A method for identifying lodging resistance of water chestnut

Abstract

The present invention discloses a method for identifying water chestnut lodging resistance in the field of water chestnut lodging resistance identification technology, comprising the steps of: selecting 10 water chestnut varieties for identification; trimming, dehydrating, embedding, slicing, staining, and sealing the selected samples to make paraffin sections; observing the prepared paraffin sections with a microscope and photographing them; a fourth step of observing the microphotographs taken after amplification by any multiple using software, and scanning and browsing the software to select the target area of ​​the tissue for 200 times imaging; a fifth step of measuring the anatomical structure index of each slice of water chestnut leaf tissue using analysis software in a unified manner with millimeters as the standard unit after imaging; and a sixth step of drawing and analyzing the data obtained from the experiment. This method provides making water chestnut into paraffin sections and observing their different indicators under a microscope, so that the lodging resistance of water chestnut can be identified.

Description

Technical Field

[0001] The invention belongs to the technical field of identification of lodging resistance of water chestnut, and specifically relates to an identification method for lodging resistance of water chestnut. Background Art

[0002] Water chestnuts, also known as water chestnuts, ground chestnuts, and black taro, are perennial herbaceous plants in the Cyperus family. Their bulbs, grown as both fruit and vegetables, are important agricultural exports in my country. In recent years, water chestnut cultivation in Guangxi has grown rapidly, reaching 20,000 hectares annually. Water chestnuts are often grown in ponds or paddy fields, where they are prone to lodging during their mature stages, leading to severe culm blight, a significant impact on water chestnut yields, resulting in a 30-70% reduction or even complete loss of production. Studies on gramineous crops such as corn, rice, and wheat have shown that the anatomical structure of the stem is closely related to its lodging resistance. Wheat, corn, rice, and buckwheat varieties with high lodging resistance have sturdy stems, a high number of mechanical tissue cell layers and vascular bundles, a large vascular bundle area, thicker vascular bundle sheaths, a larger number of cell layers, and densely packed cells. Water chestnut leaves are degenerate, and photosynthesis relies primarily on the phyllodes to provide energy for the expansion of the underground corm. Therefore, the lodging resistance of different water chestnut varieties plays a key role in their yield. However, there is no relevant identification method for the anatomical structure of the phyllodes of different water chestnut varieties and its relationship with lodging resistance. Summary of the Invention

[0003] In order to solve the above problems, the purpose of the present invention is to provide a method for identifying the lodging resistance of water chestnuts, so as to identify the lodging resistance of different water chestnut varieties.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows: a method for identifying lodging resistance of water chestnuts, comprising the following steps:

[0005] Step 1: Select 10 water chestnut varieties.

[0006] Step 2: trim, dehydrate, embed, slice, stain and seal the selected samples to make paraffin sections.

[0007] Step 3: Observe the prepared paraffin sections with a microscope and take photos.

[0008] Step 4: Use software to magnify the microscopic photos taken at any magnification of 1-400 times and observe them. Use software to scan the microscopic photos, and then select the target area of ​​the required tissue for 200x imaging. When imaging, let the tissue fill the entire field of view and ensure that the background light of each photo is consistent.

[0009] Step 5: After imaging is completed, use analysis software to standardize the micrographs with millimeters as the standard unit and measure the anatomical structure indicators of each water chestnut leaf slice.

[0010] Step six: The experimental data were processed using software, and then statistical software was used to perform statistical analysis on the leaf tissue anatomical structure indicators of different water chestnut varieties. The significance of the differences among different water chestnut varieties was tested using the new multiple range method for multiple comparisons, and the corresponding charts were drawn using the software.

[0011] Furthermore, in step one, the 10 water chestnut varieties include Guifenti No. 1, Guiti No. 2, Guiti No. 3, Guiti No. 4, Yangjiang Pearl Water Chestnut, Hezhou Fanglin, Dahongpao, Anshun, Guizhou, Zhijiang, Hubei, and Fuyang, Anhui.

