Pollen tube growth and counting in Corydalis flowers (Fumariaceae)

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Method synopsis

Fixed flowers were processed through staining and clearing to visualize and count pollen tubes that had grown down the style to assess fertilization success. This involved fixation in ethanol:acetic acid, staining with fuchsin and fast green, clearing in lactic acid, and microscopic examination.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Why are some plant-nectar robber interactions commensalisms? (2018), Oikos. Implementations in other papers (listed below) may differ.

1. Collect flowers for observational pollen tube analysis
   Up to 10 plants per site were sampled, collecting up to 5 robbed flowers (bearing at least one robbing hole) and 5 unrobbed flowers of similar age from each plant. 311 styles were collected from 32 plants across all sites during peak bloom. Flowers were fixed in 3:1 volume ratio of 95% ethanol to glacial acetic acid to arrest pollen-tube growth.
   Quantities: 311 styles from 32 plants, up to 10 plants per site, up to 5 robbed and 5 unrobbed flowers per plantDuration: Collections made during peak bloom periods in 2014Conditions: Peak bloom timing at three different elevation sites
   Equipment: 95% ethanol, glacial acetic acid, collection vials
2. Process styles for pollen tube counting
   Fixed styles were transferred to vials with 4:1 volume ratio 1% basic fuchsin in 1% fast green to stain pollen tubes for visualization via white light transmission. After 24 hours, pistils were rinsed in DI water and transferred to vials with 88% lactic acid to clear and soften tissue. After 5 days in lactic acid, styles were mounted on microscope slides using clear fingernail varnish and viewed under compound microscope at 1000× magnification to count pollen tubes at receptacle end of each style.
   Quantities: 4:1 volume ratio stain solution, 88% lactic acid, 1000× magnificationDuration: 24 hours in stain, 5 days in lactic acidConditions: Room temperature processing
   Equipment: 1% basic fuchsin, 1% fast green, DI water, 88% lactic acid, microscope slides, clear fingernail varnish, compound microscope

Typical Equipment

• 95% ethanol
• glacial acetic acid
• 1% basic fuchsin
• 1% fast green
• 88% lactic acid
• compound microscope

Output Measurements

• pollen tube count per flower

Papers Using This Protocol (1)

Species Studied With This Protocol

Places Where Used