PMC:101493 / 17372-23148 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/101493","sourcedb":"PMC","sourceid":"101493","source_url":"https://www.ncbi.nlm.nih.gov/pmc/101493","text":"Materials and methods\n\nGreenhouse spring cultivation\nTrials were carried out in central Italy (Monsampolo del Tronto-AP) and in southern Italy (Pontecagnano-SA) (approval of the Italian Ministry of Health N° B/IT/97-29). The greenhouses were rather similar and made of galvanized steel and covered with plastic polyethylene (0.12 mm thick). An apparatus for drip-irrigation was used and the soil was completely mulched. A complete randomized block design with three replicate hybrid genotypes was adopted. Each experimental plot measured 3.12 m2 and contained eight plants in a double row. Transplanting was performed on March 3rd in southern Italy and on March 27th in central Italy. The P1, P2, P5, C1, C2 and the commercial Talina hybrids were employed. Transgenic parthenocarpic hybrids P1, P2 and P5 were obtained by crossing (as male parent) the primary transgenic plant DR2 iaaM #34-1 with the line Tal 1/1 (P1), the primary transgenic plant DR2 iaaM #28-1 with Tal 1/1 (P2) and the transgenic plant Tal 1/1 iaaM #1-1 with the line Tina (P5). The hybrids P1 and P2 are homologous to C1 (DR2 × Tal1/1), except for the presence of the DefH9-iaaM gene integrated in their genome. The transgenic hybrid P5 is homologous to its untransformed control C2 (Tal1/1 × Tina). DR2 and Tina are parental lines obtained through classical breeding, Tal1/1 is a double haploid line derived from anther culture of the F1 commercial cultivar Talina. The segregation of the marker gene nptII was checked by spraying the plants with kanamycin [14] and allowed for the conclusion that the transgenes segregate as a single locus in the backcrossed progenies of the three independent events analyzed (Tal iaaM 1-1: χ2 = 0.01065, P = 0.917; DR2 iaaM 34-1: χ2 = 0.0496 P = 0.824; DR2 iaaM 28-1 χ2 = 0.06467 P = 0.799). Southern blot analysis showed that DR2 iaaM 28-1 and 34-1 had a single copy of the transgene, while Tal iaaM 1-1 had three copies of the transgene (Fig 4). Since the interaction genotype/location was not significant for the yield, the data were computed as average of the two locations and subjected to analysis of variance according to a randomized complete block design. Duncan's Multiple Range Test (P = 0.05) was used for means separations.\nFigure 4 Southern blot analysis of transgenic eggplants. Numbers above the lanes indicate the independent transgenic plant DR2iaaM#28-1 (28), DR2iaaM#34-1 (34) and Tal1/1iaaM#1-1 (Tal1/1-1). Cont indicates untransformed plants, i.e. DR2 and Tal1/1, respectively. The probe used corresponds to the DefH9 regulatory region.\n\nOpen field (summer) cultivation\nThe open field trial was carried out under open field conditions at Monsampolo del Tronto (approval of the Italian Ministry of Health B/IT/99/21). Two transgenic parthenocarpic hybrids were tested: the hybrid P1 (Tal1/1 × DR2 iaaM #34-1) with elongated fruits and the hybrid P10 (UGA × Tal1/1 iaaM #1-1) with sub-oval fruits were compared to their homologous non-transgenic controls C1 (DR2 × Tal1/1) and C10 (UGA × Tal1/1), respectively. The UGA line has oval dark purplish fruits and it has been provided by Dr. S.C. Phatak. A complete randomized block design with the hybrids replicated four times was adopted. Each experimental plot measured 11.7 m2 and contained 30 plants in a double row. Transplanting was performed on May 10th.\nEarly spring production consisted of the first four harvests (i.e. 4 out of 16 harvests during the whole production cycle), while early summer production, whose cultivation cycle consists of ten harvests, corresponds to the first three harvests. For all trials the number and weight of fruits were recorded. In addition, fruit sample for each harvest and replication was cut to check for the presence of seeds. Data were computed for the early harvesting time and for the whole harvesting season. Analysis of variance was performed according to a randomized complete block design. Duncan's Multiple Range Test (P = 0.05) was used for means separations.\n\nPlant DNA isolation and Southern blot analysis\nHigh-molecular-weight DNA was isolated from the young leaves of transgenic and untransformed eggplants by using Plant DNAzol (Invitrogen), according to the manufacturer's instructions. Ten μg of DNA was digested overnight with 80 units of KpnI (Promega) in a volume of 500 μl, separated on a 0.7% agarose gel and transferred to Hybond N (Amersham Pharmacia Biotech). A 1350 bp fragment of the DefH9 gene was used as a radiolabeled probe. The membrane was hybridized overnight in 5X SSC/50% formamide (Sigma) at 42°C and washed two times for 15 min. in 2 × SSC/0.1% SDS, and two times for 15 min. in 0.1 × SSC/0.1% SDS at 42°C. Signals were detected using Kodak X-OMAT AR5 film (Sigma).\n\nRT-PCR analysis\nSemiquantitative (competitive) PCR analysis was carried out for 38 cycles (annealing temperature 63°C) using as template cDNA (8 ng) obtained by priming poly(A)+ mRNA with an iaaM specific primer (5'-AATAGCTGCCTATGCCTCCCGTCAT-3'). The mRNA was extracted from either young flower buds (5,8,11 mm) or eggplant fruits (placental tissue from fruits either 40 or 280 mm long). As an internal standard, 0.5 fg of a 600 bp long DefH9 cDNA fragment was used in the PCR assay. To amplify the 161 bp long amplicon an iaaM specific primer (5'-GGGTGAATTAAAATGGTCATACAT-3') and a DefH9 specific primer (5'-CTTTGGAACTCGTGTTGAGCTCTCA-3') were used. For the internal standard, a 3' primer (5'-TGAGCATTGATCTCCTGAGTGGTGT-3') together with the DefH9 specific primer were used to produce the 351 bp long amplicon. The PCR assays were performed with a thermostable DNA polymerase mixture (Expand High Fidelity PCR system, Roche) in presence of 3 μCi of 32P dCTP. The intensity of the bands was quantified by using an Instant Imager (Packard, Meriden, CT).","divisions":[{"label":"title","span":{"begin":0,"end":21}},{"label":"sec","span":{"begin":23,"end":2568}},{"label":"title","span":{"begin":23,"end":52}},{"label":"p","span":{"begin":53,"end":2245}},{"label":"figure","span":{"begin":2246,"end":2568}},{"label":"label","span":{"begin":2246,"end":2254}},{"label":"caption","span":{"begin":2256,"end":2568}},{"label":"p","span":{"begin":2256,"end":2568}},{"label":"sec","span":{"begin":2570,"end":3990}},{"label":"title","span":{"begin":2570,"end":2601}},{"label":"p","span":{"begin":2602,"end":3337}},{"label":"p","span":{"begin":3338,"end":3990}},{"label":"sec","span":{"begin":3992,"end":4724}},{"label":"title","span":{"begin":3992,"end":4038}},{"label":"p","span":{"begin":4039,"end":4724}},{"label":"title","span":{"begin":4726,"end":4741}}],"tracks":[]}