CN115152628A - Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots - Google Patents
Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots Download PDFInfo
- Publication number
- CN115152628A CN115152628A CN202210749846.XA CN202210749846A CN115152628A CN 115152628 A CN115152628 A CN 115152628A CN 202210749846 A CN202210749846 A CN 202210749846A CN 115152628 A CN115152628 A CN 115152628A
- Authority
- CN
- China
- Prior art keywords
- culture
- humidity
- days
- culturing
- ginseng
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to an adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots, the method inoculates induced adventitious roots into a sterilized triangular flask containing SH culture medium, and liquid suspension culture is carried out; wherein the SH culture medium comprises 3/4SH, 3% sucrose, 3PPM IBA, 0.5PPM naphthylacetic acid and 0.3PPM kinetin; the culture conditions were: culturing at 23-25 deg.C and humidity of 35-50% for 3 days; culturing at 20-22 deg.C and humidity of 35-50% for 3 days; culturing at 17-19 deg.C and humidity of 35-50% for 3 days; culturing at 14-16 deg.C and 30-45% humidity for 4 days; culturing at 11-13 deg.C and 30-45% humidity for 4 days at 8-10 deg.C and 30-45% humidity for 4 days; all are dark culture. The cultivation method can improve the content of Rb1, rb2, rb3, rc and Rd saponins in PPD saponins in the ginseng adventitious root.
Description
Technical Field
The invention belongs to the technical field of ginseng culture, and particularly relates to an adventitious root metaphase culture method and a culture medium for improving the content of PPD saponin in ginseng adventitious roots.
Background
The ginsenoside comprises prototype ginsenoside and rare ginsenoside, and the anticancer activity of the rare ginsenoside is far stronger than that of the prototype ginsenoside. The prototype ginsenoside comprises Ra, rb1, rb2, rb3, rc, rd, rg1, rg2, rf, etc., wherein PPD saponin can be converted into rare ginsenoside with high added value through structural modification.
At present, the induction culture technology of the adventitious roots of ginseng is mature day by day, and the callus induction is generally carried out in an MS solid culture medium containing 2,4-D, 6-BA and sucrose after the fresh roots of the perennial ginseng are cleaned, disinfected and cut. And transferring the induced callus to IBA and sucrose MS for inducing and proliferating adventitious roots. However, the content of saponin in the ginseng adventitious roots cultivated in this way is low.
The prior patents or documents disclose some methods for improving the content of saponins in the ginseng adventitious roots (for example, chinese patent CN 111937748B discloses a culture method for improving the content of saponins Rg1 and Re in the ginseng adventitious roots), but the prior methods do not disclose how to improve the content of PPD saponins pertinently.
Disclosure of Invention
The invention aims to provide a middle-stage cultivation method of adventitious roots for improving the content of PPD saponin in the adventitious roots of ginseng, which induces the synthesis of PPD saponin in the adventitious roots of ginseng by a low-temperature induction mode of sequential cooling, and ensures the normal growth of the adventitious roots of ginseng during cultivation in a low-temperature environment by improving a culture medium, thereby improving the content of Rb1, rb2, rb3, rc and Rd saponin in the PPD saponin.
In order to achieve the purpose, the invention adopts the following technical scheme:
a mid-term cultivation method of adventitious root for increasing PPD saponin content in adventitious root of Ginseng radix comprises inoculating induced adventitious root into sterilized triangular flask containing SH culture medium, and performing liquid suspension culture; wherein the SH culture medium comprises 3/4SH, 3% sucrose, 3PPM IBA, 0.5PPM naphthylacetic acid and 0.3PPM kinetin;
the culture conditions were: culturing at 23-25 deg.C and humidity of 35-50% for 3 days; culturing at 20-22 deg.C and humidity of 35-50% for 3 days; culturing at 17-19 deg.C and humidity of 35-50% for 3 days; culturing at 14-16 deg.C and 30-45% humidity for 4 days; culturing at 11-13 deg.C and 30-45% humidity for 4 days at 8-10 deg.C and 30-45% humidity for 4 days; all are dark cultivations.
In a preferred aspect of the present invention, after the middle stage cultivation, the cultivated adventitious roots should be inoculated into a sterilized bioreactor to continue cultivation.
