CN111657142A

CN111657142A - Method for improving yield of starch of duckweed mutant strain induced by heavy ion radiation

Info

Publication number: CN111657142A
Application number: CN202010753898.5A
Authority: CN
Inventors: 周功克; 刘宇; 孔英珍; 于昌江
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2020-09-15

Abstract

The invention particularly relates to a method for improving the yield of heavy ion radiation induced duckweed mutant strain starch. Duckweed is an ideal plant material for starch production, and in order to further improve the efficiency of duckweed for producing starch, the invention provides a method for improving the yield of the duckweed mutant strain starch by heavy ion radiation mutagenesis, particularly, duckweed callus is subjected to heavy ion radiation mutagenesis, a regeneration plant is obtained by differentiation culture, a high-yield starch plant is screened from the regeneration plant, and certain culture conditions are combined, so that the further improvement of the yield of the starch is realized. In the mutant strain with better effect, the yield of starch is obviously improved compared with that of a control group. As an energy plant, biomass accumulation is important for bioethanol production, and the mass of the mutant is increased by 13.5% compared with that of the wild plant; biomass per unit area increased 74.0% over wild type. The mutant obtained by the method has good character genetic stability and is an ideal engineering strain for biological energy production.

Description

Method for improving yield of starch of duckweed mutant strain induced by heavy ion radiation

Technical Field

The invention belongs to the technical field of biological energy, and particularly relates to a method for improving the yield of duckweed mutant strain starch obtained by heavy ion radiation mutagenesis.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

With the continuous progress of modern society, the demand of human beings for fossil fuels such as coal, oil and natural gas is increasing day by day. The burning of these fossil fuels releases a large amount of harmful gases and solid particles, seriously threatening the health of human beings. The biomass energy is a clean energy with wide prospect, comprising bioethanol, methane, hydrogen and the like, and is considered to be one of effective ways for solving the energy crisis due to the characteristics of being renewable, environment-friendly and the like.

The duckweed is a novel aquatic crop, has the characteristics of high starch content, high growth speed, no competition with grains for people, no competition with land for the grains and the like, and is an ideal plant material for starch production. The duckweed family includes 5 genera: duckweed (Spirodela), duckweed (Landoltia), duckweed (Lemna), Wolffia (Wolffia) and Wolffia (Wolffia), about 38 varieties. The duckweed consists of two parts, namely leaves and roots, wherein the size of the leaves is generally different from 0.4 to 15mm, and the number of the roots is different from a plurality of roots to no roots. The duckweed has strong environmental adaptability, and can grow at the pH of 5-9 and the temperature of 6-33 ℃. Under proper growth conditions, the duckweed can be propagated for one generation within 2-4 days. Under the adverse stress conditions such as low temperature, nutrient starvation, heavy metal poisoning and the like, a large amount of starch is accumulated in the duckweed body, and the photosynthesis index is reduced and the growth is inhibited along with the reduction of the chlorophyll content. The duckweed can efficiently absorb inorganic nutrients such as nitrogen, phosphorus and the like in the water body, and a large amount of duckweed biomass is obtained while the eutrophic sewage is purified. The annual output of the duckweed biomass can reach 80t/hm by utilizing the duckweed pond to treat sewage²The yield of starch can reach 28t/hm²Compared with the prior art, the annual yield of the corn starch is only 5t/hm². Duckweed starch has wide application in bioenergy, agriculture and industrial production. First, using a fermenting handBio-fuels such as bio-ethanol, biogas and hydrogen are produced, so that the dependence on fossil fuels is relieved; secondly, the duckweed starch particles are relatively small, have the particle size distribution of 1-5 mu m, belong to small-particle starch and have irregular shapes, and can be used for foundation make-up, cake fat substitutes and starch capsules of cosmetics; finally, the duckweed starch can also be used for producing bio-based chemicals such as coatings, plastics, rubber and the like. In addition, the content of duckweed flavone is high, and duckweed health care products can be produced or used as traditional Chinese medicine components. The duckweed protein has high content, is rich in amino acids necessary for human bodies, is comparable to whey protein, and can be used for producing animal feed or protein powder eaten by human beings. The duckweed has high starch content and is a biological energy preparation raw material with great development potential, but the problems that the duckweed is high in large-scale culture cost and needs to be timely salvaged and the like restrict the further development of the conversion of the duckweed into the biological energy.

