CN109211910B - Simple method for observing peony flower bud differentiation process and application thereof - Google Patents
Simple method for observing peony flower bud differentiation process and application thereof Download PDFInfo
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Abstract
A simple method for observing the differentiation process of peony flower bud is characterized by that the underground bud of peony which is normally developed is selected, the bud is completely cut off from root and stem, the bud scale is removed, and the cut bud is placed in FAA fixing solution and stored at room temperature for above 12 hr. Soaking the fixed bud in anhydrous ethanol for 30min, treating in distilled water for 30min, and naturally dehydrating the material on absorbent paper at room temperature for 10-15 min. Under a stereoscopic microscope, the base part of the bud is clamped by using precise micro-forceps, the bud end is upward, all primordia of the flower bud are poked layer by layer in the formation sequence by using a forceps tip with the right hand, and the bud is smoothed out until the meristem at the top end of the bud is exposed, observed and photographed. The invention provides a complete specific method for observing the peony flower bud differentiation process by using a body type microscope, the method can greatly shorten the experimental time and the economic cost, improve the experimental efficiency and the result reliability, has simple flow and strong technical portability, and has important significance for peony flower bud differentiation and pattern classification research.
Description
Technical Field
The invention relates to the technical field of peony flower bud differentiation tissue morphology observation, and particularly relates to a simple method for observing a peony flower bud differentiation process and application thereof.
Background
Paeonia lactiflora (Paeonia lactiflora) is an important flower rooted in Chinese traditional culture and is also popular among people in the world. The occurrence pattern, the development process and the structure of the peony have stronger genetic stability, and are the most important basis for carrying out phylogenetic and developmental research on the peony. The peony flower bud differentiation process is important for basic research on flower organ development, flower type evolution and the like, is also an important reference for flowering period regulation and high yield cultivation promotion, and is closely related to whether flowering ornamental characters can be formed. Therefore, the research on the flower bud differentiation process of the Chinese herbaceous peony has important theoretical and production practical values.
As long as thousands of years of cultivation and breeding practices, the flower types of the Chinese herbaceous peony are rich in various single flower types such as single petals, half-double petals, double petals and the like, and also have a large number of pavilion type flower types formed by overlapping two or more single flowers. At present, peony flower types are classified and typed mainly from the open flower decomposition structure, and although pavilion flower types are classified and typed from the aspect of flower anatomical structure, because the flower type evolution degree is high and the flower bud differentiation process is complex, the flower development process is lack of necessary understanding at present, which depends on an efficient and reliable method.
In the last 30 years, in order to clarify the flower bud differentiation process of different flower type varieties of paeonia lactiflora, a number of methods were explored and a great deal of work was carried out, and these methods include: paraffin section, frozen section, solid dissection, scanning electron microscope, etc. However, the methods all have certain limitations, such as the freezing slicing method has great technical realization difficulty in peony flower buds and poor slicing effect; the physical anatomy method is less applicable because the complete flower structure picture cannot be stored; the scanning electron microscope method has high requirements on instruments and equipment and operation procedures, and is not popularized and used in a large range. With the increasingly mature paraffin section flaking technology, the method is applied to peony bud differentiation research and becomes the current mainstream research method, but the method has the disadvantages of complex operation flow, long experimental period, high technical requirements on operators, large use of toxic biochemical reagents, and large difference in research conclusions among different researchers in the flower bud differentiation process of the same flower type, particularly complex flower type, so that the realization efficiency is low, and the reliability of the experimental result is to be improved.
The common desire of all researchers is to use an efficient and reliable experimental method to develop scientific research, and for flower bud differentiation observation, a good experimental method is as follows: 1. the material identification degree is high, and the research result can be visually, completely and accurately displayed; 2. the research method has universality and is not limited by the development period of materials and the difference of the complexity of varieties and patterns; 3. the instrument and equipment are simple to operate and low in maintenance cost; 4. the experimental operation flow is simple, the economic and time cost is low, the efficiency is high, and the environment is protected.
