Method for screening or identifying nitrogen-efficient gleditsia sinensis seedlings and application thereof

Abstract

The invention discloses a method for screening or identifying nitrogen-efficient gleditsia sinensis seedlings and application thereof, and belongs to the technical field of seedling screening and seedling breeding. The method comprises the following steps: culturing seedlings of two months in a low-nitrogen (0.6 mM / L) and normal-nitrogen (12mM / L) environment, measuring growth indexes such as plant height, ground diameter and biomass of the gleditsia sinensis seedlings, performing principal component analysis on the growth indexes, establishing a membership function by selecting the principal component index PC1-PCi with the cumulative contribution rate greater than 80%, and calculating a comprehensive evaluation D value, finally, the seedlings with the D values larger than or equal to 0.5 under low-nitrogen treatment and normal-nitrogen treatment are nitrogen efficient seedlings, and the seedlings with the D values smaller than 0.5 are nitrogen low-efficiency seedlings. By means of the method, the gleditsia sinensis seedlings which can efficiently utilize resources when nitrogen is rich and can keep high growth capacity when nitrogen is insufficient can be screened out, and the method is an important way for reducing nitrogen fertilizer dependence and promoting sustainable artificial forest development in gleditsia sinensis seedling cultivation.

Description

Technical Field

[0001] The invention belongs to the technical field of seedling screening and seedling breeding, and in particular relates to a method for screening or identifying nitrogen-efficient Gleditsia sinensis seedlings and an application thereof in Gleditsia sinensis planting and screening or identifying nitrogen-efficient Gleditsia sinensis varieties / families. Background Art

[0002] Gleditsia sinensis is a tree species that is vigorously developed in Guizhou and even the southwest region. Although it is a leguminous plant, it cannot symbiotically fix nitrogen with rhizobia. The soil in the southwest region generally lacks nitrogen fertilizer, and a large amount of fertilizer is required during the planting process. The large-scale use of nitrogen fertilizer not only increases costs, but also has a negative impact on soil health. Therefore, screening out gleditsia varieties that can grow normally in low-nitrogen soils has become the main goal of gleditsia improvement, and it is necessary to screen out gleditsia varieties that efficiently utilize nitrogen in the seedling stage. It is also necessary to scientifically and rationally quickly evaluate the tolerance of different gleditsia seedlings to low-nitrogen soils. Summary of the invention

[0003] To solve the above-mentioned technical problems, the present invention provides a screening or identification method for nitrogen efficient Gleditsia sinensis seedlings and its application. The present invention first cultivates 2-month-old seedlings under low nitrogen (0.6mM / L) and normal nitrogen (12mM / L) environments, then measures the growth indicators of Gleditsia sinensis seedlings, such as plant height, ground diameter, biomass, etc., then performs principal component analysis on these growth indicators, selects the principal component index PC1-PCi with a cumulative contribution rate greater than 80% to establish a membership function and calculates a comprehensive evaluation D value, and finally determines that the D value under low nitrogen treatment and normal nitrogen treatment is ≥0.5 for nitrogen efficient seedlings, and the D value is <0.5 for nitrogen inefficient seedlings. Using the method in the present invention, it is possible to screen out Gleditsia sinensis seedlings that can efficiently utilize resources when nitrogen is abundant and maintain strong growth ability when nitrogen is deficient, which is an important way to reduce nitrogen fertilizer dependence and promote the development of sustainable artificial forests in the cultivation of Gleditsia sinensis seedlings.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] One of the purposes of the present invention is to provide a method for screening or identifying nitrogen-efficient Gleditsia sinensis seedlings, the screening or identification method comprising the following steps:

[0006] S1. Seedling selection: Select 55-65d old Gleditsia sinensis seedlings;

[0007] S2. Different nitrogen treatments: The seedlings were watered with low nitrogen nutrient solution and normal nitrogen nutrient solution regularly every 4-6 days for 10-15 times; the N supply in the normal nitrogen nutrient solution was 10-14mM / L, and the N supply in the low nitrogen nutrient solution was 0.5-0.7mM / L; the pH value was adjusted to 6-7;

[0008] S3. Growth index determination: After the nitrogen treatment, the growth indexes of the honey locust seedlings were determined, including root dry weight, stem dry weight, leaf dry weight, total dry weight, plant height, ground diameter and chlorophyll SPAD reading;

[0009] S4, calculation of comprehensive evaluation value: performing principal component analysis on the growth index, selecting principal component indexes PC1-PCi with cumulative contribution rates greater than 80%, establishing a membership function and calculating a comprehensive evaluation value D;

[0010] S5. Seedling determination: Seedlings with D values ​​≥ 0.5 under both low nitrogen treatment and normal nitrogen treatment were determined to be nitrogen-efficient seedlings, and seedlings with D values ​​< 0.5 under both low nitrogen treatment and normal nitrogen treatment were determined to be nitrogen-inefficient seedlings.

