Biolistic-mediated transformation of Eragrostis Curvula with the HSP 12 gene

• Main Author: Ncanana, Sandile Welcome
• Other Authors: Farrant, Jill M
• Format: Others
• Language: English
• Published: University of Cape Town 2014
• Subjects: Bioprocess Engineering
• Online Access: http://hdl.handle.net/11427/6771

Bibliography: leaves 91-107. === Eragrostis curvula is a desiccation sensitive monocotyledonous plant and an economically important forage grass in southern Africa. This species has a potential to be improved for drought and salt resistance among other important agronomic traits. In this study, E. curvula was used as a model system to explore the feasibility of producing pasture grasses with increased tolerance to water deficit. To date, no reports have been published on transformation of this species. This study reports the transformation of E. curvula with Saccharomyces cerevisiae Hsp 12 gene using biolistic-mediated transformation. Firstly, a tissue culture protocol was established for E. curvula that was suitable for transformation studies. Although this species has been previously regenerated in vitro using inflorescence tissues, this study established new protocols that utilize leaf and seed as source of material. The aim of which was to find the best regenerable tissue that could be used for transformation studies. Plant regeneration was achieved from shoot explants cultured on MS medium supplemented with either 0.5 mg/I thidiazuron (TDZ) or 0.5 mg/I 6-benzylaminopurine (BAP) through the process of direct organogenesis. It was found that TDZ was the most effective cytokinin. Plant regeneration was also achieved from callus induced from immature leaves on MS medium supplemented with 2 mg/I 2,4- 0,0.01 mg/I BAP and 6 % (w/v) sucrose. Histological experiments performed gave clear evidence that plant regeneration from callus was through a process of indirect organogenesis. The regeneration protocol was combined with an optimized biolistic mediated transformation protocol using the PDS-I00/He apparatus of which both shoot explants and callus were used as target tissues. In the molecular aspect of the study, the Hsp 12 gene was ligated in the Sac I restriction site of pCAMBIAUbeeQ vector. The successful cloning of the Hsp 12 gene was confirmed by PCR and restriction endonuclease digestion. The resultant vector pCAMBIAUbeeQ Hsp 12 was purified and subsequently used for biolistic transformation of E. curvula. The regenerable shoot explants and callus tissue were bombarded with DNA (pCAMBIAUBeeQHsp12) coated on gold particles. As a comparative study, Agrobacterium tumefaciens was transformed with pCAMBIAUBeeQHsp12 vector for subsequent transformation of Nicotiana tabacum. Transient expression of GUS gene in transformed E. curvula shoot explants was visualized 72 h after bombardment. Optimized conditions for expression of GUS gene were gold micro projectiles, 7 cm travel distance and helium pressure of 9100 kPa. Transformed tissues were cultured on the regeneration medium without antibiotic selection. Putative transformants were generated and the presence of Hsp 12 gene was verified by PCR and its expression at RNA level was confirmed by RT-PCR. The presence of Hsp 12 protein in the transformed plants was analyzed using SDS-PAGE and MALDI-TOF peptide mass spectrometry.