A method for evaluating unintended effects of gene editing soybean phenotypic traits

Abstract

The application discloses a kind of unexpected effect evaluation method of gene editing soybean phenotype character, belong to the field of biotechnology.The method includes: (1) the preparation and planting of soybean material;(2) statistics soybean germination time;(3) statistics germination rate;(4) statistics each period plant height;(5) record initial flowering time;(6) statistics compound leaf width;(7) statistics main stem node number and internode length;(8) statistics mature period yield.The application can be controlled in room, under outdoor field natural conditions, carry out relevant appearance characteristics etc. major naked-eye visible agronomic traits survey of gene editing soybean, compared with field investigation, with not easy to be influenced by external environment, survey identification period is short, survey method is simple and efficient, intuitive, result is accurate and reliable, with time cost etc. advantage of saving, it has important significance to the unexpected effect evaluation and related research work of gene editing soybean.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for evaluating the unexpected effects of gene-edited soybean phenotypic traits. Background Technology

[0002] Soybeans originated in China and are widely cultivated worldwide, serving as an important economic and oilseed crop. With economic development and rising living standards, my country's demand for soybeans is increasing daily. However, my country's total domestic soybean production is low, relying heavily on imports. In 2023, my country imported a staggering 99.41 million tons of genetically modified soybeans, demonstrating a high degree of dependence on foreign sources. To improve the overall development of my country's soybean planting industry and ensure food security, increasing soybean production capacity through genetic engineering and gene editing technologies, and accelerating the pace of bio-breeding for soybeans, is one of the important means to address the current challenges facing my country's soybean industry.

[0003] Gene editing is an emerging technology that modifies specific target sequences in the genome, enabling efficient and precise editing of gene sequences to improve plant traits. However, while improving target traits through gene editing, it may also lead to changes in important agronomic traits such as morphology due to alterations in the genome sequence and off-target effects, resulting in unintended effects. Summary of the Invention

[0004] This invention, through the cultivation of gene-edited long-juvenile soybeans, discovered changes in agronomic traits such as plant height, compound leaf width, and soybean yield, in addition to altering the growth period. Currently, there is no relevant method to evaluate the unintended effects of gene-edited soybeans. This invention establishes a simple, efficient, and intuitive identification method for the unintended effects of gene-edited soybeans, which is of great significance for evaluating these effects. The evaluation will provide suggestions and references for the application prospects of this new gene-edited soybean material.

[0005] The purpose of this invention is to fill the gap in the evaluation method of unintended effects of gene-edited soybeans, and to establish an evaluation method for unintended effects of gene-edited new varieties of long-juvenile soybeans. The experimental results can provide a reference for the evaluation of unintended effects caused by gene-edited soybeans.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for evaluating the unintended effects of gene-edited soybean phenotypic traits, the method comprising the following steps:

[0008] (1) Preparation and planting of soybean materials: Gene-edited soybeans were set up with corresponding recipient materials as controls; seeds were selected and sown.

[0009] (2) The germination time was recorded after the soybean cotyledons emerged 1-2 cm from the soil layer;

[0010] (3) After the soybean cotyledons have fully expanded, the soybean germination rate is counted.

[0011] (4) The plant height of gene-edited soybeans and recipient materials was investigated at the V3, R1, R3 and R5 stages, respectively;

[0012] (5) When a small flower opens at any node on the main stem, record the flowering time;

[0013] (6) Investigate the width of the compound leaves on the third node from the bottom of the main stem during the R6 period;

[0014] (7) Investigate the number of main stem nodes and internode length during the R7 stage;

[0015] (8) At maturity, the yield of gene-edited soybeans and their control recipient materials was statistically analyzed.

[0016] The sowing method in step (1) is as follows: select seeds for sowing, with a sowing depth of 1.5-4.5cm, a cultivation temperature of 20-30℃, a cultivation relative humidity of 40-60%, and a cultivation light cycle divided into long day period and short day period, with a light intensity of 200-400μM.

[0017] The seed sowing depth is 2-4 cm, the cultivation temperature is 23-27℃, the cultivation relative humidity is 45-55%, the photoperiod is divided into long-day and short-day periods, and the light intensity is 250-350 μM.

[0018] Furthermore, the seed sowing depth is 3 cm.

[0019] Furthermore, the culture temperature is 25°C.

[0020] Furthermore, the relative humidity of the culture is 50%.

[0021] The cultivation light cycle consists of 10-20 hours of light and 4-14 hours of darkness per day during long-day periods, and 10-14 hours of light and 10-14 hours of darkness per day during short-day periods.

