Abstract:
The battle of wheat against pests and pathogens can be strengthened by genetically engineering the wheat plant for disease tolerance, e.g. by enhancing the expression of chitinase and â-1,3-glucanase genes. In order to obtain this long-term goal, an efficient tissue culture system, as well .as an optimal transformation procedure, was produced. Fourteen spring and winter hard-red South African wheat (Triticum aestivum L.) cultivars were compared for their regeneration and transient anthocyanin expression efficiencies. Embryonic and non-embryonic callus, as well as plantlets were obtained from all the cultivars using a modified MS basal medium supplied with 5 mg/L BAP. The modified ML3 medium could not sustain callus or plantlet development from any of the cultivars. The cultivars exhibiting the most vigorous growth were 'Betta' Dn2 and 'Gamtoos' (spring wheat), and 'Palmiet' Lr29 and 'Tugela' (winter wheat). These cultivars are, therefore, the most suitable for tissue culture establishment. The particle bombardment system was efficiently optimised using the anthocyanin reporter gene cloned into the pHP 687 vector. It was found that the optimal distance between the micro-carrier assembly and target material should not exceed 13 cm. A helium pressure of 1 400 kPa produced the highest percentage of anthocyanin expression, the most foci per embryo/callus and the least amount of tissue damage. The age of the target material was found to be an important determining factor during bombardment and, thus, the age of target material should not exceed 3 weeks. The cultivars most suitable for transformation were 'Palmiet', 'Palmiet' Dn1, 'Palmiet' Dn2 and 'Palmiet' Dn5. Bombardment with small particles, namely 1 µm tungsten and 1 µm gold, resulted in efficient penetration of the target cells and relatively little tissue damage. This, in tum, enabled the bombarded tissue to express a high percentage of anthocyanin. If was further found that the cultivar bombarded is receptive towards the particles used. The material subjected to selection after bombardment should contain a large amount of cells transiently expressing anthocyanin. Plantlets could not be recovered from the material bombarded with anthocyanin since the applied selection procedure was too strict. Material bombarded with chitinase and J3-1,3¬glucanase are differentiating more efficiently and appears to survive the strict selection pressure.
