A kind of maize ms30 gene mutant and its molecular identification method and application
A MS30, corn technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of economic loss, increased cost, incompleteness, etc., and achieve the effect of huge economic value
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Embodiment 2
[0041] Example 2 M 2 Substitute Planting and Character Observation
[0042] m 1 The generation plants selfed to produce seeds, and planted M 2 Generation, in M 2 During the earing and flowering periods of the generation, the morphology of the anthers was observed in the field, and a microscope was selected for inspection. In the family numbered 4898, a plant with abnormal fertility was found. The mutant had no obvious difference from the wild type in terms of vegetative growth, heading stage, and panicle type. figure 1 The pollen iodine staining pattern of the mutant plant (Panel A) and the iodine staining pattern of the mutant male pollen (Panel B) and the comparison photos of the mutant anther (Panel C left) and the wild type anther (Panel C right). The male anthers of the mutant were significantly smaller and whiter, and the pollen grains of the mutant were small, irregular, and uncolored by iodine staining, while the pollen grains of the wild type Jingkenuo 2000 were l...
Embodiment 3
[0043] Example 3 Microscopic examination of pollen, selfing and outcrossing
[0044] The pollen fertility was counted by the ratio of iodine-stained pollen to non-colored pollen. Observing the male flower morphology of the 4898 mutant under a stereomicroscope, the anthers were smaller and lighter in color than the wild type (see figure 1 Figure C). Collect florets at the flowering stage in the field, take out the anthers with tweezers, gently squeeze the anthers in an iodine-potassium iodide solution (0.6% KI, 0.3% I2, w / w), drop them on a glass slide, cover with a cover glass, and place them under the microscope Observe the iodine staining of pollen and take pictures. The wild type has more pollen and is dyed blue-black, while the pollen grains of the mutant are small and cannot be dyed ( figure 1 Figures A and B). The mutant can bear fruit normally under open pollination, indicating that the mutant is a male sterile mutant and females are not affected. Bagging selfing w...
Embodiment 4
[0045] Example 4 Leaf Sampling and DNA Extraction
[0046] Use the CTAB method to extract DNA from rice leaves. The specific method is as follows: Weigh about 0.1g leaves, put them into a centrifuge tube, add 600 μL CTAB extraction buffer, 5 μL RNase A, oscillate to disperse, and place in a water bath at 65°C for 0.5 hr, shaking gently for 2-3 hours. Add an equal volume of chloroform / Tris-saturated phenol (1:1, v / v), mix well, and shake gently for 10 minutes; centrifuge at 10,000 rpm at 4°C for 20 minutes; transfer the supernatant to a new tube, and add 1 / 10 volume of 3M sodium acetate (pH value 5.2), 0.6-1 times the volume of cold isopropanol; gently shake and mix until flocculent precipitate appears; centrifuge at 10,000 rpm at 4°C for 10 min; discard the supernatant, and wash the precipitate with 70% ethanol by volume 2 time; air-dry, add 50 μL 1×TE to dissolve the precipitate, and store at -20°C. The DNA concentration was detected by Nanodrop2000 and diluted to 10ng / L for...
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