Lalambchop1 said:that is interesting about Stout's studies. I'd like to replicate them.
It is possible to read Stout's scientific paper if one joins JSTOR (free) at
http://www.jstor.org/stable/24....
You will be able to read how he examined the pistils and replicate his studies. Studies in other species have found that the effects of temperature on pollination are genotype-dependent so repeating his studies on several different modern diploid and tetraploid cultivars would be useful.
I have copied the relevant section of his Materials and Methods from his daylily paper directly below:
"In obtaining material for study, as a rule as many as 50 flowers of a single clone that had opened under
glassine bags were pollinated at one time, usually at or near 9:00 A.M. It is at this time that the anthers of
most flowers of the diurnal daylilies are fully dehiscing and the pistils are receptive to compatible
fertilizations. At intervals thereafter, at least three flowers were removed and their pistils preserved for
study. For premature pollination, delayed pollination and pollination of the flowers of night-blooming sorts,
applications of pollen were made at other hours.
In the study of the pollen tubes in the pistils the paraffin method was utilized, but mostly a method of direct
dissection and staining was employed as already described in considerable detail (Chandler, 1931; Stout,
1931b). The pistils are preserved in a solution of 100 cc. of 70%, alcohol and 6 to 8 cc. of commercial
formalin (37%). In staining, the pistils are dissected and a few drops of aceto-carmine (saturated solution in
45% acetic acid) are made to flow the entire distance of the style. After a few seconds a drop of magenta
(1% aqueous solution) or a drop of aceto-carmine to which has been added a trace of ferric acetate is
placed on the style. The excess stain is removed with absolute alcohol. Glycerine and a cover glass
complete a preparation which may be kept for study for a period of several months.
With the use of a microscope the extent of the growth of the pollen tubes in the various pistils is determined
and tabulated. If tubes extend into the lower part of the style the ovary is also sectioned and stained. By
this method it is rather easy to determine the position of the ends of the pollen tubes, their distribution in
the pistil, and their appearance. The pistils of the various kinds of daylilies differ somewhat in length. For
example, the styles of the H. thunbergii range from 7 to 8 cm. in length, and those of the H. fulva clone
‘Europa’ range from 9.2 to 9.6 cm. in length. In plotting the extent of pollen tube growth in these plants the
style of a pistil is divided into ten equal units and thus the data for numerous pistils differing somewhat in
length may be presented in the same type of graph."
Stout references a previous publication in 1931. That paper is also available on JSTOR and I have copied the appropriate section of those Materials and Methods directly below:
"The paraffin method of study was utilized, but for most of the studies a
method of direct dissection and staining was developed that is simple, rapid,
and especially reliable for tracing the continuous course of the pollen tubes.
As soon as the pistils were removed they were placed in a killing solution of
100 cc. of 70% alcohol and either 6 or 8 cc. of commercial formalin (37%).
With such preservation the pistils remained in excellent condition for study
for at least a year, but in most cases the material was studied as soon after
preservation as was convenient.
The dissections of pistils were made as follows: a pistil was placed on a
glass slide and slit with a razor blade lengthwise into halves which were then
arranged with the cut surfaces uppermost. Sometimes a half was also sectioned.
Another method used was to slit the pistil its entire length with a
fine sewing needle mounted in a wooden handle. The sections were made
either at right angles or parallel to the central placenta as desired.
Numerous stains were tested in various combinations and with various
methods of application before a satisfactory differential stain for the pollen
tubes in the pistils was obtained. The method found to be most fully satisfactory
for Brassica is as follows. Aceto-carmine solution made by saturating
warm 45% acetic acid with carmine is dropped on the dissected pistils
and allowed to remain for a few seconds, after which a few drops of a
saturated aqueous solution of aniline blue are added. After a few seconds,
depending on the density of stain desired, the excess stain is drawn off either
with a pipette or with blotting paper. A few drops of aqueous solution of
magenta red applied before the aniline blue sometimes improves the differentiation
of the pollen tubes. Any desired degree in the density of the stain
is then obtained by washing with absolute alcohol applied from a pipette.
A drop of glycerine and a cover glass complete the preparation.
With the use of a microscope the extent of the growth of the pollen tubes
in the various pistils was determined. If any tubes were found in the extreme
lower part of the style the ovary was also sectioned and stained. By
this method it was rather easy to determine the position of the ends of the
pollen tubes in the pistil and their appearance."
The paper by Chandler, "A method for staining pollen tubes within the pistil" is not available for free. It may be possible to read it through the library of a university near you. It can be found at
http://www.tandfonline.com/doi...
If you are interested in examining the effects of temperature on garden hybridizing then if you wish to tease apart the importance of temperature on pollen viability versus the importance of temperature on pistil viability you would need to do two sets of experiments. In one set you would need to keep the temperature the pistil experienced the same while varying the temperature that the pollen experienced and then in a second set of experiments keep the temperature the pollen experienced the same while varying the temperature that the pistil experienced.
