Method for collecting sieve tube overflow substances in bark of rubber tree

Abstract

The invention discloses a method for collecting sieve tube overflow substances in rubber tree barks, and belongs to the technical field of sieve tube overflow substance acquisition. Rubber tree coppice shoots growing within two years are selected; a section of stem is selected from the third extension unit or the fourth extension unit, the outermost epidermis and part of the cortex layer of the bark are scraped firstly, and wiping is carried out after primary latex exudates completely; after that, scraping off the remaining part of cortex, primary phloem fiber layer and primary phloem until the secondary phloem containing the sieve tube tissue is exposed, puncturing the secondary phloem, and sucking the discharged sieve tube overflow in the secondary phloem; a V-shaped opening is upwards cut in the secondary phloem part along the punctured part, and the sieve tube overflowing matter flowing out subsequently is sucked. The method is implemented on a specific rubber tree material in a specific period, a secondary milk duct is not generated in the bark, and pure sieve tube overflow of the rubber tree can be obtained by removing the primary milk duct and the latex firstly, then obtaining the sieve tube overflow and eliminating interference of the latex in the milk duct.

Description

Technical Field

[0001] This invention relates to the field of sieve tube exudate acquisition technology, specifically to a method for collecting sieve tube exudate from the bark of rubber trees. Background Technology

[0002] The sieve tubes in the phloem of rubber trees are the core channels for transporting photosynthetic products, minerals and other nutrients. The sucrose, amino acids, plant hormones and various metabolic intermediates contained in the sieve tube secretions are key materials for studying the material transport mechanism, stress resistance physiological characteristics and latex synthesis regulation mechanism of rubber trees.

[0003] Currently, common methods for obtaining plant sieve tube exudates mainly include pruning and cutting, bark girdling, and needle collection. However, because rubber trees are covered with latex cells, these methods all struggle to avoid the mixing of latex flowing from ruptured latex cells with the sieve tube exudates.

[0004] Based on this, the present invention designs a method for collecting the exudate from the sieve tubes in the bark of rubber trees to solve the problem of latex mixing with the exudate caused by the rupture of latex tubes in rubber trees. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the existing technology, the present invention provides a method for collecting the exudate from the sieve tubes in the bark of rubber trees.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for collecting phloem exudate from the bark of rubber trees includes the following steps: a) Select shoots from rubber trees that are less than two years old; b) Select a section of stem from the third or fourth elongation unit of the selected rubber tree shoot in step a). At this time, there are only primary latex ducts in the bark and no secondary latex ducts have been produced. This section can be used to collect the exudate from the sieve tubes later. c) On the selected middle section of the trunk, according to the structure of the rubber tree bark, first scrape off the outermost epidermis and part of the bark, wait for the primary latex to seep out, and then wipe it off. d) Then scrape off the remaining part of the cortex, then scrape off the primary phloem fiber layer, and then scrape off the primary phloem until the secondary phloem containing sieve tube tissue is exposed. e) Use the tip of a pipette to puncture the cavity inside the secondary phloem, including the space between the cambium and the secondary xylem, and aspirate the secretions from the sieve tubes inside the secondary phloem. f) After the sieve tube secretions in the secondary phloem have been aspirated, use a single-edged blade to cut a V-shaped opening upwards from the puncture site on the secondary phloem. The confluence point is where the pipette tip will draw the liquid, and aspirate the subsequent sieve tube secretions flowing out from this depression.

[0007] Furthermore, in step a), the rubber tree sprout must have at least 5 elongation units.

[0008] Furthermore, in step b), a section of stem is selected from the third elongation unit of the rubber tree sprout.

[0009] Furthermore, the stem length should be at least 5 cm, and the stem should be relatively straight and without buds.

[0010] Furthermore, in step b), the structure of the rubber tree bark is as follows: from the outside in, it consists of the epidermis, cortex, primary phloem fibers, primary phloem, secondary phloem, cambium, and xylem.

