Method for identifying banana cold damage
By conducting leaf structure analysis and pore or conductivity analysis on banana samples, identifying their cold damage, the problem of difficulty in cultivating new banana varieties with strong cold resistance in the existing technology is solved, and accurate identification of banana cold damage and improving cold resistance is achieved.
Patent Information
- Application Number
- CN202510068823.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-04-29
AI Technical Summary
The existing banana breeding technology is difficult to effectively cultivate new banana varieties with strong cold resistance, resulting in insufficient cold resistance in the banana industry.
By conducting leaf structure analysis on the banana samples to be tested, the samples with a sponge tissue thickness less than 50 μm and a gate-sea ratio greater than 1.8 were preliminarily determined to be cold-resistant varieties, and their cold damage was further identified through stomatal observation or conductivity analysis.
It has achieved simple and accurate identification of cold damage to bananas, provided a scientific basis for cultivating banana varieties with strong cold resistance, and improved bananas' cold resistance.
Smart Images
Figure CN119901738A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of banana identification, and particularly relates to a method for identifying banana chilling damage. Background Art
[0003] Since most of the main banana varieties are triploid, highly sterile, and the conventional breeding cycle is long, and it consumes a lot of manpower and financial resources, it is very difficult to select high-quality and highly resistant banana varieties through conventional breeding. Therefore, how to cultivate new banana varieties with strong cold resistance and improve their cold resistance is an important scientific problem that my country's banana industry urgently needs to solve. Summary of the invention
[0004] The invention provides a method for identifying banana chilling damage, which has the advantages of being simple and accurate.
[0005] The object of the present invention is to provide a method for identifying banana chilling injury, comprising the following steps:
[0006] Perform leaf structure analysis on the sample to be tested. When the spongy tissue thickness of the sample to be tested is less than 50 μm and the gate-to-sea ratio is greater than 1.8, the sample to be tested is preliminarily determined to be a cold-resistant variety.
[0007] Stomata observation or electrical conductivity analysis is performed on the cold-resistant varieties that are preliminarily determined, thereby identifying the cold damage of bananas.
[0008] Preferably, when the stomatal observation is performed, the length of the stomatal guard cells of the cold-resistant varieties is 92.3-100.15 μm; the width of the stomatal guard cells of the cold-resistant varieties is 25.8-31.5 μm.
[0009] Preferably, when performing the conductivity analysis, the relative conductivity of the cold-resistant variety at 5°C is >0.65.
[0010] Preferably, the cold-resistant temperature of the cold-resistant variety is 5-20°C.
[0011] Preferably, the varieties of the sample to be tested include: one or more of: pink banana, sour plantain, thousand-layer banana, plantain, Brazilian banana, pink banana, Hua banana, elephant leg banana, small-fruited wild banana, wild banana, emperor banana and red banana.
[0012] The present invention provides application of the method described in the above technical solution in cultivating cold-resistant banana varieties and / or improving the cold resistance of bananas.
[0013] Beneficial effects:
[0014] The present invention provides a method for identifying banana chilling damage, comprising the following steps: performing leaf structure analysis on a sample to be tested, and preliminarily determining that the sample to be tested is a cold-resistant variety when the spongy tissue thickness of the sample to be tested is <50 μm and the grid-to-sea ratio is >1.8; performing stomatal observation or electrical conductivity analysis on the preliminarily determined cold-resistant variety, thereby achieving the purpose of identifying banana chilling damage. The present invention lays a scientific foundation for preliminarily locking in cold-resistant banana varieties by performing leaf result analysis on the sample to be tested, determining the spongy tissue thickness and grid-to-sea ratio of different samples; thereafter, performing stomatal observation or electrical conductivity analysis on the preliminarily determined cold-resistant variety, thereby achieving the purpose of accurately identifying banana chilling damage.
