Method for establishing high-efficiency regeneration system by using broccoli microspore embryo as explant
A microspore and cauliflower technology, applied in the field of establishment of high-efficiency cauliflower regeneration system, can solve the problems of low genetic transformation rate, few regeneration buds, and low proliferation ability, so as to improve genetic transformation rate, reduce browning, and promote bud induction rate Effect
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Embodiment 1
[0025] Example 1: (cultivation method 1 for broccoli microspore embryo high-frequency bud regeneration)
[0026] The method proceeds as follows:
[0027] (1) Preparation of culture medium: including the culture medium of each stage of explant bud regeneration, their components and the weight of each component in each liter of medium are:
[0028] 1) Callus induction medium: MS basal medium + 2,4-D 1.0 mg / L + ZT 0.8 mg / L + AgNO 3 4.0 mg / L + 30g / L sucrose + 9g / L agar, pH 5.8, high temperature sterilization;
[0029] 2) Callus differentiation medium: MS basal medium + ZT 0.5 mg / L + BAP 2.0 mg / L + NAA 0.05mg / L + AgNO 3 4.0 mg / L + 30 g / L sucrose + 9 g / L agar, pH 5.8, autoclaved;
[0030] 3) Callus redifferentiation medium: MS basal medium + AgNO 3 4.0 mg / L + 30 g / L sucrose + 10 g / L agar, pH 5.8, autoclaved;
[0031] 4) Rooting medium: 1 / 2 MS basal medium + NAA 1 mg / L + 30 g / L sucrose + 9 g / L agar, pH 5.8, high temperature sterilization;
[0032] (2) Cultivation of...
Embodiment 2
[0039] Example 2: (cultivation method 2 for broccoli microspore embryo high-frequency bud regeneration)
[0040] In this example, step (1) medium preparation: wherein, 1) 2,4-D in the callus induction medium is 1.5 mg / L, ZT is 1.0 mg / L, AgNO 3 4.5 mg / L; 2) ZT in callus differentiation medium was 0.8 mg / L, BAP was 1.5 mg / L, AgNO 3 4.5 mg / L; 3) The agar in the callus redifferentiation medium was 10.5 g / L; Step (2) Cultivation of broccoli microspore embryo regenerated plants: 1) The selected microspores were cultured in dark at 25°C After 35 days, 180 well-grown cotyledon embryos were taken; 2) Cotyledon embryos were cultured for 1 week at 27°C with 16 hours of light per day; 3) Calli were cultured for 2-3 weeks at 28°C with 16 hours of light per day until they appeared Visible bud point; All the other step techniques are the same as in Example 1.
Embodiment 3
[0041] Example 3: (cultivation method 3 for broccoli microspore embryo high-frequency bud regeneration)
[0042] In this example, step (1) medium preparation: 1) 2,4-D in the callus induction medium is 2.0 mg / L, ZT is 0.5 mg / L, AgNO 3 5.0 mg / L; 2) ZT in callus differentiation medium was 1.0 mg / L, BAP 1.0 mg / L, AgNO 3 5.0 mg / L; 3) The agar in the callus redifferentiation medium was 11 g / L; Step (2) Cultivation of broccoli microspore embryo regenerated plants: 1) The selected microspores were cultured in the dark at 28°C for 30 Take 180 well-grown cotyledon embryos after 1 day; 2) Culture the cotyledon embryos at 28°C with 16 hours of light per day for 1 week; 3) Culture the callus at 27°C with 16 hours of light per day for 2-3 weeks until visible Bud point; All the other step techniques are the same as in Example 1.
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