Duckweed culture method for inducing large-scale accumulation of starch

Abstract

The invention discloses a duckweed culture method for inducing large-scale accumulation of starch, and belongs to the field of aquatic energy plant culture. The preparation method comprises the following steps: inoculating a water body with duckweed subjected to activation and propagation culture, performing propagation culture for several days under the condition of natural light irradiation, and performing induced culture by using an oligotrophic water body to harvest duckweed thalli. In the method, a nutrient water body of a compound fertilizer is used in an enlarged culture stage, and an oligotrophic water body is used in an induction stage. According to the technical scheme, the production efficiency can be improved while the starch yield of the duckweed dormant bodies is guaranteed, and the method is simple in nutrient water body compounding operation, energy-saving, low in cost and easy to popularize and apply.

Description

Technical Field

[0001] The invention relates to the field of aquatic energy plant cultivation, in particular to a duckweed cultivation method for inducing large-scale accumulation of starch. Background Art

[0002] Duckweed is a higher aquatic plant that grows on calm water surfaces. It mainly reproduces asexually, grows fast, has low requirements for water nutrition, and can absorb heavy metal ions. Spirodendrum multirootum is a special species of duckweed. When the growth environment lacks nutrition or the temperature is too high, it will produce a starch-rich vegetative reproduction body, i.e., a dormant body. The special structure of duckweed allows the fronds to only accumulate organic substances such as starch and protein for its own metabolism, and synthesize less lignin and cellulose, which has great application prospects for starch extraction or fermentation to produce energy substances. In addition, duckweed is rich in flavonoids such as apigenin and luteolin. In the field of traditional Chinese medicine in my country, some duckweed (dried whole grass of Spirodendrum or Green duckweed) is also used as a Chinese medicinal material. In addition, due to the genetic stability of duckweed, the current research direction gradually uses duckweed as a bioreactor for the development of animal vaccines.

[0003] Starch plays a major role in people's daily production and life as the main energy source, and the amount required is very large. Traditional starch sources are mainly grain crops. Compared with them, the main advantages of duckweed starch are fast reproduction speed, higher production efficiency; high nitrogen and phosphorus utilization rate, more environmentally friendly; unlimited culture space, good adaptability, and can be cultured in multiple layers. Although duckweed has a fast reproduction speed and high starch content, its biological mass is small, so the total output of starch production is low, which limits the application of duckweed starch and the development of new starch resources. At present, there are still some problems in the research on duckweed starch, so it is necessary to provide a duckweed culture method that induces a large amount of starch accumulation, so as to increase the biomass of duckweed and the duckweed starch yield, and provide a wider selection of raw materials for new starch resources.

[0004] Duckweed grows fast and requires a large space for cultivation, so it is more suitable for outdoor cultivation. However, in the application process of outdoor duckweed cultivation, there are still some problems in terms of cultivation methods, industrial scale and teaching costs. The initial investment in equipment is relatively large, such as the need to install water systems, nutrient supply systems and other equipment. Cultivators need to master the techniques of duckweed cultivation, including water quality management, nutrient supply and pest control, which is quite difficult. The lack of unified production standards and technical specifications in industrial scale leads to uneven quality of duckweed products among different producers. This also leads to poor economic benefits of duckweed. With high investment and operating costs, the economic return rate is not high, making it difficult for many companies or individuals to bear the risk of large-scale development. At the same time, duckweed also has many factors that affect the yield of dry matter (such as starch) and a large difference in duckweed starch content, which hinders the in-depth study of duckweed starch. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a method for cultivating duckweed that induces a large amount of starch accumulation. The method for cultivating duckweed of the present invention can effectively expand the cultivation of duckweed in an open-air environment, induce duckweed with oligotrophy, and finally harvest duckweed rich in starch, providing a new method for the research of duckweed starch resources and the large-scale cultivation practice of duckweed, which can help better solve the problems of teaching cost, method and industrial scale encountered in the process of outdoor cultivation of duckweed, and more efficiently produce duckweed rich in starch. Specifically, the present invention proposes the following technical solutions:

