Rape plant salt tolerance evaluation method and application thereof

Abstract

The invention provides a rape plant salt tolerance evaluation method and application thereof, and belongs to the technical field of rape breeding, the rape plant salt tolerance evaluation method comprises the following steps: carrying out seedling culture on seeds of salt-tolerant rape A and salt-sensitive rape B to obtain salt-tolerant rape seedlings A and sensitive rape seedlings B; the method comprises the following steps: grafting a salt-tolerant rape seedling A and a sensitive rape seedling B as a stock and a scion to obtain a B scion / A stock grafted seedling and an A scion / B stock grafted seedling; salt stress culture is conducted on the B scion / A rootstock grafted seedlings and the A scion / B rootstock grafted seedlings, the salt tolerance is measured, and if the salt tolerance of the B scion / A rootstock grafted seedlings is superior to that of the A scion / B rootstock grafted seedlings, the underground part makes great contribution to the salt tolerance; otherwise, the above-ground part contributes to the salt tolerance greatly. Through the bidirectional grafting design, the salt tolerance contribution of the overground part and the underground part of the salt-tolerant rape A can be conveniently and quickly evaluated in combination with the salt tolerance data, and the salt tolerance quality breeding efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of rapeseed breeding, and particularly relates to a method for evaluating the salt tolerance of rapeseed plants and its application. Background Art

[0002] Salt stress is one of the main abiotic stresses restricting crop production. Currently, research on crop salt tolerance mainly focuses on the root salt excretion mechanism (such as wheat and cotton) or the shoot ion compartmentalization ability (such as halophytes). For example, in horticultural crops, salt-tolerant rootstocks (such as pumpkin) are often grafted with scions (such as cucumber) to improve the salt tolerance of the scion through the salt excretion characteristics of the rootstock roots. However, Brassica napus has significant differences from other crops due to its unique salt tolerance mechanism (sodium ions can be stored in the shoot, while the root system has the ability to actively excrete sodium).

[0003] Existing research mostly evaluates the salt tolerance of plants as a whole. Traditional salt tolerance evaluation methods have the following limitations: 1. Holistic limitation: Existing technologies (such as physiological index determination and gene expression analysis) can only reflect the overall salt tolerance of plants and cannot distinguish the functional differences between the shoot (ion storage) and the root (ion excretion); 2. Lack of rootstock-scion interaction: Existing grafting research mostly focuses on the salt tolerance improvement effect of the rootstock on the scion (such as pumpkin-cucumber), but does not analyze the independent functions of different parts through reciprocal grafting of salt-tolerant / sensitive materials; 3. Insufficient control design: The salt tolerance difference between self-rooted seedlings and grafted seedlings is easily interfered by the grafting operation itself (such as stress response during the healing period). Existing methods lack self-grafting controls to exclude technical interference. Traditional methods regard plants as a whole (such as measuring the whole-plant biomass and total ion content), do not separately analyze the physiological responses of the shoot and the root, and cannot quantify the role of organ division of labor. If it is not clear which organ dominates the salt tolerance during breeding, it is difficult to optimize traits targeted, resulting in low breeding efficiency and restricting the breeding efficiency of salt-tolerant varieties. Summary of the Invention

[0004] In view of the technical problems existing in the background art, the present application provides a method for evaluating the salt tolerance of rapeseed plants and its application, aiming to solve the technical problems that the existing technology cannot evaluate the salt tolerance contributions of the shoot and the root of rapeseed plants and the breeding efficiency of salt-tolerant rapeseed varieties is low.

