Ultra-low temperature preservation method of lingonberry plant germplasm
A cryopreservation and plant technology, which is applied in the field of plant germplasm resource preservation, can solve the problems of different procedures and technologies, and achieve the effects of strong cell regeneration ability, high genetic stability, and convenient material extraction
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Embodiment 1
[0030] Example 1 Ultra-low temperature preservation technology and plant regeneration culture method of bilberry stem tip droplet vitrification method
[0031] The specific operation of the experimental method is as follows:
[0032] 1. Practicing seedlings: select 4-week-old tissue culture seedlings, and inoculate 0.5 cm stem segments with terminal buds on WPM medium for 4 ℃ dark treatment for 3 weeks;
[0033] 2. Stripping the tip of the bilberry: Under aseptic conditions, the terminal bud after the hardening is stripped under a stereo microscope, and the length of the tip is 1.5-2.0mm;
[0034] 3. Pre-cultivation: Inoculate the peeled shoot tips on WPM solid medium supplemented with 0.3M sucrose, and culture in the dark for 24h in a tissue culture room;
[0035] 4. Loading: The pre-cultured isolated shoot tips were transferred to a loading solution consisting of: WPM+2M glycerol+1.0M sucrose, and loaded at room temperature for 30min;
[0036] 5. Vitrification treatment: t...
Embodiment 2
[0041] Example 2 Effect of hardening time and loading time on ultra-low temperature preservation of bilberry shoot tips
[0042] The operation steps were the same as those in Example 1, except that the seedling hardening time was changed, and the seedling hardening time was set to 0, 1, 2, 3, 4, and 5 weeks, and the loading time was 0, 10, 20, 30, and 40 min. After 7 days of recovery, the survival rate of bilberry shoot tips and the regeneration of 30 days were counted. The results showed that the hardening time and loading time had important effects on the survival rate and regeneration rate of bilberry shoot tips after preservation. The survival rate and regeneration rate of the shoot tip increased first and then decreased with the change of seedling hardening time, such as figure 1 As shown, with the change of loading time, it basically shows a trend of first rising and then falling, such as figure 2 shown. When the seedling hardening time was 3 weeks, the survival and ...
Embodiment 3
[0043] Example 3 Effects of pre-cultivation time and vitrification time on cryopreservation of bilberry shoot tips
[0044] The operation steps are the same as in Example 1, except that the pre-cultivation time is changed, and the stripped bilberry shoot tips are inoculated into WPM solid medium containing 0.3 M sucrose, and pre-cultured in the dark in the tissue culture room for 0-48 h, and the vitrification treatment time is set. 0, 20, 40, 60, 80min. After 7 days of recovery, the survival rate of bilberry shoot tips and the regeneration of 30 days were counted. The results showed that pre-cultivation time and vitrification time had important effects on the survival rate and regeneration rate of bilberry shoot tips after preservation. The survival rate and regeneration rate of shoot tips basically showed a trend of first increasing and then decreasing with the change of pre-cultivation time, such as image 3 As shown, with the change of vitrification treatment time, there ...
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