Creating a novel petal regeneration system for function identification of colour gene of grape hyacinth

• Published in: Plant methods Vol. 17; no. 1; pp. 1 - 94
• Main Authors: Lou, Qian, Liu, Hongli, Luo, Wen, Chen, Kaili, Liu, Yali
• Format: Journal Article
• Language: English
• Published: London BioMed Central Ltd 16.09.2021; BioMed Central; BMC
• Subjects: 2,4-D; Agrobacterium; Agrobacterium transformation; Anthocyanidin 3-O-glucosyltransferase; Anthocyanins; Benzyladenine; Color; Color of plants; Dichlorophenoxyacetic acid; Enzymes; Explants; Flowers; Flowers & plants; Fruits; Gene expression; Gene function analysis; Genes; Genetic aspects; Genetic transformation; Glucosyltransferase; In vitro petal organogenesis; Muscari; Organogenesis; Ornamental plants; Petals; Physiological aspects; Pigmentation; Plant breeding; Regeneration; Regeneration (Botany); Reporter gene; RNA-mediated interference; Sucrose; Transformations; Transgenic plants; China; Japan
• ISSN: 1746-4811; 1746-4811
• DOI: 10.1186/s13007-021-00794-7

Grape hyacinth (Muscari spp.) is one of the most important ornamental bulbous plants. However, its lengthy juvenile period and time-consuming transformation approaches under the available protocols impedes the functional characterisation of its genes in flower tissues. In vitro flower organogenesis has long been used to hasten the breeding cycle of plants but has not been exploited for shortening the period of gene transformation and characterisation in flowers. A petal regeneration system was established for stable transformation and function identification of colour gene in grape hyacinth. By culturing on Murashige and Skoog medium (MS) with 0.45 [mu]M 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 [mu]M 6-benzyladenine (6-BA), during the colour-changing period, the flower bud explants gave rise to regeneration petals in less than 3 months, instead of the 3 years required in field-grown plants. By combining this system with Agrobacterium-mediated transformation, a glucuronidase reporter gene (GUS) was delivered into grape hyacinth petals. Ultimately, 214 transgenic petals were regenerated from 24 resistant explants. PCR and GUS quantitative analyses confirmed that these putative transgenic petals have stably overexpressed GUS genes. Furthermore, an RNAi vector of the anthocyanidin 3-O-glucosyltransferase gene (MaGT) was integrated into grape hyacinth petals using the same strategy. Compared with the non-transgenic controls, reduced expression of the MaGT occurred in all transgenic petals, which caused pigmentation loss by repressing anthocyanin accumulation. The Agrobacterium transformation method via petal organogenesis of grape hyacinth took only 3-4 months to implement, and was faster and easier to perform than other gene-overexpressing or -silencing techniques that are currently available.