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.

Abbreviation

ROS, Reactive Oxgyen Species; RO2, Peroxyl radical;  OH, Hydroxyl radical;  H₂O₂, Hydrgen peroxide; O-2, Super oxide radical; DNA, Deoxyribo Nucleic Acid; PCR, Polymerase chain reaction; GSH,Glutathione; TOC, Tocopherol; AA, Ascorbic acid; SN, Silver nitrate;  ECS, Embryogenic cell suspension; CTAB,Centyltrimethyl ammonium bromide; Mg, Milligram; SH, Schenk and Hildebrandt; RD1, Embryo development media; mL, Millilitre; GUS, ß-glucuronidase; G,Gram; ANOVA, Analysis of Variance; NAA, Napthalene acetic acid; IAA, Indole acetic acid.