Method for producing podophyllotoxin induced from sino podophyllum hexandrum callus
A technology of callus induction and podophyllotoxin, which is applied in horticultural methods, botany equipment and methods, plant regeneration, etc., can solve the problem of unreported callus-induced podophyllotoxin, low content, Problems such as difficulty in callus culture
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Embodiment 1
[0015] 1. Put the selected and plump peach seven seeds produced in Longnan, Gansu Province into a beaker, slowly add concentrated sulfuric acid, stir continuously with a glass rod for 10 minutes, rinse with tap water until the sulfuric acid is completely removed. Replace with 300mg / L gibberellin solution for 48 hours. The treated seeds are sterilized with 0.1% mercury liter for 5-6 minutes under aseptic conditions, rinsed with sterile water for several times, and blotted dry with sterile filter paper. Embryos were taken out and inoculated into modified MS medium (1 / 2 MS inorganic components, no hormones), and cultured in a light incubator. Other conditions are: agar 0.75%, sucrose 30g / L, pH adjustment is 5.8; Illumination (500μmol.m -2 .s -1 ) for 10 hours at a temperature of 25±1°C. The embryo begins to swell in about 3 days, two cotyledons unfold at 6 days, the cotyledons are green at 10 days, the radicle and hypocotyl elongate obviously at 20 days, and grow into complete...
Embodiment 2
[0020] 1. select four kinds of explants from the aseptic seedling that step 1-3 obtains among the embodiment 1: the aseptic seedling that grows about 40 days ① hypocotyl ② radicle ③ cotyledon and ④ true leaf petiole of about 60 days, cut into small pieces of about 0.3 cm or 0.3 cm square, connected to B 5 The induction medium (explant ①, ② and ③) is selected from B 5 Add 2,4-D 1mg / L, 6-BA 3mg / L and NAA 1mg / L; ④Choose B 5 Additional 2,4-D 5mg / L, 6-BA 1mg / L and NAA 0.5mg / L). Other conditions are: agar 0.75%, sucrose 20g / L, pH adjustment is 5.8; Illumination (500μmol.m -2 .s -1 ) for 10 hours at a temperature of 23±1°C. After about 40-50 days of culture, the four different explants all induced callus.
[0021] 2. Screen callus with loose texture and fast proliferation and subculture continuously on different subculture media respectively: callus from explant ①, ② and ③) source selection B 5 Add 2,4-D 0.2mg / L, 6-BA 1mg / L and NAA 1mg / L, callus from true leaf petiole selection...
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