Method for inducing sandalwood suspension cells to synthesize santalene

Abstract

The invention discloses a method for inducing sandalwood suspension cells to synthesize santalene. The method comprises the following steps: (1) performing induced callus culture on sandalwood explants; (2) carrying out suspension culture on the sandalwood callus to obtain a suspension cell line; and (3) adding cytokinin and auxin into the cell culture fluid of the suspension cell line, and performing induced culture to obtain the sandalwood suspension cell with high content of santalene. The method takes plant cell culture as a technical basis, and comprises the following steps: firstly, establishing a stable sandalwood suspension cell line; by adding cytokinin and auxin into the sandalwood cell culture fluid, the synthesis of the sandalene can be promoted, so that the content of the sandalene is increased. The result of the embodiment shows that according to the method provided by the invention, the biomass of the sandalwood suspension cells is improved by adding the cytokinin and the auxin. The product content in the sandalwood suspension cell line is about 3% and is obviously higher than that of a control group; gC-MS (gas chromatography-mass spectrometry) detection shows that the main components are alpha-santalene and beta-santalene.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to a method for inducing sandalwood suspension cells to synthesize santalene. Background Art

[0002] Sandalwood (Santalum album) is a plant of the Santalaceae family, Santalum genus. Wild species are mainly distributed in India, and are now widely cultivated in Australia, Indonesia, southern China and other regions. Because the heartwood of sandalwood contains high-quality essential oil, it is the species with the highest economic value in the genus Santalum. The main components of sandalwood essential oil are cis-α-santalol ((Z)-α-santalol) and cis-β-santalol ((Z)-β-santalol), accounting for more than 80% of the total essential oil. It also contains low-content components such as α-santalene, β-santalene, and trans-α-bergamotene. Sandalwood essential oil is widely used in the fragrance industry for its fragrance, and is used in the medical field for its anti-inflammatory, antibacterial and anti-tumor activities. Under natural growth conditions, it usually takes 6-8 years for the essential oil to accumulate in the heartwood of sandalwood. The essential oil content of the heartwood of sandalwood that has grown for more than 30 years can meet the international standard ((Z)-α-santalol>40%, (Z)-β-santalol>20%). Studies have shown that exogenous mechanical damage, ethylene, jasmonic acid, methyl viologen, etc. all induce the formation of sandalwood heartwood to varying degrees, and the essential oil content is higher than that of untreated. At present, the technology of artificially promoting the formation of sandalwood heartwood and the synthesis of essential oil has not been widely used in the sandalwood cultivation industry. Plant suspension cell lines have short production cycles, stable yields, high component content, and great development and utilization value. Research on significantly increasing the content of santalene in sandalwood cells through cell culture technology has not been reported. The main contents of the present invention include how to create a high-yield and stable sandalwood suspension cell line and a method for inducing cell lines to synthesize santalene. Summary of the invention

[0003] The invention provides a method for inducing sandalwood suspension cells to synthesize santalene.

[0004] The method of inducing sandalwood suspension cells to synthesize santalene comprises the following steps:

[0005] (1) inducing callus culture from sandalwood explants;

[0006] (2) suspending the sandalwood callus to obtain a suspension cell line;

[0007] (3) Adding cytokinin and auxin to the cell culture medium of the suspension cell line and inducing the culture to obtain sandalwood suspension cells with a high santalene content.

[0008] Preferably, the sandalwood explant is a sandalwood stem segment.

[0009] Preferably, the cytokinin in the cell culture medium is 6-benzylaminopurine, and the growth hormone is naphthylacetic acid.

[0010] Preferably, the cell culture medium is prepared by adding sucrose 20-30 g / L, 6-benzylaminopurine 0.2-1 mg / L, and naphthylacetic acid 0.05-0.1 mg / L to MS culture medium, and the pH value is 5.8-6.0.

[0011] Preferably, the cell culture medium is prepared by adding 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine, and 0.1 mg / L naphthaleneacetic acid to MS culture medium, and the pH value is 5.8-6.0.

[0012] Preferably, the step (1) is to select young stems of sandalwood and inoculate them on a callus induction medium for callus induction culture to obtain callus; transfer the induced callus to a proliferation medium for proliferation culture; the callus induction medium is an MS basic medium supplemented with 50-100 mg / L inositol, 1-2 mg / L glycine, 1.0-2.0 mg / L 2,4-dichlorophenoxyacetic acid, 20-40 g / L sucrose, 5-8 g / L agar, and a pH of 5.8-6.0;

[0013] Preferably, the callus induction medium comprises 100 mg / L inositol, 2 mg / L glycine, 1 mg / L 2,4-dichlorophenoxyacetic acid, 30 g / L sucrose, 7 g / L agar, and a pH of 5.8-6.0;

[0014] The proliferation medium is MS basic medium supplemented with 50-100 mg / L inositol, 1-2 mg / L glycine, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 20-30 g / L sucrose, 5-8 g / L agar, and a pH of 5.8-6.0;

[0015] Preferably, the proliferation medium is MS basic medium, supplemented with 100 mg / L inositol, 2 mg / L glycine, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 30 g / L sucrose, 7 g / L agar, and a pH of 5.8-6.0;

[0016] The callus induction and proliferation culture temperatures were both 24-26°C, the light duration was 14 h / d, and the culture time was 28-35 days.

