32. An analysis of somaclonal variation in progenies regenerated from rice calli

     Variations arising from cell culture are called somaclonal variation (Larkin and Scowcroft 1981). We have detected somaclonal variations in regenerated rice lines by using landmarkers provided by the Rice Genome Research Program of the National Institute of Agrobiological Resources (NIAR) and the Institute of the Society of Techno-Innovation in Agriculture, Forestry and Fisheries (STAFF) in Japan. Here we report the details of some variations revealed by the analysis of leaf DNA of R, progenies derived by the selfing of R0.

     In the regenerated rice plants from calli of Oryza sativa L. cv. Akihikari, R4,C lines (32 plants) originated from four month-old calli and R0E lines (21 plants) originated from seven month-old calli were analyzed by Southern blot analysis by digesting the genomic DNA with Hindffl and probed with the landmarker set 1 probes. This set has 72 markers and they are distributed in all chromosomes. Only two out of the 72 markers showed polymorphism. A landmarker of GN89-2 (Chr. No. 10) showed polymorphism in one of the R0C lines and four of the R0E lines, and GN232 (Chr. No. 3) showed polymorphism in one of the R0E lines (Fig. 1. A, C). In R0C-22, R0E-20, R0E-21 and R0E-24, GN89-2 hybridized to a 11.4 kb fragment in addition to a 3.4 kb and a 7.1 kb fragments shown in the control(lanel). These R0C-22, R0E-20, R0E-21 and R0E-24 plant lines were derived from the same callus clone. To investigate these polymorphism in detail, 30 R, progenies generated after self fertilization of R0E-20 were analyzed by the same method using Southern blot analysis (Fig. 2. A). The R,C-22, R,E-20, R,E-21 and R1E-24 segregated into three types of progenies with 3.4kb + 7.1 kb, 3.4kb + 7.1 kb + 11.4kb and 3.4kb + 11.4kb fragment patterns and fit a 1:2:1 ratio in x2 tests. These results suggest that the variations detected in R0C-22, R0E-20, R0E-21 and R0E-24 resulted from the heterozygous callus. On the other hand, all individuals of R,E-14 showed a 12 kb fragment which is present in the control (data not shown). It is assumed that all gametes of R0E14 at this region died out or were not able to be fertilized due to mutations.

     Recently, it was reported that Tosl7, a retrotransposon of rice, was activated and transposed under tissue culture conditions (Hirochika er a!. 1996). To explore the possibility that the polymorphism detected in R0 arose from the transposition of Tosl7, a reverse transcriptase domain in Tos 17 was used as a probe in the above-mentioned blots (Fig. 1. B, D and Fig. 2. B). The 11.4 kb band of R0C-22, R0E-20, R0E-21 and R0E-24 showed the same segregation pattern in both cases using the GN89-2 landmarker and the reverse transcriptase domain as probes (Fig. 2. A, B). Furthermore, the 11.4 kb band size is equal to the total band size of the retrotransposon Tosl7 (4.3 kb) and one of the control band sizes ( 7.1 kb). Thus, we believe that R0C-22, R0E-20, R0E-21 and R0E-24 arose from the insertion of the Tosl7 retrotransposon into the 7.1 kb DNA fragment.