Method for producing camptothecin and 10-hydroxycamptothecin using camptothecin suspension cells
A technology of hydroxycamptothecin and suspended cells, applied in the field of medicine and biology, can solve problems such as low product content, and achieve the effects of rapid growth and reproduction, simple and easy control, and easy cultivation.
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Embodiment 1
[0024] Example 1: Establishment of aseptic seedlings of Camptotheca acuminata
[0025] 1. Test materials
[0026] 1.1 Plant material
[0027] Choose the normal and healthy growth of Camptotheca acuminata fruit, peel, wash, and disinfect according to routine operations. In this example, the seed coat of the fruit was removed first, followed by immersion in 5% Triton for 3 minutes, and repeated washing with sterile water, with a concentration of 75% ethanol for 1 minute, 1% sodium hypochlorite solution for 3 minutes, and sterile water washing 10 times each time. 1min, finally use sterile filter paper to absorb water on the surface of the seeds of Camptotheca acuminata, and set aside.
[0028] 1.2 Medium
[0029] 1 / 2MS solid medium: 1 / 2MS medium supplemented with 0.35% agar (Sigma Chemical, MO), pH 5.8, autoclaved at 121°C for 30 min.
[0030] 2. Seed inoculation to the medium
[0031] The prepared seeds are inoculated and cultivated according to conventional aseptic methods.
[0032] Inocu...
Embodiment 2
[0033] Example 2: Establishment of Camptotheca acuminata callus
[0034] 1 Test materials
[0035] 1.1 Plant material
[0036] The sterile seedlings obtained in Example 1. According to the conventional aseptic operation, use a sterile scalpel to cut the leaves into small pieces of 10mm×10mm for use.
[0037] 1.2 Medium
[0038] 7 kinds of solid medium with MS30 (MS medium supplemented with 30mg / L sucrose) as the basic medium and growth regulators of different composition and concentration. As shown in Table 1:
[0039] Table 1 Composition of medium A-J
[0040]
[0041] 2. Inoculate the sterile leaves into the medium
[0042] The prepared leaves are inoculated and cultivated according to conventional aseptic operation methods.
[0043] Inoculate the prepared leaves on the corresponding medium in Table 1, and cultivate for 5-6 weeks at a temperature of 25°C in the dark, and wait for the callus to grow. Three groups of parallel experiments were performed on different media.
[0044] 3. Prep...
Embodiment 3
[0057] Example 3: Establishment of Camptotheca acuminata suspension cell line
[0058] 1 Test materials
[0059] 1.1 Plant material
[0060] The callus of Camptotheca acuminata obtained in Example 2 was cut into small pieces with a sterile scalpel in accordance with conventional aseptic operations and set aside.
[0061] 1.2 Medium
[0062] MS30 liquid medium is supplemented with 0.5mg / L NAA and 2mg / L BAP.
[0063] 2. Inoculation of Camptotheca acuminata callus to the culture medium
[0064] The prepared callus will be inoculated and cultivated according to the conventional aseptic method.
[0065] The callus was inoculated into 100 mL liquid medium and cultivated under continuous light. Light culture conditions: white light (25μmol m -2 s -l ), temperature 25℃, humidity 70%, rotating speed 125rpm. After 3 to 5 days, when cells grow out of the culture medium, transfer 5 mL of the cell suspension to 100 mL of a new liquid medium, culture under the same conditions, and obtain suspension ce...
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