[0006] A duckweed cultivation method for inducing a large amount of starch accumulation is as follows:

[0007] Step 1, germplasm preservation: Inoculate duckweed in a solid culture medium composed of 4.0-5.0 g / L MS solid culture medium powder, 8-12 g / L sucrose, 8-12 g / L agar, and the rest is deionized water. The light quantum flux density is 80-200 μmol / m 2 / s, continuous cultivation;

[0008] Step 2, sterile culture: inoculate the duckweed obtained in step 1 into sterile culture nutrient water, the sterile culture nutrient water is Hoagland sterile culture solution containing 10-20g / L sucrose, the rest is deionized water, the water pH is 5.0-5.5, and the light quantum flux density is 80-200μmol / m 2 / s, continued culture for 2-7 days;

[0009] Step 3, expansion culture: inoculate the duckweed obtained by the culture in step 3 into the expansion culture nutrient water, the indoor expansion culture nutrient water is a 4-6 times diluted Hoagland solution, the water pH is 5.0-5.5; or the outdoor expansion culture nutrient water is a compound fertilizer nutrient solution, the pH is 5-6, and the culture is continued for 3-5 days; the composition of the compound fertilizer nutrient solution is 19-23 mg / L of potassium dihydrogen phosphate and 6-10 mg / L of urea;

[0010] Step 4, induction stage: the duckweed obtained by culture in step 3 is cultured in deionized water or tap water for 1-8 days, and the duckweed fronds are harvested.

[0011] The indoor culture process is to cultivate duckweed under indoor conditions by controlling factors such as temperature, humidity, light, nutrition and gas to create an environment most suitable for duckweed growth and starch accumulation. The outdoor culture process is to cultivate duckweed using sunlight, climate conditions, natural water sources and easily available nutrients in the outdoor natural environment; at the same time, attention should be paid to selecting a location or water area with sufficient sunlight and relatively open space.

[0012] In one embodiment, in order to reduce the competition growth of algae, the water surface coverage rate of duckweed during the expansion and induction stages needs to reach 45-60%.

[0013] In one embodiment, the indoor environmental temperature of duckweed during the sterile culture, expansion culture and induction stages is controlled at 20-30°C.

[0014] In one embodiment, the lighting condition for outdoor cultivation is sunlight, and the lighting for indoor cultivation is LED light.

[0015] In one embodiment, LED lamps are used for additional lighting of duckweed cultivated outdoors to meet the lighting duration of 12-24 hours.

[0016] In one embodiment, the Hoagland nutrient solution is composed of 1.0-1.3 g / L calcium nitrate tetrahydrate, 1.4-1.6 g / L nitric acid, 0.12-0.15 g / L potassium dihydrogen phosphate, 2-4 mg / L tartaric acid, 4-7 mg / L ferric chloride hexahydrate, 8-10 mg / L ethylenediaminetetraacetic acid, 480-520 mg / L magnesium sulfate heptahydrate, 0.18-0.25 mg / L sodium molybdate dihydrate, 0.10-0.15 mg / L zinc sulfate heptahydrate, 0.05-0.12 mg / L copper sulfate pentahydrate, 3.30-4.00 mg / L manganese sulfate tetrahydrate, and a pH of 4.5-6.0.

[0017] Beneficial Effects

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. The present invention realizes the accumulation of starch in duckweed during the induction culture process. By utilizing the culture conditions, the duckweed fronds rich in starch can be harvested quickly and efficiently.

[0020] 2. The method of the present invention has simple process, low equipment cost, easy operation and easy industrialization.

[0021] 3. The method of the present invention realizes small, medium or large-scale indoor or outdoor cultivation of duckweed, lays a foundation for further research and application of duckweed, and expands the development prospects of duckweed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is duckweed cultivated on a large scale in an outdoor plant factory in Chengdu, Sichuan Province.

