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Adventitious regeneration in blackberry (Rubus fruticosus L.) and assessment of genetic stability in regenerants

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Abstract

Adventitious shoot regeneration from leaves of blackberry cultivar Čačanska Bestrna was examined using 30 different combinations of treatment. Young, fully expanded leaves taken from in vitro proliferating shoots were cultured on Murashige and Skoog (MS) medium containing either N 6-benzylaminopurine (BAP) (2.0 mg l−1) or thidiazuron (TDZ) (1.0 and 2.0 mg l−1) alone, or either of them combined with indol-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations (0.1, 1.0 and 2.0 mg l−1). Indirect shoot formation was observed in 12 different treatments, though the efficacy varied greatly among types and concentrations of plant growth regulators. TDZ at 1.0 mg l−1, applied either alone or combined with IBA, was significantly more effective than BAP in inducing shoot regeneration. The highest regeneration rate (41.66%) was obtained on medium containing TDZ alone. Cytological, flow cytometry and isozyme analyses were used for screening of genetic stability/instability in regenerants. Cytological study, based on chromosome counts in root tip meristems, and flow cytometry analysis indicated that adventitious shoots of Čačanska Bestrna are tetraploid with 2n = 4x = 28 as well as those derived from axillary buds. However, a study conducted on the peroxidase patterns of the different blackberry regenerating lines showed differences between some lines and control plants (in vivo plants and micropropagated plants). These differences were visible with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as hydrogenous donor for peroxidase detection.

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Abbreviations

BAP:

N6-Benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

DAPI:

4′-6-Diamidino-2-phenylindole

DNA:

Deoxyribonucleic acid

G0/G1 :

Resting phase of normal cell cycle

GA3 :

Gibberellic acid

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog (1962)

Na2EDTA:

Sodium-2-ethylenediaminetetraacetic acid

NAA:

α-Naphthaleneacetic acid

PAGE:

Polyacrylamide gel electrophoresis

PGR:

Plant growth regulator

POX:

Peroxidase

Rm :

Relative mobility

TBE:

Tris-borate-EDTA buffer

TDZ:

Thidiazuron (N-pheny-N′-1,2,3-thidiazol-5-ylurea)

TMBZ:

3,3′,5,5′-Tetramethylbenzidine

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Authors and Affiliations

  1. Fruit Research Institute, Kralja Petra I No. 9, 32000, Čačak, Serbia

    Tatjana Vujović, Đurđina Ružić & Radosav Cerović

  2. Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080, Zemun-Belgrade, Serbia

    Gordana Šurlan Momirović

Authors
  1. Tatjana Vujović
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  2. Đurđina Ružić
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  3. Radosav Cerović
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  4. Gordana Šurlan Momirović
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Correspondence to Tatjana Vujović.

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Vujović, T., Ružić, Đ., Cerović, R. et al. Adventitious regeneration in blackberry (Rubus fruticosus L.) and assessment of genetic stability in regenerants. Plant Growth Regul 61, 265–275 (2010). https://doi.org/10.1007/s10725-010-9474-9

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  • DOI: https://doi.org/10.1007/s10725-010-9474-9

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