Materials and methods

Fifteen alloplasmic lines of Triticum aestivum cv Chinese Spring (CS) with cytoplasms of Triticum
and Aegilops were used in this study (Table 1). CS was used as control. Seeds were imbibed for 5 h under tap water, placed in 4 oC overnight, planted in pots and grown under natural light with day-night temperatures of 25-20 oC. At 14th day after sowing, plants were harvested, fresh and dry weights were measured, and leaves were frozen with liquid nitrogen.

Rubisco activity of 6-9 samples per line was measured based on the rate of NaH14C03 incorporation (Keys and Parry 1990). Briefly, leaf tissues (ca. 0.5 g fresh weight) were ground in 1.5 mI of extraction buffer containing 100 mM Bicine (pH 8.2), 20mM MgCl2, 5 mM dithiothreitol and 0.025 mM polyclar AT, and centrifuged at 15,000 x g for 20 sec. The reaction was initiated in capped vials by adding 10μl of the supernatant to a reaction mixture containing 100 mM Bicine (pH 8.2), 20 mM MgCl2, 50 mM mercaptoethanol, 20 mM NaH14C03 (4.44 x 107 Bq/mmol) and 20 mM ribulose-1,5-bisphosphate. The vials were incubated at 25 oC for 10 min. The reaction was stopped by the addition of 30 mI of 4N HCl and unincorporated 14C03 was allowed to be trapped by NaOH overnight in an oven at 50 oC. The amount of incorporated14C was measured by liquid scintillation counter and means were compared by t-test. All solutions used in the experiment were prepared as C02-free using boiled-distilled water and gassed with N2 just before activity measurements.

Chlorophyll was extracted with ice-cold 80% acetone and the concentration was determined according to Arnon (1949). Protein content was determined by Bradford's method (Bradford 1976) using BioRad protein reagent. Data were based on 9 samples per line and mean separation was made by t-test.


Results

Chlorophyll content: Five alloplasmic lines with cytoplasms of Ae. caudata, Ae. umbellulata, Ae. uniaristata, Ae. speltoides and Ae. mutica showed significantly higher chlorophyll contents than CS (Fig. 1). An alloplasmic line with Ae, squarrosa cytoplasm showed a higher chlorophyll-a/b ratio than CS (data not shown).

Protein content:
Five alloplasmic lines with cytoplasms of T. boeoticum, Ae. caudata, Ae. squarrosa, Ae. heldreichii and Ae. sharonensis showed significantly higher protein contents than CS (Fig. 1). Protein content was markedly high in the alloplasmic line with cytoplasm of T. boeoticum that shows severe growth depression.

Rubisco activity:
No alloplasmic lines showed lower Rubisco activity than CS, but five alloplasmic ,lines with cytoplasms of T. boeoticum, Ae. heldreichii, Ae. uniaristata, Ae. speltoides, and T. timopheevi showed significantly higher Rubisco activity than CS, based on both fresh weight and .chlorophyll content (Fig. 2). No significant correlation was found between Rubisco activity and photosynthetic capacity measured by the rate Of 13C02 assimilation (Nakamura et al. 1991).

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