No abstract

No abstract

Plants from in vitro culture can exhibit somaclonal variation, two characteristics of which are structural rearrangements and variation in chromosome number. These characteristics were studied in barley (Hordeum vulgare L. cv. Golden Promise) callus and plants derived from nontransgenic and transgenic callus of approximately the same age; chromosomes were studied in cells from callus and root tips from plants. Analysis of these data revealed greater variation in ploidy in transgenic compared with nontransgenic plants. Of 59 independent transgenic lines, only 32 (54%) had normal diploid complements of 2n = 2x = 14, while 27 (46%) were tetraploid (2n = 4x = 28) or aneuploid around the tetraploid level (i.e., 26, 27, 29 and 30 chromosomes); no aneuploidy around the diploid number was observed. Nontransgenic plants regenerated after in vitro culture alone had a much lower percentage of tetraploids (0–4.3%). Most diploid plants had normal gross morphology, while tetraploid plants had abnormal morphological features. Ploidy determinations were made on randomly selected cells from callus of immature embryos cultured for 0 to 14 d. The number of tetraploid cells in 1‐d‐ to 7‐d‐old callus was around 2 to 4%; in callus comparable in age to that used to regenerate both the transgenic and the nontransgenic sets of plants, 23% of the cells were tetraploid. This percentage is lower than the percentage (46%) of tetraploid plants from the transgenic lines; however, it is considerably higher than the percentage (0–4.3%) of tetraploid plants from nontransgenic callus. Therefore, although chromosomal variation and abnormalities occur in callus and nontransgenic plants, the extent of ploidy changes in transgenic plants is exacerbated, perhaps due to the additional stresses that occur during transformation.

No abstract

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.