As the optimization of the invention, an SH culture medium is adopted when the bioreactor is used for cultivation, and the SH culture medium comprises 3/4SH, 3% sucrose, 5PPM IBA and 1mL of NaOH with the concentration of 3.5N; during culture, an air valve is opened, the ventilation amount is controlled to be 0.5vvm, the temperature is 23-25 ℃, the humidity is 40%, and the culture is carried out for 4 weeks, wherein all the culture is dark culture.
The second purpose of the invention is to provide a medium for the metaphase culture of the adventitious root for improving the PPD saponin content in the adventitious root of the ginseng, wherein the medium is an SH medium and comprises 3/4SH, 3% sucrose, 3PPM IBA, 0.5PPM naphthylacetic acid and 0.3PPM kinetin, and the culture adopts low-temperature liquid suspension culture with sequential cooling.
The invention has the advantages and beneficial effects that:
(1) The culture method provided by the invention obviously improves the content of Rb1, rb2, rb3 and Rd in the finally obtained adventitious roots in a low-temperature induction mode of slow cooling during the middle-stage culture of the adventitious roots, and simultaneously adds IBA, sucrose, naphthylacetic acid and kinetin into an SH culture medium, and after the IBA, the naphthylacetic acid and the kinetin are mixed according to a certain proportion, the influence on the growth of the adventitious roots of ginseng in a low-temperature environment can be avoided.
(2) The content of PPD saponin in the ginsenoside obtained by the culture method provided by the invention is obviously increased, and the PPD saponin can be converted into rare ginsenoside with high added value through structural modification; therefore, the yield of rare ginsenoside with high added value can be improved by using the adventitious roots of ginseng cultured by the invention as a raw material.
(3) The cultivation method provided by the invention is simple and short in period, and is already used for large-scale production of the ginseng adventitious roots.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments and that for a person skilled in the art, other drawings can also be derived from them without inventive effort. In the drawings:
FIG. 1 is a schematic diagram of callus induction in example 1;
FIG. 2 is a schematic diagram of adventitious root induction in example 1;
FIG. 3 is a schematic diagram of the interim adventitious-root cultivation in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below, and the following embodiments are used for illustrating the present invention and are not used for limiting the scope of the present invention.
Example 1
The invention relates to a method for cultivating adventitious roots in the middle stage, which can improve the PPD saponin content in the adventitious roots of ginseng, and the method needs to inoculate the induced adventitious roots into a sterilized triangular flask containing SH culture medium for liquid suspension culture; therefore, when the intermediate cultivation is carried out, the induced adventitious roots need to be obtained in the following specific way:
(1) Explant disinfection: washing radix Ginseng under tap water for 30s, placing in a clean white steel barrel, and adding a certain amount of ethanol with concentration of 70% to completely immerse radix Ginseng (the process is mainly for washing off dust and soil on the surface of radix Ginseng, soaking and cleaning for about 1 min); washing with sterilized sterile water for several times (mainly for washing off residual ethanol solution on the surface of ginseng), transferring to a clean bench, soaking ginseng in 2% NaClO solution for 15-20min, wherein the soaking time depends on the size and thickness of the ginseng, the color of the ginseng skin gradually turns white, washing with sterile water for several times after soaking, washing off the NaClO solution on the surface, and removing excessive water on the surface of the ginseng on sterile filter paper.
(2) Callus induction: placing Ginseng radix with surface water removed in a clean bench in a cutting disc, and slicing according to rhizome (rhizoma Phragmitis), main root (epidermis), and fine root; the size of the slices is controlled to be approximately 1cm in length. Adopting an MS culture medium which is easy to induce callus, adding a certain amount of auxin, cytokinin and sucrose into the MS culture medium, wherein the auxin is 2, 4-dichlorophenoxyacetic acid (2, 4-D), the cytokinin is 6-benzylaminopurine (6-BA), the addition amounts of the auxin, the cytokinin and the 6-BA are respectively 1PPM and 0.5PPM, and the concentration of the sucrose is 30g/L; sterilizing the culture medium in a sterilization pot (sterilization conditions: 121 deg.C, 15 min), heating to 45 deg.C, slowly pouring the culture solution into the culture medium, covering with a cover, solidifying the culture medium, placing the sliced Ginseng radix (3-8 slices) in a sterilized culture dish, and culturing in an incubator. The culture conditions were: the temperature is 23-25 ℃, the humidity is 40%, the illumination time is Light:8h, and the Dark:16h, and the result is shown in figure 1.
