Biology Reference
In-Depth Information
Open Field (Summer) Cultivation
The 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 trans-
genic 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 ran-
domized 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.
Early 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 har-
vests. 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.
Plant dnA isolation and southern blot Analysis
High-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
µ
g of DNA was digested overnight with 80
units of KpnI (Promega) in a volume of 500
µ
l, separated on a 0.7% agarose gel
and transferred to Hybond N (Amersham Pharmacia Biotech). A 1350 bp frag-
ment 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).
rt-Pcr Analysis
Semiquantitative (competitive) PCR analysis was carried out for 38 cycles (an-
nealing temperature 63°C) using as template cDNA (8 ng) obtained by prim-
ing poly(A)+ mRNA with an iaaM specific primer (5'-AATAGCTGCCTATGC-
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