Rapeseed microspore culture medium and culture method for improving cotyledon embryo production rate
A microspore culture and microspore technology, which is applied in the field of rape microspore medium and culture, can solve the problems of difficulty in one-time seedling formation, browning, and lowering the efficiency of double haploid plants in microspore culture, so as to ensure high quality, The effect of improving yield
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Embodiment 1
[0023] Three rapeseed F1 hybrid combinations (6R×ZS11), (7DH×ZS11), (ZR×R11) were planted in the field, and each combination was planted in 6 rows, with a total of 50 individual plants. Spray foliar fertilizer during the budding stage to improve stress resistance, prevent premature aging, and prevent and control Sclerotinia.
[0024] (1) Separation and collection of microspores
[0025] Pick the main inflorescence and flower buds of branches of three rapeseed F1 hybrid materials in the field, put them into a plastic box covered with wet filter paper in advance, put the plastic box in a display cabinet at 4°C for 1 day, and select the length the next day 3mm-4.2mm rape flower buds, at this time, the microspores in the stamens are in the late stage of uninucleate to the early stage of dinucleate microspores, 30-40 flower buds are sterilized in 50% (V / V) sodium hypochlorite solution for 10 minutes, and then transferred to sterile ddH 2 Soak in O 3 times, 10min each time.
[00...
Embodiment 2
[0065] Using the same method as in Example 1, 120 flower buds were selected and isolated to obtain microspores, which were cultured in the dark at 25° C. for 2 weeks, and then transferred to a petri dish containing a solid-liquid double-layer medium for culture. The difference is: when the embryos are visible to the naked eye, the petri dish is placed on a shaker with a rotation speed of 25rpm and cultured for 1 week, 2 weeks, and 3 weeks respectively, and the culture temperature is 25°C, and the spherical or heart-shaped embryos, cotyledon-shaped embryos, Similar to the number of embryonic structures, the results are shown in Table 2 and image 3 as shown ( image 3 A represents spherical or heart-shaped embryos, B represents cotyledon-shaped embryos, and C represents embryo-like structures).
[0066] Table 2
[0067]
[0068] The results showed that the total number of embryos obtained after 1 week of shaking culture was more than that of non-shaking cultured embryos, w...
Embodiment 3
[0070] The method consistent with Example 1 is adopted, except that the microspores cultivated for 2 weeks on the solid-liquid double-layer medium are transferred to a shaking table with a rotation speed of 25 rpm for 1 week respectively, and the culture temperatures are respectively: 25° C., 23°C, 21°C, 19°C, 17°C, and then continue to stand for 3 weeks at the corresponding temperature. The total number of embryos and the number of cotyledon embryos are counted as shown in Table 3.
[0071] table 3
[0072]
[0073] The results show that: with the decrease of the culture temperature, the total embryo number of 60 flower buds on average is decreasing. Reduced by 47.2%, 71.4%, 79.1%. According to the statistics of the number of cotyledon embryos under different culture temperatures, the number of normal cotyledon embryos accounted for 62.1% of the total number of embryos under the culture condition of 23 ℃, which was significantly higher than the number of cotyledon embryos...
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