A method for efficiently inducing rhizoma haleniae alcohol-producing hairy roots of iron calophyllum

Abstract

The application discloses a method for efficiently inducing Phyllanthus emblica hairy roots to produce phyllanthol, and the obtained hairy roots can be used to extract active substance phyllanthol with higher content than that in the original plant. The method comprises the following steps: 1) preparation of a sterile explant; 2) activation of Agrobacterium tumefaciens and preparation of a bacterial liquid; 3) induction of the hairy roots; 4) sterilization and induction culture; 5) subculture; and 6) expansion culture of the hairy roots. The induction and culture method can realize efficient induction and large-scale culture of Phyllanthus emblica hairy roots, and obtain Phyllanthus emblica hairy roots with rapid growth, stable state and a large amount of phyllanthol, thereby providing technical support for production of active phyllanthus emblica diterpenoid compounds and development and utilization of medicinal resources.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for inducing and culturing hairy roots of Malus bacillus using Agrobacterium rhizogenes, wherein the content of the active substance euphorbia alcohol in the hairy roots of Malus bacillus is higher than that in the original plant. Background Technology

[0002] Iron Begonia Euphorbia milii Ch. des Moulins is a genus of Euphorbia in the family Euphorbiaceae. Euphorbia *Euphorbia milii*, also known as crown of thorns or thorn beetle, is a perennial climbing shrub native to Africa and widely cultivated in tropical and temperate regions. In my country, it is primarily cultivated as an ornamental plant, commonly found in parks, botanical gardens, and courtyards. *Euphorbia milii* is also a medicinal plant, with its stems, leaves, roots, and latex used in traditional medicine. It is bitter, astringent, and cool in nature, slightly toxic, and enters the heart meridian, possessing detoxifying, blood-activating, pus-draining, and diuretic effects. Studies have shown that the chemical components of *Euphorbia milii* are mainly flavonoids, triterpenoids, diterpenoids, and phenols, rich in diterpenoid compounds, particularly the phorbol esters unique to Euphorbiaceae plants. The parent nucleus of this class of compounds, phorbol, is of significant value, serving as a raw material for the industrial semi-synthetic production of phorbol methyl butyl ester, a drug used to treat actinic keratosis. However, phorbol is only distributed in a few Euphorbiaceae plants, and the yield of phorbol isolated from plants is very low, thus the cost of semi-synthesizing phorbol methyl butyl ester remains high. In 2013, LEO Pharmaceuticals, in collaboration with the Scripps Research Institute in the United States, developed a 14-step total synthesis method for phorbolol. However, due to the high complexity of the process and the extremely low yield (only about 1%), it is difficult to industrialize.

[0003] Hairy roots are formed by the production of Agrobacterium tumefaciens (… Agrobacterium rhizogenes The T-DNA fragment on the Ri plasmid is inserted into and integrated into the plant cell genome and expressed, inducing the growth of adventitious roots. Hairy roots morphologically retain the characteristics of the original plant root and physiologically possess the complete metabolic pathways of the original root, capable of synthesizing secondary metabolites from the original plant, and the yield of secondary metabolites synthesized by hairy roots may be higher than that of the original plant. Furthermore, hairy root culture has advantages such as rapid growth, physiological and biochemical stability, genetic stability, absence of chimerism, and hormonal autotrophy. The production of bioactive secondary metabolites using hairy root culture technology has been successfully applied in many medicinal plants, such as Astragalus membranaceus, Glycyrrhiza uralensis, Uncaria rhynchophylla, and Vaccaria segetalis.

[0004] Currently, there are no reports on the induction and culture technology of hairy roots of *Crassula ovata*. This invention provides a highly efficient method for inducing and culturing hairy roots of *Crassula ovata*, from which euphorbia alcohol can be extracted at a higher content than in the original plant. This invention provides a new approach for the efficient acquisition of active euphorbia diterpenoids, including euphorbia alcohol, and lays the foundation for the further development and utilization of *Crassula ovata* medicinal resources. Summary of the Invention

