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Materials and Methods

Spring wheat cultivars, Bobwhite and Fielder, were cultivated in a greenhouse. Immature seeds were collected from panicles about 2 weeks after anthesis, sterilized with 70 % ethanol and 1 % sodium hypochlorite and then washed three times with sterilized distilled water. Immature embryos were isolated from the seeds under stereoscopic microscope.

Agrobacterium tumefaciens strain EHA101 and EHA105, and the vectors pIG121Hm (Hiei et al. 1994), pLC41bar and pLC41Hm were mainly used in this study. The vectors pLC41bar and pLC41Hm had the T-DNAs, which carried a gene for β-glucuronidase (GUS) that contained an intron in the coding sequence and a phosphinothricin (PPT) resistance gene for pLC41bar and a hygromycin (Hm) resistance gene for pLC41Hm respectively.

Isolated immature embryos were treated with centrifuging at various strength in the liquid medium and then inoculated with Agrobacterium. The embryos were placed on co-cultivation medium that contained 5 μM of AgNO3 and/or CuSO4 with the scutellum-side up and incubated at 23 °C in the dark for 2 days. Embryo axis was excised and the embryos were transferred to resting medium and incubated at 25 °C in the dark for 5 days. Some of the embryos after resting culture were examined histochemically for transient expression of GUS gene according to the procedure described by Ishida et al. (2007). The embryos without used for GUS assay were placed on the first selection medium that contained 5 mg/L of PPT or 15 mg/L of Hm and incubated for 2 weeks. Each of embryos was cut into two pieces, which were then transferred to the second selection medium that contained 10 mg/L of PPT or 30 mg/L of Hm and incubated for 3 weeks. The cell clumps proliferated from the pieces were placed on regeneration medium including 5 mg/L of PPT or 30 mg/L of Hm and incubated at 25 °C under continuous illumination (35 μmol m−2 S−1) for 2 weeks. Regenerated shoots were transferred to regeneration medium that contained 5 mg/L of PPT or 15 mg/L of Hm and incubated for 2 weeks. Regenerated plants were transferred to soil in pots and grown in a greenhouse.

Results

Preliminary Study

Agrobacterium strain EHA105 and LBA4404 were compared in the preliminary study. Because the GUS expression in the embryos infected with LBA4404 was generally weaker than that in the embryos infected with EHA101 and EHA105, LBA4404 was not examined further.

The preliminary study also revealed that centrifugation of the immature embryos at 5,000 × g or 20,000 × g for 10 min before the infection and the excision of the embryo axes from the immature embryos 2 days after the infection resulted in higher expression of GUS in the embryos after the co-cultivation. These conditions were taken into the design of the optimization experiments described below.

Immature embryos that were between 2.0 mm and 2.5 mm in length along the axis of Bobwhite and another cultivar, Fielder, were infected with EHA101 (pIG121Hm) in the optimization experiments. Firstly, further addition of salts to the co-cultivation medium was examined. Co-cultivation medium that contained 5 μM each of AgNO3 and CuSO4 was better than that without both or either one of the salts in the level of transient expression of GUS in and of callus formation from the embryos after the resting culture in both genotypes, employed in the rest of the optimization experiments, and taken into the recommended protocol.

Then, the timing of the removal of the embryo axes and the strength of centrifugation before the infection were revisited. The excision of axes 2 days after the infection and centrifugation at 20,000 × g were good in terms of both the transient expression of GUS in and the callus formation from the embryos after the resting culture in both genotypes. Thus, these processes were taken into the final protocol.

Production of Transgenic Wheat

Immature embryos that were between 1.0 mm and 3.0 mm in length along the axis of Fielder were co-cultivated with EHA105 (pLC41bar) or EHA105 (pLC41Hm) according to the protocol determined in the optimization experiments, and transgenic wheat was produced. In these experiments, the immature embryos in different size ranges were compared.

Table 18.1 Typical results of transformation

Vector

Size of immature embryo (mm)

Immature embryos inoculated (A)

Embryos produced resistant plants (B)

Frequency of transformation (B/A, %)

pLC41bar

1.5–2.0

17

8

47.1

2.0–2.5

46

34

73.9

90

67

74.4

40

38

95.0

17

13

76.5

pLC41Hm

1.0–2.0

14

3

21.4

2.0–2.5

12

7

58.3

17

14

82.4

35

24

68.6

2.0–3.0

47

28

59.6

Transformation of wheat was conducted efficiently under most of the combinations of the factors listed in Table 18.1, and the highest frequencies of transformation, ranging between 58.3 % and 95.0 %, were observed when the immature embryos of between 2.0 mm and 2.5 mm of Fielder were tested and the selection was made by the bar gene and hpt gene.

Characterization of the Transgenic Wheat

The transformants of wheat thus produced (T0 generation) were all normal in morphology and fully fertile (Fig. 18.1). The GUS was well expressed in the tissues of leaves, roots, reproductive organs and the seedlings of the next generation of the transformants (Fig. 18.1).

Some of the transformants were analyzed by Southern hybridization. The integration of the T-DNA was clearly demonstrated, and the six transformants out of the 14 shown had a single copy of the transgene. The copy number of the transgene in the other lines was mostly two or three.

The inheritance of the drug resistance was examined in the T1 generation. The hygromycin resistance segregated in 3:1 ratio in the progeny of all four Bobwhite transformants examined. The phosphinothricin resistance segregated in 3:1 ratio in the progeny of six Fielder transformants, in 15:1 in four lines and in 63:1 in three lines. One line showed none of these patterns, but, because presence of such a pattern at a low frequency among the transformants is quite normal, it was not investigated further to see whether the sensitiveness was linked to the absence of or loss of the expression of the transgene. Overall, it is evident that the expression of the transgenes was inherited to the progeny in Mendelian fashion.

Fig. 18.1 Cells and plants derived from immature embryos of cv Fielder infected with A. tumefaciens EHA105 (pLC41bar) and expression of GUS gene in transgenic Fielder. (a) Immature embryos inoculated with EHA105 (pLC41bar). (b) Immature embryos 2 days after inoculation. Upper, before excision of embryo axis; Lower, after excision of embryo axis. (c) Immature embryos at the end of 2nd selection culture. (d) Plant regeneration and rooting from phosphinothricin (PPT) resistant explants under light condition. (e) Transgenic plant at flowering. (f) Transient expression in immature embryos after the resting culture. (g) Leaves detached from young regenerants. (h) Roots detached from young regenerants. (i) Spikelet. (j) Anthers. (k) Endosperm of immature seed. (l) Leaves detached from T1 seedlings

 
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