[0023] Figure 2 It is duckweed cultivated on a small scale outdoors in Jiaxing City, Zhejiang Province.

[0024] Figure 3 It is duckweed cultivated on a large scale in an indoor environment.

[0025] Figure 4 It is duckweed cultivated on a small scale indoors.

[0026] Figure 5 It is a comparison of starch content in duckweed at different culture stages.

[0027] Figure 6 It is duckweed that has been dyed with starch.

[0028] Figure 7 The growth of duckweed water surface coverage before and after induction.

[0029] Figure 8 Growth of duckweed fresh weight before and after induction.

[0030] Fig. 9 Comparison of starch yield of duckweed by expanded culture and induced culture.

[0031] Fig.10 Comparison of the results of using 1 / 5 Hoagland nutrient solution and compound fertilizer nutrient solution to expand duckweed culture outdoors. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0033] Test method:

[0034] 1. Measurement method of duckweed water surface coverage: photograph the duckweed in the entire container from the same height directly above the water surface, convert the image to 8-bit using ImageJ software, adjust the contrast appropriately, then use the Threshold function to select the duckweed leaves on the water surface, and then use the Measure function to calculate the duckweed water surface coverage.

[0035] 2. Duckweed mass measurement and dry matter yield calculation method: Pick the duckweed out of the water, wipe the surface and root moisture with a kitchen paper towel, and measure the fresh weight of the duckweed; dry the harvested duckweed fronds in a 20-55℃ oven for 8-16 hours, then take them out and measure the dry weight of the duckweed after cooling. The duckweed yield calculation formula is as follows:

[0036] Duckweed dry matter yield = dry weight ÷ (culture container area × coverage rate)

[0037] 3. Measurement of duckweed starch content and calculation method of theoretical starch yield: Crush the dried duckweed fronds with a grinder and pass through a 0.2 mm sieve to obtain duckweed frond powder. Use the Megazyme total starch detection kit to determine the total starch content. The determination method is as described in the instructions. The calculation formula for the theoretical starch yield of duckweed is as follows based on duckweed yield and starch content:

[0038] Theoretical starch yield = duckweed dry matter yield × starch content ÷ cultivation time

[0039] 4. Duckweed starch staining and observation method: Duckweed was randomly sampled at different time points, soaked in 80% (v / v) ethanol solution, heated in a water bath at 40°C for 40-60 minutes to remove the pigment, and washed twice with deionized water. Duckweed leaves were then stained in 5% (v / v) Lugol solution for 2 minutes, and washed with deionized water to remove excess staining solution. Observed under light, the blue area is the area containing starch.

[0040] Example 1

[0041] A duckweed cultivation method for inducing large-scale accumulation of starch comprises the following steps:

[0042] (1) Germplasm selection: The duckweed germplasm used in this study was Spirogyra multiflora ZH0196, which was obtained from the duckweed germplasm resource bank of the Chengdu Institute of Biology, Chinese Academy of Sciences. Duckweed was inoculated in a solid culture medium composed of 4.0-5.0 g / L MS solid culture medium powder, 8-12 g / L sucrose, 8-12 g / L agar, and the rest was deionized water. The light quantum flux density was 80-200 μmol / m2 / s and the culture was continued.

[0043] (2) Aseptic culture stage: Several duckweeds were selected from the culture medium for germplasm preservation and inoculated into aseptic culture nutrient water. The aseptic culture nutrient water was Hoagland sterile culture solution containing 10-20 g / L sucrose and the rest was deionized water. The pH of the water was 5.0-5.5 and the light quantum flux density was 80-200 μmol / m 2 / s, and continue culturing for 2-7 days.

