阅读说明：本技术 一种快速统计植物花粉-胚珠比的方法 (Method for rapidly counting plant pollen-ovule ratio ) 是由 裴男才 孙冰 史欣 李健容 罗中莱 于 2020-04-13 设计创作，主要内容包括：本发明公开了一种快速统计植物花粉-胚珠比的方法。该方法将花药材料置于离心管中,加入适当体积的I<Sub>2</Sub>-KI溶液,挤压和旋转捣碎后使用涡旋混合仪对管中的混悬液进行震荡,用I<Sub>2</Sub>-KI溶液定容至一定体积,再次充分涡旋混合；用移液器汲取花粉混悬液置于细胞计数板的测量室中,使用全自动细胞计数仪检测花粉混悬液浓度,得到所检测植物的单花花粉量；将子房材料放入培养皿,置于研究级体视显微镜下进行解剖统计胚珠数量,解剖时培养皿中加少量PBS；根据单花花粉量和胚珠数,准确计算花粉-胚珠比。本发明的统计方法单位时间处理样品数4-6个、准确性达95％以上,显著优于传统技术手段,适用于具有不同尺寸花粉的植物类群。(The invention discloses a method for rapidly counting the ratio of plant pollen to ovule. The method comprises placing anther material in a centrifuge tube, adding appropriate volume of I 2 Squeezing KI solution, rotary mashing, shaking the suspension in the tube with vortex mixer, and adding I 2 Fixing the volume of the KI solution to a certain volume, and fully mixing by vortex again; drawing the pollen suspension by a pipettor, placing the pollen suspension in a measuring chamber of a cell counting plate, and detecting the concentration of the pollen suspension by using a full-automatic cell counter to obtain the single pollen amount of the detected plant; placing the ovary material into a culture dish, placing the culture dish under a research grade stereoscopic microscope for dissection to count the number of ovules, and adding a small amount of PBS (phosphate buffer solution) into the culture dish during dissection; and accurately calculating the pollen-ovule ratio according to the amount of the single pollen and the number of ovules. The statistical method disclosed by the invention has the advantages that the number of processed samples per unit time is 4-6, the accuracy is more than 95%, the statistical method is obviously superior to the traditional technical means, and the statistical method is suitable for plant groups with pollen of different sizes.)

1. A method for rapidly counting the ratio of pollen to ovule of a plant is characterized by comprising the following steps:

(1) obtaining plant material, fixing, cleaning and dissecting and separating;

(2) placing the dissected and separated anther material into a centrifuge tube, adding a proper amount of I₂-KI solution;

(3) mashing the anther material in the centrifugal tube, and then carrying out vortex oscillation to fully separate pollen from anther tissues; removing the anther tissue in the tube using forceps; by means of I₂Fixing the volume of the KI solution to a certain volume, and fully mixing by vortex again;

(4) sucking a proper amount of pollen suspension, placing the pollen suspension in a measuring chamber of a cell counting plate, and detecting the concentration of the pollen suspension by using a full-automatic cell counter; then, obtaining the single pollen amount of the detected plant through multiple conversion calculation;

(5) placing the dissected and separated materials into a culture dish, placing the culture dish under a stereoscopic microscope for dissection, adding a small amount of PBS into the culture dish during dissection, and counting the number of ovules;

(6) the pollen-ovule ratio is calculated based on the amount of individual pollen and the number of ovules.

2. The method of claim 1, wherein the plant material in step (1) is a developmental bud or stamen and pistil tissue taken from a mature bud.

3. The method for rapid statistics of plant pollen-ovule ratio as claimed in claim 1, wherein the fixing of the plant material in step (1) is performed by using 50% ethanol: glacial acetic acid: and fixing the FAA solution formed by mixing formaldehyde according to the volume ratio of 18:1:1 for more than 48 hours.

4. The method for rapidly counting the pollen-ovule ratio of a plant according to claim 1, wherein I in the steps (2) and (3)₂KI solution, I₂The concentration of (1) was 1g/100mL, and the concentration of KI was 8g/100 mL.

5. The method for rapidly counting the ratio of pollen to ovule of a plant according to claim 1, wherein the concentration of PBS in the step (5) is 0.1 mol/L.

Technical Field

The invention relates to the technical field of plant biology, in particular to a method for rapidly counting the ratio of plant pollen to ovule.

Background

The Pollen-ovule ratio (P/O ratio) refers to the ratio of the number of Pollen to the number of ovule in a single flower of a plant, and is generally regarded as a conservative index describing the breeding system of flowering plants and also influenced by the breeding system, pollination mechanism, life style, flower structure and other factors. The pollen-ovule ratio is an important reference index in the biological research of plant reproduction, and the corresponding numerical value fluctuates along with the difference of evolutionary branches and growth habitats. Therefore, from a larger aspect, related researches can try to break through the conventional limitation of single/few species, gradually pay attention to higher-level and more complex comprehensive researches such as class level (such as family and genus) and community level (such as forest community with more coexisting species), supplement important reproductive trait data for current (class and community level) phylogenetic researches, and build a family/order level phylogenetic evolutionary tree framework, which is helpful for exploring the reproductive trait basic pattern and corresponding network evolution process of the forest community.

