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A method and application of rapid quantification of pollen activity at room temperature or high temperature

A technology of pollen and normal temperature, which is applied in the field of plant physiological and biochemical technology, can solve problems such as not being fast, and achieve the effect of eliminating errors

Active Publication Date: 2021-06-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to overcome the defects that current various dyeing methods cannot be fast and carry out identification of pollen activity in large quantities, and to evaluate pollen activity by quantitatively extracting tritylhydrazone (TTF) after pollen or whole anther dyeing, and this This method is used for the identification of pollen activity in large quantities to simplify the quantitative operation process and improve the accuracy of detection

Method used

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  • A method and application of rapid quantification of pollen activity at room temperature or high temperature
  • A method and application of rapid quantification of pollen activity at room temperature or high temperature
  • A method and application of rapid quantification of pollen activity at room temperature or high temperature

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: optimal concentration buffer solution and TTC staining solution preparation

[0041] The 2,3,5-triphenyltetrazolium chloride (TTC) combined dyeing method adopted in the present invention is based on the method reported by the State Key Laboratory of Crop Genetics of Huazhong Agricultural University where the applicant is located (Min, L., Zhu ,L.,Tu,L.,Deng,F.,Yuan,D.,and Zhang,X.(2013).Cotton GhCKI disrupts normal male reproduction by delaying tapetum programmed cell death via inactivating starch synthase.The Plant journal: for cell and Molecular biology 75,823-835), in this document, the applicant adopted the method of dark staining in a 37°C incubator, considering that there may be no incubator conditions, referring to the list of commonly used buffer solutions, designed different concentrations Suspended pollen in phosphate buffer saline, respectively 0mol / L, 0.05mol / L, 0.1mol / L, 0.2mol / L, 0.5mol / L, 1mol / L. At the same time, referring to the concentra...

Embodiment 2

[0043] Embodiment 2: Dyeing test to different crop pollen

[0044] Using the combined dyeing solution in Example 1, the pollen of dicotyledonous crops such as rapeseed and monocotyledonous crops such as rice has been dyed. Microscopic examination of the dyed pollen found that the active pollen in the two crops can be dyed. Dye red, show that combined dyeing solution of the present invention has certain wide applicability (as Figure 5 shown).

Embodiment 3

[0045] Example 3 Optimum sample volume test on elisa plate

[0046] When preparing TTF standards of different concentrations, it was found that when the concentration of TTF of the standard was higher, the difference could not be easily distinguished by naked eyes. In order to determine the accurate measurement range of the microplate reader, the combined staining solution in Example 1 was used in this embodiment , after diluting different times, prepare a combined staining of 0μg / L (only with potassium phosphate buffer), 5mg / L, 10mg / L, 25mg / L, 50mg / L, 100mg / L, 150mg / L, and 250mg / L 1 ml of each solution (such as image 3 As shown), add excess sodium sulfoxylate (5-10mg to 1mL) for full reduction, add an equal volume of ethyl acetate to shake and extract for 5min, then centrifuge, absorb 200μL of the supernatant and use a microplate reader for measurement, and the absorbance value of each measurement Corresponding to the TTF amounts of 0 μg, 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 ...

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Abstract

The invention belongs to the application field of plant physiology and biochemistry technology. In particular, it relates to a method and application for rapid quantification of pollen activity at normal temperature or high temperature. The formula, dyeing concentration, optimum dyeing time and scope of application of pollen TTC dyeing solution at room temperature and high temperature were determined, and the formula of combined dyeing solution was prepared: 1mol / L potassium dihydrogen phosphate 5.23g, dipotassium hydrogen phosphate 14.03g, pH 7 potassium phosphate buffer solution; then 8g of 2,3,5-triphenyltetrazolium chloride (TTC) was dissolved in the resulting potassium phosphate buffer solution, and the volume was adjusted to 1L with distilled water, so that the final concentration was 8g / L. The invention can be used for quantitative detection of pollen activity of large batches of materials, omits the microscopic inspection operation after dyeing, simplifies the process of identification of pollen activity of large batches of materials, and is suitable for identification of pollen activity of various crops such as cotton and rice.

Description

technical field [0001] The invention belongs to the application field of plant physiology and biochemistry technology. In particular, it relates to a method for quickly quantifying pollen activity at normal temperature or high temperature and a scheme in which the method is used to dye and extract with a staining solution, and measure absorbance for quantification. On the one hand, the invention greatly simplifies the operation process, on the other hand, it can also accurately quantify the vigor of the pollen. Background technique [0002] With global warming, high temperature stress has become one of the main factors affecting crop production. The development of plant organs is very sensitive to changes in ambient temperature, and high temperature can lead to abnormal development of reproductive organs. Male reproductive organs are more sensitive to environmental temperature changes than female reproductive organs, especially stamens. Extreme temperatures during developm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N1/30G01N21/31G01N21/84
Inventor 闵玲马益赞张献龙
Owner HUAZHONG AGRI UNIV
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