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A direct method for genetically transforming rice seeds modelled with FHVB2, a suppressor of RNAi

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Abstract

RNA interference (RNAi) is a novel method of gene regulation and one of the potent host defense mechanisms against viruses. It however acts as a deterrent in transgene-technology by constraining the expression of the introduced gene. The virus-encoded suppressors have the ability to restrict host RNAi to promote pathogenicity. They thus have tremendous potential to ameliorate low transgene expression and have important applications in the biofarming sector. Unfortunately the suppressors severally reduce plant regeneration potentials in the standard procedures. In this study, we report a simple, fast and efficient method for in planta transformation of rice seeds that can be used for over-expressing FHVB2, a well-characterized suppressor of RNAi. The protocol involves agro-inoculation of embryos without vacuum infiltration or injury followed by their growth ex vitro. Following transformation the transgene integration, expression and stable inheritance was confirmed. We observed that the FHVB2 transgenic survival in this methodology was 15-fold higher compared to that in available callus-based methods. The protocol has the potential to be extended for transforming rice with any gene as exemplified by the use of control constructs.

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Abbreviations

FHVB2:

B2 protein of Flock House Virus

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Acknowledgments

The authors would like to thank Dr. Sunil K. Mukherjee and Dr. Raj K. Bhatnagar for providing the suppressor gene constructs. The authors would like to thank Prof. Sudhir K Sopory for critically reading the manuscript. SSD acknowledges the fellowship received from CSIR, India. The research was supported by financial grants received from the Department of Biotechnology, Government of India.

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The authors declare that they have no conflict of interest.

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  1. Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Sudhanshu Sekhar Das & Neeti Sanan-Mishra

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  1. Sudhanshu Sekhar Das
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Correspondence to Neeti Sanan-Mishra.

Electronic supplementary material

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Representative photographs of the various stages of transformation of FHVB2 (JPEG 97 kb)

11240_2014_604_MOESM2_ESM.jpg

Representative photographs to show the agro-envelope around rice seeds inoculated with Agrobacterium (EHA105) cells at a OD600 = 1.0 and b OD600 = 0.1 with 1 day and 3 days of co-cultivation period respectively (JPEG 73 kb)

11240_2014_604_MOESM3_ESM.jpg

Representative photographs to show the morphological abnormalities in rice seedlings transformed with FHVB2. a FHVB2 transformed seedling as grown on MS selection plate. Blue arrows indicate the albino seedlings, Red arrows indicate seedlings with slow growth and black arrows indicate seedlings with defective morphology. b A large proportion of seedlings transformed with FHVB2 are albino and these could not grow to maturity. c Control plants transformed with pCAMBIA1300-GUS showed normal physiology (JPEG 97 kb)

11240_2014_604_MOESM4_ESM.jpg

Genomic DNA PCR with virG gene to confirm the absence of Agrobacterium in different primary transformants growing on selection medium. Agarose gel electrophoresis of PCR amplified DNA from leaf tissue of the transformed rice lines. The virG gene amplification should result in a band of 503 bp. Lane 1. 1 kb DNA ladder (Fermentas); Lane 2-11, represents different independently transformed lines; Lane 12, negative: represents untransformed control wild type (WT) plant; Lane 13, positive: represents PCR control with 20 ng of plasmid as template (JPEG 54 kb)

11240_2014_604_MOESM5_ESM.jpg

Southern blot analysis of T1 progenies of the FHVB2 transgenics. 20 μg of BamH1 digested DNA from leaves of different plants was blotted and probed with 32P labelled FHVB2 gene. (A) Lanes 1-5. different transgenic plants; Lane 6. Wild type (WT) plant; Lanes 7 template probe (B) Lane 1-7 different transgenic plants (JPEG 132 kb)

11240_2014_604_MOESM6_ESM.jpg

RT-PCR to confirm the presence of FHVB2 transgene in the T1 generation plants. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of FHVB2 gene expression in different transgenic rice lines using the primers amplifying the region of FHVB2, GUS and Β-ACTIN genes. cDNA was prepared using 50 U of Super ScriptTM II reverse transcriptase (Invitrogen). Lanes 1-11: transgenic rice lines; Lane 12: negative: represents wild type (WT) seedlings; Lane 10: positive: represents 20 ng of plasmid used as template (JPEG 42 kb)

Genomic DNA PCR with virG gene to confirm the absence of Agrobacterium in the T1 generation plants (JPEG 33 kb)

Representative photograph of regeneration from rice callus over-expressing MYMIV-AC2 (JPEG 32 kb)

Supplementary material 9 (DOC 39 kb)

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Das, S.S., Sanan-Mishra, N. A direct method for genetically transforming rice seeds modelled with FHVB2, a suppressor of RNAi. Plant Cell Tiss Organ Cult 120, 277–289 (2015). https://doi.org/10.1007/s11240-014-0604-3

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