A method for directly inducing adventitious roots of kudzu vine by using kudzu vine leaves and root hairs and a culture medium formula

Abstract

The present application provides a method for inducing adventitious roots of kudzu directly by using kudzu leaves and root hairs and a culture medium formula. The present application belongs to the field of biotechnology, and uses kudzu leaves and root hairs as explants to directly induce adventitious roots in a MS culture medium with 0.5-5.0 mg / L IBA, 20-30 g / L sucrose, 7 g / L agar, and pH 5.5-6.5. Compared with the prior art method of generally first inducing callus by using explants and then inducing adventitious roots by using the callus, the present application has the advantages of rapid induction of adventitious roots, high repeatability, short cycle, convenient sampling, and being free from the influence of natural disasters.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and culture medium formulation for directly inducing adventitious roots of kudzu using kudzu leaves and roots. Background Technology

[0002] Kudzu (Pueraria lobata) is a perennial deciduous vine belonging to the genus Pueraria in the subfamily Papilionoideae of the legume family. It is one of the medicinal plants used in traditional Chinese medicine. Wild kudzu can accelerate blood circulation and flow, and has effects such as reducing fever, promoting rash eruption, raising yang and stopping diarrhea, and relieving hangovers. Currently, more than 70 phytochemicals have been identified from kudzu, which can be divided into several categories according to their chemical composition, including isoflavones, triterpenoid saponins, coumarins, and alkaloids. Isoflavones and triterpenoid saponins are generally considered to be the main active ingredients of kudzu. Among them, the main isoflavones are puerarin, genistein, and gentianin.

[0003] Puerarin has been extensively studied for its various effective pharmacological activities, including cardioprotection, vasodilation, cardiovascular protection, anti-inflammation, antioxidant effects, neuroprotection, pain relief, bone formation promotion, alcohol inhibition, anticancer activity, liver protection, and estrogenic activity. Experimental and clinical studies have reported that puerarin is also widely used in the treatment of cardiovascular diseases, diabetes and its complications, osteonecrosis, and cancer.

[0004] In addition, puerarin can slow gastric emptying, reduce the rate of alcohol absorption, decrease intestinal permeability, and protect the gastrointestinal mucosa. Simultaneously, puerarin can regulate the activity of enzymes related to ethanol metabolism, such as alcohol dehydrogenase and aldehyde dehydrogenase, selectively inactivating cytochromes, thereby reducing the production of free radicals and alleviating hepatocellular damage. Puerarin can also increase the activity of antioxidant enzymes in the body, reducing oxidative stress in tissues caused by liver damage.

[0005] The exploitation and unscientific harvesting of kudzu resources have severely damaged wild kudzu resources, leading to a decrease and disappearance of some high-quality resources. Currently, puerarin is mainly obtained from cultivated kudzu, resulting in high production costs, long planting cycles, and low yields. Furthermore, the limited variety of cultivation and lagging research on resource protection and variety selection have led to increased pests and diseases, genetic degradation, and a continuous decline in quality, resistance, and yield. In vitro culture of root organs is one effective means to address this problem. Adventitious roots, due to their rapid growth rate, stable production capacity of secondary metabolites, and lack of environmental constraints, coupled with the absence of highly toxic side effects and pesticide residues, have advantages in product development and have received increasing market attention in recent years. Kudzu contains abundant phenolic substances, which are prone to browning during induction, severely affecting the induction and differentiation of adventitious roots.

[0006] Therefore, it is necessary to provide a method for inducing and culturing adventitious roots of kudzu and a culture medium formulation to solve the above problems. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method and culture medium formulation for directly inducing adventitious roots in kudzu using kudzu leaves and roots. The culture medium formulation provided by this invention effectively avoids browning during the induction of adventitious roots in kudzu; and by directly inducing adventitious roots using kudzu leaves and roots, it significantly shortens the induction time, effectively maintains the superior traits of the parent plants, avoids varietal mixing and virus accumulation in the plants, and is not limited by season, among many other advantages.

