A high-efficiency induction method of liquorice adventitious roots and complete plants

Abstract

The application relates to a high-efficiency induction method of liquorice adventitious roots and complete plants, and relates to the technical field of plant regeneration, which comprises the following steps: placing liquorice stem cells or liquorice somatic embryos in an induction culture medium to obtain liquorice complete plants or liquorice adventitious roots, wherein the culture medium is an MS culture medium containing a hormone composition composed of 6-BA and KT or an MS culture medium containing a hormone composition composed of NAA and IBA. The application has high induction efficiency, and the quality of the regenerated products prepared by the method is excellent.

Description

Technical Field

[0001] This application relates to the technical field of plant regeneration, and in particular to an efficient method for inducing adventitious roots and intact plants of licorice. Background Technology

[0002] Licorice is a commonly used bulk medicinal herb, but its wild resources are dwindling, and artificial cultivation is time-consuming and susceptible to environmental influences, leading to an unstable supply. To address this resource issue, tissue culture technology has been used for the rapid propagation of licorice. However, current methods generally suffer from low induction efficiency, inconsistent quality of regenerated plants, and unclear hormone combinations, making large-scale application difficult.

[0003] Current technologies mainly rely on direct induction of regeneration using conventional explants (such as stem segments and leaves). These materials have a high degree of differentiation, inconsistent developmental pathways, and are easily affected by seasonality and individual differences, resulting in poor reproducibility. Although different explants can be used for callus induction, a systematic and efficient regeneration system is still lacking, especially in terms of stable and reproducible culture medium formulations and conditions for different differentiation pathways (adventitious roots and intact plants).

[0004] However, using pluripotent licorice stem cells or somatic embryos as starting materials for culture can significantly overcome the aforementioned bottlenecks. These materials exhibit strong cell homogeneity, clear differentiation directions, and more sensitive and consistent responses to exogenous hormones. This not only greatly improves the induction efficiency and stability of adventitious roots and plants but also facilitates the standardization and large-scale development of the culture process.

[0005] Therefore, it is necessary to provide a method for inducing adventitious roots and complete plants of licorice using stem cells or somatic embryos as materials, which has high induction efficiency, short cycle and strong stability, so as to provide reliable technical support for its large-scale production. Summary of the Invention

[0006] The purpose of this application is to provide a highly efficient method for inducing adventitious roots and intact plants of licorice, which significantly improves induction efficiency and produces excellent regeneration quality.

[0007] In a first aspect, this application provides a highly efficient method for inducing whole licorice plants or adventitious roots of licorice, the method comprising culturing licorice stem cells or licorice somatic embryos in an induction culture medium to obtain whole licorice plants or adventitious roots of licorice.

[0008] Optionally, the licorice stem cells are prepared by the following method: peeling the cambium layer of licorice stem segments, inoculating the peeled cambium layer onto MS medium containing a hormone composition consisting of 6-BA, NAA and 2,4-D, and culturing for 14-20 days in a light-dark alternating environment at 22-28°C to induce the acquisition of licorice stem cells.

[0009] Optionally, the licorice somatic embryos are prepared by the following method: the hypocotyl portion of germinating licorice seedlings is cut to a length of 0.3-0.6 cm, inoculated onto MS medium containing a hormone composition consisting of 6-BA and IBA, and cultured at 22-28°C in an alternating light and dark environment for 40-50 days to induce the formation of licorice somatic embryos.

[0010] Optionally, the induction medium is a medium for inducing complete plants, wherein the medium is MS medium containing a hormone composition consisting of NAA and IBA.

[0011] Optionally, the concentration of NAA is 0.3-0.7 mg / L.

[0012] Optionally, the concentration of the IBA is 0.3-0.7 mg / L.

[0013] Optionally, the induction medium is an adventitious root induction medium, which is an MS medium containing a hormone composition consisting of 6-BA and KT.

[0014] Optionally, the concentration of 6-BA is 0.4-0.7 mg / L.

[0015] Optionally, the concentration of KT is 0.1-0.3 mg / L.

[0016] Optionally, the hormone composition in the adventitious root induction medium may further include methyl jasmonate and / or abscisic acid.

[0017] Optionally, the cultivation conditions of the method include: culturing for 10-20 days in an alternating light and dark environment at 22-28℃.

