Effect of acetyl-coa synthetase on the biomass of physcomitrella patens

Abstract

The present application relates to the influence of acetyl-CoA synthetase on the biomass of Physcomitrella patens, and discloses application of acetyl-CoA synthetase in regulating the biomass of Physcomitrella patens, wherein the amino acid sequence of the acetyl-CoA synthetase is shown in sequence 1. The present application constructs acetyl-CoA synthetase overexpression strains, and compared with wild type, the acetyl-CoA synthetase overexpression strains can significantly improve the dry weight of Physcomitrella patens protoplast in BCDA liquid medium, and can improve the dry weight of protoplast in different degrees after adding 10 mg / L potassium acetate in the BCDA liquid medium, especially the PpACS2-1 and PpACS2-3 strains can significantly improve the dry weight of protoplast.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the effect of acetyl-CoA synthase on the biomass of *Moss sphaerocephala*. Background Technology

[0002] With its simple developmental pattern, high homologous recombination rate, and high biocompatibility, *Sphaerocarpus spp.* has become a substrate plant used in biotechnology and synthetic biology, possessing application and commercial value in biopharmaceuticals. Bioreactors for large-scale cultivation of *Sphaerocarpus spp.* have already been established. Currently, to reduce the cost of large-scale production and increase the yield of the target product, methods such as culture medium optimization and growth environment optimization are often used to enhance biomass synthesis, but the effects are not particularly significant.

[0003] Acetyl-CoA plays a crucial role in plant growth and development, serving as a pivotal intermediate metabolite. It enters the tricarboxylic acid cycle to produce energy and is converted into amino acids and carbohydrates via gluconeogenesis. The acetyl-CoA synthase gene directly regulates the amount of acetyl-CoA present. By establishing overexpressing acetyl-CoA plants through transgenic technology, biomass accumulation can be fundamentally altered.

[0004] *Sphaerocarpus spp.* possesses numerous biological advantages and holds broad research prospects and commercial value. Through biotechnology or synthetic biology, *Sphaerocarpus spp.* can be used to produce recombinant drugs, terpenoids, and specific metabolites. Cultivating *Sphaerocarpus spp.* in a standard stirred-tank glass bioreactor can improve production efficiency and reduce production costs. Studies have shown that adding appropriate amounts of acetate to the culture medium can promote algal growth, but the addition of acetate to the *Sphaerocarpus spp.* culture medium has not been reported. Summary of the Invention

[0005] This application aims to establish a highly efficient liquid culture system for *Stylos chinensis* to obtain transgenic strains of *Stylos chinensis* that absorb and utilize acetate more efficiently than wild-type *Stylos chinensis*.

[0006] This invention claims protection for the application of acetyl-CoA synthase in regulating the biomass of *Sphagnum moss*, wherein the amino acid sequence of the acetyl-CoA synthase is as shown in Sequence 1.

[0007] Furthermore, the regulation of *Stylos spp.* biomass is achieved by overexpressing the expression level of acetyl-CoA synthase in *Stylos spp.* to increase the biomass of *Stylos spp.*.

[0008] Furthermore, the method for overexpressing the expression level of acetyl-CoA synthase in *Moss simonii* is to transfer a plasmid containing the acetyl-CoA synthase encoding gene into *Moss simonii*, wherein the acetyl-CoA synthase encoding gene is shown in sequence 2.

[0009] This invention provides a method for regulating the biomass of *Sphaerocarpus septemlobus* by adjusting the expression level of acetyl-CoA synthase in *Sphaerocarpus septemlobus* and / or regulating the acetate content in the culture medium of *Sphaerocarpus septemlobus*.

[0010] Furthermore, the method to regulate the expression level of acetyl-CoA synthase in *Stylos spp.* is to increase the biomass of *Stylos spp.* by overexpressing the expression level of acetyl-CoA synthase in *Stylos spp.*.

[0011] Furthermore, the method for overexpressing the expression level of acetyl-CoA synthase in *Moss simonii* is to transfer a plasmid containing the gene encoding acetyl-CoA synthase into *Moss simonii*.

