Rapid evaluation method for salt-tolerant germplasm of paeonia lactiflora and establishment process thereof

Abstract

The invention discloses a rapid evaluation method for salt-tolerant germplasm of Chinese herbaceous peony and an establishment process thereof. The method comprises the following steps: measuring the thickness of leaves of the Chinese herbaceous peony in a natural growth state; selecting a Chinese herbaceous peony variety of which the leaf thickness is greater than or equal to 0.32 mm as a germplasm with The Chinese herbaceous peony is divided seedlings of 3-4 years old. The method can quickly and accurately predict the salt tolerance of the Chinese herbaceous peony leaves by directly measuring the thickness of the Chinese herbaceous peony leaves, is simple and convenient to operate and high in efficiency, can effectively protect germplasm resources, avoids irreversible damage to the plants caused by traditional salt stress treatment, improves objectivity and reliability of evaluation results, and is high in applicability and particularly convenient to popularize and apply.

Description

Technical Field

[0001] The present invention belongs to the technical field of germplasm resource evaluation and screening, and particularly relates to a rapid evaluation method for salt-tolerant peony germplasm based on leaf thickness and an establishment process thereof. Background Art

[0002] Soil salinization is one of the major abiotic stressors limiting plant growth and crop yields. According to statistics, the global area of saline-alkali land currently reaches 833 million hectares, of which my country has 99 million hectares, making it the third largest country in the world. With the series of climate challenges brought about by global warming, coupled with secondary salinization caused by human factors such as irrational irrigation and fertilization, the area of saline-alkali land in my country and around the world is increasing annually.

[0003] Peony (Paeonia lactiflora Pall.), a perennial herbaceous plant of the genus Paeonia in the family Paeoniaceae, holds a prominent position in both ornamental and medicinal gardening due to its rich color, high-quality, and ornamental qualities, as well as its multiple medicinal properties, including antioxidant, analgesic, anti-inflammatory, hepatoprotective, and immune-modulating properties. To meet market demand for peony and expand its cultivation, the development and utilization of saline-alkali land resources for peony cultivation is of great significance. However, saline-alkali stress can significantly inhibit the growth and development of peony, resulting in dwarf plants, yellowing leaves, reduced flowering, and even plant death, severely impacting their ornamental quality and the accumulation of medicinal components.

[0004] At present, the evaluation of salt tolerance of peony faces the following technical bottlenecks: First, traditional salt tolerance evaluation mainly relies on physiological and biochemical indicators (such as malondialdehyde content, antioxidant enzyme activity, etc.), which have limitations such as long detection cycle, complex operation, and high requirements for instruments; second, the existing evaluation system mostly requires salt stress treatment on the plants, which is not only time-consuming and labor-intensive, but may also cause irreversible damage to precious germplasm resources; in addition, research on salt tolerance morphological indicators for peony, a specific crop, is still blank, and there is a lack of rapid and non-destructive field evaluation methods. Summary of the Invention

[0005] The present invention discloses a rapid salt tolerance evaluation method for peony germplasm resources based on leaf thickness. The evaluation results obtained using this morphologically based rapid salt tolerance evaluation method can be effectively used to screen salt-tolerant germplasm. This method not only provides a scientific basis for peony cultivation in saline-alkali soils but also offers technical support for salinization prevention and control in traditional planting areas, thus having important implications for promoting the high-quality development of the peony industry.

[0006] The object of the present invention is achieved in the following ways:

[0007] A rapid evaluation method for salt-tolerant peony germplasm is provided. The method comprises measuring the thickness of peony leaves under natural growth conditions and selecting peony varieties with leaf thickness ≥0.32 mm as the most salt-tolerant germplasm. The peony is a 3-4 year-old ramet seedling.

[0008] Preferably, when the leaf thickness is 0.32≤≤0.37mm, the peony has the strongest salt tolerance; when the leaf thickness is 0.25mm<0.32mm, the peony has a moderate salt tolerance; when the leaf thickness is 0.17≤≤0.25mm, the peony has the weakest salt tolerance.

[0009] Preferably, the peony is planted in a pot, and the cultivation soil is local field soil.

