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Tomato has been considered as a potential biological production platform for plant-made pharmaceuticals (PMPs). However, information is limited for protein productivity and dynamics in tomato fruit. As part of our PMP production project, total soluble protein (TSP) content of green and red fruits was analyzed for selected six greenhouse cultivars. The six cultivars consisted of various fruit types (including cluster, cherry, mini plum, and grape types) with sizes ranging from 10 to 150 and from 14 to 188 g fresh weight for green and red fruits, respectively. In fruit TSP content per unit dry weight (DW), approximately seven- to eightfold variation was observed among cultivars for both green and red fruits. There was no significant correlation between fruit TSP content per DW and fruit DW or fruit type over the cultivars irrespective of fruit ripening stage, indicating that fruit weight or fruit type of a cultivar cannot be an indicator of the TSP content per DW. There was also an inconsistent trend in differences in fruit TSP content per whole fruit between green and red fruit among cultivars. Our results suggest that a low-yielding cultivar can produce high TSP content per unit ground area resulting from high TSP content per DW in fruit. A background tomato cultivar or genotype candidate for commercial PMP production should thus be selected from among various fruit types considering both yield potential and fruit TSP content.

Cos lettuce was grown under different spectral photon flux density distribution (SPFD) change patterns with blue- and/or red light-emitting diode (LED) irradiation with a 24-hour cycle. Twelve treatments were designed with a combination of four relative SPFD (RSPFD) change patterns and three photosynthetic photon flux density (PPFD) levels. The RSPFD change patterns were as follows: BR/BR, simultaneous blue- and red-light irradiation (BR) for 24 h; R/BR, red-light monochromatic irradiation (R) for 12 h followed by 12 hours of BR; B/BR, blue-light monochromatic irradiation (B) for 12 hours followed by 12 hours of BR; and B/R, 12 hours of B followed by 12 hours of R. Each RSPFD change pattern was conducted at three daily average photosynthetic photon flux densities (PPFD_ave) of 50, 100, and 200 µmol·m⁻²·s⁻¹. The RSPFD change patterns that included B (B/BR and B/R) resulted in elongated leaves. A low ratio of active phytochrome to total phytochrome under B was considered the reason for leaf elongation. Shoot dry weight was significantly greater under the RSPFD change patterns that included B when the PPFD_ave was 200 µmol·m⁻²·s⁻¹. The leaf elongation caused by B would have increased the amount of light received and thereby promoted growth. However, excessive leaf elongation caused the plants to fall, and growth was not promoted under the RSPFD change patterns that included B when the PPFD_ave was 50 µmol·m⁻²·s⁻¹. Thus, 12-hour B promoted growth under conditions in which leaf elongation leads to increases in the amount of light received.

Using greenhouse tomato (Solanum lycopersicum) as a model system to produce pharmaceutical proteins, electrical conductivity (EC) of hydroponic nutrient solution was examined as a possible factor that affects the protein concentration in fruit. Transgenic tomato plants, expressing F1-V protein, a plant-made candidate subunit vaccine against plague (Yersinia pestis), were grown hydroponically at high (5.4 dS·m⁻¹) or conventional EC [2.7 dS·m⁻¹ (control)] with a high-wire system in a temperature-controlled greenhouse. There was no significant difference in plant growth and development including final shoot dry weight (DW), leaf area, stem elongation rate, or leaf development rate between high EC and control. Net photosynthetic rate, transpiration rate, and stomatal conductance (g _S) of leaves were also not significantly different between EC treatments. For both EC treatments, immature green fruit accumulated DW at a similar rate, but dynamics observed in fruit total soluble protein (TSP) and F1-V during the fruit growth were different between the two ECs. Fruit TSP concentration per unit DW decreased while TSP content per whole fruit increased as fruit grew, regardless of EC. However, TSPs were significantly lower in high EC than in control. Fruit F1-V concentration per unit DW and F1-V content per whole fruit were also lower in high EC than in control. Our results found that increasing EC of nutrient solution decreased TSP including the vaccine protein in fruit, suggesting that adjusting nutrient solution EC at an appropriate level is necessary to avoid salinity stress in this transgenic tomato.

Changes in the amounts of F1-V, an antigen fusion protein and a candidate subunit vaccine against plague, and total soluble protein (TSP) in green fruit of transgenic tomato plants were investigated to identify the optimum harvest timing to maximize the F1-V yields. Two T ₂ progenies of the transgenic plant, ‘22.11.21’ and ‘22.11.5’, were grown. The F1-V concentration rapidly decreased at the beginning of the green stage and decreased to less than 5% of the initial concentration at the late green stage in ‘22.11.21’. The F1-V concentration also decreased as fruit size increased in ‘22.11.5’, but the pattern of the decrease was linear and different from that in ‘22.11.21’. The concentration of TSP also decreased with fruit growing in both plants. When calculated on a whole fruit basis, the F1-V content linearly decreased with increasing fruit size in ‘22.11.21’. In ‘22.11.5’, the F1-V content per fruit also tended to decrease from the middle to late green stage. Based on these observations, collecting small green fruits without pruning was proposed as a harvest practice that may maximize the F1-V yields. Thus, the optimum protocols for harvesting and pruning for plant-made pharmaceutical production may be substantially different from those currently used in commercial hydroponic greenhouses for fresh market tomato.

Biopharmaceutical protein production is a new application of plant biotechnology. Nevertheless, there is limited information for potential protein productivity in commercial production operation. The objective of this study was to characterize the growth and development as well as fruit and protein productivities of transgenic tomato (Solanum lycopersicum) plants in comparison with two nontransgenic reference cultivars under greenhouse conditions with commercially adopted cultural practice. Transgenic tomatoes expressing a predominant antigen fusion protein, F1-V, against plague were used as a model system. Three types of tomatoes were grown for this study: 1) a transgenic T ₂ line, ‘F1-V’; 2) the background wild-type cultivar, TA234; and 3) a commercial greenhouse cultivar well adopted in North America, Durinta. All plants were grown hydroponically in a greenhouse equipped with heating and evaporative cooling systems for 24 to 30 weeks. When comparing ‘F1-V’ with ‘TA234’, there were no significant differences in growth, cumulative fruit yield, fruit total soluble protein (TSP) concentration, nor cumulative TSP production between the two genotypes. Although there was a difference in plant leaf morphology, this suggests that the transformation event did not affect the key traits of biopharmaceutical protein production. When comparing ‘F1-V’ with ‘Durinta’, ‘Durinta’ yielded more fruit than did ‘F1-V’, although final vegetative biomass of the two genotypes was not significantly different. Cumulative fruit yield per plant of ‘Durinta’ for 13 weeks of harvest was almost twice that of ‘F1-V’. However, TSP concentration of fruits of ‘Durinta’ was only 12% to 34% of that of ‘F1-V’, making the estimated cumulative TSP production by fruits approximately half that of ‘Durinta’. Our results suggest that biomass productivity is not necessarily the high-priority trait in selecting cultivars for high-value protein production and that protein concentration of fruits may be an important factor.