[0011] Furthermore, in step b), the colors of the bark layers in the selected middle section of the stem are: The epidermis is dark green with interlaced brown lenticels; The cortex is green; The nascent bast fibers are smooth and yellowish-green; The initial phloem is fleshy and pale green; The secondary phloem is fleshy and pale yellowish-green; The cambium layer is pale yellow; The woody part is beige.

[0012] Furthermore, the dark green epidermis accounts for more than 60% of the total area.

[0013] Furthermore, in steps c) and d), the method of scraping away the rubber tree bark layer by layer is as follows: use a single-edged blade to scrape away part of the bark tissue back and forth perpendicular to the trunk direction, while the single-edged blade can also shave away part of the bark tissue along the tree's growth direction and parallel to the trunk direction.

[0014] Furthermore, in step d), after exposing the secondary phloem, some of the secondary phloem cells are scraped away.

[0015] Furthermore, in step e), when the single-edged blade scrapes back and forth the outer primary phloem fiber layer and primary phloem, the sieve tubes in the secondary phloem are squeezed, causing the cambium and woody part to separate out of the cavity, and the sieve tube secretions after the sieve tubes break seep into the cavity.

[0016] Compared with the prior art, the beneficial effects of this invention are as follows: This invention is implemented on specific rubber tree materials at a specific time, where secondary latex ducts have not yet formed in the bark. By first removing the primary latex ducts and latex, and then obtaining the sieve tube exudate, the interference of latex in the latex ducts can be eliminated, thus obtaining pure sieve tube exudate from the rubber tree.

[0017] This invention employs a method of peeling bark layer by layer from the outside in, accurately obtaining secondary phloem tissue containing only functional sieve tubes. This method yields a larger amount of sieve tube exudate and eliminates interference from latex in the latex ducts. Furthermore, by collecting sieve tube exudate only in a selected area, it minimizes damage to the entire rubber tree's shoots. After each collection, the treated area of ​​the rubber tree trunk can be wrapped with plastic wrap. After a period of time, callus tissue forms and heals at the damaged site, without affecting the survival of the rubber tree shoots or the collection of buds from other parts of the shoots. This invention offers the advantages of accurate material collection, good repeatability, and versatility. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 A schematic diagram of the collection of exudates from the sieve tubes of a rubber tree (left) and the structure of the sprouting stem of a rubber tree (right); Figure 2 A schematic diagram of the experimental steps for collecting sieve tube exudates from the bark of rubber trees: 1. Select a suitable rubber tree shoot stem; 2. Scrape off the epidermis and part of the bark of the stem, which will exude white primary latex; 3. Wait for the latex to exude completely and then wipe it off, scraping away the remaining part of the bark, leaving an intact primary phloem fiber layer; 4. The scraped primary phloem fibers; 5. Scrape open the primary phloem fiber layer, exposing a small amount of primary phloem; 6. From the opening in the primary phloem fiber layer, with the blade parallel to the trunk, shave off the primary phloem fibers to both sides, exposing the intact primary phloem; 7. With the blade perpendicular to the trunk, slowly scrape away the primary phloem tissue layer by layer, exposing the intact secondary phloem; 8. Absorb the sieve tube exudates from the rubber tree shoot at the suction point of the lancet, and use a single-edged blade to cut a V-shaped opening upwards from the puncture point on the secondary phloem, and absorb the subsequent sieve tube exudates flowing out in this depression.

[0020] Figure 3 This diagram illustrates an experiment to verify the absence of latex contamination by collecting exudates from the sieve tubes of rubber tree bark. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] Previous studies, through morphological and anatomical experiments, revealed significant differences in the location and number of primary and secondary latex ducts in the bark of one- and two-year-old rubber tree shoots at different elongation units. Primary latex ducts, differentiated from the apical meristem, are scattered and disordered from the shoot tip and down to the first elongation unit, and are located in the primary phloem. Secondary latex duct cells, sieve tube cells, and phloem ray cells, differentiated from vascular cambium cells, are secondary structures and are located in the secondary phloem. However, under natural growth conditions, secondary latex ducts are absent in the bark of one-year-old rubber tree shoots that are green or have few lenticels (first to fourth elongation units); their secondary phloem contains only sieve tube cells and phloem ray cells. Secondary latex ducts only appear in the fully browned periderm (fifth elongation unit) of one-year-old shoots. In different elongation units of one-year-old shoots of rubber trees, treatment with mechanical damage and external application of jasmonic acid can induce the differentiation of secondary latex ducts into the vascular cambium. These secondary latex ducts are arranged in concentric circles parallel to the vascular cambium. The two cell types, secondary latex ducts and sieve tubes, show significant differences in their distribution location in the bark, cell morphology, and intracellular contents.

