ABSTRACT
Background: selectable marker gene [SMG] systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression
Objectives: this study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter
Materials and Methods: in pG-IPFFDD-cre[int]-gus[int] construct, cre recombinase and selectable marker gene [nptII] cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and beta-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation
Results: the results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay
Conclusions: the established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene
ABSTRACT
Sugarcane is a monocotyledonous crop that is cultivated in the tropical and subtropical regions of the world. One of the most important criteria, influencing the efficiency of the sugarcane transformation is known to be related to physical and biological factors during the transformation procedure. The objective of this research was to study the response of callus induction and embryogenic callus production and to identify the major parameters controlling DNA delivery by particle bombardment into sugarcane [Saccharum officinarum L.] cv. NCo310. For callus induction and embryogenic callus production, leaf base segments were subjected to in vitro culture medium supplemented with two plant growth regulators [2,4-D and Dicamba]. Results showed that 1 mg.L -1 2,4-D was significantly influential in callus induction and embryogenic callus production. Considering both physical and biological factors, the efficiency of DNA [uidA gene] delivery was assessed by scoring transient GUS [gene [beta-glucuronidase]] expression in bombarded tissues. The highest transient GUS expression was obtained when callus was bombarded with the construct harboring rice Act1 promoter, and having 9 cm target distance, 25 inHg vacuum pressure, 1 microm gold particles, 12.5 micro g DNA per bombardment and one day pre-culture prior to the bombardment. A bombardment procedure suitable for elite sugarcane varieties was developed, which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.