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Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of β-glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 μF; and using 125 μg of calf thymus carrier DNA and 75 μ of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.

The amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was studied during greening in 12Pisum sativum L. genotypes. The proportion of mRNA coding for the small subunit of Rubisco (SSU mRNA) was also monitored by hybridization with a cDNA (complementary DNA) probe from one of the nuclear genes coding for SSU (rbcS). Both the SSU mRNA and Rubisco (in g·FW)(-1)) contents rapidly increased in all genotypes on illumination of dark-grown seedlings. Natural genetic variability was found in the amounts of SSU mRNA, Rubisco· (g FW)(-1), total RNA·(g FW)(-1) and mRNA·(μg total RNA)(-1). Differences among genotypes in SSU mRNA were apparently the result of differences in the rate of light-induced accumulation of therbcS gene transcripts. Genotype means for SSU mRNA and Rubisco· (g FW)(-1) amounts during greening were significantly correlated (r=0.788;P