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Ultralow-temperature preservation and recovery culture method for Heuchera micrantha cluster buds

A technology for cryopreservation and recovery culture, which is applied in the field of cryopreservation and regeneration of plants induced by alum root leaves to induce clustered buds. It can solve the problems of affecting survival rate and mechanical damage, simplify the operation steps, avoid mechanical damage, and facilitate batch processing. Effect

Active Publication Date: 2020-08-11
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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  • Ultralow-temperature preservation and recovery culture method for Heuchera micrantha cluster buds
  • Ultralow-temperature preservation and recovery culture method for Heuchera micrantha cluster buds
  • Ultralow-temperature preservation and recovery culture method for Heuchera micrantha cluster buds

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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 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 an ultralow-temperature preservation and recovery culture method for Heuchera micrantha cluster buds. The method comprises the following steps: 1, establishing an aseptic seedling system; 2, inducing leaf cluster buds; 3, pre-culturing the cluster buds; 4, mechanically fixing the cluster buds; 5, loading the cluster buds and carrying out vitrification treatment; 6, freezing, unfreezing and washing; 7, carrying out recovery culture. According to the method, the heuchera micrantha leaf induced cluster buds are successfully preserved at ultralow temperature for the first time, and the growth recovery condition of the preserved heuchera micrantha is good. According to the method, the whole basal disc with the leaves of the cluster buds serves as a cryopreservation material, the tedious technical means of stem tip stripping is omitted, operation steps are further simplified through mechanical fixing batch treatment, and finally regeneration plants of the Heuchera micrantha are obtained; therefore, the method has good reference value for establishing a convenient, stable and efficient Heuchera micrantha ultralow-temperature preservation system, being beneficial tolarge-scale preservation of Heuchera micrantha resources and construction of a resource library.

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 Applications(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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