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Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f × Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability

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Abstract

The effect of thidiazuron (TDZ) on direct multiple shoot induction in axenic seedlings of a monopodial orchid hybrid Aerides vandarum × Vanda stangeana, using a dual phase culture medium was studied. The culture system consisted of a basal agar solidified half-strength Murashige and Skoog medium overlaid by a liquid fraction of the same composition. Highest regeneration of multiple shoots (15.8 shoots per seedling) was obtained in the medium containing 2% sucrose (w/v) supplemented with 2 mgl−1 TDZ. Genetic stability of the regenerated shoots was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and restriction fragment length polymorphism of the PCR amplified (PCR-RFLP) nrITS region, as well as those of the coding (matK) and non-coding (trnL-F) regions of the cpDNA. Across the randomly selected mother plant and nine of its regenerated shoots, 2,680 bands were generated by 19 RAPD and 12 ISSR primers, exhibiting monomorphic banding profiles. Homogenous PCR-RFLP profiles were generated for nrITS using four restriction enzymes (REs), matK using five REs, and trnL-F using six REs. These molecular analyses showed no genomic alterations in all regenerated shoots obtained on medium containing 2 mgl−1 TDZ.

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Abbreviations

Av:

Aerides vandarum

BA:

N6-benzyladenine

cpDNA:

Chloroplast DNA

ISSR:

Inter simple sequence repeat

NAA:

α-Naphthalene acetic acid

MS:

Murashige and Skoog medium

nrITS:

Nuclear ribosomal internal transcribed spacer

PCR:

Polymerase chain reaction

PGRs:

Plant growth regulators

RAPD:

Random amplified polymorphic DNA

REs:

Restriction enzymes

RFLP:

Restriction fragment length polymorphism

TDZ:

Thidiazuron

Vs:

Vanda stangeana

References

  • Alvarez I, Wendel JF (2003) Ribosomal ITS sequences and plant phylogenetic inference. Mol Phylogenet Evol 29:417–434. doi:10.1016/S1055-7903(03)00208-2

    Article  PubMed  CAS  Google Scholar 

  • Arditti J (1977) Clonal propagation of orchids—by means of leaf cultures in vitro: a short review. Orchid Rev 85:102–103

    Google Scholar 

  • Arditti J, Ernst R (1993) Phalaenopsis. Micropropagation of orchids. Wiley, New York

    Google Scholar 

  • Brar DS, Jain SM (1998) Somaclonal variation: mechanisms and applications in crop improvement. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Kluwer, Boston, pp 17–37

    Google Scholar 

  • Breiman A, Felsenburg T, Galun E (1989) Is Nor region variability in wheat invariably caused by tissue culture? Theor Appl Genet 77:809–814. doi:10.1007/BF00268331

    Article  Google Scholar 

  • Campbell CS, Wojciechowski MF, Baldwin BG, Alice LA, Donoghue MJ (1997) Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae). Mol Biol Evol 14:81–90

    PubMed  CAS  Google Scholar 

  • Carlsward BS, Whitten WM, Williams NH, Bytebier B (2006) Molecular phylogenetics of Vandae (Orchidaceae) and the evolution of leaflessness. Am J Bot 93(5):770–786. doi:10.3732/ajb.93.5.770

    Article  CAS  Google Scholar 

  • Carvalho LC, Goulao L, Oliveira C, Goncalves JC, Amancio S (2004) RAPD assessment for identification of clonal identity and genetic stability of in vitro propagated chestnut hybrids. Plant Cell Tissue Organ Cult 77:23–27. doi:10.1023/B:TICU.0000016482.54896.54

    Article  CAS  Google Scholar 

  • Devarumath R, Nandy S, Rani V, Marimuthu S, Muraleedharan N, Raina S (2002) RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-India type). Plant Cell Rep 21:166–173. doi:10.1007/s00299-002-0496-2

    Article  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Dumolin S, Demesure B, Petit RJ (1995) Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theor Appl Genet 91:1253–1256. doi:10.1007/BF00220937

    Article  CAS  Google Scholar 

  • Gernandt DS, Liston A (1999) Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae). Am J Bot 86:711–723. doi:10.2307/2656581

    Article  PubMed  CAS  Google Scholar 

  • Goto S, Thakur RC, Ishii K (1998) Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers. Plant Cell Rep 18:193–197. doi:10.1007/s002990050555

    Article  CAS  Google Scholar 

  • Horn I, Walker S, Roberts AV, Mottley J, Simpkins I (1995) Micropropagation of roses: the benefits of pruned mother-plants at stage II and a greenhouse environment at stage III. J Hortic Sci 70(5):799–806

    Google Scholar 

  • Isabel N, Tremblay L, Michaud M, Tremblay FM, Bousquet J (1993) RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis derived populations of Picea mariana (Mill.) B.S.P. Theor Appl Genet 86:81–87. doi:10.1007/BF00223811

    Article  CAS  Google Scholar 

  • Jobst J, King K, Hemleben V (1998) Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae. Mol Phylogenet Evol 9:204–219. doi:10.1006/mpev.1997.0465

    Article  PubMed  CAS  Google Scholar 

  • Jones PAM, Yi Z, Murch SJ, Sazena PK (2007) Thidiazuron-induced regeneration of Echinacea purpurea L.: micropropagation in solid and liquid culture system. Plant Cell Rep 26:13–19. doi:10.1007/s00299-006-0209-3

    Article  PubMed  CAS  Google Scholar 

  • Joshi P, Dhawan V (2007) Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biol Plant 51(1):22–26. doi:10.1007/s10535-007-0005-0

