Evaluation method suitable for top-free flooding resistance of tropical water lily stock plants and fetal seedlings

Abstract

The invention discloses a method suitable for evaluating the top-free flooding resistance of a tropical water lily stock plant and a fetal seedling, and relates to the field of adversity stress, and the water lily stock plant or the fetal seedling is subjected to top-free flooding treatment with different time lengths. Determining the damage condition by taking the leaf length, the leaf width, the leaf ear length, the leaf area, the stem length, the stem diameter, the chlorophyll content and the leaf cell structure of the normally growing and waterlogged plant as evaluation indexes, and calculating the leaf rot index and the flood tolerance index of the to-be-detected water lily according to the flood damage leaf rot area to realize evaluation. According to the method, the influence of waterlogging on the external phenotype and internal structure of the water lily is considered, the difference of waterlogging treatment on plant ages is considered, the waterlogging resistance is determined through phenotype and cell structure changes, the waterlogging resistance level of the tropical fetal water lily is determined, and the method has the remarkable advantages of being short in evaluation period, visual, reliable and the like. The method is beneficial for promoting innovation of water lily flooding-resistant germplasm and breeding of a flooding-resistant fetal water lily variety, and a new method is provided for research of a water lily flooding-resistant mechanism.

Description

Technical Field

[0001] This invention relates to the field of abiotic stress, specifically to a method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings. Background Technology

[0002] Some plant flood tolerance evaluation and identification systems use quantitative grading based on changes in external morphological indicators such as leaf shape and color, stem color, stem morphology, and survival rate. They develop grading standards and evaluation schemes to comprehensively evaluate flood tolerance, establish relevant flood tolerance evaluation systems, ensure the plant's adaptability to flood stress environments, and select plants with strong flood tolerance for application.

[0003] Water lilies belong to the genus *Nymphaeus* of the family Nymphaeaceae. Nymphaea Water lilies are perennial aquatic flowering plants, classified into tropical and cold-climate types based on their environmental requirements. Compared to cold-climate water lilies, tropical water lilies boast richer flower colors, a longer flowering period, and higher ornamental value, holding an irreplaceable position in landscape design, water feature creation, and garden embellishment. As an important species for constructing aquatic purification communities, water lilies possess a strong ability to purify water pollutants and combat eutrophication. In practical production, tropical water lilies are typically propagated by seeds or bulbs. Viviparous water lily seedlings are also an important heritable propagation method, offering advantages such as a high propagation coefficient and low disease susceptibility. This facultative reproductive characteristic of viviparous seedlings makes them more adaptable to complex and changing natural environments.

