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Somatic embryogenesis and Agrobacterium-mediated transformation of Gladiolus hybridus cv. ‘Advance Red’

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Abstract

Stable and efficient Agrobacterium-mediated transformation of Gladiolus hybridus cv. ‘Advance Red’ was established via a somatic embryogenesis regeneration system in this study. Treatment with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea was sufficient to induce embryogenic callus and somatic embryogenesis in G. hybridus ‘Advance Red’. Cormel slices were used as explants and pre-cultured for 5 days prior to selection culture with 9.48 μM hygromycin and 0.52 mM cefotaxime. Treated slices were inoculated with Agrobacterium stain GV3101 harboring a binary vector, pCAMBIA1301, containing the β-glucuronidase (gus) reporter and hygromycin phosphotransferase genes (hpt). 10 min of infection was determined as the optimal time for transformation. Compared with the normal 3-day co-cultivation period, prolonged co-culture for 12 days led to significantly higher transient gus expression rate (50.52 ± 2.83 %). Permanent expression of gus was observed in leaves, roots and whole plantlets. T-DNA insertion was further confirmed using PCR and Southern blot analyses. Our data collectively indicate that extended pre-cultivation and co-cultivation improve transformation efficiency, and the somatic embryogenic regeneration system effectively facilitates the transformation of G. hybridus. This newly developed transformation system may be successfully applied for functional analysis of genes and quality modifications in G. hybridus.

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Abbreviations

AS:

Acetosyringone

CaMV 35S:

Cauliflower mosaic virus 35S promoter

gus :

β-Glucuronidase

hpt :

Hygromycin phosphotransferase

IBA:

Indole-3-butyric acid

LB:

Luria–Bertani

MES:

4-Morpholineethanesulfonic acid hydrate

MS:

Murashige and Skoog

NOS poly A:

Nopaline synthase poly A terminator

6-BA:

6-Benzylaminopurine

TDZ:

N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea

OD600 :

Optical density at a wavelength of 600 nm

PCR:

Polymerase chain reaction

vir :

Virulence genes of Agrobacterium tumefaciens

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Acknowledgments

This work was supported by the Doctoral Fund of Ministry of Education of China (20120008110006) and National Natural Science Foundation of China (31171991).

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Authors and Affiliations

  1. Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Yuan Mingyuan Western Road 2#, Beijing, 100193, China

    Jian Wu, Chen Liu, Shanshan Seng, Muhammad Ali Khan, Juanjuan Sui, Benhe Gong, Chao Liu, Chenyu Wu, Xionghui Zhong, Junna He & Mingfang Yi

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  1. Jian Wu
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Correspondence to Mingfang Yi.

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11240_2014_639_MOESM1_ESM.tif

Schematic representation of hpt and gus gene expression cassettes in the binary vector, pCAMBIA 1301, used in Gladiolus hybridus transformation. The T-DNA region of the vector contains the hygromycin expression cassette (CaMV 35S: hpt: poly A) and uid gene expression cassette (CaMV 35: gus: nos poly A). CaMV 35S cauliflower mosaic virus 35S promoter; hpt hygromycin phosphotransferase II; poly A cauliflower mosaic virus 35S poly A terminator; gus beta-glucuronidase gene; nos poly A nopaline synthase poly A terminator. The pink bar represents DIG primers and the blue bar indicates PCR primers (TIFF 1851 kb)

11240_2014_639_MOESM2_ESM.tif

Characteristics of explants and effects of hygromycin a Explant; b Callus generated from fibrovascular tissue and adjacent parenchymatous tissue; c Explant and its embryogenic callus; d Section of an explant; e, f, g, h, i Explants cultured in MS + 0, 2.5, 5, 7.5, 15 mg l−1 hygromycin at 30 days, respectively. a, b, c, d bar = 1 mm; e, f, g, h, i bar = 1 cm (TIFF 39445 kb)

11240_2014_639_MOESM3_ESM.tif

A Overall GUS-stained plantlets (no chimeras), bar = 1 cm; b and c Partially stained plantlets (chimeras), bar = 500 μm (TIFF 12200 kb)

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Wu, J., Liu, C., Seng, S. et al. Somatic embryogenesis and Agrobacterium-mediated transformation of Gladiolus hybridus cv. ‘Advance Red’. Plant Cell Tiss Organ Cult 120, 717–728 (2015). https://doi.org/10.1007/s11240-014-0639-5

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