43. Mutagenesis by means of anther culture combined with gamma irradiation

Toshiro KINOSHITA, Koh-ichi MORI and Itsuro TAKAMURE

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
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                     Number of       Number of regenerants
Treatment              calli  _ ________________________________
                              Root Albino Green spot Green plant         
                              (%)    (%)     (%)          (%)
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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)
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Table 2.Mutants obtained from gamma-irradiated anthers or anther- 
        derived calli in the R\2\ or M\2\R\2\ generation
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                No. of lines     Potentially useful variants
Treatment   __________________ _________________________________
          Total Mutant Segre- Short culm               Elongated
       observed total  gating  (dwarf)  Earliness Lateness upper 
                                                            node
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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)
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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.