[0012] Furthermore, the acetate is potassium acetate.

[0013] Furthermore, the amount of potassium acetate added is 10 mg / L.

[0014] Furthermore, the biomass of the *Sphaerophyte simulans* is reflected in its phenotype, maximum photosynthetic size, and protoplasmic dry weight.

[0015] This application aims to determine whether adding acetate to the culture medium of *Symplocos macrantha* can increase its biomass, establish a high-efficiency liquid culture system for *Symplocos macrantha*, obtain transgenic *Symplocos macrantha* strains that absorb and utilize acetate more efficiently than wild-type *Symplocos macrantha*, improve its growth and metabolic rate, and enable it to grow faster in acetate-containing liquid culture medium, thereby increasing the biomass of *Symplocos macrantha*. This provides a more efficient culture method and a more efficient acetate-absorbing strain for the large-scale cultivation of *Symplocos macrantha*. Simultaneously, it can provide a basis and ideas for the improvement of culture media for more algae and bryophytes, and provide a theoretical basis for acetate as an organic carbon source.

[0016] This invention constructs an acetyl-CoA synthase overexpression line, which significantly increases the dry weight of *Phyllostachys edulis* protonemata in BCDA liquid medium compared with the wild type. Furthermore, the addition of 10 mg / L potassium acetate to BCDA liquid medium can increase the dry weight of protonemata to varying degrees, especially significantly increasing the overexpression of PpACS2-1 and PpACS2-3 lines. Attached Figure Description

[0017] Figure 1 This is a structural diagram of the overexpression vector plasmid pTFH15.3.

[0018] Figure 2 For the identification of PpACS overexpression lines, M: 5000 DNA marker.

[0019] Figure 3For the identification of PpACS overexpression lines and wild-type lines, M: 5000 DNA marker.

[0020] Figure 4 The expression levels of the ACS gene in wild-type and PpACS overexpression lines are shown; error bars, ±SD. t-tests indicate significant differences: ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001.

[0021] Figure 5 The phenotypes of overexpressing strains at six weeks of age are: (A) WT; (B) PpACS1-3; (C) PpACS2-1; (D) PpACS2-3; (E) PpACS2-2; (F) PpACS3-3; (G) PpACS4-3.

[0022] Figure 6 The single branches of the overexpressing lines at six weeks gestation, from left to right, are WT, PpACS1-3, PpACS2-1, and PpACS2-3. Scale bar: 1 mm.

[0023] Figure 7 Single branch lengths of wild-type and PpACS-overexpressing lines are shown, with error bars and ±SD. t-tests showed significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0024] Figure 8 Chlorophyll fluorescence parameters were measured for different PpACS overexpressing lines of *Phyllostachys pubescens*. Error bars, ±SD. t-test showed significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0025] Figure 9 Dry weight measurements were taken for wild-type and PpACS-overexpressing lines; error bars, ±SD. t-tests showed significant differences: ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001.

[0026] Figure 10 To observe the growth of wild-type *Sphaerocarpus spp.* on BCD solid medium containing 0 mg / L, 5 mg / L, 10 mg / L, and 20 mg / L potassium acetate for 0, 28, and 49 days. Scale bar: 10 mm.

[0027] Figure 11 To observe the growth of wild-type *Symplocos buergeriana* on BCD solid medium containing 0 mg / L and 10 mg / L potassium acetate for 63 days. Scale bar: 10 mm.

[0028] Figure 12 chlorophyll fluorescence parameters of *Brachys wrasse* were determined; error bars, ±SD. t-test showed significant differences: *, P<0.05; **, P<0.01; ***, P<0.001. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0032] The wild-type *Physcomitrella patens* (WT) and the overexpression vector plasmid pTFH15.3 used in the following examples are both disclosed in the article *PpAKR1A, a Novel Aldo-Keto Reductase from *Physcomitrella Patens*, Plays a Positive Role in Salt Stress*, Lu Chen, 2019; the vector structure diagram is shown below. Figure 1 As shown, *Sphaerocarpus spp.* were cultured as stems and leaves on BCD solid medium and as protonema on BCDA medium. The growth conditions were 23-25℃, 50%-70% humidity, continuous light, a photoperiod of 16 / 8h, and a light intensity of 50 μmol / m². s.