Disclosure of Invention

In view of the problems described in the background art, the invention provides an engineered plant with high starch yield, which is beneficial to realizing higher yield on a limited culture scale. For this purpose, the present invention studies first the high starch mutant based on the heavy ion radiation mutagenesis of duckweed callus and further obtains higher starch yield by adjusting the culture conditions of the mutant. In addition, the high-starch duckweed mutant obtained based on the method has high genetic stability and is not easy to generate back mutation.

Based on the technical effects, the invention provides the following technical scheme:

in a first aspect of the invention, a method for improving starch yield of a duckweed mutant strain subjected to heavy ion radiation mutagenesis is provided, wherein the method comprises the following steps: the duckweed callus is subjected to heavy ion radiation mutagenesis, the survival tissue subjected to radiation mutagenesis is subjected to differentiation culture to form a regeneration plant, the regeneration plant is continuously cultured for several generations, a starch high-yield plant is screened, and the starch high-yield plant is cultured under the condition of nitrogen deficiency.

The duckweed mutant strain obtained by screening in the mode has good genetic stability, the probability of back mutation in a subsequent culture stage is low, and the genetic stability has important significance for obtaining an engineering plant. Through further research, the invention discovers that the accumulation amount of starch can be obviously improved by adopting the nitrogen-deficient culture medium to culture mutation-induced duckweed regeneration plants, and the improvement effect can reach 70-80%.

The beneficial effects of one or more technical schemes are as follows:

1. the method is based on the heavy ion mutagenesis duckweed callus, and screens the duckweed callus from the mutant after differentiation culture by combining with certain culture conditions, and the mutant obtained by the method has high genetic stability and is not easy to generate self-repairing back mutation in the subsequent culture process.

2. Under the illumination condition of the circadian rhythm of 16/8h, the starch content of the high-starch duckweed mutant strain obtained by the method is 2.91 times that of a control at the end of night time point; at the end of day time point, the starch content of the high starch plants was 1.82 times higher than that of the control, and the biomass of the mutant was increased by 13.5% per plant and 74.0% per unit area compared to the wild type. The result shows that the method can prepare the high-yield mutant strain with beneficial characters, and can obtain stable and high-yield engineering plants by matching with proper culture conditions.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows the callus and differentiation culture of duckweed induced by heavy ion irradiation in example 1;

wherein, FIG. 1A is a real object diagram of duckweed callus, and FIG. 1B is a real object diagram of differentiated tissue.

FIG. 2 is a graph showing the results of measurement of circadian starch content of wild type and mutant type in example 1.

FIG. 3 is a graph showing comparative analyses of biomass per unit area and individual plants described in example 1.

FIG. 4 is a comparative analysis of wild type and mutant sub-1 duckweed individuals and biomass per unit area.

FIG. 5 shows the results of culturing different mutants of duckweed in nitrogen-deficient medium as described in example 4.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, the invention provides a method for improving the yield of starch of a duckweed mutant strain by heavy ion radiation mutagenesis, in order to further improve the starch production capability of duckweed.

Preferably, the heavy ion radiation mutagenesis is carbon heavy ion radiation mutagenesis.

Further preferably, the carbon gravity radiation is mutagenized to a low energy carbon ion beam.

In some specific embodiments of the above preferred technical solution, the carbon gravity radiation mutagenesis is implemented by a heavy ion accelerator, and the parameter of the heavy ion radiation is that the mutagenesis energy heavy ion beam current is¹²C⁶⁺The energy was 80 MeV/u. Mixing agentThe dose ranges were set to 10Gy, 25Gy, 50Gy and 75 Gy.

Preferably, the duckweed callus is prepared in the following way: and (3) putting the duckweed fronds in the exponential growth phase into an induction culture medium, and culturing under a dark condition to obtain the callus.

More preferably, the culture time is 10 to 14 weeks.