[0005] The present invention aims to provide a method for efficiently inducing the production of megalool from the hairy roots of *Crassula ovata*, through which a large amount of megalool can be extracted from the hairy roots obtained by this method.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for efficiently inducing and culturing the hairy roots of Malus halliana includes the following steps: 1) Preparation of sterile explants: Using the flower stalks of Malus halliana as explants, sterilize them in 75% ethanol for 45 s, rinse twice with sterile water, sterilize with 0.1% mercuric chloride for 5 min, rinse 4-5 times with sterile water, cut them into 1-1.5 cm lengths, and inoculate them into 1 / 2 MS solid medium supplemented with 30 g / L sucrose and 2.5-3.0 g / L plant gel, with a pH of 5.8. After inoculation, pre-culture them in the dark at 25℃ for 5-7 days. 2) Activation of Agrobacterium and preparation of bacterial culture: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃, add it to 5 mL of YEB liquid medium containing 50 mg / L kanamycin, and culture at 28℃ with shaking at 180-200 r / min for 24 h. Then, take 1 mL of bacterial culture and inoculate it into 50 mL of YEB liquid medium containing 50 mg / L kanamycin, and culture under the same conditions with shaking until the bacterial culture reaches OD. 600 Centrifuge at 0.6–0.8, 5000 r / min for 8–10 min, discard the supernatant, and resuspend the cells in 50 mL of MS liquid medium containing 80–100 μmol / L acetylsyl syringone to obtain activated Agrobacterium rhizogenes bacterial suspension; 3) Induction of hairy roots: Place the pre-cultured explants into the above-prepared infection solution and infect for 4-6 min. Remove the explants, wipe the surface of the bacterial solution dry with sterile filter paper, and place them on the 1 / 2 MS solid medium of step 1) and co-culture at 25°C in the dark for 3 days. 4) Sterilization induction culture: The co-cultured explants were placed on sterile culture medium for sterilization induction culture. The sterile culture medium was 1 / 2 MS medium + cefotaxime sodium. The initial concentration of cefotaxime sodium was 400 mg / L. The culture medium was changed every 5-7 days and the concentration of cefotaxime sodium was gradually reduced until Agrobacterium rhizogenes was completely removed and no more cefotaxime sodium was added. 5) Subculture: The thoroughly sterilized hairy roots are inoculated onto a subculture medium for subculture. The subculture medium is 1 / 2MS solid medium. 6) Hairy root expansion culture: Transfer the hairy roots from step 5) into 1 / 2 MS liquid medium and expand the culture at 25℃ and 100-120 rpm / min. Subculture once every 10 days to obtain a large number of hairy roots of *Crassula ovata*.

[0007] The Agrobacterium rhizogenes strain used for infection in step 2) above is Ar A4, and the bacterial culture is shaken and cultured to OD. 600 Centrifuge at a concentration of 0.6–0.8 μmol / L, discard the supernatant after centrifugation, and resuspend the bacterial cells. The added MS liquid medium contains 80–100 μmol / L of acetylsuccinone.

[0008] Step 4) of the above-mentioned sterilization induction culture process specifically includes: the explants after co-culture are first placed on 1 / 2 MS solid basic medium + 400 mg / L cefotaxime sodium sterilization medium for sterilization culture. After hairy roots are visible to the naked eye and there are no visible bacterial spots on the medium and the surface of the explants, the concentration of cefotaxime sodium can be gradually reduced during subculture, each time by 50-100 mg / L, until no cefotaxime sodium is added and hairy roots are obtained after thorough sterilization.

[0009] The beneficial effects of this invention are as follows: This invention provides a highly efficient method for inducing and culturing hairy roots of Malus halliana. Using Malus halliana flower stalks as explants, hairy roots of Malus halliana that grow rapidly and are in good condition are obtained by induction with Agrobacterium rhizogenes Ar A4. After collecting and freeze-drying the hairy roots, euphorbia alcohol and various euphorbia diterpenoids can be extracted, which provides technical support for the production of active secondary metabolites of the medicinal plant Malus halliana and the further development and utilization of medicinal resources. Attached Figure Description

[0010] Figure 1 Hairy roots grow from the flower stalks of Malus bacillus Ar A4; Figure 2 Hairy roots of *Crassula ovata* grown on 1 / 2 MS solid medium; Figure 3 Hairy roots of *Crassula ovata* grown on 1 / 2 MS liquid medium; Figure 4 Hairy roots rolB and rolC Gene PCR identification results, in the figure: M is DL2000 marker; 1 is non-transformed Malus spectabilis primordial root (negative control); 2 and 3 are different root systems of Malus spectabilis hairy roots; 4 is Agrobacterium rhizogenes plasmid (positive control).

