Development of Transgenics for Improved Shelf Life
Authors: Manisha Mangal and Arpita Srivastav

The postharvest losses of fruits and vegetables in the developing countries account for almost 50% of the produce. India, the world’s second largest producer of fruits and vegetables loses 35-40% of produce because of excessive softening. The softening that accompanies ripening of fruits exacerbates damage during shipping and handling processes. It plays a major role in determining the cost factor, because it has a direct impact on palatability, consumer acceptability, shelf life, and postharvest disease/pathogen resistance. Biotechnological approaches, in particular genetic engineering, have the potential to engineer fruits with delayed ripening character. The first approval for commercial sale of a genetically engineered food product was transgenic tomato "Flavr savr" with delayed ripening trait that was developed by Calgene, USA in 1994. Fruits with improved shelf life suitable for food processing can be developed by two basic methods (1) down regulation/ modification of ethylene metabolism (2) by manipulating cell wall metabolism.

Down regulation of ethylene metabolism :

Ethylene is a plant hormone which is involved in fruit ripening and is biosynthesized from the amino acid methionine via S-adenosyl-L-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) with the help of enzymes Met Adenosyltransferase, ACC synthase (ACS) and ACC-oxidase (ACO). Ethylene biosynthesis can be induced by endogenous or exogenous ethylene. An approach to prevent fruit ripening is to inhibit ethylene production through the utilization of antisense RNA technology. An antisense gene is produced by inverting the orientation of protein coding region of a gene in relation to its promoter. The mRNA produced by this gene has the same sequence as the antisense strand of the normal gene (except for T in DNA and U in RNA) and this mRNA is called antisense RNA. When sense (endogenous gene) and antisense gene are present in the same nucleus, the transcription of the two genes yield antisense and sense RNA transcripts respectively which are complementary to each other and hence pair to form double stranded RNA molecules. The duplex formed inactivates the mRNA and no further protein can be formed.

Antisense RNA technology has been used to suppress the expression of ACC synthase and ACC oxidase gene involved in ethylene biosynthesis and fruits in the silenced transgenic plants were found to be more resistant to over ripening and shriveling than control fruits.

Modification of Ethylene Biosynthesis :

Over expression of ACC deaminase gene:

ACC deaminase metabolizes ACC to a-ketobutyrate. This gene has been isolated from from Pseudomonas sp. and was expressed in transgenic tomato plants. This approach led to 90 to 97% inhibition in ethylene production during ripening. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormalities. However, fruits from these plants showed significant delays in ripening, and they remained firm for at least 6 weeks longer than the non transgenic control fruits.

Over expression of SAM hydolase gene :

In this approach a gene from Bacteriophage T3 which encodes the enzyme S-adenosylmethionine hydrolase (SAMase) has been utilized to generate transgenic tomato plants that produce fruit with a reduced capacity to synthesize ethylene. S-adenosylmethionine (SAM) is the metabolic precursor of 1-aminocyclopropane-1-carboxylic acid, the proximal precursor to ethylene. SAMase catalyzes the conversion of SAM to methylthioadenosine and homoserine thereby reducing the synthesis of ethylene.

Control of Ethylene perception :

Ethylene signals the onset of fruit ripening therefore delayed ripening in some plants can be achieved by modifying ethylene receptors. The ETR-1 (Ethylene receptor-1) encodes for ethylene binding protein. Plants modified with ETR1 lack the ability to respond to ethylene.

Suppression of Polygalacturonase Activity:

Polygalacturonase (PG) is the enzyme responsible for the breakdown of pectin, the substance that maintains the integrity of plant cell walls. Pectin breakdown occurs at the start of the ripening process resulting in the softening of the fruit. To produce a fruit with delayed ripening trait scientists insert an anti-sense or a truncated copy of the PG gene into the plant’s genome resulting in a dramatic reduction of the amount of PG enzyme produced, thereby delaying pectin degradation. This technology was used for the production of Flavr Savr tomatoes.

Use of Polyamine genes:

Polyamines play crucial role in plant growth and development as well as basic biological processes. Polyamines and ethylene have always been considered as a part of network as the precursor SAM sits astride at a critical branch point as well as functional level. The common polyamines are putrescene (diamine), spermidine (triamine) and tetra amine (tetraamine). These two pathways (polyamines and ethylene biosynthesis) are seen to regulate each other, which is a decisive factor in determining the predominance of either of two pathways. Over expression of yeast spermidine synthase has been found to increase shelf life in tomato.

Use of n-glycan processing enzymes and cell wall modifying enzymes:

Suppression of two ripening specific N-glycoprotein modifying enzymes Alpha-mannosidase and Beta-D-N-acetylhexosaminidase resulted in down regulation of cell wall degradation and ripening related genes in transgenic fruits.

In lettuce, down regulation of a cell wall modifying enzyme Xyloglycan endotransglucosylase/ hydrolase has also resulted in extended shelf life of this salad crop.

Down regulation of Expansins:

Expansins are proteins that cause cell wall loosening, and are involved in many aspects of cell wall modification during development. In tomato, the expansin gene LeExp1 shows ripening-related accumulation of mRNA and protein, and transgenic silencing of the expression of this gene results in tomato fruit that are significantly firmer than corresponding controls throughout ripening. Examination of postharvest quality characteristics of fruit suppressed in accumulation of LeExp1 protein found that increased firmness resulted in significantly improved fruit integrity during storage at 13 °C. Based upon the first appearance of noticeable deterioration, fruit shelf life was extended by 5"10 days, depending upon the packaging.

Over expression of genes of cytokinin biosynthesis:

Isopentenyl transferase (ipt gene) is a key enzyme in cytokinin biosynthesis. Cytokinins are known to delay floral yellowing of plants. This gene was linked to senescence associated gene promoter and transferred to broccoli. The ipt transformed lines had enhanced shelf life and acceptable yield and appearance.

About Author / Additional Info:
I am working as a Senior Scientist ( Biotechnology) at IARI-New Delhi.