CN113349053B

CN113349053B - Method for producing three main ginsenosides by rapidly proliferating adventitious roots of panax notoginseng

Info

Publication number: CN113349053B
Application number: CN202010142159.2A
Authority: CN
Inventors: 由香玲; 赵越
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2020-03-04
Filing date: 2020-03-04
Publication date: 2022-03-22
Anticipated expiration: 2040-03-04
Also published as: CN113349053A

Abstract

The invention provides a method for rapidly proliferating adventitious roots of panax notoginseng and producing three main ginsenosides. Firstly, the adventitious root induced by the cotyledon of Panax notoginseng is cut into small pieces of about 1cm, inoculated in 1/2SH culture medium containing IBA and NAA with different concentrations and no NH₄NO₃1/2MS (1/2MS-N) medium. The induction and growth conditions of the adventitious roots are analyzed, the optimal culture mode is determined, the adventitious roots are firstly inoculated into 1/2SH +2.0mg/L IBA +2.0mg/L NAA culture medium to induce the formation of new adventitious roots, then the new adventitious roots are transferred to 1/2MS-N +2.0mg/L IBA culture medium to promote the elongation of the adventitious roots, and the optimal concentration of 50g/L of sucrose is determined. On the basis, inoculating adventitious roots cultured in 1/2SH +2.0mg/L IBA +2.0mg/L NAA culture medium for 3 weeks into a liquid culture medium containing 100mL 1/2MS-N for light-shielding suspension culture, analyzing the growth of the adventitious roots and the accumulation of three ginsenosides (Rg1, Re, Rb1), and determining that the optimal harvesting period is 7 weeks; the optimal sucrose concentration is 30 g/L; the optimal IBA concentration is 4.0mg/L, the optimal initial inoculation amount is 3.5g/100mL, and under the condition, the total accumulation amount of the saponin in the adventitious root can reach 11.13 mg.

Description

Method for producing three main ginsenosides by rapidly proliferating adventitious roots of panax notoginseng

Technical Field

The invention relates to a culture method for effectively accumulating three main ginsenosides (Rg1, Re and Rb1) by rapidly proliferating an adventitious root of panax notoginseng. The present invention belongs to the field of agricultural biotechnology.

Background

Panax notoginseng (Panax notoginseng), also known as Panax notoginseng (Burk.) F.H.Chen, is a plant of Panax genus of Araliaceae family, has close relationship with Panax ginseng, and many of their active saponins are the same. However, the traditional Chinese medicine is obviously different from the traditional Chinese medicine in that ginseng is slightly warm in nature, is mostly used as a qi component for strengthening the body resistance and consolidating the exterior, and panax notoginseng is warm in nature, and is mostly used as a blood component for dissipating blood stasis, stopping bleeding, reducing swelling and relieving pain (accounts for Ying, etc., Chinese medicine science and technology, 2014,21(6): 711-712). The main reason is caused by the content and the variety difference of the pseudo-ginseng and the ginsenosides. The pseudo-ginseng has the following unique components: r1, R2, R3, Rf, etc. (Zhang Chong, Ph's academic thesis, Changchun: Jilin university of agriculture, 2004). The content of the saponin in the panax notoginseng (6.2-10.32%) is much higher than that in the ginseng (4.84%) (the higher Li Dynasty, Proc. Jilin university, 1984,6(3): 95-97).

The natural distribution regions of wild Panax notoginseng in the world are China, Japan, Nepal, Burma (Baranov, Journal of ethnopharmacology,1982,6: 339-. The natural wild panax notoginseng is almost completely eradicated, and the suitable cultivation area is only concentrated in a narrow area of Yunnan and Sichuan of China. However, there are many problems in the cultivation of notoginseng: the growth period is long, and the period from sowing to harvesting is 3-5 years (Wangshuqin, etc., Kunming: Yunnan national publishing house, 1993); strong soil contraindication and serious continuous cropping obstacle; at present, the heavy metal pollution in the cultivation area is increasingly serious (Liu Yuan, etc., Jiangsu agricultural science, 2018,46(18): 298-. These problems severely restrict the yield and quality of panax notoginseng.