[0015] Based on the above technical advantages, the present invention also provides the application of the method described in the above technical solution in cultivating cold-resistant banana varieties and / or improving the cold resistance of bananas. The solution of the present invention provides a basis for cultivating cold-resistant banana varieties and improving the cold resistance of bananas. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0017] Figure 1 The cross-sectional structure diagrams of banana leaves with different cold resistance provided by the present invention, wherein each structure diagram is shown as 40 × Structural diagram;
[0018] Figure 2 Cluster analysis of 8 leaf anatomical structure indicators provided by the present invention;
[0019] Figure 3 Microscopic observation of stomata on the lower epidermis of leaves of 12 banana varieties provided by the present invention;
[0020] Figure 4 The relative conductivity variation trend of 12 kinds of banana resources provided by the present invention;
[0021] Figure 5 The present invention provides ploidy identification of 12 banana varieties. DETAILED DESCRIPTION
[0022] In order to further illustrate the present invention, the scheme provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.
[0023] Example 1
[0024] A method for identifying banana chilling injury, comprising the following steps:
[0025] Experimental materials: The 12 banana varieties tested were collected from the banana resource garden of Haikou Experimental Station, Hainan Province. The specific numbers and names are shown in Table 1.
[0026] Table 1 Numbers and names of different banana varieties
[0027]
[0028] Experimental process:
[0029] (1) Observation of leaf tissue structure
[0030] Take the mature leaves of different banana varieties in Table 1 from the middle of the main shoot, cut a 1cm 2 The leaf tissues of different sizes were fixed with Carnoy's fixative and sliced by hand. The slices with complete and thin sections were selected from the sliced materials and placed on glass slides for observation under an optical microscope. The leaf thickness, upper epidermis thickness, lower epidermis thickness, palisade tissue thickness, and spongy tissue thickness were measured with a microscope eyepiece micrometer, and the following were calculated: cell structure compactness (CTR) = palisade tissue thickness / leaf thickness; cell structure looseness (SR) = spongy tissue thickness / leaf thickness; palisade-sea ratio = palisade tissue thickness / spongy tissue thickness. The results are shown in Figure 1 .
[0031] The epidermis is the protective tissue of plants and a barrier for plants to resist adverse environments. Figure 1 It can be seen that the upper and lower epidermis of banana leaves are composed of two layers of cells. The outer layer cells are smaller and closely arranged, while the inner layer cells are larger, which is a typical complex epidermal structure. Under the microscope, it can also be observed that the outer surface of the epidermis of all test materials is covered with a cuticle. The mesophyll of bananas has obvious palisade tissue and spongy tissue, which is a heterofacial leaf.
[0032] The thickness of the upper epidermis thickness (X1), lower epidermis thickness (X2), palisade tissue thickness (X3), sponge tissue thickness (X4) and other tissues of the different test materials in Table 1 were measured and statistically analyzed. The results are shown in Table 2.
[0033] Table 2 Comparison of leaf anatomical characteristics of different banana germplasms
[0034]
[0035]
[0036] Combined with Table 2, it can be seen that there are significant differences in the mesophyll tissue structure indicators of the 12 banana varieties. The palisade tissue of Brazilian banana, plantain, pink banana, pink banana, red banana, Hua banana, Thousand-layer banana, and small-fruited wild banana is 2 layers, while the palisade tissue of Emperor Banana, Sour Plantain, Elephant Leg Banana, and Wild Banana is 1 layer. The average thickness of the palisade tissue of the 12 banana varieties is 95.359μm. Among them, the palisade tissue thickness of Pink Dai is the largest, which is above 132μm, and the Emperor Banana and Red Banana are the smallest, less than 70μm. The spongy tissue of each variety is loosely arranged. The spongy tissue thickness is 35.44-75.95μm, with an average thickness of 51.10μm. The spongy tissue of Pink Dai is the thickest, and the thickness of Hua Banana is the smallest. The spongy tissue cells are obviously denser than other varieties. The palisade / sea ratio of each variety is 1.19-3.10, with the palisade / sea ratio of Hua Banana being the largest, which is 3.10, and the palisade / sea ratio of Thousand-layer Banana being the smallest, which is 1.19. The CTR value ranges from 0.20 to 0.42, with the elephant-leg banana having the largest CTR value and the red banana having the smallest CTR value. The SR value ranges from 0.10 to 0.23, with the elephant-leg banana having the largest SR value and the Chinese banana having the smallest SR value.
