Freeze preservation method for masson pine embryonic callus
A technology of embryogenic callus and cryopreservation, which is applied to the preservation of plants, botany equipment and methods, and applications, and can solve the problems of systematic technology, poor stability, low somatic embryo induction rate, and long culture cycle , to achieve the effect of solving the difficulty of induction, solving the serious apoptosis, good economic and social benefits
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Embodiment 1
[0027] Using masson pine immature cleavage multi-embryonic zygotic embryos as explants, a large number of vigorous and vigorous masson pine embryogenic cell lines were obtained through somatic embryo induction. Among them, the faster-growing embryogenic cell line SE-17 was proliferated and maintained for 8 days, and the core part of the embryogenic callus was avoided, and the outer viscous and transparent embryogenic callus was collected as the preservation material.
[0028] Select 2 g embryogenic callus and put it in a 50 mL sterile Erlenmeyer flask, and add 7 mL of the base solution to the sterile Erlenmeyer flask, seal the sterile Erlenmeyer flask and place it on a rotary shaker for oscillating dispersion Treatment for 24 h, the treatment conditions are: dark culture, no light, temperature 20 ± 0.5 ℃; rotating oscillator speed 80 rpm / min. The raw materials and content of the base liquid are: KNO 3 200 mg·L -1 ; KH 2 PO 4 170 mg·L -1 ;KCl 625 mg·L -1 ; CaCl 2 ·2H 2 O 220 mg·L...
Embodiment 2
[0032] Using masson pine immature cleavage multi-embryonic zygotic embryos as explants, a large number of vigorous and vigorous masson pine embryogenic cell lines were obtained through somatic embryo induction. Among them, the fast-growing embryogenic cell line SE-17 was proliferated and maintained for 7 days, and the embryogenic callus was collected from the core part of the embryogenic callus, and the outer viscous and transparent embryogenic callus was collected as the preservation material.
[0033] Select 1g embryogenic callus and put it into a 25 mL sterile Erlenmeyer flask, and add 3 mL of the base solution to the sterile Erlenmeyer flask, seal the sterile Erlenmeyer flask and place it on a rotary shaker for shaking and dispersing. For 24 h, the treatment conditions are: dark culture, no light, temperature 20 ± 0.5 ℃; rotating oscillator speed 80 rpm / min. The raw materials and content of the base liquid are: KNO 3 200 mg·L -1 ; KH 2 PO 4 170 mg·L -1 ;KCl 625 mg·L -1 ; CaC...
Embodiment 3
[0037] Using masson pine immature cleavage multi-embryonic zygotic embryos as explants, a large number of vigorous and vigorous masson pine embryogenic cell lines were obtained through somatic embryo induction. Among them, the faster-growing embryogenic cell line SE-22 was proliferated and maintained for 7 days, avoiding the core part of the embryogenic callus, and collecting the outer viscous and transparent embryogenic callus as the preservation material.
[0038] Select 1 g embryogenic callus and put it into a 25 mL sterile Erlenmeyer flask, and add 4 mL of the base solution to the sterile Erlenmeyer flask. After sealing, place the sterile Erlenmeyer flask on a rotary shaker to oscillate and disperse. Treatment for 24 h, the treatment conditions are: dark culture, no light, temperature 20 ± 0.5 ℃; rotating oscillator speed 80 rpm / min. The raw materials and content of the base liquid are: KNO 3 200 mg·L -1 ; KH 2 PO 4 170 mg·L -1 ;KCl 625 mg·L -1 ; CaCl 2 ·2H 2 O 220 mg·L -1 ;...
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