[0004] As is well known, water lilies live in water and are highly tolerant of flooding. However, water lilies are aquatic floating-leaved plants. Before their young leaves rise above the water, they lack stomata. Once the leaves emerge, oxygen is primarily transported to the roots through the stomata on the leaf epidermis to maintain normal growth. Short-term submersion stress forces water lilies to exhibit an "escape phenomenon," with stems and leaves rapidly growing above the water surface. However, prolonged submersion can negatively impact the growth of stems, leaves, and viviparous seedlings due to oxygen deficiency, causing changes in internal cell structure and physiological dysfunction, ultimately leading to plant rot and death. The extent of their tolerance remains unclear. Therefore, to improve the accuracy of the evaluation results, this invention incorporates stem and leaf morphology and leaf cell tissue structure techniques to conduct flood tolerance research. This directly reflects the degree of waterlogging damage to plants under adverse conditions, improving the accuracy of the evaluation results and providing a basis for judging the future flood tolerance of water lilies and the breeding of new varieties. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings. The technical problem to be solved is how to quickly and accurately evaluate the submersion tolerance of viviparous water lilies. The specific technical solution is as follows: A method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings, using different submersion times for viviparous water lilies and seedlings of different ages as evaluation indicators, measures the phenotypic changes in stems and leaves and the cell structure of leaves; the evaluation method includes the following steps: Step 1) Select the mother plant and viviparous seedlings of the tropical water lily to be tested; Step 2) Completely submerge the mother plants and viviparous seedlings of the tropical water lilies to be tested; set the submersion duration, i.e., the number of days for complete submersion; Step 3) On the day of the complete submersion set in Step 2), observe and compare the changes in leaf and stem parameters of the tropical water lily under test; Step 4) Determine the degree of decay based on the changes in leaf parameters and stems in Step 3); Grade 0: Intact leaves, no rot; stems naturally upright; Grade 1: The area of ​​leaf decay is ≤1 / 10, with a few spots; the stem gradually turns from reddish-brown to white. Grade 2: The area of ​​leaf decay is ≤1 / 5, and the leaf edges are yellowed; decay begins in the middle section of the stem; Level 3: The area of ​​leaf decay is ≤1 / 2, brown spots appear on the entire leaf, and the middle leaves begin to decay; the stem is bent and softened; Level 4: More than half of the leaves are rotten; large areas of the leaves are withered, yellowed, and necrotic, appearing as transparent water stains; the stems are rotten or even broken. Step 5) Calculate the leaf decay index using Formula 1: Formula 1: Leaf rot index = [Σ(number of rotten leaves × rot level)] / (total number of leaves surveyed × highest rot level) × 100%; Step 6) Use Formula 2 to derive the flood tolerance index; Formula 2: Flood tolerance index (%) = 100% - Leaf decay index; Step 7) Obtain the flood resistance level based on the flood resistance index: Level I: Duration of flood tolerance ≤ 7 days, flood tolerance index > 90%; II: Tolerance duration >7 days and ≤15 days, flood tolerance index >70% and ≤90%; III: Tolerance duration >15 days, ≤30 days; flood tolerance index >50%, ≤70%; IV: Endurance duration > 30 days, flood tolerance index ≤ 50%.

[0006] The beneficial effects of this invention are as follows: Although water lilies are aquatic plants, their tolerance to submersion is not yet fully understood. The submersion tolerance evaluation index set by this invention can effectively evaluate the submersion tolerance of viviparous water lilies. This invention subjects water lily mother plants or viviparous seedlings to submersion treatment for different durations. The damage is determined by evaluating the leaf length, leaf width, auricle length, leaf area, stem length, stem diameter, chlorophyll content, and leaf cell structure of plants under normal growth and after submersion. The leaf decay index and submersion tolerance index of the tested water lily are calculated based on the area of ​​decayed leaves. This invention considers the effects of submersion on the external phenotype and internal structure of water lilies, taking into account the differences in plant age caused by submersion treatment. Submersion tolerance is determined through changes in phenotype and cell structure, clarifying the submersion tolerance level of tropical viviparous water lilies. It has significant advantages such as short evaluation cycle, intuitiveness, and reliability. This invention is beneficial for promoting the innovation of submersion-tolerant water lily germplasm and the breeding of submersion-tolerant viviparous water lily varieties, providing a new method for studying the submersion tolerance mechanism of water lilies.

