CN102676577B - Method for improving resistance of jujube trees to jujube witches broom through agrobacterium rhizogenes mediation - Google Patents

Abstract

A method for improving the resistance of jujube trees to jujube madness through the mediation of Agrobacterium rhizogenes, using the stems of jujube tissue culture seedlings infected with jujube madness as materials, infecting with Agrobacterium rhizogenes, and regenerating Select the transformed seedlings from the tissue-cultured seedlings, and then continue to culture and remove the bacteria. Molecular detection is carried out on the transformed seedlings to confirm that the transformed seedlings do not carry the pathogen of jujube madness. The disease resistance of the transformed seedlings is tested by micro-grafting technology. Through testing and verification, jujube resistant seedlings with significantly improved resistance to jujube madness were finally obtained. The method is simple and easy, and jujube tissue culture seedlings with significantly improved resistance to jujube madness can be obtained within a few months.

Description

A method for improving the resistance of jujube trees to jujube madness mediated by Agrobacterium rhizogenes

technical field

The invention relates to a method for improving the disease resistance of jujube trees to jujube madness through the mediation of Agrobacterium rhizogenes.

Background technique

Jujube witches'broom disease (JWB) is a devastating quarantine disease caused by Phytoplasma. Jujube madness manifests as arbuscules, yellowing, and distortion of flowers, leaves, and roots on the entire plant. Symptoms of diseased branches are clumps, slenderness, and shortened internodes. The arbuscular branches are caused by abnormal germination of diseased buds and reversion of flower organs. When jujube madness occurs, symptoms usually appear on local branches from the flowering stage, and then gradually spread to the whole plant. Jujube trees lose their ability to bear fruit soon after the onset of the disease, and most of them die in about 3 years. Every year, more than ten million trees die due to jujube madness in the whole country, and the annual direct economic loss is as high as hundreds of millions of yuan. Jujube madness has become a major problem that endangers the development of jujube industry and needs to be solved urgently.

The prevention and control of jujube madness can take various measures such as breeding resistant varieties, cutting out diseased trees, surgical treatment, drug treatment, prevention and control of infectious insects, heat treatment of stem tip tissue culture and detoxification. Among them, breeding disease-resistant varieties is the most economical and effective measure to control jujube madness. However, jujube flowers are small, artificial detasseling is difficult, fruit setting rate is low and embryo abortion is serious, so it is very difficult to carry out traditional hybrid breeding. With the continuous development of biotechnology, genetic engineering has increasingly become an effective means of modern plant improvement and breeding.

The Agrobacterium-mediated method is currently the most widely used genetic transformation system with the clearest mechanism, the most mature technology and the most successful cases, and it is also the most important transformation system in plant genetic engineering.

Previous studies have shown that the content of auxin (IAA) in diseased trees and diseased branches infected with jujube madness is significantly less than that of healthy trees and healthy branches, and the content of cytokinin (CK) is significantly higher than that of healthy trees and healthy branches. The IAA ratio is closely related to the incidence of jujube madness. When the ratio of CK/IAA is small, jujube trees show normal growth, but when the ratio is too large, jujube trees grow wildly. Artificially changing the ratio of CK/IAA can make the symptoms of jujube madness show "recovery". The agrobase-type Ri plasmid TR-DNA of Agrobacterium rhizogenes has auxin synthesis genes tms1 and tms2 , which guide the synthesis of auxin IAA.

This patent ingeniously uses Agrobacterium rhizogenes to infect jujube tree tissue culture seedlings infected with jujube mania (hereinafter referred to as mania tissue culture seedlings), so that mango tissue culture seedlings can produce more IAA, thereby reducing the IAA in the mango tissue culture seedlings. CK/IAA, eventually turned into normal seedlings.

Contents of the invention

The invention establishes a method for improving the resistance of jujube trees to jujube madness through the mediation of Agrobacterium rhizogenes.

The technical scheme that the present invention adopts to solve its technical problem is: use the stem section of the infected vegetative tissue cultured seedling as material, use Agrobacterium rhizogenes to infect, and screen the transformed tissue cultured seedlings in the regenerated tissue cultured seedlings, and then Continue to cultivate and remove bacteria, carry out molecular detection on the transformed seedlings to determine the transformed seedlings, use micro-grafting technology to test and verify the disease resistance of the transformed seedlings, and finally obtain jujube resistant seedlings with significantly improved resistance to jujube madness .

