Delayed ripening

1. Transgenics For Delayed Fruit Ripening
Presented By;
Anurag Patel
I.D.-2889
B.tech(biotechnology)
Guided By;
Dr. Akash Tomar
Astt. Prof.
Deptt. of RDT

2. “Ripening is a normal phase in the maturation process
of fruits and vegetables”
Highly coordinated
Genetically programmed
Irreversible phenomenon
Physiological, biochemical changes
Development of a soft and edible ripe fruit
What is Fruit Ripening?

3. The molecular mechanisms controlling the ripening of fruit

4. Increased respiration
Chlorophyll degradation
Biosynthesis of carotenoids, anthocyanins, essential
oils, Flavor and aroma Components
Increased activity of cell wall-degrading enzymes
Transient increase in ethylene production
What are the changes?

5. Major Developmental Changes during Tomato
Fruit Development and Ripening

6. Major Developmental Changes during Tomato Fruit
Development and Ripening

7. Pathway for ethylene biosynthesis
Rate limiting step

8. The expression of
ethylene biosynthesis
and ethylene
perception genes
during the transition to
climacteric in tomato
Kevany et al. 2007

9. Bleecker and Kende, 2000
Ethylene Perception and Signal Transduction

10. Tomato with a Delayed Ripening Gene
• Plasmid_PV-LERP07(pMON10117)

11. Ripe Fruit
chemical
cause
The hormone______________ initiates the ripening response:ethylene
Unripe Fruit
physical
condition
green
hard
sour
mealy
odorless
chlorophyll
pectin
acid
starch
large orgs
chemical
cause
red
soft
neutral
sweet + juicy
odor
physical
condition
anthocyanin
less pectin
neutral
sugar
small orgs
hydrolase
pectinase
kinase
amylase
hydrolases
Enzyme Produced
H2C=CH2
DNA
RNA

12. Fruit pulp or the mesocarp
parenchymatus cells
complex network of polysaccharides and proteins
The primary cell wall contains
35% pectin
25% cellulose
20% hemicellulose
10% structural, hydroxyproline-rich protein
Structural components of fruits

13. Globally cultivated fleshy fruit
World’s largest vegetable crop after potato
Indian production scenario-
3,50,000 hectares, 53,00,000 tons/year
Short generation time: 3-4 months
Simple genetics
Numerous characterized mutants
Cross fertile wild germplasm to promote genetic studies
Routine transformation technology
Postharvest losses-5 to 25% in developed countries
-20 to 50% in developing countries
Tomato: model systems for fruit development
and ripening

14. Nutrition facts of tomato

15. Worldwide Production of Tomato
In 2016, world production of tomatoes was 170.8 million tonnes

16. Export Growth

17. Disadvantages of existing methods of storage
Labor intensive
costly
Occupies a large floor space
Poor heat transfer may occur resulting in poor product quality
Excessive dehydration in unpacked products
Chemical changes during freezing
-enzyme-activated browning
-development of rancid oxidative flavors
Textural changes during freezing
-mushy and watery

18. Problem of ripening/Requirement of
Delayed Ripening
it only takes about a few days before the fruit or vegetables is
considered inedible.
• they take 20 to 30 days from blossom set to reach full size–
commonly called
“mature green”.
• they take another 15 to 20 days
to ripen.

19. Transgenic approach

20. Regulation of Ethylene Production
a. Suppression of ACC synthase gene expression.
ACC (1-aminocyclopropane-1-carboxylic acid) (ACS2)
conversion of S-adenosylmethionine (SAM) to ACC

21. Antisense Technology
• Antisense RNA is a single-stranded RNA that is complementary to
a messenger RNA (mRNA) strand transcribed within a cell
• Antisense RNA introduced into a cell to inhibit translation of a
complementary mRNA by base pairing to it and creating barrier to
the translation machinery.
• E.g.
hok/sok system of the E. coli R1 plasmid.

22. Antisense Technology
http://agbiosafety.unl.edu/flash/antisense.swf Journal of Plant Physiology.170,987– 995,2013

23. RNAi-mediated silencing
Chimeric RNAi-ACS construct designed to target ACS
homologs
Delayed ripening and extended shelf life for 45 days∼
Chimeric RNAi-ACS construct designed to target ACS
homologs
Delayed ripening and extended shelf life for 45 days∼

24. Regulation of Ethylene Production
b. Suppression of ACC oxidase gene expression.
It catalyzes the oxidation of ACC to ethylene
The last step in the ethylene biosynthetic pathway
Down regulation through anti-sense technology

25. Ripening in papaya fruit is altered by ACC
oxidase cosuppression
Fig1:Map of the construct pKYCPACOO-1 containing the ACC oxidase
fragment cloned in PKYLX80 in the sense orientation. The ACC oxidase
fragment is flanked by the CaMV 35S promoter and the RUBISCO
terminator
Fig2: Ethylene production in papaya transgenic fruits.
Rodolfo Lo´pez-Go´mezet al.Transgenic Res. 18:89–97 2009

26. c. Insertion of the ACC deaminase gene.
Regulation of Ethylene Production
The gene is obtained from Pseudomonas chlororaphis
(a common nonpathogenic soil bacterium)
It converts ACC to a different compound
Reduce the amount of ACC available for ethylene production
90-97% reduced ethylene production

27. Regulation of Ethylene Production
d. Insertion of the SAM hydrolase gene.
The gene is obtained from E. coli T3 bacteriophage
SAM is converted to homoserine
The amount of its precursor metabolite is reduced
Matto, 2002

28. Regulation of Ethylene Production

29. Regulation of Cell wall degradation
a.Polygalacturonase (PG)
degrades pectin
Antisense RNA techniques
The transgenic fruit with decreased levels of PG activity:
1)Do not get overly soft when ripe,
2)Show less damage due to fungal infection and
3)Have elevated levels of soluble solids
Chimaeric polygalacturonase (PG) gene
Produce a truncated PG transcript constitutively
Expression of the endogenous PG gene was inhibited

30. b.Pectin methylesterase (PME)
Involved in metabolism of pectin
Break large polymers into shorter molecules
Antisense RNA approach
Transgenic fruit resulted in reduced pectin depolymerization
However there was no effect on firmness during ripening
Regulation of Cell wall degradation

31. Some other techniques
1.Delayed ripening by soil bacteria-Rhodo
coccus sp.

32. Ethylene Induces Soil Microbes to Delay Fruit Ripening
• A report generated by the USDA in 2011, states American
consumption of fruits and vegetables has increased steadily since
the 1980s,
• the demand for fresh fruist and vegetables has increased by 22%
and 16.5%, respectively, (Johnson, 2014). Unfortunately nearly 20%
of all
• fruits and vegetables never reach the targeted consumer sites due
to spoilage and post-harvest loss (PHL), 19.6% of PHL is attributed
to the effects of pre-mature or prolonged climacteric ripening that
lead to senesce, apoptosis, lesions, spotting, bruising, infection,
and/ or eventual
• spoilage that render the produce unsellable (Burg, 2004; Sakimin et
al., 2012; Seymour et al., 2013).

34. 2.Delayed ripening by Sound Wave
Technology-

39. Advantages of Delayed fruit ripening
Assurance of top quality
Allowing the fruits to exude full quality
Consumers will get value for their money
Widening of market opportunities
Reduction in postharvest losses

40. THANK YOU