[0005] In a first aspect, an embodiment of the present application provides a method for evaluating the salt tolerance of rapeseed plants, including the following steps: S1. Raise seedlings from the seeds of salt-tolerant rapeseed A and salt-sensitive rapeseed B to obtain salt-tolerant rapeseed seedlings A and sensitive rapeseed seedlings B; S2. Graft the salt-tolerant rapeseed seedlings A and the sensitive rapeseed seedlings B as rootstocks and scions to each other to obtain B-scion / A-rootstock grafted seedlings and A-scion / B-rootstock grafted seedlings; S3. Salt stress culture is carried out on the B scion / A rootstock grafted seedlings and the A scion / B rootstock grafted seedlings, and the salt tolerance is measured. According to the salt tolerance data, the salt tolerance contributions of the above-ground part and the underground part of the salt-tolerant rapeseed A are evaluated. The evaluation criteria are as follows: If the salt tolerance of the B scion / A rootstock grafted seedlings is better than that of the A scion / B rootstock grafted seedlings, the underground part contributes more to the salt tolerance; otherwise, the above-ground part contributes more to the salt tolerance.

[0006] In the technical solution of the embodiment of the present application, through the two-way grafting design of the B scion / A rootstock grafted seedlings and the A scion / B rootstock grafted seedlings, the functional differences between the above-ground part (scion, ion storage) and the underground part (rootstock, ion excretion) in the same rapeseed can be distinguished, and the independent functions of different parts can be analyzed. Through the two-way grafting design of the present application, combined with the salt tolerance data, the salt tolerance contributions of the above-ground part and the underground part of the salt-tolerant rapeseed A can be conveniently and quickly evaluated, improving the efficiency of salt tolerance quality breeding.

[0007] In some embodiments, the salt tolerance is characterized by the dry weight reduction rate. The smaller the dry weight reduction rate, the stronger the salt tolerance. The calculation method of the dry weight reduction rate D is as follows: D = 100%×(W ck -W salt ) / W ck ; where W ck is the dry weight of the rapeseed seedlings after salt stress culture, and W ck is the dry weight of the rapeseed seedlings after culture in clear water.

[0008] In the technical solution of the embodiment of the present application, the dry weight reduction rate can be used to accurately quantify the growth adaptability under salt stress and is used to distinguish the contributions of the above-ground part and the underground part to the salt tolerance.

[0009] In some embodiments, the salt-tolerant rapeseed A and the sensitive rapeseed seedlings B are self-grafted to obtain self-grafted seedlings A and self-grafted seedlings B; If the salt tolerance of the B scion / A rootstock grafted seedlings and the A scion / B rootstock grafted seedlings is greater than that of the self-grafted seedlings B and less than that of the self-grafted seedlings A, it indicates that the data is normal; otherwise, it is excluded as abnormal data.

[0010] In the technical solution of the embodiment of the present application, the grafting operation will cause certain interference to the salt tolerance evaluation. Therefore, the present application also provides self-grafted seedlings A and self-grafted seedlings B. The self-grafted seedlings are obtained by grafting the rapeseed seedlings themselves as the rootstock and the scion, which can avoid the influence of the grafting operation on the salt tolerance evaluation.

[0011] In some embodiments, the seedling raising steps include: disinfecting the seeds with sodium hypochlorite, soaking the seeds, germinating them, and then sowing them, and cultivating the seedlings to the two-leaf and one-heart stage for grafting.

[0012] In some embodiments, the grafting step includes: removing the true leaves of the rootstock, longitudinally cutting the stem of the rootstock to obtain a split, retaining two true leaves on the scion, cutting the base of the scion into a wedge shape, inserting the base of the scion into the split of the rootstock and fixing it to obtain a cleft graft seedling; Placing the cleft graft seedling in an environment with a humidity of 70% - 95%, a light intensity of 85 μmol / m 2 ·s - 100 μmol / m 2 ·s, and a temperature of 20 - 28 °C for static culture for 6 - 7 d to obtain a grafted seedling.

[0013] In some embodiments, the depth of the split is 8 - 10 mm.

[0014] In the technical solution of the embodiment of the present application, regarding rapeseed grafting, in the prior art, grafting is mostly carried out during the growth period when the stem is thicker at the bolting stage or flowering stage. Since the hypocotyl of rapeseed soon after emergence is slender, it is difficult to perform grafting operations, and the survival rate is relatively low after grafting using the traditional plug grafting method. In the present application, at the two-leaf and one-heart stage, the cleft grafting method is used for grafting, and the grafting survival rate is relatively high.