    Article  CAS  Google Scholar 

  • Kishor R, Sharma GJ (2008) Multiple shoot induction in Ascocenda Kangla—a monopodial hybrid orchid. Lindleyana 21(1):6–9

    Google Scholar 

  • Kishor R, Devi HS, Jeyaram K, Singh MRK (2008) Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage. Plant Biotechnol Rep 2(2):145–152. doi:10.1007/s11816-008-0053-8

    Article  Google Scholar 

  • Larkin PJ, Srowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214. doi:10.1007/BF02342540

    Article  Google Scholar 

  • Malik KA, Saxena PK (1992) Regeneration in Phaseolus vulgaris L.: high-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and Thidiazuron. Planta 186:384–389. doi:10.1007/BF00195319

    Article  CAS  Google Scholar 

  • Martins M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496. doi:10.1007/s00299-004-0870-3

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  • Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol 34:267–275

    Article  CAS  Google Scholar 

  • Nayak NR, Rath SP, Patnaik S (1998) In vitro propagation of three epiphytic orchids, Cymbidium aloifolium (L.) Sw., Dendrobium aphyllum (Roxb.) Fisch. and Dendrobium moschatum (Buch-Ham) Sw through thidiazuron-induced high frequency shoot proliferation. Sci Hortic (Amsterdam) 71:243–250. doi:10.1016/S0304-4238(97)00075-7

    Article  Google Scholar 

  • Park SY, Murthy HN, Paek KY (2003) Protocorm-like body induction and subsequent plant regeneration from root tip cultures of Doritaenopsis. Plant Sci 164:919–923. doi:10.1016/S0168-9452(03)00019-0

    Article  CAS  Google Scholar 

  • Polonca K, Suzana S, Zalata L (2004) Direct shoot regeneration from nodes of Phalaenopsis orchids. Acta Agric Slov 83(2):233–242

    Google Scholar 

  • Pullman GS, Skryabina A (2007) Liquid medium and liquid overlays improve embryogenic tissue initiation in conifers. Plant Cell Rep 26:873–887. doi:10.1007/s00299-006-0296-1

    Article  PubMed  CAS  Google Scholar 

  • Rani V, Singh KP, Shiran B, Nandy S, Goel S, Devarumath RM, Sreenath HL, Raina SN (2000) Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae). Plant Cell Rep 19(10):1013–1020. doi:10.1007/s002990000228

    Article  CAS  Google Scholar 

  • Sala F, Arencibia A, Castiglione S, Christou P, Zheng Y, Han Y (1999) Molecular and field analysis of somaclonal variation in transgenic plants. In: Altman A, Ziv M, Izhar S (eds) Plant biotechnology and in vitro biology in the 21st century. Kluwer, Dordrecht, pp 259–262

    Google Scholar 

  • Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am J Bot 84(9):1120–1136. doi:10.2307/2446155

    Article  CAS  Google Scholar 

  • Sim GE, Loh CS, Goh CJ (2007) High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae). Plant Cell Rep 26:383–393. doi:10.1007/s00299-006-0242-2

    Article  PubMed  CAS  Google Scholar 

  • Sujatha G, Kumari RBD (2007) High-frequency shoot multiplication in Artemisia vulgaris L. using Thidiazuron. Plant Biotechnol Rep 1:149–154. doi:10.1007/s11816-007-0028-1

    Article  Google Scholar 

  • Sun Y, Skinner DZ, Liang GH, Hubert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theor Appl Genet 89:26–32. doi:10.1007/BF00226978

    Article  CAS  Google Scholar 

  • Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109. doi:10.1007/BF00037152

    Article  PubMed  CAS  Google Scholar 

  • Thomas TD, Michael A (2007) High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid. Plant Biotechnol Rep 1:243–249. doi:10.1007/s11816-007-0038-z

    Article  Google Scholar 

  • Thomson ID, Edwards JT, Staden JV (2007) A novel dual phase medium promotes germination and seedling establishment from immature embryos in South African Disa (Orchidaceae) species. Plant Growth Regul 53:163–171. doi:10.1007/s10725-007-9215-x

    Article  CAS  Google Scholar 

  • Van Winkle SC, Pullman GS (2005) Achieving desired plant growth regulator levels in liquid plant tissue culture media that include activated carbon. Plant Cell Rep 24:201–208. doi:10.1007/s00299-005-0931-2

    Article  PubMed  CAS  Google Scholar 

  • Venkatachalam L, Sreedhar RV, Bhagyalakshmi N (2007) Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers. Plant Growth Regul 51:193–205. doi:10.1007/s10725-006-9154-y

    Article  CAS  Google Scholar 

  • Vinocur B, Carmi T, Altman T, Ziv M (2000) Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media. Plant Cell Rep 19:1146–1154. doi:10.1007/s002990000243

    Article  CAS  Google Scholar 

  • Visure J (1987) Micropropagation of pear, Pyrus communis, in a double-phase culture medium. Acta Hortic 212:117–124

    Google Scholar 

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Acknowledgments

This research was supported by the Department of Biotechnology, Government of India, in the form of a Post-doctoral fellowship to Rajkumar Kishor. The authors wish to thank Director, IBSD, Imphal, India for extending the research facilities and critically reading the manuscript.

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  1. Medicinal Plants and Horticultural Resources Division, Institute of Bioresources and Sustainable Development, Takyelpat Institutional Area, Imphal, 795001, Manipur, India

    Rajkumar Kishor & H. Sunitibala Devi

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  1. Rajkumar Kishor
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  2. H. Sunitibala Devi
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Correspondence to Rajkumar Kishor.

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Kishor, R., Devi, H.S. Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f × Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability. Plant Cell Tiss Organ Cult 97, 121–129 (2009). https://doi.org/10.1007/s11240-009-9506-1

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