Huperzia serrata hairy root system preparation and cultivation method
A cultivation method, the technology of the Melaleuca tower, which is applied in the field of preparation and cultivation of the hairy root system of the Melaleuca tower, to achieve the effect of alleviating market demand and solving the problem of dependence on imports
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Embodiment 1
[0025] Embodiment 1 Mannoline paper chromatography analysis
[0026] (1) Material: Melaleuca hair root (NTR) newly induced by the present invention; hair root (TR) of the present invention through subculture; organ root (NR), leaf (L) or callus cell (C) ; Agrobacterium rhizogenes A4 bacterial liquid;
[0027] (2) Extraction of agromine: Take 1g of the above samples and add 1ml (0.1M) HCl to grind at room temperature, centrifuge at 12,000rpm for 5min, take 10μl of the supernatant, and apply the sample with a micro-sampler.
[0028](3) Chromatography: preparation of chromatographic filter paper: cut chromatographic filter paper into 8×8 (cm) size, soak in 0.4mol / L HCl solution for 24 hours, then rinse with distilled water, and then wash with ethanol and diethyl ether in turn After washing, lay flat on a tray, dry at 30°C, and set aside.
[0029] (4) Sample spotting: draw a straight line 1-1.5 cm away from the bottom edge of the chromatography filter paper, and mark the equidis...
Embodiment 2
[0034] (1) Cut the young stems of Melaleuca pagoda into small sections of 2cm, and use HgCl in the aseptic operation table 2 Treat for 7 minutes, soak in alcohol for 6 seconds, rinse with sterile water for 4 times, then transfer to B 5 +6-BA0.5mg / L+NAA0.3mg / L culture medium, cultured in the dark at 25°C±2 for 12d, and obtained Melaleuca callus.
[0035] (2) Dilute the Agrobacterium rhizogenes ACCC10060 strain with 1 μL of sterile water, spread it on YEB solid medium, culture in the dark at 25°C±3 for 4 days, and grow a single clone to obtain Agrobacterium rhizogenes A4.
[0036] (3) Pick the Agrobacterium rhizogenes A4 clone and place it in YEB medium, culture it with shaking at 25°C±2, the OD value is 0.5, take 1ml of the bacterial liquid and place it in 100ml YEB liquid medium for shaking culture, the OD value is 0.5 , collect the bacterial liquid, and centrifuge at 3000 rpm for 7 minutes to collect the bacterial cells.
[0037] (4) Place the thalline that step (3) collect...
Embodiment 3
[0042] (1) Cut the young stems of Melaleuca pagoda into small sections of 4cm, and use HgCl in the aseptic operation table 2 Treat for 10 minutes, soak in alcohol for 8 seconds, wash with sterile water for 7 times, then transfer to B5+6-BA0.5mg / L+NAA0.3mg / L medium, culture in dark at 25℃±2 for 30d, and obtain Melaleuca japonica damage tissue.
[0043] (2) Dilute the Agrobacterium rhizogenes ACCC10060 strain with 1 μL of sterile water, spread it on YEB solid medium, culture in the dark at 25°C±3 for 5 days, and grow a single clone to obtain Agrobacterium rhizogenes A4.
[0044] (3) Pick Agrobacterium rhizogenes A4 clone and place it in YEB medium, culture it with shaking at 25°C±2, the OD value is 1, take 1ml of the bacterial liquid and place it in 100ml YEB liquid medium for shaking culture, the OD value is 0.7 , collect the bacterial liquid, and centrifuge at 3000rpm for 10min to collect the bacterial cells.
[0045] (4) Place the thalline that step (3) collects into 100ml ...
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