Acta Physiologiae Plantarum

, Volume 34, Issue 3, pp 1207–1215

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

Authors

    • Department of BiotechnologySchool of Life Sciences, Mizoram University
  • Lingaraj Sahoo
    • Department of BiotechnologyIndian Institute of Technology Guwahati
Original Paper

DOI: 10.1007/s11738-011-0917-3

Cite this article as:
Thangjam, R. & Sahoo, L. Acta Physiol Plant (2012) 34: 1207. doi:10.1007/s11738-011-0917-3

Abstract

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS+ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR+ plants.

Keywords

Parkia timoriana Cotyledonary node In vitro regeneration Agrobacterium-mediated genetic transformation

Abbreviations

MS

Murashige and Skoog (1962)

B-5

Gamborg et al. (1968)

NAA

α-Naphthalene acetic acid

BA

6-Benzylaminopurine

IBA

Indole-3-butyric acid

GUS

β-Glucuronidase

CaMV

35S 35S promoter of the cauliflower mosaic virus

nptII

Neomycin phosphotransferase

PCR

Polymerase chain reaction

OD600

Optical density at 600 nm

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2011