An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

AbstractBackgroundFunctional foods can help prevent metabolic diseases, and it is essential to evaluate functional characteristics of foods through in vitro and in vivo experimental approaches.ObjectiveWe aimed to use the bicameral cell culture system combined with the in vitro digestion to evaluate glucose bioavailability.Materials and methodsCake, almond paste, and pudding were modified by adding fiber and replacing sugar with sweeteners and polyols. Digestion process was modeled in test tubes. Rat enterocyte cells (IEC-6) were grown in a bicameral cell culture system to mimic the physiological characteristics of the human intestine. The glucose bioaccessibility and cellular glucose efflux were measured by glucose oxidase assay.Results and discussionThe glucose bioaccessibilities of modified foods were significantly lower (cake: 2.6 fold, almond paste: 9.2 fold, pudding 2.8 fold) than the controls. Cellular glucose effluxes also decreased in the modified cake, almond paste, and pudding by 2.2, 4, and 2 fold respectively compared to their controls.ConclusionOur results suggest that combining in vitro enzymatic digestion with cell culture studies can be a practical way to test in vitro glucose bioaccessibility and bioavailability in functional food development.

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Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant

Plants ◽

10.3390/plants10040664 ◽

2021 ◽

Vol 10 (4) ◽

pp. 664

Author(s):

M. Moniruzzaman ◽

Yun Zhong ◽

Zhifeng Huang ◽

Huaxue Yan ◽

Lv Yuanda ◽

...

Keyword(s):

Transgenic Plants ◽

Suspension Culture ◽

Cell Suspension ◽

Cell Suspension Culture ◽

Culture System ◽

Transformation Rate ◽

Suspension Cell Culture ◽

Citrus Reticulata ◽

Agrobacterium Mediated Transformation ◽

Solid Media

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.

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