Biotechnological Approaches to Improve Nutritional Quality and Shelf life of fruits and vegetables

1. Biotechnological Approaches to
Improve Nutritional Quality and
Shelf Life of
Fruits and Vegetables
Anila Sajjad

2. Introduction
• The importance of fruits and vegetables (F&V) in the diet of
mankind cannot be over emphasized. Many reviews have
reported the wide range of determinants of desirable quality
attributes in fruits and vegetables such as nutritional value,
flavor, colour, texture, processing qualities and shelf-life.
• Typically, biotechnology technique such as genetic modification is
used in F& V to enable plants tolerate the biotic and abiotic stresses,
and plant resistances to problematic pests and disease, which
may provide higher nutritional contents, and extend the shelf-
life of the produce.

3. The need for biotechnology in fruits and vegetable
production
• A number of challenges have called for the application of
biotechnology in the production of fruits and vegetables. These
are population increase, water shortages, climate change, high
perishability or postharvest decays, and short shelf-life
associated with fruits and vegetables. Fruits and vegetables by
their intrinsic properties require more water and in the face of
water scarcity throughout the world, biotechnology will be
required to develop fruits and vegetables that can withstand
water stress and still be able to produce good crop of high
quality and yield.

4. • World population is anticipated to rise to 10 billion by 2050.
Freshwater, vital for agricultural productivity, is becoming scarce and
climate change could increase temperature, drought.
• New crop varieties need to be developed quickly to meet these
challenges and biotechnology will be needed to enhance existing
technologies to achieve this.
• So far, biotechnology has been successfully used to develop insect
and herbicide resistance in a limited number of crops such as corn.

5. Post harvest Loss & Waste
• Postharvest loss is unintentional. It describes the incidental losses
that result from events occurring from farm-to-table, such as physical
damage, internal bruising, premature spoiling, and insect damage,
among others. Produce loss is also described as quantitative because
it is measurable.
• Postharvest waste, in contrast, is intentional. It describes when
produce is discarded because it does not meet buyer expectations,
even though it is edible. Produce may be rejected by growers,
distributors, processing companies, retailers, and consumers for
failing to meet desired or established preferences. Produce waste is
described as qualitative because it is difficult to measure and assess
the waste.

6. Strategies to combat Ph L & W

7. Figure: Linking quality to physio-biological characteristics

8. Figure: Determinants of produce quality
• (A) Extrinsic environmental factors such as
season, irrigation, soil nutrition and minerals,
climate, stress, pathogens and pests, and
agronomic practices as well as physiological
genetic factors together determine fruit quality at
harvest. Postharvest intervention, including
refrigeration, chemical treatment, radiation, and
modified atmospheres and pressure aims to
maintain that quality through shipping and
storage. Minor injury, ranging from mechanical or
pathogenic damage to temperature, light, or
pressure-induced damage, lowers the quality of
fruit. More extensive injury renders produce
inedible and contributes to the quantitative loss.
• (B) Potential postharvest outcomes for
produce. Harvesting fruit prior to full ripeness will
increase its shelf-life [a], but compromises quality
during and after ripening [2a]. Fruit harvested at
ripe [b] has a limited shelf-life before it declines in
quality or rots [1b]. Postharvest intervention
delays senescence and typically also results in
some compromise of quality [2b]. The goal of gene
editing is to extend shelf-life without loss of
quality [3] and therefore reduce postharvest loss
and waste.

9. • Postharvest decay of fruits and vegetables are a major challenge
throughout the world.
• In the industrialized countries, about 20– 25% of the harvested fruits and
vegetables are decayed by pathogens during postharvest handling.
• In the developing countries, where postharvest decays are often times over
35% , due to inadequate storage, processing and transportation facilities.
• The use of synthetic fungicides such as benomyl and iprodione to control
postharvest diseases of fruits and vegetables.
• The health and environmental concerns associated with the continuous use
of synthetic fungicides have alarmed legal enforcers and consumers to
demand greener technology and quality products from the food industry as
well as the scientific community.

10. Potential for improving postharvest quality of
horticultural crops by gene editing
• The current gene-editing tool of choice is CRISPR–Cas9. The researcher is able to
generate mutations in narrowly defined regions of the genome, and it has been
successfully applied to induce valuable traits in many crop species.
• CRISPR (clustered regularly interspaced short palindromic repeats) is a
prokaryotic system that protects organisms from viral infection. This naturally
occurring mechanism in bacteria has been co-opted by scientists to remove
unwanted nucleotides or to insert new or altered ones to promote traits seen as
desirable in an organism of interest. For CRISPR editing, a synthetic guide RNA
(gRNA) is designed to an identified protospacer adjacent motif (PAM) in the
sequence of interest, and this, along with the Cas protein sequence, is inserted
into a cell where they are processed using the cell’s gene expression apparatus.
The Cas protein synthesized in the plant produces a double-stranded break (DSB)
at the bases identified by the gRNA. Repair of the DSB in DNA is usually not
faithful to the original sequence, and thus, mutations may be introduced into the
genome. DSB repairs occur naturally in almost all plant tissues, so this is not an
inherently foreign process.

11. • The expression of a gene may also be varied by changing its DNA
methylation status. In tomato, orange, and bell pepper, DNA
methylation regulates ripening by controlling ripening-related TFs or
genes. Binding a methylation modifying protein to a CRISPR complex
with a deactivated Cas9 may be a feasible approach to edit regions
targeted for demethylation in ripening-related genes, thus controlling
shelf-life.