[0007] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0008] Figure 1 Map showing the planting areas of perennial viviparous water lilies and viviparous seedlings; Figure 2 Comparison images of plants submerged in water and those growing normally; Figure 3 The graphs show the growth of plants after different periods of flooding; A-D. Phenotypic changes of mother water lilies with varying flooding time; E-G. Phenotypic changes of viviparous water lily seedlings with varying flooding time. Figure 4 This is a diagram showing the changes in leaf cell structure of the 'Black Beauty' mother plant under different flooding durations in Example 1; Figure 5 This is a diagram showing the changes in leaf cell structure of the 'Ruby' mother plant under different flooding durations in Example 1; Figure 6 This is a graph showing the changes in leaf thickness of the mother plants of 'Ruby' and 'Black Beauty' under different flooding durations in Example 1; Figure 7 This is a graph showing the changes in epidermal thickness of 'Ruby' and 'Black Beauty' mother plants under different flooding durations in Example 1; Figure 8 This is a graph showing the changes in the lower epidermal thickness of 'Ruby' and 'Black Beauty' mother plants under different flooding durations in Example 1; Figure 9 This is a graph showing the changes in spongy tissue thickness of 'Ruby' and 'Black Beauty' mother plants under different flooding durations in Example 1; Figure 10This is a graph showing the changes in the thickness of the palisade tissue of 'Ruby' and 'Black Beauty' mother plants under different flooding durations in Example 1; Figure 11 This is a graph showing the changes in the porosity of the spongy tissue structure of the mother plants of 'Ruby' and 'Black Beauty' under different flooding durations in Example 1; Figure 12 This is a graph showing the changes in the density of the fence tissue structure of the mother plants of 'Ruby' and 'Black Beauty' under different flooding durations in Example 1; Figure 13 This is a diagram showing the changes in leaf cell structure of 'Black Beauty' viviparous seedlings under different flooding durations in Example 2; Figure 14 This is a diagram showing the changes in leaf cell structure of 'Ruby' viviparous seedlings under different flooding durations in Example 2; Figure 15 This is a graph showing the changes in leaf thickness of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding times in Example 2; Figure 16 This is a graph showing the changes in epidermal thickness of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding time treatments in Example 2; Figure 17 This is a graph showing the changes in the lower epidermal thickness of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding time treatments in Example 2; Figure 18 This is a graph showing the changes in spongy tissue thickness of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding times in Example 2; Figure 19 This is a graph showing the changes in the thickness of the palisade tissue in viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding time treatments in Example 2; Figure 20 This is a graph showing the changes in the porosity of the spongy tissue structure of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding time treatments in Example 2; Figure 21 This is a graph showing the changes in the density of the fence tissue structure of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding time treatments in Example 2.

[0009] Note: Figures 6-12 , Figures 15-21 In the text, R: 'Ruby'; M: 'Black Beauty'; different lowercase letters indicate differences between different treatments for the same indicator within the same variety. P <0.05). Detailed Implementation

[0010] The specific embodiments of the present invention are described in detail below. The given examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The technical means used are conventional means well known to those skilled in the art. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present invention in any way.

[0011] A method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings uses different submersion times for viviparous water lilies and seedlings of different ages as evaluation indicators, and measures the phenotypic changes of stems and leaves and the cell structure of leaves. The evaluation method includes the following steps: Step 1) Select the mother plant and viviparous seedlings of the tropical water lily to be tested; Step 2) Completely submerge the mother plants and viviparous seedlings of the tropical water lilies to be tested; set the submersion duration, i.e., the number of days for complete submersion; Step 3) On the day of the complete submersion set in Step 2), observe and compare the changes in leaf and stem parameters of the tropical water lily under test; Step 4) Determine the degree of decay based on the changes in leaf parameters and stems in Step 3); Grade 0: Intact leaves, no rot; stems naturally upright; Grade 1: The area of ​​leaf decay is ≤1 / 10, with a few spots; the stem gradually turns from reddish-brown to white. Grade 2: The area of ​​leaf decay is ≤1 / 5, and the leaf edges are yellowed; decay begins in the middle section of the stem; Level 3: The area of ​​leaf decay is ≤1 / 2, brown spots appear on the entire leaf, and the middle leaves begin to decay; the stem is bent and softened; Level 4: More than half of the leaves are rotten; large areas of the leaves are withered, yellowed, and necrotic, appearing as transparent water stains; the stems are rotten or even broken. Step 5) Calculate the leaf decay index using Formula 1: Formula 1: Leaf rot index = [Σ(number of rotten leaves × rot level)] / (total number of leaves surveyed × highest rot level) × 100%; Step 6) Use Formula 2 to derive the flood tolerance index; Formula 2: Flood tolerance index (%) = 100% - Leaf decay index; Step 7) Obtain the flood resistance level based on the flood resistance index: I: Duration of tolerance ≤ 7 days, flood tolerance index > 90%; II: Tolerance duration >7 days and ≤15 days, flood tolerance index >70% and ≤90%; III: Tolerance duration >15 days, ≤30 days; flood tolerance index >50%, ≤70%; IV: Endurance duration > 30 days, flood tolerance index ≤ 50%.