[0033] The competent Escherichia coli cells used in the following examples are Trelief™ 5α Escherichia coli purchased from Beijing Qingke Biotechnology Co., Ltd.

[0034] The commonly used solutions and culture media in the following examples are shown in Table 1 below: Table 1. Commonly used stock solutions for *Sphagnum moss* culture medium.

[0035] All mother liquors were stored at 4°C.

[0036] Table 2. Preparation of BCD solid culture medium for *Moss simulans*

[0037] Table 3. Preparation of BCDA solid culture medium for *Moss simulans*

[0038] After the culture medium solidifies, place a piece of cellophane slightly larger than the culture medium on its surface to facilitate the subsequent transfer of protonema to BCD solid culture medium.

[0039] Example 1: Construction of overexpression lines 1. Carrier Construction The sequence of the Arabidopsis thaliana ACS gene was obtained from the NCBI website and compared with the whole genome of *Sphaerocephalum spp.* to obtain the sequence of the acetate-coagulase synthase gene in *Sphaerocephalum spp.*. The CDS sequence of this gene was obtained (as shown in Sequence 2, and its encoded amino acid sequence is shown in Sequence 1). Primers were designed using Primer 5 software and synthesized by Beijing Qingke Biotechnology Co., Ltd. PCR was performed using Green Taq Mix from Nanjing Novizan Biotechnology Co., Ltd. The target sequence fragment (full length 2052 bp) was ligated into the pTFH15.3 vector and transformed into *E. coli*. Single colonies of *E. coli* were selected for colony PCR, and positive clones were selected for sequencing identification. Plasmids were extracted from colony number 8 with the completely correct colony sequence for subsequent transformation of *Sphaerocephalum spp.*.

[0040] 2. Construction and identification of PpACS overexpression lines of *Phyllostachys pubescens* Plasmid from colony #8 was extracted, linearized by enzyme digestion, and transformed into wild-type protoplasts of *Bryum simonii*. After culture and two rounds of resistance selection, genomic DNA was extracted from the selected positive plants. PCR identification was performed using the primer pair 15.3-TY-S / 15.3-TY-A, with the wild-type as a control. The results are as follows: Figure 2 and Figure 3 As shown, PpACS overexpression lines were obtained.

[0041] To further screen for strains of *Moss simonii* that highly express acetyl-CoA synthase, RNA was extracted from wild-type and seven overexpression-positive lines, and the transcriptional level of the ACS gene was detected by qRT-PCR (real-time quantitative PCR). Results are as follows: Figure 4 As shown, the relative expression levels of the PpACS1-2 and PpACS2-2 lines were slightly lower than those of the wild type, while the relative expression levels of the other lines were higher than those of the wild type. The three overexpression lines (PpACS1-3, PpACS2-1, and PpACS2-3) with the highest expression levels and significant differences compared to the wild type were selected for subsequent experiments.

[0042] Table 4 Primer Information Table (5'-3')

[0043] Example 2: Phenotypic and chlorophyll fluorescence parameter analysis of PpACS overexpression lines of *Phyllostachys pubescens* 1. Phenotypic Analysis The phenotypes of several ACS gene overexpression lines were observed, such as Figure 5 As shown, PpACS2-2 grew more slowly than the wild type. The plants of PpACS1-3, PpACS2-1, and PpACS2-3 were slightly larger than the wild type, while the plants of PpACS3-3 and PpACS4-3 showed no significant difference from the wild type. To verify whether the growth of PpACS1-3, PpACS2-1, and PpACS2-3 was slightly faster than the wild type, the single-branch length of the wild type and these three lines was compared after six weeks. Figure 6 For each strain, 50 individual branches at the base of the *Mammillaria pulcherrima* were selected for measurement and statistical analysis. The results are as follows: Figure 7 As shown, the average length of a single branch in the wild type was 4.36 mm, the average length of a single branch in PpACS1-3 was 4.90 mm, the average length of a single branch in PpACS2-1 was 4.65 mm, and the average length of a single branch in PpACS2-3 was 4.60 mm. There was a significant difference in the length of single branches between the wild type and PpACS1-3, with the PpACS1-3 branch being approximately 12.3% longer than the wild type branch.