16. The Dynamics of Ripening and Perishability in Fruits and
Vegetables
• Fruit ripening and softening are major attributes that contribute to perishability in both
climacteric (ripen after harvest) and non-climacteric fruits.
• Fruits and vegetables such as tomato, banana, mango, avocado etc. take about a few
days after which it is considered inedible due to over-ripening.
• The spoilage includes excessive softening and changes in taste, aroma and skin color. This
unavoidable process brings significant losses to both farmers and consumers alike. Even
though ripening in F&V can be delayed through several external procedures, the
physiological and biochemical changes associated with ripening is an irreversible process
and once started cannot be stopped.
• Ethylene has been identified as the major hormone that initiates and controls ripening in
fleshy fruits and vegetables. Influencing ethylene biosynthesis during ripening in fleshy
commodities has been the foremost attempt for combating post-harvest deterioration.

17. Ripening studies with tomato as a model
system • Ethylene biosynthesis occurs in two enzymatic steps,
catalyzed by ACC (1-aminocyclopropane-1-carboxylic
acid) synthase (ACS) and ACC oxidase (ACO). In
climacteric fruit, such as tomato, banana, mango, and
apple, there is a rapid rate of increase in ethylene at the
onset of ripening and continued production leads to
ripening and senescence.
• Enzymatic inhibitors AVG (aminoethoxyvinylglycine) or
AOA (aminooxyacetic acid) inhibit ACS.
• Cobalt ions (Co2+), high temperatures (T°), and low
oxygen concentration inhibit ACO.
• Silver ion, silver thiosulfate (STS), carbon dioxide, and 1-
methylcyclopropene (1-MCP) inhibit ethylene binding to
the receptor for activation of ethylene signaling pathway.
For example, 1-MCP, a synthetic growth regulator
structurally related to ethylene, is commercially used in
fruit crops such as apple, kiwifruit, pear, avocado, melons
and others, and it has also shown biological benefits in a
range of other species. SAM S-adenosyl methionine

19. • In climacteric fruit, ethylene synthesis, regulation, and perception lead to
the transcription of ripening-regulated genes that determine quality
attributes desired by consumers. When ACO and ACS expression is
genetically suppressed or silenced in a range of species, e.g., petunia,
tomato, melon, papaya, and kiwifruit, ethylene production is decreased
and shelf-life is extended due to slowed ripening processes.
• The recent use of CRISPR to induce targeted deletions or substitutions in
CNRand NOR, and in other transcription factors, AP2a, FUL1, and FUL2
revealed regulation in the ripening pathway.
• This knowledge would allow us to control ethylene production so that
ripening proceeds at the rate and with the timing that is optimal for supply
chain dynamics while maintaining quality. This would directly mitigate PLW.

20. Produce postharvest attributes that would
minimize PLW
• Longer shelf-life with maximal quality retention
• Many of the approaches for extending the life of produce often lead to poor taste and flavor, and this link
must be broken to increase consumer satisfaction and repeat purchases. Longer shelf life is one of the most
key traits for fleshy fruits, vegetables, and ornamentals, and it has a greater impact on market potential.
Maintaining and prolonging the shelf life is a major challenge in breeding and genome engineering
programs. The plant hormone ethylene, also known as the ripening hormone, plays a vital role in the
ripening process of fruits and vegetables; therefore, its production needed to be controlled to maximize the
shelf-life. Hence, it becomes imperative to regulate the expression of shelf-life-related genes to maintain the
taste, aroma, and quality features. Recently, advanced genome editing technology tools have been
potentially utilized to enhance the quality as well as post-harvest traits of horticultural crops
• Convenience
• Quality attributes needed to provide safe, long-lasting, visually and texturally appealing fresh-cut products
can be challenging to maintain since cut produce often respires faster and is prone to browning and
premature senescence.
• Better quality
• The criteria for a favorable appearance include produce of the right color and color uniformity, correct shape
and dimensions, and often a glossy surface area free from defects. Identifying and manipulating the genes
determining these pathways could improve quality.

21. • Fruit Texture Quality Improvement
• Texture-related number of enzymes such as polygalacturonase (PG), pectin
methylesterase (PME), endo-b-(1,4)-glucanase (EGs), β-galactosidase (β-gal), and
expansin (EXPs) and N-glycoprotein-modifying enzymes, e.g., α-mannosidase (α-
Man) and β-D-N-acetylhexosaminidase (β-Hex) are responsible for firmness and
softening processes in these crops. Various research reported the suppression of
relevant gene expression in strawberries and tomatoes. In tomato, PG gene
suppression had no obvious effect on fruit softening, but this gene also influences
the firmness of strawberries. However, another gene, i.e., pectate lyase (PL) gene,
which is a cell wall-related protein has been silenced (asRNA approach)
effectively to enhance fruit firmness without changing its physical (size and color)
and biochemical (metabolites) parameters, ultimately influencing the sensory
characteristics in strawberry and tomato, respectively. In this process, utilizing
CRISPR/Cas9 editing of PL gene resulted in the mutants exhibiting a beneficial
effect on fruit firmness while maintaining the fruit color, aroma, and flavor in
tomato.