[0010] The process of establishing the above-mentioned rapid evaluation method for salt-tolerant peony germplasm includes the following steps: the peony is subjected to salt treatment after the morphological indicators are measured; on the 15th day after the start of salt treatment, the phenotypic traits of each peony are phenotypic scored according to the evaluation criteria; the salt tolerance of the peony is classified by cluster analysis using the phenotypic score; the salt tolerance evaluation index of the peony is screened by path analysis, and it is found that the leaf thickness of the peony is positively correlated with the salt tolerance of the peony.

[0011] Preferably, the peony is selected from 20 3-4 year old ramet seedlings of different peony varieties, with 10-20 plants of each variety.

[0012] Preferably, the salt treatment is to irrigate each plant with 1 L of 400 mM NaCl solution each time, once every 3 days, for a total of 5 times, and end on the 13th day.

[0013] Preferably, the 1L salt treatment solution is irrigated in two slow pouring steps, 500mL each time.

[0014] Preferably, the morphological indicators of peony include plant height, stem diameter, leaf thickness, number of branches, leaf length, leaf width, and leaf area. The plant height is measured from the potting soil surface to the top of the plant; the stem diameter is measured at the width of the stem base; the leaf thickness is measured at the thickness of the terminal leaflets; the leaf length is measured from the base of the petiole to the tip of the leaf; the leaf width is measured at the lateral distance of the widest part of the leaf; and the leaf area is calculated by photographing the leaf and analyzing the image using ImageJ software.

[0015] The above evaluation criteria divide the phenotypic traits of peony into 5 levels: Level I external morphology has no obvious symptoms of salt stress damage, with a score of 1; Level II external morphology has a small number of leaves wilting and drooping, and the leaf tips and leaf edges turn yellow, with a score of 2; Level III external morphology has about 1 / 2 of the leaf tips and leaf edges turn yellow, curl and dry, with a score of 3; Level IV external morphology has most of the leaves curling and drying up, with a score of 4; Level V external morphology has leaf and branch withering until the entire plant dies, with a score of 5.

[0016] It is accurate and effective to preliminarily screen salt-tolerant peony germplasm resources by measuring the thickness of peony leaves.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] Aiming at the increasingly serious problem of soil salinization in current peony planting areas (such as coastal areas, saline-alkali land and facility cultivation), the present invention proposes a method for rapid evaluation of salt tolerance of peony germplasm resources based on leaf thickness and screening of salt-tolerant germplasm.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects:

[0020] (1) High scientificity and accuracy: Through systematic experiments, the present invention found that peony leaf thickness is significantly positively correlated with salt tolerance, that is, varieties with thicker leaves have stronger salt tolerance. This method can quickly and accurately predict peony salt tolerance by directly measuring its leaf thickness, avoiding the irreversible damage to plants caused by traditional salt stress treatment, while improving the objectivity and reliability of the evaluation results.

[0021] (2) Simple operation and high efficiency: Traditional salt tolerance identification requires salt stress treatment and the measurement of multiple physiological and biochemical indicators (such as MDA content and SOD activity), which is time-consuming and labor-intensive. However, the present invention only requires the use of a vernier caliper to precisely measure the thickness of peony leaves under natural growth conditions, which can achieve rapid identification, significantly shorten the evaluation cycle, reduce experimental costs, and is particularly suitable for large-scale germplasm resource screening.

[0022] (3) Non-destructive testing to protect germplasm resources: Existing salt tolerance evaluation methods usually require applying salt stress to plants, which may lead to growth inhibition or even death, affecting the preservation of precious germplasm. The present invention does not require salt treatment and can complete the evaluation only by non-destructive measurement of leaf thickness, thus maximizing the protection of the integrity of peony germplasm resources.

[0023] (4) Strong applicability and easy to promote: This method has low requirements for instruments (such as basic tools such as vernier calipers) and does not require complex experimental conditions or molecular biology techniques. It is particularly suitable for use by scientific research institutions, breeding companies, and grassroots growers. It can be widely used in the selection and breeding of salt-tolerant peony varieties, the layout of saline-alkali land cultivation, and the exploration of stress-resistant germplasm resources.

