A hormone treatment method for increasing the content of rotenone in the roots of Derris elliptica

Abstract

The present invention relates to the field of Derris truncatum cultivation technology, and more particularly to a hormone treatment method for improving the rotenone content in Derris truncatum roots. The method comprises the following steps: in a 1-2 year old field-cultivated Derris truncatum cultivation process, using a kinetin solution having a mass concentration of 100-200 mg / L to irrigate the roots of the Derris truncatum plants, with an irrigation amount of 1 L / plant, irrigating the plants twice at an interval of 7 days, and then allowing the plants to grow normally. The Derris truncatum roots are harvested 30 days after irrigating the kinetin solution. The hormone treatment method for improving the rotenone content in the Derris truncatum roots provided by the present invention can significantly increase the rotenone content in the Derris truncatum roots by 9-21% at a relatively low cost.

Description

Technical Field

[0001] The invention relates to the technical field of Derris tangutica cultivation, and in particular to a hormone treatment method for increasing the rotenone content in Derris tangutica roots. Background Art

[0002] Derris elliptica is a plant of the genus Derris in the Leguminosae family. It is found in Asia, including India, Indochina, and the Malay Peninsula, and was later introduced to my country. Its roots are rich in rotenone, an isoflavone that can be used as an insecticide, acaricide, and fish poison. Since the 1950s, Derris elliptica has been widely cultivated in my country as a raw material for the rotenone industry. Currently, the cost of chemically synthesizing rotenone remains high, and Derris elliptica remains the primary source of raw materials for the rotenone industry. The yield and active ingredient content of raw materials are the main constraints on the development of rotenone pesticides. The lack of cultivation techniques and quality grading standards has resulted in low yields and low and unstable rotenone content in cultivated plants, severely hindering the development of the Derris elliptica industry.

[0003] Rotenone accumulation in Derris tricholoma is tissue-specific, with the highest accumulation occurring in its roots. Industrial extraction primarily uses Derris tricholoma roots as raw material. Cultivation experiments with Derris tricholoma have demonstrated that light, nutrient content, and lime application affect rotenone content and root yield (Moreau, 1945). Root size, soil type, and harvest season all influence rotenone content (Ren Mingdao, 1953). Rotenone content varies significantly across harvest periods (Pan Yanlin et al., 2023). Grafting has no significant effect on rotenone content in Derris tricholoma roots, suggesting that the primary controlling factor for rotenone content lies in the roots, not in the stems and leaves (Jones et al., 1946). Research into the regulatory mechanisms of rotenone metabolism and the development of methods to increase rotenone content will significantly promote the development of the rotenone industry and bring significant social and economic benefits.

[0004] Plant hormones are naturally occurring small signaling molecules that play important roles in many aspects of plant physiology, metabolism, and defense. Exogenous hormones of varying concentrations have varying degrees of influence on various plant tissues, regulating endogenous hormone levels and, in turn, modulating plant growth and development. Numerous reports have been published on the use of exogenous hormones or fertilizers formulated with hormone additives to promote the accumulation of plant secondary metabolites (Zhao Yue et al., 2012; Yu Jiangshan et al., 2021; Liang Hao et al., 2013). However, there are very few reports on the use of exogenous hormones to increase the production of the secondary metabolite rotenone. For example, Xu Hanhong et al. (2010) used naphthaleneacetic acid (NAA) to treat stem segments of Derris tricolor, significantly increasing their rotenone content by 10–20 times. However, there have been no reports on successfully increasing the rotenone content in Derris tricolor roots, the primary raw material for rotenone extraction, thus creating an urgent need to address this gap. Summary of the Invention

[0005] The object of the present invention is to provide a hormone treatment method for increasing the rotenone content in the roots of Derris ternata to address the above-mentioned problems, which can significantly increase the rotenone content in the roots of Derris ternata.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A hormone treatment method for increasing the rotenone content in the roots of Derris truncatum is provided. During the cultivation process of Derris truncatum, a rotenone solution is used to irrigate the roots of the Derris truncatum plants, and then the plants are normally managed and grown.

[0008] Furthermore, the mass concentration of the kinetin solution is 100-200 mg / L, and the irrigation amount is 1 L / plant.

[0009] Furthermore, the number of times and time intervals for watering the roots of the Derris ternata plant with the kinetin solution were: a total of 2 waterings, with an interval of 7 days.

[0010] Furthermore, the roots of the Herba Lycopodii were harvested 30 days after watering with the kinetin solution.

[0011] Furthermore, the Derris ternata is a 1-2 year old field-cultivated Derris ternata.