[0022] Furthermore, the cultivation light cycle is 15 hours of light and 9 hours of darkness per day during long-day periods, and 12 hours of light and 12 hours of darkness per day during short-day periods.

[0023] Furthermore, the light intensity is 300 μM.

[0024] Furthermore, in step (2), the germination time is recorded after the soybean cotyledons emerge 1 cm from the soil layer.

[0025] Gene-edited soybeans are a novel material for biological breeding. Currently, there is no method for evaluating the unintended effects of gene-edited soybeans. This invention provides an evaluation method for the unintended effects of gene-edited soybeans. Compared with existing technologies, this invention has the following advantages:

[0026] The experimental materials were grown in an artificial climate chamber, where the environment was highly controllable and reproducible, allowing for the simultaneous identification of multiple materials. Compared to post-planting field surveys, field identification is time-consuming and labor-intensive, and planting and growth conditions are easily affected by the environment, leading to inconsistent soybean growth and interfering with the accuracy of the survey results.

[0027] This invention evaluates the unintended effects of gene-edited soybeans by investigating the main agronomic traits during soybean growth and the related phenotypes caused by prolonged juvenile period. The investigation method is simple and efficient, and the results are intuitive, accurate, and reliable. The experimental results have good stability and usability, providing an efficient and stable method for evaluating and studying the unintended effects of gene-edited soybeans. Attached Figure Description

[0028] Figure 1 Example 1 shows the phenotypic survey of gene-edited soybean plants at the initial flowering stage during long juvenile period (where the left figure is the wild-type soybean control group and the right figure is the gene-edited soybean experimental group).

[0029] Figure 2 Example 1 shows the phenotypic survey of long-juvenile gene-edited soybean plants at the grain-filling stage (where the left figure is the wild-type soybean control group and the right figure is the gene-edited soybean experimental group). Detailed Implementation

[0030] To better illustrate the present invention, the following embodiments are provided. Obviously, the described embodiments are merely a part of the present invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are all within the scope of protection of the present invention.

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] Example 1

[0033] Using "Huachun No. 1" soybean as the recipient, and employing the CRISPR-Cas9 gene editing vector, the GmFT gene of soybean was edited through Agrobacterium-mediated genetic transformation, resulting in multiple mutants. Among them, the lines with significantly delayed growth period (long juvenile stage) were selected as materials for the study of the unexpected effects evaluation method of this invention.

[0034] (1) Preparation and planting of soybean materials:

[0035] a) Set up gene-edited soybeans and corresponding recipient materials as controls;

[0036] b) Before sowing, select gene-edited soybeans and corresponding recipient materials, removing shriveled, damaged, and moldy seeds, and selecting plump and round seeds.

[0037] c) Sow the seeds in flowerpots filled with uniform soil (ordinary field soil taken from the field) at a depth of 3 cm. Cultivate the seeds under controlled conditions such as in an artificial climate chamber. Set the cultivation temperature to 25℃, the relative humidity to 50%, and the cultivation light cycle to 15 hours of light and 9 hours of darkness per day for long-day cultivation and 12 hours of light and 12 hours of darkness per day for short-day cultivation. The light intensity is 300 μM.

[0038] (2) The germination time was recorded after the soybean cotyledons emerged 1 cm from the soil layer.

[0039] (3) After the soybean cotyledons have fully expanded, the germination rate of soybeans will be counted.

[0040] (4) The plant height of gene-edited soybeans and recipient materials was investigated at the V3, R1, R3 and R5 stages, respectively.

[0041] (5) When a small flower opens at any node on the main stem of the plant, record the flowering time.

[0042] (6) Investigate the width of the compound leaves on the third node from the bottom of the main stem during the R6 period.

[0043] (7) Investigate the number of main stem nodes during the R7 period.

[0044] (8) At maturity, the yield of gene-edited soybeans and their control recipient materials was statistically analyzed.

[0045] In this experiment, 20 individual plants were set up in each group to measure the germination time, germination rate, plant height, flowering time, compound leaf width, and number of main stem nodes of soybeans in the experimental and control groups. 30 individual plants were set up to count the yield of soybeans in the experimental and control groups.

[0046] The above statistical results were summarized and compiled into tables. The specific statistical results are shown in Table 1 and Table 2 below.

[0047] Table 1. Plant height statistics (cm)

[0048] Material V3 R1 period R3 R5 Receptor control 19.00±0.47a 38.9±1.60a 56.78±0.65a 66.94±0.85a Gene-edited soybeans 18.28±0.70a 37.72±1.04a 60.76±1.13b 81.36±1.09b

[0049] Note: Data in the table are mean ± standard error; letters after the values ​​indicate the significance of differences between treatments at the α = 0.05 level. Differences between treatments with the same lowercase letter are not significant, while differences between treatments with different lowercase letters are significant.

