Method for in-vitro rapid propagation of clivia miniata by using mature embryos as explants
A technology for explants and Clivia, applied in the field of plant reproduction, can solve the problems of low dedifferentiation rate and regeneration rate, limit the application of Clivia tissue culture technology, etc., achieves good growth state, low cost, and overcomes the effects of transgenic silence.
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Embodiment 1
[0012] 1. Explant detoxification, inoculation and callus induction: get the seed embryo in the mature fruit, soak it in 75% (volume / volume) alcohol for 30 seconds in a sterile ultra-clean bench, and use 0.1% (mass / volume) liter of Soak in mercury solution for 2 minutes, rinse with sterile water for 3 times, cut off the dense yellow end of about 2 mm in length on the naked embryo, and inoculate it in a medium containing 2,4-D 2 mg / L, 3% sucrose (mass / volume), 0.45 agar % (mass / volume) MS medium, 20 ℃, 40W fluorescent lamp every day for 8 hours. Subculture once every 40 days, and induce differentiation when the callus (yellow) or green swelling grows to a diameter of about 5 mm.
[0013] 2. Induction of adventitious buds: transfer the yellow callus or green swollen tissue in a good growth state to the induction differentiation medium: MS+KT0.25mg / L+2, 4-D0.75+3% (mass / volume) Sucrose + 0.45% (mass / volume) agar, subculture once every 40 days, and light for 10 hours a day at 20°C...
Embodiment 2
[0017] 1. Explant detoxification, inoculation and callus induction: get the embryo in the mature fruit in a sterile ultra-clean bench, soak it for 60 seconds with 75% (volume / volume) alcohol, and use 0.2% (mass / volume) liter of Soak in mercury solution for 5 minutes, rinse with sterile water 5 times, cut off the dense yellow end of the naked embryo with a length of about 2 mm, and inoculate it on a medium containing 2,4-D 4 mg / L, 4% sucrose (mass / volume), 0.7 agar % (mass / volume) MS medium, irradiated with 40W fluorescent lamp for 9 hours every day at 30°C. Subculture once every 50 days, and induce differentiation when the callus (yellow) or green swelling grows to a diameter of about 5 mm.
[0018] 2. Induction of adventitious buds: the yellow callus with good growth state or the green expanded tissue are transferred to the induction differentiation medium: MS+KT0.5mg / L+2, 4-D1.5mg / L+4% (quality / volume) sucrose+0.7% (mass / volume) agar, subculture once every 50 days, and lig...
Embodiment 3
[0022] 1. Explant detoxification, inoculation and callus induction: get the seed embryo of ripe fruit and soak in 75% (volume / volume) alcohol for 40 seconds in a sterile ultra-clean bench, and use 0.15% (mass / volume) mercuric chloride Soak in the solution for 4 minutes, rinse with sterile water 4 times, cut off the end of the dense yellow about 2 mm in length on the naked embryo, and inoculate it in a medium containing 2,4-D 3 mg / L, 3.5% sucrose (mass / volume), 0.6% agar (mass / volume) MS culture medium, 25 ℃ 40W daylight lamp irradiation 8.5 hours every day. Subculture once every 48 days, and induce differentiation when the callus (yellow) or green swelling grows to a diameter of about 5 mm.
[0023] 2. Induction of adventitious buds: transfer the yellow callus or green swollen tissue in good growth state to the induction differentiation medium: MS+KT (0.4mg / L)+2,4-D (1.0mg / L)+3.5 % (mass / volume) sucrose+0.5% (mass / volume) agar, subculture once every 45 days, and light for 8 h...
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