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Embryo culture
Because crosses made in 1997 between T. urartu as female and emmer wheat did not produce any viable F1 seeds, embryo culture was employed for the same cross in 1998. In this case, ovaries of 13-17 days after pollination were dissected and sterilized with 70% ethanol for 2 min, then embryos were excised from them and cultured on the N6 medium that was supplemented with casein hydrolysate (400 mg/l), GA3 (0.5 mg/l), IBA (0.5 mg/l), and sucrose (50 g/l), keeping the cultures at 26C under artificial illumination of a 16-hour photoperiod, after Koba et al. (1991). Culture was carried out for about 50 days, then the cultures were discarded because they all withered.


Results

Transfer of T. boeoticum cytoplasm to emmer wheat
Table 1 shows the results of the crosses, T. boeoticum x emmer wheats, and two successive backcrosses of the F1 and B1 hybrids to emmer wheats as recurrent pollen parent (Hori and Tsunewaki 1967, unpubl). One hundred and fifty florets of T. boeoticum pollinated with T. dicoccum cv. Hokudai produced 93 F1 seeds, the seed setting rate being 62%. From those seeds 73 F1 hybrids were obtained, of which germination rate was 78.5%. Some of the F1's were backcrossed as female to four accessions of emmer wheat. Crossed seed fertility was only 0.5% when all F1's data were pooled. Germination rate of the B1 seeds was very high (70% in total). The B1 hybrids were backcrossed further to three accessions of emmer wheat. Seed setting rate (61%) was improved greatly. The B2 seeds obtained germinated well at the overall frequency of 93%. Almost all B2 plants cytologically checked had 2n = 28 chromosomes, with an exceptional plant that had 2n 29 (Hori and Tsunewaki 1967). Thus, transfer of the T. boeoticum cytoplasm to emmer wheat was achieved rather easily.

Transfer of T. urartu cytoplasm to emmer wheat
Crosses between T. urartu (female) and an emmer wheat (male) did not set any viable F 1 seeds, as shown in the last line of Table 1. To overcome this cross incompatibility, embryo culture was performed (Table 2). Ovaries of T. urartu, which. were pollinated with the pollen of T. durum, were excised on the 13th to 17th day after pollination, and the embryos dissected were cultured as described above.

The embryos dissected from the ovaries of 16 or 17 days after pollination rarely showed any sign of development, with one exception. About 60% of ovaries which were on the 13th to 15th day after pollination contained embryo. When cultured, about 55% of those embryos showed some growth, differentiating shoot and/or roots. Most of the remainders showed no sign of development. However, all shoots developed were abnormal (Fig. 1), and sooner or later they stopped growing and finally withered out. Thus, even with the aid of embryo culture, we could not get any F1 hybrid from the cross, T. urartu (female) x emmer wheat. It appears very difficult, if not impossible, to transfer the T. urartu cytoplasm to polyploid wheat.

Discussion

Yamagishi (1987) made a large number of crosses between different accessions of T. boeoticum and T. urartu. He demonstrated that these two species have the identical genome A, although their reciprocal F1 hybrids showed deep sterility, indicating that the sterility barrier has developed between the two species in the course of their speciation. Dvorak et al. (1988) and Takumi et al. (1993) showed that their nuclear genomes have greatly been differentiated to each other, based on the magnitude of RFLP between their nuclear genomes. They used repeated DNA and genomic DNA sequences as probes, respectively. On the other hand,T. boeoticum and T. urartu have identical chloroplast genome so far as the restriction fragment patterns of their chloroplast DNAs obtained by the use of 13 restriction endonucleases were concerned (Ogihara and Tsunewaki 1988).

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