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Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell.

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Abstract

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD600, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.

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Abbreviations

ANOVA:

Analysis of variance

AS:

Acetosyringone

BA:

6-Benzyladenine

CaMV 35S:

Cauliflower mosaic virus 35S promoter

GUS:

β-Glucuronidase

gusA:

β-Glucuronidase

IBA:

Indole-3-butyric acid

MES:

2-[N-Morpholino]ethanesulfonic acid

MS:

Murashige and Skoog

NAA:

a-Naphthaleneacetic acid

nptII:

Neomycin phosphotransferase

OD:

Optical density

PCR:

Polymerase chain reaction

RH:

Relative humidity

Rpm:

Rotation per minute

YEP:

Yeast extract and peptone medium

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Acknowledgments

This work was supported by a grant from the Agriculture Research Center (Grant No. 06362010-0010) from the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Forestry and Fisheries, Republic of Korea. Prof. A. Ganapathi is grateful to the University Grants Commissions (UGC), Government of India, for the award of UGC-Emeritus Fellowship. C. Arunachalam and A. Alharbi Sulaiman would like to extend their sincere appreciation to the deanship of Scientific Research at King Saud University for its funding of this research through the research group No: RG-1435-081.

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Authors and Affiliations

  1. Molecular Genetics and Genomics Laboratory, Department of Horticulture, College of Agriculture and Life Sciences, Chungnam National University, Daejeon, 305 764, South Korea

    Ganeshan Sivanandhan & Yong Pyo Lim

  2. Department of Biotechnology and Genetic Engineering, School of Life Sciences, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Venkatachalam Vasudevan, Gnanajothi Kapildev & Andy Ganapathi

  3. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

    Chinnathambi Arunachalam & Ali Alharbi Sulaiman

  4. Department of Botany, Periyar E. V. R College (Autonomous), Tiruchirappalli, 620 023, Tamil Nadu, India

    Natesan Selvaraj

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  1. Ganeshan Sivanandhan
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  2. Chinnathambi Arunachalam
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  3. Venkatachalam Vasudevan
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  4. Gnanajothi Kapildev
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  5. Ali Alharbi Sulaiman
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  6. Natesan Selvaraj
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  7. Andy Ganapathi
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  8. Yong Pyo Lim
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Correspondence to Ganeshan Sivanandhan or Yong Pyo Lim.

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Sivanandhan, G., Arunachalam, C., Vasudevan, V. et al. Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell.. Plant Biotechnol Rep 10, 49–60 (2016). https://doi.org/10.1007/s11816-016-0385-8

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