[0008] The technical means employed in this invention are as follows:

[0009] A method for directly inducing adventitious roots of kudzu using leaves and roots is proposed. Kudzu leaves and roots are used as explants for adventitious root induction and cultured in MS medium containing 0.5–5.0 mg / L IBA, 20–30 g / L sucrose, 7 g / L agar, and pH 5.5–6.5.

[0010] Furthermore, the specific steps include the following:

[0011] S1. Establishing sterile seedlings from seeds: Weigh 20g MS medium powder, 30g sucrose, and 7g agar per liter of culture medium, add them to 1L of water, stir well, sterilize, and then use them as seedling culture medium.

[0012] The seeds were rinsed to remove surface mud and sand, soaked in sterile water for 4 hours, rinsed with 75% alcohol for 30 seconds, rinsed 3 times with sterile water, soaked in 5.5% sodium hypochlorite solution for 6 minutes, rinsed 5 times with sterile water, dried with sterile filter paper, inserted into the culture medium with sterile forceps, and cultured at (25±1)℃ for 12 hours under light for 30 days to obtain sterile kudzu seedlings;

[0013] S2. Preparation of adventitious root induction medium: Weigh 20g MS medium powder, 20-30g sucrose, and 7g agar per liter of medium, add to 1L of water, stir well, add 0.5-5.0mg / L IBA, adjust the pH to 5.5-6.5, stir well, and sterilize to obtain the adventitious root induction medium.

[0014] S3. Adventitious root induction: Select leaves and roots from sterile seedlings that have grown for more than 30 days as described in step S1.

[0015] The leaves were scratched with a sterile knife, and the underside of the leaves was placed in close contact with the culture medium obtained in step S2 to induce the formation of adventitious roots.

[0016] The roots are scratched with a sterile knife and placed in the culture medium obtained in step S2 for culture to induce the formation of adventitious roots.

[0017] Further, in step S2, 20g of MS medium powder, 30g of sucrose, and 7g of agar are weighed per liter of culture medium, added to 1L of water, stirred evenly, and then 1.5mg / L IBA or 4.0mg / L IBA is added to adjust the pH to 6.0. After stirring evenly, the mixture is sterilized to obtain the adventitious root induction medium.

[0018] Furthermore, the culture conditions in step S3 are: dark culture for 30 days at 20-30℃.

[0019] Furthermore, the induction rate of adventitious roots in leaves was ≥45%. The induction rate of adventitious roots in roots was ≥85%.

[0020] The present invention also provides a culture medium for directly inducing adventitious roots of kudzu using leaves and roots. The culture medium is MS medium, which consists of 0.5-5.0 mg / L IBA, 20-30 g / L sucrose and 7 g / L agar, with a pH of 5.5-6.5. The culture is carried out in the dark at 20-30°C.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. This invention utilizes the leaves and roots of kudzu to directly induce adventitious roots of kudzu. The induction rate of adventitious roots from leaves is ≥45%, and the induction rate of adventitious roots from roots is ≥85%.

[0023] 2. This invention takes the direct induction of adventitious roots from kudzu leaves and roots as the research object. By statistically analyzing the induction rate and observing the growth changes of adventitious roots, the growth and dynamic changes of adventitious roots are explored. IBA hormone regulation is used to promote the induction of adventitious roots from leaves and roots.

[0024] 3. The adventitious roots of kudzu obtained by this invention can be used for suspension culture to produce puerarin.

[0025] Compared with the existing technology of first inducing callus tissue from explants and then using callus tissue to induce adventitious roots, this invention has the advantages of rapid adventitious root induction, high repeatability, short production cycle, high efficiency, convenient material sourcing, industrial production capability, and immunity to natural disasters. It provides a new method for producing physiologically active substances such as puerarin, and a new approach for the rational development and utilization of kudzu. It can effectively protect wild Chinese herbal medicine resources and provide opportunities for the modernization of traditional Chinese herbal medicine.