[0018] Optionally, the cultivation conditions of the method include: culturing at 22-28℃ in an alternating light and dark environment for 30-40 days.

[0019] Secondly, this application provides the use of an adventitious root induction medium in inducing licorice stem cells or licorice somatic cells to differentiate into adventitious roots, said medium being an MS medium containing a hormone composition consisting of 6-BA, KT and optionally methyl jasmonate or abscisic acid.

[0020] Thirdly, this application provides the use of a medium for inducing complete plant differentiation in licorice stem cells or licorice somatic embryos into complete plants, wherein the medium is an MS medium containing a hormone composition consisting of NAA and IBA.

[0021] In summary, this application includes at least one of the following beneficial technical effects:

[0022] 1. Significantly improved induction efficiency: This invention breaks through the limitations of traditional explants (such as stem segments and leaves) and innovatively uses pluripotent licorice stem cells or licorice somatic embryos as starting materials. These materials have uniform cell state, high differentiation potential, and are sensitive and consistent in response to exogenous hormones. Experimental data show that, using the optimal induction scheme, the induction rate of adventitious roots can reach as high as 98.34%, and the differentiation rate of complete plants reaches 52.7%, far exceeding the control and other hormone combinations, solving the core bottleneck of generally low induction rates in existing technologies.

[0023] 2. Controllable differentiation direction and excellent regeneration quality: This invention achieves precise control of the differentiation pathway by using two specific induction culture media; not only is the induction efficiency high, but the number of adventitious roots obtained is large and the roots are long; the regenerated complete plants are robust rather than weak and poor, which significantly improves the survival rate of regenerated seedlings and the feasibility of subsequent transplanting.

[0024] 3. Shortened production cycle and huge application potential: Compared with traditional cultivation or existing tissue culture methods, the induction method provided by this invention has a shorter cycle (about 35 days for adventitious root induction and about 14 days for complete plant induction), and is not limited by seasons, enabling year-round, factory-scale production, providing a new and efficient technical path to meet the huge market demand for licorice medicinal materials and their active ingredients.

[0025] 4. Providing a new strategy for the production of the secondary metabolite glycyrrhizin: The induced adventitious root or complete plant system can be directly used as a wild-type alternative system for large-scale cultivation to produce glycyrrhizin, which has broad application prospects in the industrial biosynthesis of medicinal components. Attached Figure Description

[0026] Figure 1 These are adventitious roots obtained from somatic cell embryonic induction differentiation in this application;

[0027] Figure 2 These are adventitious roots obtained through stem cell-induced differentiation in this application;

[0028] Figure 3 It is a complete plant obtained by somatic cell embryonic induction differentiation according to this application;

[0029] Figure 4 It is a complete plant obtained by stem cell-induced differentiation in this application;

[0030] Figure 5 These are the results of the detection of glycyrrhizin content in adventitious roots and intact plants. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific implementation examples, but the invention is not limited thereto.

[0032] Experimental materials

[0033] MS: Cooler Master PM10101-100L; NAA: Solarbio N8010-5g; 6-BA: Solarbio A8170-5g; KT: Solarbio K8010-250mg; BAP: Solarbio SB8840-100mg; 2,4-D: Solarbio D8100-5g; IAA: Solarbio IAA14030-5mg; IBA: Merck 57310-5G-F.

[0034] Example 1: Preparation of licorice stem cells

[0035] The stem segments of licorice (from the licorice root collected from Ningxia and kindly donated by the South China Botanical Garden) were disinfected with 75% alcohol for 1 minute, rinsed 3 times with sterile water, disinfected with 10% sodium hypochlorite for 5 minutes, disinfected with 5% sodium hypochlorite for 5 minutes, rinsed 3 times with sterile water, and then dried with filter paper.

[0036] The cambium layer of sterilized licorice stem segments was peeled off under a microscope, and the peeled cambium layer was inoculated onto a solid culture medium. The segments were then cultured at 25°C with alternating light and dark conditions for 14-20 days to induce the production of licorice stem cells. The solid culture medium consisted of MS basal medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L 2,4-D, and 3 g / L plant gel, with a pH of 5.8-6.0.

