CN112900126B - Method for separating vascular bundle from dicotyledonous plant root and hypocotyl - Google Patents
Method for separating vascular bundle from dicotyledonous plant root and hypocotyl Download PDFInfo
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- CN112900126B CN112900126B CN202110055550.3A CN202110055550A CN112900126B CN 112900126 B CN112900126 B CN 112900126B CN 202110055550 A CN202110055550 A CN 202110055550A CN 112900126 B CN112900126 B CN 112900126B
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- D21B1/00—Fibrous raw materials or their mechanical treatment
- D21B1/04—Fibrous raw materials or their mechanical treatment by dividing raw materials into small particles, e.g. fibres
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Abstract
The invention discloses a method for separating vascular bundles from dicotyledonous plant roots and hypocotyls, which comprises the following steps: plant cultivation, fixation, vascular bundle stripping, cellulase and pectinase digestion and impurity contamination removal by using Triton X-100. According to the invention, tea saponin is utilized to assist the separation of the vascular bundle and adjacent tissues in the fixing step, and then cellulase and pectinase are utilized to carry out combined digestion treatment, so that cell walls, intercellular residues, fibrous tissues, membrane-like substance residues and the like in the vascular bundle and the adjacent tissues are effectively removed, Triton X-100 is further utilized to elute tissues around the vascular bundle, and adhesion pollution of membrane-like soft tissues is effectively removed, so that the vascular bundle is separated from roots and hypocotyls, and the vascular bundle separated by the method can be observed under a microscope with clear morphological structure and no impurity influence.
Description
Technical Field
The invention belongs to the technical field of vascular bundle separation, and particularly relates to a method for separating vascular bundles from dicotyledonous plant roots and hypocotyls.
Background
Vascular bundles are the transport and conduction system of vascular plants, often present bundle-like structures in the plant body, penetrating through various organs of the plant body. The device mainly comprises a phloem and a xylem, wherein the phloem comprises a sieve tube and a companion cell and is mainly responsible for transporting an assimilate; xylem is mainly composed of vessels for transporting water and inorganic salts. Besides the transportation function, the vascular bundle also plays a mechanical supporting role for the plant organs.
The research on the formation and development of vascular bundles has been widely regarded by the academia, and in the prior art, the occurrence, development and structure of some parts of plants are generally explored in multiple dimensions by means of microscopic and ultra-microscopic techniques, but the prior known microscopic technique for observing high-definition vascular bundle morphological structures has not appeared yet, and the main reason is that the prior art lacks a vascular bundle complete separation technique, thereby limiting the deep research on the morphological development of vascular bundles.
Disclosure of Invention
The invention aims to provide a method for separating vascular bundles from dicotyledonous plant roots and hypocotyls, which comprises the following steps: soaking, rinsing and fixing roots and hypocotyls of plants, separating vascular bundles from adjacent tissues under a microscope, performing combined digestion treatment on the dissected materials by using cellulase and pectinase, and further eluting membrane-like soft tissues around the vascular bundles by using Triton X-100 so as to realize the separation of the vascular bundles, wherein the vascular bundles obtained by the separation method can be observed with a clear morphological structure under the microscope.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method of isolating vascular bundles from dicot roots and hypocotyls comprising the steps of:
p1: selecting plant seeds, performing surface disinfection, sowing the seeds into a culture medium, and culturing the seeds;
p2: selecting a robust complete plant growing in P1, removing leaves, reserving roots and hypocotyls, and placing in a centrifuge tube;
p3: adding a PEM (proton exchange membrane) buffer solution containing 0.1 volume percent of tea saponin into a centrifugal tube of P2, soaking for 30-40 minutes, then rinsing with the PEM buffer solution, fixing the rinsed material in a PBS buffer solution containing 4 volume percent of paraformaldehyde for 30-40 minutes, and then rinsing with the PEM buffer solution again; the step adds tea saponin into PEM buffer solution, and the tea saponin can quickly infiltrate into root and hypocotyl tissues to reduce the connection between vascular bundle and adjacent tissues, thereby facilitating the stripping of vascular bundle.
P4: under a dissecting microscope, separating the vascular bundle from adjacent tissues by using a dissecting needle, taking out the vascular bundle, and putting the vascular bundle into a centrifuge tube filled with a PEM buffer solution; the dissecting material separated by the dissecting needle contains not only the vascular bundle but also various cells such as residual walls of vascular bundle sheath cells, fibrous tissues, membrane-like substances, and intercellular residues, and thus cannot be directly used for observing the morphological structure of the vascular bundle.
