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

Abstract

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.