Genetic intelligent seed breeding and production system for crossbreeding and production of crops and application thereof

A hybrid breeding and crop technology, applied in the field of genetically intelligent breeding system, can solve the problems of inability to industrialize utilization, inability to distinguish fertile seeds, and inability to maintain male sterility of offspring on a large scale.

Pending Publication Date: 2021-11-09
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the male sterility of the "three-line method" male sterile line is caused by the interaction of genes between the nucleus and the cytoplasm, this method has a flaw: only the restorer line with a specific restorer gene in the nucleus can be restored to the sterile line after crossing with the sterile line Fertility to produce hybrid rice seeds
The discovery of recessive sterility has a very long history. When it is crossed with wild-type material, its F1 generation will be in a heterozygous state and will be fertile, and the F2 generation will segregate for fertility (in the case of single gene control, the segregation ratio is 3:1), but when the seeds of the F1 generation are self-fertilized and harvested (ie F2), because the appearance of the seeds is exactly the same, it is impossible to distinguish which part of the seeds will be fertile after developing into an adult plant in the future, and which part will be sterile
Therefore, it has long been limited by the inability to maintain the male sterility of its offspring on a large scale and cannot be used industrially

Method used

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  • Genetic intelligent seed breeding and production system for crossbreeding and production of crops and application thereof
  • Genetic intelligent seed breeding and production system for crossbreeding and production of crops and application thereof
  • Genetic intelligent seed breeding and production system for crossbreeding and production of crops and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1 GAT carrier construction and verification

[0112] 1. Construction of GAT vector

[0113] The GAT vector adopts the strategy of segmented construction with the expression cassette as the unit and unit splicing. First in the transition vector pC1300 ( Figure 9 ) and pUC57-Simple ( Figure 10 ) to construct an expression cassette and verify it by digestion and sequencing, and then splicing the expression cassette into the final vector. The specific steps of vector construction are as follows:

[0114] 1. MCS pC0307 fragment (SEQ ID NO.38) was synthesized, and pC0307 was obtained after being connected into pC1300 with Sac II and Pme I ( Figure 6 ).

[0115] 2. Synthesize the MCS pC0308 fragment (SEQ ID NO.39), connect pC1300 with Sac II and Sph I to obtain pC0308 ( Figure 7 ).

[0116] 3. MCS pC0309 fragment (SEQ ID NO.40) was synthesized, and pC0309 ( Figure 8 ).

[0117] 4. Synthesize the DNA fragment NSPT-Construct V1.81-Marker 1, cut it with Kp...

Embodiment 2

[0152] Example 2 Agrobacterium-mediated genetic transformation of rice with GAT vector

[0153] 1. Transformation of the GAT vector constructed in Example 1 into Agrobacterium and verification

[0154] Agrobacterium EHA105 stored at -80°C was streaked on a YEP plate containing rifampicin (25 μg / ml) + streptomycin (50 μg / ml), and cultured at 28°C. Pick a single colony and inoculate it in 5 ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 220 rpm and shake at 28° C. for 12-16 hr. Take 2ml of bacterial liquid and transfer it to 100ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 28°C and shake at 220rpm until OD 600 = 0.5. Pre-cool on ice for 10 min, centrifuge at 5000 rpm for 10 min at 4°C (refrigerated centrifuge is pre-cooled to 4°C). Wash twice with sterile deionized water (10 ml each time), wash once with 10% sterile glycerol, centrifuge at 5000 rpm for 10 min at 4° C., and resuspend the bacteria in 3 ml of 10% s...

Embodiment 3

[0162] Example 3 Molecular Identification of GAT T0 Transformation Materials

[0163] At the five-leaf stage, the transgenic plant leaves obtained in Example 2 were extracted with the CTAB method to extract the total DNA of the genome, and the extraction method was as follows:

[0164] Take 2-4cm leaves and put them into a mortar, add 800-900ul of 1.5% CTAB solution, grind, then transfer the ground liquid into a 1.5ml centrifuge tube, put it on ice or in a low-temperature refrigerator for use; sample Water bath at 65°C for 30 minutes, during which time it was inverted and mixed several times; in the fume hood, use a glass pipette to add chloroform and isoamyl alcohol solution (chloroform: isoamyl alcohol = 24:1, that is, 500ml chloroform is added to 22ml isoamyl alcohol, Gently mix) 650ul, shake on the shaker for 30min or shake by hand for about 10min after mixing, at this time, obvious stratification can be seen; centrifuge the shaken sample at 8000-10000rpm for 8min; suck th...

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Abstract

The invention provides a genetic intelligent seed breeding and production system for crossbreeding and production of crops and application thereof. The system contains a GAT system vector, and the vector comprises five functional element expression cassettes: a plant male fertility restoration gene element expression cassette used for restoring male fertility of recessive genic male sterility mutants, a plant pollen abortion gene element expression cassette used for removing pollen containing GAT and keeping a heterozygotic state or a hemizygotic state of a GAT maintainer line, a chemical herbicide positive selection expression cassette used for gene transformation and GAT maintainer line impurity removal and purification, a chemical herbicide negative selection expression cassette used for removing herbicide-sensitive GAT maintainer line pollen and seed escape and removing impurities and purifying a GAT sterile line, and a seed screening element expression cassette used for mechanically sorting seeds. The system can be used for crossbreeding and hybrid seed production of plant recessive genic male sterility materials, so that new varieties of plants with high quality, high yield, wide applicability and high resistance and seeds of the plants are obtained.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a genetic intelligent seed production system for crop hybrid breeding and seed production and its application. Background technique [0002] Heterosis is a phenomenon in which offspring of hybrids surpass their parents in one or more traits, and it generally exists in the biological world. The utilization of crop heterosis is an important means to increase agricultural production, and the core of the industrialization of heterosis utilization lies in the male sterility or male sterile line of the female parent. [0003] Taking rice as an example, China is the most successful country in the world in the industrialization of hybrid rice, and it is in a leading position in the field of hybrid rice breeding research. At present, the breeding methods of hybrid rice in my country are mainly divided into "three-line method" and "two-line method", each has its advan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H5/02A01H6/46A01H1/02A01C1/06A01G13/00
CPCC12N15/8289C12N15/8274C12N15/8212A01H1/02A01C1/06A01G13/00A01H5/00A01H5/02A01H6/46C12N15/82C12N15/8218
Inventor 安保光龙湍李新鹏曾翔吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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