P5: taking the dissected material in P4, placing into enzyme solution containing cellulase and pectinase, and digesting at 28 deg.C; in the step, cellulase and pectinase are used for combined digestion treatment, so that residual cell walls, intercellular residues, fibrous tissues, partial membrane-like substances and the like in the anatomical material can be effectively removed.
P6: the completely digested material of P5 was washed in Triton X-100 for 15-20 min, and then washed with PEM buffer solution to complete the separation of vascular bundles in plant roots and hypocotyls. In the step, Triton X-100 is further adopted to clean the vascular bundle so as to remove tissues around the vascular bundle and effectively eliminate adhesion pollution of membrane-like soft tissues.
Further, in step P5, the components of the enzyme solution include: 5mM MES buffer, 0.5% (w/v) cellulase, 0.2% (w/v) pectinase, 0.12M sucrose and 1mM CaCl2And the pH was adjusted to 5.5.
Further, the PEM buffer comprises: 50mM PIPES (piperazine-1, 4-diethylsulfonic acid), 5mM EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), 2.5mM MgSO4And the pH was adjusted to 6.9.
Further, in step P1, the plant seeds are surface sterilized with chlorine in a fume hood.
Further, in step P1, the surface-sterilized seeds were sown on MS medium and cultured for 7-30 days under 16h light/8 h dark at 22-23 ℃.
Further, step P3 is specifically: adding 20mL of PEM buffer solution containing 0.1% by volume of tea saponin into a centrifugal tube of P2, soaking for 30-40 minutes, rinsing with PEM buffer solution for 3 times for 20 minutes each time, fixing the rinsed material in PBS buffer solution containing 4% by volume of paraformaldehyde under vacuum drying at-20 ℃ for 30-40 minutes, and rinsing with PEM buffer solution for 3 times for 5 minutes each time.
Further, the step P4 is repeated to obtain a plurality of dissected materials, and the number of dissected materials is not more than 6. Too much of this can lead to the material becoming entangled with one another, interfering with subsequent processing, causing the material to be damaged and unusable for viewing.
Further, during the digestion process of step P5, microscopic examination was performed at any time until complete digestion of the remaining tissue on the vascular bundle was observed. The microscopic examination is carried out at any time in the process of carrying out digestion treatment by using enzyme, so that the influence on the observation of the vascular bundle caused by residual substances due to incomplete digestion is avoided, and meanwhile, the damage to the vascular bundle caused by the digestion time process can also be avoided.
Further, the concentration of Triton X-100 in step P6 was 5%.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for separating vascular bundles from dicotyledonous plant roots and hypocotyls, which utilizes the characteristic that tea saponin can be quickly infiltrated into the tissues of the roots and the hypocotyls in the fixing step to reduce the connection between the vascular bundles and adjacent tissues so as to be convenient for stripping, then cellulase and pectinase are used for combined digestion treatment, cell walls, intercellular residues, fibrous tissues, membrane-like substance residues and the like in the vascular and adjacent tissues are effectively removed, Triton X-100 is further used for eluting the tissues around the vascular bundle, the adhesion pollution of the membrane-like soft tissues is effectively removed, so that the vascular bundle is separated from the root and hypocotyl, the vascular bundle separated by the method can be observed with a clear morphological structure under a microscope without impurity influence, the method is simple, rapid and easy to implement, has good separation effect, and can be used for research on formation and development of vascular bundles.
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FIG. 1 is an SEM picture of Arabidopsis vascular bundles isolated in example 1 of the present invention;
FIG. 2 is a SEM photograph of tobacco vascular bundles separated in example 1 of the present invention;
FIG. 3 is an SEM picture of Arabidopsis vascular bundle isolated in comparative example 1 of the present invention;
FIG. 4 is an SEM photograph of tobacco vascular bundles separated in comparative example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a method for separating vascular bundles from root and hypocotyl of dicotyledonous plants, taking arabidopsis thaliana and tobacco as examples, the specific steps are as follows:
p1: selecting a proper amount of arabidopsis thaliana and tobacco seeds, respectively placing the arabidopsis thaliana and tobacco seeds in a 1.5mL centrifuge tube, marking the names of the arabidopsis thaliana and tobacco seeds on the tube wall by using a pencil, carrying out surface disinfection on the seeds for 40 minutes in a fume hood by using chlorine, and respectively sowing the arabidopsis thaliana and tobacco seeds with the surfaces disinfected on a 1 × MS culture medium. Placing the culture medium in a culture room, and vertically growing for 15 days under long-day conditions (16h light/8 h dark) at 22-23 deg.C;
p2: respectively selecting healthy and complete arabidopsis thaliana plants and tobacco plants from P1, removing leaves, taking off hypocotyls and root parts, respectively placing the hypocotyls and the root parts in 50mL centrifuge tubes, and marking the names of arabidopsis thaliana and tobacco by using pencils on the walls of the centrifuge tubes.
