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Method for establishing high frequency regenerating system of perennial ryegrass

A technology of high-frequency regeneration and ryegrass, which is applied in plant regeneration, horticultural methods, botanical equipment and methods, etc., can solve the problems of poor ability to regenerate plants, difficult cultivation, slow progress of perennial ryegrass, and achieve the goal of obtaining materials Convenience and the effect of increasing the induction rate

Inactive Publication Date: 2006-02-15
BEIJING FORESTRY UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Young leaves, immature embryos, mature embryos and shoot apical meristems of perennial ryegrass can produce callus, but it is difficult to differentiate plants from calluses from young leaves and shoot apical meristems, and the operation of immature embryo culture is difficult , only a few genotypes of mature embryo culture can produce callus with strong regenerative ability
[0004] Perennial ryegrass seeds mostly contain endophytic bacteria that are resistant to diseases and insects. The infection rate of endophytic bacteria in the seeds of individual varieties is as high as 90%, so it is difficult to thoroughly disinfect the seeds
Due to the difficulty in culturing perennial ryegrass tissue and cells, the established transgenic receptor system is limited to individual genotypes, and the ability to regenerate plants is poor, so that the use of transgenic technology to improve perennial ryegrass is progressing slowly

Method used

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  • Method for establishing high frequency regenerating system of perennial ryegrass
  • Method for establishing high frequency regenerating system of perennial ryegrass
  • Method for establishing high frequency regenerating system of perennial ryegrass

Examples

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Embodiment 1

[0022] Utilize the present invention to obtain the high-frequency regeneration system of perennial ryegrass Aishote and Super Derby

[0023] Plant Material: Mature seeds of perennial ryegrass Eroste and Super Derby.

[0024] Pretreatment before inoculation: immerse an appropriate amount of seeds in a sodium hypochlorite stock solution (7% active chlorine) with a drop of Tween-20, stir and disinfect for 40 minutes, and wash with sterile water more than 6 times. Soak at low temperature at 4°C for 12 hours, wash again with sterile water, and then inoculate some seeds directly on the callus induction medium; some of the seeds were inoculated on the same medium by stripping the embryos with tweezers; the rest were directly inoculated on the MS basic medium. When it grows for 3 days, its hypocotyl and radicle (both about 5 mm in length) are inoculated on the callus induction medium. The results are shown in Table 1. It can be seen that the callus rate and embryogenic callus rate o...

Embodiment 2

[0044] Transforming DREB1A gene into perennial ryegrass to obtain transgenic plants by particle gun method

[0045] Perennial ryegrass Juventus embryogenic callus and a suitable medium for callus induction and differentiation were obtained according to the method in Example 1. After 2-3 passages, the embryogenic callus was picked and pre-cultured for 3 days before being bombarded. The DREB1A gene was coated on the gold powder microprojectiles, and the microprojectiles were uniformly coated on the carrier film by using the PDS-1000 / He gene gun of Biorad Company of the United States. The flying distance of the microprojectiles was 6cm, and each dish was bombarded twice.

[0046] After bombardment overnight on medium containing osmotica. The next day, the transient expression of the gene was observed by gusA histochemical staining, and the callus was transferred to the induction medium for 10 days in the dark, so that the transferred gene could be fully expressed. Then it is tr...

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Abstract

The invention discloses a method for establishing a high-frequent regenerating system for the perennial ryegrass, providing good receptor material for the work of genetic conversion, comprising: sterilizing the seeds, inducing the embryo callus on the traumatic inducing culture medium by employing the mature embryo as explant, shifting it to the regenerative culture medium after subculture for the regenerative sprout growth, then shifting it to the root-growing culture medium for the root growth and replanting it after getting the complete plant. The draw material of explant is easy and free of seasonal constrain, the sterilizing mode is safe and effective, by regulating the concentration of the hormone and addictive in the traumatic inducing culture medium, growing culture medium and root-growing culture medium, the inductivity of embryo callus is increased effectively, and the callus embryo structure is kept in the course of subculture; the regenerative rate and root-growing rate is very high and the survival rate of replanting is 100%.

Description

Technical field: [0001] The invention relates to a method for establishing a high-frequency regeneration system of perennial ryegrass, which belongs to a plant tissue culture method and can be used for the cultivation of receptor materials for gene transformation such as gene gun method and agrobacterium-mediated method. technical background: [0002] At present, using genetic engineering to breed and improve plant varieties is a convenient and practical way. Successful plant gene transformation firstly depends on a good plant receptor system. Such plant receptor systems for transgenics must have high-frequency, stable regeneration capabilities, and good sensitivity to transformation screening agents. The establishment and improvement of plant tissue culture and regeneration system is the basis and premise of genetic transformation. However, due to the characteristics of gramineous plants, the tissue culture and regeneration systems of many forage grasses and turfgrasses a...

Claims

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Application Information

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IPC IPC(8): A01H4/00C12N5/04
Inventor 韩烈保李雪曾会明信金娜刘君
Owner BEIJING FORESTRY UNIVERSITY
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