CN1108373C - Isolated biological synthesis of elliptone - Google Patents

Abstract

The present invention relates to the biotechnology of plant isolated tissue culture to produce useful secondary metabolites. The invention includes callus induction, callus growth culture, differentiation and synthesis of callus and extraction of rotenone, and proposes the medium and culture conditions (such as light intensity and temperature) suitable for in vitro cultured cell growth and rotenone synthesis and accumulation and cultivation cycle. The invention also proposes the application of rotenone biosynthesis precursors in in vitro biosynthesis. The production of rotenone through in vitro culture can avoid the shortcomings of the production method of live cultivation in the field, save arable land, shorten production time, and reduce costs, and can achieve the purpose of sustainable industrial production without being limited by external environmental conditions.

Description

Production method of rotenone in vitro biosynthesis

The invention relates to biotechnology, in particular to the technology of plant isolated tissue culture to produce useful secondary metabolites.

Rotenone is a well-known plant-derived insecticidal active ingredient, which has the characteristics of high efficiency, low toxicity, low resistance and no environmental pollution. It can be used to control hundreds of pests and mosquitoes on fruit trees, vegetables, tea, flowers and food crops Good insecticide for other sanitary pests. At present, the source of rotenone is mainly obtained by cultivating rotenone roots in the field. The yield is limited by region and season, and the content is unstable. The production time is long and the cultivated land is occupied, resulting in low yield and relatively high production cost.

The object of the present invention is to provide a kind of in vitro biosynthetic production method of rotenone, utilize the plant that can produce rotenone, as explants such as leguminous rotenone, edamame etc. to carry out in vitro culture biosynthetic rotenone, overcome current rotenone only Obtained by planting deer vine roots in the field, it takes up arable land, is greatly affected by environmental conditions, and has limited resources.

The present invention includes several procedures of callus induction, callus growth and culture, callus differentiation and synthesis, and extraction of rotenone, and the specific implementation steps are as follows:

1. Callus induction: take the explants of plants that can produce rotenone, such as deer vine and edamame of the leguminous family, cut them into small pieces under aseptic conditions after sterilization, and place them in the There are 6-BA 2.253μg/ml+2, 4-D 2.210μg/ml, agar 0.8%, sucrose 30g/L, and pH value 5.8 on the MS basic medium, when the light is light: dark=12:12 hours, The light intensity is about 2000 lux, the relative humidity is 80±5%, and the temperature is 28±2.5° C. in a culture room for in vitro culture to obtain callus pieces.

2. Callus growth culture: After the above-mentioned callus pieces are cut, they are inserted into the improved MS medium, and the light is light: dark=12:12 hours, and the light intensity is about 4200 lux. The maximum biomass can be obtained by culturing under the conditions of relative humidity 80±5%, temperature 30±2.0°C, and ventilation. The formula of the improved MS medium is: the concentration of trace elements and macroelements is the same as that of MS medium, the concentration of organic matter is twice that of MS medium, sucrose is 50g/L, 0.8% agar, and the combination of hormones is 0.225μg of 6-BA /ml+2, 4-D 6.630 μg/ml, pH value is 6.0.

3. Differentiation and synthesis culture of callus: the callus after the growth culture growth is transferred to the medium for callus differentiation and synthesis, and the light is light: dark=12:12 hours, and the light intensity is about 1100 lux. The relative humidity is 80 ± 5%, the temperature is 30 ± 2.0 ° C, and the conditions of ventilation are cultivated to make the callus differentiate into roots and synthesize rotenone, and then transfer to dark conditions to continue culturing for 5-10 days, so that the in vitro biosynthetic Rotenone accumulation.

The medium formula used for callus differentiation synthesis is the same as the modified MS medium in step 2, but the hormone is changed to 0.1 μg/ml NAA, and the pH value is changed to 6.5. To promote rotenone biosynthesis in vitro, 100-150 μg/ml phenylalanine was added to the medium as a precursor compound for rotenone biosynthesis.

4. Extraction of rotenone: using organic solvent Soxhlet extraction or immersion extraction. A certain amount of organic solvent is used to extract the dried and ground in vitro culture, and the obtained extract is filtered, concentrated, and purified by chromatography to obtain rotenone.

