Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths

• Published in: Plant cell reports Vol. 39; no. 9; pp. 1143 - 1160
• Main Authors: Castellanos-Arévalo, Andrea P., Estrada-Luna, Andrés A., Cabrera-Ponce, José L., Valencia-Lozano, Eliana, Herrera-Ubaldo, Humberto, de Folter, Stefan, Blanco-Labra, Alejandro, Délano-Frier, John P.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2020; Springer Nature B.V
• Subjects: Acidification; Agrobacterium - genetics; Amaranth; Amaranthus - genetics; Amaranthus - physiology; Amaranthus hypochondriacus; Biomedical and Life Sciences; Biotechnology; Calcium chloride; Cell Biology; Cotyledon - genetics; Culture Media - chemistry; Cytokinins; Embryos; Explants; Fluorescence; Gene Expression Regulation, Plant; Genetic Markers; Genetic transformation; Germination; Glucuronidase - genetics; Green fluorescent protein; Green Fluorescent Proteins - genetics; Hairy root; Life Sciences; Original Article; Osmotic stress; Phosphoric acid; Plant Biochemistry; Plant Roots - chemistry; Plant Roots - cytology; Plant Roots - growth & development; Plant Roots - microbiology; Plant Sciences; Plant Somatic Embryogenesis Techniques - methods; Plantlets; Plants, Genetically Modified; Polymerase Chain Reaction; Regeneration; Roots; Somatic embryos; Species; Sucrose; Sugar; Transformation, Genetic; Transformations; Transgenic plants; Red light; Grain amaranths; Ethanol-inducible expression; Calcium; Plant regeneration; Hairy roots; Amaranthus hybridus; Agrobacterium rhizogenes
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-020-02553-9

Key message Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus , their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus . Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes- mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD 600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus , respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or β-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5 g/L gelrite, and 0.2 mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100 mg/L CaCl 2 . These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.