[0012] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0013] The present invention treats Derris truncatum plants of different ages and growth conditions by selecting hormone types and concentrations, identifying a hormone treatment method that significantly increases the rotenone content in the roots. The hormone treatment method provided by the present invention for increasing the rotenone content in Derris truncatum roots can significantly increase the rotenone content by 9-21% at a low cost. Increasing the active ingredient content in Derris truncatum roots can reduce extraction costs for enterprises and improve economic benefits. The present invention has important reference and application significance in the fields of agricultural biotechnology and plant growth regulator application technology. DETAILED DESCRIPTION

[0014] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0015] The hormone treatment method of the present application for increasing the rotenone content in the roots of Derris ternata mainly involves irrigating the roots of 1-2 year-old Derris ternata plants with a kinetin solution with a mass concentration of 100-200 mg / L during the cultivation process of the plants in the field, with an irrigation volume of 1 L / plant, for a total of 2 irrigations with an interval of 7 days, after which the plants are normally maintained for growth, and the roots of Derris ternata are harvested 30 days after the irrigating with the kinetin solution.

[0016] The plant materials used in the following examples are all from the same source of Derris ternata, and are propagated by branch cuttings. The field cultivation site and management conditions are consistent; the cultivation soil and management of the potted materials are consistent with those of the field, but the lighting conditions are different from those of the field, and they are placed on the edge of the field with trees for shade.

[0017] Example 1

[0018] Two-year-old Derris volvacea cultivated in the field from the same batch were selected. Kinetin, salicylic acid, naphthaleneacetic acid (NAA), abscisic acid (ABA), ethephon [(2-chloroethyl)phosphonicacid], and methyl jasmonate (MeJA) were used as exogenous hormones. The mother solutions of the hormones were prepared according to the instructions of the hormones, and then the mother solutions of each hormone were diluted into three concentrations with clean water.

[0019] Derris truncatula was divided into several experimental groups and one control group, with 6-8 Derris truncatula plants in each group. The experimental groups were watered along the roots of the plants using a measuring cup, with a diluted hormone solution (1 L per plant, 7 days per treatment, for a total of two waterings); the control group was watered along the roots of the plants with clean water (1 L per plant, 7 days per treatment, for a total of two waterings). From the first watering until the 30th day, Derris truncatula roots were harvested, and multiple plants in each group were combined into a single sample for rotenone determination.

[0020] The specific steps of the rotenone determination experiment are as follows:

[0021] The harvested roots of Derris tricuspidata were rinsed with clean water, then cut and dried in a 70°C oven for 10 h. The powder was then ground in a sample grinder and passed through a 60-mesh sieve. Weigh 1.00 g of dry powder and accurately add 50 ml of methanol. The mixture was soaked for 12 h, refluxed in an 80°C water bath for 1 h, cooled, topped up with methanol, shaken, and filtered through a 0.45 μm filter. 10 μL of the filtrate was injected and analyzed using an Agilent 1200 high-performance liquid chromatograph (Agilent Technologies, USA). The rotenone content (g / g dry weight) was calculated based on the peak area of the rotenone standard and samples at 299 nm. The mean of the data from three technical replicates was analyzed, and the variation in values was calculated.

[0022] The results of rotenone determination are shown in Table 1.

[0023] Table 1 Changes in rotenone content in the roots of Derris pubescens treated with different hormones and concentrations

[0024]

[0025]

[0026] As can be seen from the results in Table 1, Kinetin and ABA irrigation treatment of Derris ternata has a great effect on the accumulation of rotenone in the roots of Derris ternata. Among them, 100 mg / L Kinetin and 10 mg / LA ABA irrigation treatment of Derris ternata increased its rotenone content by 21.44% and 18.47%, respectively. Although the concentration of Kinetin is greater than that of ABA, the price of Kinetin is much lower than that of ABA. Therefore, the present invention chooses to use Kinetin to irrigate Derris ternata, which can not only significantly increase the content of rotenone in the roots of Derris ternata, but also save costs.

[0027] Example 2

[0028] Two-year-old Derris truncatula plants from the same field cultivation batch were treated with kinetin at concentrations of 0 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L. The plants were divided into four experimental groups, each containing 6-8 Derris truncatula plants. Kinetin solutions of varying concentrations were irrigated along the root surface using a measuring cup, at a rate of 1 L per plant, every 7 days, for a total of two treatments. From the first irrigating period until the 30th day, roots of the Derris truncatula plants were harvested, and multiple plants from each group were combined into a single sample for rotenone determination.

[0029] The specific steps of the rotenone determination experiment are consistent with those in Example 1. The rotenone determination results are shown in Table 2.

[0030] Table 2 Changes in rotenone content in the roots of Derris pubescens treated with different concentrations of Kinetin

[0031]

[0032] The results are expressed as mean ± standard deviation, and the superscripts are t-test results: d indicates P < 0.0001, and ns indicates P ≥ 0.05, indicating no statistical significance.

