Agrobacterium-mediated transformation of avocado (Persea americana Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

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• 作者：Elena Palomo-Ríos ; Sergio Cerezo…
• 关键词：Avocado ; Fluorescent markers ; Plant regeneration ; Transgenic plants
• 刊名：Plant Cell, Tissue and Organ Culture (PCTOC)
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：128
• 期：2
• 页码：447-455
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Plant Sciences; Plant Physiology; Plant Genetics & Genomics; Plant Pathology;
• 出版者：Springer Netherlands
• ISSN：1573-5044
• 卷排序：128

文摘

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l−1 TDZ and 1 mg l−1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l−1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l−1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.