Phaseolus polytene chromosomes

Phaseolus polytene chromosomes from embro suspensor

Schnittfolge RC1

Coating of microscope glass slides

  • rinse slides in a staining jar for 5 min in aceton and then air dry them
  • apply a drop of 4 µl Poly-L-Lysin in the first third of the slide
  • spread the drop using a second slide and let the coated slide air dry

Materials, chemicals and solutions

  • 10 µl pipette
  • staining jar, glass vessel
  • aceton, technical
  • Poly-L-Lysin (mw 100.000, Sigma, P-1274), 1 mg/ml in H20

Seed fixation

  • 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).
  • Längsschnitt Längsschnitt
    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

Preparation

  • 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)
  • Riesenchromosomen2 Riesenchromosomen3
    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
  • (if one suspeonsor does not contain enough large cells, collect the biggest cells from multiple suspensors)
  • 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.
  • Alternatively for people with very cam hand:

    Very carefully squeeze out the content of each suspensor cell, where the major part represent the nucleus, with gentle pressure of a preparation needle. Take the nuclei using a pipette and transfert onto a 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
  • Squashing 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
  • Quality 1 Quality 2 Quality 3
    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!

Material

Instruments Light microscopes
Instruments Light microscopes
  • Scalpel, mounted pins, tweezers with fine tip, Pasteurpipette
  • cover slips, 18x18mm
  • microscope slides, microscope slides with 1 cavitiy
  • filter paper
  • cooling plate, razor blade, glass writing diamond
  • plastic jars with with tight-closing lid (100 ml)
  • 20µl-pipette, cutted 200µl-tips

Chemicals and solutions

  • MES (2[N-Morpholino] ethanesulfonic acid) (Sigma, M-3023)
  • sucrose (SERVA, 35580)
  • pectinase from mould, 0.0095 units/mg (Fluka, 76290)
  • acetic acid, 45%
  • propionic acid, 99.5%
  • DL-lactic acid
  • methylated ethanol, 99%
  • MES-Sucrose-Medium: 25 mM MES, pH 5.5 with 6% (w/v) sucrose
11/17/2018 15:08  Phaseolus polytene chromosomes by Mario Nenno. © 2008- All rights reserved.
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