Single step direct transgenic plant regeneration from adventive embryos of agro-infected sugarcane ( Saccharum spp.) spindle leaf roll segments with assured genetic fidelity

Single step direct transgenic plant regeneration from agro-infected sugarcane (Saccharum spp.) spindle leaf roll segments with assured genetic fidelity is reported. The pre-cultured (96 h) leaf segments of cultivar CoJ 83 were incubated (10 min) with intermittent shaking in Agrobacterium suspension (OD 1.0–1.2) carrying antifungal gene encoding β-1,3-glucanase under the control of CaMV 35S promoter and NOS terminator, in presence of acetosyringone. Thereafter, the segments were co-cultivated (72 h) and transferred for shoot regeneration. The scanning electron micrographs of sectioned leaf segments undergoing in vitro shoot regeneration revealed occurrence of globular structures in clusters near cut leaf edges within 10 days of incubation. In each cluster, two to ten structures of varying sizes occurred concurrently. The sectioning of globular structures confirmed that these were arising directly from pre-embryogenic determined palisade cells, had unicellular origin and were referred to as adventive embryos. The adventive embryos successively expressed their potential for direct shoot regeneration without callus interphase in 3 weeks and transgenic plant formation within a short period of 8 weeks. The integration of β-1,3-glucanase transgene and its expression in T0 plants were determined by reverse transcription-PCR and quantitative real time-PCR, respectively. Simple sequence repeat DNA markers did not detect any polymorphism in the transgenic plants, pointing towards assured genetic fidelity of the plants.