[0011] Figure 5 A comparative bar chart showing the determination of euphorbia alcohol content in the hairy roots and various parts of the original plant of *Crassula ovata*. Implementation

[0012] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example

[0013] 1. Preparation of sterile explants: The flower stalks of *Crassula ovata* were used as explants. They were sterilized in 75% ethanol for 45 s, rinsed twice with sterile water, sterilized in 0.1% mercuric chloride for 5 min, rinsed five times with sterile water, cut into 1 cm lengths, and inoculated into 1 / 2 MS medium with 30 g / L sucrose and 2.8 g / L plant gel added. The pH value was 5.8, and the medium was pre-cultured at 25℃ in the dark for 5 days. 2. Preparation of inducing hairy root infection solution: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃, add it to 5 mL of YEB liquid medium containing 50 mg / L kanamycin, and culture at 28℃ with shaking at 180 r / min for 24 h. Take 1 mL of the bacterial solution and inoculate it into 50 mL of YEB liquid medium containing 50 mg / L kanamycin, and culture under the same conditions with shaking until the bacterial solution OD... 600 Centrifuge at 5000 r / min for 8 min at 0.6, discard the supernatant, and resuspend the cells in 50 mL of MS liquid medium containing 80 μmol / L acetylsuccinone to obtain activated Agrobacterium rhizogenes bacterial suspension; 3. Induction of hairy roots: The pre-cultured flower stalks of Malus halliana were placed in the inoculation solution and inoculated for 6 min. After inoculation, the explants were removed, the surface of the bacterial solution was dried with sterile filter paper, and the explants were placed on 1 / 2 MS solid medium and co-cultured in the dark at 25°C for 3 days. 4. Sterilization and induction culture of hairy roots: The explants after co-culture were transferred to sterile culture medium and cultured in the dark at 25°C. The sterile culture medium was 1 / 2 MS medium + cefotaxime sodium, with an initial concentration of 400 mg / L. The medium was changed every 7 days, and the concentration of cefotaxime sodium was reduced by 100 mg / L each time. After 19 days of sterile culture, white hairy roots were observed to grow (see Appendix). Figure 1 After four subcultures, cefotaxime sodium was no longer added to the culture medium, resulting in thoroughly sterilized hairy roots (see appendix). Figure 2 ); 5. Subculture: Take thoroughly sterilized hairy roots and inoculate them into 1 / 2 MS solid medium for subculture, subculturing once every 10 days; 6. Expansion culture of hairy roots: Hairy roots of *Malus spectabilis* were transferred to 1 / 2 MS liquid medium and cultured in the dark at 25°C with shaking at 130 rpm / min. Subculture was performed every 10 days to obtain a large number of rapidly growing hairy roots of *Malus spectabilis* (see appendix). Figure 3 ); 7. PCR detection of hairy roots of *Malus spectabilis*: Genomic DNA was extracted from hairy roots and untransformed protoroots of *Malus spectabilis* using the TIANGEN kit (DP305). PCR amplification was performed on the T-DNA of the Ri plasmid from *Agrobacterium rhizogenes*. rol B and rol C gene sequence, used rol The upstream primer sequence for gene B is 5'-CGAGGGGATCCGATTTGCTTT-3', and the downstream primer sequence is 5'-GACGCCCTCCTCGCCTTCCT-3'. rol The upstream primer sequence for the C gene was 5'-CTCCTGACATCAAACTCGTC-3', and the downstream primer sequence was 5'-TGCTTCGAGTTATGGGTACA-3'. PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 10 min. The amplified products were analyzed by 1.0% agarose gel electrophoresis. PCR amplification results showed that DNA from the hairy roots of *Malus spectabilis* was amplified... rol B and rol C gene fragment, and amplified from Agrobacterium rhizogenes Ar A4 plasmid DNA rol B and rol The C gene fragments are of uniform size. rol B: 622bp; rol C: 586bp), DNA from the original root of normal plants was not amplified. rol B and rol The gene fragment of C proves that the T-DNA of the Ri plasmid in Agrobacterium rhizogenes Ar A4 has been successfully integrated into the hairy root gene of Malus bacillus (see appendix). Figure 4 );

[0014] 8. Determination of phorbol content in hairy roots of *Crassula ovata*: Hairy roots of *Crassula ovata* were rapidly frozen in liquid nitrogen and then freeze-dried in a freeze dryer. After freeze-drying, they were ground into a uniform powder. 50 mg of the freeze-dried powder was weighed and extracted with 10 mL of methanol using ultrasonic extraction for 30 min. 5 mL of the supernatant was collected, and 5% KOH solution was added to adjust the pH to 13. The mixture was then hydrolyzed in a 45℃ water bath for 30 min. The pH was adjusted to neutral with 0.2 mol / L acetic acid solution to obtain an alkaline hydrolysate. Water was added to 25 mL, and the solution was passed through an SPE column. After elution with 10 mL of methanol, the solution was concentrated to dryness under reduced pressure. The solution was dissolved in 5 mL of chromatographic methanol to prepare a sample with a concentration of 50 mg / mL for liquid chromatography-mass spectrometry (LC-MS). Simultaneously, standard curves were plotted using phorbol standard solutions of different concentrations to calculate the phorbol content in the hairy roots of *Crassula ovata*. The results showed that the phorbol content in the hairy roots of *Crassula ovata* was significantly higher than that in other tissues of the original plant (see Appendix). Figure 5 The concentration reached 593.14 mg / kg (dry weight). Example