[0044] (3) Expanded culture of duckweed: After the duckweed in the germplasm preservation stage is activated and cultured for 7 days in the sterile culture stage, it is placed in 1 / 5 times Hoagland culture medium for open expansion culture. The culture medium composition is 0.236g / L calcium nitrate tetrahydrate, 0.304g / L potassium nitrate, 0.0272g / L potassium dihydrogen phosphate, 0.6mg / L tartaric acid, 1.08mg / L ferric chloride hexahydrate, 1.8mg / L ethylenediaminetetraacetic acid, 0.1g / L magnesium sulfate heptahydrate, 0.024mg / L sodium molybdate dihydrate, 0.024mg / L zinc sulfate heptahydrate, 0.016mg / L copper sulfate pentahydrate, 0.724mg / L manganese sulfate tetrahydrate; the rest is deionized water, and the water pH is adjusted to 5.0-5.5 with KOH or HCl. In the initial stage of expansion culture, at least 50% of the duckweed is covered by water. The culture environment temperature was 25°C and the light quantum flux density was 80-200 μmol / m 2 / s, 24h full light, continuous cultivation for 3-5d.

[0045] (4) Inducing duckweed starch accumulation indoors: Take the duckweed out of the nutrient solution, wipe off the residual water on the surface, and transfer it to a culture container filled with deionized water. In the initial stage, ensure that the duckweed coverage rate on the water surface is about 50%, and the fresh weight of duckweed is about 2.00g. The induction environment temperature is 25℃, and the light quantum flux density is 80-200μmol / m 2 / s, 24h full light, and induced culture for 2 days.

[0046] (5) Harvesting duckweed: Before harvesting, take photos to calculate the coverage of duckweed; remove the duckweed from the water, wipe off the residual water on the surface, and weigh the fresh weight as soon as possible; then dry it in an oven at 40°C for 12 h, and weigh it until it reaches a constant weight to obtain the dry weight.

[0047] Example 2

[0048] (1) Expanding duckweed culture: duckweed in the germplasm preservation stage was activated by aseptic culture for 7 days, and then placed in 1 / 5 times Hoagland culture medium for open expansion culture. The composition of the culture medium was the same as that in Example 1. In the initial stage of expansion culture, the duckweed was guaranteed to have at least 50% water surface coverage. The culture environment temperature was 25°C, and the light quantum flux density was 80-200 μmol / m 2 / s, 24h full light, continuous cultivation for 3-5d.

[0049] (2) Inducing duckweed starch accumulation indoors: Take the duckweed out of the nutrient solution, wipe off the residual water on the surface, and transfer it to a culture container filled with deionized water. In the initial stage, ensure that the duckweed coverage rate on the water surface is about 50%, and the fresh weight of duckweed is about 2.00g. The induction environment temperature is 25℃, and the light quantum flux density is 80-200μmol / m 2 / s, 24h full light, and induction time is 20d.

[0050] (3) Harvesting duckweed: Before harvesting, take photos to calculate the duckweed coverage; remove the duckweed fronds on the water surface, wipe off the residual water on the surface, and weigh the fresh weight as soon as possible; then dry it in an oven at 40°C for 12 h, and weigh it until it reaches a constant weight to obtain the dry weight.

[0051] Example 3

[0052] (1) Expanded culture of duckweed: After the duckweed in the germplasm preservation stage is activated by aseptic culture for 7 days, it is placed in 1 / 5 times the Hoagland culture medium for open expansion culture. The composition of the culture medium is the same as that in Example 1; or the duckweed is placed in a compound fertilizer nutrient solution for open expansion culture. The culture medium composition is 21.7 mg / L of potassium dihydrogen phosphate, 8.8 mg / L of urea, and the pH is 5.5. In the initial stage of expansion culture, ensure that the duckweed has at least 50% water surface coverage. The culture environment temperature is 25°C, and the light quantum flux density is 80-200μmol / m 2 / s, 24h full light, continuous cultivation for 3-5d.