[0012] Furthermore, in step 2, 3 small pieces of each sample tissue were collected, and a total of 30 slices of the sample tissue were prepared.

[0013] Furthermore, in step five, the measured indicators include epidermal thickness, stratum corneum thickness, tissue thickness, number of vascular bundles, perimeter, and number of vascular bundles per unit length.

[0014] Furthermore, in step five, each indicator was randomly measured 20 times at different locations on each slice.

[0015] The above scheme achieves the following beneficial effects: when making paraffin sections from water chestnuts, the water chestnuts need to be trimmed, dehydrated, embedded, sliced, stained, and sealed. When slicing, water chestnut sections of appropriate size can facilitate subsequent staining operations on the water chestnuts, so that the water chestnuts can be more fully and evenly colored during staining. At the same time, after staining is completed, the structure of each tissue of the water chestnut will be more obvious, which makes it easier to select the required tissue for observation. When the paraffin sections are completed, each sample group will be Three small pieces of tissue were collected, totaling 30 sections. By making multiple paraffin sections of the same water chestnut, multiple tissues of the same water chestnut can be observed at the same time, thereby improving the observation efficiency to a certain extent. When observing microscopic photos, the water chestnut tissue must fill the entire field of view, and the background light of each photo must be consistent, so that in the observation of water chestnut, the influence of different light sources on the observation is reduced, thereby reducing errors and making the observation results of various water chestnut tissues more objective. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a microscopic diagram of the cross-section of a water chestnut leaf stem during the experimental process of the method for identifying lodging resistance of water chestnut in an embodiment of the present invention. (Here, 1 represents assimilated tissue, 2 represents thin-walled vascular bundle sheath, 3 represents thick-walled vascular bundle sheath, 4 represents phloem, 5 represents metaxylem, 6 represents primary xylem, 7 represents central air cavity, 8 represents air cavity, 9 represents posterior wall zone, 10 represents cuticle, and 11 represents epidermis)

[0017] Figure 2This is a diagram of the vascular bundle structure of the phyllodes of different water chestnut varieties during the experimental process of the water chestnut lodging resistance identification method in the embodiments of the present invention. (A represents Guifenti No. 1, B represents Guiti No. 2, C represents Guiti No. 3, D represents Guiti No. 4, E represents Yangjiang Zhenzhu Ma Ti, F represents Hezhou Fanglin, G represents Dahongpao, H represents Anshun, Guizhou, I represents Zhijiang, Hubei, and J represents Fuyang, Anhui)

[0018] Figure 3 This diagram shows the vascular bundle structure of the 'Guiti No. 3' leaf-like stem at different locations from the base during the experimental process of the water chestnut lodging resistance identification method in the embodiments of the present invention. (A represents 18 cm from the base, B represents 35 cm from the base, C represents 50 cm from the base, and D represents 75 cm from the base.) DETAILED DESCRIPTION

[0019] The following is further described in detail through specific implementation methods:

[0020] The embodiment is basically as shown in the attached Figure 1 、 Figure 2 and Figure 3 As shown: A method for identifying water chestnut lodging resistance comprises the following steps:

[0021] Step 1: Select 10 water chestnut varieties for identification. The 10 water chestnut varieties include Guifenti No. 1, Guiti No. 2, Guiti No. 3, Guiti No. 4, Yangjiang Pearl Water Chestnut, Hezhou Fanglin, Dahongpao, Anshun, Guizhou, Zhijiang, Hubei, and Fuyang, Anhui, as shown in Table 1.

[0022] Table 1 Information of the 10 water chestnut varieties tested

[0023]

[0024] Step 2: The selected samples are trimmed, dehydrated, embedded, sliced, stained, and sealed to make paraffin sections. Three small pieces of each sample tissue are collected and 30 sections are prepared.

[0025] Step 3: The prepared paraffin sections were observed using a NIKON ECLIPSE E100 upright optical microscope and photographed using a NIKON DS-U3 imaging system.