[0015] 1. Preparation of sterile explants: The flower stalks of Malus halliana were used as explants. They were sterilized in 75% ethanol for 45 s, rinsed twice with sterile water, sterilized in 0.1% mercuric chloride for 5 min, rinsed 6 times with sterile water, cut into 1.5 cm lengths, and inoculated into 1 / 2 MS medium with 30 g / L sucrose and 2.6 g / L plant gel added. The pH value was 5.8, and the medium was pre-cultured at 25℃ in the dark for 7 days. 2. Preparation of inducing hairy root infection solution: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃, add it to 5 mL of YEB liquid medium containing 50 mg / L kanamycin, and culture at 28℃ with shaking at 200 r / min for 24 h. Take 1 mL of bacterial solution and inoculate it into 50 mL of YEB liquid medium containing 50 mg / L kanamycin. Culture under the same conditions with shaking. When the OD600 of the bacterial solution is 0.8, centrifuge at 5000 r / min for 8 min, discard the supernatant, and resuspend the bacterial cells in 50 mL of MS liquid medium containing 100 μmol / L acetylsyl syringone to obtain activated Agrobacterium rhizogenes bacterial solution; 3. Induction of hairy roots: Place the pre-cultured flower stalks of Malus halliana into the inoculation solution and infect for 8 min. After inoculation, remove the stalks, wipe the surface of the bacterial solution dry with sterile filter paper, and place them on 1 / 2 MS solid medium. Incubate in the dark at 25°C for 3 days. 4. Sterilization induction culture of hairy roots: The explants after co-culture were transferred to sterile medium and cultured in the dark at 25°C. The sterile medium was 1 / 2 MS medium + cefotaxime sodium, with an initial concentration of 400 mg / L. The medium was changed every 5 days, and the concentration of cefotaxime sodium was reduced by 50 mg / L each time. After 20 days of sterilization culture, white hairy roots were observed to grow. After 7 subcultures, cefotaxime sodium was no longer added to the medium, and thoroughly sterilized hairy roots were obtained. 5. Subculture: Thoroughly sterilized hairy roots were inoculated onto subculture medium and subcultured in the dark at 25°C. The subculture medium was 1 / 2 MS solid medium. 6. Expansion culture of hairy roots: The hairy roots of Malus halliana were transferred into 1 / 2 MS liquid medium and cultured in the dark at 25°C with shaking at 110 rpm / min. Subculture was carried out every 14 days to obtain a large number of rapidly growing hairy roots of Malus halliana. 7. PCR detection of hairy roots of *Malus spectabilis*: Genomic DNA was extracted from hairy roots and untransformed protoroots of *Malus spectabilis* using the TIANGEN kit (DP305). PCR amplification was performed on the T-DNA of the Ri plasmid from *Agrobacterium rhizogenes*. rol B and rol C gene sequence, used rol The upstream primer sequence for gene B is 5'-CGAGGGGATCCGATTTGCTTT-3', and the downstream primer sequence is 5'-GACGCCCTCCTCGCCTTCCT-3'. rol The upstream primer sequence for the C gene was 5'-CTCCTGACATCAAACTCGTC-3', and the downstream primer sequence was 5'-TGCTTCGAGTTATGGGTACA-3'. PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 10 min. The amplified products were analyzed by 1.0% agarose gel electrophoresis. PCR amplification results showed that DNA from the hairy roots of *Malus spectabilis* was amplified... rol B and rol C gene fragment, and amplified from Agrobacterium rhizogenes Ar A4 plasmid DNA rol B and rol The C gene fragments are of uniform size. rol B: 622bp; rol C: 586bp), DNA from the original root of normal plants was not amplified. rol B and rol The gene fragment of C proves that the T-DNA of the Ri plasmid in Agrobacterium rhizogenes Ar A4 has been successfully integrated into the hairy root gene of Malus bacillus.