Further preferably, the induction medium is an MS induction medium; the MS induction culture medium comprises an MS solid culture medium added with a plant growth regulator; the plant growth regulators include, but are not limited to, 2, 4-dichlorophenoxyacetic acid (2,4-D) and N-phenyl-N' -1,2, 3-thiadiazole-5-urea (tdiazuron, TDZ).

In some embodiments of the above technical solution, the MS induction medium is an MS sucrose agar medium added with 2,4-D and TDZ, wherein the mass ratio of 2,4-D to TDZ is 10: 0.8-1.2.

Further preferably, the callus culture temperature is 25 ± 1 ℃.

In some embodiments of the above technical solution, the duckweed fronds are cultured in the induction medium for 6-8 weeks and then replaced with a new induction medium for further culture.

Preferably, the differentiation culture medium is a B5 medium comprising phytohormones including, but not limited to, Kinetin (KT) and indoleacetic acid (IAA).

In some embodiments of the above preferred embodiment, the differentiation medium is B5 sucrose agar medium added with KT and IAA.

In some embodiments of the above preferred technical scheme, the mass ratio of KT to IAA in the differentiation medium is 1: 4-5.

Preferably, the regenerated plant is cultured by using an SH nitrogen-deficient culture medium.

Further preferably, the SH nitrogen-deficiency medium comprises potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, manganese sulfate, boric acid, zinc sulfate, sodium molybdate, potassium iodide, copper sulfate, cobalt chloride, ferrous sulfate, disodium ethylenediaminetetraacetate, inositol, ammonium sulfate, vitamins, nicotinic acid and sucrose.

The research of the invention finds that the starch content of the mutant strain can be effectively improved by culturing the mutant strain obtained after heavy ion radiation induction by adopting a nitrogen-deficient culture medium, and in some embodiments with better effects, the starch content can be improved by 73% by adopting the nitrogen-deficient culture medium to culture the mutant strain for 5 days.

In a second aspect of the invention, the duckweed mutant strain screened by the method for improving the yield of starch of the duckweed mutant strain subjected to heavy ion radiation mutagenesis in the first aspect is provided for application as an engineering plant.

In a third aspect of the present invention, a method for preparing an engineered plant with high starch yield is provided, wherein the method for preparing the engineered plant with high starch yield comprises the steps described in the method for improving starch yield of a duckweed mutant strain mutagenized by heavy ion radiation in the first aspect.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

In this embodiment, a method for increasing starch yield of a duckweed mutant strain subjected to heavy ion radiation mutagenesis is provided, wherein the method comprises the following steps: the duckweed fronds (Lemna aequinoctialis 6002) in the exponential growth phase are paved on an MS induction culture medium, dark culture is carried out for 7 weeks, the culture is transferred to a new MS induction culture medium, and dark culture is continued for 5 weeks to obtain faint yellow spherical callus serving as a radiation mutagenesis starting material.

Putting the duckweed callus under a vertical irradiation terminal device of a heavy ion accelerator, and carrying out carbon heavy ion radiation mutagenesis on the duckweed callus, wherein the mutagenesis energy heavy ion beam is¹²C⁶⁺The energy was 80 MeV/u. The dose ranges were set to 10Gy, 25Gy, 50Gy, and 75 Gy.

And differentiating and culturing the callus subjected to radiation mutagenesis into a regeneration plant, continuously culturing for several generations after obtaining the regeneration plant, and screening the high-starch plant.

In this example, the (Lemna aequinoctialis 6002) radiation-induced culture conditions were as follows:

1. culture conditions and use of culture Medium

Inducing duckweed callus in dark, wherein the temperature between plant cultures is 25 +/-1 ℃, and the used culture medium and hormone formula are as follows: MS +2, 4-D1.0 mg/L + TDZ 0.1mg/L + sucrose 30g/L + agar 8.0 g/L.

Callus differentiation is carried out in a plant culture room, the temperature is 25 +/-1 ℃, the illumination period is 16/8 hours, and the used culture medium and hormone formula are as follows: b5+ KT 1mg/L + IAA 4.5mg/L + sucrose 10g/L + agar 8.0 g/L.