[0053] (2) Inducing duckweed starch accumulation outdoors: Take the duckweed out of the nutrient solution, wipe off the residual water on the surface, and transfer it to a culture container filled with deionized water. In the initial stage, ensure that the duckweed covers about 50% of the water surface and the fresh weight of the duckweed is about 2.00 g. The induction environment is outdoors, the temperature is 16-23°C, and the sunlight is used. The induction time is 2 days.

[0054] (3) Harvesting duckweed: Before harvesting, take photos to calculate the coverage of duckweed; remove the duckweed from the water, wipe off the residual water on the surface, and weigh the fresh weight as soon as possible; then dry it in an oven at 40°C for 12 h, and weigh it until it reaches a constant weight to obtain the dry weight.

[0055] Duckweed was cultivated in outdoor environments at different locations, such as Figure 1 , Figure 2 As shown: Figure 1 Duckweed was induced in a pool in an outdoor plant factory in Chengdu, Sichuan Province. The pool was 2m wide, 50m long, and 2m deep, with an isolation area in the middle of the pool. Figure 2Duckweed was induced outdoors in Jiaxing City, Zhejiang Province. The size of the culture container was 15cm*10cm, and the induction liquid was 400ml. Duckweed was harvested after 2 days, and the starch content was measured to be 18.45%-32.35%.

[0056] Example 4

[0057] (1) Expanded culture of duckweed: After the duckweed in the germplasm preservation stage is activated by aseptic culture for 7 days, it is placed in 1 / 5 times the Hoagland culture medium for open expansion culture. The composition of the culture medium is the same as that in Example 1; or the duckweed is placed in a compound fertilizer nutrient solution for open expansion culture. The culture medium composition is 21.7 mg / L of potassium dihydrogen phosphate, 8.8 mg / L of urea, and the pH is 5.5. In the initial stage of expansion culture, ensure that the duckweed has at least 50% water surface coverage. The culture environment temperature is 25°C, and the light quantum flux density is 80-200μmol / m 2 / s, 24h full light, continuous cultivation for 3-5d.

[0058] (2) Inducing starch accumulation in duckweed outdoors: The duckweed was removed from the nutrient solution and transferred to an outdoor plant factory culture pool. In the initial stage, the duckweed coverage rate on the water surface was ensured to be at least 70%. The outdoor ambient temperature was 25-35°C, sunlight was used, and the induction time was 20 days.

[0059] (3) Harvesting duckweed: Use a fishing net to scoop the duckweed out of the water, use a spin dryer to spin off the remaining water, place it outdoors in a clean, ventilated place to dry, and further dry it in an oven at 40°C to constant weight.

[0060] The outdoor induced culture environment is similar to Figure 1 , duckweed was induced in a pool at an outdoor plant factory in Chengdu, Sichuan Province. The water depth during the induction stage was 10-15 cm. Duckweed was harvested 28 days later, and the starch content was 6.32%-8.54%.

[0061] Example 5

[0062] (1) Expanded culture of duckweed: duckweed in the germplasm preservation stage was activated by aseptic culture for 7 days, and then placed in 1 / 5 times Hoagland culture medium for open expansion culture. The composition of the culture medium was the same as that in Example 1. The culture environment temperature was 25°C, and the light quantum flux density was 80-200 μmol / m 2 / s, 24h full light, continuous cultivation for 3-5d.

[0063] (2) Inducing duckweed starch accumulation outdoors and indoors: Take the duckweed out of the nutrient solution, wipe off the residual water on the surface, and transfer it to a culture container filled with deionized water. In the initial stage, ensure that the duckweed covers the water surface at about 50% and the fresh weight of the duckweed is about 2.00 g. During the daytime, when the weather is clear, the duckweed induction environment is outdoors, the temperature is 16-23°C, and the sunlight is used; at night and on cloudy days, the induction environment is indoors, the induction environment temperature is 25°C, and the light quantum flux density is 80-200 μmol / m 2 / s, 24h full light, and induction time is 2d.