[0015] In some embodiments, the salt stress culture step includes: culturing the grafted seedling to the four-leaf stage, and applying 3 L of salt solution to the grafted seedling every 7 d for culture, and continuously culturing for 14 d.

[0016] In some embodiments, the salt solution is an NaCl solution, and the concentration of the NaCl solution is 150 mmol / L.

[0017] In some embodiments, the fresh water culture step includes: culturing the grafted seedling to the four-leaf stage, and applying 3 L of fresh water to the grafted seedling every 7 d for culture, and continuously culturing for 14 d.

[0018] In a second aspect, the embodiment of the present application provides the application of the rapeseed plant salt tolerance evaluation method in the field of rapeseed breeding.

[0019] In the technical solution of the embodiment of the present application, in the field of rapeseed breeding, through the rapeseed plant salt tolerance evaluation method of the present application, the following methods can be used for breeding: (1) preferentially select plants with strong above-ground Na + storage capacity or high root Na + efflux efficiency as salt-tolerant parents for subsequent cross-breeding; (2) combining the differences in the salt tolerance contributions of the above-ground and underground parts, localize molecular markers related to ion transport genes (such as SOS1, NHX family) or stress response genes to accelerate marker-assisted selection of salt tolerance traits; (3) formulate a differential breeding strategy according to the proportion of salt tolerance contribution: for "above-ground dominant" varieties, focus on breeding the leaf Na + compartmentalization ability; for "root dominant" varieties, strengthen the root salt excretion-related traits.

[0020] Distinct from the existing technical solutions, the beneficial effects of this application include: 1. Through the two-way grafting design of B scion / A rootstock grafted seedlings and A scion / B rootstock grafted seedlings, the functional differences between the above-ground part (scion, ion storage) and the underground part (rootstock, ion excretion) in the same kind of rapeseed can be distinguished, and the independent functions of different parts can be analyzed. The grafting operation will cause certain interference to the salt tolerance evaluation. Therefore, this application also provides self-grafted seedling B, which is obtained by grafting the sensitive rapeseed seedling B itself as both the rootstock and the scion, and can avoid the influence of the grafting operation on the salt tolerance evaluation. By using the two-way grafting design and self-grafting as a control, combined with the salt tolerance data, it is convenient and fast to evaluate the salt tolerance contributions of the above-ground part and the underground part of the salt-tolerant rapeseed A, and the efficiency of salt tolerance quality breeding is improved.

[0021] 2. In the prior art, grafting is mostly carried out during the growth period when the stem is thicker at the bolting stage or flowering stage. Since the hypocotyl of rapeseed is slender soon after emergence, it is difficult to perform the grafting operation, and the survival rate is low after grafting using the traditional plug grafting method. In this application, at the two-leaf and one-heart stage, the split grafting method is used for grafting, and the grafting survival rate is relatively high.

[0022] The above description is only an overview of the technical solution of this application. In order to be able to understand the technical means of this application more clearly, it can be implemented according to the content of the specification. And in order to make the above and other purposes, features and advantages of this application more obvious and understandable, the specific embodiments of this application are specifically given below. Brief Description of the Drawings

[0023] In order to more clearly illustrate the technical solution of this application, the drawings used in this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a graph showing the detection results of the dry weight decrease rate in the embodiment of this application. Detailed Description of the Embodiments

[0025] The embodiments of the technical solution of this application will be described in detail below with reference to the drawings. The following embodiments are only used to more clearly illustrate the technical solution of this application, so they are only examples and cannot be used to limit the protection scope of this application.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion.

[0027] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in connection with the embodiments can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0028] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain this application and should not be construed as a limitation of this application. For those embodiments where specific techniques or conditions are not indicated, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0029] Embodiment Select the seeds of salt-tolerant rapeseed C71 and salt-sensitive rapeseed C272. The seeds are disinfected with 0.5% sodium hypochlorite, soaked at 25°C for 12 hours, and germinated with wet gauze until the seeds show white. Sow them in a 32-hole plug tray (substrate pH 6.0, EC 0.8 mS / cm) under greenhouse conditions (day / night temperature 25°C / 18°C, light intensity 300 μmol·m -2 ·s -1 ), and cultivate the seedlings until the two-leaf and one-heart stage to obtain salt-tolerant rapeseed seedlings C71 and salt-sensitive rapeseed seedlings C272 for grafting.

