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An easy and efficient protocol in the production of pflp transgenic banana against Fusarium wilt

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Abstract

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing N 6-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.

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Abbreviations

ANOVA:

One-way analysis of variance

AS:

Acetosyringone

Atfd3 :

Arabidopsis root-type ferredoxin gene

BA:

N 6-benzylaminopurine

BI:

Bud induction medium

CaMV 35S:

Cauliflower mosaic virus 35S promoter

CC:

Co-cultivation medium

ECS:

Embryogenic cell suspension

FOC:

Fusarium oxysporoum f. sp. cubense

HR:

Hypersensitive response

IAA:

Indole-3-acetic acid

MBC:

Multiple bud clumps

MS:

Murashige and Skoog

NOS:

Nopaline synthase promoter

nptII :

Neomycin phosphotransferase gene

PCR:

Polymerase chain reaction

PFLP:

Plant ferredoxin-like protein

PP333:

Paclobutrazol

TDZ:

Thidiazuron

YEB:

Yeast extract broth

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Acknowledgments

The authors are especially thankful to Dr. Swee-suak Ko and Ms. Mei-jin Huang for their technical support. We also give a deep appreciation to the Taiwan Banana Research Institute for the supply of banana cultivars and to the Academia Sinica Biotechnology Experimental Center in southern Taiwan for providing the transgenic-qualified greenhouse and net-house. Special thanks are expressed to Mr Conmar Malmis (National Chung Hsing University) for critical evaluation of the manuscript and valuable suggestions. This work was supported by grants from Academia Sinica and the Science and Technology Development Foundation, Taiwan.

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Authors and Affiliations

  1. Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, Republic of China

    Mei-Kuen Yip, Kuei-Ching Su, Yi-Hsien Lin, Tai-Yang Chen & Teng-Yung Feng

  2. Tissue Culture Section, Taiwan Banana Research Institute, Chiuju, Pingtung, 90442, Taiwan, ROC

    Sin-Wan Lee

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  1. Mei-Kuen Yip
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  2. Sin-Wan Lee
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  3. Kuei-Ching Su
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  4. Yi-Hsien Lin
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  6. Teng-Yung Feng
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Correspondence to Teng-Yung Feng.

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Yip, MK., Lee, SW., Su, KC. et al. An easy and efficient protocol in the production of pflp transgenic banana against Fusarium wilt. Plant Biotechnol Rep 5, 245–254 (2011). https://doi.org/10.1007/s11816-011-0179-y

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  • DOI: https://doi.org/10.1007/s11816-011-0179-y

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