15. Somatic reversion induced by ion beam irradiation in stable yellow leaf mutant of rice

1)Research Institute for Bioresources, Okayama University, Kurashiki, 710-0046 Japan
2)Department of Radiation Research for Environment and Resources, JAERI, Takasaki, 370-1292 Japan

Transposon (mobile element) is very useful for gene isolation in several plant species. Since class II type active transposons have not been discovered in rice, Ac/Ds system in maize has been introduced and utilized for gene-tagging in rice (Izawa et al. 1997). If endogenous active transposons were discovered in rice, it could easily be used as a powerful tool for gene-tagging in open environments. As En/Spm was discovered in a population of maize exposed to atomic bomb at Bikini (Peterson 1953), active transposons may be found in mutagenized rice. As ion beams are a type of high linear energy transfer (LET) radiation and can deposit high energy on a target compared to low LET radiations, the novel mutants or large DNA rearrangements are expected to be induced by ion beam irradiation (Tanaka, 1999). Hence, it is expected that inactive transposon may be induced by ion beam irradiation in rice. Visualization of transposon activity is made by variegation in chlorophyl mutant or anthocyanin accumulation. Maekawa (1995) found a variegated yellow leaf (yl-v) mutant in F2 of a cross between distantly related rice varieties. Genetic analysis showed that the yellow leaf character was controlled by a nuclear recessive gene. The F2 mutant also segregated stable yellow leaf plant and revertants (Maekawa et al. 1996). These results suggest that the variegation of yellow leaf mutant might be caused by a transposable element and the stable phenotype was surmised to be caused by inactivation of the transposon. Although a near isogenic line (NIL) for this mutant gene with T-65 genetic background was bred, this line did not show any variegations through generations. If variegation could be induced by mutation in this NIL, and the variegation could be inherited together with segregation of revertants, there is a strong possibility that the activation of the transposon occurred. Thus, this study aims to induce somatic mutation at M1 in this stable chlorophyl mutant line by ion beams.

Seeds of a chlorophyl mutant (referred yl) were derived from a yl-stable (stable yl) plant in T-65 yl-stb BC3F2. Ion beams used were helium and carbon. Irradiated seeds were sterilized with 70% EtOH and sown in planters containing commercial substrate. After three weeks, the presence/absence of variegations were screened because yl mutants start withering by this time.

Carbon ion beam irradiation drastically reduced the germination rate of T-65 yl-stb plants irradiated with increasing dose, and the median lethal dose with carbon ion was estimated to be 30 Gy (Table. 1). In M1 plants irradiated with 50 Gy of 220 MeV C ions, a variegated yl plant was generated (Fig. 1b). This plant showed small or large sectors in leaves expanded later (Fig. 1c, d). As a result, one variegated plant was obtained out of 992 M1 plants

germinated. On the other hand, helium ion beam irradiation slightly reduced the germination rate of yl-stb plants with increasing dose (50 Gy to 150 Gy), and the median lethal dose was estimated to be more than 150 Gy. In M1 plants irradiated with 4He2+ with 100 MeV, yl plants showing clear variegation were not observed though 9 vague type yl plants were generated (Table. 1). These vague type yl plants were also obtained at frequencies of 0.1 % in control. The frequency of vague type yl plants generated with helium ion beam irradiation was 0.3%. This frequency was slightly higher than that of control, suggesting that helium ion beam irradiation might induce vague type variegation.

The variegated yl plant at M1 bore 9 panicles. The frequencies of clear variegation were examined in panicle-row lines. Spikelet fertilities in 9 panicles were low, varying from 0% to 41.7%. Most of panicle-row lines (no. 2 to 6 and no. 9)segregated into variegated and stable yl plants (Table. 2). For panicle no. 5 line, 2 revertants segregated. In total, the ratio of variegated to stable yl plants showed a 3:1 ratio, suggesting that mutable dominance gene conversion might be induced by carbon ion beam irradiation. In addition, panicle lines no. 2 and 5 segregated for albino. On the other hand, all yl plants showing vague type variegation obtained by helium ion beam irradiation segregated only for stable yl pedigrees at M2 (data not shown). This result showed that vague type variegations observed at M1 plants by helium ion beam irradiation were transient. However, the causative mechanism for vague type variegations remains unknown.

Our results indicate that clear variegation induced by carbon ion beam irradiation on M1 plants is heritable and the inactive state of yl is mutable by stress, such as ion beam irradiation. This suggests that stable yl phenotype might be caused by the inactivation of a transposon.


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