Moens (1965, Can. J. Genet. Cytol. 7:296-303) reported on the occurrence of an isochromosome of the short arm of chromosome 2 (2S) of the tomato. The 2S, being an entirely heterochromatic arm and thus genetically inert, the sporophyte can tolerate 7-8 copies of this isochromosome without any notable hindrance for growth and reproduction (Quiros, 1976, Genetics 84:43-50). In view of this, plants with isochromosomes are of much value for the study of certain cytogenetic phenomena such as the effects of extra heterochromatin, pairing behavior of chromosomes and dosage effect of RDNA etc. In an attempt to make use of some of the isochromosome lines in our cytogenetic studies it was necessary to define the structure of these isochromosomes. Therefore a Giemsa c-banding method was successfully applied, and the procedures are described below.
For mitotic chromosomes:
- Collect root tips from a young healthy plant and pre-treat with 0.002 M hydroxyquinoline for 2 1/2-3 h at room temperature.
- Fix root tips in 1:3 acetic acid:ethanol solution for 48 h.
- Wash in distilled water for 20 min.
- Hydrolyse in 1N Hydrochloric acid for 5 min at room temperature.
- Macerate in an enzyme solution of 1% cellulase and 0.5% pectinase in 0.01 M citrate buffer (pH 4.0) at 380 for 25-30 min.
- Transfer to 45% acetic acid for 25-30 min at room temperature.
- Squash the root tip in 45% acetic acid on a clean slide. Cells can be observed with 40x phase contrast objective at this stage.
- Freeze the slides in liquid nitrogen and remove the cover glass with a sharp blade.
- Air dry the slides and wait 2 days bef ore use.
- Incubate the slides in 45% acetic acid for 20 min. at 600C.
- Denature the preparation in 6% solution of Barium hydroxide for 6-7 min. at room temperature.
- Wash thoroughly in running tap water for 1 h.
- Incubate slides in 2x SSC buffer (pH 6.8) at 600C for 2 h.
- Rinse the slides in 0.04 M phosphate buffer (pH 6.8) and stain in 4% Giemsa stain (in phosphate buffer pH 6.8) for 10-15 min.
- Rinse the slides in phosphate buffer, air dry and mount in euparal.
For meiotic chromosomes the procedure is the same as above, but with a slight modification in squashing. Without any pretreatment, the anthers are fixed as above, hydrolysed for 5 min. in 1N HCl at room temperature and directly squashed in 45% acetic acid.
In somatic cells two types of isochromosomes were detected: (i) those with both distal ends with c-heterochromatin and (ii) those with only one distal end having c-heterochromatin. The latter were the most predominant. Except for the telomeres, the normal somatic chromosomes did not reveal any large blocks of c-heterochromatin.
During pachytene, the large blocks of c-heterochromatin were confined exclusively to satellite region of chromosome 2, and, as in somatic cells, either both or one of the distal ends of isochromosomes. The latter was most predominant.
The block of c-heterochromatin serves as a good cytological marker for the determination of the pairing behavior of the isochromosomes among themselves as well as with other bivalents in the complement. At metaphase I of meiosis, the isochromosomes can be selectively identified because of the presence of c-heterochromatin, an observation that agrees with a previous report by Lamm (1980, TGC 30:25).