LIBRO-CONGRESO-CITRUS

S05
tomato constitutively express Lycb-1 was engineered in this study. The β-carotene level of transformant increased
4.1 fold, and the total carotenoid content increased by 30% in the fruits. In the transgenic line, the downstream
α-branch metabolic fluxes were repressed during the three developmental stages while α-carotene content
increased in the ripe stage. Microarray analysis in the ripe stage revealed that the constitutive expression of Lycb-
1 affected a number of pathways including the synthesis of fatty acids, flavonoids and phenylpropanoids, the
degradation of limonene and pinene, starch and sucrose metabolism and photosynthesis. This study provided
insight into the regulatory effect of Lycb-1 gene on plant carotenoid metabolism and fruit transcriptome.
S05P17
Role of the sweet orange tau glutathione transferases (csgstu) in transgenic tobacco plant
detoxification from xenobiotics
Lo Cicero L. 1 , Madesis P. 2 , Tsaftaris A. 3 , Lo Piero A.R. 4
1 University of Catania (DISPA), Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, Italy; 2 The Centre for Research and
Technology Hellas (CERTH), Institute of Applied Biosciences, Greece; 3 The Centre for Research and Technology Hellas (CERTH),
Institute of Agrobiotechnology, Greece; and 4 University of Catania (DISPA), Dipartimento di Fitopatologia e genetica vegetale, Italy.
lociceroluca1981@libero.it
The glutathione S-transferases (GSTs) catalyse the attack of glutathione (GSH) on a variety of hydrophobic
toxic chemicals. The conjugation of GSH to such molecules increases their solubility and facilitates further
metabolic detoxifying processes. Two csgst genes (gstu1 and gstu2) encode for proteins which differ only for
three amino acids located in the hydrophobic co-substrate binding site (gstu1: R89, E117, I172; gstu2: P89,
K117, V172). Previously, site-directed mutagenesis experiments generated several cross-mutate enzymes and
among them the GST-RKV mutant derives from the P89R substitution upon the gstu2 isoform. In this work,
both wild-type (gstu1 and gstu2) and the GST-RKV mutant were incorporated into the tobacco genome under
the control of the strong constitutive CaMV 35S promoter via Agrobacterium mediated transformation. PCR
and Southern blot analysis was performed to confirm the presence of the nptII gene only in the genomic DNA
from transformed tobacco plants. The expression of the transgenes was also assessed by RT-PCR experiments.
The GST activity of tobacco leaves transformed with wild type GSTs turned out to be similar with that exhibited
by untransformed plants, whereas plants harbouring the GST-RKV mutant gained a 2-fold increase of the GST
activity assayed against several pollutants, thus suggesting that the RKV-transformed tobacco plants acquired
novel favourable traits which might provide a more efficient contribution in the detoxification system.
S05P18
An alternative transformation method in citrus using cell-penetrating peptides (CPPs)
Jensen S.P., Febres V.J., and Moore G.A.
University of Florida (UF), Plant Molecular and Cellular Biology, United States of America. jensensp@ufl.edu
Citrus transformation is typically Agrobacterium tumefaciens-mediated, in which citrus tissues are cultivated
with the bacterium and regenerated on selection media. Part of the bacterial plasmid, with the gene of
interest and resistance genes, is then inserted into the citrus genome. Due to the slow growth of citrus,
this long process must be optimized for each cultivar and ultimately the transformation efficiency in citrus
is substantially less than with other model systems. Commercialization of transgenic citrus is even slower
because of the regulations on genetically modified produce worldwide. In order to decrease the dependence
upon bacterial transformation and increase transformation efficiency, we propose an alternative method of
transformation using cell penetrating peptides (CPPs) that does not involve Agrobacterium. CPPs are positively
charged short amino acid sequences able to simultaneously bind proteins and nucleic acids and deliver them
across cellular membranes and cell walls. CPPs are used currently in plants in transient expression assays and
gene silencing, but have not been used in citrus or in stable transformation experiments. We have developed
a standard method for the transient expression of reporter genes (gus and gfp) in citrus. Our data indicate
that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have
been tested and the efficiency is increased to 100% when CPPs are used in conjunction with a lipid reagent.
We have transformed several explant segments which survived kanamycin selection, produced shoots and
roots, and were planted. PCR and reporter gene analysis will confirm stable integration. Further experiments
with CPPs, comprising RNAi and protein trafficking, will also be performed.
86 - VALENCIA CONFERENCE CENTER, 18th-23rd NOVEMBER 2012