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Optimisation of Adventitious Shoot Regeneration and Agrobacterium-mediated Transformation in Canna × generalis (Canna Lily)

Singh, Rani, Dubey, Arvind Kumar, Sanyal, Indraneel
Horticultural plant journal 2019 v.5 no.1 pp. 39-46
Agrobacterium, Canna, Southern blotting, ascorbic acid, benzyladenine, cefotaxime, cultivars, cutin, cysteine, dithiothreitol, excision, explants, fibrous roots, food industry, genotype, greenhouses, horticultural crops, indole acetic acid, indole butyric acid, kanamycin, kinetin, market value, ornamental plants, perianth, plantlets, polymerase chain reaction, protocols, rhizomes, shoots, thidiazuron, tissue culture, transgenes, vase life
Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine (6-BA), thidiazuron (TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata’ was the best responding cultivar. And 2 mg · L−1 6-BA or 1.5 mg · L−1 TDZ along with 0.1 mg · L−1 IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata’ (36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L−1 ascorbic acid (AsA), while, 100 mg · L−1 of l-cysteine or 100 mg · L−1 dithiothreitol (DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L−1 indole-3-butyric acid (IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100% survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in Agrobacterium suspension. The explants were washed with 500 mg · L−1 cefotaxime solution, subjected to 100 mg · L−1 kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis.