[0025] Example 1
[0026] (1) Medium preparation: The medium including the different culture stages of microspores. The components and the weight of each component in each liter of medium are:
[0027] ①NLN-13 liquid medium: 1L of NLN liquid medium + 130g sucrose, pH 6.0, filter sterilized;
[0028] NLN liquid medium, based on 1L, composition: KNO 3 125mg, Ca(NO 3 ) 2 ·4H 2 O 500mg, MgSO 4 ·7H 2 O 125mg, KH 2 PO 4 125mg, H 3 BO 3 6.2mg, MnSO 4 ·H 2 O 18.95mg, ZnSO 4 ·7H 2 O 8.6mg, Na 2 MoO 4 ·2H 2 O 0.25mg, CuSO 4 ·5H 2 O 0.025mg, CoCl 2 ·6H 2 O 0.025mg, vitamin B1 0.5mg, vitamin B6 0.5mg, biotin 0.05mg, folic acid 0.5mg, Na 2 EDTA 37.3mg, FeSO 4 ·7H 2 O 27.8mg, Inositol 100mg, Glycine 2mg, Niacin 5mg, L-glutamine 800mg, Glutathione 30mg, Vitamin B5 5mg, Serine 100mg and the balance of sterile water.
[0029] ②Embryoid body differentiation medium: B5 medium 1L + sucrose 30g + agar 9g, pH 6.0, high temperature and high pressure sterilization;
[0030] B5 medium, based on 1L, composition: NaH 2 PO 4 ·2H 2 O 169.5mg, KNO 3 2500mg, (NH 4 ) 2 SO 4 134mg, MgSO 4 ·7H 2 O 500mg, MnSO 4 ·4H 2 O 10mg, H 3 BO 3 3mg, ZnSO 4 ·7H2O 2mg, KI 0.75mg, Na 2 MoO 4 ·2H 2 O 0.25mg, CuSO 4 ·5H 2 O 0.025mg, CoCl 2 ·6H 2 O 0.025mg, Na 2 -EDTA 37.3mg, FeSO 4 ·7H 2 O 27.8mg, CaCl 2.2H 2 O 150mg, VB1 10mg, VB6 1mg, VPP 1mg, Inositol 100mg and the remainder of distilled water.
[0031] ③Rooting medium: MS medium 1L+sucrose 30g+agar 7g, pH 5.8, autoclaved;
[0032] MS medium, based on 1L, composition: NH 4 HO 3 1650 mg, KNO 3 1900 mg, CaCl 2 ·2H 2 O 440 mg, KH 2 PO 4 170 mg, MgSO 4 ·7H 2 O 370 mg, FeSO 4 ·7H 2 O 27.8 mg, Na 2 EDTA 37.3 mg, H 3 BO 3 6.2 mg, MnSO 4 ·H 2 O 16.9 mg, ZnSO 4 ·7H 2 O 8.6 mg, Na 2 MoO 4 ·2H 2 O 0.25 mg, CuSO 4 ·5H 2 O 0.025 mg, CoCl 2 ·6H 2 O 0.025 mg, KI 0.83 mg, inositol 100 mg, glycine 2 mg, niacin 0.5 mg, vitamin B1 0.1 mg, vitamin B6 0.5 mg and the balance of sterile water.
[0033] (2) Methods to promote microspore embryogenesis of stubborn kale genotype:
[0034] ① Flower bud selection: Take kale (the petal to anther length ratio on the inflorescence is 0.7) and rape (the petal to anther length ratio is 0.5) in the late mononuclear to the early binuclear, healthy, disease-free buds, as the microspore culture supply body;
[0035] ②Sterilization of flower buds: Put the flower buds of rapeseed and kale into a sterile petri dish, add 0.1% (mass percentage concentration) mercury solution, seal, put on a shaker and shake the surface to disinfect for 8 minutes, then sterilize Shake and wash 5 times with sterile water on the workbench for use;
[0036] ③Place 12 sterile kale flower buds in a sterile beaker on an ultra-clean workbench, add 10 mL of NLN-13 liquid medium, and grind the flower buds into a suspension with a glass rod; use a 300-mesh aseptic suspension for the suspension Filter the filter into a 50 mL centrifuge tube, centrifuge the resulting filtrate at 700 rpm for 5 minutes, discard the supernatant, and add 40 mL of NLN-13 liquid medium and 0.5 ml of NLN liquid medium 1L + agarose 5g + activated carbon 1g to the precipitate. The aseptic activated carbon mixture prepared and sterilized at high temperature is mixed into a microspore suspension; the suspension is divided into 10 plastic or glass sterile petri dishes with a diameter of 60 mm, and each petri dish is divided 4 mL;
[0037] ④Under aseptic conditions, peel off the aseptic rape buds, take out the whole anthers and add them to the above petri dishes, put 6 in each petri dish, and seal with parafilm film after capping;
[0038] ⑤Put the petri dish containing the suspension of cabbage microspores and rape anthers in a 33°C biochemical incubator and culture for 2 days in the dark; then take it out and place it in a constant temperature incubator at 25°C and incubate it for 10-15 days in the dark to the naked eye Visible cell clusters appeared, and then cultured with shaking at 50 rpm in the dark at 25°C for 10 days until the cotyledon-type embryoid bodies were formed and matured;
[0039] ⑥ Under aseptic conditions, transfer mature cotyledon-type embryoid bodies to embryoid body solid differentiation medium, culture for 15-20 days at 23±2℃ under 14 hours of light every day to form callus or until differentiation Sprout; then cut the callus into 2 to 4 pieces according to the size and place them on the same medium and conditions to cultivate until they differentiate to form regenerated plants;
[0040] ⑦Cut healthy regenerated buds and inoculate them on the rooting medium. Carry out rooting culture under 14 hours of light per day and 25℃; 3 weeks later, the seedlings with strong roots will be tempered for 3 days; the group should be washed with water when transplanting Cultivate the root medium for seedlings, then plant the plants on a special substrate plug for vegetable seedlings, cover them with a plastic film for moisture, cultivate in the greenhouse for 10 days and then transplant them to obtain regenerated plants;
[0041] ⑧According to the morphological characteristics of the plants, the method of field observation is used to distinguish between the kale plants and the rape plants; take the tender leaves of the regenerated plants, observe the number of chloroplasts in the stoma guard cells, and detect the ploidy of each plant.
[0042] (3) Except that in step ①, only the healthy, disease-free inflorescence of the kale is used as the donor plant for the microspore culture, the other operations are the same as "(2) The method to promote the embryogenesis of the stubborn kale genotype microspore", As a control kale.
[0043] Results: The embryonic rate of mixed culture kale was 9 per bud, while the embryo rate of control kale was only 1.5 per bud. Field observations showed that 20 kale microspore seedlings were obtained by co-cultivation with rape anthers. The number of control strains of Brassica oleracea microspores cultured alone was one.