[0023] Example 1: A method for collecting exudates from the sieve tubes of rubber tree bark, comprising the following steps: a) Select shoots from rubber trees that are less than two years old; b) Select a section of stem from the third or fourth elongation unit of the selected rubber tree shoot in step a) for subsequent collection of sieve tube exudate; c) On the selected middle section of the trunk, according to the structure of the rubber tree bark, first scrape off the outermost epidermis and part of the bark, wait for the primary latex to seep out, and then wipe it off. d) Then scrape off the remaining part of the cortex, then scrape off the primary phloem fiber layer, then scrape off the primary phloem until the secondary phloem is exposed, and scrape off some of the secondary phloem cells.

[0024] e) Use the tip of a pipette to puncture the cavity inside the secondary phloem, including the space between the cambium and the secondary xylem, and aspirate the secretions from the sieve tubes inside the secondary phloem. f) After the sieve tube secretions in the secondary phloem have been aspirated, use a single-edged blade to cut a V-shaped opening upwards from the puncture site on the secondary phloem. The confluence point is where the pipette tip will draw the liquid, and aspirate the subsequent sieve tube secretions flowing out from this depression.

[0025] Preferably, in step a), the rubber tree sprouts must have at least 5 elongation units.

[0026] Preferably, in step b), a section of stem is selected from the third elongation unit of the rubber tree sprout, with a stem length of at least 5 cm, and the stem is selected to be relatively straight and without buds.

[0027] Preferably, in step b), the structure of the rubber tree bark is as follows: from the outside to the inside, it consists of the epidermis, cortex, primary phloem fibers, primary phloem, secondary phloem, cambium, and xylem.

[0028] Preferably, in step b), the colors of the bark layers in the selected middle section of the stem are: The epidermis is dark green with interspersed brown lenticels, and the area of ​​the dark green epidermis should reach more than 60%.

[0029] The cortex is green; The nascent bast fibers are smooth and yellowish-green; The initial phloem is fleshy and pale green; The secondary phloem is fleshy and pale yellowish-green; the sieve tubes that transport organic nutrients longitudinally in the bark are mainly concentrated in the secondary phloem. The cambium layer is pale yellow; The woody part is beige.

[0030] Preferably, in steps c) and d), the method of scraping off the bark tissue of the rubber tree layer by layer is as follows: a single-edged blade is used to scrape off part of the bark tissue back and forth perpendicular to the trunk direction. At the same time, the single-edged blade can be used to remove part of the bark tissue along the tree growth direction and parallel to the trunk direction.

[0031] Preferably, in step e), when the sieve tubes in the secondary phloem are scraped back and forth by a single-edged blade to remove the outer primary phloem fiber layer and primary phloem, the sieve tubes in the secondary phloem are squeezed, causing the cambium and the woody part to separate out of the cavity. The sieve tube secretions after the sieve tubes are broken have already seeped into the cavity. Initially, a 200 μl pipette tip can be used to puncture the cavity and quickly aspirate the flowing sieve tube secretions.

[0032] Example 2: Collecting sieve tube exudate from the third elongation unit of a one-year-old rubber tree shoot. One-year-old shoots of the vigorous rubber tree clonal variety Reyan 7-33-97 with five elongation units were selected. At the third elongation unit of the shoots, sieve tube exudates were obtained using the method of this invention. A 10 cm long, straight stem without buds on the bark was selected from the third elongation unit of the selected rubber tree shoots for subsequent collection of sieve tube exudates.