[0012] The tropical water lily to be tested in this invention is Nymphaea 'Black Beauty' Black Beauty Water Lily or Nymphaea 'Ruby' Ruby Water Lily. Preferred 3-year-old small plant 'Black Beauty' (…) N 'Black Beauty') and medium to large-sized plants 'Ruby' N 'Ruby') and its corresponding 1-year viviparous seedlings.

[0013] The flooding duration of the mother plants of the tropical water lilies to be tested in this invention was set to 0d, 3d, 7d, 11d, and 15d, with plants with normal floating leaf growth serving as the control (CK).

[0014] The water immersion time of the viviparous seedlings of tropical water lilies to be tested in this invention was set to 0d, 7d, 14d, 21d, and 28d, with normally growing floating-leaf viviparous seedlings as the control (CK).

[0015] The complete submersion treatment in step 2) of the present invention includes: keeping the top of the tropical water lily to be tested 10-15cm away from the water surface.

[0016] The complete flooding treatment in step 2) of the present invention includes: the treatment water temperature is 22±3℃.

[0017] The leaf parameters in step 3) of this invention include: leaf length, leaf width, leaf area, auricle length, and leaf cell structure; the leaf length and leaf width are measured with a ruler centered on the leaf navel under different flooding durations; the leaf area is calculated by measuring the longest and shortest sides; the auricle length is the length from the leaf navel along the leaf lobes to the edge of the leaf blade, measured with a ruler.

[0018] The stem parameters in step 3) of this invention include stem length and stem diameter; the stem length and stem diameter are measured under different flooding durations using a ruler and vernier calipers.

[0019] The leaf cell structure described in this invention includes leaf thickness, upper epidermal thickness, lower epidermal thickness, spongy tissue thickness, palisade tissue thickness, spongy tissue structure looseness, and palisade tissue structure density. The leaf cell structure is observed under a microscope using paraffin sectioning. Compared to the control (CK), at least two (including two, three, or four) parameter values ​​of the leaf thickness, upper epidermal thickness, lower epidermal thickness, spongy tissue thickness, palisade tissue thickness, spongy tissue structure looseness, and palisade tissue structure density show significant differences under the set flooding duration. P <0.05.

[0020] Compared with the control (CK), the leaf chlorophyll content described in this invention shows significant differences in at least two (including two, three, or four) parameter values ​​under the set flooding duration. P <0.05. Example

[0021] A method for evaluating the flood tolerance of tropical water lily mother plants and viviparous seedlings is proposed. The method uses leaf length, leaf width, leaf area, auricle length, stem length, and stem diameter of two types of viviparous water lily mother plants under different flooding durations as phenotypic evaluation indicators, combined with cell structure evaluation methods, to identify the strength of water lily flood tolerance.

[0022] Material selection: Select 3-year-old mother plants of viviparous water lily, such as the small 'Black Beauty' and the medium-to-large 'Ruby', which are growing in the same way and free from pests and diseases. Each plant has about 25 to 30 leaves, and each leaf has a viviparous bud.

[0023] Submerged Flooding Experiment Design: All water lily mother plants were planted in 60×60 cm large vats, with one plant in each vat. The submerged cultivation method was uniformly adopted, and all stems and leaves of the water lily mother plants were submerged in water, so that the stems and leaves were completely immersed in water and the water level was maintained 10-15 cm above the plant. The water temperature was 22±3℃. Submerged flooding was carried out for 0 days, 3 days, 7 days, 11 days and 15 days, with 5 plants in each treatment and 3 replicates. A mother plant with normal floating leaf growth was used as a control (CK). Water was added at any time during the experiment.

