The explant is the tender stem of a new branch extracted from the old root of chrysanthemum, or the tender stem of a plant that survived by cutting in the same year. Remove the leaves and petioles from the tender stems together.
① Sterilization of explants. Rinse the tender stems in clean water for several times, then rinse them with detergent and enter the inoculation room; Soak in 0. 1% Tween for 2 ~ 3 minutes, mainly to remove the fluff on the surface of the shoots; Then soak in 70% alcohol for 30 seconds, disinfect with 10% bleaching powder solution for 8 ~ 10 minutes, and finally rinse with sterile water for 6 ~ 7 times, which can be used as inoculation material.
② Inoculation. The inoculation material was cut into small pieces with the length of 0.5 ~ 1 cm and inoculated on the prepared culture medium within the flame control range of the ultra-clean table.
(2) culture medium.
Chrysanthemum culture medium is MS plus 6-Ba2mg/L, NAA 0.2mg/L (or KT2mg/L plus NAA 0.2mg/L), and the hydrogen ion concentration of the medium is 316.3 ~1000 mmol/l (ph 6 ~ 6.5). Sterilize for 20min under conventional sterilization conditions.
(3) Culture conditions.
Cultured at room temperature of 25 1℃ and irradiated by fluorescent lamp for 8 hours, callus and green seedlings can be produced successively after about 2 weeks. After about 1 month of transfer, green seedlings continue to grow and elongate, and then transfer and proliferate.
(4) Rooting culture.
Rooting medium is ms+NAA1~ 2 mg/l. Transfer the stem segments of green seedlings to rooting medium and take root after about 10 days.
(5) Transplanting test-tube seedlings.
Transplant rooting test-tube seedlings in time (root length 1 cm). Take the test-tube seedlings out of the test tube, wash off the culture medium attached to the seedlings and transplant them into vermiculite (or perlite, fine peat particles, etc.). ) culture medium, watered with clear water, and after new roots are produced in about 10 days, they can be moved into culture soil for conventional cultivation.