Manipulation of Fruit Softening | Genetics

In this article we will discuss about the manipulation of fruit softening by antisense RNA.

Plant cell wall is responsible for providing mechanical strength to plant organs. Tissue and cells due to their complex structure and biomolecules are involved. During fruit ripening several hydrolytic enzymes such as PG, PME and cellulase cleaves various polymers that con­stitute cell wall and consequently cell changes are responsible for textural softening of fruits such as tomato which greatly retard their use by the agro-based processing industries and consumers.

Therefore, inhibition of these hydrolytic enzymes through antisense RNA technol­ogy will boost economy of the agro-based industry.

Antisense Polygalacturonase (PG):

One of the major components of cell wall is polygalactonic acid and particularly abun­dant in fruit cells. Increase in softening of fruit was evidenced by increase in PG activity, which degrades polygalactoronic acid to galacturonic acid monomers at the onset of ripening. One of the earliest attempts on extending shelf-life of fruit has been attempted in transgenic toma­toes with reduced PG.

In the initial experiments, transgenic plants expressing antisense PG, A730 base pair fragments from 5′ end of the PG cDNA including 50 bp untranslated region and the translation start site was fused in the reverse orientation to the 35S RNA promoter and the 3′ end of nopaline synthese (nos) gene (Fig. 22.8). This hybrid antisense gene was introduced to produce transformed tomato.

Transgenic tomatoes expressing anti PG shows PG activity to as little as 0.5% of wild type level having little effect on softening but fruits are more resistant to spliting. In the subsequent experiments with antisense PG reported that PG inhibition is in­creased upto 1% of the control level and impair softening of the fruit. The fruit from PG antisense plants exhibit increased soluble polyuronide content and consequently improved processing quality of the fruit over control.

Antisense Pectin Methyl Esterase (PME):

Plant cell wall contains pectin material and its galactoronysyl residue undergoes esterification by frequent methoxylation even before incorporation into the cell wall. The cell wall pectin can be de-esterified to various degrees by pectin esterase. Long polymers of pectins are generally described in green fruit.

Pectin esterase is expressed throughout the fruit devel­opment and increased substantially to three-fold during ripening. The PME during fruit ripen­ing break the long polymers into short molecules. The role of PE has been addressed by antisense gene mediated suppression. Transgenic tomato expressing antisense PE results in reduction to less than 10% of control wild plants.

The PE antisense fruits contains less polyuronids com­pared with wild evidenced that Ca⁺ bridge theory of pectin cross linkage, which is generally blocked by esterification due to antisense inhibition. Reduced level of PME can be observed in transgenic plants. Pectin will not broken down into shorter molecules and remains as larger molecules.

Antisense fruit contains longer polymer pectin increases viscosity of solution and juice extracted from this has high viscosity than normal fruits and consequently facilitated processing. There have been reports on increased resistance to softening by simultaneous inhi­bition of both PG and PE with homologous suppressing transgenes. Classic example is the fusion of PG and PE sense-sense inhibited expression of both genes and have additional im­provement in food processing industry.

Flavr Savr Tomato:

Calgene, one of the small biotechnology company based in California advanced antisense technology for marketing of tomato fruit. They proposed that reducing the production of polygalactoronase substantially decrease softening of the fruit. The tomato fruits are harvested first before fully ripens.

This condition is to ensure firmness of the fruit during harvesting. Construction of vector in which antisense PG gene is placed in between CaM35 promoter and nopaline synthase (NOS) termination sequence. Kanamycin marker gene was placed for screening antisense gene.

This gene construct was then transformed to produce flavr savr tomato. Genomic southern blotting was to ensure presence of antisense PG transgene and confirmed that each transformed plants contain only a single copy of inserted antisense gene. It was claimed that reduced softening of flavr savr allowing them to attach on the vine longer.

Flavr savr tomato was launched and commercialized in 1994 and were initially sold un­der the brand name “Macgreger tomatoes”. In the subsequent years it was withdrawn from the market. The lack of commercial success due to several reasons like high price, undesirable texture of tomato fruit, skin, compositional changes and more importantly backlash from envi­ronmentalist. Although convincing evidence clearly suggests that there is no human health risk from flavr savr, but received considerable resistance from the sector of the society.