Browsing by Author "Mbambalala, Nelisa"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemAnalysis of the role of transcription factors in enhancing drought tolerance in sugarcane (Saccharum spp.)(Stellenbosch : Stellenbosch University, 2020-12) Mbambalala, Nelisa; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: Sugarcane is a large perennial grass of the genus Saccharum. Economically, this grass species is an important source of sugar for food purposes and biomass for biofuel production. However, the sustainability of production is greatly constrained by drought, which directly affects crop yield. Drought can lead to modification of metabolic processes in the plant, membrane disorder, disruptions and instabilities of many physiological and biochemical process, including photosynthesis and increased production of reactive oxygen species (ROS), which causes oxidative stress and can ultimately lead to plant death. It is therefore essential to continue developing cultivars with improved drought tolerance, which can possibly be achieved through the identification and introduction of genes that confer tolerance in crops. Transcription factors (TF) are gene regulators that control gene expression and consequently stress responses in plants. A single TF can regulate the expression of many target genes. This study aims at analysing the role of two TFs namely, BBX (B-Box Zinc Finger) and NAC2 (NAM, ATAF and CUC) in enhancing drought tolerance in sugarcane. For this, sugarcane was independently genetically transformed via particle bombardment with a BBX TF from Arabidopsis thaliana and a NAC2 TF from tomato. Attempts were also made to determine the subcellular localization of the AtBBX29 and SINAC2 genes but results were inconclusive due to poor microscopic imaging and faint GFP reporter signals. Transgene insertion was confirmed in putative transformed sugarcane through PCR analysis and transgene expression through semi-quantitative reverse transcriptase PCR. Transgenic sugarcane plantlets were planted ex vitro and drought pot trials were setup in the glasshouse. Once plants were deprived of water, phenotypic changes in transgenic sugarcane lines were compared to non-transgenic control sugarcane plants. Under drought conditions, both AtBBX29 and SINAC2 overexpression in sugarcane enhanced drought tolerance. All transgenic plants exhibited higher survival and recovery rates than wild-type (WT) plants. Transgenic plants overexpressing AtBBX29 maintained relative water content (RWC) at levels not significant different from the WT plants. However, these plants maintained significantly higher chlorophyll fluorescent rates and stomatal conductance under mild and severe drought conditions. Under severe water-deficit stress, oxidative damage was reduced in BBX transgenic plants which exhibited low malondialdehyde (MDA) levels and less accumulation of reactive oxygen species (ROS) throughout the water-deficit stress period. The scavenging activity of antioxidants, which was present at significantly higher levels in the transgenic plants under severe water-deficit stress, most likely played a role in reducing the ROS levels. Transgenic plants also accumulated significantly more proline under mild and severe stress conditions compared to the WT plants. Abscisic acid levels varied between WT and transgenic plants exposed to drought. In addition, BBX transgenic sugarcane was grown to maturity (8 months) under a normal watering regime in the glasshouse where these plants displayed normal phenotypes and no significant difference in carbohydrate content compared to non-transgenic control plants. A preliminary drought trial was conducted with the SINAC2 transgenic sugarcane plants. Overexpression of SINAC2 enhanced drought tolerance in transgenic sugarcane plants exposed to water-deficit stress with higher survival rates seen in the transgenic lines compared to the WT plants. Transgenic plants overexpressing SINAC2 maintained significantly higher RWC levels and displayed less visual damaged such as leaf wilting and yellowing than the WT plants. Over the course of water-deficit period the root biomass increased in all genotypes, but less so in the transgenic plants, SINAC2 overexpression however enhanced root elongation. Transgenic plants also upheld photosynthesis, with high chlorophyll fluorescence and stomatal conductance seen in most transgenic plants under severe water-deficit stress.