[0033] The results in Table 2 show that among the three experimental groups, the rotenone content in the roots of Derris truncatum was increased the most after being irrigated with 100 mg / L Kinetin, reaching 18.64%; while the rotenone content in the roots of Derris truncatum was increased by only 16.43% after being irrigated with 200 mg / L Kinetin.

[0034] Example 3

[0035] The same batch of one-year-old Derris truncatula cultivated in the field was selected, and the exogenous hormones were 100 mg / L kinetin, 10 mg / L abscisic acid ABA, and a mixed solution of 100 mg / L kinetin and 10 mg / L abscisic acid ABA respectively;

[0036] Derris truncatula was divided into three experimental groups and one control group, with 6-8 plants in each group. The experimental groups were irrigated along the roots of the plants using a measuring cup, with an exogenous hormone solution (1 L per plant, 7 days apart) for two irrigations. The control group was irrigated along the roots of the plants with clean water (1 L per plant, 7 days apart) for two irrigations. From the first irrigation until the 30th day, roots of Derris truncatula were harvested and pooled into a single sample for rotenone determination. Three biological replicates were performed for each experimental and control group.

[0037] The specific steps of the rotenone determination experiment were consistent with those in Example 1. The experimental results of three biological replicates were similar. The specific results are shown in Table 3.

[0038] Table 3 Changes in rotenone content in the roots of one-year-old field-grown Derris chinensis treated with hormones

[0039]

[0040] The results are expressed as mean ± standard deviation, and the superscripts are t-test results: d indicates P < 0.0001, and ns indicates P ≥ 0.05, indicating no statistical significance.

[0041] From the results in Table 3, it can be seen that when one-year-old Derris chinensis was used as the treatment material, only the rotenone content in the roots of Derris chinensis treated with 100 mg / L Kinetin increased significantly, with an increase of 17.00%.

[0042] Example 4

[0043] The hormone treatment method for increasing the rotenone content in the roots of Derris ternata in this embodiment is the same as that in Example 3, with the only difference being that the Derris ternata used in this embodiment is a two-year-old Derris ternata cultivated in the field.

[0044] The specific steps of the rotenone determination experiment were consistent with those in Example 1, and the experimental results of biological replicates were similar. The specific results are shown in Table 4.

[0045] Table 4 Changes in rotenone content in the roots of two-year-old Derris dasyphylla treated with hormone irrigation

[0046]

[0047] The results are expressed as mean ± standard deviation, and the superscripts are t-test results: d indicates P < 0.0001, and ns indicates P ≥ 0.05, indicating no statistical significance.

[0048] The results in Table 4 show that when two-year-old field-cultivated Derris volvacea was used as the treatment material, the rotenone content was significantly increased after treatment with 100 mg / L Kinetin or 10 mg / LABA, with increases of 9.34% and 10.64%, respectively. The difference between the two increases was not significant, but the cost of 100 mg / L Kinetin was much lower, so 100 mg / L Kinetin was preferred for irrigation treatment.

[0049] Example 5

[0050] The hormone treatment method for increasing the rotenone content in the roots of Derris ternata in this embodiment is the same as that in Example 3, with the only difference being that the Derris ternata used in this embodiment is a 2-year-old potted Derris ternata cultivated in the shade.

[0051] The specific steps of the rotenone determination experiment were consistent with those in Example 1, and the experimental results of biological replicates were similar. The specific results are shown in Table 5.

[0052] Table 5 Changes in rotenone content in the roots of two-year-old potted Derris odorata treated with hormone irrigation

[0053]

[0054] The results are expressed as mean ± standard deviation, and the superscripts are t-test results: c indicates P < 0.001, d indicates P < 0.0001, and there is no statistical significance.

[0055] The results showed that when two-year-old potted Derris odorata was used as the treatment material, the rotenone content increased after Kinetin, ABA and Kinetin+ABA treatment, with the increases being 6.34%, 4.16% and 8.99% respectively. There are problems of small increase or increased cost.

[0056] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A hormone treatment method for increasing the rotenone content in the roots of Derris ternata, characterized in that: During the cultivation of Derris truncatula, the roots of the Derris truncatula plants were irrigated with a kinetin solution, and then the plants were grown normally. The mass concentration of the kinetin solution was 100-200 mg / L, and the irrigation amount was 1 L / plant. The number of times and time intervals for irrigating the roots of the Derris ternata plants with the kinetin solution were as follows: 2 times in total, with an interval of 7 days; and the roots of the Derris ternata were harvested 30 days after the start of irrigating the kinetin solution.

2. The processing method according to claim 1, characterized in that The Derris ternata is a 1-2 year old Derris ternata cultivated in the field.

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