Structural Deterioration in Produce: Phospholipase D, Membrane Deterioration, and Senescence - Postharvest Biology and Technology of Fruits, Vegetables, and Flowers - page 222

that PLD beta and gamma isoforms could not replace the decreased PLD alpha activity
induced by antisense suppression may have arisen because of the naturally low PLD beta
and gamma activity (nearly 1,000-fold) in Arabidopsis (Fan et al., 1999).

9.7.1 Generation of antisense PLD α 1 tomato plants

Transformation of “Celebrity” tomatoes was conducted through Agrobacterium- mediated
transfer of the antisense construct to excised cotyledons. For this, a binary vector designated
pBIN-mgfp5-ER (Pinhero et al., 2003) was used. The GFP (cDNA sequence for green
fluorescent protein) was dissected out and a 2,427-bp PLD cDNA was introduced in the
antisense orientation between a Bam H I site and a Sac I site. The control of transcription
initiation and termination was achieved by a CaMV 35S promoter (constitutive) and an NOS
terminator, respectively. The transformed cotyledons were grown in vitro in the presence of
kanamycin. Kanamycin-resistant calli and seedlings that possess a positive transformation
were passed through two additional generations in the presence of kanamycin, and further
transferred and grown in a regular MS rooting medium. Rooted seedlings were further
transferred to sterile perlite and grown in a greenhouse. The presence of the NPT gene
(kanamycin resistance) in transformed plants was confirmed by PCR amplification of the
DNA using primers specific for the NPT gene, which resulted in the amplification of a 700-
bp NPT gene segment. The transformed plants were propagated clonally to eliminate the
possibility of the loss of antisense PLD cDNA in subsequent generations. The cotyledons
were also transformed with the vector alone and plants regenerated. These plants could
serve as controls to eliminate the physiological repercussions of the transformation process.
The transformed plants were always compared with control plants that originated from
cotyledons through the tissue culture procedure.

9.7.2 Characteristics of Celebrity antisense PLD α 1 tomatoes

The transformation and regeneration efficiency of Celebrity tomatoes was very low ( 2%).

The transformed plants were very vigorous in vegetative growth. Many of the transformed
plants produced flowers as usual, but did not set fruit. The plants transformed with the vector
alone showed slow vegetative growth and did not set flower. Among several antisense
PLD strains grown, one strain produced normal flowers and set fruits. The fruits from
the antisense PLD plants were smaller than the control fruits. The pictures of a set of
control (C) and antisense PLD (T) Celebrity tomatoes at harvest are given in Fig. 9.13.
During subsequent storage for 2 weeks, the control fruits developed wrinkles indicative of
senescence and dehydration (Fig. 9.13), whereas the fruits from the antisense PLD plants
appeared relatively normal. The loss of fresh weight was more or less identical in fruits from
control and transgenic plants. Thus, the appearance of wrinkles may be indicative of a loss
of structural integrity. The transgenic fruits were also firmer and possessed a higher level of
red pigmentation (Table 9.4) as indicated by a higher level of lycopene, a + (red) value and
a + /b + (red/yellow) color ratio. The antisense PLD fruits also possessed an increased level
of soluble solids and vitamin C (Table 9.4). A cross section of control and transgenic fruits
is shown in Fig. 9.13 (C, T), respectively. The control fruits are multilocular, whereas the
transgenic fruits are bilocular.

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