[0024] Determination of flood resistance evaluation method: First, observe the growth status of the mother plant's stems and leaves. Damage includes yellowing leaves with brown spots, water-soaked rotten spots on the edges, normal or necrotic bud growth points, elongated or rotten and broken stems, etc. Finally, comprehensively determine the level of flooding damage to a single plant. See the reference photos for each level. Figure 3 .

[0025] Statistical analysis of phenotypic results of water lily submersion flood treatment After being submerged, the stem and leaf phenotypes of both water lily varieties showed significant changes. Figure 3 As the duration of flooding increased, the color of the leaves first deepened, then gradually faded and brown patches appeared. After 7 days of flooding, the edges of the leaves began to wither and rot. The growth point of the buds did not grow significantly due to the decay of the leaves. The stem of the mother plant was thin. On the 15th day of flooding, most of the stems and leaves were rotten and waterlogged, and the plant eventually died.

[0026] Table 1. Phenotypic changes in stems and leaves of two types of water lily mother plants under different flooding durations.

[0027] Table 1 shows that, as the control plants grew, submersion had varying degrees of promoting effects on the stems and leaves of both water lily varieties. Among them, leaf length, leaf width, leaf area, and auricle length all showed a trend of first increasing and then decreasing, reaching their peak after 7 days of submersion. However, with the increase in submersion time, the stems elongated rapidly, and the increase in stem diameter was related to the swelling and decay of the stems.

[0028] Table 2. Chlorophyll content of leaves from 'Ruby' and 'Black Beauty' mother plants under different flooding durations.

[0029] After the submersion treatment, the damage to the water lily plants was counted, and their chlorophyll content was measured. As shown in Table 2, the chlorophyll content of both varieties showed a trend of first increasing and then decreasing. The maximum value was reached on the 7th day of both normal flooding and submersion.

[0030] Changes in cell structure in cross-sections of leaves from two types of water lily mother plants submerged for different durations are shown below. Figure 4 , Figure 5 As shown, on the day of flooding, the epidermal cells of the upper and lower leaves are tightly packed. With increasing duration of submersion, the porosity of the palisade tissue increases and its structural density decreases, while the intercellular spaces of the spongy tissue increase and its looseness increases. Furthermore, the leaf structure exhibits varying thickness. From a quantitative perspective ( Figures 6-12 Compared to the control (CK), the overall leaf thickness of both water lily species initially increased and then decreased. After 7 days of submersion, the thickness of the upper and lower epidermis, palisade tissue, and spongy tissue all reached their maximum. At this point, the leaves began to show varying degrees of rotting, but the affected area was small and had little impact on the mesophyll tissue. In the following 4 days, the number of rotting leaves increased dramatically. The thickness of the leaf epidermis, palisade tissue, and spongy tissue all decreased sharply. After 15 days of submersion, more than 88% of the leaves were in a state of rotting. At this point, the leaf thickness, upper and lower epidermis, palisade tissue, and spongy tissue thickness all reached their minimum. The density of the palisade tissue structure decreased, while the looseness of the spongy tissue increased. Both the palisade tissue and spongy tissue became granular, and the mesophyll cells at the junctions began to disintegrate. This indicates that the tolerance limit of submerged leaves of perennial water lilies is approximately 7 days; after 7 days, their phenotypic characteristics and internal cellular structure are severely damaged.

[0031] Calculate the leaf decay index and flood tolerance index using formulas 1 and 2: Leaf rot index = [Σ(number of rotten leaves × rot level)] / (total number of leaves surveyed × highest rot level) × 100% Flood tolerance index % = 100% - Leaf rot index % Statistics on leaf rot index and flood tolerance index of water lilies after submersion in waterlogging treatment Table 3. Calculation of decay index and flood tolerance index of viviparous water lily mother plants based on leaf decay area.