[0044] 2. Chlorophyll fluorescence parameter analysis This embodiment measured the chlorophyll fluorescence parameters of the wild type and these three lines during the vigorous growth stage (four weeks), and the results are as follows: Figure 8 As shown, the maximum photosynthetic efficiency (Fv / Fm) of the three lines PpACS1-3, PpACS2-1, and PpACS2-3 was significantly higher than that of the wild type. Among them, the average maximum photosynthetic efficiency of PpACS2-1 was the highest, increasing the maximum photosynthetic efficiency by nearly 8.8%. The actual photosynthetic efficiency of all three lines was higher than that of the wild type, except for PpACS2-1 and PpACS2-3, which showed significant differences from the wild type. Among them, PpACS2-3 had the highest actual photosynthetic efficiency.

[0045] In the above experiment, the chlorophyll fluorescence parameter was measured using a modulated chlorophyll fluorescence imager and PAM technology. Before measurement, the samples were kept in darkness (protected from light for 30 minutes) to ensure that the PSII reaction centers were fully open. The photosynthetically active radiation (PAR) was set to 55 μmol / m² during measurement. The system activates a saturation pulse every 20 seconds. Multiple measurement sites are selected based on experimental requirements. Other settings such as Gain and Meas.Light are adjusted according to the Ft value, and measurements begin immediately. When the experimental sample is protonema, it needs to be centrifuged for 30 minutes before measurement, and a suitable amount of water should be absorbed using filter paper. The protonema should then be evenly spread on a petri dish before the experiment. After measurement, the system automatically displays all values. Select the required data and save the file. Excel and statistical analysis software are used to analyze the chlorophyll fluorescence parameters of the samples to determine whether their photosynthetic efficiency (F0) and maximum photosynthesis (Fv / Fm) are as shown in italics.

[0046] Example 3: Growth of protonema from the overexpression line Protonema stock was prepared by adding 10 mL ddH2O to 2 g of mature protonema. 1 mL of protonema homogenate was added to 50 mL BCDA medium and BCDA liquid medium containing 10 mg / L potassium acetate. After 10 days of culture, the dry weight was measured as follows: Figure 9 As shown, the dry weight of protonemata of the three strains PpACS1-3, PpACS2-1, and PpACS2-3 was higher than that of the wild-type protonemata in different culture media. In particular, in BCDA liquid medium, the dry weight of protonemata of the PpACS overexpression strains was significantly higher than that of the wild-type protonemata. Figure 9 A) The dry weight of PpACS1-3 increased by 27.6% compared to the wild type, PpACS2-1 increased by 80.7% compared to the wild type, and PpACS2-3 increased by 31.7% compared to the wild type. The dry weight of the PpACS overexpression lines in BCDA liquid medium containing 10 mg / L potassium acetate was superior to that of the wild type. Figure 9 (B) The culture conditions were consistent with those in BCDA medium. The dry weight of PpACS2-1 and PpACS2-3 was significantly higher than that of the wild type. The dry weight of PpACS1-3 increased by 23.9% compared to the wild type, PpACS2-1 by 90.1%, and PpACS2-3 by 34.3%. Both the wild type and the PpACS overexpression lines showed better dry weight accumulation in BCDA liquid medium containing 10 mg / L potassium acetate than in BCDA medium. Specifically, the addition of 10 mg / L potassium acetate increased the dry weight of the wild type by 12.4%, PpACS1-3 by 9.1%, PpACS2-1 by a significant 18.2%, and PpACS2-3 by 14.6%.

[0047] The method for weighing the dry weight of the protonema in the above experiment was as follows: The protonema in the conical flask was transferred to a 50 mL centrifuge tube, centrifuged at 7800 rpm for 30 min, the supernatant (liquid culture medium) was discarded, and the protonema at the bottom of the centrifuge tube was collected, placed on filter paper, and pressed to absorb excess moisture. The protonema was then placed in a 2 mL centrifuge tube and dried using a vacuum centrifuge concentrator (Eppendorf Concentrator Plus) at 60°C for 4 h. Finally, it was weighed using an analytical balance.

