Abstract
Callus growth and development, a crucial process in plant propagation, is involved in hormonal balance and abundant gene regulation. MiRNAs are key regulators in the process of cell differentiation and development. MiR397 was identified as participating in plant growth, development, and response to stress, and it was regulated by targeting the LAC gene. The regulatory function of miR397 during callus growth and development was not clear in Liriodendron. In this study, LhmiR397a and its targets were identified, and its regulatory function between LhmiR397a and LhLAC11 was shown using qRT-PCR and transient expression in protoplasts. Furthermore, to clarify the regulatory function of LhmiR397a-LhLAC11, transgenic calli overexpressing LhMIR397a, LhLAC11, and mLhLAC11 were separately obtained by Agrobacterium-mediated transfer. The results showed that overexpressing LhMIR397a might retard callus proliferation, while overexpressing LhLAC11 or mLhLAC11 could promote callus proliferation. Genes associated with the cell cycle had decreased expression when LhMIR397a was overexpressed, while increased expression was observed when LhLAC11 or mLhLAC11 was overexpressed. Additionally, the calli overexpressed with LhMIR397a could generate early cotyledons 21 days after induction, and the somatic embryo induction time was short compared with other genotypes. This study identified LhmiR397a and its targets and provided a functional characterization of LhmiR397a in callus growth and development by regulating its target in Liriodendron.
Highlights
Liriodendron sino-americanum is an excellent woody tree species and garden afforestation tree species
To further identify LhmiR397a, the sequence of pre-miR397a was obtained with BLAST; the secondary structure was predicted with RNAfold
Four miR397s were identified in L. sino-americanum, and LhLAC was confirmed as a target of LhmiR397a
Summary
Liriodendron sino-americanum is an excellent woody tree species and garden afforestation tree species. The somatic embryogenesis system is a powerful tool for massive propagation and character improvement of higher plants. The determination of the mechanism of somatic embryogenesis in Liriodendron sino-americanum is urgently needed. The in vitro plant regeneration process often begins with callus induction. Callus refers to a disorganized cell mass and can be induced by wounding stress, which activates a set of reprogramming-related genes, including ERF115, WIND1–WIND4, PLT3, PLT5, and PLT7 [2,3,4]. Calli can be formed in vitro when cultured with an optimal ratio of auxin/cytokinin [5]. In Liriodendron, a balance between auxin and cytokinin successfully induced callus formation [6]
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