Abstract
Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.
Highlights
The importance of Citrus spp. is linked to their enormous economic and nutritional values [1]
Solutions to the problems will rely mostly on breakthroughs in breeding, which is hindered by problems, such as sterility, self- and cross-incompatibility [6], widespread nucellar embryony, and long juvenile periods that are associated with traditional breeding practices [7]
We chose undeveloped ovules as callus induction material because previous studies showed that undeveloped ovule is a preferable material for somatic embryogenesis for having higher regeneration capacity and for being mostly virus-free [52]
Summary
The importance of Citrus spp. is linked to their enormous economic and nutritional values [1]. The classical conception of somatic embryogenesis (SE) is based on the unicellular origin of somatic embryos [30], and this mode of somatic embryo development was the most frequently noticed in embryogenic cell suspensions of D. carota [31] Both a multicellular and a unicellular origin of somatic embryos in the same regeneration system is quite a common phenomenon, as was observed in several species, including Musa spp. By following the general procedure (establishment and maintenance of the cell suspension, transformation of the cells, and subsequent plant organogenesis from putative transgenic cells), we successfully established a cell suspension culture and an associated Agrobacterium tumefaciens-mediated transformation system for the three Citrus cultivars was established. The developed methods should be useful for Citrus genetic improvements through genome engineering experiments
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