[0011] Furthermore, 2-month-old Gleditsia sinensis seedlings are selected in S1.

[0012] Furthermore, the N source in the low nitrogen nutrient solution and the normal nitrogen nutrient solution in S2 is provided by Ca(NO3)2·4H2O, and the insufficient Ca is supplemented by CaCl2; other nutrient formulas are: KCl 1.8-2.2mM·L -1 , K2HPO4·3H2O 1.8-2.2mM·L -1 , MgSO4·7H2O 0.8-1.2mM·L -1 , C 10 H 12 FeN2NaO828-32 uM·L -1 , H3BO324-26 uM·L -1 ,MnSO4·H2O18-22uM·L -1 ,CuSO46-8 uM·L -1 , ZnSO4·H2O 28-32uM·L -1 ,Na2MoO44-6 uM·L -1 .

[0013] Furthermore, in S2, the seedlings are watered with low nitrogen nutrient solution and normal nitrogen nutrient solution once every 5 days, respectively, for 12 times.

[0014] Furthermore, the N supply in the normal nitrogen nutrient solution in S2 is 12mM / L, and the N supply in the low nitrogen nutrient solution is 0.6mM / L; other nutrient formulas are: KCl 2mM·L -1 , K2HPO4·3H2O 2mM·L -1 , MgSO4·7H2O 1mM·L -1 , C 10 H 12 FeN2NaO830 uM·L -1, H3BO325 uM·L -1 ,MnSO4·H2O 20uM·L -1 ,CuSO47uM·L -1 , ZnSO4·H2O 30uM·L -1 ,Na2MoO45 uM·L -1 .

[0015] Furthermore, the pH value in S2 is adjusted by NaOH or HCl, specifically, using 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl.

[0016] Furthermore, the total dry weight in S3 = root dry weight + stem dry weight + leaf dry weight.

[0017] Furthermore, the D value in S4 is calculated by the following formula (1) to formula (3):

[0018]

[0019] D = ∑ [U (Xj) × W (j)] (3)

[0020] Among them, U(Xj) represents the membership function value of the j-th comprehensive indicator, Xj represents the value of the j-th comprehensive indicator, Xmax and Xmin represent the maximum and minimum values ​​of the comprehensive indicator respectively; W(j) is the weight of each comprehensive indicator, which represents the proportion of the j-th comprehensive indicator in all comprehensive indicators, and Pj represents the contribution rate of the j-th principal component.

[0021] The second object of the present invention is to provide an application of the screening or identification method in Gleditsia sinensis planting.

[0022] The third object of the present invention is to provide an application of the screening or identification method in the screening or identification of Gleditsia sinensis varieties / families with high nitrogen utilization efficiency.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The method of the present invention proposes to comprehensively evaluate and screen multiple growth indicators of seedlings (such as plant height, ground diameter, biomass, etc.). The comprehensive multi-indicator evaluation can reduce the misjudgment caused by the fluctuation of a single indicator. This can not only more comprehensively reflect the nitrogen utilization efficiency of the seedlings, but also ensure that the selected seedlings show good growth performance and nitrogen utilization ability under a variety of growth conditions. At the same time, the use of principal component analysis and membership function analysis simplifies the screening process and ensures the quality of the comprehensive evaluation.

[0025] 2. The nitrogen-efficient honey locust seedlings comprehensively identified by the method of the present invention under two very different nutritional conditions of low nitrogen and normal nitrogen are a highly forward-looking strategy. This strategy not only takes into account the growth efficiency when nitrogen is sufficient, but also focuses on evaluating the adaptability and growth potential of seedlings under nitrogen-restricted conditions. Through double screening, honey locust seedlings that can efficiently utilize resources when nitrogen is abundant and maintain strong growth ability when nitrogen is deficient can be screened out. The honey locust seedlings bred by this method can reduce the amount of nitrogen fertilizer applied while maintaining the quality of the seedlings in the planting of future honey locust forests, which is beneficial to the sustainable development and environmental protection of honey locust plantations.