[0048] This application constructed an overexpression line (PpACS) that overexpresses acetyl-CoA synthase. Phenotypic observation, single-branch length measurement, and chlorophyll fluorescence parameter determination showed that the growth of the three lines (PpACS1-3, PpACS2-1, and PpACS2-3) was slightly faster than that of the wild type. Increased acetyl-CoA synthase expression increases the content of acetyl-CoA in the metabolism of *P. spp.*, thereby increasing the metabolic rate and promoting the growth and development of *P. spp.*. The single-branch length of the three lines (PpACS1-3, PpACS2-1, and PpACS2-3) was longer than that of the wild type, and their maximum and actual photosynthetic efficiencies were also higher, indicating that the growth of the overexpressing acetyl-CoA synthase line was slightly faster than that of the wild type. The protonema biomass of the PpACS overexpression lines increased by 27.6%, 80.7%, and 31.7% compared to the wild type, respectively. In a liquid culture system with 10 mg / L potassium acetate added, the protonema biomass of the wild-type and overexpression lines increased by 12.4%, 9.1%, 18.2%, and 14.6%, respectively.

[0049] Example 2: Application of potassium acetate in promoting the accumulation of dry weight of protonematter Wild-type and overexpression lines were cultured in BCDA liquid medium and BCDA liquid medium containing 10 mg / L potassium acetate, respectively, with other culture conditions unchanged, and the dry weight of protonema was measured.

[0050] The results showed that in BCDA liquid medium containing 10 mg / L potassium acetate, the dry weight accumulation of both wild-type and PpACS overexpression lines was superior to that in BCDA medium. Specifically, the dry weight of wild-type increased by 12.4%, PpACS1-3 by 9.1%, PpACS2-1 by a significant 18.2%, and PpACS2-3 by 14.6%. Consistent with the culture in BCDA medium, the dry weight of PpACS2-1 and PpACS2-3 was significantly higher than that of wild-type. The dry weight of PpACS1-3 increased by 23.9% compared to wild-type, PpACS2-1 by 90.1%, and PpACS2-3 by 34.3%. Example 4: Effect of potassium acetate addition to culture medium on the growth of *Sphaerocarpus septemlobus*. Wild-type *Phyllostachys pubescens* were cultured on BCD medium without potassium acetate, on BCD medium containing 5 mg / L potassium acetate, on BCD medium containing 10 mg / L potassium acetate, on BCD medium containing 20 mg / L potassium acetate, and on BCD medium containing 30 mg / L potassium acetate. After 7 days of culture, no significant difference in the size of *Phyllostachys pubescens* was observed in each dish. After 28 days of culture, no significant difference was found between *Phyllostachys pubescens* cultured on media containing 5 mg / L to 30 mg / L potassium acetate and those cultured without potassium acetate. Potassium acetate concentrations of 5 mg / L to 30 mg / L did not inhibit the growth of *Phyllostachys pubescens*. Figure 10 As shown. After 49 days of culture, it was observed that the lateral stems and leaves of *Mammillaria pulcherrima* on culture medium containing 10 mg / L potassium acetate were slightly longer than those on culture mediums with other methods. At the same time, it was observed that the growth of *Mammillaria pulcherrima* on culture medium containing 30 mg / L potassium acetate was slightly slower than that on culture medium without potassium acetate.

[0051] To further observe whether 10 mg / L potassium acetate could promote the growth of *Phyllostachys nigra*, the culture time was extended to 63 days. This allowed for a more obvious observation that the lateral stems and leaves of *Phyllostachys nigra* on the 10 mg / L potassium acetate medium were slightly longer than those on the medium without potassium acetate, and the leaf color was slightly greener than that on ordinary BCD medium. Figure 11 As shown. To verify whether 10 mg / L potassium acetate could promote the growth of *Sphaerocarpus spp.*, chlorophyll fluorescence parameters of *Sphaerocarpus spp.* grown on potassium acetate medium for four weeks were measured.