Therefore, the production of pseudo-ginseng cells, tissues and the like by using biotechnology is an effective way for obtaining various secondary metabolic products of pseudo-ginseng (Zhengguang planting and the like, plant science and report, 1978, 20(4): 373-375). In the field of cell culture of Panax notoginseng, clones of stem callus with high content of Panax notoginseng saponins have been selected (Zhengguang et al, Yunnan plant research, 1989,11: 255-. Generally, the content of ginsenoside in adventitious roots is high and the synthesis is stable as compared to cell culture. Researchers of Gao et al who have been in Shanghai province first induced adventitious roots of Panax notoginseng from young buds of Panax notoginseng (Gao et al, Biotech. letters,2005,27: 1771-1775). Then, researchers in the same laboratory studied various factors affecting the growth of adventitious roots of Panax notoginseng, and confirmed that 10 types of saponins with higher content in the adventitious roots of Panax notoginseng (thin ink, Shanghai: Shanghai traffic university, 2008; Yao et al, appl.Microbiol.Biotechnol.2019, 103, 4405-. The invention aims to provide a culture technology for rapidly proliferating the adventitious roots of panax notoginseng and effectively accumulating three main ginsenosides (Rg1, Re and Rb1), and lays an early foundation for the mass production of the ginsenosides by utilizing the adventitious roots of panax notoginseng.

Disclosure of Invention

The invention aims to provide a culture technology for rapidly proliferating panax notoginseng adventitious roots and effectively accumulating three main ginsenosides (Rg1, Re and Rb1), which comprises the following specific contents:

inoculating the germinated pseudo-ginseng seed after disinfection into a culture medium: 1/2SH +2.0mg/L IBA +30g/L sucrose to induce adventitious roots, and the adventitious roots are utilizedThe root is explant material, and the rapid proliferation is realized by a two-step culture method: firstly, inducing new adventitious roots under the conditions of 1/2SH +2.0mg/LIBA +2.0mg/L NAA +30g/L sucrose for 3 weeks, transferring to 1/2MS containing 2.0mg/LIBA without NH₄NO₃(1/2MS-N) for 3 weeks. The effect of sucrose concentration on adventitious root formation and elongation was then examined and 50g/L sucrose was found to be optimal. Then, 1.5g of the induced new adventitious roots for 3 weeks were inoculated into a 250mL glass culture flask containing 100mL of a liquid elongation medium for suspension culture for 8 weeks, the material was taken every 1 week, and Fresh Weight (FW) was weighed and dried to weight (DW). The contents of three main saponins (Rg1, Re, Rb1) in these adventitious roots were analyzed by High Pressure Liquid Chromatography (HPLC). From these statistics, it was determined that the optimal time for one cycle of suspension culture was 7 weeks. Finally, the optimal sucrose concentration in suspension culture was determined to be 30g/L, IBA at 4.0mg/L with an initial inoculum size of 3.5 g/L.

The culture medium used in the invention is treated according to the conventional method: after adjusting the pH to 5.8, the mixture was sterilized at 121 ℃ under 1.5 atmospheres for 15 minutes and then used. The culture environment is a conventional tissue culture chamber: the temperature is 24-26 ℃, the humidity is 60% -70%, and dark culture is carried out. The rotational speed of the shaker for suspension culture was 110 rpm/min.

Drawings

FIG. 1 Induction and proliferation of adventitious roots of Panax notoginseng: A. b-is the adventitious root generated by inducing the cotyledon of the seed germinated from the pseudo-ginseng under the conditions of 1/2SH +2.0mg/L IBA +30g/L sucrose for 6 weeks and 10 weeks respectively; C. d-, culturing the adventitious roots in the D-under the conditions of 1/2SH +2.0mg/L IBA +2.0mg/L NAA +30g/L sucrose and 1/2MS-N +2.0mg/L IBA +30g/L sucrose for 6 weeks to induce new adventitious roots respectively; E. f-is the adventitious root which is induced by culturing the adventitious root under the conditions of 1/2SH +2.0mg/L IBA +2.0mg/L NAA +30g/L respectively and adding 70g/L and 50g/L of sucrose, and then transferring to 1/2MS-N +2.0mg/L IBA +30g/L of sucrose for culturing 3 weeks; scale bar: 1.0 cm.