The conventional flower bud differentiation observation method cannot completely meet the above conditions, so that a novel reliable and efficient peony flower bud differentiation observation method is needed. The method provided by the invention can accurately and efficiently track the whole peony flower bud differentiation process, and the obtained map is three-dimensional, visual and accurate, the operation is time-saving, the economic investment is low, and the method is simple and easy to learn, so that the method is particularly suitable for morphological observation and research in the peony flower bud differentiation process.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a simple method for observing the differentiation process of peony flower buds and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
1. a simple method for observing the differentiation process of peony flower buds comprises the following steps:
(1) obtaining underground buds of the peony which normally develop, peeling off scales, and then placing the buds in a stationary liquid for storage;
(2) carrying out gradient treatment on the buds obtained in the step (1) by using absolute ethyl alcohol and clear water, and dehydrating at room temperature until the water content is 65-70%;
(3) and (3) placing the bud obtained in the step (2) under a body type microscope, and smoothing the floral organ primordia with precise micro forceps until the apical meristem is completely exposed, thus obtaining the bud.
The invention solves the problems of complex experimental treatment process, long period, use of a large amount of chemical reagents and consumables, excessive investment, limited tissue observation range, low tissue identification degree and the like of the traditional tissue observation method, greatly shortens the experimental time and the economic cost, improves the experimental efficiency and the result reliability, has few operation steps, simple flow, easy operation and strong technical transportability, and provides a reference method for the flower bud differentiation observation research of peony plants and other species with similar tissue morphological structures.
In the above technical scheme, in the step (1), the obtaining of the underground buds of the peony comprises: digging downwards by an iron shovel at a position which is about 15cm away from the root of the peony stem and is vertical to the ground, and digging to a depth of about 15cm, so as to ensure that large soil layers around the buds are removed, simultaneously reduce the damage to underground buds and roots as far as possible, and ensure the structural integrity of the underground buds. After the bud scales are exposed out of the soil layer, slightly removing soil around the buds by hands to ensure that the buds are completely exposed, and completely cutting off the buds from the roots and the stems in a direction vertical to the longitudinal axis of the buds by using an art designer when the length of the underground buds is less than or equal to 2 cm; when the length of the underground bud is more than 2cm, the top of the bud is cut with a utility knife along a direction perpendicular to the longitudinal axis of the bud for a length of about 2 cm. When the length of the bud is small, the bud is integrally cut from the base part, so that the integrity of the meristem structure of the top of the obtained bud can be ensured. When the length of the bud is larger than 2cm, the bud base is properly cut off, the apical meristem form is not influenced, and meanwhile, the length of the bud can be ensured to be in a proper range, so that the subsequent observation operation is facilitated.
Preferably, in the above technical solution, the underground bud in step (1) develops normally, specifically: the underground bud scale has complete structure, plump bud body and no obvious plant diseases and insect pests, and ensures that the top bud tissue form is complete.
Further preferably, in the above technical solution, in the step (1), the bud is stored in a fixing solution, specifically: removing bud scales layer by layer according to the growth sequence of the bud scales by using micro-forceps until terminal buds are exposed, placing the bud scales in a centrifuge tube with a cover, pouring a fixative solution into the centrifuge tube with the cover, completely immersing the materials in the fixative solution, covering the centrifuge tube with the cover, and standing for 12 hours at room temperature for later use. The scale outside the bud is removed, so that the full contact between the flower organ primordial tissues at the top of the flower and the fixing liquid can be promoted, the fixing efficiency is improved, and the fixation softening and subsequent treatment time can be shortened.
Still further preferably, in the above technical solution, the fixing solution is FAA fixing solution, and the specific mixture ratio is: 70% ethanol: glacial acetic acid: formaldehyde 90:5:5 (volume ratio). The formaldehyde contained in the fixing liquid can effectively sterilize and is convenient for long-term storage of materials. In addition, the fixing liquid can effectively remove redundant pigments in the buds, improve the identification degree of meristems at the top ends of the buds, promote the softening of materials and facilitate subsequent operation.
