Method for observing embryo sac of paddy rice by using stone peculiar fluorescent dye, and transparent technique of whole ovary

A technique of fluorescence staining and ovary, applied in fluorescence/phosphorescence, preparation of test samples, material analysis by optical means, etc., can solve the problems of difficult operation, limited success rate, cumbersome steps, etc., and achieve clear cell contours. , the effect of strong layering

Inactive Publication Date: 2007-02-21
代西梅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the operation is difficult and the success rate is limited
However, this method has cumbersome steps and is only suitable for observing the smaller embryo sacs before flowering, but not for the larger embryo sacs after flowering.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The inventive method of the present invention is to observe the embryo sac of each developmental stage of rice and the embryonic development process of each stage after fertilization with nuclear fluorescent staining and ovary integral transparent technology; Rinse the fixed florets with 50% alcohol, then separate the ovary under a dissecting microscope, and store it in 70% alcohol for later use; The solution was treated with distilled water, 20min each time; then treated with NaOH solution or HCl solution for 1h, rinsed with distilled water, and then stained with 0.5μg / ml DAPI solution or Hoechst solution for 10h; then dehydrated with ethanol solution gradient, 20min at each level; then Dehydrate the ovary in absolute ethanol at least twice, each dehydration time is at least 1 hour, and then transfer to absolute ethanol for dehydration for more than 12 hours; then dehydrate the dehydrated ovary material in a volume ratio of 1:1 Transition in the mixed solution composed...

Embodiment 2

[0017]The inventive method of the present invention is to observe the embryo sac of each developmental stage of rice and the embryonic development process of each stage after fertilization with nuclear fluorescent staining and ovary integral transparent technology; Rinse the fixed spikelets with 60% alcohol, then separate the ovary under a dissecting microscope, and store it in 72% alcohol for later use; The solution was treated with distilled water for 23 minutes each time; then treated with NaOH solution or HCl solution for 1.2 hours, rinsed with distilled water, and then stained with 0.6 μg / ml DAPI solution or Hoechst solution for 15 hours; then dehydrated with ethanol solution gradient, 23 minutes per step; Then put the ovary into absolute ethanol for dehydration twice, each dehydration time is 2h, and then transfer to dehydrated in absolute ethanol for more than 13h; Transition in the mixed solution composed of absolute ethanol and methyl salicylate for 2 hours, and then ...

Embodiment 3

[0019] The inventive method of the present invention is to observe the embryo sac of each developmental stage of rice and the embryonic development process of each stage after fertilization with nuclear fluorescent staining and ovary integral transparent technology; Rinse the fixed florets with 65% alcohol, then separate the ovary under a dissecting microscope, and store it in 73% alcohol for later use; The solution was treated with distilled water, 25min each time; then treated with NaOH solution or HCl solution for 1.5h, rinsed with distilled water, and then stained with 0.7μg / ml DAPI solution or Hoechst solution for 20h; then dehydrated with ethanol solution gradient, 25min at each level; Then put the ovary into dehydrated ethanol twice for dehydration, each dehydration time is 3h, and then transfer it to dehydrated in dehydrated ethanol for more than 14h; Transition in the mixed solution composed of absolute ethanol and methyl salicylate for 3 hours, and then transparently...

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PUM

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Abstract

A method of utilizing nuclear specific fluorescent staining and ovary being made to be completely transparent technique to observe blastocyst of rice includes using certain concentration of sodium hydroxide solution to carry out softening treatment on blastocyst of rice before staining then utilizing specific combination of nucleus fluorescent staining DAPI with nucleus to observe structure of cell and nucleus in blastocyst, enabling to use laser scan confocal microscope to observe internal structure of cell clearly.

Description

technical field [0001] The invention relates to a method for observing rice embryo sacs, in particular to a method for observing rice embryo sacs by utilizing nuclear-specific fluorescent staining and ovary overall transparency techniques. Background technique [0002] Embryo sac is a unique female gametophytic structure of angiosperms, which participates in many processes of sexual reproduction. The structure and function of the embryo sac is a major aspect of plant reproductive biology. However, the embryo sac of angiosperms is surrounded by layers of ovule tissue, and some plants have thicker ovary walls outside the ovule, which brings great difficulties to the research work of the embryo sac. So far, many methods and techniques for observing the embryo sac have been proposed, such as paraffin section method; microdissection method; enzymatic separation method; overall transparent method; [0003] The traditional method of observing the embryo sac is the paraffin sectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/84G01N21/17G01N21/64G01N1/30
Inventor 代西梅黄群策秦广雍李国平
Owner 代西梅
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