Plant cytogenetics lab
Plant evolution often involves cytogenetic phenomena.
The first lab in this section will involve determining pollen size and fertility to compare
them among ploidy levels. We will also prepare root tips so we can examine
chromosomes in the following lab.
1. Pollen fertility as assessed by pollen stainability (lab 3).
A standard and simply technique in plant biology is to assess pollen stainability which
provides and estimate of pollen fertility/sterility. Pollen may be sterile as a result of a
number of causes including genic sterility, or cytogenetically induced sterility. For
example, if meiosis is disrupted (as it usually is in triploids) little or no pollen will be
fertile because pollen will receive unbalanced numbers of chromosomes. Likewise, some
polyploids, known as autopolyploids might exhibit some sterility because of unusual
chromosome pairing and disjunction. Pollen sterility in synthetic or naturally occurring
hybrids also provides insight into whether two taxa represent biological species.
Collect fresh pollen from diploid, triploid, tetraploid, hexaploid and octoploid species of
Turnera and mount each in a small drop of lactophenol cotton blue. Allow pollen to stain
in drop for approx. 10 minutes. Then cover with coverslip and view. Count 100 pollen
grains on the slide and classify whether they are uniformly stained blue (fertile) or not
uniformly stained and typically smaller and more irregularly shaped than the uniformly
stained grains (i.e. are sterile).
Also, measure the diameter of 10 pollen grains for each slide you prepare to compare
pollen sizes among the ploidy levels.
Calculate the percent pollen fertility for diploid, tetraploid, hexaploid and octoploid
species.
In the lab following lab (lab 4) we will use standard chromosome staining techniques to
examine and count chromosomes in root tips, and examine meiosis in flower buds. We
will also assess pollen fertility using standard staining techniques. Bring lab coat and
dissection kit.
Lab 2. Chromosome numbers and their collective morphology (Karyotype) provide a
wealth of useful information that is relevant both to plant taxonomy and evolution.
Species exhibiting differences in chromosome number are usually intersterile with one
another and therefore chromosome number alone might provide information on whether
two morphologically similar entities represent biological species (i.e. independently
evolving lineages).
Root tip squashes of broad bean, corn, onion:
Seeds or bulbs were germinated for 5 days. Roots were excised, washed in water and
placed on filter paper saturated with 0.05% colchicine for approx. 4 hours. Roots were
fixed in fresh fixative(3 ethanol : 1 acetic acid) overnight. Roots were hydrolyzed in 1 N
HCl at 60C for 10 minutes and then stained in basic fuschin.
Alternatively they may be hydrolyzed and then stained immediately in a drop of
acetocarmine or acetorceine.
Dissect a very small amount of tissue from a root tip and prepare a mitotic squash.
You should be able to count the number of chromosomes and draw their morphology.
The best preparations should be completely flat and none of the chromosomes should be
overlapping.
3. Meiotic squashes of anthers.
An examination of meiosis can also provide information on chromosome numbers.
More importantly, meiosis can be used to examine the causes of sterility in some plants,
or in hybrids. It can help identify the mode of origin of various polyploids, and in
hybrids, it can be used to identify the progenitor species of polyploids.
Meiotic squashes of Rhoeo versicolor.
Dissect the anthers out of very small buds of Rhoeo versicolor and squash them on a
microscope slide in a drop of acetocarmine or acetorceine. Determine the stage of
meiosis you are examining. You should make a squash when you find a bud that is at
metaphase I of meiosis. Examine the chromosome configurations (i.e. how many are
paired with one another). Note the this species has a very unusual meiosis.