The callus and differentiated tissue are shown in figure 1.

Duckweed culture is carried out in a plant culture room, the temperature is 25 +/-1 ℃, the illumination period is 16/8 hours, and the culture formula of a liquid culture medium is as follows: SH nitrogen-deficient culture medium + sucrose 10g/L,

the SH nitrogen deficiency culture medium comprises the following formula:

TABLE 1 SH Nitrogen-deficient culture medium recipe

The following measurements were made for starch content, individual biomass of regenerated plants:

2. starch content determination

The starch content in duckweed was determined using the method reported by Smith and Zeeman (2006). The specific operation steps are as follows:

1) weighing about 10mg of dried duckweed sample, placing into a 2ml centrifuge tube, adding steel balls, and grinding the duckweed sample using a high throughput tissue grinder (TissueLyser II, Qiagen, Germany);

2) adding 1.5ml 80% ethanol, water bathing at 70 deg.C for 15min, centrifuging at 12000rpm for 5min, and removing supernatant; 3) repeating the previous step once;

4) after the ethanol is volatilized, distilled water is used for fixing the volume to 2.0ml, after the ethanol is uniformly mixed, 0.5ml is taken and added into a new 2ml centrifugal tube, and water bath is carried out for 10min at the temperature of 100 ℃;

5) after cooling, 0.5ml of a mixed enzymatic hydrolysate of 200mM sodium acetate buffer (pH5.8), 1. mu.l of amylolytic enzyme (SigmaA4582) and 1. mu.l of amylotransglucosidase (Sigma A7095) was added, and after mixing, water bath at 37 ℃ was carried out for 4 hours;

6) after the hydrolysis is finished, centrifuging at 12000rpm for 5min, wherein the glucose in the supernatant is the hydrolysate of the duckweed starch;

7) the glucose content in the supernatant is determined by a 3-5-dinitrosalicylic acid (DNS) method, and the method comprises the following specific steps:

[1] adding 200 mul of supernatant into a 2ml centrifuge tube, and then adding 150 mul of DNS solution;

[2] boiling for 5min for color development, quickly transferring to cold water for cooling, and adding distilled water to 2 ml;

[3] the absorbance at 540nm was measured with a spectrophotometer, and the glucose content was calculated from the standard curve. H) Finally, the glucose content was converted to starch content according to the formula (starch content 0.909). And finally dividing the dry weight of the sample to obtain the starch content of the duckweed.

And (3) after starch content is determined, screening to obtain a mutant strain obtained by the induction method, and naming the mutant strain as sub-1.

3. Biomass of single plant and biomass per unit area

Taking 20 vigorous duckweed mutant strains sub-1, sucking water on the surfaces of leaves, weighing by using a one-ten-thousandth balance, and finally calculating the biomass of each plant.

And pouring clear water into a culture dish with the diameter of 6cm, flatly laying the grown duckweed layer by layer, and calculating the biomass in unit area.

As shown in FIG. 2, under the illumination culture condition of 16/8h, the starch content of the high-starch plants is 2.91 times of that of the control at the End of night time point; at the End of day time point, the starch content of the high starch plants was 1.82 times higher than that of the control.

As can be seen from FIG. 3, the mass of the mutant is improved by 13.5% compared with that of the wild type, the mass per unit area is improved by 74.0%, duckweed is a plant growing on the surface of a flat water body, the mutant can accumulate more biomass per unit area, and the characteristics determine that the mutant is expected to become an ideal engineering plant for bioenergy production.

Example 2

In this embodiment, a method for mutagenizing a starch high-yield duckweed mutant strain based on heavy ion radiation is provided, which comprises the following steps: and (3) flatly paving the duckweed fronds in the exponential growth phase on an MS induction culture medium, culturing in the dark for 6 weeks, transferring to a new MS induction culture medium, and continuously culturing in the dark for 6 weeks to obtain a faint yellow spherical callus serving as a radiation mutagenesis starting material.

