Chromosome preparation

Phaseolus polytene chromosomes from embro suspensor

harvest the pods (mostly mornings) in a big, lockable plastic bag and store them at +4°C for 1-3 days.
open the pods along the joint using a sharp blade and dissect the tissue containing the embryo suspensor. For this, open the seed with a angular superficial cut (figure a) and cut out only the embroy tissue (figure b).


Fig. a: surface section (longitudinal cut)	Fig. b: disection of the embryo tissue with suspensor below the microphyle (cross section)

store the disected tissues at +4°C for 3-7 days and change fixation solution after the first 24h

transfer fixed seeds/tissue from 70% Ethanol at - 20°C in 70% ethanol at room temperature and wash on slow rotation shaker for 5 min at min
exchange ethanol with aqua dest. and wash 2x 10 min, like above. Note: Seeds/tissues should precipiate at the bottom within 20-30 min. If rotation is too fast, it takes more time for them to sink to the bottom!
collect about 30 tissues in 10 ml MES-Sucrose buffer, rinse 3x und transfer them in plastic container with 15 ml 10% pektinase solution
macerate tissues on a shaker at 120 rpm for 3 h at 37°C
decant the pectinase solution and wash tissues 3x with MES-Sucrose buffer
fix in 5 ml in solution of propanoic acid and lactic acid (1:1) for 3 h at room temperature
rinse 3x in aqua dest. and leave at +4°C for 1-2 h
transfer each tissue in some drops of 45% acetic acid onto a hollow slide
separate the biggest cells of the suspensor with needles and tweezers (s. Foto RC2 und RC3)


RC2: Suspensor cells liberated from enclosing tissue	RC3: Big suspensor cells (10) in which the giant chromosomes appear as dark threads

use only the biggest suspensor cells with very clear transparent cytoplasm
take 8-10 large suspensor cells using a 20 µl pipette (with cutted tip) in 15 µl of 45% acetic acit and transfer onto poly-L-lysin coated slide.

distribute single suspensor cells at sufficient distance from each other on the slide (cells must not overlap when the coverslip is landing with a smack, see next step)
take a coverslip and hold it angular against the slide with a tweezers(1); create some tention by pressing the needle against the cover slip (2)
removing the tweezers suddenly, the coverslip is landing with a smack on the slide and squeezing the chromsomes out of the suspensor cells

cover the cover slip with a filter paper (same size) and squash additionally by tapping the rounding back of a needle at right angle on the cover slip in order to seperate the chromosomes even more


good squashing	medium	insufficient

remove coverslip by dry-ice method of Conger and Fairchild (1953)
dehydrate preparations by immersion in 99% ethanol and air-dry over night
lable the position of the squashed cells at the border of the slide using a glass writing diamond!


Instruments	Light microscopes