Abstract
Gene transfer into the plant cell is a key step towards its successful genetic modification and its efficiency is heavily dependent on plant and bacterial cell biological status and a wide array of physical conditions. Gene transfer efficiencies in East African Highland Banana (EAHB) cell lines remains low compared to other monocotyledonous crops like rice and wheat due to factors such as high oxidative stress. The use of antioxidants is fundamental in influencing gene transfer events during Agrobacterium-plant cell co-cultivation. Here we report significant enhancement of gene transfer efficiency in the EAHB cultivar ‘Nakinyika’ (EA-AAA) by supplementing co-cultivation medium with antioxidants; ascorbic acid (AA), glutathione (GSH), tocopherol (TOC) and silver nitrate (SN). The most enhancing antioxidant by number of blue foci after histochemical assay, as a parameter of gene transfer efficiency, was ascorbic acid (174 cells) at a concentration of 40 mg/L, followed by glutathione (91 cells) and tocopherol (91 cells) both at 50 mg/L. The least enhancement was observed when ascorbic acid (39 cells), silver nitrate (41 and 31 cells) were used at concentrations of 20, 4 and 6 mg/L compared to 72 cells in controls (no anti-oxidants). Regeneration efficiency increased from 29 cells in controls to 46% for SN at 8 mg/L; 43% for GSH at 100 mg/L; 30% for TOC at 75 mg/L; and 48% for AA at 20 mg/L. Polymerase chain reaction (PCR) results using gusA specific primers showed that these regenerants were putative transformants and grew normally during regeneration, rooting and multiplication. GSH and TOC significantly enhanced gene transfer efficiency while AA and SN showed significant increases in shoot regeneration compared to controls. The current results show that antioxidants significantly enhance gene transfer and regeneration efficiency in recalcitrant banana cell lines and could significantly enhance the overall transformation efficiencies in cases where numerous transgenic lines are required in a short time. Key words: Highland bananas, transgenic, browning, transformation efficiency, agrobacterium.
Highlights
Banana ranks among the 10 most important crops in the tropical and sub-tropical zones of the world (Ortiz and Swennen, 2014)
Different concentrations of ascorbic acid, glutathione, silver nitrate and tocopherol added to MA3 media resulted into variable numbers of GUS stained cells (Figure 3)
Transformation efficiency results indicate that, cultivar „Nakinyika‟ Embryogenic cell suspensions (ECS) were successfully transformed based on GUS expression assays
Summary
Banana ranks among the 10 most important crops in the tropical and sub-tropical zones of the world (Ortiz and Swennen, 2014). Enters the commercial market suggesting that the crop is more important as food for local consumption than for export (Brown et al, 2017). Genetic engineering provides a promising alternative strategy for the improvement of commercial banana varieties with resistance to biotic and abiotic stresses (Ortiz and Swennen, 2014). Agrobacterium tumefaciens mediated transformation is the outstanding method for plant genetic transformation due to several advantages in the T-DNA transfer process, including high efficiency, low-copy number inserts, large DNA segments, low rearrangement rate and low cost (Gelvin, 2012)
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