Nabila Mohamad Ali Bacha
Bio-Technology Division, General Commission for Scientific Agricultural Research (GCSAR)
Faculty of Agriculture, Damascus University
Abstract
The aim of the present study was to develop an efficient direct shoot regeneration approach from leaf explants for apple cvs. ‘Golden Delicious’, ‘Royal Gala’ and ‘MM111’, ‘M26’ rootstocks as a method for rapid clonal multiplication and also as a prerequisite for genetic transformation with antifungal genes of the studied apples to reach the final desired aim of establishing an efficient and practical reproducible approach of Agrobacterium-mediated transformation which harbour g2ps1 gene for apple cultivars and rootstocks studies for improving their fungal resistance.
Adventitious shoot formation from leaf pieces of apples studied was achieved using middle leaf segments taken from the youngest leaves from in vitro-grown plants. Optimum conditions for ‘direct’ shoot organogenesis resulted in high regeneration efficiency of 90%,95%,92%,94% producing one or more shoot per explant with high regeneration rates of 4.5, 5.6, 4.0 and, 4.1 new shoots in the studied apples respectively on MS basal medium with B5 vitamins, 1.0 g/l MES, 2.0 mg/l TDZ with 0.2 mg/l NAA. While, no regeneration could be observed on media free of cytokinins.
The organogenic capacity of leaf pieces was related to the leaf maturity and the origin of the leaf piece with the youngest light green expanding leaves being more regenerative than the older ones. Middle leaf segments were more responsive than the upper or lower part of the leaf. Adventitious shoots were regenerated from cut sides and leaf surfaces. Therefore, middle leaf parts were used afterwards for organogenesis using leaf tissues of the studies apples. The most efficient regeneration media consisted of: MS+ B5 vit. + 1.0 g/l MES+ 30 g/l sucrose+ 1 mg/l BAP+ 0.3 mg/l IBA+ 0.2 mg/l GA3 + 6 g/l agar
Subcultures were carried our every 4 weeks to provide sufficient amount of leaves needed for transformation.
Genetic transformation conditions of the studied apples were then optimized using g2ps1 gene from Gerbera hybrida coding for 2-pyrone synthase which contribute for fungal and insect resistance was used.
Putative transgenic shoots could be obtained on MS media with B5 Vitamins, 5.0 mg l-1 BAP, or 2.0 mg l-1 TDZ with 0.2 mg l-1 NAA in the presence of the selection agent “PPT” at 3.0 mgl-1.
Shoot multiplication of transgenic shoots was achieved on: MS + B5 vitamins + 1.0 mg l-1 BAP + 0.3 mg l-1 IBA, 0.2 mg l-1 GA3+1.0 g/l MES+ 30 g/l sucrose + 7.0 g/l Agar, with the selection agent PPT at 3 mg l-1 and were sub-cultured every 4 weeks in order to get sufficient material to confirm transformation of the putative shoots obtained.
Transgenic clones of the apples studied respectively have been obtained and confirmed by selection on the media containing the selection agent “PPT” and by PCR analysis using the suitable primers in all clones obtained for the presence of the selection” bar gene (447 bp) and the gene-of- interest “g2PS1” (1244 bp), with transformation efficiency of 0.4%, 0.6%, 0.1% and 0.3% respectively.
Results of DNA sequence analysis of the transgenic plants also proved the successful transformation and had 97 to 99% sequence homology with the gerbera hybrida mRNA for 2-pyrone synthase g2ps1 gene (accession no. Z38097.2). These transgenic clones were multiplied further and rooted in vitro by transferring 2-3 cm long shoot tips to rooting ½ MS basal medium supplemented with 1.0 mg/l indole-3-butyric acid (IBA), in the presence of the selection agent ‘PPT’. Rooted transgenic plantlets were successfully acclimatized and are being kept under-containment conditions according to the biosafety by-law in Syria to evaluate their performance for fungal resistance.