[0026] Step 4: The microscopic photographs were magnified at any magnification of 1-400 times using CaseViewer 2.2 software for observation, and the target area of ​​the tissue was selected using CaseViewer 2.2 software for scanning and browsing to perform 200x imaging. During imaging, the tissue was allowed to fill the entire field of view as much as possible to ensure that the background light of each photo was consistent.

[0027] Step 5. After imaging is completed, Image-Pro Plus 6.0 analysis software is used to measure the anatomical structure indicators of each water chestnut leaf slice using millimeters as the standard unit. The measured indicators include epidermal thickness, cuticle thickness, tissue thickness, vascular bundle number, circumference, and number of vascular bundles per unit length. Each indicator is randomly measured 20 times at different locations on each slice.

[0028] Step 6: The experimental data were processed using Microsoft Excel 2007 software, and the leaf tissue anatomical structure indicators of different water chestnut varieties were statistically analyzed using DPSv14.10 statistical software. The Duncan multiple comparison test was used to test the significance of the differences, and the corresponding charts were drawn using Microsoft Excel 2007 software.

[0029] When making paraffin sections of water chestnuts, the water chestnuts need to be trimmed, dehydrated, embedded, sliced, stained, and sealed. When slicing, the water chestnut slices are of appropriate size to facilitate subsequent staining operations, so that the water chestnuts can be more fully and evenly colored during staining. At the same time, when staining is completed, the structure of each tissue of the water chestnut will be more obvious, which makes it easier to select the required tissue for observation. When the paraffin sections are completed, 3 small pieces will be collected from each sample tissue, for a total of 30 sections. By preparing multiple paraffin sections of the same water chestnut, multiple tissues of the same water chestnut can be observed at the same time, thereby improving the observation efficiency to a certain extent. When observing microscopic photos, the water chestnut tissue must fill the entire field of view, and the background light of each photo must be consistent. In this way, the influence of different light sources on the observation is reduced, thereby reducing errors and making the observation results of various water chestnut tissues more objective.

[0030] Experimental process:

[0031] 1. Anatomical structural characteristics of the leaf-like stem tissue of water chestnut plants and differences among different varieties

[0032] Water chestnuts lack leaves, and their stems are commonly called leaf-like stems. Their cross-section is very similar to that of other cultivars of the same genus, such as the shell-leaved water chestnut and the transparent-scaled water chestnut. The cross-section is circular or nearly circular, consisting of the epidermis, assimilated tissue, vascular bundles, and air cavities.

[0033] Comparison of the anatomical structure of the phyllodes of 10 water chestnut varieties revealed circumferences ranging from 13.295 to 17.677 mm, with 'Dahongpao' being the smallest and 'Guiti No. 3' being the largest. Thickness ranged from 0.404 to 0.661 mm, with 'Yangjiang Zhenzhu Ma Ti' being the thinnest and 'Guiti No. 3' being the thickest. The outer epidermis of all varieties showed varying degrees of cuticle differentiation, with thickness ranging from 11.699 to 14.691 μm, with Dahongpao being the thinnest, 'Guiti No. 3' being the second thinnest, and 'Guiti No. 2' being the thickest. Cuticle thickness ranged from 1.436 to 2.133 μm, with 'Guifenti No. 1' being the thinnest and 'Guiti No. 2' being the thickest. Within the epidermis, assimilated tissue was present, arranged in a palisade pattern, as shown in Table 2.

[0034] The assimilation tissue is located inwards of the vascular bundle, which is composed of the vascular bundle sheath, phloem and xylem. The number of vascular bundles is between 46 and 56, and the average number of vascular bundles per unit length is between 2.979 and 3.987. The metaxylem is on both sides of the xylem. There are air cavities between the vascular bundles, and inwards is the central air cavity. Figure 2 It can be seen that the arrangement patterns of the vascular bundles of the leaf-like stems of different varieties are not the same. The vascular bundles of most varieties are arranged in a circular shape and alternately in the parenchyma tissue of the leaf-like stems.