[0016] 8. Determination of phorbol content in hairy roots of *Euphorbia milii*: The determination method is the same as in Example 1. First, the hairy roots of *Euphorbia milii* were rapidly frozen in liquid nitrogen, then freeze-dried in a freeze dryer. After freeze-drying, they were ground into a uniform powder using a pulverizer. 50 mg of the freeze-dried hairy root powder was weighed, and 10 mL of methanol was added for ultrasonic extraction for 30 min. 5 mL of the supernatant was collected, and 5% KOH solution was added to adjust the pH to 13. The mixture was then hydrolyzed in a 45℃ water bath for 30 min. The pH was adjusted to neutral with 0.2 mol / L acetic acid solution to obtain an alkaline hydrolysate. Water was added to 25 mL, and the solution was passed through an SPE column. After elution with 10 mL of methanol, the solution was concentrated to dryness under reduced pressure. The solution was dissolved in 5 mL of chromatographic methanol to prepare a sample with a concentration of 50 mg / mL for liquid chromatography-mass spectrometry (LC-MS). Simultaneously, standard curves were plotted using phorbol standard solutions of different concentrations to calculate the phorbol content in the hairy roots of *Euphorbia milii*. The results showed that the content of phorbol in the hairy roots of *Crassula ovata* was significantly higher than that in other tissues of the original plant, reaching 536.82 mg / kg (dry weight).

[0017] The above embodiments are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for efficiently inducing the production of phorbol from the hairy roots of *Malus speciosa*, characterized in that... Includes the following steps: 1) Preparation of sterile explants: Using the flower stalks of Malus halliana as explants, sterilize them in 75% ethanol for 45 s, rinse twice with sterile water, sterilize with 0.1% mercuric chloride for 5 min, rinse 5-6 times with sterile water, cut them into 1-1.5 cm lengths, and inoculate them into 1 / 2 MS solid medium supplemented with 30 g / L sucrose and 2.5-3.0 g / L plant gel, with a pH of 5.

8. After inoculation, pre-culture them in the dark at 25℃ for 5-7 days. 2) Activation of Agrobacterium and preparation of bacterial culture: Take 50 μL of Agrobacterium rhizogenes strain Ar A4 stored at -80℃ and add it to 5 mL of YEB liquid medium containing 50 mg / L kanamycin. After incubating at 28℃ with shaking at 180-200 r / min for 24 h, take 1 mL of bacterial culture and inoculate it into 50 mL of YEB liquid medium containing 50 mg / L kanamycin. Incubate under the same conditions with shaking until the bacterial culture reaches OD. 600 Centrifuge at 0.6–0.8, 5000 r / min for 8 min, discard the supernatant, and resuspend the cells in 50 mL of MS liquid medium containing 80–100 μmol / L acetosyringone to obtain activated Agrobacterium rhizogenes bacterial suspension, i.e., infection suspension; 3) Induction of hairy roots: Place the pre-cultured explants into the above-prepared bacterial inoculum and infect for 4-6 min. Remove the explants, wipe the surface of the bacterial inoculum dry with sterile filter paper, and place them on the 1 / 2 MS solid medium of step 1) and co-culture at 25°C in the dark for 3 days. 4) Sterilization induction culture: The co-cultured explants were placed on sterile medium and subjected to sterilization induction culture at 25°C in the dark. The sterile medium was 1 / 2 MS medium + cefotaxime sodium. The initial concentration of cefotaxime sodium was 400 mg / L. The medium was changed every 5-7 days and the concentration of cefotaxime sodium was gradually reduced until Agrobacterium rhizogenes was completely removed and no more cefotaxime sodium was added. 5) Subculture: Take thoroughly sterilized hairy roots and inoculate them onto a subculture medium, which is 1 / 2 MS solid medium. Subculture once every 7 to 10 days and culture at 25°C in the dark. 6) Hairy root expansion culture: Transfer the hairy roots from step 5) into 1 / 2 MS liquid medium and culture them under 110-130 rpm / min shaking conditions at 25℃ in the dark. Subculture once every 10-14 days to obtain a large number of rapidly growing hairy roots of *Crassula ovata*.

2. The method according to claim 1, characterized in that... Step 4) The sterilization induction culture process specifically includes: the explants after co-culture are first placed on 1 / 2 MS solid basal medium + 400 mg / L cefotaxime sodium sterilization medium for sterilization culture. After hairy roots are visible to the naked eye and there are no visible bacterial spots on the medium and the surface of the explants, the concentration of cefotaxime sodium can be gradually reduced during subculture, each time by 50-100 mg / L, until no cefotaxime sodium is added and hairy roots are obtained after thorough sterilization.

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