Development of Transgenic Papaya

with Delayed Ripening Characteristics

Containing the ACC Oxidase Gene Via
Agrobacterium-Mediated
Transformation
(Aug. 2002-June 2008)

PM Magdalita, AC Laurena, RL Comia and MTM Perez

Crop Science Cluster - Institute of Plant Breeding,

College of Agriculture, University of the Philippines,
Los Baños, College, Laguna

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

 One of the targets in

engineering the ethylene
biosynthesis pathway

 Antisense RNA reduces

translation of ACC oxidase
thereby blocking ethylene
production

 ACC oxidase gene was cloned

from „Davao Solo‟ papaya (Perez,
1999).

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)

Regenerated Transformed somatic

T0 plantlets embryos undergoing
selection

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

Zygotic embryo Somatic embryos-1 mo. Proliferating somatic

embryos-2 mos.

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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

>transformed plasmid pGA643 with the antisense

ACC oxidase gene into E. coli strain DH5α

Electrophorated E. coli colonies with

antisense ACC oxidase gene

Plasmid pGA643 with antisense

ACC oxidase gene

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 >purified plasmid pGA643 with ACC oxidase gene by
plasmid maxiprep

>transformed pGA643 with antisense ACC oxidase

gene into Agrobacterium tumefaciens strain LBA 4404
by electrophoration

>purified pGA643 with antisense ACC oxidase gene

by Agrobacterium maxiprep
1 2 3 4 5 10 12 14

Purified pGA643 with ACC oxidase

by Agrobac miniprep

1: MW marker, 2: pGA643 w/ACC oxidase, 3-

14: purified samples using miniprep

>compared ACC oxidase gene sequence with other papaya

ACO of other countries

Cuba
95-98% Philippines
homology
Indonesia

Taiwan-chi-
tsai
Taiwan
Malaysia
74%
homology Hawaii

India cv Singapore

India cv
Surya

T-Tainung 2 H-Sunset Solo P-Solo

M-Eksotika C-Maradol Roja I-Samangka

Phylogenetic tree of ACO genes of different papaya

varieties from other countries

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 Accomplishments

C. Transformation by Agrobacterium tumefaciens

> previously established transformed

somatic embryo cultures were
contaminated due to typhoon “Milenyo”

> 295 new set of golden yellow somatic

embryos were transformed using
Agrobacterium w/ pGA643 + antisense
ACC oxidase gene

Modifications of Regeneration

D. Regeneration of transformed somatic embryos

●Transformed tissues are difficult to regenerate due to
strong selection pressure (ie. carbenicillin to kill Agro &
kanamycin to kill untransformed tissues)
●2,925 somatic embryo clusters & calli that passed
kanamycin selection were regenerated
●Different media formulations were tried for
regeneration.
>Varying the levels of hormones (BAP, NAA, GA3)
sugar, nurse culture and some macro and
micronutrients plus brassinolide were used

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 Modifications of Regeneration
White fingerlike structures

½ MS + 10µM GA3 + 0.5µM NAA + ½ MS + 20µM GA3 + 01.0µM NAA +

0.5µM BAP + 3g/L Casein 1.0µM BAP + 3g/L Casein
hydrolysate hydrolysate

Modifications of Regeneration

Light brown Greening of the

somatic embryo

DF + 0.16% brassinolide DF + 0.08% brassinolide

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 Modifications of Regeneration
Partial greening of the Yellow fingerlike structure
somatic embryo w/ single shoot

DF without hormone, DF without hormone

+ 0.08% brassinolide (plantlet died)

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)

Source of chemical structure: members.tripod.com/~mzullo/brassinolide.gif

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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

Initial green transformed somatic embryos obtained 180 days after

transformation and grown on DF + 0.5µM NAA + 0.5µM BA + low
Brassinolide (0.08%)

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 DF + 0.5 µM NAA + 0.5 µM BA + 0.08% Brassinolide
2 weeks old somatic embryos

180 days after

transformation 205 days 264 days 299 days

3 weeks old somatic embryos

180 days after

transformation 205 days 264 days 299 days

92 putative transgenic lines (T0) with antisense ACO gene were regenerated

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 Micropropagation of putative transgenic lines

Regulatory, Licensing & IPR

PCARRD-DOST holds the license for Delayed

Ripening Project

NCBP approval obtained for “contained use”

experiment (2002)

IP Audit completed in cooperation with

PCARRD (January 17 to April 11, 2002)

Patent application for the ACC oxidase gene

filed with the Phil. IPO, Makati City

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Title of Invention: Papaya ACC Oxidase Gene and its
Use

Assignee: UPLB and PCARRD

Inventors: PM Magdalita, MTM Perez, AC Laurena, RL

Comia

Application no.: 1-2008-000215

Date of filing: June 27, 2008

Description of the Invention: (i.e. technical field,

background, summary of invention, object of invention,
detailed description of invention, commercial use and
patent claims)

Problems met

• Slow growth of transformed tissues once co-cultivated

with Agrobacterium tumefaciens

• Delayed release of funds and procurement procedures

• Natural calamities – Typhoon ‘Milenyo’ (Sept. 2006) &

Fire at Module A, IPB (Aug. 2007)

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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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