Agrobacterium Mediated Transformation Optimizations for Sugarcane (Saccharum Officinarum L.) Cultivar SPF-234 with Direct Organogenesis

Abstract

Sugarcane (Saccharum officinarum L.) is the most important food and energy crop worldwide. In the present study, an efficient Agrobacterium mediated transformation and regeneration system for sugarcane cultivar SPF-234 was established. Agrobacterium tumefaciens strains EHA101and LBA4404 using vector pIG121 Hm, having GUS, HPTII and NPTII genes were used. Polymerase chain reaction (PCR) and histochemical assays confirmed the GUS gene expression. A 620 bp fragment from GUS positive plants was amplified. The GUS expressing putative transformants were 35% of the total plants formed under 30 minute immersion time and 72 hr of incubation period. The co-cultivation media having 60 μM acetosyringone produced 66% GUS expressing plants for LBA4404 and 58% for EHA101. The maximum average number of directly produced shoot (59.5%) from leaf explant was in M6 media having 1.00 mg/l 6-Benzylaminopurine (BAP) and 2.5 mg/l Naphthaleneacetic acid (NAA). A significant decrease (17%) was observed when auxin (NAA) concentration was increased to 4.0 mg/l. The best response of shoot elongation was observed in SE4 media having equal concentration (2.00 mg/l) of both kinetin and BAP. Increased concentrations of kinetin significantly decreased shoot elongation of the subject cultivar. Agrobacterium strain LBA4404 performed better for genetic transformation of the said sugarcane cultivar.This quick and less expensive transformation and direct regeneration system could be exploited for sugarcane on commercial scale in general, and for this elite cultivar in particular.