Details about need and methods for Genetic Engineering of Papaya for Ring Spot Virus.

1. Genetic Engineering of
Papaya for Ring Spot
Virus
Amandeep Kaur
L-2014-A-12-D

2. The Papaya Story
 Christopher Columbus: "Fruit of the Angels"
 Produce fruit year round, with a slight seasonal
peak in early summer and fall.
 Rich source of:
Antioxidant nutrients such as carotenes, vitamin C and
flavonoids;
B vitamins, folate and pantothenic acid;
Minerals, potassium, copper, and magnesium;
Fiber , etc.
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3. The Papaya Story
 Ranked third (15.36%)in total tropical fruit
production after mango (52.86%) and pineapple
(26.58%).
 Hawaii: the major U.S. producer since1920s.
 Exports 85% of crop Japan
http://www.whfoods.com/genpage.php?tname=foodspice&dbid=47
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4. Papaya Ring Spot Disease4
 Major problem of papaya industry
 Cause: Papaya Ring Spot Virus (PRSV)
 The virus occurs as two strains :
 PRSV - type P (for papaya) infects both
papaya and cucurbits (e.g. squash, pumpkin,
cucumber, and melons).
 PRSV- type W (for watermelon), which infects
cucurbits only.

5. Papaya Ring Spot Disease5
 Transmission:
 Aphids [green peach aphid (Myzus persicae)]
 Mechanical means
 There is no cure for infected trees

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7. Emergence of PRSV7
 D.D. Jensen(1945): first report in Hawaii of PRSV
 PRSV was first discovered on Oahu causing severe
damage
 Industry relocated to Puna in the late 1950s and early
1960s.
 In May 1992, PRSV was discovered in Puna and was
widespread throughout the growing region by 1995.

8. Controlling PRSV prior to GE
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 Rouging
 Spraying with aphicides
 Interplanting rows of non-host crops
 Cross protection by inoculation of mild or mutated
strain of PRSV
 Breeding techniques
 Hybridization with wild relatives

9. Pathogen Derived Resistance
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 Concept conceived in 1980s
 Protection against the detrimental effects of
pathogens by expression of genes or sequences
of the same or related pathogens
 First demonstrated by Beachy’s group in
transgenic tobacco resistant to TMV

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Coat Protein Mediated Resistance
 Accumulation of the CP confers resistance to
infection and/or disease development by the virus
from which the CP gene was derived and by
related viruses.
 CP affects infecting virus:
 Interfering with release of encapsidated RNA
 Preventing its translation and replication
 Re-encapsidation.

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 Transformed PRSV-CP gene into tobacco
 To determine the cross protection against other
potyvirus
 Source of CP gene: mild mutant strain PRSV HA
5-1

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 CP gene: encoded as polyprotein
 Lack 5’ UTR regions and initiation codon
 Translational and transcriptional elements
needed to be added
 pUC1813cpCMV expression vector:
 35S CaMV promoter
 70bp CMV 5’ UTR region
 Initiation codon
 Codons for first 16 amino acid of CMV-CP
Engineering PRSV-CP gene

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Nucleotide Sequence of pUC1813cpPRV-4

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The Binary Vector Construct

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 Explant used: -Hypocotyl
-Embryonic calluses
-Zygotic embryos
 Cultivars used: ‘Sunset’ and ‘Kapoho’
 Transformation method: Biolistics

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General Strategy

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GUS Expression
Immature zygotic embryo after 4 weeks of
bombardment
A total of 12 plants
were found to be
GUS(+)

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GUS expression
 Using leaf samples: more
reliable than embryos
 Varied expression between
individual plants as well as
within a plant
Fig: A) S55-1
B) S60-3

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 2.0kb BamHI/HindIII
fragment containing Nos-
NPTII gene in six out of
seven plant samples.
 Larger fragments:
incomplete digestion or
rearranged genes
Southern Blot Analysis for NOS-
NPTII gene

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 Primers amplifying PRSV-CP
genes
 10 out of 12 GUS(+) plants
were PRSV-cp gene (+)
PCR detection of PRVcp gene
Fig.: a) EtBr stained gel
b)blotted and hybridized with
PRSVcp gene probe
GUS(+) but PRSV cp gene(-)

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 K39-1: no
protection
 K19-1 and S60-3:
intermediate
protection (25-
33%)
 S55-1: Complete
protection
Reaction of subcloned transgenic
R0 papaya to inoculation with
PRV HA

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 1.35kb transcript observed
in both S55-1(lanes 2,3)
and S60-3(lanes 4,5)
 Predicted transcript size
Northern Blot of PRV cp gene+

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Virus Screening of transgenic
papaya plants
A) Uninoculated S55-1, B) PRSV HA inoculated S55-1, C)
Untransformed Sunrise inoculated with PRSV HA , D) S55-1 after 6
months of inoculation

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Conclusions
 Most efficient recovery of transgenic by
bombardment method is from immature zygotic
embryos
 No transgenics from hypocotyl explant
 Neither GUS expression nor PRVcp gene assays
were reliable predictors of resistant plants.
 Only reliable indicator: functional analysis

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 APHIS issued a permit for field trials of the new
transgenic plants in 1991
 First trails designed to asses resistance to
mechanical and aphid inoculations of PRSV
 1992: Two new transgenic cultivars were developed
‘SunUp’ and ‘Rainbow’
Field Trails and Commercialization

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 ‘SunUp’ is a transgenic red-fleshed Sunset that is
homozygous for the coat protein gene.
 ‘Rainbow’ is a yellow-fleshed F1 hybrid developed
by crossing ‘SunUp’ and non-transgenic yellow-
fleshed ‘Kapoho’.
 Licenses to commercialize the transgenic papaya
were obtained in April 1998.
Field Trails and Commercialization

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