[0032] Significant differences in flood tolerance were observed between water lilies of different sizes and between different ages of the same type. The results of leaf rot index and flood tolerance level identification are shown in Table 3. At the beginning of submersion, the stems and leaves were intact. As the duration of submersion increased, the stems and leaves showed varying degrees of rot. Among them, the leaf rot index of 'Ruby' and 'Black Beauty' mother plants reached 22.01% and 17.80% respectively on the 7th day of submersion, with flood tolerance indexes both >70%. In the following week, the rot of stems and leaves rapidly increased to 95.94% and 88.33%, mainly affecting plants with about 1 / 2 of their leaves rotted. The flood tolerance index rapidly dropped to below 12%, indicating that the maximum submersion tolerance time for perennial mother plants was about 7 days, with a flood tolerance level of I. The time for plant death was 15 days of submersion, with a flood tolerance level of II. However, the leaf rot rate of the small 'Black Beauty' was slower than that of the medium and large 'Ruby'. Example

[0033] The method described in Example 1 was used to evaluate the viviparous seedlings formed from the buds on the leaves of the viviparous water lily mother plant again by submerging them. The specific method is as follows: Material selection: Select viviparous water lily buds with uniform growth from the leaves and propagate them into viviparous seedlings with stems about 10cm long.

[0034] Submerged Flooding Experiment Design: Viviparous seedlings were planted in small pots (15×15cm in diameter) filled with an equal amount of pond mud, one seedling per pot, with three pots placed in each large tank. A uniform submerged cultivation method was adopted, with all stems and leaves of the water lily seedlings completely submerged in water, maintained at a water level 10-15cm above the plant. The water temperature was 22±3℃. Submerged flooding was conducted for 0, 7, 14, 21, and 28 days, with 15 seedlings in five tanks per treatment, replicated three times. Viviparous seedlings with normal floating leaf growth served as the control (CK). Water was continuously added as the seedlings grew throughout the experiment.

[0035] Determination of flood resistance evaluation method: First, observe the growth status of the stems and leaves of the viviparous seedlings. Symptoms include yellowing and brown spots on the leaves, water-soaked rotten spots on the edges, normal or necrotic growth points of the viviparous buds, elongated or rotten and broken stems, etc. Finally, combine this with the leaf anatomy to comprehensively determine the level of flooding damage to a single plant. See the reference photos for each level. Figure 3 .

[0036] Statistical analysis of phenotypic results of water lily viviparous seedlings submerged in waterlogging treatment After being submerged, there were no significant differences in the stems and leaves of the viviparous seedlings of the two water lily varieties within one week. Figure 3One week later, the stem elongation accelerated. The viviparous seedlings experienced accelerated stem and leaf growth after 14 days of flooding, but still maintained a good growth status. As the flooding time increased, the leaves began to turn yellow after 21 days of flooding. By the 28th day of flooding, some stems and leaves began to rot and become transparent and water-soaked, but new leaves still grew.

[0037] Table 4. Phenotypic changes in stems and leaves of two types of viviparous water lily seedlings under different flooding durations.

[0038] Table 4. Phenotypic changes in stems and leaves of two types of viviparous water lily seedlings under different flooding durations. Table 4 shows that, with the increase of the duration of submersion, the phenotypic changes of the two viviparous water lily seedlings differed from those of the mother plant. Compared with the control (CK), leaf length, leaf width, leaf area, auricle length, and stem diameter decreased, but stem length generally increased and the differences were significant. Among them, the phenotypic indicators all reached their maximum values ​​after 28 days of submersion.

[0039] Table 5. Chlorophyll content of leaves of viviparous seedlings of 'Ruby' and 'Black Beauty' under different flooding durations.

[0040] As shown in Table 5, after the submersion treatment, the damage to the water lily plants was counted and their chlorophyll content was measured. The chlorophyll content of the viviparous seedlings of both varieties showed a trend of first increasing and then decreasing. The chlorophyll content reached its maximum value on the 21st day of both normal flooding and submersion.

