Abstract
This work was carried out to optimize the conditions for highly effective embryogenic callus induction from mature seeds, plantlet regeneration and genetic transformation of Citrus sinensis L. by Agrobacterium tumefaciens strain EHA105 (pCAMBIA 1305.1). Embryogenic calli could be successfully induced from mature seeds employing the MT medium supplemented with 500 mg/l malt extract. The percentage of embryogenic callus induction was 85. With the same medium, the high proliferation rate of embryogenic callus was achieved. The liquid MT medium containing 500 mg/l malt extract in combination with 50 mg/l lactose could be used as the embryoid development medium. Somatic embryos, however, could be regenerated with normal shoots and roots in the MS medium, with the regeneration percentage of 60. The delivery of an antisense ACC oxidase gene into the species C. sinensis mediated by Agrobacterium tumefaciens strain EHA 105 was successful by co-cultivating explants with the strain EHA 105 for 10 min, following that by eliminating the bacterium with 200 mg/l cefotaxime, and subsequently selecting transformed embryoid with 20 mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into plant genome.
Highlights
Citrus is the most widely grown fruit crop throughout the world [1]
After being rinsed 3 times with sterile distilled water, seeds were cultured on Murashige and Tucker (MT) medium [17] supplemented with varied concentrations of malt extract (0, 200, 300, 400 and 500 mg/l), 50 g/l sucrose and 8 g/l agar, pH 5.8, kept at 25 ̊C ± 2 ̊C in dark condition for 8 weeks to determine the optimal concentration of malt extract for embryogenic callus induction
The development of seeds into friable calli was distinct 4 weeks after being cultured on the MT medium supplemented with malt extract
Summary
Citrus is the most widely grown fruit crop throughout the world [1]. It is the number one fruit of the world on account of its high nutritional value. Harvested fruits which are usually stored before they reach the market for fresh consumption [3] are subjected to biotic and abiotic stress conditions, especially stress from excess ethylene production during the degreening process of citrus fruits [4,5,6,7,8]. Ethylene treatment during the degreening process of citrus fruits is involved in ethylene production as well as enhances chlorophyllase activity [11,12,13] and the synthesis of carotenoids [13].
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