By using shoot tips as explants, various media and culture conditions for callus induction and proliferation, shoot differentiation, root induction and plantlet transplantation to develop an efficient and reliable regeneration system with Dendrocalamus hamiltonii were tested. Murashige and Skoog (MS) medium supplemented with 3 mg/l 2, 4-dichlorophenoxyacetic acid, 1 mg/l benzyladenine (BA), 500 mg/l glutamine, 500 mg/l proline, and 500 mg/l casein hydrolysate yielded the best rates of callus induction and granular-compact callus induction. MS medium supplemented with 1 mg/l BA, 0.3 mg/l kinetin and 0.3 mg/l naphthaleneacetic acid conferred the highest differentiation rate of calli. The maximum rooting rate was obtained in 1/2 MS medium supplemented with 3 mg/l indole-3-butyric acid, and the roots were long and thick. All hardened plantlets survived after transfer to an equal ratio mixture of peat, vermiculite and perlite. The regeneration system of D. hamiltonii developed is efficient and provides a useful tool for genetic transformation in bamboo species.

BackgroundBamboo is a very important forest resource. However, the prolonged vegetative stages and uncertainty of flowering brings difficulties in bamboo flowers sampling. Until now, the flowering mechanism of bamboo is still unclear.ResultsIn this study, three successive stages of flowering buds and the corresponding vegetative buds (non-flowering stage) from Lei bamboo (Phyllostachys violascens) were collected for transcriptome analysis using Illumina RNA-Seq method. We generated about 442 million clean reads from the above samples, and 132,678 unigenes were acquired with N50 of 1080 bp. A total of 7266 differentially expressed genes (DEGs) were determined. According to expression profile and gene function analysis, some environmental stress responsive and plant hormone-related DEGs were highly expressed in the inflorescence meristem formation stage (TF_1) while some floral organ development related genes were up-regulated significantly in floral organs determination stage (TF_2) and floral organs maturation (TF_3) stage, implying the essential roles of these DEGs in flower induction and maturation of Lei bamboo. Additionally, a total of 25 MADS-box unigenes were identified. Based on the expression profile, B, C/D and E clade genes were more related to floral organs development compared with A clade genes in Lei bamboo.ConclusionsThis transcriptome data presents fundamental information about the genes and pathways involved in flower induction and development of Lei bamboo. Moreover, a critical sampling method is provided which could be benefit for bamboo flowering mechanism study.

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Grafting, cutting, and pruning are important horticultural techniques widely used in the establishment of clonal forestry. After the application of these techniques, some properties of the plants change, however, the underlying molecular mechanisms are still unclear. In our previous study, 27 age-related transcripts were found to be expressed differentially between the juvenile vegetative (1-and 2-year-old) and adult reproductive (25-and 50-year-old) phases of Larix kaempferi.Here, we re-analyzed the 27 age-related transcripts, cloned their full-length cDNA sequences, and measured their responses to grafting, cutting, and pruning. After sequence analysis and cloning, 20 transcription factors were obtained and annotated, most of which were associated with reproductive development, and six (LaAGL2-1, LaAGL2-2, LaAGL2-3, LaSOC1-1, LaAGL11, and LaAP2-2) showed regular expression patterns with L. kaempferi aging. Based on the expression patterns of these transcription factors in L. kaempferi trees subjected to grafting, cutting, and pruning, we concluded that (1) cutting and pruning rejuvenate the plants and change their expression, and the effects of cutting on gene expression are detectable within 14 years, although the cutting seedlings are still maturing during these years; (2) within three months after grafting, the rootstock is more sensitive to grafting than the scion and readily becomes mature with the effect of the scion, while the scion is not readily rejuvenated by the effect of the rootstock; and (3) LaAGL2-2 and LaAGL2-3 are more sensitive to grafting, while LaAP2-2 is impervious to it. These findings not only provide potential molecular markers to assess the state of plants but also aid in studies of the molecular mechanisms of rejuvenation.

Dormancy release and reactivation of temperate-zone trees involve the temperature-modulated expression of cell-cycle genes. However, information on the detailed regulatory mechanism is limited. Here, we compared the transcriptomes of the stems of active and dormant larch trees, emphasizing the expression patterns of cell-cycle genes and transcription factors, and assessed their relationships and responses to temperatures. Twelve cell-cycle genes and 31 transcription factors were strongly expressed in the active stage. Promoter analysis suggested that these 12 genes might be regulated by transcription factors from 10 families. Altogether, 73 cases of regulation between 16 transcription factors and 12 cell-cycle genes were predicted, while the regulatory interactions between LaMYB20 and LaCYCB1;1, and LaRAV1 and LaCDKB1;3 were confirmed by yeast one-hybrid and dual-luciferase assays. Last, we found that LaRAV1 and LaCDKB1;3 had almost the same expression patterns during dormancy release and reactivation induced naturally or artificially by temperature, indicating that the LaRAV1-LaCDKB1;3 module functions in the temperature-modulated dormancy release and reactivation of larch trees. These results provide new insights into the link between temperature and cell-cycle gene expression, helping to understand the temperature control of tree growth and development in the context of climate change.

SCR-LIKE-6 (SCL6), a member of the GRAS transcription factor family, plays important roles in many aspects of plant growth and development. In a previous study, we showed that the post-transcriptional regulation of Larix (La)SCL6 by the microRNA miR171 functions in regulating the mode of cell division and the maintenance of embryogenic potential during somatic embryogenesis in larch. To investigate its expression pattern during tree aging, which has been studied by comparative transcriptomic analysis, we reexamined the annotation and expression of every transcript and found one that was longer; we annotated it as SCL6, indicating that it might be a variant of LaSCL6. To verify this, we cloned the DNA and cDNA sequences of this transcript and found that alternative splicing indeed occurred in its expression. Moreover, both variants were detectable in embryogenic cultures and several organs. Notably, the DNA sequence of LaSCL6 contained simple sequence repeats and a single-nucleotide polymorphism which might result in the expression of two variants with a change in their tertiary structures. Regulation of LaSCL6 by miR171 was also found. Taken together, the expression of LaSCL6 is controlled at several levels, and this study not only provides further information about the expression of LaSCL6 but also offers a means to study the regulation of gene expression.

Key message Expression analysis of Larix kaempferi mature miR171s and their primary transcripts and target gene LaSCL6 during somatic embryogenesis revealed the transcriptional and post-transcriptional regulation of the miR171-LaSCL6 module. Abstract Somatic embryogenesis provides a useful experimental system for studying the regulatory mechanisms of plant development. The level and activity of microRNA171 (miR171) fluctuate during somatic embryogenesis in Larix kaempferi, but the underlying mechanisms are still unclear. Here, in L. kaempferi we identified five members of the miR171 family, which cleave LaSCL6 mRNA at different sites. In addition, we improved the method of measuring miRNA activity in a more direct way. Furthermore, we measured the expression patterns of mature miR171s and their primary transcripts during somatic embryogenesis in L. kaempferi and found that their patterns differed, indicating that the transcription of MIR171 genes and the subsequent cleavage of their intermediate products are regulated. Taken together, our findings not only offer a means to study the regulation of miRNA activity, but also provide further insight into the regulation of L. kaempferi somatic embryogenesis by miR171-LaSCL6.

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