[0064] (3) Harvesting duckweed: Use a fishing net to scoop the duckweed out of the water, use a spin dryer to spin off the remaining water, place it outdoors in a clean, ventilated place to dry, and further dry it in an oven at 40°C to constant weight.

[0065] Indoor duckweed cultivation environment Figure 3 and Figure 4 The duckweed was harvested 2 days later, and the starch content was 34.60±0.47%.

[0066] Example 6: Duckweed cultured outdoors using compound fertilizer nutrient solution

[0067] (1) After the duckweed in the germplasm preservation stage is activated by aseptic culture for 7 days, it is placed in a compound fertilizer nutrient solution for open expansion culture. The culture solution consists of 21.7 mg / L potassium dihydrogen phosphate and 8.8 mg / L urea, and the pH is 5.5. In the initial stage of expansion, the duckweed is guaranteed to have at least 50% water surface coverage, and the fresh weight of the duckweed is about 2.00 g. The expansion environment is outdoors, the temperature is 16-23°C, and the sunlight in the environment is used.

[0068] (2) After harvesting duckweed, measure coverage and fresh weight yield.

[0069] Comparative Example 1 Duckweed in the germplasm preservation stage:

[0070] Prepare solid MS culture medium, the composition of which is: 4.0-5.0g / L MS solid culture medium powder, 8-12g / L sucrose, 8-12g / L agar, and the rest is deionized water. Put the prepared culture medium into an autoclave and sterilize it at 115°C for 20 minutes. When the temperature of the MS culture medium drops to about 50°C, pour it into the plate while it is hot, with about 25mL of culture medium poured into each plate. After it cools and solidifies in the clean bench, pick healthy sterile duckweed into the plate and seal it with sealing film. The ambient temperature is 25°C, the light-dark cycle is 16:8, and the photon flux density is 80-200μmol / m 2 / s, and the culture was continued for 14 days. The duckweed was dried at 40℃ for 12h, ground into powder, and the starch content was measured by the kit method.

[0071] Comparative Example 2 Duckweed in the sterile culture stage:

[0072] Prepare liquid Hoagland medium, the nutrient solution composition is 1.18g / L calcium nitrate tetrahydrate, 1.52g / L potassium nitrate, 0.136g / L potassium dihydrogen phosphate, 3mg / L tartaric acid, 5.4mg / L ferric chloride hexahydrate, 9mg / L ethylenediaminetetraacetic acid, 0.5g / L magnesium sulfate heptahydrate, 0.12mg / L sodium molybdate dihydrate, 0.12mg / L zinc sulfate heptahydrate, 0.08mg / L copper sulfate pentahydrate, 3.62mg / L manganese sulfate tetrahydrate. Add 1.5% (w / w) sucrose to the culture medium, the rest is deionized water, and adjust the water pH to 5.0-5.5 with KOH or HCl. Put the prepared culture medium into an autoclave and sterilize it at 115°C for 20min. After the culture solution is cooled, sterile duckweed germplasm is inoculated. The culture environment temperature is 25°C and the light quantum flux density is 80-200 μmol / m 2 / s, and continue to culture for 2-7 days. The duckweed was dried at 40℃ for 12 hours, ground into powder, and the starch content was measured by kit method.

[0073] Comparative Example 3 Duckweed in the indoor expansion culture stage:

[0074] Prepare a nutrient culture solution, which is 1 / 5 of the Hoagland culture solution, and the culture solution composition is 0.236 g / L of calcium nitrate tetrahydrate, 0.304 g / L of potassium nitrate, 0.0272 g / L of potassium dihydrogen phosphate, 0.6 mg / L of tartaric acid, 1.08 mg / L of ferric chloride hexahydrate, 1.8 mg / L of ethylenediaminetetraacetic acid, 0.1 g / L of magnesium sulfate heptahydrate, 0.024 mg / L of sodium molybdate dihydrate, 0.024 mg / L of zinc sulfate heptahydrate, 0.016 mg / L of copper sulfate pentahydrate, and 0.724 mg / L of manganese sulfate tetrahydrate; the rest is deionized water, and the pH of the water body is adjusted to 5.0-5.5 with KOH or HCl. Take out the aseptically cultured duckweed from the nutrient solution, and carry out open culture in a container of appropriate size, maintaining at least 50% of the water surface coverage. The culture environment temperature was 25°C and the light quantum flux density was 80-200 μmol / m 2 / s, and the culture was continued for 4 days. The duckweed was dried at 40℃ for 12 hours, ground into powder, and the starch content was measured by the kit method.