Still further preferably, in the above technical scheme, in the step (2), the terminal bud is subjected to gradient treatment with absolute ethyl alcohol and clear water, specifically: pouring the fixing solution for preserving bud in the centrifuge tube into a waste liquid bottle, adding anhydrous ethanol with the same amount for treatment for 30min, pouring off anhydrous ethanol, adding distilled water with the same amount, and soaking for treatment for 30 min. The absolute ethyl alcohol can rapidly remove the fixing liquid in the material, so that the phenomenon that pungent odor and toxic substances generated by the fixing liquid in the subsequent operation process generate adverse effects on an operator is avoided, and on the other hand, the absolute ethyl alcohol is favorable for further removing redundant pigments in the material, so that the tissue identification degree is improved. The ethanol in the tissue can be fully removed by soaking with distilled water, so that the tissue is prevented from browning, becoming brittle and hard in the operation process, and the operation and observation are prevented from being influenced.
Still further preferably, in the above technical solution, in the step (2), the room-temperature dehydration specifically includes: taking out the bud from the centrifuge tube with forceps, and placing on absorbent paper at room temperature for 10-15 min. The excessive moisture content not only easily reflects light during observation to influence photographing, but also has small tissue plasticity, and the floral organs are easy to rebound after deformation to influence subsequent operation; the water content is too low, the tissue is lack of water and shrinks, the tissue is easy to brown, and the tissue is easy to break or damage during operation, which is not beneficial to maintaining the integrity of the tissue structure.
Still preferably, in the above technical solution, in step (3), the micro forceps are used to smooth out the flower organ primordia, specifically: the forceps valve of the precision micro forceps is slightly opened by the thumb and the forefinger of the left hand, the bud is placed into the opened forceps valve by the thumb and the forefinger of the right hand, the end of the bud faces upwards, the base part of the bud is clamped by the resilience force of the forceps valve, and the bud is placed under a body type microscope. The left hand moves the tweezers for clamping the buds, the focal distance is adjusted to enable the terminal buds to completely appear in the visual field range of the microscope, the right hand holds another pair of precision micro tweezers, and the forceps tips are used for poking the flower organ primordium of the buds layer by layer to the periphery according to the occurrence sequence until the apical meristem is completely exposed. The bud base is clamped by the natural resilience force of the forceps valve of the precise micro forceps, so that the position of the material can be ensured to be fixed in the photographing and operating processes, and the material is not easy to shake. The flower organ primordium is pulled apart according to the sequence, so that the integrity of the whole tissue structure can be ensured, and the identification and analysis are convenient.
Further preferably, in the technical scheme, the micro-forceps are straight-head precise micro-forceps, the forceps body of the precise micro-forceps is hard and firm, the elasticity is good, the forceps tip is extremely fine, and the bud body fixation and the observation operation of the meristem of the bud tip are facilitated.
The invention also provides application of the simple method for observing flower bud differentiation, namely, the flower buds are observed under a body type microscope and photographed, the photographing is completed within 10-15min after the processing operation is completed, and if the material is dehydrated too much due to overlong standing time, the flower buds can be placed in distilled water again for water supplement and dehydration operation.
The invention has the advantages that:
the method has the advantages of simple operation flow, easy reference and copy, low learning difficulty, little practice, over 95 percent of experiment success rate, saving of a large amount of toxic chemical reagents, common instruments and convenient popularization and application. According to the operation flow shown in the invention, fixed time is removed, the observation experiment period of one bud can be controlled within 2 hours theoretically, while the experiment period of the mainstream paraffin section technology is about one week, and corresponding experiment conditions need to be explored aiming at materials in different development periods, so that the experiment effect of the invention is obvious in advantage and the efficiency can be improved by dozens of times. Meanwhile, the reagents and consumables used by the invention are relatively common and can be easily obtained in common laboratories. Compared with the traditional section observation method, the three-dimensional stereoscopic impression is stronger, and the primordia and the meristem states of the flower buds can be reflected more intuitively, comprehensively and accurately, so that the reliability is higher.
