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Optimized tissue culture method for Columbia type arabidopsis thaliana
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A technology of tissue culture and optimization of medium, applied in the field of bioengineering, can solve problems such as poor growth, slender taproots, narrow rosette leaves, etc.
Inactive Publication Date: 2016-05-04
畲茂云
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However, our team found that Arabidopsis thaliana seedlings grown on ordinary 1 / 2 MS or MS medium grew poorly after transplanting, showing slender and weak main roots, sparse lateral roots, and narrow and thin rosette leaves, which directly affected The progress of related plant gene function research on Arabidopsis
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Embodiment 1
[0026] Embodiment 1, wild Columbia type Arabidopsis seed tissue culture
[0027] 1. Preparation and sterilization of culture medium
[0028] MS: 10×MS large amount 100ml, 100×MS trace amount 10ml, 200×MS iron salt 5ml, sucrose 30g, set the volume to 1000ml, then adjust the pH to 6.0 with HCl and NaOH, then add 10g of domestic agar, the above ingredients are used at 121℃ Autoclave for 20 minutes.
[0029] 1 / 2 MS: 10×MS large amount 50ml, 100×MS trace amount 10ml, 200×MS iron salt 5ml, sucrose 30g, set the volume to 1000ml, then adjust the pH to 6.0 with HCl and NaOH, then add 10g of domestic agar, the above ingredients are Autoclave at 121°C for 20 minutes.
[0030] 1 / 4MS: 25ml of 10×MS large amount, 10ml of 100×MS trace amount, 5ml of 200×MS iron salt, 30g of sucrose, set the volume to 1000ml, then adjust the pH to 6.0 with HCl and NaOH, then add 10g of domestic agar. Autoclave at 121°C for 20 minutes.
[0047] Embodiment 2, genetic transformation of wild Arabidopsis thaliana
[0048] 1. Preparation and sterilization of culture medium
[0049] 1 / 2 nitrogen optimized medium: KH 2 PO 4 0.17g, MgSO 4 ·7H 2 O0.37g, CaCl 2 2H 2 O0.44g, KOH1.053g, NH 4 NO 3 0.825g, HNO 3 0.163ml, 100×MS trace amount 10ml, 200×MS iron salt 5ml, sucrose 30g, set the volume to 1000ml, then adjust the pH to 6.0 with HCl and NaOH, then add 10g of domestic agar, the above ingredients are sterilized by high pressuredamp heat at 121℃ for 20 minute.
[0050] 2. Sterilization of seeds
[0051] Colombian wild Arabidopsis seeds were sterilized by 70% ethanol for 1 min, 12% sodiumhypochlorite solution for 10 min, and finally rinsed with sterile water for 5 times before use.
[0052] 3. Planting of seeds
[0053] The sterilized seeds were sown on-demand on 1 / 2 nitrogen-optimized medium. Cultivate at 4°C for 3 days, then transfer to light incubator, culture condition is 16 / 8h photoperiod, constant t...
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Abstract
The invention discloses an optimized tissue culture method for Columbia type arabidopsis thaliana and an application. The tissue culture method comprises steps as follows: KNO3 in a culture medium is replaced with KOH and HNO3 on the basis of an original MS macroelement formula, the content of HNO3 is reduced half, and a 1 / 2 nitrogen element optimized culture medium is obtained, wherein the culture medium comprises ingredients as follows: 1.25mM KH2PO4, 10.3mM NH4NO3, 1.5mM MgSO4*7H2O, 3 Mm CaCl2*2H2O, 18.8mM KOH and 9.4mM HNO3. The method is mainly used for improving the growth vigor of tissue cultureArabidopsis thaliana at the seedling stage, the survival rate of plants transplanted in the later period is ensured, and robust plants are cultured. The method can provide technical support for plantgenefunctional identification and research and accelerates the research progress of the plantfunctional genomics.
Description
technical field [0001] The present invention relates to the culture method and culture medium of carrying out tissue culture to plant in the field of bioengineering, particularly relate to the culture method and culture medium of Arabidopsis thaliana seed tissue culture, especially relate to a significantly improved Columbia type Arabidopsis tissue culture Key technologies for seedling growth status. Background technique [0002] Arabidopsis thaliana (Arabidopsis thaliana) is not only a model species for the study of plant genomics, but also has important reference value for the analysis of basic issues such as eukaryotic structure and function due to its short growth cycle, many seeds, and small genome. Researchers pay attention. MS medium was designed by Murashige and Skoog for tobacco tissue culture in 1962. Because of its relatively stable ion balance and appropriate proportion of nutrients, it is the basic medium for rapid propagation of most plant tissue cultures. Ar...
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