The document discusses various topics related to aging and senescence in plants and methods to enhance shelf life using biotechnology. It provides information on: 1) The maximum lifespan of different organisms ranging from months in fruit flies to over 1000 years in some trees. 2) Senescence and programmed cell death processes in plants and their relationship to aging. 3) Methods used in biotechnology to manipulate genes responsible for senescence and enhance shelf life, including gene identification, suppression/overexpression, and genetic engineering. 4) Several studies documenting successful enhancement of shelf life in crops like litchi, tomato, and banana by modifying genes involved in ripening.

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2. Maximum life span:
• The range is tremendous.
• Drosophila = 3 months,
• Humans = 120 years,
• some turtles and lake trout = 150 years,
• some trees = 1000 years or more.
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What is the oldest living organism on earth?
Celebrating its 4,645th birthday in 2002,
a bristlecone pine in the White
Mountains of California is the world's
oldest-known living organism
Jeanne Calment, the oldest
confirmed human, died in 1997
at 122 years of age.
Guinness World Records. 2014

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Perishable in nature
Unripe stage or immature stage
Still post harvest loses

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Decrease in shelf life

6. Causes heavy Losses

7. Enhancement of shelf life in
horticultural produce by using
biotechnological tools
Shweta
UHS15PGM551
Dpt. Of BCI

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SHWETA
UHS 15PGM 551
Date: 3/12/2016
Enhancement of shelf life in horticultural produce by
using biotechnological tools
Seminar- I
On
KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE,
ARABHAVI
UNIVERSITY OF HORTICULTURAL SCIENCES, BAGALKOT
DEPT. OF BIOTECHNOLOGY & CROP IMPROVEMENT

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Aging
Senescence
Programmed cell death: Apoptosis
The developmental processes leads to……..
Tripathi and Tuteja, 2007

10. Aging
The primary causes of ageing are
1)Telomere shortening in reproductive cells,
2) Non-enzymatic glycosylation
3) DNA damage and oxidative damage due to reactive
oxygen species
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Tripathi and Tuteja, 2007

11. 13-02-2017 Dept.BCI 11Tripathi and Tuteja, 2007
Fig1 :Apoptosis (Programmed cell death)

12. • Senescence represents the sequence
of metabolic events occurring in the
final stage of development and
ultimately culminating in the
programmed death of whole plants,
organs, tissues or cells.
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13. Shelf life:- It is the length of time that a
commodity may be stored without becoming
unfit for use, consumption, or sale.
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14. Heat
Moisture
Contamination
by
microorganisms
Mechanical
stresses
Transmission of
gases,
light
Decrease
in shelf
life
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PCD
signals
Environmental signals
Pollination
Drought
Mechanical injury
Developmental signal
Reproduction
Growth regulators
Hormonal regulation
Ethylene↑ Cytokinin↓
ABA ↑ GA ↓
Non hormonal regulation
Polyamines ↑ sugar ↓
Initiation of senescence
(SAGs Expression)
proteases
Nucleases
Dnase/RNase
Cell wall
modifiers
Accelerate
senescence
(symptoms visible)
Oxidative
enzymes
ROS
Signaling phase
Regulatory phase
Transcriptional
regulation
Execution phase
Cell
death
Tripathi and Tuteja, 2007

16. Senescence
Protein degradation
Nucleic acid breakdown
Chlorophyll breakdown
Membrane and lipid breakdown
Biosynthesis of Ethylene
Biosynthesis of Cytokinin
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17. Protein degradation
• Decreases in total protein during senescence results from
increase in proteolytic enzyme activity.
• Cysteine proteases are the main proteases involved in protein
hydrolysis.
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Tripathi and Tuteja, 2007

18. Membrane and lipid breakdown
• The cause of membrane deterioration in leaves and
petals is a decreases in phospholipids and enrichment
of fatty acid.
13-02-2017 Dept.BCI 18Tripathi and Tuteja, 2007
The activity of phosopholipase A, C and D have been
detected during in flower petals, only PLC and PLA
activities increased during petal senescence.

19. Nucleic acid breakdown
• Level of DNA remain relatively constant, total RNA has been
shown to decrease in senescing tissues.
13-02-2017 Dept.BCI 19Brady and Green, 1994
The RNA is an important source of C, N, and P,
which is recycled to metabolically active tissues
during cell death and when P is limiting. This is
accomplished through the activity of
ribonucleases (RNases).
The activity of RNases will be increased during
senescence

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Chlorophyll breakdown
Matile et al., 1996
NCCs have ben localized in
the vacuoles of senescent
mesophyll cells.
Under mild acidic conditions
of the vacuolar sap.
Tautomerization
Non enzyme

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Biosynthesis of cytokinin
Adenosine -mono phosphate
Adenine phosphate isopentenyl transferase
N6 – Isopentenyl pyrophosphate- 5- phosphate
Ribose
zeatin
Cytokinin degradation
Tripathi and Tuteja, 2007

