Regeneration and transformation systems for improving resistance to weevils in Ugandan sweetpotato cultivars.

Abstract

Productivity of sweetpotato [Ipomoea batatas (L.) Lam.] in Sub-Saharan Africa is significantly reduced due to damage caused by weevils, Cylas puncticollis Boheman and C. brunneus Fabricius. The improvement of weevil resistance in sweetpotato using classical breeding has been limited because no sources of resistance have been identified in the crop germplasm. Additionally, the application of genetic transformation has been hampered since different cultivars respond differently to both regeneration and transformation conditions. The main objective of the reported research was to optimise regeneration and transformation conditions for recalcitrant Uganda sweetpotato cultivars in order to pave the way for genetic transformation with weevil resistance genes. ‘Whole leaf’ explants of twenty Ugandan sweetpotato cultivars were screened for their embryogenic response using a common auxin 2,4-Dichlorophenoxyacetic acid (2,4-D) (0.2 mg/L) supplemented to Murashige and Skoog (MS) basal medium. Cultivar showed a highly significant (P < 0.001) effect on frequency of embryogenic callus and efficiency of both shoot and root regeneration. The best responding Ugandan cultivars Bwanjule, Kyebandula, Magabali, New Kawogo and Semanda were included in a follow-up study where three different auxins were investigated; 4-fluorophenoxyacetic acid (4FA) (1 mg/L), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (1.3 mg/L) and 2,4-D. This experiment was laid in a completely randomised design. The three factors namely auxin type, plant organ and cultivar had a significant (p ≤ 0.05) effect on the frequency of embryogenic callus. 2,4-D was found to be the best (p ≤ 0.05) auxin for induction of embryogenic callus while cultivar Bwanjule had the highest (20.2%) embryogenic callus frequency. Although some Ugandan cultivars demonstrated high ability to regenerate roots, shoot regeneration was restricted to non-African cultivars. Cultivars, Kyebandula and Bwanjule, were further investigated on medium supplemented with different levels of thidiazuron (0.5, 2.0 and 4.0 μM) and 0.25 μM α-Naphthalene acetic acid. The best response (P < 0.001) was from stem explants of cv. Kyebandula. The conversion of adventitious buds into shoots was improved significantly (P < 0.001) when thidiazuron (TDZ) was reduced or completely removed in subsequent stages of culture. Stem internode pieces excised from internode position 3 from the apex were the best (70.0 %) in adventitious bud formation. An efficient Agrobacterium-mediated transformation method was developed using readily accessible explants of cv. Kyebandula. Transformation efficiency was optimized by screening for transient β-glucuronidase (GUS) expression using histochemical localization of GUS activity after co-culture of primary roots, petioles, whole leaves and stem internode segments under various conditions. Concentration of Agrobacterium at an optical density (OD) of 1.0 at 600 nm gave significantly (p < 0.001) high transformation frequency for all the sweetpotato organs. The best (p < 0.001) transformation results were obtained when explants were co-cultured for 4 days. Kanamycin concentration of 100 mg/L significantly (p < 0.001) inhibited survival of non-transformed explants and callus. When coupled with the established thidiazuron-based regeneration protocol, the optimized transformation conditions led to regeneration of plants from explants co-cultured with Agrobacterium strain EHA 105. This Agrobacterium harbours the pCIP84 plasmid construct which contains cry7Aa1, cry3Ca1 and nptII genes in its transferred DNA (T-DNA). PCR for cry7Aa1 gene gave an early indication of transformation for 10 randomly selected plants among the regenerated 18 plants. Six of these plants showed that they were transformed with cry7Aa1 gene representing a 2.0 % transformation efficiency. Southern hybridization and Northern blot are the next steps to fully determine the transgenic nature of the plants. Taken together, the present data demonstrate that it is possible to genetically transform recalcitrant cultivars, including important African cultivars