[0017] Preferably, the step (2) is to select callus tissue, inoculate it into a liquid culture medium for shaking culture, then filter the suspension culture, remove large cell clumps, retain small cell clumps and single cells with good dispersion, and then add fresh liquid culture medium for subculture, and repeat this process to establish a stable sandalwood cell suspension culture system; the liquid culture medium is MS medium, inositol 50-100 mg / L, glycine 1-2 mg / L, 2,4-D 0.5 mg / L, sucrose 20-40 g / L, pH 5.8-6.0

[0018] Preferably, the liquid culture medium is MS medium, supplemented with 100 mg / L inositol, 2 mg / L glycine, 0.5 mg / L 2,4-D, 30 g / L sucrose, and a pH of 5.8-6.0;

[0019] The culture temperature is 24-26°C, the culture is shaken at a rotation speed of 100 rpm, and the culture time is 12-14 days.

[0020] The present invention takes plant cell culture as the technical basis, firstly establishes a stable sandalwood suspension cell line ( Figure 1 ); by adding cytokinins and auxins to the sandalwood cell culture medium, the synthesis of santalene can be promoted, thereby increasing its content. The results of the examples show that the method provided by the present invention increases the biomass of sandalwood suspension cells by adding cytokinins and auxins ( Figure 2 The product content in the sandalwood suspension cell line was about 3%, significantly higher than that in the control group ( Figure 3 ); GC-MS analysis showed that the main components were α-santalene and -β-santalene ( Figure 4 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a sandalwood suspension cell line;

[0022] Figure 2 This is the fresh weight change curve of sandalwood suspension cell line after treatment;

[0023] Figure 3 is the change of the content of the products of sandalwood suspension cell line after treatment, A, the products obtained at different treatment times, B, the comparison of the content of the products obtained at different treatment times;

[0024] Figure 4 Figure GC-MS analysis of the components of the sandalwood suspension cell line products treated for 32 days. A, treatment group, Peak 1, α-santalene, 2, α-bergamotene, 3, epi-β-santalene, 4, β-santalene; B, relative contents of the four components; C, control group; D, standard, Peak 1, α-santalene, 2, α-bergamotene, 3, epi-β-santalene, 4, β-santalene. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Embodiment 1:

[0027] A method for inducing sandalwood suspension cells to synthesize santalene comprises the following steps:

[0028] (1) Select young stems of sandalwood and inoculate them on a callus induction medium for callus induction culture to obtain callus; transfer the induced callus to a proliferation medium for proliferation culture; the callus induction medium is an MS basic medium, supplemented with 100 mg / L inositol, 2 mg / L glycine, 1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g / L sucrose, 7 g / L agar, and a pH of 5.8-6.0; the proliferation medium is an MS basic medium, supplemented with 100 mg / L inositol, 2 mg / L glycine, 0.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g / L sucrose, 7 g / L agar, and a pH of 5.8-6.0. The callus induction and proliferation culture temperatures are both 24-26°C, the light exposure time is 14 h / d, the culture time is 28-35 days, and the grown callus is then subcultured every 1-2 months.

[0029] (2) Selecting the callus tissue that has been proliferated and cultured for one month in step 1), which has vigorous growth and loose texture, and inoculating it into a liquid culture medium, the inoculation amount, i.e., the fresh weight of the callus tissue / the volume of the liquid culture medium is 50 g / L, placing it on a shaker, and shaking and culturing it for 12-14 days, then filtering the suspension culture with a 60-mesh and 30-mesh sterile nylon mesh to remove large cell clumps, retaining small cell clumps and single cells with good dispersion, and then adding fresh liquid culture medium for subculture, repeating this process for 3-4 times, and finally establishing a stable sandalwood cell suspension culture system; the liquid culture medium is MS medium, supplemented with 100 mg / L inositol, 2 mg / L glycine, 0.5 mg / L 2,4-D, and 30 g / L sucrose, and the pH is 5.8-6.0; the culture temperature is 24-26° C., the shaking culture is carried out at a speed of 100 rpm, and the culture time is 12-14 days.

[0030] (3) harvesting the sandalwood cells in the sandalwood suspension culture system described in step 2), transferring them to an induction culture medium, placing them in a shaker, and shaking and culturing them for 30-35 days; obtaining suspension cells that accumulate santalene; the induction culture medium is an MS culture medium, supplemented with 30 g / L sucrose, and a pH of 5.8-6.0; 0.5 mg / L 6-benzylaminopurine (6-BA), and 0.1 mg / L naphthaleneacetic acid (NAA); the control group culture medium is an MS culture medium, supplemented with 30 g / L sucrose, and a pH of 5.8-6.0; the volume ratio of the suspension cell system to the liquid culture medium is 1:9; the culture temperature is 24-26°C, and the light intensity is 80-120 μmol / m 2 s -1 , shaking culture was carried out at a speed of 110 rpm, and the induction culture was carried out for 32 days.