FIG. 2 suspension culture of adventitious roots in the presence of 1/2MS-N +2.0mg/L IBA +50g/L sucrose: a-adventitious roots induced by culturing for 3 weeks under the conditions of 1/2SH +2.0mg/L IBA +2.0mg/L NAA +50g/L sucrose; B. the adventitious roots in C-A were suspended for 7 weeks and 8 weeks under the conditions of 1/2MS-N +2.0mg/L IBA +50g/L sucrose, respectively; scale bar: 1.0cm

FIG. 3 growth curves of adventitious roots during 8 weeks of suspension under the conditions of 1/2MS-N +2.0mg/L IBA +50g/L sucrose

Detailed Description

EXAMPLE 1 Induction and proliferation of adventitious roots of Panax notoginseng

1) Induction of adventitious roots of pseudo-ginseng: the seeds of Panax notoginseng are collected from Yunnan Wenshan. Laminating the seeds in wet sand for 1 month, sterilizing with 70% ethanol for 3 min, and adding 5% NaClO₃Sterilizing for 10 min, and washing with sterilized distilled water for 5 times. The seed coat was removed from the sterilized seed, about 200 seed cotyledons were cut, and inoculated into 1/2SH +2.0mg/L IBA medium supplemented with 30g/L sucrose and 3.0g/L gelrite to induce the formation of adventitious roots.

2) Proliferation of adventitious roots of panax notoginseng: cutting the above adventitious root into small segments of about 1cm, inoculating into two types of culture media containing IBA and NAA with different concentrations, i.e. 1/2SH culture medium and NH-free culture medium₄NO₃1/2MS medium (1/2 MS-N). After 6 weeks of culture, the growth of new adventitious roots was statistically induced as shown in Table 1. As can be seen from the table, when the explants are inoculated to 1/2SH +2.0mg/L IBA +2.0mg/L NAA culture medium, the induction rate of the new adventitious roots reaches 100%, and the number of the new adventitious roots induced by each explant is the largest and reaches 11.57, but the length of the new adventitious roots is shorter and is only 4.45mm on average. Under the culture condition of 1/2MS-N +2.0mg/L IBA, the induction number of the new adventitious roots is small, but the length is longest, and the average length can reach 9.86 mm. Therefore, the induction of adventitious roots was carried out in 1/2SH +2.0mg/L IBA +2.0mg/L NAA medium, and then elongation culture was carried out on the adventitious roots in 1/2MS-N +2.0mg/L IBA medium.

TABLE 1 Induction and growth of New adventitious Roots in 1/2MS-N and 1/2SH media at different concentrations of IBA and NAA

Note: the same letters were not significantly different (p <0.05) using Duncan multiple comparisons. The same applies below.

3) Determining the proper sucrose concentration in the process of culturing the adventitious roots of the panax notoginseng: on the basis of the above, different sucrose concentrations (30,50,70g/L) were set, and the effect on adventitious root proliferation was analyzed. After culturing adventitious roots in 1/2SH +2.0mg/L IBA +2.0mg/L NAA medium for 3 weeks, the adventitious roots are transferred to 1/2MS-N +2.0mg/L IBA to continue culturing, and the adventitious roots grow in an elongation mode. The statistical adventitious root growth is shown in Table 2. When the concentration of the sucrose is 50g/L, the induction rate of the adventitious roots, the number of newly induced adventitious roots and the length of the adventitious roots are all the highest, the number of the newly induced adventitious roots generated by each explant is 12.62 on average, and the average length of the newly induced adventitious roots is 14.23 mm.