[0037] The average thickness of the upper epidermis of banana leaves (47.89 μm) is significantly greater than that of the lower epidermis (22.76 μm). Among them, the upper epidermis of the thickest banana is the Zinnia officinalis (65.039 μm), and the thinnest is the Musa acuta (29.097 μm); the thickest lower epidermis is the S. huaensis, and the thinnest is the Musa acuta.
[0038] In order to reasonably use various anatomical structure indicators to evaluate the cold resistance of banana germplasm, it is necessary to first select representative indicators from multiple indicators and then use them as the basis for evaluation. The 8 indicators measured and statistically analyzed were systematically clustered using the Ward's method. The results are shown in Figure 2 .
[0039] Combination Figure 2 It can be seen that the eight leaf structure and organization indicators are clustered into three categories, of which the first category includes X2, X6 (grid-sea ratio P / S), X7 (leaf structure compactness), and X8 (leaf structure density), the second category includes X1 and X2, and the third category includes X3 and X5 (leaf thickness). Among them, the selection of typical indicators in each category is determined according to the size of the relevant index. The larger the relevant index of the indicator, the stronger the representativeness.
[0040] After classifying the indicators, the correlation coefficients between them (see Table 3) are used to calculate the correlation index of each indicator. In the same category, the larger the correlation index of the indicator, the more representative it is in the category. The correlation index and ranking of each indicator are calculated, and the results are shown in Table 4.
[0041] As can be seen from Table 3, among the four indicators in the first category, X8 (leaf structure density) has the largest correlation index, but its coefficient of variation is small, so it is not selected as the typical indicator of this category, and the grid / sea ratio with a larger correlation index is selected as the typical indicator; among the four indicators in the second category. Similarly, the spongy tissue thickness in the second category is selected as the typical indicator.
[0042] Table 3 Correlation matrix of leaf structure indicators
[0043]
[0044] Table 4 Correlation index and ranking of various indicators
[0045]
[0046] As can be seen from Table 4, among the four indicators in the first category, X8 (leaf structure density) has the largest correlation index, but its coefficient of variation is small, so it is not selected as the typical indicator of this category, and the "gate / sea ratio" with a larger correlation index is selected as the typical indicator; among the four indicators in the second category. Similarly, the "spongy tissue thickness" in the second category is selected as the typical indicator.
[0047] Based on the two banana leaf structure indicators (spongy tissue thickness and gate-to-sea ratio) selected above, the membership function method was used to comprehensively evaluate the cold resistance of 12 banana germplasms. Since the spongy tissue thickness is negatively correlated with the cold resistance of plants, its membership degree was calculated using the inverse membership function formula, while the gate-to-sea ratio is positively correlated with the cold resistance, so its membership function was calculated using the positive membership function formula. The results are shown in Table 5.
[0048] Table 5 Membership degree and cold resistance evaluation of 12 banana germplasms
[0049]
[0050] Combined with the average membership degree in Table 5, the cold resistance of the 12 germplasms was ranked as follows: Pink banana > Sour banana > Thousand-layer banana > Large banana > Brazilian banana > Pink banana > Chinese banana > Elephant leg banana > Small-fruited wild banana > Wild banana > Emperor banana > Red banana.
[0051] (2) Stomatal observation and measurement
[0052] The samples were collected at 10 am when the weather was clear. The mature leaves in the middle and lower parts of the main shoots were selected and quickly fixed with Carnoy's fixative. The lower epidermis of the leaves was torn off and placed on a slide. Two drops of 1% gentian violet stain were added. After staining for two minutes, the coverslip was covered. The excess stain was absorbed with filter paper and examined under an optical microscope. The results were shown in Table 1. Figure 3 and Table 6.
[0053] Combination Figure 3It can be seen that there are no stomata on the upper epidermis of bananas, but there are a large number of stomata scattered on the lower epidermis. The stomata are flat, indefinite, oval or oblong. The stomatal apparatus is composed of two kidney-shaped guard cells, which are similar in shape.
