Plant cell engineering process of producing puerarin
A technology of plant cells and puerarin, applied in plant cells, sugar derivatives, organic chemistry, etc., can solve the problems of inability to expand cultivation, shortage of kudzu resources, unsatisfactory, etc., to achieve small changes in active ingredients, good growth, The effect of meeting the requirements of suspension culture
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Embodiment 1
[0023] (1) Induce callus formation in isolated organs: take mature seeds and carry out surface disinfection, place them on wet filter paper for dark germination for 5 days to obtain aseptic seedlings, take the leaves of aseptic seedlings and cut them into 0.3 cm long, and put them in the following formula Cultivate in solid medium for 10 days: MS+0.5mg / L NAA+1.0mg / L6-BA, the culture conditions are temperature (25±2)°C, dark or weak light.
[0024] (2) Subculture of callus: the callus produced in step (1) is moved into the subculture medium of the following formula for subculture: B 5 +1.0mg / L NAA+1.0mg / L6-BA+100mg / L hydrolyzed casein, the culture conditions are the same as step (1), the culture time is 8 days, and the fresh medium is replaced 3 times during the period. Dilated and loosened.
[0025] (3) Suspension culture of callus: prepare the new liquid culture medium of following formula: B 5 +1.0mg / L NAA+1.0mg / L 6-BA+5000mg / L hydrolyzed casein; mix the prepared fresh med...
Embodiment 2
[0027] Others are with embodiment 1, and the time of dark germination of seed in step (1) is 3 days, and the leaf of aseptic seedling is cut into 1.0 centimeter section, and medium formula is: B 5 +2.0mg / L IAA+0.5mg / LCPPU; medium in step (2) adopts MS+2.0mg / L 2,4-D+0.1mg / L KT+200mg / L hydrolyzed casein, and the culture time is 5 days , during which the fresh medium was replaced twice; the newly prepared medium in step (3) was MS+2.0mg / L 2,4-D+0.1mg / L KT+1000mg / L hydrolyzed casein, and 30ml of mixed In the medium, the inoculation density of callus is 1.5 g per 45 ml, and the culture time is 5 days. The desired puerarin is obtained as a result.
Embodiment 3
[0029] Others are with embodiment 1, in the step (1) with the stem segment of healthy plant as explant, adopt 75% ethanol surface disinfection for 30 seconds, then use 0.1% mercuric chloride surface disinfection for 5 minutes (add surfactant few drops), Medium formula is B 5 +1.0mg / L 2,4-D+1.0mg / L TDZ, the culture time is 15 days; the medium in step (2) uses MS+0.5mg / L 2,4,5-T+0.5mg / L KT + 400mg / L hydrolyzed casein, the culture time is 5 days, and the fresh medium is replaced once during the period; the newly prepared medium in step (3) is MS+0.5mg / L 2,4,5-T+0.5mg / L KT+100mg / L Chitosan, take 100ml of mixed culture medium, callus inoculation density is 3g per 45ml, culture time is 3 days. The desired puerarin is obtained as a result.
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