Scale tissue culture quickly reproducing method of saffron crocus seedball (sprout)
A technology of tissue culture rapid propagation and saffron, which is applied in the field of biochemical engineering, can solve problems such as the shortage of saffron resources, and achieve the effect of solving the shortage of resources
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Embodiment 1
[0018] 1. Add 2,4-dichlorophenoxyacetic acid 2mg / L, N 6 - Benzyl adenine 0.25mg / L, thiadizuron 0.5mg / L, then add 30g / L sucrose, adjust the pH to 5.9 with acid or alkali, add 7g / L agar and heat to dissolve, then pack in the Erlenmeyer flask 121 After sterilizing at ℃, cool to room temperature as basic medium;
[0019] 2. The saffron corm is sterilized by 70% ethanol for 0.5 minutes, then sterilized with 2% active chlorine sodium hypochlorite solution for 10 minutes, washed three times with sterile water, and cut into 1cm 2 as explants on the above medium at 23°C and cultured in the dark for 4 weeks, a light yellow callus can be induced;
[0020] 3. The pale yellow callus was added with 0.25 mg / L of 2,4-dichlorophenoxyacetic acid and N 6 -Benzyl adenine 2mg / L MS basic medium 23 ℃ dark culture for 3-6 weeks can be partially transformed into white tumor-like callus, the white tumor-like callus is picked out, after adding naphthaleneacetic acid 2mg / L, N 6 -Benzyladenine 2mg / L, t...
Embodiment 2
[0024] 1. Add naphthaleneacetic acid 1mg / L, N 6 - 2 mg / L benzyl adenine and 0.5 mg / L thiadizuron, then add 30 g / L sucrose, adjust the pH to 5.9 with acid or alkali, add 7 g / L agar and heat to dissolve, then pack in a Erlenmeyer flask at 121°C After disinfection, cool to room temperature as basic medium;
[0025] 2. The saffron corm is sterilized by 70% ethanol for 0.5 minutes, then sterilized with 2% active chlorine sodium hypochlorite solution for 10 minutes, washed three times with sterile water, and cut into 1cm 2 As explants, the cut pieces were cultured on the above medium at 23°C, 2000lux, 12h / d light conditions for 4 weeks, and white tumor-like tissues could be induced;
[0026] 3. The tumor-like tissue was cultured and proliferated in the dark at 23°C on 1 / 2 MS basic medium supplemented with 0.5 mg / L naphthaleneacetic acid, 1 mg / L kinetin, 0.5 mg / L thiadizuron and 300 mg / L acid hydrolyzed casein;
[0027] 4. The white tumor-like material was cultured in the dark at 2...
Embodiment 3
[0030] 1. The white nodular tissue that obtains as embodiment two, add 0.2mg / L of naphthaleneacetic acid, zeatin 2mg / L, thiadizuron 0.5mg / L and 300mg / L on the B5 culture medium of acid hydrolyzed casein 21 ℃ Proliferation in dark culture;
[0031] 2. The proliferating tumorous tissue is transferred to a periodic immersion reactor and an atomization reactor, the inoculation amount is 50g / L, and the medium is changed to indole-3-acetic acid 0.5mg / L, N 6 - 1 / 2MS medium of benzyl adenine 0.5mg / L, 300mg / L acid hydrolyzed casein, 18℃ light 12h / d, 2000lux culture, periodic immersion reactor immersion cycle 5min / 2h, ventilation 0.1vvm, mist The atomization cycle of the chemical reactor is 10min / 2h, the ventilation rate is 0.1vvm, and corms are formed in 6-8 weeks, and the induction rate is 22% and 28%. Step 5, the step 4 method of example two are cultivated to produce more bulbs;
[0032] 3. After the bulbs or root seedlings are hardened under natural conditions indoors for a week, ...
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