Drawings
FIG. 1 is a schematic representation of the meristem morphology of the apical end of the flower bud of Paeonia lactiflora of the present example;
FIG. 2 is a diagram showing the meristem morphology of the apical end of the flower bud of Paeonia lactiflora of the present example;
FIG. 3 is a diagram showing the meristem morphology of the apical bud of peony 'Dahongpao' in the present example;
FIG. 4 is a diagram showing the meristem morphology of the apical end of the flower bud of peony 'Edulis Superba' in the present example;
FIG. 5 is a diagram showing the meristem morphology of the apical bud of the ` Border Charm ` flower bud of Italian Paeonia in an example of the present invention;
Detailed Description
The following describes in further detail embodiments of the present invention with reference to the accompanying drawings. The following drawings are included to illustrate and not to limit the scope of the invention, which is defined by the claims.
Collecting underground buds of different peony type varieties (Yiteng peony 'Border Charm' (unipetal type), peony 'edlis Superba' (Tougui type), peony 'Coral Sunset' (lotus type), peony 'Dahongpao' (Taige type) and peony 'Karl Rosenfield' (Taige type)) from the end of 8 months to the middle of 4 months of 2018, taking 15 buds of each variety, taking the buds every 20 days from 27 days of 11 months to 6 days of 3 months of 2018 years of 2017, and taking the buds every 10 days for the rest time. The test material was planted in the small Tangshan germplasm resource garden (Beijing, Changruo district, small Tangshan town) of the national floriculture center.
As described above, the present invention provides a simple method for observing the differentiation process of peony flower buds, which specifically comprises the following steps:
(1) obtaining underground buds of the peony which normally develop, peeling off scales, and then placing the buds in a stationary liquid for storage, wherein the method specifically comprises the following steps: digging downwards by an iron shovel at a position which is 15cm away from the root of the peony stalks and is vertical to the ground surface, and digging to a depth of about 15 cm. Slightly removing soil around the buds by hands after bud scales of the underground buds are exposed out of a soil layer to ensure that the buds are completely exposed, and completely cutting off the buds from rhizomes in a direction vertical to the longitudinal axis of the buds by using a cutter when the length of the underground buds is less than or equal to 2cm to ensure that the meristem structure of the top ends of the obtained buds is complete; when the length of the underground bud is more than 2cm, the length of about 2cm of the top of the bud is cut by an art knife along the direction vertical to the longitudinal axis of the bud, the length of the bud body is ensured to be in a proper range on the basis of ensuring the integrity of the apical meristem structure, and the subsequent observation operation is facilitated. Removing the outer scales of the cut buds by using micro-tweezers, placing the buds into a centrifuge tube with a cover and containing FAA fixing liquid, wherein the volume of the FAA solution is determined by the size of the material so that the buds can be completely immersed into the fixing liquid, and placing the buds for later use at room temperature for 12 hours after the bottle cover is closed.
(2) After the material is fixed, pouring the fixing liquid in the centrifuge tube into a waste liquid bottle, pouring the fixing liquid into absolute ethyl alcohol with the same volume for soaking for 30min, pouring out the absolute ethyl alcohol, then adding distilled water with the same volume for soaking for 30min, taking out the buds from the centrifuge tube by using tweezers after the treatment is finished, and placing the buds on absorbent paper to dry for 10-15min at room temperature.
(3) The forceps valve of the microscope forceps is slightly opened by the thumb and the forefinger of the left hand, the bud is placed into the opened forceps valve by the thumb and the forefinger of the right hand, the end of the bud faces upwards, the base part of the bud is clamped by the resilience force of the forceps valve, and the bud is placed under the body type microscope. The left hand moves the tweezers for clamping the buds, the right hand adjusts the focal length to enable the terminal buds to be completely displayed in the visual field range of the microscope, the right hand holds another pair of precision micro tweezers to poke the flower organ primordium of the buds layer by layer to the periphery according to the occurrence sequence of the flower organ primordium, and each flower organ is guaranteed to be complete in structure, fixed in position and not rebound until the meristem at the top is completely exposed.
The invention also provides an application of the simple method for observing the peony flower bud differentiation process, which comprises the following steps: and (4) observing the apical meristem of the bud with the well stroked floral organ tissue and the intact apical meristem under a microscope, and taking a picture. And the photographing is completed within 10-15min, and if the tissue is deformed due to excessive water loss of the buds caused by overlong standing time, the buds are placed in distilled water again for water supplement and subsequent dehydration operation.
