Method for extracting pear pollen tube vacuoles

A pollen tube and vacuole technology, applied in the field of plant cell biology, can solve the problems of large amount of materials, unsuitable application of pear pollen, difficulty in obtaining materials, etc., and achieve the effect of less pollution

Active Publication Date: 2015-03-04
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 3. Mechanical crushing: Although the vacuoles are released directly from the sheared flake tissue in the plasmodium separation medium without protoplasts, this method requires a large amount of materials, and it is not suitable for pear pollen, which is difficult to obtain.

Method used

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  • Method for extracting pear pollen tube vacuoles
  • Method for extracting pear pollen tube vacuoles
  • Method for extracting pear pollen tube vacuoles

Examples

Experimental program
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Effect test

Embodiment 1

[0046] 1) Pollen collection: collect anthers at the big bud stage and dry them with silica gel.

[0047] 2) Pollen culture: separate the pollen from the dried anther, take 3 mg of pollen, add it to 2 ml of pollen medium, and add 0.005% neutral red dye. Under dark conditions, culture at 23-27°C, 140-180rpm, for 3-4 hours. The formula of pollen medium: 100g / L sucrose, 0.3g / L Ca(NO 3 ) 2 4H 2 O, 30mM MES (α-sodium sulfomethyl ester sulfonate), 0.1g / L boric acid were dissolved in distilled water and fixed to volume, and the pH value was adjusted to 5.9-6.4 with Tris (trishydroxymethylaminomethane).

[0048] 3) Release of protoplasts: mix the cultured pollen and extracellular fluid at a volume ratio of 1:3, add 1% cellulase, 1% isolated enzyme, 23-27°C, under dark conditions, 60- 80rpm, react for 10-15 minutes. Extracellular fluid formula: 100mM glucose, 10mM CaCl 2 , 5mM MES (sodium α-sulfomethyl ester sulfonate), 0.8-1M sorbitol, and adjust the pH to 5.5-6.0 with Tris (tris...

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Abstract

The invention discloses a method for separating and purifying pear pollen tube vacuoles. The method mainly comprises the steps of pollen collection, pollen culture, protoplast release, protoplast purification, vacuole separation, vacuole purification and the like. A neutral red two-staining method is adopted, and pollen grains, vacuoles, protoplasts and water vacuoles can be clearly distinguished under an optical microscope. The method provided by the invention is high in speed and simple, not only can be applied to patch clamp researches, but also can be further applied to vacuole proteome researches, and has the advantages that vacuole membranes have smooth surfaces, and are low in pollution degree, and fewer materials and medicaments are required.

Description

technical field [0001] The invention belongs to the technical field of plant cell biology, in particular to a method for extracting and purifying vacuoles from pear pollen tubes. Background technique [0002] Plant vacuole is a unique large organelle in plant cells, which plays an important role in maintaining and regulating cell osmotic pressure, storing and transporting various ions, nutrients and metabolic intermediates, and also has functions similar to lysosomes, removing harmful Substances and stress resistance are regulated. Pollen vacuoles play an important role in pollen formation, germination and pollen tube growth, but due to the difficulty in extracting pollen vacuoles, there are few studies on the role of vacuoles in pollen germination and pollen tube growth. [0003] There are many ion channels in the plant vacuole, which are involved in many physiological processes. Pollen tube vacuole ion channels play a huge role in the maturation and growth of pollen and i...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 张绍铃周宏胜吴巨友高永彬陶书田齐开杰陈建清
Owner NANJING AGRICULTURAL UNIVERSITY
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