We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9 kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.

ABSTRACr MATERIALS AND METHODSThe effect of chilling temperatres upon cell cultures of tomato (Lycopersicon eaculektum Mill cv 'VF36,' and cv 'VFNT Cherry,' and L hirsutam Humb. & Bonpl.) was tested. Doubling times for L escuketum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No (12,14). L. hirsutum is reported to show greater tolerance to chilling temperatures than the domestic tomato (3, 7, 12-15, 18, 20 (11), 100 LM iron EDTA, 3% sucrose, 2 mg/l 2,4-D, and 0.1 mg/l BA or isopentenyl adenine. The medium is modified from the procedures of S. E. Barsel (personal communication). Cultures were maintained on orbital shakers at 180 to 200 rpm or on a reciprocating shaker at 64 cycles min-'. All stock cultures were maintained at 28C. 'VF36' and L. hirsutum stocks were maintained in 200 ml of medium in 500 ml flasks and transferred every 7 to 10 d by transferring 25 ml of cells to 175 ml of fresh medium. Stock cultures of 'VFNT Cherry' were maintained in 125 ml flasks.Callus from all tissues was initiated on NH4NO3 medium. Suspension cultures were initiated from 2 to 6 months after initiation of callus, and used for experiments from 2 months to 1 year afterwards. Suspension cultures of L. esculentum cv 'VENT Cherry' were maintained in NH4N03 medium, while cultures of cv VF36 and L. hirsutum were maintained in ammonium medium. The cultures grew well in either medium, results were similar using either medium, and choice ofNH4NO3 or ammonium media was not significant to the behavior of the cultures.Growth Measurements. Several methods were used to assess the effect of temperature on growth.A. Weight Determinations. Stock cultures were maintained in 200 ml of medium in 500 ml flasks at 28C. Inoculum from a culture in the early stationary phase of growth was diluted 1 to 5 into 30 ml of medium in a series of 125 ml flasks. The flasks were immediately transferred to the indicated temperatures. At 2 d intervals, flasks were harvested by vacuum filtration onto Whatman No. 4 filter paper on a Buchner funnel, and fresh weight and dry weight were determined.B. SCM3 Determinations Using Stationary Phase Cells. A nondestructive means ofmeasuring the growth ofthe cell cultures was needed. The cultures were thick; also the cells settled rapidly when not agitated, so measurements of the optical density of the cells in a side arm did not seem appropriate. Instead, a method was developed to measure the settled cell volume. Inoculum from a culture in the early stationary phase ofgrowth was diluted 1 to 9 into 45 ml ofmedium in a series of 300 ml side arm flasks, 3Abbreviation: SCM, settled-cell-to-medium ratio.

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