[0026] 3. The method of the present invention is simple and easy to implement. It only needs to measure the growth indicators of the plants and does not require destructive sampling. It can realize large-scale seedling evaluation in a short time, is suitable for large-scale seedling screening, and improves the identification and cultivation efficiency of nitrogen-efficient Gleditsia sinensis seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is an overall flow chart of the method in Example 1 of the present invention;

[0028] Figure 2 is the D value of the nitrogen high-efficiency seedlings and the nitrogen low-efficiency seedlings after the normal nitrogen treatment and the low nitrogen treatment in Example 1 of the present invention;

[0029] Figure 3 The growth of seedlings under normal nitrogen treatment in Example 1 of the present invention, wherein C-NN represents a nitrogen-efficient family under normal nitrogen treatment, and L-NN represents a nitrogen-inefficient family under normal nitrogen treatment;

[0030] Figure 4 The growth conditions of seedlings under low nitrogen treatment in Example 1 of the present invention are shown, wherein C-LN represents a nitrogen-efficient family under low nitrogen treatment, and L-LN represents a nitrogen-inefficient family under low nitrogen treatment. DETAILED DESCRIPTION

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The products, equipment, etc. used in the following examples are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0032] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0033] Example 1

[0034] This embodiment provides a method for quickly identifying or screening nitrogen-efficient Gleditsia sinensis seedlings at the seedling stage, and the specific steps are as follows:

[0035] S1. Select 2-month-old honey locust seedlings of 30 test families V1-V30. The honey locust is a plant of the genus Gleditsia sinensis Lam. The 30 test families are conventionally grown, and when the seedlings grow for 2 months, 60 plants are randomly selected from each family for this test treatment.

[0036] S2. Different nitrogen treatments: A randomized block design was used, with 30 test families of Gleditsia sinensis seedlings, 60 plants in each family. Two nitrogen levels were set, with a normal nitrogen supply of 12 mM / L and a low nitrogen supply of 0.6 mM / L. Other nutrient formulas were KCl 2 mM·L -1 , K2HPO4·3H2O 2mM·L -1 , MgSO4·7H2O 1mM·L -1 , C 10 H 12 FeN2NaO830 uM·L -1 , H3BO325uM·L -1 ,MnSO4·H2O 20uM·L -1 ,CuSO47 uM·L -1 , ZnSO4·H2O 30uM·L -1 ,Na2MoO45uM·L -1 , where the N source is provided by Ca(NO3)2·4H2O, and the insufficient Ca is supplemented by CaCl2 (specifically, Low nitrogen nutrient solution formula for :Ca(NO3)2·4H2O 0.3mM·L -1 , KCl 2mM·L -1 , K2HPO4·3H2O 2mM·L -1 , MgSO4·7H2O 1mM·L -1 , C 10 H 12 FeN2NaO830 uM·L -1 , H3BO325 uM·L -1 ,MnSO4·H2O 20uM·L -1 ,CuSO47 uM·L -1 , ZnSO4·H2O 30uM·L -1 ,Na2MoO45 uM·L -1 , 5.7mM·L -1 CaCl2; The formula of normal nitrogen nutrient solution is :Ca(NO3)2·4H2O 6mM·L -1 , KCl 2mM·L -1, K2HPO4·3H2O 2mM·L -1 , MgSO4·7H2O 1mM·L -1 , C 10 H 12 FeN2NaO830 uM·L -1 , H3BO325 uM·L -1 ,MnSO4·H2O 20uM·L -1 ,CuSO47 uM·L -1 , ZnSO4·H2O 30uM·L -1 ,Na2MoO45 uM·L -1 ; Note: Because 1 mol of Ca(NO3)2·4H2O contains 2 mol of N, the amount of Ca(NO3)2·4H2O used in the specific formulas of normal nitrogen nutrient solution and low nitrogen nutrient solution is half of the normal nitrogen supply and low nitrogen supply). Use 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl was used to adjust the pH of the solution to about 6.5. Seedlings were irrigated with 50 ml of nutrient solution every 5 days. Nitrogen treatment started on July 18, 2022 and lasted for 60 days. Each family had three replicates, with 15 seedlings in each replicate. Water was added as needed throughout the culture period to maintain appropriate substrate humidity.

