CN102495037B - A Fluorescent Tracing Method for the Unloading Path of Phloem Assimilates in Pear Fruit - Google Patents

Abstract

The invention discloses a fluorescent tracing method of an assimilation substance unloading path of a phloem in a pear fruit, comprising the following steps of: preparing a fluorescent solution, introducing the fluorescent solution, sampling, preparing slices, observing and photographing and so on. The invention provides an effective method for researching sugar transportation in the fruit. According to the invention, 5(6)-carboxyfluoresein diacetate CFDA is used as a fluorescent tracing substance and a CFDA solution is introduced into the phloem through a fruit stem part without special equipment and devices, thereby achieving the aim of rapidly and effectively marking the assimilation substance unloading path of the phloem in the development process of the pear fruit. The method provided by the invention has the advantages of no need of the special devices, simplicity in operation, uneasiness of damaging xylems and uneasiness of evaporating the fluorescent solution, no need of special treatments of samples in a fluorescent observation process, and low cost.

Description

A Fluorescent Tracing Method for the Unloading Path of Phloem Assimilates in Pear Fruit

technical field

The invention relates to a fluorescent tracing method for the unloading path of phloem assimilates in pear fruit. The method uses fluorescent dye 5(6) carboxyfluorescein ester (CFDA) to quickly mark the unloading path of photosynthetic products from source leaves in sink organs during pear fruit development, and belongs to the field of plant cell physiology.

Background technique

Pear is one of the main fruit trees planted in the world and in China, and is also a traditional fruit for my country's foreign exchange earnings. In my country, there are pear trees cultivated in all provinces (cities, districts) except Hainan Province, and it is a fruit tree in many provinces (such as Hebei, Jiangsu, Anhui, etc.). etc.), has become a pillar industry for rural economic development in many places in my country. According to the China Statistical Yearbook, by the end of 2010, the total cultivated area of pear trees in China was 1.105 million hm ² , with a total output of 14.263 million tons, accounting for 62% and 64% of the world's total cultivated area and total output of pears respectively, ranking first in the world. Large pear producing country.

The fruit is the economic "storage" organ of the pear tree, and the quality of the fruit is directly related to whether the pear industry can make great progress in the market competition. The photosynthetic products from the source leaves are transported through the phloem in the form of sorbitol and sucrose into the fruit for storage or further utilization. They not only become the basic raw materials for the synthesis of fruit quality and other nutrients and aromatic substances, but also serve as important signal molecules to participate in genes related to fruit development. The expression and regulation of the whole plant source-sink activity have been recognized by everyone. Material transport in higher plants is mainly carried out in vascular bundles, and phloem is the main organization responsible for the transport of assimilants in the body. The pathways of phloem assimilate unloading in sink organs mainly include symplastic pathway and apoplast pathway. The symplast pathway is carried out through plasmodesmata, and the apoplast pathway is transmembrane movement, which requires specific carrier mediation and energy drive. At present, it is generally believed that the unloading path largely determines a series of transport processes of assimilates from the 'source' to the 'sink', plays a key role in regulating the unloading efficiency, and ultimately affects the quality of the fruit. Revealing the path of assimilate unloading during fruit development has important theoretical significance and practical value for promoting the accumulation and transformation of sugar in fruit and achieving high quality and high yield, and has attracted more and more attention.

There is a well-developed vascular tissue system in pear fruit. Except for the lateral carpel bundle, other major vascular bundles are located in the parenchyma. The branches of these vascular bundles spread throughout the pulp, forming a network-like system, which flows continuously. Provide essential substances for fruit growth and development. For a long time, due to the limitations of research conditions, people's understanding of the unloading pathway of phloem assimilates during pear fruit development has been severely restricted. In recent years, 5(6) carboxyfluorescein ester (CFDA), as an ideal fluorescent tracer substance, has been used to study the unloading path of assimilates in different types of bank organs, and its transport mode is assimilated by autoradiography. The unloading method of objects is similar. The principle is: when CFDA is loaded into the cell, it will be decomposed by endogenous esterase into 5(6) carboxyfluorescein (CF) with fluorescent signal, which becomes a membrane-impermeable symplast assimilate unloading fluorescent indicator. The pathways of phloem assimilate unloading can be understood by observing the distribution of CF within the sink organ under a fluorescence microscope, which is safer and simpler than conventional microautoradiography.

However, when we took pear fruit as the research material, we found that in the process of CFDA labeling, if the current injection method, pressure injection method and microinjection method used in other crops need special equipment and high technical requirements, while Eppendorf tube cotton thread Although the introgression method and leaf smearing method do not require special equipment, the operation process is cumbersome, the xylem and phloem are easily damaged, and the fluorescent dye solution evaporates, which cannot achieve the ideal marking effect. To this end, we have established a more effective fluorescent labeling method that can quickly trace the assimilate unloading path in the phloem of pear fruit through experiments, and achieved ideal results, which has important practical application value.

