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Genetic transformation method of agrostis stolonifera

A genetic transformation method and genetic transformation technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., to achieve the effects of simple and easy operation, promotion of molecular breeding and genetic improvement, and high transformation positive rate

Active Publication Date: 2021-01-05
武汉天问生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the prior art, there is no successful case of genetic transformation of creeping bentgrass through glufosinate-resistant screening combined with transgenic technology. Therefore, it is necessary to study a new genetic transformation method to promote the molecular breeding and development of creeping bentgrass. Genetic improvement, cultivating fine varieties with more market value

Method used

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  • Genetic transformation method of agrostis stolonifera
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  • Genetic transformation method of agrostis stolonifera

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Embodiment 1

[0035] The disinfection treatment of embodiment 1 seed

[0036] The seeds of creeping bentgrass are very small and covered with fluff. Sandpaper should be used to gently rub the fluff to remove the fluff. At the same time, it serves the purpose of shelling and is convenient for subsequent disinfection treatment. Disinfect the seeds on the ultra-clean workbench: soak in 75% (v / v) alcohol for 1 minute, pour off the alcohol, add 50% (v / v) 84 solution to soak for 6, 10, 14 minutes, wash with sterile water After 5 times, transfer the seeds to a sterilized 0.1% agarose solution, pipette evenly, spread them evenly on the MS medium with a pipette, seal them with a parafilm and place them in a culture room at 25-28°C in the dark After 2 weeks of cultivation, the germination rate was counted. The results showed that the 84 solution disinfection for 10 minutes could effectively control the pollution, and the germination rate reached 79.3%. With the prolongation of the treatment time, th...

Embodiment 2

[0039] Embodiment 2 induces callus

[0040]After the seeds have been subjected to the above-mentioned optimal disinfection treatment, the seeds are transferred to a sterilized 0.1% agarose solution, pipetted evenly, and spread evenly on the MS medium with a pipette. Medium composition: 4.43g / L MS, 30g / L sucrose, 500mg / L casein hydrolysate, 0~8.8mg / L 2,4-D, 0~1mg / L 6-BA, 2g / L phytagel, PH=5.7 , sealed with a parafilm and placed in a culture room at 25°C in the dark for 6 weeks. Among them, the concentration of 2,4-D is set to 2.2mg / L, 4.4mg / L, 6.6mg / L and 8.8mg / L, and the concentration of 6-BA is set to 0.5mg / L and 1.0mg / L. After 4 weeks, the statistics of callus induction showed that when the concentration of 2,4-D was 6.6mg / L and the concentration of 6-BA was 0.5mg / L, the callus induction rate reached 92.3%. Bright yellow is a relatively good growth state. Accordingly, this concentration combination is selected as the optimal formula for callus induction.

[0041] Table 2....

Embodiment 3

[0043] The determination of embodiment 3 resistant callus screening concentration

[0044] The callus with better growth state and the same size was selected and transferred to the subculture medium (medium composition: 4.43g / LMS, 30g / L sucrose, 500mg / L casein hydrolysate, 6.6mg / L 2,4-D, 0.5mg / L 6-BA, 2-10mg / L phosphinothricin, 2g / L phytagel, pH=5.7). The concentrations of glufosinate-ammonium are 2mg / L, 4mg / L, 6mg / L, 8mg / L, 10mg / L respectively. Observe the growth state of the callus after cultivating in the dark at 25-28°C for two weeks. It is found that when the concentration of glufosinate-ammonium reaches 8 mg / L, the callus turns brown and growth stagnates, which is a more suitable concentration for screening.

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Abstract

The invention relates to a genetic transformation method of agrostis stolonifera, and relates to the field of plant culture by utilizing genetic engaineering. According to the method, glufosinate ammonium serves as a screening agent, target plasmids with glufosinate ammonium resistance genes are used, and the glufosinate ammonium resistance genes are successfully transformed into agrostis stolonifera plants. The method realizes regeneration and genetic transformation of the agrostis stolonifera, is simple and easy to operate, successfully transforms glufosinate ammonium resistance genes into agrostis stolonifera plants through an optimized regeneration system, obtains glufosinate ammonium-resistant positive strains, and promotes molecular breeding and genetic improvement of the agrostis stolonifera so as to cultivate excellent varieties with higher market value.

Description

【Technical field】 [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for genetic transformation of creeping bentgrass. 【Background technique】 [0002] Creeping bentgrass (Agrostis. Stolonifera L.) belongs to the genus Bentgrass of Gramineae, and is a perennial herb. It is distributed in Northeast my country, North China, Northwest China, Jiangxi, Zhejiang and other regions. Because of its strong cold tolerance, drought tolerance, barren tolerance, shade tolerance, and pruning tolerance, as well as small and dense leaves and strong horizontal spreading ability, it is widely used in sports field lawns and ornamental lawns. However, creeping bentgrass is usually threatened by a variety of weeds, some of which are highly competitive, and severe infestation will lead to a sharp drop in yield and even loss of economic benefits. [0003] Glufosinate ammonium (phosphinothricin) is a broad-spectrum contact-killing herbicide s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65A01H4/00A01H5/00A01H6/46
CPCC12N15/8205C12N15/65A01H4/00
Inventor 凌飞徐庆
Owner 武汉天问生物科技有限公司
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