[0052] F0 is the fluorescence yield (initial fluorescence yield) when the PSII reaction center is fully open, reflecting the degree of permanent damage to PSII in plant leaves. When the photosynthetic structure of the plant leaf is disrupted, F0 tends to increase. Results are as follows... Figure 12 As shown in Figure A, compared to the acetate-free medium, the F0 value of *Phyllostachys nigra* was significantly decreased in both the 5 mg / L and 10 mg / L acetate media, indicating reduced damage to PSII. However, the F0 value of *Phyllostachys nigra* in the 20 mg / L acetate medium was slightly higher than in the other groups, demonstrating that PSII was damaged in this medium. Fv / Fm represents the maximum photochemical quantum yield, also known as the maximum light energy conversion efficiency of PSII, and also indicates the maximum photosynthetic efficiency of PSII under dark reaction conditions. Figure 12 B shows that the light energy conversion efficiency of *Phyllostachys edulis* in 10 mg / L acetate medium is significantly higher than that in acetate-free medium. Y(II) is the actual photosynthetic efficiency of PSII, representing its effective light energy conversion capacity over a period of time. A higher value indicates a higher PSII light energy conversion rate and stronger PSII activity. Figure 12As shown in Figure C, there was no significant difference in maximum photosynthetic efficiency between *Phyllostachys edulis* grown in 5 mg / L acetate medium and those grown in acetate-free medium. However, the maximum photosynthetic efficiency of *Phyllostachys edulis* grown in 10 mg / L acetate medium was significantly higher than that grown in acetate-free medium, demonstrating that adding 10 mg / L potassium acetate to BCD solid medium can improve the actual photosynthetic efficiency of PSII. ETR reflects the apparent electron transport efficiency under actual light intensity conditions, i.e., electron transport efficiency. Figure 12 As shown in D, there was no significant difference in electron transfer efficiency among the four treatment groups. Adding 10 mg / L potassium acetate to the BCD solid medium could slightly increase electron transfer efficiency by about 4.3%.

[0053] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of acetyl-CoA synthase in regulating the biomass of *Moss sphaerocephala*, characterized in that, The amino acid sequence of the acetyl-CoA synthase is shown in Sequence 1.

2. The application according to claim 1, characterized in that, The regulation of *Styrax simonii* biomass is achieved by overexpressing the level of acetyl-CoA synthase in *Styrax simonii* to increase its biomass.

3. The application according to claim 1, characterized in that, The method for overexpressing the expression level of acetyl-CoA synthase in *Moss simonii* is to transfer a plasmid containing the acetyl-CoA synthase encoding gene into *Moss simonii*, the acetyl-CoA synthase encoding gene being shown in sequence 2.

4. The application according to any one of claims 1-3, characterized in that, The biomass of *Sphaerophyte simulans* is reflected in its phenotype, maximum photosynthetic size, and protoplasmic dry weight.

5. A method for regulating the biomass of *Sphagnum moss*, characterized in that, By regulating the expression level of acetyl-CoA synthase in *Sphagnum moss* and / or controlling the acetate content in the *Sphagnum moss* culture medium.

6. The method according to claim 5, characterized in that, The method to regulate the expression level of acetyl-CoA synthase in *Sphaerocera dorsalis* is to increase the biomass of *Sphaerocera dorsalis* by overexpressing the expression level of acetyl-CoA synthase.

7. The method according to claim 6, characterized in that, The method for overexpressing the expression level of acetyl-CoA synthase in *Moss simonii* is to transfer a plasmid containing the gene encoding acetyl-CoA synthase into *Moss simonii*.

8. The method according to claim 8, characterized in that, The acetate is potassium acetate.

9. The method according to claim 5, characterized in that, The amount of potassium acetate added is 10 mg / L.

10. The method according to any one of claims 5-9, characterized in that, The biomass of *Sphaerophyte simulans* is reflected in its phenotype, maximum photosynthetic size, and protoplasmic dry weight.

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