[0037] S3. Growth index determination: After 60 days of cultivation, the growth indexes of Gleditsia sinensis seedlings were determined, including root dry weight, stem dry weight, leaf dry weight, total dry weight, plant height, ground diameter and chlorophyll SPAD reading;

[0038] S4, calculation of comprehensive evaluation value: perform principal component analysis on the growth index, select principal component indexes PC1-PCi with cumulative contribution rate greater than 80% to establish a membership function and calculate the comprehensive evaluation D value, specifically according to the following formula;

[0039]

[0040] D = ∑ [U (Xj) × W (j)] (3)

[0041] Among them, U(Xj) represents the membership function value of the jth comprehensive index, Xj represents the value of the jth comprehensive index, Xmax and Xmin represent the maximum and minimum values ​​of the comprehensive index respectively. The weight W(j) of each comprehensive index represents the proportion of the jth comprehensive index in all comprehensive indexes, and Pj represents the contribution rate of the jth principal component.

[0042] First, U(Xj) and W(j) are obtained according to equations (1) and (2);

[0043] Then the comprehensive evaluation index D is obtained according to formula (3).

[0044] S5. Seedlings with comprehensive evaluation D values ​​≥ 0.5 under low N treatment and normal N treatment were determined to be nitrogen-efficient families, and seedlings with D values ​​< 0.5 were determined to be nitrogen-inefficient families.

[0045] The overall flow chart of the method in this embodiment is as follows Figure 1 The D values ​​and growth indices of nitrogen-efficient seedlings and nitrogen-inefficient seedlings after normal nitrogen treatment and low nitrogen treatment are shown in Tables 1 and 2, respectively. Figure 2 The growth of nitrogen-efficient and nitrogen-inefficient seedlings under normal nitrogen treatment and low nitrogen treatment is shown in Figure 3-Figure 4 shown.

[0046] Table 1 D values ​​and growth indices of nitrogen-efficient and nitrogen-inefficient seedlings under normal nitrogen treatment

[0047]

[0048] Table 2 D values ​​and growth indices of nitrogen-efficient and nitrogen-inefficient seedlings under low nitrogen treatment

[0049]

[0050] Based on the growth indicators of the seedlings after normal nitrogen treatment and low nitrogen treatment, the comprehensive D value was calculated, and finally V3, V5, V7, V9, V10, V11, V13, V18, V22, and V26 were determined to be nitrogen-efficient soapberry seedlings; V1, V4, V6, V8, V12, V16, V17, V19, V21, V23, V24, V25, V27, and V30 were nitrogen-inefficient soapberry seedlings.

[0051] Under normal nitrogen treatment conditions, the biomass, plant height and ground diameter of the nitrogen-efficient Gleditsia sinensis seedlings were higher than those of the nitrogen-inefficient Gleditsia sinensis seedlings, showing their good growth potential. In the low nitrogen environment, despite the limited nitrogen supply, the nitrogen-efficient seedlings can still maintain relatively high biomass, plant height and ground diameter, indicating that they have strong nitrogen utilization efficiency and adaptability to low nitrogen environments.

[0052] Example 2

[0053] S1. Select two seedlings with significant difference in nitrogen efficiency (nitrogen high efficiency V3 and nitrogen low efficiency V24), the soapberry variety is soapberry, Latin name is Gleditsia sinensis Lam.. The two test families are raised by conventional methods, and when the seedlings grow for 6 months, the seedlings are transplanted to the field.

[0054] S2, field soil pH is 5.43, total nitrogen, phosphorus, potassium, organic matter content is 1.29mg / kg, 0.92mg / kg, 24mg / kg, 11.12mg / kg, alkaline nitrogen content is 43.16mg / kg. Uniform weed control management is carried out during the cultivation period.

[0055] S3. Growth index determination: After 5 months of cultivation, the growth indexes of the Gleditsia sinensis seedlings were determined, including plant height, ground diameter, and total dry weight of the seedlings. The results are shown in Table 3.

