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Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system

A technique of genetic transformation of Agrobacterium rhizogenes, applied in the field of production of salidroside, which can solve problems such as increasing costs and affecting the quality of salidroside, and achieve the effects of increasing production, not occupying arable land area, and reducing costs

Inactive Publication Date: 2011-09-14
JILIN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is equivalent to the content of salidroside in wild alpine rhodiola. Moreover, the addition of hormones not only increases the cost, but also affects the quality of salidroside

Method used

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  • Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system
  • Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system
  • Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0037] 1 Obtaining sterile vaccines

[0038] Rhodiola rosea seeds collected from Changbai Mountain at an altitude of 1700-2500 meters were washed with tap water, soaked in 75% alcohol for 1 min, and then sterilized with 15% NaClO for 12 min. Then rinse with sterile water for 3 to 5 times, dry the water and inoculate on hormone-free 1 / 2MS+Pro700mg / L medium (0.6% agar) for germination and culture for 10 to 18 days. 25°C, light intensity 2000lux, 16 hours light, 8 hours dark culture. After 15 days, the aseptic seedlings grow out, and the green cotyledons (leaving about 2 mm of cotyledon petiole) of the 3-day seedling age are taken to be subcultured on MS+0.5mg / LNAA+3.0mg / L 6-BA medium. figure 1 shown.

[0039] 2 pre-culture

[0040] Get the cotyledon nodes, stem segments and leaves of the subcultured aseptic seedlings, lightly scratch the dorsal and ventral surfaces of the leaves and cotyledons with petioles with a knife, cut the stem segments into 1 cm long as explants, inocu...

Embodiment 2

[0085] 3. Culture of Agrobacterium rhizogenes

[0086] Five kinds of Agrobacterium rhizogenes LBA9402 were inoculated on YEB solid medium for streak culture, activated three times at 27°C, picked a single colony on the plate, and inoculated in 25ml LB liquid medium. Cultured on a shaking table at 27°C, 4000r / min in the dark, and subcultured for 3 times, Agrobacterium rhizogenes liquid A 600 When it is 0.55, it is used to infect explants.

[0087] 4. Infection, co-cultivation, sterilization and screening of Agrobacterium rhizogenes

[0088] The above-mentioned pre-cultivated explants were immersed in the logarithmic phase bacterial solution A 600 rhizogenes LBA9402 bacterial solution with a concentration of 0.45-0.60, infect in the dark for 25 minutes, take out the excess bacterial solution with sterile filter paper, and place it in MS+NAA (0.05mg / L)+Pro 700mg / L medium After 2.5 days of co-cultivation, transfer to 1 / 2MS + kanamycin 50mg / L + cephalosporin 450mg / L + 80mmol / L a...

Embodiment 3

[0090] 3. Culture of Agrobacterium rhizogenes

[0091] Inoculate 5 species of Agrobacterium rhizogenes A4 on YEB solid medium for streak culture, activate 3 times at 27°C, pick a single colony on the plate, and inoculate it in 25ml LB liquid medium. Cultured on a shaking table at 27°C, 4000r / min in the dark, and subcultured for 3 times, Agrobacterium rhizogenes liquid A 600 When it is 0.50, it is used to infect explants.

[0092] 4. Infection, co-cultivation, sterilization and screening of Agrobacterium rhizogenes

[0093] The above-mentioned pre-cultivated explants were immersed in the logarithmic phase bacterial solution A 600 rhizogenes A4 bacterial solution with a concentration of 0.50, infect in the dark for 24 minutes, take out the excess bacterial solution with sterile filter paper, and place it on MS+NAA (0.05mg / L)+Pro 700mg / L medium for total After culturing for 2 days, transfer to 1 / 2MS + kanamycin 50mg / L + cephalosporin 450mg / L + 90mmol / L acetosyringone medium, a...

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Abstract

The present invention discloses a method using Agrobacterium rhizogenes (Ar) gene to transform Rhodiola sachalinensis A.Bor to achieve a hairy root system for Salidroside production. By preparation of explant and cultivation in Ar bacteria liquid, Rhodiola sachalinensis A.Bor is induced to grow hairy roots by genetic transformation; the PCR testing proves that hairy roots are generated by transformation; a fermenter is used for massive production of hairy roots, and precursor substances and elicitors are added to improve the Salidroside content. The method is applied to produce Salidroside to substitute the drying-up Rhodiola sachalinensis A.Bor resources.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. Specifically, it is a method for producing salidroside by using Agrobacterium rhizogenes to genetically transform rhodiola rosea to establish a hairy root culture system. Background technique [0002] Rhodiola sachalinensis A.Bor, also known as Rhodiola sachalinensis A.Bor, is a perennial dicotyledonous herbaceous plant belonging to the genus Rhodiola L. in the Sedum family. It is one of the rare medicinal plants. In traditional Chinese medicine, it has the effect of "consolidating the root and strengthening the body", and has the function of "adaptogen". It also has significant functions such as anti-hypoxia, anti-cold, anti-fatigue, anti-microwave radiation, etc., and has the effects of delaying body aging and preventing senile diseases. Due to the industrial development of Rhodiola rosea, a large number of wild plants have been excavated, and the resources are becoming increasing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/87C12P19/44
Inventor 周晓馥徐洪伟
Owner JILIN NORMAL UNIV
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