Abstract
An efficient transformation protocol was developed for vanilla (Vanilla planifolia) using protocorm-like bodies (PLBs) derived from shoot tips as explants. Of the ten media tested, Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron (TDZ) produced maximum PLBs per shoot tip. Genetic fidelity of PLB-derived plantlets was confirmed by random amplified polymorphic DNA (RAPD) using 23 random primers. PLBs were co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pBI121 containing the β-glucuronidase (gusA) and neomycin phosphotransferase II (npt II) genes for 3 days in MS medium supplemented with acetosyringone and transferred to selective regeneration medium containing 4.43 μM benzyladenine (BA), 2.68 μM naphthalene acetic acid (NAA) supplemented with 50 mg l−1kanamycin and 250 mg l−1 cefotaxime. After 15 days of culture, the surviving explants were transferred to the same regeneration medium but with a higher concentration of kanamycin (75 mg l−1). Finally, explants surviving after 30 days were subjected to more stringent selection in the regeneration medium supplemented with 100 mg l−1 kanamycin. Strong β glucuronidase activity was detected in the transformed plantlets by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and Southern hybridization, while expression of transgene was confirmed by northern hybridization. This protocol allows effective and high frequency transformation of vanilla.
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Abbreviations
- BA:
-
6-Benzyladenine
- GUS:
-
β-Glucuronidase
- NAA:
-
α-Naphthalene acetic acid
- MS:
-
Murashige and Skoog
- npt II:
-
Neomycin phosphotransferase II
- PLB:
-
Protocorm-like body
- PCR:
-
Polymerase chain reaction
- RAPD:
-
Random amplified polymorphic DNA
- TDZ:
-
Thidiazuron
- X-glcA:
-
5-bromo-4-chloro-3-indolyl-β-d-glucuronide
- YEP:
-
Yeast extract peptone medium
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Acknowledgment
The authors are grateful to the Director and Head, Division of Crop Protection, IISR, Calicut for providing facilities. This piece of work forms part of the PhD research work of R.S.T.
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Retheesh, S.T., Bhat, A.I. Genetic transformation and regeneration of transgenic plants from protocorm-like bodies of vanilla (Vanilla planifolia Andrews) using Agrobacterium tumefaciens . J. Plant Biochem. Biotechnol. 20, 262–269 (2011). https://doi.org/10.1007/s13562-011-0057-2
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DOI: https://doi.org/10.1007/s13562-011-0057-2