[0030] Adopt the split grafting method: Remove the true leaves of the rootstock, make a longitudinal cut of 8 mm on the stem; retain two true leaves on the scion, cut the base into a wedge shape (8 mm), insert it into the split of the rootstock, and fix it with a grafting clip.

[0031] Management during the healing period: Place the grafted seedlings in an environment with a humidity of 85%, weak light (50 μmol·m -2 ·s -1 ), and a constant temperature of 25°C for 7 days, and gradually restore to normal growth conditions. Repeat 3 times, with 3 pots in each repeat, and retain 16 plants in each pot after grafting.

[0032] Using salt-tolerant rapeseed seedling C71 as the rootstock and salt-sensitive rapeseed seedling C272 as the scion, the C272 / C71 grafted seedling was obtained; using salt-tolerant rapeseed seedling C71 as the scion and salt-sensitive rapeseed seedling C272 as the rootstock, the C71 / C272 grafted seedling was obtained; using sensitive rapeseed C272 itself as the rootstock and scion, the C272 / C272 self-grafted seedling was obtained; using salt-tolerant rapeseed C71 itself as the rootstock and scion, the C71 / C71 self-grafted seedling was obtained.

[0033] The salt-tolerant rapeseed seedling C71 and the salt-sensitive rapeseed seedling C272 without grafting treatment were cultured under the same conditions as the grafted seedlings to obtain C272 rapeseed seedlings and C71 rapeseed seedlings.

[0034] Salt stress culture: The above grafted seedlings and the non-grafted C272 rapeseed seedlings and C71 rapeseed seedlings were irrigated with 150 mmol / L NaCl solution, and 3 L of NaCl solution was irrigated every 7 days, and cultured under salt stress conditions for 14 days.

[0035] Fresh water culture: The above grafted seedlings and the non-grafted C272 rapeseed seedlings and C71 rapeseed seedlings were irrigated with fresh water, and 3 L of fresh water was irrigated every 7 days, and cultured under salt stress conditions for 14 days.

[0036] Evaluation of salt tolerance of rapeseed plants: The salt tolerance of C272 / C71 grafted seedlings, C71 / C272 grafted seedlings, C71 / C71 self-grafted seedlings, C272 / C272 self-grafted seedlings, and non-grafted C272 rapeseed seedlings and C71 rapeseed seedlings was detected. The salt tolerance was characterized by the dry weight decrease rate. The smaller the dry weight decrease rate, the stronger the salt tolerance. The calculation method of the dry weight decrease rate D is as follows: D = 100%×(W ck -W salt ) / W ck ; where W ck is the dry weight of the rapeseed seedling after salt stress culture, and W ck is the dry weight of the rapeseed seedling after fresh water culture.

[0037] The test results are as follows Figure 1 , from Figure 1 it can be seen that the total dry weight of the non-grafted C71 rapeseed seedlings is larger, indicating that it is a salt-tolerant rapeseed seedling, and the total dry weight of the C71 rapeseed seedlings is smaller, indicating that it is a salt-sensitive rapeseed seedling. Under salt stress, the dry weight decrease rates of the C272 / C71 grafted seedlings and the C71 / C272 grafted seedlings are similar, both higher than those of the salt-tolerant C71 / C71 self-grafted seedlings and lower than those of the salt-sensitive C272 / C272 self-grafted seedlings. Both the above-ground and underground parts of rapeseed C71 play a role under salt stress, and they are equally important in terms of contribution rate.

[0038] It should be noted that this application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and embodiments with the same composition and the same effect as the technical idea within the scope of the technical solution of this application are included in the technical scope of this application. In addition, within the scope of not departing from the gist of this application, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways constructed by combining some of the constituent elements in the embodiments are also included in the scope of this application.