22. Biosynthesis of Ethylene
13-02-2017 Dept.BCI 22Saltveit, 1998
Some of the intrinsic and extrinsic factors that promote (+) or inhibit (-)
ethylene (C₂H₄) synthesis in higher vascular plants.
Antisense gene of ACC oxidase
Antisense gene of ACC synthase
Insertion of ACC deaminase gene
Manipulation of Ethylene Biosynthesis

23. Enhanced shelf life: biotechnology
• Biotechnology : A set of tools (molecular)
used to modify the genetic make up of an
organism to that:
It produces a new product
The product perform new function(s)
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24. The basic techniques of biotechnology
Plant tissue culture :
Mutation
Genetic engineering
• Manipulation done at molecular level
• Specific genes may be changed
• Genes can be transferred between species.
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25. Mutation breeding
The change in base sequence of a gene may occurs
due to
• Base substitution
• Base insertion and deletion
• Transposition
• Trinucleotide repeat expansion
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Biotechnology
2)Gene manipulation
1)Gene identification

27. Gene identification
Identification of genes which are responsible for
senescence :
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28. Gene manipulation
Gene manipulation in crop is altered in various
ways:
Suppression ( a deleterious gene)
Over expression (favorable gene)
Modification (enhancing a characteristic)
Transgene expression (introducing new function)
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29. Advances in Bioscience and Biotechnology, 2016, 7, 300-310 Published
Online June 2016 in SciRes.
Objective : To obtaining the
role of polyamines
during the fruit ripening.
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30. • S-adenosyl-1-methionine decarboxylase (SAMDC)
gene was cloned from Datura stramonium and used
for litchi transformation.
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32. figure:1 PCR analysis of SAMDC gene sequence.
Lane 1, 2, 3, 4, 6, 7 & 8 primer based SAMDC gene sequence ;
Lane 5 molecular size marker
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Das et al., 2016

33. Fig: 2 RT-PCR analysis for transgene expression at
transcript level using primers specific to Datura SAMdc
gene. The RNA from wild-type plant and different transgenic
plants ((A), (B), (C) & (D)).
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Fig 3 : Northern-blot analysis of DNA from Transgenic
plant. hybridized with a [32P]dATP-labelled transgenic
plant SAMDC cDNA. The expected sizes (1.9 kb) of the
hybridizing fragments are indicated on the left.
Das et al., 2016

34. Table 1: Polyamine level in leaves from in vivo-grown litchi
plants of non-trnsgenic lines B2 and Q5 without high temperature
treatment
Plants Spermidine
(Spd)
Spermine
(Spm)
Putrescine
(Put)
Non-transgenic 165 ±25 51 ± 7.8 193 ± 34
Transgenic A 379 ± 52 85 ±19 225 ± 30
Transgenic B 424 ± 65 91 ± 14 253 ± 41
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Values (nmol/g fresh weight) represent average of data from three independent
experiments and are shown as means ± S.E.
Das et al., 2016
The S-adenosylmethionine decarboxylase acts on SAM (S- adenosyl metionine)
and converts it into polyamines so that very small amount of SAM is available
to produce ethylene and consequently ripening process can be delayed and
consequently fruit’s shelf life can be enhanced.

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Objective :To find a novel
regulatory function
of SlMSI1 in fruit
ripening.

36. • Based on the sequence in the tomato genome
full-length cDNA of SlMSI1 was cloned from
Alisa Craig tomato.
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Fig 4: Expression and protein profiles of SlMSI1 in tomato. (a,b) Expression and protein level analysis of
SlMSI1 in different organs of tomato. (c,d) Transcript and protein level analysis of SlMSI1 during fruit
development.
Liu et al., 2016

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Figure 5 : Expression profile of SlMSI1 in L1,L2,L29
Over expressed lines: L1,L2,L29 with SlMSI1
Control without SlMSI1
Liu et al., 2016

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SL1,SL2,SL3 : Suppressed lines
Liu et al., 2016
Figure 6 : Expression profile of SlMSI1 in SL1,SL2,SL3

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fig 6 : Ripening comparison among the control, mutant rin, and SlMSI1 overex pression
lines L1, L2 and L29. (B) The dehydration statistics in the transgenic and
control fruits at 30, 40, and 50 days after the breaker stage.
Liu et al., 2016

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Fig 7:SlMSI1 inhibits fruit ripening by repressing the expression of RIN and
its regulon genes.
Found that the PcG protein SlMSI1 negatively regulates RIN and other ripening genes
demonstrating that SlMSI1 is a novel regulator suitable for use in genetic engineering
modification of fruit shelf life.

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To know the roles of MaMADS1 and
MaMADS2 genes in banana fruit
ripening process.
Elitzur et al., 2016

43. Banana (Musa acuminate, AAA Cavendish
subgroup, Grand Nain) was used for
transformation
Gene sections used :RNAi or AS constructs for
MaMADS1 or MaMADS2
developed the 3 transgenics they are
RNAi MaMADS1
RNAi MaMADS2
AS MaMADS2
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Fig 8: Determination of MaMADS1 and MaMADS2 expression levels in the repressed
lines.