Contents of the invention

The purpose of the present invention is to provide a fluorescent tracing method for the unloading path of phloem assimilates in pear fruit, which can be used to quickly understand the path of photosynthetic products from the source leaf in the sink organ during the development of pear fruit. It has the advantages of simplicity, convenience, practicality and ideal results.

The purpose of the present invention can be achieved through the following technical solutions:

A fluorescent tracing method for the unloading path of phloem assimilates in pear fruit, comprising the following steps:

(1) Prepare 1-2 mg/mL fluorescent dye 5(6) carboxyfluorescein ester (5(6)-carboxyfluoresein diacetate, CFDA) solution;

(2) Introduction of the CFDA solution prepared in step (1): Gently rub the epidermis of the fruit stalk without injuring the xylem → quickly wind the cotton ball around the fruit stalk → seal the cotton ball with a sealing film → use a micro-syringe to inject the prepared Slowly inject the good CFDA solution into the cotton, and inject 150-250 μl of the above-prepared CFDA solution into each fruit → immediately seal the pinhole with a parafilm to prevent the solution from leaking → then cover the fruit stalk with tin foil to prevent CFDA from decomposing when exposed to light ;

(3) Sampling: pick the fruit after being treated with CFDA solution for 48-72 hours;

(4) Preparation of slices: Cut the fresh fruit treated with CFDA solution into pulp tissue blocks with vascular bundles, and then slice them into slices. When each slice is completed, use a glass slide to directly dip the slices, and spread the slices on the slide glass the center of the piece;

(5) Observation and photographing: the prepared slices were immediately observed with a fluorescence microscope for distribution images of 5(6) carboxyfluoresin (CF) in the fruit to prevent fluorescence quenching.

For the fluorescent tracing method of the phloem assimilate unloading path in the above-mentioned pear fruit, the method for preparing a 1-2 mg/mL fluorescent dye CFDA solution is as follows: dissolve CFDA with a DMSO aqueous solution with a volume ratio of 1:1, and prepare a 1-2 mg/mL solution. The CFDA solution was protected from light for later use.

For the fluorescent tracing method of the unloading path of phloem assimilates in the above-mentioned pear fruit, the size of the tissue block cut in step (4) is 1cm×1cm×2cm; The glue was chilled for 20-30 minutes, the chilling temperature was -20°C, and the slice thickness was 20 μm; or the observed part of the fruit (that is, the cut tissue block) was directly cut crosswise or longitudinally by hand to prepare freehand slices.

In the above-mentioned fluorescence tracing method for the unloading path of phloem assimilates in pear fruit, a 488nm laser is used for excitation in step (5) observation, and a blue filter is used to detect the image, the eyepiece is 2.5×, and the objective lens is 10×.

The technical scheme mainly includes: preparation of fluorescent dye CFDA solution, introduction of CFDA solution, sampling, preparation of slices, observation and photographing and other main links.

Beneficial effects of the present invention:

(1) The fluorescent dye CFDA used in this method is non-toxic to cells, chemically inert, and has a relatively high quantum yield. Under the condition of not damaging the unloading mechanism as much as possible, the assimilates in pear fruit at different developmental stages can be tracked in a timely manner from the level of living cells The unloading path is safer and simpler than conventional microautoradiography.

(2) This method has advantages that existing methods do not have, such as the introduction of fluorescent tracers does not require special equipment and devices, simple operation, less damage to xylem, and less easy evaporation of fluorescent solutions. It also does not require special treatment of samples, is low in cost, and can quickly and effectively obtain ideal marking results.

(3) Applying this method to trace the unloading path of assimilates during pear fruit development will help us understand the mechanism of transport and accumulation of assimilates in sink cells in the formation of pear fruit quality, and provide a theoretical basis for high-quality and high-yield pears.

Description of drawings

Figure 1 takes 'Aigan Shui' as an example, the method of introducing CFDA solution in pear fruit.

Among them, 1- Epidermis 2- Phloem 3- Xylem 4- Cotton 5- Parafilm 6- Tin foil.

Fig. 2 Sampling sites of frozen sections of pear fruit.

Among them, 7-vascular bundle 8-fruit stem, namely the introduction site of CFDA solution 9-epidermis 10-pulp 11-ventral carpel vascular bundle 12-seed 13-main sampling site

Fig. 3 CF distribution image in pear fruit observed by fluorescence microscope.