[0075] Comparative Example 4 Duckweed grown outdoors using 1 / 5 Hoagland culture solution:

[0076] After the duckweed in the germplasm preservation stage is activated and cultured for 7 days in the sterile culture stage, it is placed in 1 / 5 times the Hoagland culture medium for open expansion culture, and the composition of the culture medium is the same as that of comparative example 3. Open culture is carried out in a container of appropriate size, maintaining at least 50% of the water surface coverage, and the fresh weight of duckweed is about 2.00g. The expansion environment is outdoor, the temperature is 16-23℃, and the sunlight in the environment is used for continuous cultivation for 4 days. After harvesting the duckweed, the water surface coverage and fresh weight yield are measured.

[0077] Figure 5 The following is a comparison chart of duckweed starch content between comparative examples 1, 2, 3 and the embodiment. Figure 5 It can be seen that the starch content is very low in the germplasm culture, sterile culture and expansion culture stages, and the starch content ranges from 9.76% to 11.11%, 4.85% to 5.52% and 1.94% to 4.51% respectively. In the embodiment, the starch content of duckweed after induction can reach 45.03%, which is 4.9-23 times that of the comparative example. This shows that the starch content of duckweed starch can be significantly increased after induction.

[0078] Figure 6 This is a comparison of duckweed starch dyeing in Comparative Example 3 and Example 1. Figure 6 It can be seen that duckweed starch accumulates in large quantities in the leaves in the early stage of induction, indicating that the starch content of duckweed starch can be significantly increased after induction.

[0079] Figure 7 The water surface coverage of duckweed before and after induction in Example 1, Example 3, and Example 5 is used to determine the growth of duckweed. Figure 7 It can be seen that within 2 days of induction, although the nutrition was reduced, duckweed was still growing in the three cases. Moreover, the growth of duckweed cultured outdoors and indoors was close to that of duckweed cultured indoors; at the same time, the growth rate of duckweed cultured outdoors alone was slightly lower than that of the other two cases, but it should be considered that the poor outdoor lighting conditions may have a certain negative impact on the growth of duckweed. The above shows that outdoor cultivation and induction of duckweed is feasible and can maintain the strong growth vitality of duckweed.

[0080] Figure 8 The changes in the fresh weight of duckweed before and after induction in Example 1, Example 3, and Example 5 are shown, which can further quantify the growth of duckweed. Figure 8 It can be seen that within 2 days of induction, the accumulation of duckweed substances increased again. Similar to the water surface coverage, the growth of duckweed in outdoor + indoor culture was close to the fresh weight of duckweed in indoor culture; while the growth rate of duckweed in outdoor culture alone was lower than the other two conditions. This shows that outdoor + supplementary light induction has a better effect.

[0081] Fig. 9The figure is a comparison of the starch yield of duckweed in the expanded culture (Comparative Example 3) and the induced culture (Example 1, Example 3, Example 5). It shows that oligotrophic induction can significantly increase the starch yield of duckweed fronds, whether indoors or outdoors. In comparison, although the outdoor + indoor induction form is not as good as the complete indoor culture, it is also better than the complete outdoor culture.