[0041] After the water lily plants were submerged, the extent of damage was assessed, and the cell structure of the leaves was examined.

[0042] Changes in cell structure in cross-sections of leaves from two types of viviparous water lily seedlings at different times after being submerged in water are as follows: Figures 13-14 Compared to the control (CK), submersion increased the porosity of the palisade and spongy tissues, decreased their structural density, and increased the looseness of the spongy tissue, indicating a positive correlation between leaf cell structure and leaf decay index. From a quantitative perspective (… Figures 15-21The thickness of the upper epidermis of both viviparous seedlings gradually decreased with increasing submersion time, while the thickness of the lower epidermal cells first increased and then decreased, reaching its maximum on day 7. After 7 days of submersion, the stem and leaf morphology and cell structure of the viviparous seedlings remained intact. After 14 days of submersion, a few brown spots began to appear on the leaves, and the upper and lower epidermis thinned, but the mesophyll and spongy tissue structures remained intact. After 21 days of submersion, the leaves gradually yellowed and a small portion rotted, but the area of ​​rotten leaves was less than 1 / 2. The overall leaf thickness and upper epidermis thickness were lower than the control (CK), while the lower epidermis thickness increased, indicating that the upper epidermal cells were gradually damaged, and the thickness of the palisade and spongy tissues decreased. After 28 days of submersion, the stems and leaves of both viviparous seedlings began to rot and become waterlogged. The leaf thickness decreased, the palisade tissue thickness increased and the porosity increased, the structural density decreased, and the spongy tissue became loose and collapsed, with increased looseness, all higher than the CK. The mesophyll cells began to disintegrate, and more than 75% of the leaves were damaged, showing obvious signs of flood tolerance and impending death.

[0043] Statistics on leaf rot index of water lily viviparous seedlings after being submerged in waterlogging Table 6. Calculation of decay index and flood tolerance index of viviparous water lily seedlings based on leaf decay area.

[0044] The results of the identification of leaf rot index and flood tolerance level of water lily viviparous seedlings are shown in Table 6. At the beginning of submersion, similar to the mother plant, the stems and leaves of the viviparous seedlings were intact. However, as the duration of submersion increased, varying degrees of rot appeared in both stems and leaves. Under the same duration, the leaf rot index of 'Ruby' and 'Black Beauty' viviparous seedlings was 0% on day 7 of submersion. In the following week, the rot indices of the two varieties were 2.6% and 4.2%, respectively, with a flood tolerance level of I. When submerged for 21 days, the leaf rot index was <25%, and the flood tolerance index was >75%, with a flood tolerance level of II. When submerged for 28 days, the rot area of ​​most viviparous seedlings was still <1 / 2, but the flood tolerance index dropped below 25%, with a flood tolerance level of IV. It can be seen that the maximum submersion tolerance time for viviparous seedlings is approximately 21 days, leading to plant death within approximately one month. The leaf rot rate was comparable for both plant types of viviparous seedlings.

[0045] Combining the phenotypic traits, leaf rot index, flood tolerance index, and leaf cell structure of water lilies, the flood damage results are consistent with the flood damage levels listed in step 7. Tables 3 and 6 can clearly distinguish the submersion limit and lethal duration of perennial mother plants and viviparous seedlings. The larger the plant and the older the plant, the worse the flood tolerance. This indicates that the experimental method of using submerged water lilies for flood treatment and the identification of submerged water lily tolerance is feasible.