Concrete content of the present invention, that is, the specific method for improving jujube mad disease resistance through the mediation of Agrobacterium rhizogenes is as follows:

⑴ Infection of Agrobacterium rhizogenes with Agrobacterium rhizogenes: Cut the tender stems of Agrobacterium rhizogenes into about 1 cm long, infect with Agrobacterium rhizogenes with an OD ₆₀₀ of 0.6-1.0 for 5-15 minutes, and then transfer to co-culture medium MS+5.5g/L agar+30g/L sucrose+200μΜ acetosyringone (AS) (pH 5.8) co-cultured for 3-7d, then transferred to medium MS+5.5g/L agar+30g/L sucrose+100mg /L ceftriaxone sodium (CRO) (pH 5.8), cultured for 60 days at 25±2°C, light/darkness 14/10h, and screened out the transformed seedlings whose symptoms of jujube madness disappeared;

⑵Degerm-free subculture of transformed seedlings Transfer the transformed seedlings into MS+5.5g/L agar+30g/L sucrose+0.2mg/L BA+0.1mg/L IBA (pH value) added with CRO 50-100mg/L 5.8) Continue to cultivate in the medium, change the medium every 20 days, and gradually reduce the concentration of CRO until the finally transformed healthy seedlings are completely free of Agrobacterium rhizogenes, and the sterilized transformed healthy seedlings are obtained;

(3) Molecular detection of the germ-free and healthy seedlings Extract the genomic DNA of the germ-free and healthy seedlings for PCR detection of rolC , rolD and tms genes and the detection of jujube mad disease pathogens, and screen out those carrying agrobacterium rhizogenes rolC , rolD and tms genes Transformed plantlets of Agrobacterium rhizogenes carrying the pathogen of jujube madness;

(4) Identification of the resistance of Agrobacterium rhizogenes transformed seedlings to jujube madness The micro-grafting method of tissue culture reverse split grafting was adopted, that is, on the ultra-clean workbench, the lower end of the stem section of the transformed seedlings to be identified was split and inserted into the rootstock In the mad tissue culture seedlings, the cambium of the rootstock and the scion are bonded together, and the sterilized aluminum foil is bound outside the grafting interface to fix it, and then the grafted seedlings are transferred to the grafting medium for cultivation, and the grafting seedling medium is MS +30g/L sucrose+8g/L agar (pH value 5.8), observe the growth and disease of grafted seedlings, and the transformed seedlings without disease show that their resistance has been improved, that is, resistant seedlings.

The beneficial effect of the patent of the present invention is that through the infection of Agrobacterium rhizogenes, the jujube tissue culture seedlings with improved disease resistance are finally obtained. The method is simple and easy to operate, and jujube tissue culture seedlings with improved disease resistance can be obtained within a few months. Compared with field breeding that takes many years to obtain disease-resistant plants, the test is not affected by environmental conditions and has strong controllability ,save time and energy.

Description of drawings

Figure 1 Tissue culture seedlings of Dongzao chinensis

Figure 2 Regenerated Crazy Crazy seedlings from the stem section of Dongzao Crazy tissue cultured seedlings (as a control)

Fig. 3 Transgenic seedlings regenerated from the stems of Dongzaofeng tissue cultured seedlings infected by Agrobacterium rhizogenes

Figure 4 Transformed winter jujube seedlings that do not carry the pathogen of jujube madness obtained after molecular detection

Figure 5 Verification of micrografting using healthy seedlings of Dongzao as scion and tissue cultured seedlings of Dongzao as rootstock (as a control)

Figure 6 The control showed madness after 45 days of micro-grafting, with arbuscular branches, stems becoming thin and weak, and leaves turning yellow

Fig. 7. Using the transformed seedlings of Dongzao that do not carry the pathogen of Jujube as scion and the tissue-cultured seedling of Dongzao as rootstock, the disease resistance was verified by micro-grafting

Fig. 8 After 45 days of micro-grafting, the transformed seedlings of Dongzao as scions that do not carry the pathogen of jujube madness still maintain a healthy seedling state.

