Abstract
Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L−1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1–1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.
Highlights
Tetraploid Robinia pseudoacacia L. (R. pseudoacacia L.) is a leguminous, deciduous, ornamental tree species that is artificially produced by doubling the chromosomes, with the application of colchicine, in diploid cells of the congeneric species, known as black locust (2n = 92)
The primary adventitious root (AR) meristems began to develop after 48 h after excision (HAE), and several root primordia were formed after 72 HAE (Figure 1B)
Only the 0 HAE time point was selected from control micro-shoot cuttings, as no histological observations were made at the other time points for the control cuttings
Summary
R. pseudoacacia L. is native to south Korea, and was introduced into China in 1997 [1,2]. This species has a significant role in supplying wood production, honey production, and feed for animals; it favors the rapid fixation of elite genotypes [3,4]. Tetraploid R. pseudoacacia L. is highly adaptable to harsh environments, including cold, drought, salt, pest infestation, and nutrient deficiencies [5], which increases its economic value for further research studies [6]. The propagation of these species is difficult due to its long generation periods, and life cycle, which is a limiting factor. Several reports have shown that in vitro propagation of tetraploid R. pseudoacacia L. is an effective method to produce large numbers of clonal plants [8], because woody species are usually more difficult to root than herbaceous plants [9]
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