[0024] (5) Significant ecological and economic value: Through precise screening of salt-tolerant varieties, the cost of improving salinized soil can be reduced, the loss of ornamental quality and medicinal components caused by salt damage can be reduced, the success rate of peony cultivation in saline-alkali areas can be improved, and the efficient utilization of saline-alkali land resources can be promoted, which is in line with the sustainable development needs of ecological agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is a comparison chart of the phenotypic traits of the peony germplasm materials in Example 20 before and after salt treatment.

[0026] Figure 2 This is a cluster analysis diagram of the salt tolerance of peony germplasm materials in Example 20.

[0027] In the figure, the English letters are the numbers of the peony germplasm materials, and each letter represents a variety, which has been marked in Table 1. DETAILED DESCRIPTION

[0028] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0029] Example 1

[0030] (1) Experimental materials: including 20 peony cultivars. These peony varieties were divided three years ago and transplanted to the experimental base of the Gardening Department of Jiangsu Agricultural and Animal Husbandry Science and Technology Vocational College for potted cultivation in April 2023, with one plant per pot. The specifications of the flower pots are 32 cm in upper diameter, 26 cm in lower diameter, and 35 cm in height, and the cultivation soil is local field soil. In April 2024, 3-4 year-old division plants with relatively consistent growth potential were selected from each variety as experimental materials, and the flower buds of all plants were removed. The names, numbers and sources of the experimental materials are detailed in Table 1;

[0031] Table 1 Names and numbers of tested peony varieties

[0032]

[0033]

[0034] (2) Determination of indicators before salt treatment: Morphological indicators of peony were measured, including plant height, stem diameter, leaf thickness, number of branches, leaf length, leaf width, and leaf area. Plant height: measured with a ruler, i.e., the vertical distance from the surface of the potting soil to the top of the plant; stem diameter: measured with a vernier caliper, the width of the stem base; leaf thickness: measured with a vernier caliper, the thickness of the terminal leaflet; leaf length: measured from the base of the petiole to the tip of the leaf; leaf width: measured from the horizontal distance at the widest part of the leaf; leaf area: calculated by photographing the leaves and analyzing the images using ImageJ software (see Table 5).

[0035] (3) Salt treatment: Salt treatment started on May 9, 2024, and that day was designated as day 0. Every day from 10:00 to 12:00, each plant was irrigated with 1L of 400mM NaCl solution, and the treatment was repeated every 3 days (a total of 5 times, ending on the 13th day). The irrigated soil was irrigated in two slow pours (500mL each time) to ensure that the soil was fully saturated. If there was exudate in the plastic tray, it was poured back into the pot. The phenotypic characteristics of the peony germplasm before and after salt treatment are shown in the table. Figure 1 In the figure: the varieties numbered AT are the same as those in Table 1, which are photos before salt treatment, and the varieties numbered (A)-(T) are photos after salt treatment.

[0036] (4) Salt tolerance identification: On the 15th day after the first irrigation of NaCl solution, the phenotypic traits of each peony variety after salt treatment were photographed and scored according to the standards in Table 2 (refer to Wang Qi, Yuan Yanbo, Yu Xiaonan. Study on physiological characteristics and salt-alkali tolerance of two peony varieties under salt-alkali stress [J]. Journal of Hebei Agricultural University, 2013, 36(6): 52-60.);

[0037] Table 2 Phenotypic trait scoring standards for peony

[0038]

[0039] (5) Based on the phenotypic scores in step (4), cluster analysis was used to classify the salt tolerance of peony: the salt tolerance of the 20 peony varieties was divided into three groups: I, II, and III (see Figure 2 ), among which Group I is salt-tolerant germplasm resources: including 3 varieties, D (Red Charm), M (Basra), and T (Salad), accounting for 15%; Group II is moderately salt-tolerant germplasm resources, including 10 varieties, K (Black Sea Waves), R (Imperial Performance), I (Blue Fuji), L (Purple Hydrangea), O (Red Fuji), Q (Qingwen), F (Coral Sunset), H (Pillow Talk), J (Gaoganhong), and C (Peach Blossom Snow), accounting for 50%; Group III is salt-sensitive germplasm materials, including 7 varieties, N (Red Rose), S (Seed Powder), A (Great Fortune), B (Moyu Tower), E (Pouch), G (Purple Phoenix Feather), and P (Colorful), accounting for 35%.

