Sterilization method of rape seeds for tissue culture

A sterilization method and technology for seeds, applied in the field of plant tissue culture, can solve the problems of damaged seeds, inability to germinate, and inability to obtain, and achieve the effects of saving use costs, saving operation time, and improving efficiency

Inactive Publication Date: 2019-01-18
FUJIAN AGRI & FORESTRY UNIV
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AI Technical Summary

Problems solved by technology

[0003] Sodium hypochlorite (NaClO) is currently the most used sterilant for rapeseeds. It is worth noting that if the concentration of NaClO used is too low or the sterilization time is not enough, the seeds will not be sterilized completely, and pathogenic bacteria will easily breed after sowing, and the seedlings will be easily infected by pathogenic bacteria. If the concentration is too high or the sterilization time is too long, the seeds will be damaged, the seeds will germinate abnormally or even fail to germinate, and it will not be possible to obtain enough seedlings for transgenic research. The incubation time ranges from 6 minutes to 45 minutes, which brings great difficulties to the application and promotion of transgenic rapeseed research

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  • Sterilization method of rape seeds for tissue culture
  • Sterilization method of rape seeds for tissue culture
  • Sterilization method of rape seeds for tissue culture

Examples

Experimental program
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Effect test

Embodiment 10

[0020] Example 1 0% - 30% NaClO sterilized seeds wide range screening

[0021] Pick seeds that are plump and without obvious scars and soak them in water for 1 hour. After removing the seeds floating on the water surface, rinse them with tap water several times, and then transfer them to an ultra-clean workbench for operation. Disinfect them with 75% alcohol for 1 minute and rinse them with sterile water for 2 minutes. Again, use different concentrations of NaClO (0%, 0.1%, 1%, 5%, 10%, 20%, 30%; set another without 75% alcohol, only use sterile H 2 O (control treatment group) was sterilized for 10 min, then rinsed with sterile water for 6 times, transferred to sterile dry filter paper to dry, and sowed in MS seed germination medium, each group treated 32 seeds, a total of 4 replicates .

[0022] The above seeds were sown in MS seed germination medium: MS medium 2.22 g / L + sucrose 30 g / L + agar powder 8 g / L, pH value 5.8. Cultivation environment: 25°C, relative humidity 60% ...

Embodiment 21

[0029] Example 2 Small-scale screening of 1% - 5% NaClO sterilized seeds

Embodiment 2

[0030] As can be seen from Example 1, 1% concentration NaClO is the best for seed sterilization, but it is worth noting that the germination rate and healthy germination rate of 1% and 5% NaClO sterilized seeds are significantly different, so there is reason to suspect that at 1 There is a concentration more suitable for seed sterilization between %-5% concentration, in order to verify this hypothesis, we have done embodiment 2, specifically as follows:

[0031] Pick seeds that are plump and without obvious scars and soak them in water for 1 hour. After removing the seeds floating on the water surface, rinse them with tap water several times, and then transfer them to an ultra-clean workbench for operation. Disinfect them with 75% alcohol for 1 minute and rinse them with sterile water for 2 minutes. sterilized with different concentrations of NaClO (1%, 2%, 3%, 4%, 5%) for 10 min, rinsed with sterile water for 6 times, transferred to sterile dry filter paper to dry, and sowed o...

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Abstract

The invention discloses a sterilization method of rape seeds for tissue culture. The sterilization method comprises the following steps: firstly, putting seeds in water for socking for 1 hour, removing seeds floating on the water surface, washing with tap water twice, transferring into a clean bench, sterilizing for 1 minute with 75% alcohol, sterilizing the seeds for 15 minutes with 3% NaClO (keeping a shaking and rotating state in the process), further washing with sterile water 5-6 times, and finally transferring the seeds onto sterile filter paper for airing, wherein the 3% NaClO has the optimal sterilization concentration screened from seed sterilization effects of 0-30% NaClO, and the sterilization time of 15 minutes is the optimal sterilization time screened from 10-30 minutes of seed sterilization with the optimal concentration of 3% NaClO. By adopting the sterilization method, maximum healthy seedlings can be cultured for the production of transgenic oilseed rape, so that thesuccess rate of oilseed rape gene transformation is increased.

Description

technical field [0001] The invention relates to the field of plant tissue culture, in particular to a method for sterilizing rape seeds used for tissue culture. Background technique [0002] Rapeseed rape belongs to Brassica genus Brassica and is an important oil crop. Its products can be processed into human edible oil, industrial lubricating oil, and can also be used as green manure and compost. According to reports, between 2014 and 2015, the global rapeseed oil production exceeded 2.698 million tons, making it the third largest oil-producing plant. Rapeseed cultivars with ideal traits can be quickly obtained through transgenic technology, which is now widely used. According to the statistics of the International Agricultural Biotechnology Application Service (ISAAA), the planting area of ​​genetically modified crops reached 185.1 million hectares in 2016, and 24% of the rapeseed planted globally is Transgenic varieties (http: / / www.isaaa.org), the common way to obtain tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00A61L2/18
CPCA01H4/001A01H4/008A61L2/18
Inventor 傅淑杨广刘昭霞陈金芝孙庚晓尤民生
Owner FUJIAN AGRI & FORESTRY UNIV
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