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Introduction
Papaya
A climacteric fruit which
ripens 1-2 wks from the time of
harvest
“Solo” varieties exported to
Hong Kong, Japan & United
Arab Emirates
Tropical fruit like papaya
deteriorates quickly after
harvest
Ripening is controlled by
ethylene where ACC oxidase
gene is involve
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ACC Oxidase Gene
One of the key enzymes
involved in ethylene biosynthesis
pathway
Objectives
General
> To develop a transgenic papaya with delayed ripening
characteristics containing the antisense ACC oxidase
gene
Specific
> Prepare gene construct for transformation
> Characterize the construct containing the antisense
ACC oxidase gene
> Generate somatic embryos and transform by
Agrobacterium tumefaciens
> Regenerate putative transgenic plants
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Methodology
Agrobacterium transformation
pGA643 w/ ACO
Agrobacterium w/ Somatic embryos
pGA643 w/ ACO
Agrobacterium suspension (w/ ACO)
Transgenic papaya
Accomplishments
A. Somatic embryogenesis
>17,145 somatic embryos clusters were produced
>27-88% somatic embryogenesis was obtained
>770 immature somatic embryos isolated after
typhoon „Milenyo‟ (Sept. 2006)
> 370 proceeded to somatic embryogenesis
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Accomplishments
B. Cloning & molecular characterization
>cloned the ACC oxidase gene from „Davao Solo‟
papaya
TCCNTCACTGACGTAGGGACTGACGCACANTTT
ACCCACTATCCTTCGCAAGACCTTCCTCTATATAA
GGAAGTCATTTCATTTGGAGAGGACCCTCGACC
AAGCTTCTAGAGTTCCTTCGCCTGGAACTTCAAC
CCAGCATACAGCTTCATGTAATCCTCGAACACGA
ATTTCGGGTACGCTGTTTTCTTCTCCTCTGTTTCT 800
TTCTCCACCAATATCGGCGCCGGATAAATCACGG
CGTCGCTTCCGGGGTTGTAGAAAGAAGCTATCG bp
ACATCCTCGTCCCGTCGGTTTGTGCCACCACTCT
GTGCTCCACGCTCTTGTATTTCCCGTTGGTAATC
ACCTCGAGTTGGTCGCCGAGGTTGACGACAATG
GAGTGGCGCATTGGTGGAACATCAACCCATTTG
CCGTCTTTGAGGAGTTGGAGGCCGCTGACTTTG Partial ACC oxidase gene
TCGTCCTGGAAGAGCAAGATGATGCCGCCGGC obtained from cDNA using
GTCGGTGTGTGCCCGGAGCCCTTTGATCAAGTT
TGGTTTACGGGCATGGAGGGTAGTTGCTGACTTT primers VF01 & VF02
GGTGCCGAAAGTTGGACCTCTCGACCCGTAAAA
TGCTTATTTCAATACCCTTTTTCCAGCCCGAGAT 1: 200 bp ladder, 2: early ripe, 3:
fully ripe,4: + control, 5: - control
Sequence of ACC oxidase gene (834
bp) by Waikato Sequencing, NZ
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>purified plasmid pGA643 with ACC oxidase gene by
plasmid maxiprep
Cuba
95-98% Philippines
homology
Indonesia
Taiwan-chi-
tsai
Taiwan
Malaysia
74%
homology Hawaii
India cv Singapore
India cv
Surya
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Accomplishments
Modifications of Regeneration
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Modifications of Regeneration
White fingerlike structures
Modifications of Regeneration
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Modifications of Regeneration
Partial greening of the Yellow fingerlike structure
somatic embryo w/ single shoot
Brassinolide
• Belongs to brassinosteroids
• A steroidal hormone extracted from Brassica napus
pollen
• Helps in regulating certain cellular activities like division
and differentiation thereby controlling overall
developmental processes in plant morphogenesis
(Clouse, 2002)
• In Helianthus tuberosus, brassinolide can stimulate cell
division in the presence of auxin and cytokinin (Clouse
and Zurek, 1991)
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Accomplishments
Regeneration using brassinolide (BL) treatments
Untransformed Transformed
Control
DF + 0.5µM NAA +
0.5µM BA
High BL
DF + 0.5µM NAA +
0.5µM BA + 0.16%
Brassinolide
Low BL
DF + 0.5µM NAA +
0.5µM BA + 0.08%
Brassinolide
Accomplishments
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DF + 0.5 µM NAA + 0.5 µM BA + 0.08% Brassinolide
2 weeks old somatic embryos
92 putative transgenic lines (T0) with antisense ACO gene were regenerated
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Micropropagation of putative transgenic lines
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Title of Invention: Papaya ACC Oxidase Gene and its
Use
Problems met
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Acknowledgement
1. PCARRD/GIA-DOST
2. ISAAA
3. BIOTECH, IPB & UPLB
4. MARDI
5. Papaya Biotechnology Network of SEAsia
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