45. Reducing the transcript levels of either
MaMADS1 or MaMADS2. which leads to
decrease in ripening progression.
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46. Fig 9 :Schematic diagram of the TILLING and EcoTILLING techniques.
Muhammad et al., 2011
TILLING Targeting Induced Local Lesions in Genomes
TILLING is a technique that can identify polymorphisms (more
specifically point mutations) resulting from induced mutations in a target
gene by heteroduplex analysis.
Development of a mutagenized
population
DNA preparation and pooling
Mutation discovery

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To engineer melon fruit with improved shelf-
life, by TILLING platform in a monoecious
and climacteric melon line.

48. • Experiments were carried out using the
melon inbred line CharMono, a monoecious
climacteric cultivar.
• CharMono seeds for EMS treatment.
• M2 population for TILLING.
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49. Table 2: Impact of EMS concentration on
germination
EMS dose
M1 plants
Seed
germination (%)
1% 100 99
1.50% 100 99
12% 100 98
3% 40 97
13-02-2017 Dept.BCI 49Mardas et al., 2010

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CmDET1 light signaling pathway 3193 35.11 24 2538 1/337 kb
CmDHS eIF5A activation 2434 36.48 19 2538 1/325 kb
CmACO1 ethylene biosynthesis 1469 40.16 7 3306 1/694 kb
CmNOR fruit ripening process 1272 39.70 9 3306 1/467 kb
CmEXP1 cell-wall modification 1168 46.23 11 3306 1/351 kb
CmSP1 Inflorescence development 1055 33.93 3 4023 1/1415 kb
CmTCTP cell growth 1512 36.24 11 4023 1/553 kb
CmCNR fruit ripening process 1333 40.36 11 4023 1/487 kb
CmSGR Chlorophyll degradation 1383 37.24 13 4023 1/428 kb
CmACS7 sex determinism 1680 38.57 8 4023 1/845 kb
CmWIP1 sex determinism 1812 31.95 18 4023 1/405 kb
Targets Function
Amplicon
size (pb)
GC
content(%)
Identified
mutants
Screened M2
families
Mutation
frequency
Table 3. Tilled genes and mutation frequency in CharMono mutant population.
Mardas et al., 2010

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Missense nonsense silent
Expected 64.0% 5.0% 29.3%
observed 65.1% 2.4% 31.3%
Table 4: Expected and observed frequencies of induced
mutation types in tilled gene-coding regions
Mardas et al., 2010
Fig 11: Schematic diagram of CmACO1 gene structure
Intronic :
Silent :
L124F :
G194D :

52. • Identified a TILLING mutant with enhanced
fruit shelf life.
• And the effectiveness of TILLING as a reverse
genetics tool to improve crop species.
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53. Objective: To characterise sACO-1
transgenic line.
73

54. • Naturally in carnation , ethylene is first
produced from the gynoecium; the evolved
ethylene, acting as a diffusible signal which is
perceived by petals, induces the expression of
DC-ACO1 and DC-ACS1 genes that results in
ethylene production in petals.
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55. Kosugi et al., 2002
Fig 13. Senescence profile of carnation flowers of the non-transformed
(NT) control (top) and the sACO-1 transgenic line (bottom).
Non-transformed line showing vase life 5.8 days, whereas
sACO-1 Transgenic showing vase life 9.5 days

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Figure 14: Ethylene production from cut carnation flowers during the senescence
period in the NT control and the sACO-1 lines.Five flowers each of both lines were
harvested at full opening stage (day 0) and their ethlene production monitored daily. Data
are shown by the mean + SE of 5 flowers.
Kosugi et al., 2002
NT
sACO-1 x

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Kosugi et al., 2002
Fig 15: RNA gel blot analysis of mRNA or DC-ACO-1 and DC-ACS-1 in petals and
gynoecium of the NT control and the SACO-1 lines during natural senescence.
Indicated that sACO transgene inhibits the expression of DC-ACO1, by cosuppression in the
gynoecium.

58. Sl.no crop gene functions author
1 Lillipot carnation Antisense ACC oxidase Reduced ethylene
production
Kosugi et al., 2002
2 Carnation MR and 532-6 DcACS1 and DcACO1 Ethylene biosynthetic
genes
Tanase et al., 2014
Carnation MR and 532-6 DcbGa1 ,DcGST1 and
DcLip
Senescence related
genes
Tanase et al., 2014
3 Pelargonium pSAG12::ipt Delayed leaf
Senescence
Sogo et al., 2012
4 Arabidopsis FYF homolog from
Oncidium orchid
Delay of flower
senescence and
abscission
Chen et al., 2011
5 Transgenic Lettuce1 PSAG12-IPT Delayed postharvest
leaf senescence
McCabe et al., 2001
6 Indifferent crops Senescence associated
genes
Tripathi andTuteja
2007
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59. Conclusion
• Use of biotechnological approaches offers the
opportunity to enhance yield, shelf life,
quality of produce, fetches more price to
farmers and helps to meet out present market
demand for horticultural produce.
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