International Rice Research Institute, P.O. Box 933, Manila, Philippines
A number of methods described for the preparation of somatic chromosomes of Oryza sativa L. differ in mode of pretreatment, fixation, and maceration (Fukui et al. 1988; Khan 1975; Kurata and Omura 1978; Kurata et al. 1981; Quy and Phai 1985). Khan (1975) reported that a single root could yield 10-15 well-spread metaphase cells. Other authors do not mention the number of countable cells to expect. At IRRI, these methods have proven to be either time-consuming or to yield only a few prometaphase cells. The method described here yields approximately 40 countable cells from a single rice root tip. The number of countable cells has been as high as 250, with a minimum of 10 per slide.
Germinate dehulled seeds on filter paper moistened with tap water, in a petridish stored at 30 deg.C in an incubator. Excise roots of about 1 cm long and pretreat in 0.002M Hydroxyquiline at room temperature for 2-3 hrs, to arrest/ accumulate cells at metaphase. Fix pretreated roots in Carnoy's fixative (6: 3: 1- absolute ethanol: chloroform: glacial acetic acid; two changes of fixative are advisable). Store the roots at 4 deg.C for at least 2 days; storage may be extended to 60 days.
Wash the fixed roots in distilled water 2-3 times (5-6 minutes each time) and then in ice cold 0.01M citrate buffer (0.01M citric acid, monohydrate in 0.01M sodium citrate, pH 4.6) 2-3 times (2-3 minutes each time). Cut off the root tips, transfer to an eppendorf tube containing an enzyme solution (2-6% cellulase [Onozuka R-10] and 1-2% Pectolyase [Yakul Y-23] in citrate buffer) held in a 37 deg.C water bath. Digest the root tips for 45-75 minutes. Wash the root tips in 0.01 M citrate buffer 2- 3 times (10 minutes each time) and in distilled water twice (5-10 minutes each time) to remove the enzyme solution.
Transfer a single root tip to an acid-cleaned and ethanol-washed slide. Remove excess water without disturbing the specimen. Place a drop of 3: 1 absolute ethanol: glacial acetic acid onto the root tip and squash immediately with a needle. Add another drop of 3: 1 fixative and spread the material over the slide. Air-dry the slides and store overnight or longer in a dessicator.
Examine the slide using phase-contrast microscopy. For better contrast, stain the chromosomes by placing a drop of 1% acetocarmine or 1% propionic carmine over the air-dried cells, cover with a coverslip, and heat briefly over a spirit flame. Remove excess stain solution before applying light pressure to the coverslip.
References
Fukui, K., K. Kakeda, K. lijima and K. Ishiki, 1988. Computer-aided identification of rice chromosomes. RGN 5: 31-34.
Khan, S. H., 1975. A technique for staining rice chromosomes. Cytologia 40: 595-598.
Kurata, N. and T. Omura, 1978. Karyotype analysis in rice. I. A new method for identifying all chromosome pairs. Jpn. J. Genet. 53: 251-255.
____, N. lwata and T. Omura, 1981. Karyotype analysis in rice. II. Identification of extra chromosomes in trisomic plants and banding structure on some chromosomes. Jpn. J. Genet. 56: 41-50.
Quy, T. D. and P. Phai, 1985. An improved technique for rice karyotype study. RGN 2: 102- 103.