[0054] Table 6 Stomatal analysis of 12 banana varieties
[0055]
[0056] Combined with Table 6, it can be seen that there are extremely significant differences in the stomatal characteristics of each variety. The banana pores are small and scattered, the average length of the stomatal guard cells is 111.32μm, the average width of the stomatal guard cells is 29.77μm, and the average stomatal density is 274 / mm2. The stomatal guard cells of the pink banana are the longest, which is 133.84μm, and the stomatal guard cells of the small fruit wild banana are the shortest, which is 87.49μm. The largest stomatal guard width is the thousand-layer banana, which is 42.4μm, and the smallest stomatal guard cell width is the small fruit wild banana, which is 23.02μm. The stomatal density of the sour banana, small fruit wild banana, and wild banana is the largest, which is above 350㎜2, and the stomatal density of the Chinese banana is the smallest, which is below 186㎜2. It can be seen that compared with other varieties of bananas, the small fruit wild banana has small and densely distributed stomata.
[0057] (3) Relative conductivity measurement
[0058] Wipe the dust off the surface of different banana leaves in Table 1 with gauze, rinse with deionized water three times, and dry with clean filter paper. Divide the banana leaves to be treated into 4 groups, and place them in artificial climate boxes at 5°C, 10°C, 15°C and 20°C for cold treatment for 12 hours, and set a control group without cold treatment (room temperature condition);
[0059] The materials after different low temperature treatments (including control treatment) were cut into 2 mm small sections, the midribs were removed, 3 g of the sample was weighed and put into a triangular flask, and 30 ml of double distilled water was added. After soaking for 24 hours, the conductivity of the extract (S1) and the ion water conductivity (S0) were measured at a constant temperature of 25°C. Then the test tube was placed in a 100°C boiling water bath for 15 minutes to kill the plant tissue. After taking it out, the outer wall was rinsed with tap water and cooled for 10 minutes, and its boiling conductivity (S2) was measured at a constant temperature of 25°C. The calculation formula of relative conductivity is: L = [(S1-S0) / (S2-S0)] × 100%, and each sample was biologically repeated 5 times.
[0060] The relative conductivity of plant branch tissue at different low temperatures was analyzed by logistic equation. The regression model was y = k / (1 + ae bt ), where y is the relative conductivity, t is the treatment temperature, k is the saturation capacity of the cell damage rate, and a and b are equation parameters. Assuming the k value is 100, to determine the a and b values, the Logistic equation is transformed into: Then the equation is transformed into a linear equation between y and t, and The half-lethal temperature is Results Figure 4 .
[0061] Depend on Figure 4 It can be seen that with the decrease of temperature, the relative conductivity of the 12 banana resources showed a trend of gradual increase, but the increase was different. When the treatment temperature was between 25℃ and 5℃, the relative conductivity of red banana, wild banana, small-fruited wild banana, and emperor banana increased greatly, increasing from 0.507, 0.416, 0.410, and 0.451 to 0.775, 0.610, 0.595, and 0.652, respectively, with an increase of 26.80%, 18.30%, 19.78%, 17.90%, and 20.10%. Moreover, there were significant differences between the treatment temperatures and the control group (P<0.05); when the treatment temperature was between 25℃ and 20℃, the relative conductivity of each variety increased relatively slowly, with increases of 1.2%, 4.6%, 1.5%, 2.7%, 2.2%, 1.2%, 1.2%, 1.3%, 0.1%, 0.29%, 1.3%, and 1.7%, respectively, and there was no significant difference between the two temperature treatments (P<0.05).
[0062] The relative conductivity of the 12 banana resources under different low temperature treatments was fitted with a Logistic equation using EXcel software, and the semi-lethal temperatures of the 12 banana resources were obtained as shown in Table 7.