(3) Induction of adventitious roots: the callus with good growth is divided into small sections of about 5mm in a super clean bench and transferred to a culture medium for inducing adventitious roots, the culture medium for inducing the adventitious roots adopts an MS culture medium, a certain amount of growth hormone IBA and sucrose are added into the MS culture medium, the addition amount of the growth hormone IBA is 2PPM, and the concentration of the sucrose is 30g/L. Sterilizing the culture medium in a sterilizing pot (sterilization condition: 121 deg.C, 15 min), slowly pouring the culture solution into a culture dish at 45 deg.C, covering with a cover, solidifying the culture medium, and placing the callus small segments on the sterilized culture dish for culturing. The culture conditions were: the temperature is 23-25 deg.C, humidity is 40%, and adventitious root is induced by dark culture, and the result is shown in FIG. 2.
In the invention, when the ginseng is cultivated in the middle stage of adventitious roots, induced adventitious roots are transferred to a sterilized 250mL triangular flask containing an SH culture medium (containing 80-100mL of SH culture medium) from a culture dish in a super clean workbench for liquid suspension culture;
the SH culture medium is 3/4SH +3% of sucrose + IBA (3 PPM) + naphthylacetic acid (0.5 PPM) + kinetin (0.3 PPM), and the culture medium is used after being sterilized in a sterilization pot (sterilization conditions: 121 ℃,15 min).
The culture conditions are as follows: culturing at 23-25 deg.C and humidity of 40% for 3 days; culturing at 20-22 deg.C and humidity of 35% for 3 days; culturing at 17-19 deg.C and 35% humidity for 3 days; culturing at 14-16 deg.C and 30% humidity for 4 days; culturing at 11-13 deg.C and 30% humidity for 4 days at 8-10 deg.C and 30% humidity for 4 days; all the results are dark culture, and the results are shown in FIG. 3.
After the cultivation of the ginseng in the middle stage of adventitious roots is finished, the cultivation of the adventitious roots is continued, and the specific cultivation mode is as follows:
inoculating adventitious roots in about 20-30 triangular flasks into a sterilized bioreactor in a clean bench, connecting the bioreactor system, opening an air valve, and allowing the air flow to be 0.5vvm, so as to culture by using the bioreactor system. The bioreactor used SH medium (3/4 SH) +3% sugar + IBA (5 PPM) + NaOH (3.5N 1mL). The culture conditions are as follows: culturing at 23-25 deg.C and 40% humidity in dark condition for four weeks.
Comparative example 1
The difference from the example 1 lies in the middle-stage cultivation of the adventitious roots of the ginseng, which comprises the following specific steps:
transferring the induced adventitious roots from the culture dish to a sterilized 250mL triangular flask containing SH culture medium (containing 80-100mL SH culture medium) in an ultra-clean workbench, carrying out liquid suspension culture, wherein the SH culture medium is 3/4SH +3% sugar + IBA (3 PPM)), and placing the culture medium in a sterilization pot for sterilization (sterilization conditions: 121 ℃,15 min). The culture conditions are as follows: culturing at 23-25 deg.C and humidity of 40% in dark condition for three weeks.
The method for measuring the content of the ginsenoside in the adventitious root of the ginseng comprises the following steps:
(1) Preparation of a standard solution: accurately weighing 1.5mg of each standard substance of the ginsenosides Rbl, rb2, rb3, rc, rd, re, rg1, rg3 and Rh1, placing the standard substances into a 10mL volumetric flask, dissolving the standard substances with chromatographic pure methanol, and fixing the volume to the scale for later use. Ginsenoside standards are purchased by Wodsman Biotech, inc.
(2) Preparation of sample solution: accurately weighing 0.2g of dry powder of ginseng adventitious roots, placing the powder in a 100ml triangular flask, adding 20ml of methanol, carrying out water bath at 60 ℃ for 1h, carrying out suction filtration while the powder is hot, repeatedly extracting the powder once, combining filtrates and volatilizing the filtrate; dissolving the extract with 10ml distilled water, extracting with 20ml n-butanol twice, collecting n-butanol layer, rotary steaming, diluting methanol to 1ml, and filtering with 0.22 μm microporous membrane to obtain sample solution.
