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A method for cryopreservation and recovery culture of clustered buds of alum roots

A technology of ultra-low temperature preservation and recovery culture, which is applied in the field of ultra-low temperature preservation and regeneration of plants for inducing cluster buds from alum root leaves, can solve problems such as affecting the survival rate, mechanical damage, etc., achieves simplified operation steps, avoids mechanical damage, and is conducive to batch processing Effect

Active Publication Date: 2022-05-03
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its good genetic stability, the shoot tip tissue is generally used as cryopreservation materials. However, because the shoot tip tissue is small and tender, it requires a high level of technical staff. Vulnerable to mechanical damage, affecting survival rate

Method used

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  • A method for cryopreservation and recovery culture of clustered buds of alum roots
  • A method for cryopreservation and recovery culture of clustered buds of alum roots
  • A method for cryopreservation and recovery culture of clustered buds of alum roots

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Experimental program
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Effect test

Embodiment 1

[0036] The acquisition of embodiment 1, alum root aseptic seedling and and multiplication

[0037] Stir and clean the base tissue of the alum rhizome with detergent solution, rinse it with tap water for 1 hour, and place it on an ultra-clean workbench; soak it in 75% ethanol for 30 seconds, and then soak it in 10% sodium hypochlorite solution with Tween for 12 minutes after washing ;Finally rinsed with sterile water for 5 times, blotted the surface moisture with sterile gauze, inoculated on the medium containing 6-BA 2.0mg / L+NAA0.1mg / LMS to induce aseptic buds, and obtained aseptic vaccines ( figure 1 The alum root sterile seedlings subcultured for 3 months) were subcultured on MS medium containing 6-BA 0.1 mg / L, the culture temperature was 24±2°C, and the light intensity was 30-35 μmol·m -2 ·s -1 , the light time is 12h / d. Passage once every 30 days ( figure 2 The alum-root bushy seedlings of subculture propagation 30d).

Embodiment 2

[0038] The cluster bud induction of embodiment 2, alum root

[0039] Get the aseptic tissue culture plantlet of embodiment 1 gained, get the aseptic tissue culture plantlet blade of 5mm, put into cluster bud induction culture medium (MS medium+6-BA 2mg / L+NAA 0.1mg / L) upside down , after 15 days, light green buds began to appear and a small amount of small buds grew out ( image 3 Leaf basal plate with clustered buds), clustered buds formed after 30d.

Embodiment 3

[0040] The cryopreservation process of embodiment 3, the ultra-low temperature freeze-preservation process of clustered buds of alum roots:

[0041]Get the leaf base disc ( image 3 (leaf discs with clustered buds) were transferred to 0.3mol / LMS medium containing sucrose and cultured for 3 days, and the leaf base discs with clustered buds were connected in series with sterilized 12-15mm pins with tweezers, and each needle was connected in series 4 leaf base discs to form a leaf base disc string ( Figure 4 Leaf base disk string), the leaf base disk string is transferred to the culture bottle containing the pretreatment solution (MS medium+1.2mol / L glycerol+0.4mol / L sucrose) containing 0.4mol / L sucrose and 1.2mol / L glycerol After slowly shaking and culturing at room temperature for 1 hr, transfer to a cryotube filled with precooled vitrification agent PVS2 (MS medium + 15% DMSO + 15% ethylene glycol + 30% glycerol + 0.4mol / L sucrose) 20-60 minutes of ice bath treatment, when ...

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Abstract

The invention discloses a method for ultra-low temperature preservation and recovery cultivation of alum root cluster buds; comprising the following steps: 1. establishment of a sterile seedling system; 2. induction of leaf cluster buds; 3. pre-cultivation of cluster buds; 4. cluster bud cultivation Mechanical fixation; 5. Bulk loading and vitrification treatment; 6. Freezing and thawing washing; 7. Recovery culture. The invention proposes for the first time that the clustered buds induced by the leaves of the alum root are successfully preserved at ultra-low temperature, and the alum root recovers and grows in good condition after preservation. The present invention uses the clustered buds with leaves as a whole as a frozen storage material, saves the tedious technical means of stem tip stripping, and further simplifies the operation steps through mechanical fixing and batch processing, and finally obtains the regenerated plants of alum roots; therefore, for The establishment of a convenient, stable and efficient ultra-low temperature preservation system for alum root is conducive to the large-scale preservation of alum root resources and the construction of a resource bank, which has a good reference value.

Description

technical field [0001] The invention belongs to the technical field of plant cell engineering and relates to a method for ultra-low temperature preservation and recovery cultivation of clustered buds of alum root, in particular to a method for ultra-low temperature preservation and regeneration of plants induced by clustered buds of alum root leaves. Background technique [0002] Heuchera micrantha (Heuchera micrantha) is a perennial colorful herbaceous flower of the saxifrage family Heuchera genus. Because of its colorful evergreen leaves and its characteristics of cold resistance, shade tolerance, drought resistance, and salt-alkali tolerance, it is widely used in gardens and has good development prospects. There are more than 40 species and many horticultural hybrids in the genus Alumia, and the effective preservation of its germplasm resources is very important for the cultivation of new varieties with independent intellectual property rights. The conventional propagati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N3/00A01H4/00
CPCA01N3/00A01H4/001A01H4/005A01H4/008
Inventor 林田殷丽青腾小英李天菲韩静周丽杨华刘鸿艳龙萍罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT
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