P3, fixing the material, which comprises the following steps:
A. adding 20mL of PEM buffer solution containing 0.1% by volume of tea saponin into a centrifugal tube of P2, and soaking for 30 minutes, wherein the formula of the PEM buffer solution is as follows: 50mM PIPES (piperazine-1, 4-diethylsulfonic acid), 5mM EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), 2.5mM MgSO4And the pH was adjusted to 6.9. Wherein the tea saponin can rapidly infiltrate into root and hypocotyl tissues to reduce the connection between vascular bundle and adjacent tissues, thereby facilitating the stripping of vascular bundle.
B. Then rinsed 3 times with PEM buffer for 20 minutes each time;
C. fixing the rinsed material in 1 XPBS buffer solution containing 4% paraformaldehyde, and performing vacuum infiltration treatment for 30 minutes in a low vacuum drier (from CHRIST) at-20 deg.C;
D. the immobilized material was washed 3 times for 5 minutes each with PEM buffer;
p4: under a dissecting microscope, the vascular bundle is separated from adjacent tissues by using the needle point of a dissecting needle, the vascular bundle is taken out by using tweezers and is respectively placed into 2mL centrifuge tubes filled with 1mL of PEM solution, and the names of Arabidopsis and tobacco are marked by using pencils on the centrifuge tube walls. And the number of the dissecting materials in each centrifugal tube must not exceed 6, so that the dissecting materials are prevented from being wound together to influence the subsequent treatment and damage the materials. The dissecting material obtained by separation with a dissecting needle contains not only the vascular bundle but also various cells or intercellular residues such as cell residual walls, membrane-like substances and the like, cannot be directly used for observing the morphological structure of the vascular bundle, and needs to be subjected to subsequent treatment.
P5 enzymatic digestion of dissected material, the specific steps are as follows:
A. 2 centrifuge tubes of 2mL are prepared, the tube wall marks the names of arabidopsis and tobacco by a pencil, and 1mL of prepared enzyme solution is placed in each centrifuge tube, wherein the enzyme solution specifically comprises the following components: 5mM MES buffer, 0.5% (w/v) cellulase, 0.2% (w/v) pectinase, 0.12M sucrose and 1mM CaCl2And the pH was adjusted to 5.5. The cellulase and the pectinase are utilized for combined digestion treatment, so that the residual cell walls, intercellular residues, fibrous tissues, partial membrane-like substances and the like attached to the vascular bundles can be effectively digested and removed;
B. taking out the dissected material in the step P4 by using tweezers, correspondingly putting the dissected material into a 2mL centrifuge tube filled with enzyme solution, and carrying out digestion treatment, wherein the centrifuge tube is placed on a shaking table in the digestion treatment process, the temperature of the shaking table is maintained at 28 ℃, and the rotating speed is maintained at 120 rpm. In the digestion treatment process, microscopic examination is carried out at any time until the residual tissues on the vascular bundle are completely digested, so that incomplete digestion of the residual tissues is avoided, and meanwhile, the complete structure of the vascular bundle is prevented from being damaged in the digestion time process.
P6, taking out the digested material, further soaking and rinsing in 5% Triton X-100 for 15 minutes, then rinsing with PEM solution for 2 times, 5 minutes each time, and completing the separation of the vascular bundles of Arabidopsis, tobacco roots and hypocotyls after rinsing.
