Method for mechanically producing seed by using female sterile hybrid rice
A hybrid rice and female technology, applied in the field of agricultural biology, can solve problems such as large-scale commercial application, and achieve the effects of reducing seed production procedures, improving efficiency, and easy isolation
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[0028] Example 1: Construction of a rice expression vector carrying three gene expression cassettes of rice female fertility gene, pollen lethal gene and fluorescent selection marker gene
[0029] In the expression vector pCAMBIA1300, three expression cassettes were inserted: wild-type PTB1 expression cassette, pollen lethal amylase gene ZM-AA1 expression cassette, and fluorescent screening marker gene RP expression cassette. Wild-type PTB1 carries its own promoter and terminator (see SEQ ID NO. 2 for its nucleotide sequence); the elements of the expression cassette of the amylase gene ZM-AA1 include: corn’s late pollen development specific promoter PG47 (its nuclear See SEQ ID NO. 3 for the nucleotide sequence, the transit peptide ZM-BT1 of corn (see SEQ ID NO. 4 for its nucleotide sequence), and the coding sequence of the corn amylase gene ZM-AA1 (see SEQ ID for its nucleotide sequence) ID NO. 5), the terminator IN2-1 of corn (see SEQ ID NO. 6 for its nucleotide sequence); the ...
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[0036] Example 2: Obtaining transgenic rice
[0037] 2.1 Plant material selection
[0038] The rice (Oryza sativa L.) variety used for genetic transformation was selected as the corresponding female sterile rice line carrying the PTB1 gene (SEQ ID NO.1).
[0039] 2.2 Induction of rice embryogenic callus
[0040] Take the immature rice seeds 10-15 days after pollination, peel off the seed coat, soak in 70% alcohol for 3 minutes, then soak in 0.1% mercury for 15 minutes (or transfer to 20% sodium hypochlorite solution, shake and sterilize for 25 minutes on a shaker) ), clean the sterile water in the ultra-clean workbench for 3-5 times, squeeze the sterilized immature embryos of the seeds with tweezers, inoculate them on the induction medium, put about 35 grains in each dish, culture in the dark at 28°C for 4-5 days, The radicle was removed, and the culture was continued for 12-15 days. After the callus grew up, it was subcultured, subcultured once every two weeks for a total of 2-3 sub...
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[0053] Example 3: Molecular detection of transgenic plants
[0054] 3.1 Large-scale DNA extraction
[0055] T 0 The CTAB method (Murry and Thompson, 1980) was used to extract DNA from the generation of transgenic rice. Weigh 2-4 grams of fresh leaves, cut them into pieces, put them in a mortar pre-cooled at -20°C, grind them into powder with liquid nitrogen, and transfer them to a pre-cooled sample flask at -20°C for later use. During extraction, add 10ml 1.5X CTAB preheated to 100℃, stir quickly, shake in a water bath insulated shaker at 56℃ for 20 minutes, and then extract with an equal volume of chloroform:isoamyl alcohol (24:1). After centrifugation, the supernatant was extracted once with 1 / 10 volume of 10% CTAB buffer preheated to 56°C and an equal volume of chloroform:isoamyl alcohol (24:1), and then the supernatant was added with 1% CTAB to precipitate DNA After centrifugation, add 1mol / L NaCl with RNase A to dissolve the DNA. After completely dissolving the alcohol preci...
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