Among them, a-result of frozen section b-result of freehand transverse section c-result of freehand longitudinal section

Detailed ways

1. Dissolve CFDA with DMSO aqueous solution with a volume ratio of 1:1, make a 1-2 mg/mL CFDA solution, and keep away from light for later use. Fluorescent dye CFDA is insoluble in water and can be dispensed according to the dosage and stored in a refrigerator at -20°C in the dark for ready-to-use.

2. Choose a healthy fruit with a sunny part on a sunny day, rinse the fruit stalk and dry it → gently rub the skin of the fruit stalk without hurting the xylem → wrap the cotton ball around the fruit stalk quickly → seal the cotton ball with a sealing film , the parafilm should not be too tight → slowly inject the prepared CFDA solution into the cotton with a micro-syringe carefully, and inject about 150-250 μl of the above-mentioned CFDA solution into each fruit → immediately seal the pinhole with a parafilm to prevent the solution from leaking → then Cover the fruit stalk with tinfoil to prevent CFDA from decomposing when exposed to light, and ensure that the solution slowly enters the fruit along with the bast flow.

3. After being treated with CFDA solution for 48-72 hours, pick the fruit and bring it back to the laboratory in an ice box.

4. With reference to Fig. 2, cut the pulp tissue block with vascular bundles from the fresh fruit processed by CFDA, the size is 1cm * 1cm * 2cm, and use a frozen microtome (for example, with a Leica CM1850 frozen microtome) to carry out serial sections , before slicing, freeze the tissue block with ordinary glue for 20-30 minutes, the freezing temperature is -20°C, and the slice thickness is 20 μm; or directly cut the observed part of the fruit by hand crosswise or longitudinally to prepare freehand slices. Every time a section is completed, use a glass slide to directly dip the section, so that the section is flat on the center of the slide.

5. Immediately observe the distribution image of CF in the fruit with a fluorescence microscope on the prepared slices to prevent fluorescence quenching. 488nm laser excitation is used for observation, the blue filter is used to detect the image, the eyepiece is 2.5×, and the objective lens is 10×. The CF distribution image in pear fruit observed by fluorescence microscope is shown in Fig. 3. The results show that the fluorescent tracer CF is always confined in the phloem, without any signs of diffusion into the pulp tissue, indicating that the unloading of assimilates in pear fruit is qualitative. Exosome pathway, and vice versa for the symplast pathway.

Claims (4)

1. the fluorescent tracing method in pear fruit endophloem assimilation quotient unloading path is characterized in that may further comprise the steps:

(1) preparation 1 ~ 2 mg/mL fluorescent dye CFDA solution;

(2) introducing of the CFDA solution of step (1) preparation: carpopodium epidermis gently rubs, do not injure that xylem → rapidly cotton mass is wrapped on the carpopodium → cotton mass being sealed → adopted micro syringe with sealed membrane slowly injects cotton with the CFDA solution for preparing, the above-mentioned CFDA solution for preparing of each fruit injection 150 ~ 250 μ l → prevent that with sealed membrane sealing pin hole solution from exosmosing → then with masking foil the carpopodium position being covered immediately prevents that CFDA from seeing that light decomposes;

(3) sampling: behind CFDA solution-treated 48 ~ 72 h, pluck fruit;

(4) preparation section: the above-mentioned fruit of crossing through the CFDA solution-treated that will be fresh, cut with fascicular pulp organization piece, then section is whenever finished a section and is directly picked section with microslide, and making cuts into slices is tiled in the central authorities of microslide;

(5) observation is taken pictures: the section for preparing, use immediately the distributed image of fluorescence microscope 5 (6) Fluoresceincarboxylic acids in fruit, and prevent fluorescent quenching.

2. pear fruit endophloem assimilation quotient according to claim 1 unloads the fluorescent tracing method in path, it is characterized in that the method for preparing 1 ~ 2 mg/mL fluorescent dye CFDA solution is: be the DMSO aqueous solution dissolving CFDA of 1:1 with volume ratio, be made into the CFDA solution of 1 ~ 2 mg/mL after lucifuge for subsequent use.

3. the fluorescent tracing method in pear fruit endophloem assimilation quotient according to claim 1 unloading path is characterized in that in the step (4) that institute cuts that to organize block size be 1 cm * 1 cm * 2 cm; During the preparation section, use freezing microtome to carry out serial section, section is front to be-20 ℃ with cold solid 20 ~ 30 min of common glue, cold solid temperature, slice thickness 20 μ m; Perhaps direct look-out station with fruit carries out free-hand crosscut or rip cutting, preparation free-hand section.

4. the fluorescent tracing method in pear fruit endophloem assimilation quotient according to claim 1 unloading path, it is characterized in that step (5) adopt when observing 488 nm laser excitations, blue color filter detected image, eyepiece be 2.5 *, object lens are 10 *.

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