Claims

1. A method for evaluating salt tolerance of rapeseed plants, characterized in that: The steps include: S1. Raising seeds of salt-tolerant rapeseed A and salt-sensitive rapeseed B to obtain salt-tolerant rapeseed seedlings A and salt-sensitive rapeseed seedlings B; S2, grafting the salt-tolerant rapeseed seedlings A and the sensitive rapeseed seedlings B as rootstocks and scions to obtain B scion / A rootstock grafted seedlings and A scion / B rootstock grafted seedlings; S3. The B scion / A stock grafted seedlings and the A scion / B stock grafted seedlings are cultured in clear water and under salt stress, and the salt tolerance is measured. The salt tolerance contribution of the aboveground and underground parts of the salt-tolerant rapeseed A is evaluated based on the salt tolerance data. The evaluation criteria are as follows: If the salt tolerance of the B scion / A stock grafted seedling is better than that of the A scion / B stock grafted seedling, the underground part contributes more to the salt tolerance; otherwise, the aboveground part contributes more to the salt tolerance.

2. The method for evaluating salt tolerance of rapeseed plants according to claim 1, characterized in that: The salt tolerance is characterized by the dry weight reduction rate. The smaller the dry weight reduction rate, the stronger the salt tolerance. The calculation method of the dry weight reduction rate D is as follows: D=100%×(W ck -W salt ) / W ck ; Among them, W ck is the dry weight of rapeseed seedlings after salt stress culture, W ck It is the dry weight of rapeseed seedlings after culture in clean water.

3. The method for evaluating salt tolerance of rapeseed plants according to claim 1, characterized in that: The salt-tolerant rapeseed A and the sensitive rapeseed seedling B are self-grafted to obtain self-grafted seedlings A and self-grafted seedlings B; If the salt tolerance of the B scion / A rootstock grafted seedlings and the A scion / B rootstock grafted seedlings is greater than that of the self-grafted seedlings B and less than that of the self-grafted seedlings A, it means that the data is normal; otherwise, it is eliminated as abnormal data.

4. The method for evaluating salt tolerance of rapeseed plants according to claim 1, characterized in that: The seedling raising step comprises: sterilizing the seeds with sodium hypochlorite, soaking the seeds, germinating and then sowing, and cultivating the seedlings to the two-leaf and one-heart stage for grafting.

5. The method for evaluating salt tolerance of rapeseed plants according to claim 4, characterized in that: The grafting step comprises: removing the true leaves of the rootstock, longitudinally cutting the stem of the rootstock to obtain a cleft, retaining two true leaves of the scion, cutting the base of the scion into a wedge shape, inserting the base of the scion into the cleft of the rootstock and fixing it, so as to obtain a cleft-grafted seedling; The cleft-grafted seedlings were placed in a humidity range of 70% to 95% and a light intensity of 85 to 100 μmol / m 2 ·s, and culture them statically in an environment with a temperature of 20-28°C for 6-7 days to obtain grafted seedlings.

6. The method for evaluating salt tolerance of rapeseed plants according to claim 4, characterized in that: The split depth is 8 to 10 mm.

7. The method for evaluating salt tolerance of rapeseed plants according to claim 1, characterized in that: The salt stress culturing step comprises: culturing the grafted seedlings to the four-leaf stage, applying 3L of salt solution to the grafted seedlings every 7 days for culturing, and continuing the culturing for 14 days.

8. The method for evaluating salt tolerance of rapeseed plants according to claim 7, characterized in that: The salt solution is a NaCl solution, and the concentration of the NaCl solution is 150 mmol / L.

9. The method for evaluating salt tolerance of rapeseed plants according to claim 1, characterized in that: The clean water culturing step includes: culturing the grafted seedlings to the four-leaf stage, applying 3 L of clean water to the grafted seedlings for culturing every 7 days, and continuing the culturing for 14 days.

10. Application of the method for evaluating salt tolerance of rapeseed plants according to any one of claims 1 to 9 in the field of rapeseed breeding.

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