In the last decade, tremendous progress has been made through various techniques of genetic engineering in the development of transgenic plants. One of them is to improve the shelf life of the fruits.

Advantages of delayed fruit ripening:-
• It extends the shelf life, keeping the quality of the fruit intact.
• Long distance transport becomes easy without damage to fruits.
• Slow ripening improves the flavor.
• Delayed fruit ripening work has been extensively carried out in tomatoes.

Biochemical changes during fruit ripening
Fruit ripening is an active process characterized by increased respiration accompanied by a rapid increase in ethylene synthesis. As the chlorophyll gets degraded, the green color of fruit disappears and a red pigment, lycopene is synthesized. The fruit gets softened as a result of the activity of cell wall degrading enzymes namely polygalacturonase (PG) and methyl esterase. The phytohormone ethylene production is linked to fruit ripening as the same is known to trigger the ripening effect. The breakdown of starch to sugars and accumulation of large number of secondary products improves the flavor, taste and smell of the fruits.

3 distinct genes involved in tomato ripening have been isolated and cloned.
i) pTOM5 encodes for phytoene synthase which promote lycopene synthesis that gives red coloration
ii) pTOM6 gene encodes for polygalacturonase. This enzyme degrades the cell wall, resulting in fruit softening.
iii) pTOM gene encodes for ACC oxidase. This enzyme catalyzes the ethylene formation that triggers the fruit ripening.

Genetic manipulation of fruit ripening:-

This involves antisense RNA approach. Few examples are:-
1) Manipulation of enzyme polygalacturonase
2) Manipulation of ethylene biosynthesis.

1) Manipulation o enzyme polygalacturonase (development of flavr Savr tomato):- softening of fruits is largely due to degradation of cell wall (pectin) by enzyme polygalacturonase (PG). The gene encoding PG has been isolated and cloned (pTOM6).

Procedure involves: -

1) Isolation of DNA from tomato plant that encodes the enzyme polygalacturonase (PG).
2) Transfer of PG gene to a vector bacteria and production of complementary DNA (cDNA) molecules.
3) Introduction of cDNA into a fresh tomato plant to produce transgenic plant.

Mechanism of PG antisense RNA approach:-
In normal plants, PG gene encodes a normal or sense mRNA that produce the enzyme PG and it is actively involved in fruit ripening. The cDNA of PG encodes for antisense mRNA, which is complementary to sense mRNA. The hybridization between sense and antisense mRNA render the sense mRNA ineffective. Consequently, no polygalacturonase is produced hence fruit ripening is delayed.

Rise and fall of flavr savr tomato:-
Genetically engineered tomato known as flavr savr was approved by US Food and Drug Administration on 18th may 1994. FDA ruled that flavr savr tomatoes are as safe as normal tomatoes. The new tomatoes could be shipped without refrigeration to far off places as it was capable of resisting rot for more than 3 weeks (double the time of a conventional tomato). But there are some limitations:-
1) Transgenic tomatoes could not be grown properly in different parts of United States of America.
2) Yield of tomatoes was low.
3) Cost was high.
Therefore unfortunately, within a year after its entry, flavr savr was withdrawn and it is now almost forgotten.

2) Manipulation of ethylene biosynthesis:-
Ethylene plays a key role in ripening of fruits. Ethylene is synthesized from S-adenosyl methionine via the formation of an intermediate namely 1-aminocyclopropane 1-carboxylic acid (ACC) catalyzed by ACC synthase. Next step is conversion of ACC to ethylene by ACC oxidase. 3 different strategies have been developed to block ethylene biosynthesis. These are:-
1) Antisense gene of ACC oxidase: - transgenic plants with antisense gene of ACC oxidase have been developed. In these plants production of ethylene was reduced by about 97%.
2) Antisense gene of ACC synthase: - ethylene biosynthesis was inhibited to an extent of 99.5% by inserting antisense gene of ACC synthase and the tomato ripening was markedly delayed.
3) Insertion of ACC deaminase gene: - ACC deaminase is a bacterial enzyme. It acts on ACC (remove amino group) and consequently substrate availability for ethylene biosynthesis is reduced. Bacterial gene encoding ACC deaminase has been transferred and expressed in tomato plants. Ethylene biosynthesis is inhibited up to 90% in these plants. Fruit ripening is delayed about 6 weeks.

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