[0026] Based on the above reasons, this invention can be widely applied in the field of induced adventitious root technology of kudzu. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a physical image of a sterile seedling established from seeds in this invention.

[0029] Figure 2 A physical image showing the induction of adventitious roots using leaves in accordance with the method of this invention.

[0030] Figure 3 A physical diagram of adventitious roots induced by rootlets, used in accordance with the method of this invention. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] This invention provides a method for directly inducing adventitious roots of kudzu using kudzu leaves and roots as explants.

[0033] Example 1

[0034] Taking the direct induction of adventitious roots from leaves as an example, the specific steps include:

[0035] Establishing sterile seedlings from seeds: Weigh 20g MS medium powder, 30g sucrose, and 7g agar per liter of culture medium, add them to 1L of water, stir well, sterilize, and then use them as seedling culture medium.

[0036] The seeds were rinsed to remove surface mud and sand, soaked in sterile water for 4 hours, rinsed with 75% alcohol for 30 seconds, rinsed three times with sterile water, then soaked in 5.5% sodium hypochlorite solution for 6 minutes, rinsed five times with sterile water, and the surface moisture was absorbed with sterile filter paper. The seeds were then inserted into the culture medium using sterile forceps and cultured at (25±1)℃ for 12 hours under light for 30 days to obtain sterile kudzu seedlings. Figure 1 As shown.

[0037] Weigh 20g MS medium powder, 30g sucrose, and 7g agar per liter of culture medium, add them to 1L of water, stir well, and then add 0.5, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mg / L of IBA respectively. Adjust the pH to 6.0, sterilize, and the resulting culture medium for inducing adventitious roots is obtained. Scratch the leaves with a sterile knife, place the back of the leaves in close contact with the culture medium, and culture in the dark at (25±1)℃ for 30 days. Observe the induction and growth of adventitious roots of kudzu, and count the induction rate. The results are shown in Table 1.

[0038] Table 1 Results of the experiment on optimizing adventitious root hormone induction from leaves

[0039] Experimental protocol IBA (mg / L) Induction rate % Average number of roots Average root length (mm) 1 0.5 24.8 1.9 43 2 0.8 11.7 1.1 20 3 1.0 37.9 2.6 47 4 1.5 49.5 2.1 79 5 2.0 41.7 2.9 38 6 3.0 21.9 3.8 53 7 4.0 11.3 1.8 23 8 5.0 19.7 3.1 23

[0040] Table 1 shows that in group 4, with MS + IBA 1.5 mg / L, the adventitious root induction rate was 49.5%, the average number of roots was 2.1, and the root length was 79 mm. Leaf induction yielded the best results (e.g., ...). Figure 2 (As shown).

[0041] Example 2

[0042] Taking the direct induction of adventitious roots by root hairs as an example, the specific steps include the following:

[0043] Weigh 20g MS medium powder, 30g sucrose, and 7g agar per liter of culture medium, add them to 1L of water, and add 0.5, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mg / L of IBA respectively. Adjust the pH to 6.0, sterilize, and obtain the adventitious root induction medium. Take sterile seedling roots, make cuts with a sterile knife, and place them on the surface of the adventitious root induction medium. Incubate in the dark at (25±1)℃ for 30 days, observe the adventitious root induction growth of kudzu, and count the induction rate. The results are shown in Table 2.

[0044] Table 2 Results of the Root-Induced Adventitious Root Hormone Optimization Experiment

[0045] Experimental protocol IBA (mg / L) Induction rate % Average number of roots Average root length (mm) 1 0.5 54.9 1.8 26 2 0.8 73.2 2.1 24 3 1.0 27.5 3.2 21 4 1.5 73.9 1.7 12 5 2.0 53.1 2.1 13 6 3.0 25.1 1.0 28 7 4.0 87.4 1.9 85 8 5.0 23.7 1.7 31

[0046] Table 2 shows that in group 4, with MS + IBA 4.0 mg / L, the adventitious root induction rate was 87.4%, the average number of roots was 1.9, and the root length was 85 mm. The optimal state for inducing adventitious roots using rootlets was observed (e.g.,...). Figure 3 (As shown).