[0037] Example 2: Preparation of Licorice Somatic Cell Embryos

[0038] Select plump, disease-free *Glycyrrhiza glabra* seeds. Disinfect with 75% alcohol for 3 minutes, rinse three times with sterile water, then disinfect with 10% sodium hypochlorite for 15 minutes, rinse three times with sterile water, blot dry with filter paper, and inoculate into MS medium. Incubate at 25°C in the dark for 3-5 days to induce germination. Cut the hypocotyl of the germinated licorice seedlings to a length of 0.5 cm, inoculate onto solid medium, and incubate at 25°C with alternating light and dark for 45 days to induce licorice somatic embryos. The solid medium consists of MS basal medium, 0.5 mg / L 6-BA, 2 mg / L IBA, and 3 g / L plant gel, with a pH of 5.8-6.0.

[0039] Example 3: Method for inducing differentiation of adventitious roots of licorice

[0040] Take 9 pieces of 0.3cm 3Licorice stem cells and licorice somatic embryos were inoculated into 90 mm culture dishes containing solid adventitious root induction medium to induce adventitious roots. Six different culture media were tested, and the culture conditions were alternating light and dark (2000 lx, 8 h dark / 16 h light) at a temperature of 25 °C. High induction rate, short root emergence time, long root length, and a large number of adventitious roots were the preferred indicators. The culture media (all containing 3 g / L of plant gel) for the six different hormone combinations for adventitious root induction are as follows:

[0041] 1. MS + 1 mg / L IBA;

[0042] 2. MS + 1 mg / L NAA;

[0043] 3. MS + 1 mg / L IBA + 1 mg / L NAA;

[0044] 4. 1 / 2 MS+3mg / L 2,4-D+1mg / L KT+0.1% CH;

[0045] 5. 1 / 2 MS+1mg / L 2,4-D+1mg / L NAA;

[0046] 6. MS+0.5mg / L 6-BA+0.1mg / L KT.

[0047] The experimental results are shown in Table 1. The optimal culture medium was experimental group 6 (MS as the basal medium, 6-BA concentration of 0.5 mg / L, KT concentration of 0.1 mg / L, plant gel addition of 3 g / L, pH 5.8-6.0). The results of obtaining licorice adventitious roots after 37 days of somatic embryo culture and 35 days of stem cell culture are shown in Table 1. Figure 1 and Figure 2 As shown.

[0048] Table 1

[0049]

[0050] Example 4: Method for inducing differentiation of complete licorice plants

[0051] Take 9 pieces of 0.3cm 3 Licorice stem cells and licorice somatic embryos were inoculated into 90 mm culture dishes containing solid adventitious root induction medium to induce differentiation into complete plants. Culture conditions included alternating light and dark (2000 lx, 8 h dark / 16 h light) at 25 °C. High differentiation rate, short budding time, and good growth status were the preferred indicators. Eight different hormone combinations were tested in the culture medium (all containing 3 g / L of plant gel) as follows:

[0052] 1. MS+2mg / L NAA+0.5mg / L 6-BA;

[0053] 2. MS+0.5mg / L NAA+0.5mg / L IBA;

[0054] 3. MS+0.5mg / L KT+0.5mg / L 6-BA+0.1 mg / L IBA;

[0055] 4. MS+0.5mg / L IAA+5mg / L NAA+2.5 mg / L IBA+0.1% CH;

[0056] 5. MS+0.5mg / L 2,4-D+1mg / L BAP;

[0057] 6. MS + 0.5 mg / L 2,4-D;

[0058] 7. MS + 0.2 mg / L 2,4-D;

[0059] 8. MS+0.1mg / L KT+0.5mg / L 6-BA.

[0060] The experimental results are shown in Table 2. The optimal treatment group was experimental group 2 (MS as basal medium, IBA concentration 0.5 mg / L, NAA concentration 0.5 mg / L, plant gel addition 3 g / L, pH 5.8-6.0). The results of obtaining intact licorice plants after 14 days of culture are shown in Table 2. Figure 3 and Figure 4 As shown.

