Screening and selection of high carotenoid producing in vitro tomato cell culture lines for [13C]-carotenoid production

Abstract

Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors' knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture.

Figure 1

Simplified study design for the derivation and selection of tomato cell lines for (A) phytoene production from the ghost tomato cell line and (B) lycopene, phytoene, and phytofluene production from a high lycopene tomato cell line.

Figure 2

Lycopene concentrations (A) and yields (B) from different tomato cell line suspension cultures treated with CPTA for a 14 day growth cycle and harvested approximately 8.5, 10.5, or 11 months postinitiation of cell suspension cultures from callus cultures. Each point represents the average of two samples.

Figure 3

Combined carotenoid (LYC, PF, and PE) yields from hp-1 tomato cell suspension cultures treated with different herbicides (n = 3 trials). Error bars represent the SEM of combined carotenoids. Significantly different combined carotenoid yields (α = 0.05) detected by Tukey’s studentized range test compared to the control are noted with an asterisk (*).

Figure 4

HPLC chromatograms (λ = 472 nm) of (A) the LYC analytical standard with all-E (peak 3) and 5-Z (peak 4) lycopene isomers eluting at 28 min with other preceding Z-isomers (RT = 22.5 and 24.5 min; peaks 1 and 2) and (B) purified ¹³C-all-E LYC from an hp-1 tomato cell suspension culture.

Figure 5

Mass isotopomers present in purified ¹³C-lycopene from the hp-1 tomato cell culture treated with CPTA. Percent isotopomer in the sample is calculated from mass chromatogram peak signal areas obtained from four analytical injections of ¹³C-lycopene (1000 ng each in 40 µL of diethyl ether); each injection yielded peak area signals for 6 masses selected, and sequential injections were normalized. Signals for <m/z = 557 were not quantifiable. In vitro labeling yields 45% uniformly labeled (m/z = 577) ¹³C-lycopene and a distribution of less enriched isotopomers (m/z = 557–576).