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A method for cryopreservation and recovery culture of wild rice stem tips

A technology of ultra-low temperature preservation and preservation method, which is applied to the preservation of wild rice shoot tips, the ultra-low temperature preservation of ordinary wild rice shoot tips, and the recovery culture field after ultra-low temperature preservation, and can solve the problem of small size, damage and difficulty of shoot tip meristems. Survival and other problems, to achieve the effect of restoring good growth condition, reducing damage and simplifying operation

Inactive Publication Date: 2018-06-15
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The cryopreservation of intact organs of wild rice will be the focus of future research. However, due to the small size of the shoot tip meristem of wild rice, the small and tender characteristics of the shoot tip tissue of wild rice, the physiological state is fragile, and it is protected by external protection. Leaf layers are tightly packed, easy to break and be damaged during stem tip stripping, pre-cultivation and vitrification operations, and difficult to survive during storage

Method used

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  • A method for cryopreservation and recovery culture of wild rice stem tips
  • A method for cryopreservation and recovery culture of wild rice stem tips
  • A method for cryopreservation and recovery culture of wild rice stem tips

Examples

Experimental program
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Effect test

Embodiment 1

[0020] The acquisition of embodiment 1 wild rice aseptic seedling

[0021] refer to figure 1 As shown, the induction and subculture steps of wild rice aseptic seedlings are as follows: take the underground stems of wild rice with germinated stems, wash them repeatedly for about 1 hour under running water, and then disinfect them with 70% alcohol for 1 min, 0.1% mercurial disinfection for 15 minutes, rinsed several times with sterile water, stripped about 2mm of the shoot apex under the dissecting microscope, and inoculated it with 4 mg L -1 BA and 0.5mg·L -1 Cluster shoots were induced on MS medium with NAA. The culture temperature was 25±2°C, and the light was 12 h d -1 , light intensity 36µmolm -2 the s -1 . After the clustered shoots were induced, the clustered shoots were cut off and inoculated in a medium containing 2 mg·L -1 BA and 0.5 mg·L -1 NAA was subcultured on MS medium. Subculture every 3 months.

Embodiment 2

[0022] Gradient pre-cultivation of embodiment 2 wild rice

[0023] refer to figure 1 As shown in the gradient domestication culture of sterile seedlings, the steps of peeling off the shoot tip and shoot tip pre-cultivation are: get the tissue culture seedling gained in Example 1, cut off the upper plant, leave about 3 cm of the base, and put about 1 cm of the root into the plant that does not contain After 7 days of culture in the MS medium of hormones, take the buds that sprout 1 cm from the base, and transfer them to 0.3 molL sucrose -1 and abscisic acid 5mgL -1 cultured on culture medium. After 7 days, transfer to 0.7molL -1 and abscisic acid 5mgL -1 pre-cultured on medium for 5 days. Under the dissecting microscope, the shoot tip of 2-3mm was stripped, and the shoot tip containing 0.4 molL -1 and glycerol 2moL -1 , without NH4 + Pre-culture on MS solid medium for 16 hours.

Embodiment 3

[0024] The device fixing of embodiment 3 wild rice stem tips

[0025] refer to figure 1 with Figure 5 As shown, the steps of fixing the shoot tip to the loading strip and pretreatment and vitrification treatment are: lightly dip the shoot tip pre-cultured in Example 2 with tweezers in 2-4% calcium ion-free sodium alginate solution , that is, neatly placed on a 5mm×20mm metal mesh loading strip, with 10 stem tips on each loading strip. Dip the loading strip with the shoot tip into a solution containing 0.2% CaCl, 0.4 molL -1 Sucrose and 2moL -1 Glycerin, NH4 Free +After being fixed for 20 min in MS liquid medium, place the loading strip on sterile filter paper to absorb excess liquid.

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Abstract

A method for ultralow-temperature storage and renewal cultivation of wild rice stem tips includes steps: taking a subcultured sterile tissue culture seedling, keeping the base portion, culturing in a culture medium free of hormone MS, cutting new sprouts of the base portion, transferring to a culture medium containing 0.3molL<-1> of sucrose and 0.5-5mgL<-1> of abscisic acid to realize culturing, transferring to a culture medium containing 0.7molL<-1> of sucrose and 0.5-5mgL<-1> of abscisic acid to realize preculture, stripping the stem tip, pre-culturing in an MS solid culture medium containing 0.4molL<-1> of sucrose and 2molL<-1> of glycerin, gently dipping in 2-4% calcium-free sodium alginate solution, putting on a loading strip, immersing in an MS liquid culture medium containing 0.1-0.3% of calcium chloride, 0.4molL<-1> of sucrose and 2molL<-1> of glycerin, immobilizing, sucking excessive liquid, treating in a pretreatment solution, transferring to a vitrification protective agent PVS2 to realize ice-bath treatment, and putting into liquid nitrogen to realize storage.The method has the advantages of simplicity, convenience and feasibility in storage, stability, reliability and favorable recovery growth conditions of stored wild rice.

Description

technical field [0001] The invention relates to a method for preserving the stem tip of wild rice, in particular to a method for ultra-low temperature preservation of the stem tip of common wild rice. At the same time, the invention also relates to a recovery culture method after cryopreservation of wild rice stem tip, which belongs to the technical field of plant cell engineering. Background technique [0002] Wild rice resources are an important part of rice seed resources. It preserves the genes that have not been or disappeared in cultivated rice, and has special excellent traits and high resistance to diseases and insect pests. Yield and quality are of inestimable value. The current rapidly deteriorating environment and unfavorable climatic conditions are eroding the genetic diversity of rice; the rapid expansion of human economic activities in modern times has led to the loss and deterioration of wild rice habitats, and the sharp reduction of habitats; The biological...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00A01N3/00
CPCA01H4/005A01N3/00
Inventor 林田杨华王飞魏士伟张前荣王国军石群芳李天菲龙萍罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT
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