[0033] The specific steps are as follows: Using a sharp single-edged blade perpendicular to the trunk, with moderate and rapid force, scrape away the outermost bark and part of the outer layer of bark from top to bottom, waiting for the primary latex to seep out and then wiping it away; then scrape away the overflowing latex while scraping the bark until reaching the harder, more bast fiber layer; then, with slightly increased force and a slightly slower speed, use the blade perpendicular to the trunk to scrape away part of the primary bast fiber layer back and forth, repeatedly scraping and breaking through part of the primary bast fiber layer, and then using the single-edged blade... Following the direction of the fiber layer damage, parallel to the trunk, carefully slice away the entire primary phloem fiber layer, reaching the pale green primary phloem. Then, using a blade perpendicular to the trunk, gently and slowly scrape away the fleshy primary phloem until the pale yellowish-green secondary phloem is exposed. Due to the abundance of sieve tubes and the compression of the sieve tubes during scraping, the sieve tube secretions have seeped into the cambium region between the bark and xylem. Since the volume of sieve tube secretions in the secondary phloem is approximately 30-50 μl, use the tip of a 200 μl pipette to puncture the secondary phloem and aspirate the sieve tube secretions quickly. After aspirating the sieve tube secretions from the secondary phloem, use a single-edged blade to cut two V-shaped openings in the secondary phloem, the confluence of which is the puncture point of the pipette tip. Aspirate any subsequent sieve tube secretions flowing out from this depression.

[0034] Results: Approximately 50-60 μl of sieve tube exudate could be collected from the third elongation unit of a robust one-year-old shoot of the clonal rubber tree cultivar Reyan 7-33-97.

[0035] Example 3: Collecting sieve tube exudate from the fourth elongation unit of sprouts in two-year-old rubber trees. Since robust, one-year-old rubber tree shoots with five complete elongation units require nearly a year to mature and are limited in number, two-year-old shoots of the clonal rubber tree variety Reyan 7-33-97 with five to eight elongation units are selected. The method of this invention is used to obtain sieve tube exudates at the fourth elongation unit. A 15 cm long, straight stem without buds on the bark is selected from the fourth elongation unit of the chosen two-year-old rubber tree shoot for subsequent collection of sieve tube exudates.

[0036] The specific steps are the same as in Example 2. Using this method, the volume of secretions from the sieve tubes in the secondary phloem is approximately 30-60 μl.

[0037] Results: Approximately 40-80 μl of sieve tube exudate could be collected from the fourth elongation unit of a two-year-old shoot of the clonal rubber tree cultivar Reyan 7-33-97.

[0038] Experiments in Examples 2 and 3 demonstrate that the present invention, employing a method of peeling bark layer by layer from the outside in, accurately obtains secondary phloem tissue containing only functional sieve tubes. This method yields a greater amount of sieve tube exudate and eliminates interference from latex in the latex ducts. Furthermore, by collecting sieve tube exudate only in a selected area, it minimizes damage to the entire rubber tree's shoots. After each collection, the treated area of ​​the rubber tree trunk can be wrapped with plastic wrap. After a period of time, callus tissue forms and heals at the damaged site, without affecting the survival status of the rubber tree shoots or the collection of buds from other parts of the shoots.

[0039] Experimental example: such as Figure 3 As shown, Figure 3 A represents the sieve tube exudate obtained from the sprouts of five rubber trees; 1 represents the total RNA extraction buffer containing the sieve tube exudate from the rubber trees; and 2 represents the total RNA extraction buffer control. It can be seen that the sieve tube exudate is light yellow in color.