In this example, the mutant plants were cultured under the following conditions:

1. culture conditions and use of culture Medium

Inducing duckweed callus in dark, wherein the temperature between plant cultures is 25 +/-1 ℃, and the used culture medium and hormone formula are as follows: MS +2, 4-D1.0 mg/L + TDZ 0.12mg/L + sucrose 30g/L + agar 8.0 g/L.

Callus differentiation is carried out in a plant culture room, the temperature is 25 +/-1 ℃, the illumination period is 16/8 hours, and the used culture medium and hormone formula are as follows: b5+ KT 1mg/L + IAA 4mg/L + sucrose 10g/L + agar 8.0 g/L.

Duckweed culture is carried out in a plant culture room, the temperature is 25 +/-1 ℃, the illumination period is 16/8 hours, and the culture formula of a liquid culture medium is as follows: SH nitrogen-deficient medium + sucrose 10g/L from Table 1 in example 1 was used

Example 3

In this embodiment, a method for mutagenizing a starch high-yield duckweed mutant strain based on heavy ion radiation is provided, which comprises the following steps: and (3) flatly paving the duckweed fronds in the exponential growth phase on an MS induction culture medium, culturing in the dark for 7 weeks, transferring to a new MS induction culture medium, and continuously culturing in the dark for 6 weeks to obtain a faint yellow spherical callus serving as a radiation mutagenesis starting material.

In this example, the conditions for inducing and culturing the mutant strain are as follows:

1. culture conditions and use of culture Medium

Inducing duckweed callus in dark, wherein the temperature between plant cultures is 25 +/-1 ℃, and the used culture medium and hormone formula are as follows: MS +2, 4-D1.0 mg/L + TDZ 0.08mg/L + sucrose 30g/L + agar 8.0 g/L.

Callus differentiation is carried out in a plant culture room, the temperature is 25 +/-1 ℃, the illumination period is 16/8 hours, and the used culture medium and hormone formula are as follows: b5+ KT 1mg/L + IAA 5mg/L + sucrose 10g/L + agar 8.0 g/L.

Duckweed culture is carried out in a plant culture room, the temperature is 25 +/-1 ℃, the illumination period is 16/8 hours, and the culture formula of a liquid culture medium is as follows: SH nitrogen-deficient medium + sucrose 10g/L described in Table 1 in example 1 was used.

Example 4

In this example, the culture methods of duckweed mutant strains after heavy ion radiation mutagenesis were compared, and mutant strains FS25-8, FS50-7, and sub-1 were screened under the heavy ion radiation mutagenesis conditions described in example 1, and were divided into a control group and an experimental group, and cultured under the following culture conditions, respectively, to measure the starch content:

control group culture conditions: SH Medium, shown in Table 2

Experimental group culture conditions: SH nitrogen deficiency medium is described in Table 1 in example 1. The SH nitrogen-deficiency culture medium adopts potassium chloride and dipotassium phosphate to replace potassium nitrate and ammonium dihydrogen phosphate in the SH culture medium.

TABLE 2 SH Medium formulation

Composition (I)	mg/L
		Potassium nitrate KNO₃	2500.00
Magnesium sulfate MgSO₄	195.34
		Ammonium dihydrogen phosphate NH₄H₂PO₄	300.00
Calcium chloride dihydrate CaCl₂·2H₂O	200.00
		Manganese sulfate monohydrate MnSO₄·H₂O	10.00
Boric acid H₃BO₃	5.00
		Zinc sulfate heptahydrate ZnSO₄·7(H₂O)	1.00
NaMoO·2H₂O	0.10
		Potassium iodide KI	1.00
Blue vitriod CuSO₄·5H₂O	0.20
		Cobalt chloride hexahydrate CoCl₂·6(H₂O)	0.10
Ferrous sulfate heptahydrate FeSO₄·7H₂O	15.00
		Na₂ EDTA	20.00
Inositol myo-Inositol	1000.00
		Thiamine hydrochloride	5.00
Vitamin Pyridoxine hydrochloride	0.50
		Nicotinic acid niacin	5.00
Sucrose	10000.00

As can be seen from FIG. 4, the starch content of the mutant strain sub-1 was increased by 73% when cultured in SH nitrogen-deficient medium for 5 days, compared to SH medium.