[0082] Fig.10 The following is a comparison chart of the results of expanding the cultivation of duckweed outdoors using 1 / 5 Hoagland nutrient solution (Comparative Example 4) and compound fertilizer nutrient solution (Example 6). Under outdoor conditions, the effect of expanding the cultivation using the compound fertilizer nutrient solution after 4 days is better, with the water surface coverage reaching 100% and the fresh weight being higher. This may be due to the large changes in factors such as light, temperature, humidity and wind speed under outdoor conditions, which may cause the nutrients in the Hoagland nutrient solution to be quickly consumed or degraded, and unable to continuously provide the nutrients required by the plants. Compound fertilizers may be more adaptable to this dynamic environment and can release nutrients more sustainably. In addition, in outdoor environments, 1 / 5 Hoagland nutrient solution is more susceptible to the influence of microorganisms and algae, which may compete with duckweed for nutrients; while compound fertilizers may inhibit the growth of these organisms to a certain extent, thereby better supporting the growth of duckweed.

[0083] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A duckweed cultivation method for inducing a large amount of starch accumulation, characterized in that: The method is as follows: Step 1, germplasm preservation: Inoculate duckweed in a solid culture medium composed of 4.0-5.0 g / L MS solid culture medium powder, 8-12 g / L sucrose, 8-12 g / L agar, and the rest is deionized water. The light quantum flux density is 80-200 μmol / m 2 / s, continuous cultivation; Step 2, sterile culture: inoculate the duckweed obtained in step 1 into sterile culture nutrient water, the sterile culture nutrient water is Hoagland sterile culture solution containing 10-20g / L sucrose, the rest is deionized water, the water pH is 5.0-5.5, and the light quantum flux density is 80-200μmol / m 2 / s, continued culture for 2-7 days; Step 3, expansion culture: inoculate the duckweed obtained by the culture in step 3 into the expansion culture nutrient water body, the indoor expansion culture nutrient water body is a 4-6 times diluted Hoagland solution, the water pH is 5.0-5.5; the outdoor expansion culture nutrient water body is a compound fertilizer nutrient solution, the pH is 5-6, and the culture is continued for 3-5 days; the composition of the compound fertilizer nutrient solution is 19-23 mg / L of potassium dihydrogen phosphate and 6-10 mg / L of urea; Step 4, induction stage: the duckweed obtained by culture in step 3 is cultured in deionized water or tap water for 1-8 days, and the duckweed fronds are harvested.

2. The duckweed cultivation method for inducing a large amount of starch accumulation according to claim 1, characterized in that: The water surface coverage of duckweed during the expansion and induction stages needs to reach 45-60%.

3. The duckweed cultivation method for inducing a large amount of starch accumulation according to claim 1, characterized in that: The indoor environmental temperature of duckweed during the sterile culture, expansion culture and induction stages was controlled at 20-30℃.

4. The duckweed cultivation method for inducing a large amount of starch accumulation according to claim 1, characterized in that: The lighting conditions for outdoor cultivation are sunlight, and the lighting conditions for indoor cultivation are LED lights.

5. The duckweed cultivation method for inducing a large amount of starch accumulation according to claim 4, characterized in that: Duckweed cultivated outdoors uses LED lights for additional lighting to ensure a lighting duration of 12-24 hours.

6. The duckweed cultivation method for inducing a large amount of starch accumulation according to claim 1, characterized in that: The Hoagland nutrient solution is composed of 1.0-1.3 g / L of calcium nitrate tetrahydrate, 1.4-1.6 g / L of nitric acid, 0.12-0.15 g / L of potassium dihydrogen phosphate, 2-4 mg / L of tartaric acid, 4-7 mg / L of ferric chloride hexahydrate, 8-10 mg / L of ethylenediaminetetraacetic acid, 480-520 mg / L of magnesium sulfate heptahydrate, 0.18-0.25 mg / L of sodium molybdate dihydrate, 0.10-0.15 mg / L of zinc sulfate heptahydrate, 0.05-0.12 mg / L of copper sulfate pentahydrate, 3.30-4.00 mg / L of manganese sulfate tetrahydrate, and has a pH of 4.5-6.0.

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