[0046] The above descriptions are merely some specific embodiments of the present invention (since the present invention encompasses numerical ranges, the embodiments cannot be exhaustive; the scope of protection described in the present invention includes the numerical range and other technical aspects of the present invention). Specific details or common knowledge in the solutions are not described in detail here (including but not limited to abbreviations, acronyms, units commonly used in the art, experimental methods, parameter conditions, etc.). It should be noted that the above embodiments do not limit the present invention in any way. For those skilled in the art, any technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of protection of the present invention. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings, characterized in that, The evaluation method includes the following steps: Step 1) Select the mother plant and viviparous seedlings of the tropical water lily to be tested; Step 2) Completely submerge the mother plants and viviparous seedlings of the tropical water lilies to be tested; set the submersion duration, i.e., the number of days for complete submersion; Step 3) On the day of the complete submersion set in Step 2), observe and compare the changes in leaf and stem parameters of the tropical water lily under test; Step 4) Determine the degree of decay based on the changes in leaf parameters and stems in Step 3); Grade 0: Intact leaves, no rot; stems naturally upright; Grade 1: The area of ​​leaf decay is ≤1 / 10, with a few spots; the stem gradually turns from reddish-brown to white. Grade 2: The area of ​​leaf decay is ≤1 / 5, and the leaf edges are yellowed; decay begins in the middle section of the stem; Level 3: The area of ​​leaf decay is ≤1 / 2, brown spots appear on the entire leaf, and the middle leaves begin to decay; the stem is bent and softened; Level 4: More than half of the leaves are rotten; large areas of the leaves are withered, yellowed, and necrotic, appearing as transparent water stains; the stems are rotten or even broken. Step 5) Calculate the leaf decay index using Formula 1: Formula 1: Leaf rot index = [Σ(number of rotten leaves × rot level)] / (total number of leaves surveyed × highest rot level) × 100%; Step 6) Use Formula 2 to derive the flood tolerance index; Formula 2: Flood tolerance index (%) = 100% - Leaf decay index; Step 7) Obtain the flood resistance level based on the flood resistance index: Level I: Duration of flood tolerance ≤ 7 days, flood tolerance index > 90%; II: Tolerance duration >7 days and ≤15 days, flood tolerance index >70% and ≤90%; III: Tolerance duration >15 days, ≤30 days; flood tolerance index >50%, ≤70%; IV: Endurance duration > 30 days, flood tolerance index ≤ 50%.

2. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The tropical water lily to be tested is Nymphaea 'Black Beauty' or Black Beauty Water Lily Nymphaea 'Ruby' Water Lily.

3. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The flooding duration of the mother plants of the tropical water lilies to be tested was set to 0 days, 3 days, 7 days, 11 days, and 15 days.

4. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The immersion time for the viviparous seedlings of the tropical water lily to be tested was set to 0 days, 7 days, 14 days, 21 days, and 28 days.

5. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The complete submersion treatment in step 2) includes keeping the top of the tropical water lily to be tested 10-15cm above the water surface.

6. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The complete flooding treatment in step 2) includes: the treatment water temperature is 22±3℃.

7. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The leaf parameters in step 3) include: leaf length, leaf width, leaf area, auricle length, leaf cell structure, and leaf chlorophyll content. The leaf length and width are measured with the leaf navel as the center under different flooding durations. The leaf area is calculated using the longest and shortest sides. The auricle length is the length from the leaf navel along the leaf lobes to the leaf edge. Compared to the control, the leaf chlorophyll content shows significant differences in at least two parameter values ​​under the set flooding durations. P < 0.

05.

8. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 1, characterized in that, The stem parameters in step 3) include stem length and stem diameter; the stem length and stem diameter are measured under different flooding durations.

9. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 7, characterized in that, The leaf cell structure includes leaf thickness, upper epidermal thickness, lower epidermal thickness, spongy tissue thickness, palisade tissue thickness, spongy tissue structure looseness, and palisade tissue structure density.

10. The method for evaluating the submersion tolerance of tropical water lily mother plants and viviparous seedlings according to claim 9, characterized in that, Compared with the control, at least two parameters of the leaf thickness, upper epidermis thickness, lower epidermis thickness, spongy tissue thickness, palisade tissue thickness, spongy tissue structure looseness, and palisade tissue structure density showed significant differences in variation under the set flooding duration. P < 0.05.

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