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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

Active Publication Date: 2014-06-04
HUNAN TAOHUAYUAN SEED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned methods have many defects and cannot fundamentally realize the mechanized seed production of hybrid rice, and there is no large-scale commercial application in production at present.

Method used

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  • Method for mechanically producing seed by using female sterile hybrid rice
  • Method for mechanically producing seed by using female sterile hybrid rice
  • Method for mechanically producing seed by using female sterile hybrid rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of rice expression vector carrying three gene expression cassettes of rice female fertility gene, pollen lethal gene and fluorescent selection marker gene

[0029] On the expression vector pCAMBIA1300, three expression cassettes were inserted: wild-type PTB1 expression cassette, pollen lethal amylase gene ZM-AA1 expression cassette, 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 amylase gene ZM-AA1 expression cassette elements include: maize pollen development late specific promoter PG47 (its nuclear See SEQ ID NO.3 for the nucleotide sequence), corn transit peptide ZM-BT1 (see SEQ ID NO.4 for its nucleotide sequence), ZM-AA1 for the amylase gene coding sequence of corn (see SEQ ID NO.4 for its nucleotide sequence) ID NO.5), corn terminator IN2-1 (see SEQ ID NO.6 for its nucleotide sequence); red fluorescent protein gene RP express...

Embodiment 2

[0036] Embodiment 2: Transgenic rice obtains

[0037] 2.1 Plant material selection

[0038] The rice (Oryza sativa L.) variety used for genetic transformation was selected as the corresponding rice female sterile line carrying the PTB1 gene (SEQ ID NO.1).

[0039] 2.2 Induction of rice embryogenic callus

[0040] Take 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 chloride for 15 minutes (or transfer to 20% sodium hypochlorite solution and sterilize on a shaking table for 25 minutes ), washed 3-5 times with sterile water in the ultra-clean workbench, extruded the sterilized immature embryos with tweezers, inoculated them on the induction medium, put about 35 seeds in each dish, cultured in dark at 28°C for 4-5 days, Cut off the radicle, continue to culture for 12-15 days, and subculture after the callus grows up, once every two weeks, for a total of 2-3 times.

[0041] The mature embryos...

Embodiment 3

[0053] Example 3: Molecular detection of transgenic plants

[0054] 3.1 DNA large-scale extraction

[0055] T 0 Transgenic rice DNA was extracted using the CTAB method (Murry and Thompson, 1980). Weigh 2-4 grams of fresh leaves, cut them into pieces, put them into a pre-cooled mortar at -20°C, grind them into powder with liquid nitrogen, transfer them to a pre-cooled grinding bottle and store them at -20°C for later use. When extracting, add 10ml of 1.5X CTAB preheated to 100°C, stir quickly, shake in a water bath at 56°C 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 1% CTAB was added to the supernatant to precipitate DNA , after centrifugation, add 1mol / L NaCl with RNase A to dissolve the DNA, completely dissolve the alcohol precipitate at 56°C overnigh...

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Abstract

The invention discloses a method for mechanically producing seed by using female sterile hybrid rice. The method is characterized in that female sterile hybrid rice is transferred into three closely-linked gene expression cassettes, namely, 1) female fertile rice gene expression cassette, 2) pollen lethal gene expression cassette, and 3) fluorescent sieving marking gene expression cassettes. According to the method, the pollen grains that carry transgenosis cannot participate in fertilization during self-fertility of hybrid transgenic rice, and the pollen grains that do not carry transgenosis can normally fertilize with a female gamete to seed; and on that basis, photoelectric separating and sorting is carried out to obtain homozygous female sterile rice which can be used for mechanically producing seed for the hybrid rice, and the hybrid seeds that carry transgenosis is continuously used for breeding female sterile rice. With the adoption of the method, the problem of breeding of female sterile rice is solved, and the mechanical seed production of hybrid rice, in particular the mechanical seed production by a mixed sowing and mixed harvesting manner, is realized.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology and discloses a hybrid rice mechanized seed production method utilizing female sterility. Background technique [0002] The annual planting area of ​​hybrid rice in my country is about 16 million hm2, the seed production area needs about 150,000 hm2, and about 240 million kg of commercial hybrids are needed every year. my country's current hybrid rice seed production technology mainly relies on manual operations from sowing to harvesting. The parents are sown in stages, mostly the male parent is sown first, and the female parent is sown later; the parent parent is planted separately in a box-type, the female parent has multiple rows, and the male parent has two rows; when harvesting, the parent parent is harvested separately, or the parent parent is cut off after pollination and before maturity. Book. The whole process of seed production is complicated, it is difficult to realize mechan...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H1/02C12N15/29A01H5/00
CPCC12N15/8287C12N15/829A01H1/024
Inventor 曹孟良夏玉梅沈春修方真蔡立湘
Owner HUNAN TAOHUAYUAN SEED
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