Detailed ways

Using the tissue-cultured seedlings of Dongzao chinensis as the test material, cut the tender stems into about 1cm long stems, infect with Agrobacterium rhizogenes 30148 with an OD ₆₀₀ of 0.6-1.0 for 5-15min, and then transfer to the co-culture medium MS+5.5 g/L agar + 30g/L sucrose + 200μΜ acetosyringone (AS) co-cultured for 3-7d, and then transferred to medium MS + 5.5g/L agar + 30g/L sucrose + 100mg/L ceftriaxone sodium (CRO ), cultured at 25±2°C for 60 days under the condition of light/darkness of 14/10h, and screened out the transformed seedlings whose symptoms of jujube madness disappeared. Transfer healthy seedlings to MS+5.5g/L agar+30g/L sucrose+0.2mg/L BA+0.1mg/L IBA (pH value 5.8) supplemented with CRO 50-100mg/L to continue culturing. The culture medium was changed once every 20 days, and the transformed seedlings were completely sterilized by culturing for 10 generations. The genomic DNA of the transformed seedlings was extracted for PCR detection of rolC , rolD , tms genes and detection of jujube mad disease pathogen. Finally, the transformed seedlings carrying the genes of agrobacterium rhizogenes rolC , rolD and tms but not the pathogen of jujube madness were screened out, and the transformation rate reached 10.6%. Then, the tissue culture micro-grafting technique was used to detect the resistance of the transformed seedlings to jujube madness. The rootstock was selected from the vigorous tissue-cultured seedlings of Dongzao madrassica, the scion was selected from the transformed seedlings with good growth, and the scion of healthy winter jujube tissue-cultured seedlings was selected as a control. After 45 days of micrografting culture, it was found that the healthy seedlings of winter jujube in the control had shown obvious symptoms of jujube madness, the leaves turned yellow, the stems became thin and thin, and arbuscular branches appeared, while the transformed seedlings remained healthy, which were resistant seedlings. .

Claims (1)

1. a kind of method that improves jujube tree to jujube mad disease resistance by Agrobacterium rhizogenes mediation, is characterized in that comprising the following steps: (1) Infection with Agrobacterium rhizogenes and the acquisition of healthy seedlings Cut the tender stems of jujube tissue-cultured seedlings infected with jujube mania to a length of about 1 cm, and infect them with Agrobacterium rhizogenes with an OD ₆₀₀ of 0.6-1.0 for 5 -15min, then transferred to the co-culture medium MS+5.5g/L agar+30g/L sucrose+200μM acetosyringone with a pH value of 5.8 for co-cultivation for 3-7d, and then transferred to the screening medium MS+5.5g with a pH value of 5.8 /L agar + 30g/L sucrose + 100mg/L ceftriaxone sodium, cultivated for 60 days at 25±2°C, light/darkness 14/10h, and screened out the transformed seedlings whose symptoms of jujube madness disappeared; (2) Germ-free subculture of transformed seedlings Transfer the transformed seedlings into MS+5.5g/L agar+30g/L sucrose+0.2mg/L BA with ceftriaxone sodium 50-100mg/L and pH 5.8 Continue culturing in +0.1mg/L IBA medium, change the medium every 20 days, and gradually reduce the concentration of ceftriaxone sodium until the transformed seedlings completely remove Agrobacterium rhizogenes, and obtain the sterilized transformed seedlings; (3) Detection of germ-free and healthy seedlings Extract the genomic DNA of the germ-free and healthy seedlings for PCR detection of rolC, rolD, and tms genes and detection of jujube mad disease pathogens, and screen out the genes carrying Agrobacterium rhizogenes rolC, rolD, and tms Transformed seedlings that do not carry the pathogen of jujube madness; (4) Identification of the resistance of transformed seedlings to jujube madness Using tissue culture micro-grafting method, the transformed seedlings to be identified were grafted onto the jujube tissue cultured seedlings. Cultivate in L agar medium, observe the growth and pathogenesis of grafted seedlings, and the transformed seedlings without disease show that their resistance has been improved, that is, resistant seedlings.

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