[0035] Table 2 Comparison of anatomical indices of phyllodes of different water chestnut varieties

[0036]

[0037] Note: Data are the mean ± standard deviation of 3 replicates. Different size letters in the same column indicate significant differences between species (P < 0.05\P < 0.01)

[0038] 2. Differences in vascular bundle structure among different water chestnut varieties

[0039] In the microscopic structure of rice and wheat stems, vascular bundles play a crucial role in lodging resistance. The vascular bundle sheath, which primarily supports the stem, acts as a skeleton. The length and width of the vascular bundles are good indicators of stem quality. There is no significant difference in the number of vascular bundles between lodging-resistant and lodging-prone varieties, with Yangjiang Pearl Horseshoe having the fewest.

[0040] The width and length of the vascular bundles of the lodging-resistant varieties 'Guiti No. 3', 'Guiti No. 2', 'Guiti No. 4', 'Hezhou Fanglin', and 'Anhui Fuyang' were larger than those of the lodging-prone varieties 'Dahongpao', 'Yangjiang Zhenzhu Mati', 'Guifenti No. 1', 'Hubei Zhijiang', and 'Guizhou Anshun', as shown in Table 3.

[0041] The vascular bundle consists of three parts: the bundle sheath, phloem, and xylem. The bundle sheath primarily supports the vascular bundle, and its thickness indirectly contributes to its flexural strength. Lodging-resistant varieties have thicker, more densely packed bundle sheaths, while lodging-resistant varieties have thinner bundle sheaths.

[0042] Table 3 Vascular bundle structural characteristics of different water chestnut varieties

[0043]

[0044] Note: Data are the mean ± standard deviation of 3 replicates. Different size letters in the same column indicate significant differences between species (P < 0.05\P < 0.01)

[0045] 3. Differences in the anatomical structure of the tissues at different locations at the base of the leaf-like stem of the water chestnut variety 'Guiti No. 3'

[0046] 3.1 Anatomical differences

[0047] Figure 3 This image shows the anatomical structure of the 'Guiti No. 3' leaf-like stem at different locations from the base. The differences are in the structure of the assimilated tissue, vascular bundles, and air cavities. The assimilated tissue becomes denser and denser in its palisade-like arrangement as it moves further from the base, particularly at 50 and 75 cm, while the vascular bundles and air cavities become more oblate.

[0048] Table 4 shows that the phyllodes of Guiti No. 3 are thickest at the base and thinner as they get further away, with similar circumferences. Epidermal thickness ranks 35 cm at the base, 50 cm at the base, 18 cm at the base, and 75 cm at the base, with thicker layers in the middle. The cuticle is thickest at the base 50 cm, followed by the base 75 cm, and thinnest at the base 18 cm.

[0049] Table 4 Anatomical indices of tissues at different positions of the base of the phyllodes of 'Guiti 3'

[0050]

[0051] Note: Data are the mean ± standard deviation of 3 replicates. Different size letters in the same column indicate significant differences between species (P < 0.05\P < 0.01)

[0052] 3.2 Differences in vascular bundle structure

[0053] As can be seen from Table 5, the number of vascular bundles in the leaf-like stem tissue of Guiti No. 3 is the largest at 75 cm from the base; its width is the largest at 50 cm from the base, followed by 18 cm from the base, and finally 75 cm from the base; its length is the longest at the lower part of the base and becomes shorter as the distance becomes farther, and the thickness of the vascular bundle sheath is also similar.

[0054] Table 5 Characteristics of vascular bundle structure at different locations of the base of the phyllodes of 'Guiti 3'

[0055]

[0056] Note: Data are the mean ± standard deviation of 3 replicates. Different size letters in the same column indicate significant differences between species (P < 0.05\P < 0.01)

[0057] Experimental conclusion:

[0058] The field growth cycle of water chestnuts consists of four phases: seedling establishment, tillering (vegetative growth), corm formation (a period of concurrent vegetative and reproductive growth), and corm expansion. During the corm formation phase, nutrients from the aboveground portion of the plant rapidly transfer to the underground to fuel corm expansion, reducing resistance. Furthermore, during this phase, field density is high, and the phyllodes of water chestnut varieties are less resistant to lodging, which can easily lead to large-scale lodging in the field. This significantly reduces the effective photosynthetic area of ​​the phyllodes and accelerates pest and disease damage, severely impacting corm expansion and resulting in reduced or even complete crop failure. Therefore, in the variety breeding process, lodging resistance should be considered as a key indicator to ensure high and stable yields.

