Molecular breeding in fig ( Ficus carica ) by the use of genetic transformation.

摘要

An efficient and reproducible system for regeneration and agrobacterium-mediated transformation of the common fig (Ficus carica) cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption) was developed. Optimal shoot regeneration (up to 100%) was obtained on MS basal salt mixture supplemented with 100 mg L-1 myo-inositol, 1 mg L-1 thiamine HCl and addition of 2.0 mg L-1 Thidiazuron (TDZ), 2 mg L-1 Indole-3-butyric acid (IBA), 4% sucrose and 0.8% agar. Regeneration was highly dependent on the dorsoventral orientation of the explants: When explants were cultured with the adaxial surface up, 100% regeneration was achieved with more than 5 shoots per regenerating explant in both studied cultivars. Leaf explants of in vitro propagated plants were co-cultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504 that carried the uidA-intron, bar and nptII genes. Transformation efficiencies were in a range of 1.7-10.0% for cv. Brown Turkey and 2.8-7.8% for cv. Smyrna. The transgenic nature of the regenerated plants was confirmed by molecular analyses (PCR and Southern blot), as well as by GUS staining and Basta resistance. Similar to regeneration, the orientation of the leaf surface during organogenesis was a key factor for successful transformation. To introduce health-beneficial compounds into the Brown Turkey cultivar it was transformed with grapevine (Vitis vinifera L.) cDNA encoding stilbene synthase, transcriptionally regulated by an enhanced cauliflower mosaic virus (CaMV) 35S promoter. The gene encoding stilbene synthase is responsible for the synthesis of the compound resveratrol. Resveratrol considered to have beneficial effects on health, including anti-ageing, anti-inflammatory, anti-platelet and anti-carcinogenic activities. Three transgenic cv. Brown Turkey plants were obtained and resveratrol was identified in these three-month-old rooted transgenic plants. Successful transformation of commercial fig cultivars provides a new promising tool for the introduction of desired genes into transgenic fig cultivars. The regeneration and transformation methodologies described here may pave the way for transgenic varieties with improved agronomic characteristics, such as storability and disease resistance, and will provide a means for the production of desired proteins in the edible parts of fig, leading to improved nutritional and/or pharmaceutical composition.