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Development of Transgenic Papaya

with Delayed Ripening Characteristics

Containing the ACC Oxidase Gene Via

Agrobacterium-Mediated Transformation

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

(Aug. 2002-June 2008)

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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Agrobacterium transformation

Agrobacterium w/ pGA643 w/ ACO

pGA643 w/ ACOSomatic embryos

Agrobacterium suspension (w/ ACO)

Transformed somaticembryos undergoing selection

Regenerated T0 plantlets

Transgenic papaya

Methodology

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

TTCTCCACCAATATCGGCGCCGGATAAATCACGG

CGTCGCTTCCGGGGTTGTAGAAAGAAGCTATCG

ACATCCTCGTCCCGTCGGTTTGTGCCACCACTCT

GTGCTCCACGCTCTTGTATTTCCCGTTGGTAATC

ACCTCGAGTTGGTCGCCGAGGTTGACGACAATG

GAGTGGCGCATTGGTGGAACATCAACCCATTTG

CCGTCTTTGAGGAGTTGGAGGCCGCTGACTTTG

TCGTCCTGGAAGAGCAAGATGATGCCGCCGGC

GTCGGTGTGTGCCCGGAGCCCTTTGATCAAGTT

TGGTTTACGGGCATGGAGGGTAGTTGCTGACTTT

GGTGCCGAAAGTTGGACCTCTCGACCCGTAAAA

TGCTTATTTCAATACCCTTTTTCCAGCCCGAGAT

Sequence of ACC oxidase gene (834 bp) by Waikato Sequencing, NZ

800 bp

Partial ACC oxidase gene obtained from cDNA using

primers VF01 & VF02

1: 200 bp ladder, 2: early ripe, 3: fully ripe,4: + control, 5: - control

>transformed plasmid pGA643 with the antisense

ACC oxidase gene into E. coli strain DH5α

Plasmid pGA643 with antisense ACC oxidase gene

Electrophorated E. coli colonies 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

Purified pGA643 with ACC oxidase

by Agrobac miniprep

1: MW marker, 2: pGA643 w/ACC oxidase, 3-14: purified samples using miniprep

1 2 3 4 5 10 12 14

>compared ACC oxidase gene sequence with other papaya

ACO of other countries

Cuba

Philippines

Indonesia

Taiwan-chi-

tsai

Taiwan

Malaysia

Hawaii

India cv Singapore

India cv

Surya

Phylogenetic tree of ACO genes of different papaya varieties from other countries

95-98%

homology

74%

homology

T-Tainung 2

M-Eksotika

H-Sunset Solo

C-Maradol Roja

P-Solo

I-Samangka

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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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½ MS + 10µM GA3 + 0.5µM NAA +

0.5µM BAP + 3g/L Casein

hydrolysate

½ MS + 20µM GA3 + 01.0µM NAA +

1.0µM BAP + 3g/L Casein

hydrolysate

White fingerlike structures

Modifications of Regeneration

DF + 0.16% brassinolide DF + 0.08% brassinolide

Modifications of Regeneration

Light brown Greening of the

somatic embryo

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DF without hormone,

+ 0.08% brassinolide

DF without hormone

(plantlet died)

Partial greening of the

somatic embryo

Yellow fingerlike structure

w/ single shoot

Modifications of Regeneration

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

Control

High BL

Low BL

DF + 0.5µM NAA +

0.5µM BA

DF + 0.5µM NAA +

0.5µM BA + 0.16%

Brassinolide

DF + 0.5µM NAA +

0.5µM BA + 0.08%

Brassinolide

Untransformed Transformed

Initial green transformed somatic embryos obtained 180 days after

transformation and grown on DF + 0.5µM NAA + 0.5µM BA + low

Brassinolide (0.08%)

Accomplishments

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DF + 0.5 µM NAA + 0.5 µM BA + 0.08% Brassinolide

2 weeks old somatic embryos

3 weeks old somatic embryos

A

B

180 days after

transformation 205 days 264 days

180 days after

transformation 205 days 264 days

299 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