(3) A chromatographic column YMC J' sphere ODS-H80 (4.6mmx250 mm,4 um), a UV detector, a detection wavelength of 203nm, a sample injection amount of 20uL, a column temperature of 35 ℃, and a flow rate of 1mL/mim; the mobile phase A is acetonitrile, and the mobile phase B is water.
Gradient elution procedure of 0 min-10min, A; 10 min-20min, A; 20 min-25min A; 25-45min, A; 45-45.10 min, A; 45.10 min-60min, A is 60 min-60.10 min.
The invention completes the determination of the saponin content in the ginseng adventitious roots obtained in the example 1 and the comparative example 1 according to the mode, and the result is as follows:
TABLE 1 PPD Saponin content (mg/g) in adventitious roots of Ginseng radix
| Sample (I) | Rb1 | Rb2 | Rb3 | Rd | Rc |
| Example 1 | 9.91±0.24 | 3.87±0.14 | 4.52±0.21 | 0.87±0.03 | 0.53±0.02 |
| Comparative example 1 | 5.61±0.18 | 1.12±0.22 | 0.34±0.05 | 0.36±0.04 | 0.45±0.08 |
And (4) conclusion: the culture method provided by the invention can obviously improve the content of saponins such as Rb1, rb2, rb3, rd and the like by inducing in a low-temperature induction mode of sequential cooling when the ginseng adventitious roots are cultured in the middle stage.
Claims (4)
1. A mid-term cultivation method of adventitious roots for improving the PPD saponin content in the adventitious roots of ginseng is characterized in that the induced adventitious roots are inoculated into a sterilized triangular flask containing SH culture medium for liquid suspension culture; wherein the SH culture medium comprises 3/4SH, 3% sucrose, 3PPMIBA, 0.5PPM naphthylacetic acid and 0.3PPM kinetin;
the culture conditions were: culturing at 23-25 deg.C and humidity of 35-50% for 3 days; culturing at 20-22 deg.C and humidity of 35-50% for 3 days; culturing at 17-19 deg.C and humidity of 35-50% for 3 days; culturing at 14-16 deg.C and 30-45% humidity for 4 days; culturing at 11-13 deg.C and 30-45% humidity for 4 days at 8-10 deg.C and 30-45% humidity for 4 days; all are dark cultivations.
2. The mid-term cultivation method of adventitious roots for improving PPD saponin content in ginseng adventitious roots according to claim 1, wherein after the mid-term cultivation is finished, the cultivated adventitious roots are inoculated into a sterilized bioreactor to continue cultivation.
3. The mid-term cultivation method of adventitious roots for improving PPD saponin content in the adventitious roots of ginseng according to claim 2, characterized in that an SH culture medium is adopted during the cultivation in the bioreactor, wherein the SH culture medium comprises 3/4SH, 3% sucrose, 5PPM IBA, and 1mL NaOH of 3.5N; during culture, an air valve is opened, the ventilation amount is controlled to be 0.5vvm, the temperature is 23-25 ℃, the humidity is 40%, and the culture is carried out for 4 weeks, wherein all the culture is dark culture.