[0047] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for directly inducing adventitious roots of kudzu using its leaves and roots, characterized in that, The leaves and root hairs of the kudzu vine are used as explants to induce adventitious roots in a MS medium containing 0.5-5.0 mg / L IBA, 20-30 g / L sucrose, 7 g / L agar and having a pH of 5.5-6.

5.

2. The method of directly inducing adventitious roots of Pueraria montana var. lobata using leaves and root hairs of Pueraria montana var. lobata according to claim 1, wherein the leaves and root hairs of Pueraria montana var. lobata are directly applied to the roots of Pueraria montana var. lobata. Specifically, the method comprises the following steps: S1, seed establishment of aseptic seedlings: 20 g of MS medium powder, 30 g of sucrose and 7 g of agar are weighed out per liter of medium, added into 1 L of water, stirred uniformly, sterilized and used as a seed seedling culture medium; The seeds are washed to remove surface dirt, soaked in sterile water for 4 hours, washed with 75% alcohol for 30 seconds, washed with sterile water for 3 times, soaked in 5.5% sodium hypochlorite solution for 6 minutes, washed with sterile water for 5 times, the surface water is absorbed with sterile filter paper, the culture medium is inserted with a sterile forceps and cultured under the condition of 25±1℃ and 12 hours of light for 30 days to obtain aseptic seedlings of the kudzu vine; S2, preparation of an adventitious root induction medium: 20 g of MS medium powder, 20-30 g of sucrose and 7 g of agar are weighed out per liter of medium, added into 1 L of water, stirred uniformly, 0.5-5.0 mg / L IBA is added, the pH is adjusted to 5.5-6.5, stirred uniformly, sterilized and an adventitious root induction medium is obtained; S3, induction of adventitious roots: the leaves and root hairs of the aseptic seedlings which have been grown for more than 30 days in step S1 are selected, the leaves are cut with a sterile knife and the back of the leaves is tightly attached to the medium obtained in step S2 for culture to induce adventitious roots; the root hairs are cut with a sterile knife and the root hairs are placed in the medium obtained in step S2 for culture to induce adventitious roots.

3. The method of directly inducing adventitious roots of Pueraria montana var. lobata using leaves and root hairs of Pueraria montana var. lobata according to claim 2, characterized in that, In step S2, 20 g of MS medium powder, 30 g of sucrose and 7 g of agar are weighed out per liter of medium, added into 1 L of water, stirred uniformly, 1.5 mg / L IBA or 4.0 mg / L IBA is added, the pH is adjusted to 6.0, stirred uniformly, sterilized and an adventitious root induction medium is obtained.

4. The method of directly inducing adventitious roots of Pueraria montana var. lobata using leaves and root hairs of Pueraria montana var. lobata according to claim 2, wherein the roots are induced in the medium containing 0.5 to 2 mg / L of 2,4-D and 0.5 to 2 mg / L of IAA. In step S3, the culture condition is 20-30℃ and dark culture for 30 days.

5. The method of directly inducing adventitious roots of Pueraria montana var. lobata using leaves and root hairs of Pueraria montana var. lobata according to claim 2, characterized in that, The induction rate of adventitious roots of the leaves is ≥45% and the induction rate of adventitious roots of the root hairs is ≥85%.

6. A medium for directly inducing adventitious roots of Pueraria montana var. lobata (Willd.) Am. using leaves and rootlets of Pueraria montana var. lobata (Willd.) Am., characterized by comprising a plant growth regulator. The medium is a MS medium, additionally containing 0.5-5.0 mg / L IBA, 20-30 g / L sucrose and 7 g / L agar, having a pH of 5.5-6.5 and being cultured under the condition of 20-30℃ and dark culture.

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