Main equipment used in the present invention has autoclave, aseptic operating table. The content of rotenone biosynthesized in vitro can be analyzed by HPLC.

The use of in vitro culture technology to produce rotenone in a biosynthetic way has unique advantages and significance: the production of rotenone through in vitro culture can avoid the unfavorable factors brought by field cultivation, save arable land, shorten production time, reduce costs, and can be used without Restricted by external environmental conditions, the purpose of sustainable industrial production is achieved.

Example:

Take healthy young leaves of deer vine as explants, dip in 0.1% mercuric chloride solution for 8-10 minutes, after sterilization, cut into small pieces under aseptic conditions, place in 2.253μg/ml+2,4-D 2.210μg/ml, agar 0.8%, sucrose 30g/L, pH value 5.8 on the MS basic medium, when the light is light: dark = 12:12 hours, the light intensity is about 2000 lux, relative humidity 80 ± 5%, and temperature 28 ± 2.5 ° C in a culture room in vitro culture for 25-30 days to obtain loose cells, milky white, well-growing callus pieces.

Take the above-mentioned callus block, cut it into small pieces, and insert it into the improved MS culture medium. The light is light: dark=12:12 hours, the light intensity is about 4200 lux, the relative humidity is 80 ± 5%, and the temperature is 30°C. The maximum biomass can be obtained by culturing at ±2.0°C and aeration for 30 days, and the average dry weight increases by 7.03 times. The formula of the improved MS medium is: the concentration of trace elements and macroelements is consistent with that of MS medium, the concentration of organic matter is twice that of MS medium, sucrose is 50g/L, 0.8% agar, hormone combination is 0.225μg of 6-BA /ml+2, 4-D 6.630μg/ml, pH 6.0.

Transfer the enlarged callus pieces after growth and culture into the medium for callus differentiation and synthesis. The formula: the ingredients are the same as the above-mentioned modified MS medium, but the hormone is changed to 0.1 μg/ml NAA, pH Change the value to 6.5 and add 100-150 μg/ml phenylalanine as a precursor compound for rotenone biosynthesis. The light is light: dark=12:12 hours, the light intensity is about 1100 lux, the relative humidity is 80 ± 5%, the temperature is 30 ± 2.0 ° C, and the condition of ventilation is cultivated for 25 days to make the callus differentiate into roots and synthesize Rotenone, and then transferred to dark conditions to continue culturing for 5-10 days, so that the rotenone biosynthesized in vitro can accumulate.

The obtained in vitro culture was dried and ground, put into a container, added 10 times the amount of analytically pure acetone, and then placed on a shaker for immersion extraction for 48 hours, filtered out the extract, and concentrated to obtain a light brown solid. The extraction rate is 3.96%. The extract was dissolved in methanol, and purified by reverse-phase column chromatography to obtain the rotenone product. As a result of HPLC determination, the yield of rotenone in the in vitro culture reaches 0.56 grams of rotenone per 100 grams of cultured product.

After the callus was transferred to the growth medium, it was a lag phase in the first 5 days, and its growth rate was slow; it was a slow growth phase between the 5th day and the 10th day; it entered the logarithmic growth phase on the 10th day, and the callus The growth rate of the tissue was very fast; after 20 days, the growth rate of the callus began to slow down, and the cells entered the slow growth period. After 30 days, the growth of the callus almost stopped; on the 35th day, the growth of the callus entered the stationary phase. The synthesis and accumulation of rotenone in callus also showed a certain regularity. The callus accumulated rotenone from the beginning of culture. From 5 days to 15 days, the synthesis and accumulation of rotenone in callus showed an increasing trend. The content of rotenone in the callus was the highest at day 1; the content of rotenone showed a decreasing trend after 15 days, and the content of rotenone was almost undetectable at day 35.

