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Koelreuteria paniculata genetic transformation method

A method of genetic transformation, the technology of eucalyptus, applied in the field of genetic transformation of eucalyptus, can solve the problem of unsatisfactory effect of adventitious bud differentiation and rooting medium formula, germination rate and germination speed, callus induction medium, slow callus growth rate, rooting medium Problems such as insufficient rooting rate

Active Publication Date: 2021-03-19
CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, if the eucalyptus seed germination medium in patent 2018113475844 is adopted, the seed germination rate and germination speed, the callus growth rate of the callus induction medium are relatively slow, and the rooting rate of the rooting medium is far lower than that of the present invention. Patents 201410267835.3; 201410463255.1 are adopted; 201410607050.6; 201510124023.8; 201510231119.4; 201610697486.8; 201711019129.7 The effects of adventitious bud differentiation, proliferation medium, rooting medium and other formulas are not ideal
In short, there is currently no complete set of rapid tissue culture methods for eucalyptus transgenics with outstanding effects, which can not only successfully construct a eucalyptus transgenic system, but also be able to culture from seed germination, explant stem induction, callus differentiation to adventitious buds, and rooting. Efficiency and other aspects to achieve very good results

Method used

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  • Koelreuteria paniculata genetic transformation method
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  • Koelreuteria paniculata genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation of Sano Seeds Seeds

[0068] The seed seed seed seed seed seed is selected from the 5, 6, 10 mesh sieve, and the seeds greater than the mesh screen is soaked for 3-4 minutes, 50 g / L sodium bicarbonate solution repeatedly cleaned seeds thoroughly and concentrated sulfuric acid, and then 50g Sodium hydrogencarbonate solution of sodium hydrogen carbonate seeds 10-12 min, disinfectant treatment: 70% ethanol 45S, 0.1% liter concentrate 2-10 min, sterile water is rinsed three times to obtain a sterile seed, and the sprout culture is carried out after disinfection, where eucalyptus Seed promoted sputum solid medium DKW basic medium, according to Table 1: 6BA, 1.5mg / L, 2.0mg / L, NaA, 0.2mg / L, 0.4mg / L, GA 3 , 0.4 mg / l, 0.6 mg / L, agar powder 4g / L and the tandem 1.2 g / L fixed addition. Placed in a 25 ± 2 ° C environment, 16 / 8h light cycle environment, light intensity is 60 μmol m -2 s -1 .

[0069] Table 1

[0070]

[0071] After obtaining a ...

Embodiment 2

[0072] Example 2 induction of foreign plants and callus

[0073] (1) Select the stem segment of sleep, soak for 12h, flush for 3 hours, cut into 1-2 buds, sterilization: 70% ethanol 45S, 0.1% liter with more 8 minutes, sterile water Rinse three times more than the aseptic epillaa, transferred to YD medium to culture until the sprout (silassic seedlings). Aseptic seedlings were transferred to the subsequent medium (also YD medium) to grow to 5-8 cm. The YD medium is MS base medium, attached to 6-BA 2 mg / L, IBA 0.1 mg / L, TDZ 0.5 mg / L. Placed in a 25 ± 2 ° C environment, 16 / 8h light cycle environment, light intensity is 60 μmol m -2 s -1 .

[0074] (2) Select 5-8 cm Aseptic seedling or aseptic seedlings in Example 1, cutting it into a stem of about 0.5 cm with sterilized blades, transfer to the rapid induction medium of eucalyptus cryogenic tissue. 1 / 2MS basic medium, followed by Table 2: IBA, 0.2mg / L, 0.25mg / L, 2.4D, 0.5mg / L, 1.0mg / L, Coconut juice, 10%, 15%; and 6...

Embodiment 3

[0087] Example 3 Agrobacterium hydrochloride preparation

[0088] (1) The transformed has been converted with a 35S promoter of -80 ° C, super rich, Southeast Sky HMA3 (AJF37113.1) CD transporter protein gene, GFP gene (for conversion detection) and resistance genes (for use in Screening) plasmid PBI1302 Agrobacterium LBA4404 bacterial ice melts (plasmid PBI1302 is purchased from Shanghai Baige Biocuri, converted to Agrobacterium by chemical conversion), draw 200 μL coated to LB plate (no antibiotics), 28 ° C, 200 rpm 24-48h, until a single colony.

[0089] (2) Picking the single colonies, inoculated to 2 ml of LB liquid medium containing an appropriate amount of antibiotics (including 50 mg / l kana, 25mg / l rif), 200 rpm in 28 ° C shock and cultured overnight to OD 600 0.6-0.8.

[0090] (3) Incoocataion into a 40 mLLB liquid medium (containing 50 mg / l kana, 25 mg / l RIF), 200 rpm at 28 ° C, 200 rpm, to the bacterial liquid OD 600 The value is between 0.5-0.8.

[0091] (4) Ca...

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Abstract

The invention discloses a koelreuteria paniculata genetic transformation method. The method comprises the following steps of (1) preparing a koelreuteria paniculata explant; (2) performing rapid induction of koelreuteria paniculata calluses; (3) performing infection and co-culture of agrobacterium liquid; (4) performing degerming and screening culture of the calluses; (5) performing adventitious bud differentiation culture; and (6) performing rooting culture. According to the koelreuteria paniculata genetic transformation method, aseptic seedlings are obtained from koelreuteria paniculata seedgermination; genetic transformation is carried out through an agrobacterium-mediated method with the calluses induced by all tissue parts as receptors and GFP genes as reporter genes; positive transformation receptor calluses are screened in a gradient mode through hygromycin resistance loci; the calluses are developed into a complete plant through differentiation culture, so that plant regeneration is achieved; and an efficient and rapid koelreuteria paniculata genetic transformation system is established.

Description

Technical field [0001] The present invention relates to the field of eucalyptus genetic transformation and composition culture, and in particular, a genetic conversion method of anemylomycin (CEF) gradient screening, which can be used to translate the positive transformation of the cemillomycin (CEF) gradient screening. It also includes rapid acquisition of eucalyptus rapid access and the high-efficiency culture medium proportional ratio such as callus, differentiation, and rooting required. Background technique [0002] Eucalyptus is unpracored to be a tree plant, which grows in calcium-based soil produced by limestone wind, and the plant isaten salt, drought, and short-term floods, with a deep root, which has strong adaptability to the environment. These characteristics have made eucalyptus into an important pioneer species of plant restoration in southern my country's heavy metal pollution zone. In addition, there is a strong resistance and control of dust, sulfur dioxide and ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/00
CPCC12N15/8205C12N15/8212A01H4/008A01H4/001
Inventor 王平周韬白磊何其浩孙吉康
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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