[0056] Table 3 Seedling growth indicators of field nitrogen high efficiency and nitrogen low efficiency

[0057]

[0058]

[0059] In the field, the biomass, plant height and ground diameter of the nitrogen-efficient Gleditsia sinensis seedlings were higher than those of the nitrogen-inefficient Gleditsia sinensis seedlings, showing their good growth potential, indicating that the screening or identification method of the present invention is indeed effective.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for screening or identifying nitrogen-efficient Gleditsia sinensis seedlings, characterized in that: The following steps are involved: S1. Seedling selection: Select 55-65d old Gleditsia sinensis seedlings; S2. Different nitrogen treatments: The seedlings were watered with low nitrogen nutrient solution and normal nitrogen nutrient solution regularly every 4-6 days for 10-15 times; the N supply in the normal nitrogen nutrient solution was 10-14mM / L, and the N supply in the low nitrogen nutrient solution was 0.5-0.7mM / L; the pH value was adjusted to 6-7; S3. Growth index determination: After the nitrogen treatment, the growth indexes of the honey locust seedlings were determined, including root dry weight, stem dry weight, leaf dry weight, total dry weight, plant height, ground diameter and chlorophyll SPAD reading; S4, calculation of comprehensive evaluation value: performing principal component analysis on the growth index, selecting principal component indexes PC1-PCi with cumulative contribution rates greater than 80%, establishing a membership function and calculating a comprehensive evaluation value D; S5. Seedling determination: Seedlings with D values ​​≥ 0.5 under both low nitrogen treatment and normal nitrogen treatment were determined to be nitrogen-efficient seedlings, and seedlings with D values ​​< 0.5 under both low nitrogen treatment and normal nitrogen treatment were determined to be nitrogen-inefficient seedlings.

2. The screening or identification method according to claim 1, characterized in that In the S1, 2-month-old Gleditsia sinensis seedlings were selected.

3. The screening or identification method according to claim 2, characterized in that The nitrogen source in the low nitrogen nutrient solution and the normal nitrogen nutrient solution in S2 is provided by Ca(NO3)2·4H2O, and the insufficient Ca is supplemented by CaCl2; other nutrient formulas are: KCl 1.8-2.2mM·L -1 , K2HPO4·3H2O 1.8-2.2mM·L -1 , MgSO4·7H2O 0.8-1.2mM·L -1 , C 10 H 12 FeN2NaO828-32uM·L -1 , H3BO324-26 uM·L -1 ,MnSO4·H2O 18-22uM·L -1 ,CuSO46-8 uM·L -1 , ZnSO4·H2O28-32uM·L -1 ,Na2MoO44-6 uM·L -1 .

4. The screening or identification method according to claim 3, characterized in that In the S2, the seedlings were watered with a low nitrogen nutrient solution and a normal nitrogen nutrient solution once every 5 days, respectively, for 12 times.

5. The screening or identification method according to claim 4, characterized in that The N supply in the normal nitrogen nutrient solution in S2 is 12mM / L, and the N supply in the low nitrogen nutrient solution is 0.6mM / L; other nutrient formulas are: KCl 2mM·L -1 , K2HPO4·3H2O 2mM·L -1 , MgSO4·7H2O 1mM·L -1 , C 10 H 12 FeN2NaO830 uM·L -1 , H3BO325uM·L -1 ,MnSO4·H2O 20uM·L -1 ,CuSO47 uM·L -1 , ZnSO4·H2O 30uM·L -1 ,Na2MoO45 uM·L -1 .

6. The screening or identification method according to claim 5, characterized in that The pH value in S2 is adjusted by NaOH or HCl.

7. The screening or identification method according to claim 6, characterized in that The total dry weight in S3 = root dry weight + stem dry weight + leaf dry weight.

8. The screening or identification method according to claim 7, characterized in that The D value in S4 is calculated by the following formula (1) to formula (3): D = ∑ [U (Xj) × W (j)] (3) Among them, U(Xj) represents the membership function value of the j-th comprehensive indicator, Xj represents the value of the j-th comprehensive indicator, Xmax and Xmin represent the maximum and minimum values ​​of the comprehensive indicator respectively; W(j) is the weight of each comprehensive indicator, which represents the proportion of the j-th comprehensive indicator in all comprehensive indicators, and Pj represents the contribution rate of the j-th principal component.

9. Application of the screening or identification method according to any one of claims 1 to 8 in Gleditsia sinensis planting.

10. Use of the screening or identification method according to any one of claims 1 to 8 in screening or identifying varieties / families of Gleditsia sinensis with high nitrogen utilization efficiency.

Images