The content of product rotenone is determined by HPLC method, instrument name: U.S. Hewlett-Packard 1100 high performance liquid chromatograph (configuration: quaternary gradient pump, ultraviolet-visible light wavelength detector, manual injector, Hewlett-Packard Chemical Workstation); Column: HEWLWTT PACKARD (ODS HYPERSIL, 5μm, 125×4mm) Determination conditions: detection wavelength: λ = 295nm; mobile phase: 75:25 = methanol: water; flow rate: 1ml/min; detection sensitivity: AUFS = 0.001; column temperature: normal temperature; injection volume : 20 μl. The retention time of the standard sample was 5.661 minutes. The rotenone content of the sample can be obtained according to the ratio of the ratio of the absorption peak of the rotenone absorption peak of the standard sample of known concentration to the area of the rotenone absorption peak of the test sample and the ratio of their respective contents.

Tested with acetone extract, it was found that _the biological activity of the rotenone biosynthesized in vitro to the larvae of P. cabbage was equivalent to that obtained from field planting. ml, the 24-hour lethal dose (LD ₅₀ ) is 0.283 μg/head, and the 24-hour effective medium concentration (EC ₅₀ ) for the inhibition of body weight growth is 2.050 μg/ml; the 48-hour EC ₅₀ is 1.170 μg/ml, The egg hatching rate was 14.56%, the adult eclosion rate was 22.22%, and the larva pupation deformity rate was 87.50%. The indoor pot test results show that the rotenone concentration of 40μg/ml sample treatment can well control the damage of cabbage butterfly larvae.

Claims (3)

1. The production method of rotenone biosynthesis in vitro, which is characterized in that the biosynthesis of rotenone in vitro includes callus induction, callus growth culture, differentiation synthesis of callus and extraction of rotenone; (1) Callus induction: take plants capable of producing rotenone, such as the explants of deer vine and bean bean of leguminous family, after sterilization, under aseptic conditions, cut into small pieces, place in Add 6-BA 2.253μg/ml+2, 4-D 2.210μg/ml, agar 0.8%, sucrose 30g/L, pH value 5.8 on the MS basic medium, in the light to light: dark = 12: 12 hours , a callus mass obtained from in vitro culture in a culture room with a light intensity of about 2000 lux, a relative humidity of 80±5%, and a temperature of 28±2.5°C; (2) Callus growth culture: After the callus piece is cut, it is inserted into the MS medium after the improvement, and the light is light: dark=12:12 hours, the light intensity is about 4200 lux, and the relative humidity 80±5%, the temperature is 30±2.0℃, and the maximum biomass can be obtained by culturing under the condition of ventilation; the improved MS medium formula is: the concentration of trace elements and macroelements is consistent with MS medium, and the organic matter is MS medium 2 times the medium concentration, sucrose is 50g/L, 0.8% agar, hormone combination is 6-BA 0.225μg/ml+2, 4-D 6.630μg/ml, pH value is 6.0; (3) Callus differentiation and synthesis culture: the callus pieces will be inserted into the medium for callus differentiation and synthesis, and the light will be light: dark=12:12 hours, the light intensity is about 1100 lux, and the relative humidity 80 ± 5%, temperature 30 ± 2.0 ℃, and cultured under the condition of ventilation, so that the callus can differentiate into roots and synthesize rotenone, and then transfer to the dark condition to continue the culture, so that the rotenone biosynthesized in vitro can be accumulated; used for callus The composition of the culture medium formula for wound tissue differentiation is the same as the modified MS medium of (2), but the hormone is changed to 0.1 μg/ml NAA, and the pH value is changed to 6.5; (4) Extraction of rotenone biosynthesized in vitro: use organic solvent Soxhlet extraction or immersion extraction, use a certain amount of organic solvent to extract the in vitro culture after drying and grinding, and obtain the extract, after Filtration, concentration, and purification by chromatography to obtain rotenone. 2. according to the said rotenone biosynthesis production method in vitro of claim 1, it is characterized in that adding rotenone biosynthesis precursor compound phenylalanine in the culture medium of callus differentiation synthesis culture, and its add-on is 100-150 μ g/ ml. 3. according to the said rotenone biosynthesis production method in vitro of claim 1, it is characterized in that after the callus differentiates into roots, it is transferred to the dark condition to continue culturing for 5-10 days.