Institute of Botany, Academia Sinica, Nankang, Talpei, Taiwan 115, ROC
Microscopic analysis of the pachytene chromosomes of rice was first
carried out by Shastry et al. (1960), and this technique was further used by
Sen (1963), Wu (1967), Khan (1975) and others with some modifications. These
workers found that rice pachytene chromosomes were not easily stained by 1%
aceto-carmine after the plain acetic alcohol fixation, and some of them added
a trace of ferric chloride to there fixatives (Shastry et al. 1960; Das and
Shastry 1963; Misra and Shastry 1967; Ranganadhacharyulu and Yesoda Rai 1974;
Khan 1975; Reddi and Reddi 1977; Dolores et a]. 1979; Sato et ai. 1980).
This treatment gave a deeper staining than the use of fixatives without iron
(Yao et al. 1958; Bouharmont 1962; katayama 1966). In many cases, fixed
materials were soon moved to 70% alcohol, but Khan (1975) kept his materials
in the fixative with iron for two months or even longer and obtained improved
staining of pachytene chromosomes.
Another difficulty has been insufficient spreading of the pachytene chromosomes. Good spreading seemed to depend on the choice of an appropriate stage of cell division and sufficient swelling of the cell. Pollen mother cells fixed in a fixative with iron do not swell well upon heating.
Wu (1967) developed a double mordant technique, by which these difficulties were circumvented. In this technique, a trace of ferric hydroxide is added to 1% aceto-carmine and to the fixative to which a trace of ferric chloride is also added. Not only chromosomes but cytoplasm also are overstained, especially when the slide is gently heated, and the residual stain can be removed by adding several drops of 45% acetic acid to one side of cover slip and blotting the excess fluid from the other side. Swelling of the cell and differential staining of chromosomes are then carried out simultaneously by heating the slide again evenly and gently up to a point just before boiling. No intended pressure needs to be applied directly to the cover slip except turning the slide upside down over a piece of blotting paper as soon as the heating is completed.
The pachytene chromosomes prepared in this way are well spread and reasonably differentiated (Chen et al. 1982). Among those who had studied rice pachytene chromosomes, Ranganadhacharyulu et al. (1974) was the only ones who used 45% acetic acid to remove excess stain.
The technique developed by Kurata et a]. (1981) is quite different from the ones mentioned above. By their technique, anthers of proper size are treated with 75 mM KC1 or 0.5 mM uridine and macerated in a mixture of pectinase and cellulase. After being rinsed with distilled water, the anthers are smashed in a drop of fixative (3 parts methanol + 1 part acetic acid), flame-dried and Giemsa stained. Their micrographs have shown well differentiated configurations of chromosomes with prominence of centromeres. This technique may also be highly recommended for analysis of rice pachytene chromosomes.
References
Bouharmont, J., 1962. Observations on somatic and meiotic chromosomes of Oryza spieces. Cytologia 27: 258-275.
Chen, J. T., H. C. Lai, Y. H. Hwang, M. C. Chung and H. K. Wu, 1982. Identification of rice reciprocal translocation and the location of lazy gene. Bot. Bull. Acad. Sinica 23: 71-87.
Das, D. C. and S. V. S. Shastry, 1963. pachytene analysis in Oryza. VI. karyomorphology of O. perennis Moench. Cytologia 28: 36-43.
Dolores, R. C., T. T. Chang and D. A. Ramirez, 1979. The cytogenetics of F\1\ hybrids from Oryza nivara Sharma et Shastry x O. sativa L. Cytologia 44: 527-540.
Katayama, T. 1966. Cytogenetical studies on the genus Oryza, 2. Chromosome pairing in the interspecific hybrid with the ABC genomes. Jpn. J. Genet. 41: 309-316.
Khan, S. H., 1975. A technique for staining rice chromosomes. Cytologia 40: 595-598.
Kurata, N., T. Omura and N. Iwata, 1981. Studies on centromere, chromomere and nucleolus in pachytene nuclei of rice, Oryza sativa. Cytologia 46: 791-800.
Misra, R. M. and S. V. S. Shastry, 1967. Pachytene analysis in Oryza, VIII. Chromosome morphology and karyotypic variation in O. sativa. Ind. J. Genet. Pl. Breed. 27: 349-368.
Ranganadhacharyulu, N. and A. Yesoda Ral, 1974. Pachytene analysis in an interspecific hybrid, Oryza punctata Kotschy ex Steud. x O. eichingeri A. Peter. Cytologia 39: 233-243.
Reddi, V. R. and T. V. V. Seetharami Reddi, 1977. Chromosome pairing at pachytene and meiosis in autotetraploid rice. Cytologia 42: 189-196.
Sato, S., T. Kinoshita and M. Takahashi, 1980. Location of centromere and interchange breakpoints in the pachytene chromosome of rice. Genetical studies on rice plant, LXXI. Jpn. J. Breed. 30: 387-398.
Sen, S.K., 1963. Analysis of rice pachytene chromosomes. The Nucleus 6: 107-120.
Shastry, S. V. S., D. R. Ranga Rao and R. N. Misra, 1960. Pachytene analysis in Oryza, I. Chromosome morphology in Oryza sativa.. Ind. J. Genet., Pl. Breed. 20: 15-21.
Wu, H.K., 1967. Note on the preparing of pachytene chromosomes by double mordant. Scientific Agriculture (ROC) 15: 40-44.
Yao, S.Y., M.T. Henderson and N.E. Jodon, 1958. Cryptic structural hybridity as a probable cause of sterility in intervarietal hybrids of cultivated rice, Oryza sativa L. Cytologia 23: 46-55.