TABLE 2 Effect of different sucrose concentrations on adventitious root Induction and growth

EXAMPLE 2 determination of the cultivation time for suspension cultivation of Panax notoginseng adventitious roots

1) Suspension culture of adventitious root Biomass

Adventitious roots growing for 3 weeks in 1/2SH +2.0mg/L IBA +2.0mg/L NAA culture medium added with 50g/L of cane sugar are inoculated into a 250mL glass triangular flask filled with 1/2MS-N +2.0mg/L IBA liquid culture medium added with 5% of cane sugar for shake flask suspension culture, the rotation speed of a shaking table is 110rpm/min, the shake flask is cultured in a dark place at 25 ℃, and the fresh weight of the initial inoculation amount is 1.5 g. Taking materials once every week, taking materials 8 times, repeating 3 times each time, weighing fresh weight after the filter paper absorbs water, drying in a 65 ℃ oven to constant weight, weighing dry weight, counting biomass of adventitious roots, namely Fresh Weight (FW) and Dry Weight (DW), drawing a suspension culture growth curve of the adventitious roots, and determining the growth period of the adventitious roots.

2) Accumulation of three saponins (Rg1, Re, Rb1) in adventitious roots by suspension culture

The extraction method of three saponins in adventitious roots comprises the following steps: 0.50g of dry powder of adventitious roots of panax notoginseng is precisely weighed and soaked in 5mL of 75% ethanol overnight. The next day, ultrasonic extraction is carried out for 30min at room temperature. Ultrasonic extracting with 5mL 75% ethanol for 2 times, each for 30 min. Mixing extractive solutions, filtering with 0.45 μm organic filter membrane, and evaporating to dryness under reduced pressure in 80 deg.C water bath. Redissolving with 20% acetonitrile water solution, and fixing the volume to 2.0 mL.

Three saponin High Performance Liquid Chromatography (HPLC) analysis methods: the mobile phase was acetonitrile (a): water (B) linear gradient elution (v/v); flow rate: 1.0 mL/min^-1(ii) a Column temperature: room temperature; detection wavelength: 203 nm. Sample introduction amount: 20 μ L. The gradient change of the mobile phase with time is shown in table 3. The method is used for determining ginsenoside Rg1, Re, Rb1 standard substances and samples. The regression curves and coefficients of the three saponins were: rg 1: 4288.8X +15376, R²＝0.9997；Re：Y＝5622.9X+25158，R²＝0.9999；Rb1：Y＝4924.3X-49153；R²0.9994. The total content is Rg1 content + Re content + Rb1 content; the saponin yield is the saponin content multiplied by the dry weight of the sample; the total yield is Rg1 yield + Re yield + Rb1 yield.

Table 3 acetonitrile (a): water (B) mobile phase gradiometer

The accumulation of saponins in adventitious roots is shown in Table 4. As can be seen from the table, the total yield of three saponins in the adventitious roots in suspension culture reached the highest at week 7. And (3) comprehensively considering the growth cycle of the adventitious roots and the saponin yield, and finally determining the optimal harvesting time of the adventitious roots of the pseudo-ginseng in suspension culture to be 7 weeks.

TABLE 4 cumulative changes in saponin during the growth cycle of adventitious roots

EXAMPLE 3 determination of suitable sucrose concentration for suspension culture of Panax notoginseng adventitious roots

1.5g of adventitious roots of Panax notoginseng were inoculated into liquid media with sucrose concentrations of 10,30,50,70g/L, 1/2MS-N +2.0mg/L IBA, with 3 replicates per treatment set-up. After 7 weeks of suspension culture, the cultures were harvested and counted for biomass and saponin accumulation as in Table 5. When the concentration of sucrose is 70g/L, the fresh weight and the dry weight of the adventitious roots reach the maximum value, and are respectively 3.91g and 0.54 g. The total saponin yield of the adventitious root has no obvious difference when the sucrose concentration is 30,50 and 70g/L, and the total saponin content is in a higher level when the sucrose concentration is 30 and 50 g/L. Therefore, from the economical point of view, it was finally determined that the optimum sucrose concentration in the medium for suspension culture of adventitious roots of Panax notoginseng was 30 g/L.