Then the vascular bundle is dehydrated in a gradient way, the vascular bundle is dehydrated in 15 percent, 30 percent, 50 percent and 75 percent ethanol for 15 minutes in turn, and then dehydrated by absolute ethyl alcohol for 3 times, each time for 15 minutes. The dehydrated material was dried at-20 ℃ for 1 hour in a Low vacuum desiccator (CHRIST). After drying, the samples were fixed on the conductive gels of two sample platforms respectively, and then observed by a tiram MIRA3 field emission scanning electron microscope. The observation results are shown in fig. 1 and fig. 2, wherein fig. 1 is an SEM picture of arabidopsis thaliana vascular bundle, and fig. 2 is an SEM picture of tobacco vascular bundle, and it can be seen from the SEM pictures that vascular bundle tissues can be effectively separated by the method of the present invention, and the separation effect is excellent without impurity residue.
Comparative example 1
This comparative example differs from example 1 in that: no steps P5 and P6 were taken, i.e.the vascular bundle was separated from its adjacent tissue directly under the dissecting microscope with a dissecting needle without digestion with cellulase and pectinase and impurities were removed with Triton X-100.
The obtained vascular bundle was observed by an electron microscope in the same manner as in example 1, and the results are shown in fig. 3 and 4, where fig. 3 is an SEM picture of an arabidopsis vascular bundle and fig. 4 is an SEM picture of a tobacco vascular bundle, and it was found from the SEM pictures that treatment without cellulase, pectinase and Triton X-100 resulted in adhesion of many impurities on the obtained vascular bundle, and the structure of the vascular bundle could not be clearly observed.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (9)
1. A method for isolating vascular bundles from dicot roots and hypocotyls, said method comprising the steps of:
p1: selecting plant seeds, performing surface disinfection, sowing the seeds into a culture medium, and culturing the seeds;
p2: selecting a robust complete plant growing in P1, removing leaves, reserving roots and hypocotyls, and placing in a centrifuge tube;
p3: adding a PEM buffer solution containing 0.1% of tea saponin by volume fraction into a centrifugal tube of P2, soaking for 30-40 minutes, then rinsing with the PEM buffer solution, fixing the rinsed material in a PBS buffer solution containing 4% of paraformaldehyde by volume fraction for 30-40 minutes, and then rinsing with the PEM buffer solution again;
p4: under a dissecting microscope, separating the vascular bundle from adjacent tissues by using a dissecting needle, taking out the vascular bundle, and putting the vascular bundle into a centrifuge tube filled with a PEM buffer solution;
p5: taking the dissected material in P4, placing into enzyme solution containing cellulase and pectinase, and digesting at 28 deg.C;
p6: the completely digested material of P5 was washed in Triton X-100 for 15-20 min and then washed with PEM buffer to complete the separation of vascular bundles in plant roots and hypocotyls.
2. The method according to claim 1, wherein in step P5, the composition of the enzyme solution comprises: 5mM MES buffer, 0.5% (w/v) cellulase, 0.2% (w/v) pectinase, 0.12M sucrose and 1mM CaCl2And the pH was adjusted to 5.5.
3. The method of claim 1, wherein the PEM buffer comprises: 50mM PIPES, 5mM EGTA, 2.5mM MgSO4And the pH was adjusted to 6.9.
4. The method as claimed in claim 1, wherein in step P1, the plant seeds are surface sterilized with chlorine in a fume hood.
5. The method according to claim 1, wherein in step P1, the surface-sterilized seeds are sown on MS medium and cultured for 7-30 days under conditions of 16h light/8 h dark at 22-23 ℃.
6. The method according to claim 1, wherein step P3 is specifically: adding 20mL of PEM buffer solution containing 0.1% by volume of tea saponin into a centrifugal tube of P2, soaking for 30-40 minutes, rinsing with PEM buffer solution for 3 times for 20 minutes each time, fixing the rinsed material in PBS buffer solution containing 4% by volume of paraformaldehyde under vacuum drying at-20 ℃ for 30-40 minutes, and rinsing with PEM buffer solution for 3 times for 5 minutes each time.
7. The method according to claim 1, wherein step P4 is repeated to obtain a plurality of dissected materials, and the number of dissected materials does not exceed 6.
8. The method according to claim 1, wherein the digestion process of step P5 is performed by microscopic examination at any time until complete digestion of the remaining tissue in the vascular bundle is observed.
9. The method of claim 1, wherein the concentration of said Triton X-100 in step P6 is 5%.
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| JP2009166397A (en) * | 2008-01-17 | 2009-07-30 | Takex Labo:Kk | Assembly of long vascular bundle of bamboo, its manufacturing method, and block material using the same |
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