[0063] Table 7 Semi-lethal temperatures of 12 banana resources
[0064]
[0065]
[0066] Combined with the data in Table 7, it can be seen that the semi-lethal temperature of sour plantain is 9.48℃, the semi-lethal temperature of Hua banana is 1.68℃, the semi-lethal temperature of Brazilian banana is 3.34℃, the semi-lethal temperature of pink banana is 3.57℃, the semi-lethal temperature of Thousand-layer banana is 3.846℃, the semi-lethal temperature of pink banana is -2℃, the semi-lethal temperature of emperor banana is 10.65℃, the semi-lethal temperature of plantain is 14℃, the semi-lethal temperature of small-fruited wild banana is 1.98℃, the semi-lethal temperature of elephant-leg banana is 6.13℃, the semi-lethal temperature of wild banana is 1.66℃, and the semi-lethal temperature of red banana is 14.9℃. The lower the temperature, the stronger the cold resistance of bananas. Based on this, the cold resistance is ranked as follows: pink banana>Hua banana>Thousand-layer banana>sour plantain>plantain>Brazilian banana>pink banana>emperor banana>sour plantain>emperor banana>wild banana>red banana.
[0067] (4) Ploidy analysis
[0068] The ploidy of banana resources was identified by flow cytometry, and the banana germplasm with known ploidy was used as the control material. The Partec Cy S-tain Cy Flow Space was used for detection. The specific experimental process is as follows: the young leaves of the experimental material banana were cut into about 0.5 cm 2 Size, put into a clean culture dish, add 400μL of cell lysis extract (HRA), and then use a sharp blade to quickly chop the banana leaves as finely as possible. Then add 120μL of propidium chloride and 60μL of RNase A stock solution to 20mL of staining buffer for staining for 2min; then filter into a test tube with a 30wm filter and quickly test on the machine. The ploidy identification peak graph of the tested variety is automatically generated by the flow cytometer and stored in the computer.
[0069] In this experiment, using wild banana (BB) as the internal reference, FCM technology was used to identify the ploidy of 12 banana germplasm resources. The results of FCM analysis are shown in Figure 5 (exist Figure 5 In the histogram, the number of nuclei in each channel directly reflects the relative fluorescence intensity value. The horizontal axis in the figure represents the relative content of DNA. The fluorescence intensity is proportional to the content of A and T bases, and is also proportional to the amount of chromosome content; the vertical axis represents the number of cells).
[0070] Other results of FCM analysis of ploidy identification of 12 banana germplasm resources are shown in Table 8.
[0071] Table 8 Ploidy identification results of 12 banana varieties
[0072]
[0073]
[0074] The results of FCM analysis reflect the complexity of germplasm ploidy during evolution and hybridization, and also indicate the necessity of banana germplasm karyotype analysis. FCM technology is simple to operate, highly reliable, and highly repeatable. It can reveal the relationship between DNA content and ploidy. It is an accurate means of analyzing the ploidy of cultivated plants for vegetative propagation. It provides important information for banana breeding and genetic improvement, and provides strong guidance for the subsequent cultivation and screening of cold-resistant varieties.
[0075] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for identifying banana chilling damage, characterized in that: The steps include: Perform leaf structure analysis on the sample to be tested. When the spongy tissue thickness of the sample to be tested is less than 50 μm and the gate-to-sea ratio is greater than 1.8, the sample to be tested is preliminarily determined to be a cold-resistant variety. Stomata observation or electrical conductivity analysis is performed on the cold-resistant varieties that are preliminarily determined, thereby identifying the cold damage of bananas.
2. The method according to claim 1, characterized in that When the stomatal observation is performed, the length of the stomatal guard cells of the cold-resistant varieties is 92.3-100.15 μm; the width of the stomatal guard cells of the cold-resistant varieties is 25.8-31.5 μm.
3. The method according to claim 1, characterized in that When conducting the conductivity analysis, the relative conductivity of the cold-resistant varieties at 5°C is >0.
65.
4. The method according to any one of claims 1 to 4, characterized in that: The cold-resistant temperature of the cold-resistant variety is 5-20°C.
5. The method according to claim 1, characterized in that The varieties of the samples to be tested include: one or more of: pink banana, sour banana, thousand-layer banana, plantain, Brazilian banana, pink banana, Hua banana, elephant-leg banana, small-fruit wild banana, wild banana, emperor banana and red banana.
6. Use of the method according to any one of claims 1 to 5 in cultivating cold-resistant banana varieties and / or improving the cold resistance of bananas.