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Method for rapid quantification of pollen activity at normal temperature or high temperature, and application

A pollen, room temperature technology, applied in the application field of plant physiology and biochemical technology, can solve problems such as inability to quickly and achieve the effect of eliminating errors

Active Publication Date: 2019-05-14
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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  • Method for rapid quantification of pollen activity at normal temperature or high temperature, and application
  • Method for rapid quantification of pollen activity at normal temperature or high temperature, and application
  • Method for rapid quantification of pollen activity at normal temperature or high temperature, and application

Examples

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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 field of application of plant physiological and biochemical technology, and specifically relates to a method for rapid quantification of pollen activity at normal temperature or high temperature, and application. The method comprises the steps of determining formulations, dyeing concentrations, optimum dyeing times and applicable ranges of pollen TTC staining solutionat a normal temperature and a high temperature, preparing a combined dyeing solution formula: 5.23g of 1 mol / L potassium dihydrogen phosphate, 14.03g of dipotassium hydrogen phosphate and potassium phosphate buffer with pH of 7; dissolving 8g of 2,3,5-triphenyltetrazolium chloride (TTC) in the obtained potassium phosphate buffer, and adjusting the volume to 1L with distilled water to a final concentration of 8g / L. The invention can be used for quantitative detection of pollen activity of large quantities of materials, omits the microscopic examination operation after dyeing and simplifies theprocess of pollen activity identification of large quantities of materials, which is suitable for pollen activity identification of cotton, rice and other crops.

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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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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