[0059] Many factors influence the mechanical strength of plant stems, including morphological traits, anatomical characteristics, and chemical composition. Studies in stem-bearing plants such as rice, wheat, maize, and buckwheat have shown that stem lodging resistance is closely related to stem anatomical structure. The number and thickness of mechanical tissue layers, stem wall thickness, number of large vascular bundles, vascular bundle area, and vascular bundle sheath thickness are correlated with lodging resistance. Lodging-resistant cultivars have greater numbers of mechanical tissue cell layers, stem wall thickness, number of large vascular bundles, vascular bundle area, and vascular bundle sheath thickness at the stem base than lodging-prone cultivars. This study focused on analyzing the microstructure of the water chestnut (Cardiff chub) phyllodes, the differences between cultivars, and their relationship to lodging resistance. Results showed that the phyllodes are circular or nearly circular in cross section and consist of the epidermis, assimilated tissue, vascular bundles, and air cavities. Among the 10 tested varieties, there were differences in epidermal thickness, cuticle thickness, assimilated tissue, and vascular bundle structures. The lodging-resistant varieties 'Guiti 3,' 'Guiti 2,' and 'Guiti 4' had vascular bundle widths and lengths greater than those of the lodging-susceptible varieties. Their vascular bundle sheath cells were thicker and more densely packed. The vascular bundle sheaths of the lodging-resistant varieties were thinner, consistent with research findings in rice. However, there was no significant relationship between vascular bundle number between lodging-resistant and lodging-susceptible varieties, which differs from studies in other crops [9, 10, 15, 16, 21, 22] and may be closely related to the growth characteristics of water chestnuts. 'Guiti 3,' a recently developed variety, has demonstrated lodging resistance in the field for three consecutive years. Its anatomical structure at different locations near the base of the spur showed differences in assimilated tissue, vascular bundles, and air cavities. The farther away from the base, the denser the palisade arrangement of the assimilated tissue, especially at 50 and 75 cm from the base, while the vascular bundles and air cavities become more oblate.

[0060] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0061] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for identifying lodging resistance of water chestnuts, characterized in that: The following steps are included: Step 1, selecting 10 water chestnut varieties, wherein the 10 water chestnut varieties include Guifenti No. 1, Guiti No. 2, Guiti No. 3, Guiti No. 4, Yangjiang Pearl Water Chestnut, Hezhou Fanglin, Dahongpao, Anshun, Guizhou, Zhijiang, Hubei, and Fuyang, Anhui; Step 2: Trimming, dehydrating, embedding, slicing, staining, and mounting the selected samples to prepare paraffin sections. Three small pieces of each tissue sample were collected, and a total of 30 sections were prepared from the tissue sample. Step 3: Observe the prepared paraffin sections with a microscope and take photos. Step 4: The micrographs were magnified at any magnification of 1-400 times using software and then observed. The micrographs were scanned using software, and then the target area of ​​the desired tissue was selected for imaging at 200 times. The tissue was imaged so that it filled the entire field of view and the background light was consistent in each photo. Step 5: After imaging is completed, use analysis software to standardize the micrographs in millimeters and measure the anatomical structure indicators of each water chestnut leaf slice. The measured indicators include epidermal thickness, cuticle thickness, tissue thickness, vascular bundle number, perimeter, and number of vascular bundles per unit length. Each indicator is randomly measured 20 times at different locations on each slice. Step six: The experimental data were processed using software, and then statistical software was used to perform statistical analysis on the leaf tissue anatomical structure indicators of different water chestnut varieties. The significance of the differences among different water chestnut varieties was tested using the new multiple range method for multiple comparisons, and the corresponding charts were drawn using the software.

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