Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

74 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
Numerous ethylene-insensitive mutants, such as Arabidopsis thaliana etr1-1 or ein-2,
or never ripe tomato mutants exist (Zacarias et al., 1999). Flowers could be engineered to
produce reduced levels of ethylene by introduction of an antisense ACC-oxidase transgene,
as occurs in tomatoes (FLAVR SAVR R○ ), driven by a flower or senescence-specific promoter
(John et al., 1995; Wilkinson et al., 1997; Bleecker et al., 1998; Zacarias et al., 1999).
Transgenic fruits containing ACC deaminase and antisense ACC synthase, ACC oxidase,
and polyphenol oxidase have been produced, the first three reducing ethylene production
and slowing ripening, the last reducing browning of damaged tissue (Flores et al., 2001).
When the endogenous cytokinin status is manipulated through transgenic intervention,
a stay-green phenotype can be obtained, as occurred in the fusion of ipt, an Agrobacterium
gene encoding a limiting step in cytokinin biosynthesis, to an Arabidopsis See (SAG12)
promoter (Gan and Amasino, 1997). Greenness can also be altered (delay in leaf senescence)
by downregulating the production of a senescence-promoting hormone, as seen in tomato
plants in which ethylene biosynthesis is inhibited by antisense suppression of the gene for
ACC oxidase (John et al., 1995).
4.16.1 Genetic manipulation
Within flower species and cultivars, there is great variability in ethylene sensitivity (of the
flowers). This implies that breeding toward less sensitive flowers is possible. In fact, almost
all modern carnation cultivars are much less sensitive to ethylene than the cultivar
“White Sim” that has been used over the years to study ethylene-induced senescence. In
many flowers, breeding programs aiming at a better vase life may (unintentionally) target
the ethylene biosynthesis and perception processes. To the best of our knowledge, breeding
of plants that are highly insensitive to ethylene in fully developed flowers has not been
successful so far. A few attempts to produce plants with ethylene insensitive flowers, or
with low production of endogenous ethylene, have been performed (Onozaki et al., 2001).
Facilitated by the detailed knowledge of ethylene production and perception in plants,
several attempts have been made to produce plants with prolonged flower life by genetic
transformation. The first experiments in this line of work were based on ACC oxidase
(ACO), the last enzyme in the ethylene biosynthetic pathway. Savin et al. (1995) transformed
carnation with a construct in which an antisense sequence of the carnation ACO
gene was placed under control of a constitutive promoter. This resulted in a few plants
with dramatically reduced ethylene production during flower senescence, and with flower
longevity of 8–9 days for cut flowers compared to 5 days for the nontransformed flowers.
An attempt to use this technique in Begonia failed (Einset and Kopperud, 1995). Transgenic
plants were obtained, but they did not show prolonged flower life. The ornamental Torenia
fournieri has been successfully transformed using both sense and antisense ACC-oxidase
gene constructs (Aida et al., 1998). The transgenic plants showed slight but significant
enhancement of flower longevity. Carnations with reduced ACC-synthase activity using
a cosuppression technique were produced at Florigene (Michael et al., 1993; patent no.
WO9635792, transgenic carnations exhibit prolonged postharvest life). Transgenic flowers
had a much longer vase life than wild type, but also showed problems related to a decreased
resistance to fungal pathogens. Till now, these products have not yet entered the market.
The first attempt to block the function of the ethylene receptor was done by Hua et al.
(1995) using a mutated Arabidopsis ers gene. Transgenic Arabidopsis plants showed strong