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Transfer of transformed Lesquerella fendleri (Gray) Wats. chloroplasts into Orychophragmus violaceus (L.) O.E. Schulz by protoplast fusion

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Abstract

Asymmetric intergeneric hybrid plants were obtained through protoplast fusion between Orychophragmus violaceus (L.) O.E. Schulz and Lesquerella fendleri (Gray) Wats. The latter carried chloroplasts transformed with the fused aadA16gfp gene construct, conferring streptomycin–spectinomycin resistance and UV-induced green fluorescence. The somatic hybrids were selected using the properties of spectinomycin-induced plastid defects in “albino” O. violaceus plants (chloroplast recipient) combined with the γ-irradiation-induced inactivation of nuclei in plastid donor L. fendleri. The morphology and esterase isozyme pattern of the hybrid plant as well as the results of the PCR analysis of internal transcribed spacer of nuclear ribosomal DNA proved that the regenerated hybrids carried O. violaceus nuclei, while PCR amplification of the atpB– rbcL spacer and aadA16gfp gene fragments confirmed the presence of the transformed L. fendleri chloroplasts in these plants. Expression of the fused aadA16gfp gene construct was confirmed by sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis and the resistance of the obtained plants to both streptomycin and spectinomycin.

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Abbreviations

aadA:

Aminoglycoside 3′-adenylyltransferase gene

atpB :

β subunit of the chloroplast ATP synthase gene

BA:

6-Benzyladenine

2,4-D:

2,4-Dichlorophenoxyacetic acid

gfp :

Green fluorescent protein gene

IAA:

Indole-3-acetic acid

ITS:

Internal transcribed spacer of nuclear ribosomal DNA

Kin:

Kinetin

NAA:

α-Naphthalene acetic acid

ndh4 :

NADH-dehydrogenase ND4 subunit gene

PEG:

Polyethylene glycol

rbcL :

Rubisco large subunit gene

SDS–PAGE:

Sodium dodecylsulfate–polyacrylamide gel electrophoresis

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Acknowledgments

The authors are grateful to Prof. P. Maliga, Waksman Institute, Rutgers, The State University of New Jersey, USA, for providing plant material of the transplastomic L. fendleri used in this research. We thank the editor and two anonymous reviewers from the journal for their valuable comments on the manuscript. We greatly appreciate the help of Dr. Andrei Golovko, Texas Institute for Genomic Medicine, in revising this paper. This work was partly supported by grant for young scientists (No 0206U000381) of the Ukrainian National Academy of Science.

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Authors and Affiliations

  1. Institute of Cell Biology and Genetic Engineering, Zabolotnogo str. 148, 03680, Kyiv-143, Ukraine

    O. Ovcharenko, V. Momot, N. Cherep, Y. Sheludko, I. Komarnitsky, V. Rudas & N. Kuchuk

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  1. O. Ovcharenko
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  2. V. Momot
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  3. N. Cherep
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  4. Y. Sheludko
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  5. I. Komarnitsky
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  6. V. Rudas
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  7. N. Kuchuk
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Correspondence to O. Ovcharenko.

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Ovcharenko, O., Momot, V., Cherep, N. et al. Transfer of transformed Lesquerella fendleri (Gray) Wats. chloroplasts into Orychophragmus violaceus (L.) O.E. Schulz by protoplast fusion. Plant Cell Tiss Organ Cult 105, 21–27 (2011). https://doi.org/10.1007/s11240-010-9833-2

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  • DOI: https://doi.org/10.1007/s11240-010-9833-2

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