32. Estimation of nuclear DNA content in Oryza by flow cytometry
Cesar P. MARTINEZ1, K. ARUMUGANATHAN2 , H. KIKUCHI3 and E.D. EARLE2
1) Centro Internacional de Agricultura Tropical, Apartado Aereo No. 6713,
2) Department of Plant Breeding and Biometry, Cornell University, 252 Emerson Hall, Ithaca, N.Y. 14853-1902
3) Kamikawa Agricultural Experiment Station, Nagayama, Asahikawa-shi, Hokkaido, 079 Japan
Rice is the most important food crop of the developing world and it has been considered by many people as an ideal species for genetic and molecular studies. Because rice is used extensively for mapping, isolation, and transfer of genes and is one of the leading candiates for complete genome sequencing, accurate estimations of nuclear DNA content are important. The objectives of this study were to use flow cytometry to estimate nuclear DNA content in several rice species and to identify ploidy levels in anther culture-derived rice plants.
Ten rice species (Table 1) were selected for estimation of nuclear DNA content by flow cytometry. The seeds were obtained from the International Rice Research Institute, Manila, Philippines. Pre-geminated rice seeds were sown in plastic trays containing soil which was kept wet throughout the experiment. Trays were placed in a growth chamber with 12 hours of florescent light and a day/night temperature setting of 27/25°C, respectively. Samples (15-20 mg) were taken from the two youngest leaves of 20-30 day-old seedlings for analysis. This experiment was repeated twice on different days.
Young rice plantlets (2-3 weeks old) obtained through anther culture were provided by Dr. T. Croughan (Louisiana State University at Crowley, LA). After DNA determination, plantlets were potted and kept in the growth chamber for observations on flowering, sterility, plant height, and tiller number.
Samples were prepared for flow cytometric analysis using the procedures of Arumuganathan and Earle (1991). Samples of suspended plant nuclei stained with propidium iodide (PI) were analyzed using EPICS profile analyser (Coulter Electronics, Hialeah, Florida) equipped with an Argon ion laser emitting at 488nm. Cell clumps and debris were excluded from analysis using red fluorescence and forward angle light scatter gates. The red fluorescence emission signal of PI was processed of 457-502 nm long-pass laser blocking and 601nm long pass absorbance fitters. The resulting DNA content distributions of particles were determined on a linear scale.
Table 1. Nuclear DNA content of 10 rice species as determided by flow cytometry
=============================================================================== Genome Nuclear DNA Content (pg/2C) Scientific Name Group This study Previous work pg/2C Mbpa /1C pg/2C (reference)b =============================================================================== Oryza glaberrinma AgAg 0.73,0.76 352-366 1.37 (1) (15 accessions) 5.33 (7) Oryza A1A1 0.78 376 0.78 (6c) longistaminata 1.25 (1) Oryza sativa AA 0.87-0.96 419-463 0.87-0-96 (6c) ssp. Indica 1.01 (8) (49 cultivars) 1.20 (2) 1.67 (1) 2.10 (3) 3.40 (4) Oryza sativa AA 0.86-0.91 415-439 0.86-0.91 (6c) ssp. Japonica 0.80 (8) (20 cultivars) 1.20 (2) 1.55 (1) 1.90 (3) 3.30 (4) Oryza sativa AA 0.88 424 0.88 (6c) ssp. Javanica (3 cultivars) Oryza officinalis CC 1.14 550 2.02 (1) 4.7-6.07 (5) Oryza elchingeri CC 1.17 564 ND Oryza atistralietisis EE 1.99 960 1.78 (1) 11.41 (7) Oryza ridleyi unknown 1.31,1.85 632-931 2.65 (1) (5 plants) tetraploid 1.93 Oryza grandigliimis CCDD 1.99 960 ND Oryza latifolia CCDD 2.32 1124 ND Oryza minuta BBCC 2.33 1124 2.83 (1) =============================================================================== LST species (5%)=O.05; MSE=0.001; C.V.=2.94%. a 1 picogram (pg)=965 million base pairs (Mbp) (Bennet and Smith 1976). b Except where noted, Feulgen microdensitometry was used for previous work. ND: not determined; 1: Iyengar and Sen (1978); 2: Bennet and Smith (1976); 3: Bennet et al. (1982); 4: Nagato et al. (1981); 5: Katayama (1971); 6: Arumuganathan and Earle (1991); 7: Katayama (1967); 8: Bennet and Smith (1991). c Determined by flow cytometry.
Two determinations were done per isolation, and at least 2000 nuclei were examined each time. Values for nuclear DNA content were estimated by comparison of the rice nuclear peak on the linear scale with the peak for chicken red blood cells (CRBC) included as internal standards in each run. A value of 2.33 pg/2C was used for the CRBC (Galbaith et al., 1983).
For comparisons of DNA content among rice species a standard analysis of variance model under a completely randomized design with one factor (rice species) was carried out. For comparisons of DNA content among anther culture-derived plants a combined analysis of variance was used considering the following factors: cross, ploidy level, and individual plants (cross/ploidy). One tetraploid plant found in cross D was not included in the analysis.
Flow cytometric analysis of nuclei isolated from rice leaves shows only one peak corresponding to 2C level (G0+G1 phase). Peaks corresponding to 4C level (G2+M phase) were not detected. This indicated that there were no dividing cells in rice leaves. Figure 1 shows frequency histograms of relative fluorescence of nuclei in samples as measured by flow cytometry. Nuclei from test plants were mixed with nuclei of O. sativa cv IR36 and CRBC except in the case of O. latifolia and O. minuta. The 2C peaks of O. minuta (Fig. 1f) and O. latifolia (Fig. 1h) coincided with CRBC peak indicating that they have similar DNA content.
Significant differences in genome size among rice species were found. O. glaberrima had the smallest genome size (0.73-0.76 pg/2C), while O. minuta and O. latifolia had the largest values (ca. 2.33 pg/2C). Tetraploid species had more nuclear DNA than diploid ones. The nuclear DNA content in genome AA (0.86-0.96pg/2C) is smaller than in genomes CC (1.14-1.17pg/2C) and EE (1.99pg/2C). Significant differences in nuclear DNA content were found be- tween IR36 (AA genome) with a value of 0.90pg/2C, and the AgAg (O. glaberrima) and AIAI (O. longistaminata) genomes which had mean values of 0.73-0.76 and 0.78 pg/2C, respectively. Significant differences were also found between IR36 (Indica), and Yukihikari (Japonica) cultivars. Significant differences were found in nuclear DNA content (1.93, 1.85, and 1.31 pg/2C) among five plants analyzed in O. ridleyi.
Our results also showed that flow cytometry gave a quick and very reliable determination of the ploidy level of anther culture-derived plants, and the ploidy status of rice cell suspension cultures. There were significant differences in nuclear DNA content among haploid (0.45±0.0l pg/2C), diploid (0.91±0.03 pg/2C), and tetraploid (1.80±0.004 pg/2C) plants derived from anther culture.
We have also used flow cytometry to determine the ploidy status of rice cell suspension cultures kept in our lab for different lengths of time. The profile analysis indicated that the ploidy level of cell suspension cultures changes over time, resulting in a mixture of cells having different ploidy levels (data not shown). This can be very important in cases where cell suspensions are used in transformation experiments aimed at regeneration of normal diploid plants.
We conclude that flow cytometry has several useful applications to manipulation of rice by conventional or biotechnological methods.
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