[0061] Table 2

[0062]

[0063] Example 5: Detection of glycyrrhizin

[0064] Sample preparation: Take respectively Figures 1-2 adventitious roots and Figures 3-40.1 g of whole plant samples were added to 2 ml of 90% ethanol (ensuring a solid-liquid ratio of 1:20), and the samples were extracted ultrasonically at 50℃ for 60 min. After ultrasonic extraction, the supernatant was transferred to a centrifuge tube, centrifuged, and then filtered through a filter membrane into a chromatographic injection bottle for analysis. The glycyrrhizin in the samples was determined by HPLC under the following chromatographic conditions: column: Shim-pack GISTODS (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) - 0.05% phosphoric acid (B) aqueous solution; gradient elution (elution program shown in Table 3); flow rate: 0.8 mL / min; detection wavelength: 280 nm; column temperature: 30 ℃; injection volume: 10 μL.

[0065] Table 3

[0066] T / min A / % B / % 0 46 54 10 46 54 30 53 47 50 55 45 55 80 20 60 46 54

[0067] Test results as follows Figure 5 As shown, the content of glycyrrhizin, a secondary metabolite, in adventitious roots and intact plants obtained by the method of this application is significantly higher than that in wild licorice (usually less than 50 μg / g).

[0068] Example 6: Optimization of the method for inducing stem cell differentiation into licorice adventitious roots

[0069] To further increase the content of the secondary metabolite glycyrrhizin, the applicant added 10 μM methyl jasmonate or abscisic acid to experimental group 6 of Example 3 (MS basal medium, 6-BA concentration of 0.5 mg / L, KT concentration of 0.1 mg / L, plant gel addition of 3 g / L, pH of 5.8-6.0), and the culture method was the same as in Example 3. Using the detection method of Example 5, the glycyrrhizin content of licorice adventitious roots cultured in the medium supplemented with 10 μM methyl jasmonate or 10 μM abscisic acid was found to be 521.34 μg / g and 430.58 μg / g, respectively.

[0070] Of course, the above description is only a specific embodiment of this application and is not intended to limit the scope of the invention. All equivalent changes or modifications made in accordance with the features and principles described in the claims of this invention should be included in the scope of the claims of this invention.

Claims

1. A highly efficient method for inducing licorice root growth in whole plants or adventitious roots, characterized in that, The method includes culturing licorice stem cells or licorice somatic embryos in an induction medium to obtain complete licorice plants or adventitious roots of licorice. The licorice stem cells are derived from the cambium layer of licorice stem segments. The licorice stem cells are prepared by the following method: the cambium layer of licorice stem segments is peeled off, and the peeled cambium layer is seeded onto MS medium containing a hormone composition consisting of 6-BA, NAA and 2,4-D. The licorice stem cells are induced to be obtained by culturing in an alternating light and dark environment at 22-28℃ for 14-20 days. The licorice somatic embryos are derived from the hypocotyl of licorice seedlings. The licorice somatic embryos are prepared by the following method: the hypocotyl of germinating licorice seedlings is cut to a length of 0.3-0.6 cm and inoculated onto MS medium containing a hormone composition composed of 6-BA and IBA. The licorice somatic embryos are induced by culturing at 22-28℃ in a light-dark alternating environment for 40-50 days. Wherein, when the induction medium is a medium for inducing complete plants, it is an MS medium containing a hormone composition consisting of NAA and IBA, wherein the concentration of NAA is 0.3-0.7 mg / L and the concentration of IBA is 0.3-0.7 mg / L; When the induction medium is an adventitious root induction medium, it is an MS medium containing a hormone composition of 6-BA and KT, wherein the concentration of 6-BA is 0.4-0.7 mg / L and the concentration of KT is 0.1-0.3 mg / L.

2. The method according to claim 1, characterized in that, The hormone composition in the adventitious root induction medium also includes methyl jasmonate and / or abscisic acid.

3. The method according to claim 1, characterized in that, The culture conditions for obtaining complete licorice plants include: culture at 22-28℃ in an alternating light and dark environment for 10-20 days.

4. The method according to claim 1, characterized in that, The culture conditions for obtaining adventitious roots of licorice include: culture at 22-28℃ in an alternating light and dark environment for 30-40 days.

5. The application of the efficient induction method for licorice adventitious roots as described in claim 1 in inducing licorice stem cells or licorice somatic cells to differentiate into adventitious roots.

6. The application of the efficient induction method for complete licorice plants as described in claim 1 in inducing differentiation of licorice stem cells or licorice somatic embryos into complete plants.

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