TABLE 5 influence of sucrose on growth of adventitious roots and saponin accumulation of suspended Panax notoginseng

EXAMPLE 4 determination of suitable IBA content in suspension-cultured adventitious roots of Panax notoginseng

1.5g of adventitious roots of Panax notoginseng were inoculated into 1/2MS-N liquid medium supplemented with IBA (1.0-5.0mg/L) of different concentrations and cultured in suspension, 3 replicates were set for each treatment, and after 7 weeks of culture, the material was harvested and counted for fresh weight, dry weight and saponin accumulation as shown in Table 6. As can be seen from the table, the biomass of the adventitious roots increased with the increase of the IBA content and reached the maximum at an IBA content of 5.0mg/L, with a fresh weight of 4.18g and a dry weight of 0.47 g. The total glycoside content reaches a higher level when the IBA content is 2.0mg/L and 4.0mg/L, and the total saponin yield reaches the highest level when the IBA addition amount is 4.0mg/L and reaches 1.97 mg. Therefore, the most suitable IBA content of the suspension culture panax notoginseng adventitious roots is finally determined to be 4.0 mg/L.

TABLE 6 influence of IBA on growth of adventitious roots and saponin accumulation of suspended Panax notoginseng

EXAMPLE 5 determination of suitable initial inoculum size for suspension culture of Panax notoginseng adventitious roots

Adventitious roots of panax notoginseng were inoculated in 100mL of liquid medium containing 1/2MS-N +4mg/L IBA supplemented with 3% sucrose at different initial inoculum sizes (1.5, 2.5, 3.0, 3.5, 4.0g), and after 7 weeks of suspension culture, the growth rate and saponin accumulation of adventitious roots were counted, as shown in table 7. When the amount of the adventitious roots to be inoculated is 1.5-3.5g, the growth amount of the adventitious roots increases with the increase of the inoculation amount, and reaches the maximum at 3.5g, the fresh weight is 7.79g, and the dry weight is 0.67 g. When the inoculation amount is more than 3.5g, the biomass of adventitious roots begins to decrease. The total saponin production in adventitious roots also reached a maximum at an initial inoculum size of 3.5g, 11.13 mg. The total content of saponin reaches the maximum when the inoculation amount is 3.0g, and is 18.58 mg/g. Compared with the root of the three-year-old cultivated panax notoginseng, when the inoculation amount is 3.0g, the content of the ginsenoside Re is 8.05mg/g, which is 1.8 times of the content of Re (4.44mg/g) in the cultivated panax notoginseng.

TABLE 7 Effect of initial inoculum size on growth of suspended Panax notoginseng adventitious roots and saponin accumulation

Claims

1. A method for producing three main ginsenosides by rapidly proliferating adventitious roots of panax notoginseng is characterized in that: inoculating pseudo-ginseng cotyledons in 1/2SH culture medium containing 30g/L of sucrose and 2.0mg/L of IBA to induce the generation of adventitious roots, cutting the pseudo-ginseng cotyledons into small root segments of about 1.0cm, and inoculating the small root segments in the culture conditions of adventitious root induction: the culture was carried out in 1/2SH medium containing 50g/L sucrose, 2.0mg/L IBA, 2.0mg/L NAA for 3 weeks to produce new adventitious roots, which were then inoculated into elongation culture conditions: containing 50g/L sucrose, 2.0mg/L IBA, no ammonium Nitrate (NH)₄NO₃) Culturing in 1/2MS culture medium for 3 weeks to realize rapid growth of adventitious root; after 3.5g of the proliferated adventitious roots were cultured under induction culture conditions for 3 weeks, they were inoculated in a medium containing 100mL of a liquid medium: containing 30g/L sucrose, 4.0mg/L IBA, and no ammonium Nitrate (NH)₄NO₃) The culture flask of 1/2MS culture medium is suspended and cultured for 7 weeks in the absence of light, the rotation speed of a shaking table is 110rpm/min, and the rapid growth of adventitious roots and the rapid accumulation of three main ginsenosides Rg1, Re and Rb1 are realized.