4. A medium for culturing the adventitious root in middle stage for increasing the content of PPD saponin in the adventitious root of ginseng features that said medium is SH medium containing 3/4SH, 3% cane sugar, 3PPM IBA, 0.5PPM naphthylacetic acid and 0.3PPM kinetin, and the culture is performed by low-temp liquid suspension culture with sequential cooling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210749846.XA CN115152628B (en) | 2022-06-29 | 2022-06-29 | Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210749846.XA CN115152628B (en) | 2022-06-29 | 2022-06-29 | Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115152628A true CN115152628A (en) | 2022-10-11 |
| CN115152628B CN115152628B (en) | 2023-04-11 |
Family
ID=83489219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210749846.XA Active CN115152628B (en) | 2022-06-29 | 2022-06-29 | Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115152628B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020142463A1 (en) * | 2001-01-19 | 2002-10-03 | Kee-Yoeup Paek | Method for the mass propagation of adventitious roots of ginseng, camphor ginseng and wild ginseng by tissue culture and the improvement of their saponin content |
| CN108739377A (en) * | 2018-04-28 | 2018-11-06 | 刘汉石 | A kind of ginseng adventitious root inducing culture and its application |
| CN109729974A (en) * | 2019-02-12 | 2019-05-10 | 天津大学 | A kind of method for tissue culture of Low- temperature culture ginseng adventitious root |
| CN111134010A (en) * | 2020-01-06 | 2020-05-12 | 烟台大学 | Method for preparing American ginseng adventitious roots with high antioxidant activity |
| CN112806267A (en) * | 2021-04-02 | 2021-05-18 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng adventitious roots by using biological reaction device |
| CN112997884A (en) * | 2021-04-02 | 2021-06-22 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng adventitious roots by using biological reaction device |
| CN113647324A (en) * | 2021-01-18 | 2021-11-16 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
-
2022
- 2022-06-29 CN CN202210749846.XA patent/CN115152628B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020142463A1 (en) * | 2001-01-19 | 2002-10-03 | Kee-Yoeup Paek | Method for the mass propagation of adventitious roots of ginseng, camphor ginseng and wild ginseng by tissue culture and the improvement of their saponin content |
| CN108739377A (en) * | 2018-04-28 | 2018-11-06 | 刘汉石 | A kind of ginseng adventitious root inducing culture and its application |
| CN109729974A (en) * | 2019-02-12 | 2019-05-10 | 天津大学 | A kind of method for tissue culture of Low- temperature culture ginseng adventitious root |
| CN111134010A (en) * | 2020-01-06 | 2020-05-12 | 烟台大学 | Method for preparing American ginseng adventitious roots with high antioxidant activity |
| CN113647324A (en) * | 2021-01-18 | 2021-11-16 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
| CN112806267A (en) * | 2021-04-02 | 2021-05-18 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng adventitious roots by using biological reaction device |
| CN112997884A (en) * | 2021-04-02 | 2021-06-22 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng adventitious roots by using biological reaction device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115152628B (en) | 2023-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112931216B (en) | Biological reaction device and culture method for ginseng adventitious roots | |
| CN112997884B (en) | Method for culturing ginseng adventitious roots by using biological reaction device | |
| CN108271689A (en) | A kind of method of the indefinite Root tissue culture of wild ginseng | |
| CN112369324B (en) | Tissue culture method for sedum aizoon | |
| CN115152628B (en) | Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots | |
| CN115053810B (en) | Culture method for simultaneously increasing contents of multiple ginsenosides in ginseng adventitious roots | |
| CN112806267B (en) | Method for culturing ginseng adventitious roots by using biological reaction device | |
| CN113025555B (en) | Method for separating and culturing ginseng stem cells by using biological reaction device | |
| CN112956415B (en) | Liquid culture medium for culturing ginseng adventitious roots and culture method | |
| CN113647327B (en) | Method for converting ginsenoside in adventitious roots of ginseng | |
| CN113647324B (en) | Liquid culture medium for culturing ginseng adventitious roots and culture method | |
| CN113151144B (en) | Isolated culture method of ginseng stem cells | |
| CN115109739A (en) | Method for rapidly culturing desert rose suspension cells at high density to produce chlorogenic acid at high yield | |
| CN113647326A (en) | Method for culturing adventitious roots of ginseng | |
| CN113046296A (en) | Method for culturing ginseng stem cells by using biological reaction device | |
| CN113046297A (en) | Isolated culture method of ginseng stem cells | |
| CN106699561B (en) | Method for quickly obtaining chlorogenic acid crude extract | |
| CN113046292B (en) | Method for culturing ginseng stem cells by using biological reaction device | |
| CN113025554B (en) | Method for culturing ginseng stem cells by using biological reaction device | |
| CN113647325B (en) | Liquid culture medium for culturing ginseng adventitious roots and culture method | |
| CN113151146B (en) | Ginseng stem cell separation culture method using biological reaction device | |
| CN112806266B (en) | Method for culturing ginseng adventitious root by using biological reaction device | |
| CN113186150B (en) | Ginseng stem cell separation culture method using biological reaction device | |
| CN117448254B (en) | Preparation method and application of Glycyrrhiza glabra stem cells | |
| CN118360233A (en) | Induction culture method of ginseng embryonic stem cells and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |