Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

Abstract

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l^-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l^-1), indole-3-acetic acid (IAA) (0.01 mg l^-1) plus 6-BAP (1.0 mg l^-1), and IAA (0.5 mg l^-1) plus (0.5 mg l^-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l^-1) plus 6-BAP (0.5 mg l^-1). MS medium containing indole-3-butylric acid IBA (1 mgl^-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 Rif^R (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l^-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes.

Keywords: Chicory, regeneration, transformation, 1-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), 6-Benzylaminopurine (6-BAP)