Most of the studies have been directed to a single species, and only about not more than 5% of the cases contain more than 2 species at the same time. In addition, no review or systematic study on the important index of pollen ovule ratio is seen for the moment. The statistic method of the pollen ovule ratio mainly comprises a blood counting plate method and a microscope-glass slide method, but the existing method has lower efficiency, poorer accuracy and narrower applicability. Therefore, pollen estimation and ovule calculation are limited in research and practical application in more plant groups.

The plant pollen-ovule ratio statistical method with higher efficiency, better accuracy and wider applicability is established, and plays an important role in developing plant reproduction biology research and practical application.

Disclosure of Invention

In order to solve the existing problems, the invention provides a method for rapidly counting the number of plant pollen and ovule, which reduces the time consumption of unit sample counting, improves the counting accuracy, is simple and convenient to operate and has strong universality.

The invention aims to provide a method for rapidly counting the ratio of pollen to ovule of a plant.

The technical scheme adopted by the invention is as follows:

a method for rapidly counting the pollen-ovule ratio of a plant comprises the following steps:

(1) obtaining plant material, fixing, cleaning and dissecting and separating;

(2) placing the dissected and separated anther material into a centrifuge tube, adding a proper amount of I₂-KI solution;

(3) mashing the anther material in the centrifugal tube, and then carrying out vortex oscillation to fully separate pollen from anther tissues; removing the anther tissue in the tube using forceps; by means of I₂Fixing the volume of the KI solution to a certain volume, and fully mixing by vortex again;

(4) sucking a proper amount of pollen suspension, placing the pollen suspension in a measuring chamber of a cell counting plate, and detecting the concentration of the pollen suspension by using a full-automatic cell counter; then, obtaining the single pollen amount of the detected plant through multiple conversion calculation;

(5) placing the ovary material subjected to dissection into a culture dish, placing the culture dish under a stereoscopic microscope for dissection, adding a small amount of PBS into the culture dish during dissection, and counting the number of ovules;

(6) the pollen-ovule ratio is calculated based on the amount of individual pollen and the number of ovules.

The plant material in the step (1) is a bud in a development stage, or stamen and pistil tissues taken out from a mature bud.

The fixation of the plant material in step (1) is performed using 50% ethanol: glacial acetic acid: and fixing the FAA solution formed by mixing formaldehyde according to the volume ratio of 18:1:1 for more than 48 hours.

I in steps (2) and (3)₂KI solution, I₂The concentration of (1) was 1g/100mL, and the concentration of KI was 8g/100 mL. Use of I₂the-KI solution dilutes pollen, has a dyeing effect, and is convenient to use an automatic cell counter in pollen suspensionThe pollen grains can be quickly identified, and the accurate detection and counting effect can be achieved.

In the step (3), the anther material in the centrifuge tube is smashed by using a tissue grinding pestle, and the tissue grinding pestle is made of polypropylene and has a conical tip and a reticulate pattern. The method comprises the steps of extruding and rotationally mashing the anther material by using a tissue grinding pestle which is made of polypropylene and has a reticulate pattern at the head part, and oscillating suspension in the tube by using a vortex mixer to fully separate pollen from anther tissue and form uniform suspension.

The cell counting plate in the step (4) is made of transparent plastic, and the volume of the measuring chamber is 20 mu L.

The concentration of PBS in step (5) was 0.1 mol/L. The ovules were immersed in 0.1mol/L PBS to facilitate the experimental work and reduce tissue deformation.

Compared with the prior art, the invention has the following beneficial effects:

1. the method for rapidly counting the ratio of the plant pollen to the ovule can enable the number of processed samples per unit time to reach more than 4, the accuracy to reach more than 95 percent, and the experimental efficiency and the accuracy are obviously improved.

2. The method can be suitable for counting the number of the plant pollen with different types such as large size (the average pollen diameter is more than 35 mu m), medium size (the average pollen diameter is 25-35 mu m), small size (the average pollen diameter is less than 25 mu m) and the like, and the applicability of the method is obviously improved.

3. The statistical method is improved, and the ovule is immersed in 0.1mol/L PBS phosphate buffer solution, so that the experimental operation is facilitated, the tissue deformation is reduced, and the working efficiency and the accuracy are also obviously improved.

Drawings

FIG. 1 shows a common peony ovary. After dissection, a large number of ovules were shown to grow on the placenta.

FIG. 2 is a micrograph of plant pollen (Melastoma esculentum) with smaller dimensions. The pollen is large in quantity and small in diameter; average pollen diameter: 20.3 μm.

FIG. 3 is a micrograph of plant pollen with medium size (Colon Tree). Pollen belongs to the common size range and is approximately circular in shape; average pollen diameter: 31.2 μm.

FIG. 4 is a micrograph of plant pollen (Gardenia jasminoides Ellis) with a larger size. The pollen quantity is less, and the diameter is large; average pollen diameter: 44.7 μm.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The method is supported by a youth talent project (subtropical forest woody plant reproduction character and community pedigree research; number: CAFYBB2017QB002), Guangdong province forestry scientific and technological innovation project (Lingnan maple improved variety breeding research and demonstration; number: 2015KJCX017, 2017KJCX022) and a plant resource protection and sustainable utilization key laboratory open fund funding project (south Asia tropical evergreen broad leaf forest important plant group reproduction character monitoring; number: PCU201903) of a central public welfare scientific research institute of China academy.