As can be seen from FIG. 5, the starch content of the mutants FS25-8, FS50-7 and sub-1 was further increased compared to that of conventional SH culture medium after the mutants FS25-8, FS50-7 and sub-1 were cultured in nitrogen-deficient medium.

Under the condition of nitrogen-deficiency culture, the starch contents of wild duckweed and mutant strains are rapidly and massively accumulated, and compared with the wild duckweed, the starch content of the mutant strains is also obviously improved.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing starch yield of a duckweed mutant strain mutagenized by heavy ion radiation, the method comprising the steps of: the duckweed callus is subjected to heavy ion radiation mutagenesis, the survival tissue subjected to radiation mutagenesis is subjected to differentiation culture to form a regeneration plant, the regeneration plant is continuously cultured for several generations, a starch high-yield plant is screened, and the starch high-yield plant is cultured under the condition of nitrogen deficiency.

2. The method for increasing starch production in a duckweed mutant by heavy ion radiation mutagenesis of claim 1, wherein the heavy ion radiation is induced into a low energy carbon ion beam.

3. The method for increasing starch production in duckweed mutant strains by heavy ion radiation mutagenesis of claim 2, wherein the carbon gravity radiation mutagenesis is performed by a heavy ion accelerator, and the heavy ion radiation is characterized by a mutagenesis energy heavy ion beam current of¹²C⁶⁺The energy was 80 MeV/u. The dose ranges were set to 10Gy, 25Gy, 50Gy and 75 Gy.

4. The method for increasing starch production in a duckweed mutant by heavy ion radiation mutagenesis of claim 1, wherein the duckweed callus is prepared by: and (3) putting the duckweed fronds in the exponential growth phase into an induction culture medium, and culturing under a dark condition to obtain the callus.

5. The method for increasing starch production of a duckweed mutant strain by heavy ion radiation mutagenesis according to claim 4, wherein the cultivation time is 10-14 weeks;

or, the induction culture medium is an MS induction culture medium; the MS induction culture medium comprises an MS solid culture medium added with a plant growth regulator; such plant growth regulators include, but are not limited to, 2, 4-dichlorophenoxyacetic acid and N-phenyl-N' -1,2, 3-thiadiazole-5-urea;

preferably, the MS induction culture medium is an MS sucrose agar culture medium added with 2,4-D and TDZ, wherein the mass ratio of the 2,4-D to the TDZ is 10: 0.8-1.2.

6. The method for increasing starch production in a duckweed mutant by heavy ion radiation mutagenesis of claim 4, wherein the callus culture temperature is 25 ± 1 ℃;

preferably, the duckweed fronds are placed in an induction culture medium for culturing for 6-8 weeks, and then the induction culture medium is replaced with a new induction culture medium for continuous culture.

7. The method for increasing starch production in a duckweed mutant by heavy ion radiation mutagenesis according to claim 1, wherein the differentiation culture medium is B5 medium comprising plant hormones including but not limited to kinetin and indoleacetic acid;

preferably, the differentiation medium is B5 sucrose agar medium added with KT and IAA.

8. The method for increasing starch production in a duckweed mutant by heavy ionizing radiation mutagenesis according to claim 1, wherein the regenerated plant is cultured in SH nitrogen-deficient medium;

preferably, the SH nitrogen-deficiency culture medium comprises potassium chloride, magnesium sulfate, monopotassium phosphate, calcium chloride, manganese sulfate, boric acid, zinc sulfate, sodium molybdate, potassium iodide, copper sulfate, cobalt chloride, ferrous sulfate, disodium ethylene diamine tetraacetate, inositol, ammonium sulfate, vitamins, nicotinic acid and sucrose.

9. Use of a duckweed mutant selected according to any one of the methods for increasing starch production in a duckweed mutant by heavy ionizing radiation mutagenesis of claim 1 to 8 as an engineered plant.

10. A method for producing an engineered plant with high starch yield, said method comprising the steps of any one of claims 1-8 of said method for increasing starch yield of duckweed mutant strain mutagenized by heavy ionizing radiation.