[0040] (6) Path analysis was used to screen the salt tolerance evaluation index of peony: Path analysis of the salt tolerance scores and morphological indicators of 20 peony varieties (see Table 4) showed that among the growth indicators measured, only leaf thickness had a significant effect on the salt tolerance score of peony. Y = 7.637-16.274*X, where X represents leaf thickness. That is, the thicker the leaf thickness of the peony, the lower the salt tolerance score, indicating that the peony is more salt-tolerant.

[0041] (7) Based on the data in Table 5, the optimal peony salt tolerance classification and leaf thickness comparison table was developed (see Table 6).

[0042] Table 4 Path analysis of salt tolerance scores and morphological indices of 20 peony varieties

[0043]

[0044] Table 5 Morphological indicators of 20 peony varieties before salt treatment.

[0045]

[0046] Table 6 Comparison of salt tolerance level and leaf thickness of peony

[0047]

Claims

1. A rapid evaluation method for salt-tolerant peony germplasm, characterized in that The method is to measure the thickness of peony leaves in a naturally growing state, and select peony varieties with leaf thickness ≥0.32 mm as the most salt-tolerant germplasm; the peony is a 3-4 year old ramet seedling.

2. The rapid evaluation method for salt-tolerant peony germplasm according to claim 1, characterized in that When the leaf thickness is 0.32 mm or less and is less than 0.37 mm, the peony has the strongest salt tolerance; when the leaf thickness is 0.25 mm or less and is less than 0.32 mm, the peony has a moderate salt tolerance; when the leaf thickness is 0.17 mm or less and is less than 0.25 mm, the peony has the weakest salt tolerance.

3. The rapid evaluation method according to claim 1 or 2, characterized in that The herbaceous peony is planted in a pot, and the cultivation soil is local field soil.

4. A process for establishing the evaluation method according to claim 1 or 2, characterized in that: After the morphological indicators of peony were measured, salt treatment was carried out. On the 15th day after the start of salt treatment, the phenotypic traits of each peony were phenotypic scored according to the evaluation criteria. The salt tolerance of peony was classified by cluster analysis based on the phenotypic score. The evaluation indicators of peony salt tolerance were screened by path analysis, and the leaf thickness of peony was positively correlated with the salt tolerance of peony.

5. The establishment process according to claim 4, characterized in that: The peony is selected from 20 3-4 year old ramet seedlings of different peony varieties, with 10-20 plants of each variety.

6. The establishment process according to claim 4, characterized in that The salt treatment was to irrigate each plant with 1 L of 400 mM NaCl solution each time, once every 3 days, for a total of 5 times, and end on the 13th day.

7. The establishment process according to claim 6, characterized in that The 1L salt treatment solution was slowly poured twice, 500mL each time.

8. The establishment process according to claim 4, characterized in that The morphological indicators of the peony include plant height, stem thickness, leaf thickness, branch number, leaf length, leaf width and leaf area.

9. The establishment process according to claim 8, characterized in that The plant height is measured by the vertical distance from the soil surface to the top of the plant; the stem diameter is measured by the width of the stem base; the leaf thickness is measured by the thickness of the terminal leaflet; the leaf length is measured by the maximum distance from the base of the petiole to the tip of the leaf; the leaf width is measured by the lateral distance at the widest part of the leaf; and the leaf area is calculated by photographing the leaf and analyzing the image using ImageJ software.

10. The establishment process according to claim 4, characterized in that The evaluation criteria are to divide the phenotypic traits of peony into 5 levels: Level I has no obvious symptoms of salt stress damage in the external morphology, with a score of 1; Level II external morphology has a small number of leaves wilting and drooping, and the leaf tips and edges turning yellow, with a score of 2; Level III external morphology has about 1 / 2 of the leaves turning yellow at the tips and edges, curling and drying up, with a score of 3; Level IV external morphology has most of the leaves curling and drying up, with a score of 4; Level V external morphology has withered leaves and branches until the entire plant dies, with a score of 5.

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