[0031] (4) The suspended cells cultured in step 3) are filtered to separate the cells and the culture medium. The samples of the treatment group and the control group are dried at low temperature, extracted with n-hexane and dried with a nitrogen concentrator. The volume ratio of the obtained product to the fresh weight of the suspended cells is used to obtain the amount of the suspended cell product (v / w %).

[0032] The present invention takes plant cell culture as the technical basis, firstly establishes a stable sandalwood suspension cell line ( Figure 1 ); by adding cytokinins and auxins to the sandalwood cell culture fluid, the synthesis of santalene can be promoted, thereby increasing its content. The results of the examples show that the method provided by the present invention increases the biomass of sandalwood suspension cells by adding cytokinins and auxins (compared with the control group) ( Figure 2 The product content in the sandalwood suspension cell line was about 3%, significantly higher than that in the control group ( Figure 3 ); GC-MS test shows that the main components are α-santalene and -β-santalene, with a relative content of more than 80% ( Figure 4 ).

Claims

1. A method for inducing sandalwood suspension cells to synthesize santalene, It is characterized in that The following steps are involved: (1) inducing callus culture from sandalwood explants; (2) suspending the sandalwood callus to obtain a suspension cell line; (3) Adding cytokinin and auxin to the cell culture medium of the suspension cell line and inducing the culture to obtain sandalwood suspension cells with a high santalene content.

2. The method according to claim 1, It is characterized in that The sandalwood explant is a sandalwood stem segment.

3. The method according to claim 1, It is characterized in that The cytokinin in the cell culture fluid is 6-benzylaminopurine, and the growth hormone is naphthylacetic acid.

4. The method according to claim 3, It is characterized in that The cell culture fluid is prepared by adding sucrose 20-30 g / L, 6-benzylaminopurine 0.2-1 mg / L and naphthaleneacetic acid 0.05-0.1 mg / L to MS culture medium, and the pH value is 5.8-6.

0.

5. The method according to claim 4, It is characterized in that The cell culture fluid is prepared by adding 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine and 0.1 mg / L naphthaleneacetic acid to MS culture medium, and the pH value is 5.8-6.

0.

6. The method according to claim 1, It is characterized in that The step (1) is to select young stems of sandalwood and inoculate them on a callus induction medium to perform callus induction culture to obtain callus; and transfer the induced callus to a proliferation medium to perform proliferation culture; the callus induction medium is an MS basic medium, supplemented with 50-100 mg / L of inositol, 1-2 mg / L of glycine, 1.0-2.0 mg / L of 2,4-dichlorophenoxyacetic acid, 20-40 g / L of sucrose, and 5-8 g / L of agar, and the pH value is 5.8-6.0; the proliferation medium is an MS basic medium, supplemented with 50-100 mg / L of inositol, 1-2 mg / L of glycine, 0.5 mg / L of 2,4-dichlorophenoxyacetic acid, 20-30 g / L of sucrose, and 5-8 g / L of agar, and the pH value is 5.8-6.0; the callus induction and proliferation culture temperatures are both 24-26°C, the light exposure time is 14 h / d, and the culture time is 28-35 days.

7. The method according to claim 6, It is characterized in that The callus induction medium contains 100 mg / L inositol, 2 mg / L glycine, 1 mg / L 2,4-dichlorophenoxyacetic acid, 30 g / L sucrose, 7 g / L agar, and has a pH of 5.8-6.

0.

8. The method according to claim 6, It is characterized in that The proliferation medium is MS basic medium, supplemented with 100 mg / L inositol, 2 mg / L glycine, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 30 g / L sucrose, 7 g / L agar, and a pH of 5.8-6.

0.

9. The method according to claim 1, It is characterized in that The step (2) is to select callus tissue, inoculate it into a liquid culture medium for shaking culture, then filter the suspended culture, remove large cell clusters, retain small cell clusters and single cells with good dispersion, and then add fresh liquid culture medium for subculture, and repeat this process to establish a stable sandalwood cell suspension culture system; the liquid culture medium is MS culture medium, inositol 50-100 mg / L, glycine 1-2 mg / L, 2,4-D 0.5 mg / L, sucrose 20-40 g / L, pH 5.8-6.0; the culture temperature is 24-26°C, the shaking culture is carried out at a rotation speed of 100 rpm, and the culture time is 12-14 days.

10. The method according to claim 9, It is characterized in that The liquid culture medium is MS culture medium, which is supplemented with 100 mg / L inositol, 2 mg / L glycine, 0.5 mg / L 2,4-D, and 30 g / L sucrose, and has a pH of 5.8-6.0.

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