[0040] Figure 3 B is the electrophoresis diagram of total RNA from the sieve tube exudate obtained from the sprouts of rubber trees. 1 is the sieve tube exudate obtained from the sprouts of 5 rubber trees, and 2 is the sieve tube exudate obtained from the sprouts of 3 rubber trees. This shows that the method can obtain qualified sieve tube exudate and can be used for subsequent molecular experiments. The sieve tube exudate obtained from the sprouts of 5 rubber trees (1) and 3 rubber trees (2) showed clear 28sRNA and 18sRNA bands on the electrophoresis diagram after electrophoresis. The brightness of the 28sRNA band was about 2:1 compared with the 18sRNA band, indicating that the sieve tube exudate collected by this sampling method contains RNA and the quality of the extracted total RNA is high, which can be used for subsequent molecular experiments.

[0041] Figure 3 C represents the rubber elongation factor (REF) gene, which is specifically expressed in latex. HbREF, The expression of this substance was detected in primary latex and sieve tube exudate, and its expression level can reflect whether latex contamination exists in the collected sieve tube exudate. This method yields sieve tube exudate from rubber trees containing this substance. HbREF The gene expression level was extremely low, showing a significant difference compared to the control group (based on the latex content of the same plant). HbREF (Gene expression level is used as a control), thus proving that this sampling method can avoid latex contamination of rubber tree latex cells during the extraction of sieve tube cell exudates.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for collecting exudates from the sieve tubes of rubber tree bark, characterized in that, Includes the following steps: a) Select shoots from rubber trees that are less than two years old; b) Select a section of stem from the third or fourth elongation unit of the selected rubber tree shoot in step a) for subsequent collection of sieve tube exudate; c) On the selected middle section of the trunk, according to the structure of the rubber tree bark, first scrape off the outermost epidermis and part of the bark, wait for the primary latex to seep out, and then wipe it off. d) Then scrape off the remaining part of the cortex, then scrape off the primary phloem fiber layer, and then scrape off the primary phloem until the secondary phloem containing sieve tube tissue is exposed. e) Use the tip of a pipette to puncture the cavity inside the secondary phloem, including the space between the cambium and the secondary xylem, and aspirate the secretions from the sieve tubes inside the secondary phloem. f) After the sieve tube secretions in the secondary phloem have been aspirated, make a V-shaped opening on the secondary phloem along the puncture site upwards. The confluence point is where the pipette tip will draw the liquid, and aspirate the subsequent sieve tube secretions flowing out from this depression.

2. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In step a), the rubber tree sprout must have at least 5 elongation units.

3. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In step b), a section of stem is selected from the third elongation unit of the rubber tree sprout.

4. The method for collecting exudates from the sieve tubes of rubber tree bark according to claim 1, characterized in that, The stem should be at least 5 cm long, and should be relatively straight and without buds.

5. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In step b), the structure of the rubber tree bark is as follows: from the outside to the inside, it consists of the epidermis, cortex, primary phloem fibers, primary phloem, secondary phloem, cambium, and xylem.

6. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In step b), the colors of the bark layers in the selected middle section of the stem are: The epidermis is dark green with interlaced brown lenticels; The cortex is green; The nascent bast fibers are smooth and yellowish-green; The initial phloem is fleshy and pale green; The secondary phloem is fleshy and pale yellowish-green; The cambium layer is pale yellow; The woody part is beige.

7. The method for collecting sieve tube exudates from rubber tree bark according to claim 6, characterized in that, In the epidermis, the dark green epidermis accounts for more than 60% of the area.

8. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In steps c) and d), the method of scraping off the bark tissue of the rubber tree layer by layer is as follows: use a single-edged blade to scrape off part of the bark tissue back and forth perpendicular to the trunk direction. At the same time, the single-edged blade can also be used to remove part of the bark tissue along the tree's growth direction and parallel to the trunk direction.

9. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In step d), after the secondary phloem is exposed, some of the secondary phloem cells are scraped away.

10. The method for collecting sieve tube exudates from rubber tree bark according to claim 1, characterized in that, In step e), when the single-edged blade scrapes back and forth the outer primary phloem fiber layer and primary phloem, the sieve tubes in the secondary phloem are squeezed, causing the cambium and woody part to separate out of the cavity, and the sieve tube secretions after the sieve tubes break seep into the cavity.

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