[0050] Table 2 Soybean germination time, germination rate, compound leaf width, and number of main stem nodes

[0051] Material Germination time (d) Germination rate (%) Compound leaf width (cm) Number of nodes on the main stem (nodes) Yield per plant (g) Receptor control 3.4±0.55a 95.2±3.27a 6.04±0.30a 11.2±0.45a 39.3±4.1a Gene-edited soybeans 4.6±0.55b 96.2±1.79b 7.65±0.45b 14.2±0.45b 46.2±3.5b

[0052] Note: Data in the table are mean ± standard error; letters after the values ​​indicate the significance of differences between treatments at the α = 0.05 level. Differences between treatments with the same lowercase letter are not significant, while differences between treatments with different lowercase letters are significant.

[0053] The material used in this invention is gene-edited soybean with a delayed growth period (long juvenile stage soybean). Observations over multiple growing seasons revealed that, except for the delayed growth period, the gene-edited soybean was consistent with our initial expectations. Furthermore, at stages V3 and R1, the plant height of the gene-edited soybean was not significantly different from that of the wild type. However, at stages R3 and R5, the plant height of the gene-edited soybean was significantly higher than that of the wild type. In terms of germination rate, compound leaf width, and number of main stem nodes, the gene-edited soybean was significantly higher than the wild type; and in terms of yield, which is of paramount concern in production, the yield per plant of the gene-edited soybean was significantly higher than that of the wild type. These results demonstrate the significant changes and unexpected effects of gene-edited soybean in agronomic traits. Therefore, the implementation of this project allows for the concentrated evaluation and prediction of relevant materials before the promotion of new varieties, providing important guidance for both production and breeding.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for evaluating the unintended effects of gene-edited soybean phenotypic traits, characterized in that, The evaluation method includes the following steps: (1) Preparation and planting of soybean materials: Gene-edited soybeans were set up and the corresponding recipient materials were used as controls; seeds were selected for sowing, with a sowing depth of 1.5-4.5cm, a culture temperature of 20-30℃, a culture relative humidity of 40-60%, and a culture photoperiod divided into long day period and short day period, with a light intensity of 200-400μM. (2) The germination time was recorded after the soybean cotyledons emerged 1-2 cm from the soil layer; (3) After the soybean cotyledons have fully expanded, the soybean germination rate is counted. (4) The plant height of gene-edited soybeans and recipient materials was investigated at the V3, R1, R3 and R5 stages, respectively; (5) When a small flower opens at any node on the main stem, record the flowering time; (6) Investigate the width of the compound leaves on the third node from the bottom of the main stem during the R6 period; (7) Investigate the number of main stem nodes and internode length during the R7 stage; (8) At maturity, the yield of gene-edited soybeans and their control recipient materials was statistically analyzed.

2. The method for evaluating the unintended effects of gene-edited soybean phenotypic traits as described in claim 1, characterized in that, The seed sowing depth is 2-4 cm, the cultivation temperature is 23-27℃, the cultivation relative humidity is 45-55%, the photoperiod is divided into long-day and short-day periods, and the light intensity is 250-350 μM.

3. The method for evaluating the unexpected effects of gene-edited soybean phenotypic traits as described in claim 2, characterized in that, The seed is sown at a depth of 3 cm.

4. The method for evaluating the unintended effects of gene-edited soybean phenotypic traits as described in claim 2, characterized in that, The culture temperature is 25℃.

5. The method for evaluating the unintended effects of gene-edited soybean phenotypic traits as described in claim 2, characterized in that, The relative humidity for cultivation is 50%.

6. The method for evaluating the unintended effects of gene-edited soybean phenotypic traits as described in claim 2, characterized in that, The cultivation light cycle consists of 10-20 hours of light and 4-14 hours of darkness per day during long-day periods, and 10-14 hours of light and 10-14 hours of darkness per day during short-day periods.

7. The method for evaluating the unexpected effects of gene-edited soybean phenotypic traits as described in claim 6, characterized in that, The cultivation light cycle consists of 15 hours of light and 9 hours of darkness per day during long-day periods, and 12 hours of light and 12 hours of darkness per day during short-day periods.

8. The method for evaluating the unintended effects of gene-edited soybean phenotypic traits as described in claim 2, characterized in that, The light intensity is 300 μM.

9. The method for evaluating the unintended effects of gene-edited soybean phenotypic traits as described in claim 1, characterized in that, In step (2), the germination time is recorded after the soybean cotyledons emerge 1 cm from the soil layer.

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