Plant Breeding Institute, Faculty of Agriculture, Hokkaido University, Sapporo 060 Japan
Anther culture is useful in mutagenesis since both dominant and recessive mutants are detected effectively after in vitro selection of microspores (Swanson et al. 1988). In our first experiment,anthers taken from the rice variety Kitaake were irradiated with gamma rays, just after inoculation on the callus-forming medium. In our second experiment, anther-derived calli consisting of a mixture of haploid and diploid cells were subjected to irradiation. Irradiation was carried out using a gamma room in Faculty of Engineering, Hokkaido University. Both anthers and anther-derived calli were treated for one minute at two exposure rates, 500 and 1000 R/min, respectively (acute irradiation). Beside this, the same materials were also irradiated at the rate of 200 R/hr (chronic irradiation) to make the total administered exposure 20 kR or 30 kR.
Anther culture was initiated and maintained on the N6 medium supplemented with 2mg/l 2, 4-D. The rate of callus formation from the anthers irradiated with a relatively low dose, in both the acute and chronic treatments, showed a slight reduction (36-38%) as compared with that from the non-irradiated control (44.7%). Callus formation rates in plots of higher doses were lower, being 16.7% in 1000 R/min (acute) and 18.7% in 30 kR (chronic, 200 R/hr) treatment.
The effects of irradiation on plant regeneration and organogenesis are shown in Table 1. The rate of green-plant regeneration through callus from irradiated anthers was relatively uniform among different irradiation treatments, but that from irradiated calli was lowered except in the 500 R/min plot. In those plots showing a reduced rate of green-plant regeneration, the rate of root differentiation and occurrence of albino plants increased. The rate of green-spot formation varied widely among different treatments.
Different kinds of mutants obtained from these irradiation experiments are summarized in Table 2. In the R\2\ (second generation after plant regeneration) or M\2\R\2\ (R\2\ after irradiation) generation, visible mutants were counted by growing 10 to 15 plants per line.
In the control raised from the seed of Kitaake, there was no variants among 23 lines each consisting of 10 plants. In contrast, in the anther-cultured but not irradiated R\2\ lines, variants were found in 11 out of 36 lines (Table 2). In the chronic irradiation plots with 20 kR and 30 kR total exposures, mutation frequen-
Table 1.Effect of gamma irradiation on the rate of regeneration from anther-derived calli ________________________________________________________________ Number of Number of regenerants Treatment calli _ ________________________________ Root Albino Green spot Green plant (%) (%) (%) (%) ________________________________________________________________ Exp. 1. (Anther irradiation) 50OR (acute) 532 86(19.2) 72(13.5) 70(13.5) 292(54.9) 1OOOR(acute) 206 27(13.1) 42(20.4) 11(5.3) 119(57-8) 20kR (chronic) 431 63(14.6) 43(10.0) 76(17.6) 224(52.0) 3OkR (chronic) 256 56(21.9) 28(10.9) 26(10.2) 147(57.4) Control 541 25(4.6) 33(6.1) 53(9.8) 425(78.6) Exp. 2. (Callus irradiation) 50OR (acute) 400 55(13.8) 42(10.5) 38(9.5) 261(65.3) 1OOOR(acute) 400 80(20.0) 93(23.2) 47(11.8) 174(43.5) 20kR (chronic) 400 104(26.0)124(31.0) 18(4.5) 144(36.0) 3OkR (chronic) 400 118(29.5)151(37.8) 20(5.0) 100(25.0) Control 400 26(6.5) 72(18.0) 32(8.0) 260(65.0) ________________________________________________________________ Table 2.Mutants obtained from gamma-irradiated anthers or anther- derived calli in the R\2\ or M\2\R\2\ generation ________________________________________________________________ No. of lines Potentially useful variants Treatment __________________ _________________________________ Total Mutant Segre- Short culm Elongated observed total gating (dwarf) Earliness Lateness upper node ________________________________________________________________ Anther culture 36 11 5 6 1 3 1 Exp. 1.(Anther irradiation) 50OR(acute) 57 20 6 7 0 0 0 100OR(acute) 9 5 3 3 1 0 0 2OkR(chronic)31 17 6 10 0 1 3 3OkR(chronic)13 7 4 4 0 4 0 Exp. 2. (Callus irradiation) 50OR(acute) 51 10 6 6 0 0 0 1OOOR(acute) 21 8 2 3 0 0 0 20kR(chronic)23 18 15 12 1 1 0 3OkR(chronic)30 24 17 15 2 0 0 Control 23 0 0 0 0 0 0 (no treatment) ________________________________________________________________cies were much higher irrespective of anther or callus irradiation.
Of 120 lines which showed at least one mutant, 64 lines (53.3%) exhibited segregation for normal and recessive phenotypes clearly. This suggests that some of the mutations occurred in diploid cells of anther calli as heterozygotes. Mutant characters useful for breeding, such as earliness, semidwarfism and elongated upper internode were obtained frequently (Table 2).
Reference
Swanson, E. B., M. P. Coumans, G. L. Brown, J. D. Patel and W. D. Beversdolf, 1988. The characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection of microspores and protolasts. Plant Cell Reports 7: 83-87.