Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato

Cahya Prihatna, Ruolan Chen, Martin John Barbetti, Susan Jane Barker

Research output: Contribution to journalArticle

Abstract

Genetic transformation of tomato was first accomplished around 30 years ago. However, variability in transformation efficiency of distinct cultivars exists and to some extent remains a bottleneck for transgenic research. This study reports strategies to improve transformation efficiency in tomato and investigates regeneration capacity of transgenic plants under different selection regimes and hormonal applications. Tomato cv. Rio Grande was used as plant material and hygromycin and phosphinothricin (PPT) were used as selection agents. We found that cv. Rio Grande inherently produced a significant number of abnormal (“blind”) shoots lacking an apical meristem. Replacing cytokinin zeatin riboside with 6-benzylaminopurine (BAP) reduced the number of blind shoots although it slightly prolonged regeneration time. Survival rate of calli and shoots was very low using PPT as selection, whereas regeneration was achieved using hygromycin. Delayed application of hygromycin selection following co-cultivation with Agrobacterium tumefaciens improved the overall callus and shoot production. In vivo GFP fluorescence was detected to investigate the development of transgenic tissues using different hygromycin selection regimes. Higher transformation frequency was achieved when explants were continuously exposed to selection agents immediately following the pre-selection stage. Reducing the selection period followed by a non-selection stage increased the number of shoots, but these shoots were mostly non-transgenic. Thus, although less stringent selection, as expected, encouraged regeneration of shoots from calli, it did not improve transformation efficiency. Omitting selection altogether greatly reduced the efficiency of transformation. It was concluded that BAP is more suitable for normal shoot development, and that delayed selection followed by continuous selection results in higher transformation frequency.

Original languageEnglish
Pages (from-to)473-483
Number of pages11
JournalPlant Cell, Tissue and Organ Culture
Volume137
Issue number3
DOIs
Publication statusPublished - 1 Jun 2019

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tomatoes
shoots
callus
glufosinate
benzyladenine
genetically modified organisms
shoot meristems
zeatin
genetic transformation
apical meristems
Agrobacterium radiobacter
cytokinins
transgenic plants
explants
survival rate
fluorescence
cultivars

Cite this

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title = "Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato",
abstract = "Genetic transformation of tomato was first accomplished around 30 years ago. However, variability in transformation efficiency of distinct cultivars exists and to some extent remains a bottleneck for transgenic research. This study reports strategies to improve transformation efficiency in tomato and investigates regeneration capacity of transgenic plants under different selection regimes and hormonal applications. Tomato cv. Rio Grande was used as plant material and hygromycin and phosphinothricin (PPT) were used as selection agents. We found that cv. Rio Grande inherently produced a significant number of abnormal (“blind”) shoots lacking an apical meristem. Replacing cytokinin zeatin riboside with 6-benzylaminopurine (BAP) reduced the number of blind shoots although it slightly prolonged regeneration time. Survival rate of calli and shoots was very low using PPT as selection, whereas regeneration was achieved using hygromycin. Delayed application of hygromycin selection following co-cultivation with Agrobacterium tumefaciens improved the overall callus and shoot production. In vivo GFP fluorescence was detected to investigate the development of transgenic tissues using different hygromycin selection regimes. Higher transformation frequency was achieved when explants were continuously exposed to selection agents immediately following the pre-selection stage. Reducing the selection period followed by a non-selection stage increased the number of shoots, but these shoots were mostly non-transgenic. Thus, although less stringent selection, as expected, encouraged regeneration of shoots from calli, it did not improve transformation efficiency. Omitting selection altogether greatly reduced the efficiency of transformation. It was concluded that BAP is more suitable for normal shoot development, and that delayed selection followed by continuous selection results in higher transformation frequency.",
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Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato. / Prihatna, Cahya; Chen, Ruolan; Barbetti, Martin John; Barker, Susan Jane.

In: Plant Cell, Tissue and Organ Culture, Vol. 137, No. 3, 01.06.2019, p. 473-483.

Research output: Contribution to journalArticle

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AU - Prihatna, Cahya

AU - Chen, Ruolan

AU - Barbetti, Martin John

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