As shown in fig. 1-5, the body type observation pictures of different flower type varieties at different development periods are respectively shown, from which the morphological characteristics of the organ tissues of each flower can be clearly distinguished and identified, thus further confirming the feasibility and reliability of the simple method for observing the flower bud differentiation process of the invention.
Finally, the above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A simple method for observing a peony flower bud differentiation process is characterized by comprising the following steps:
(1) obtaining underground buds of the peony which normally develop, peeling off scales, and then placing the buds in a stationary liquid for storage;
(2) treating the buds obtained in the step (1) with absolute ethyl alcohol for 30min, pouring out the absolute ethyl alcohol, adding distilled water with the same amount, soaking for 30min, and dehydrating at room temperature until the water content is 65-70%;
(3) placing the bud obtained in the step (2) under a body type microscope, and using micro forceps to smooth the floral organ primordia until the apical meristem is completely exposed;
the underground peony bud is obtained by the following method: digging downwards at a position 15cm away from the root of the peony stem by using an iron shovel, digging to a depth of about 15cm, slightly removing soil around the buds by using hands after bud scales are exposed out of a soil layer, and completely cutting off the buds from the root and stem in a direction vertical to the longitudinal axis of the buds by using an art designer when the length of the underground buds is less than or equal to 2 cm; when the length of the underground bud is more than 2cm, cutting the top of the bud by an art designing knife along the direction vertical to the longitudinal axis of the bud for about 2cm, and ensuring that the cut is smooth.
2. The simple method for observing the differentiation process of peony flower bud according to claim 1, wherein the normally developed underground bud in step (1) is specifically: underground bud scale has complete structure, plump bud body and no obvious diseases and insect pests.
3. The simple method for observing the differentiation process of peony flower buds as claimed in claim 1, wherein in the step (1), the buds are preserved in a stationary liquid, specifically: removing the outer scales of the cut buds by using micro-tweezers, placing the buds into a centrifuge tube with a cover and containing FAA fixing liquid, wherein the FAA fixing liquid is prepared by 70% ethanol, glacial acetic acid and formaldehyde according to the volume ratio of 90:5, the volume of the FAA solution is determined by the material size so that the buds can be completely immersed into the fixing liquid, and after the bottle cover is covered, placing the buds for later use at room temperature for 12 hours.
4. The simple method for observing the differentiation process of peony flower buds as claimed in claim 1 or 2, wherein the underground buds are obtained from plants grown in a plant division manner for 2 years or more.
5. The simple method for observing the differentiation process of peony flower buds as claimed in claim 1, wherein the buds soaked in distilled water are taken out by tweezers and placed on absorbent paper for natural dehydration at room temperature for 10-15min to keep the water content of buds at 65-70%.
6. The simple method for observing peony bud differentiation process according to claim 1, wherein in step (3), floral organ primordia are smoothed by using micro forceps, specifically: the forceps valve of the micro forceps is slightly opened by the thumb and the forefinger of the left hand, the base part of the bud is clamped by the resilience force of the forceps valve, the end of the bud is upward and is placed under the body type microscope, the forceps for clamping the bud is moved by the left hand, the focal length is adjusted to enable the apical bud to completely appear in the visual field range of the microscope, and the other precise micro forceps is held by the right hand to poke the flower organ primordium of the bud layer by layer around according to the occurrence sequence until the apical meristem is completely exposed.
7. The simplified method for observing differentiation process of peony flower bud according to claim 1, wherein said method of step (3) further comprises the step of analyzing the shoot after apical meristem treatment by taking a photograph under a microscope, wherein said photograph is completed within 10-15min after apical meristem treatment of the shoot.
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| CN201811365635.6A CN109211910B (en) | 2018-11-16 | 2018-11-16 | Simple method for observing peony flower bud differentiation process and application thereof |
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| CN201811365635.6A CN109211910B (en) | 2018-11-16 | 2018-11-16 | Simple method for observing peony flower bud differentiation process and application thereof |
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| CN109211910B true CN109211910B (en) | 2021-10-01 |
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