 2 O 21mg / L+Na 2 MoO 4 .2H 2 O...

Embodiment 2

[0076] 1. Pre-culture treatment

[0077] According to the pre-culture method commonly used in coniferous trees, this study was carried out by adding high concentrations of sucrose or sorbitol for gradient culture. The pre-cultivated culture medium adopts the liquid proliferation medium, and carries out gradient pre-cultivation at the concentration of 0.2 mol / L and 0.4 mol / L respectively, and cultures for 24 hours at each concentration.

[0078] 1-1) The first stage of pre-cultivation treatment

[0079] Take the embryogenic suspension of subculture for 3-4 days as the test material, add 30ml of the first preculture medium in a 100ml Erlenmeyer flask, and carry out the first stage of preculture treatment; wherein:

[0080] The initial inoculation amount is 2% (w / v), that is, 0.6 g of embryogenic callus is added to the medium;

[0081] The first pre-medium is: 1 / 2LV+2,4-D 0.2mg / L+6-BA 0.1mg / L+sucrose 30g / L+hydrolyzed casein 400mg / L+glutamine 500mg / L+0.2mol / L Sorbet alcohol;

...

Embodiment 3

[0098] 1. Pre-culture treatment

[0099] 1-1) The first stage of pre-cultivation treatment

[0100] Same as embodiment 2;

[0101] 1-2) The second stage of pre-cultivation treatment

[0102] All the other are identical with embodiment 2;

[0103] 2. Freezing treatment

[0104] Except cryoprotectant solution is sorbitol 0.4mol / L+DMSO 10%, all the other are the same as embodiment 2.

[0105] 3. Thawing treatment

[0106] Same as Example 2.

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Abstract

The invention discloses a method for ultralow-temperature preservation of embryonic tissues of larix principis-rupprechtii. The method comprises the steps of carrying out liquid gradient pre-culture treatment with gradually increased penetrant concentration on the embryonic tissues of the larix principis-rupprechtii (cell line embryonic calli of the larix principis-rupprechtii), and carrying out ultralow-temperature cryopreservation on the embryonic tissues of the larix principis-rupprechtii subjected to the gradient pre-culture treatment, and carrying out unfreezing treatment during use. Themethod is suitable for long-term ultralow-temperature preservation of excellent embryonic cell lines of the larix principis-rupprechtii; and after embryonic cells of the larix principis-rupprechtii are preserved for a long time by adopting the method, the cell survival rate is high, embryonic tissue recovery culture is rapid, and the recovery growth rate of the embryonic calli is high and reaches66% or above.

Description

technical field [0001] The invention relates to a method for maintaining plant callus, in particular to a low-temperature preservation method for cell line embryogenic callus of North China larch, and belongs to the technical field of tissue culture of tree breeding. Background technique [0002] Under normal temperature conditions, long-term subculture of embryogenic cultures often has many unavoidable problems: first, long-term repeated subculture, high subculture costs; and, if there is human or environmental interference during the operation, Contamination of the culture is likely to cause a major loss of embryogenic cell lines; what is more noteworthy is that after a long period of subculture, it may cause somatic clonal variation, resulting in loss of embryogenic ability of the embryogenic culture. decline or even loss. Ultra-low temperature cryopreservation technology is currently a very widely used and successful long-term storage method for germplasm. It can effect...

Claims

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Application Information

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IPC IPC(8): A01H4/00A01N3/00
CPCA01H4/005A01N3/00Y02P60/40
Inventor 赵健陈晓艺江帅菲张金凤孔立生崔莹李珊珊
Owner BEIJING FORESTRY UNIVERSITY
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