Figures (5)  Tables (2)

    • Figure 1. 

      Propagation by root-sprouting and examples of black locust plants produced with different propagation methods. (a) Exposure of a woody root. (b) Girdling one quarter of the root circumference. (c) Hormone treatment. (d) Moisturizing treatment. (e) Treated root after 15 d. (f) Germinated plantlets in root-sprouting treatment. (g) Seed-derived seedlings. (h) Plantlets obtained by root-sprouting. (i) Plantlets obtained from root-cuttings. (j) Plantlets obtained from shoot-cutting. Scale bars: (a) 50 cm, (b)−(f) 2 cm, (g), 5 cm, (h)−(j) 2 cm.

    • Figure 2. 

      Growth and leaf traits of plantlets, seedlings and mother trees. (a) Basal diameter. (b) Seedling height. (c) Leaflet area. (d) Leaflet length/width. (e) Leaflet length. (f) Leaflet perimeter. (g) Leaflet width. (h) Leaf relative water content. Different lowercase letters above the bars indicate significant differences between plant materials (p < 0.05, ANOVA and Tukey's honestly significant difference test). Abbreviations: SS, seed-derived seedlings; RS, root-sprouting plantlets, RC, root-cutting plantlets; SC, shoot-cutting plantlets; MT, mother trees.

    • Figure 3. 

      Physiological traits of juvenile plantlets/seedlings and mother trees. (a) Total soluble protein. (b) Total soluble sugars. (c) Phenolic content. (d) MDA content. Different lowercase letters above the bars indicate significant differences among plant materials. Abbreviations: SS, seed-derived seedlings; RS, root-sprout plantlets; RC, root-cutting plantlets; SC, shoot-cutting plantlets; MT, mother trees. MDA, malondialdehyde.

    • Figure 4. 

      Shoot cross-sectional anatomy and leaf-cell ultrastructure of plantlets. (a)–(d) Stem cross sections of (a) SS, (b) RC, (c) RS, and (d) SS. (e)–(s) Ultrathin sections of leaves. SS, seed-derived seedlings; RS, root-sprout plantlets; RC, root-cutting plantlets; SC, shoot-cutting plantlets; MT, mother trees. Abbreviations: Ep, Epidermis; Co, Cortex; Ph, Phloem; Xy, Xylem; Ve, Vessel; Pi, Pith; CW, cell wall; Ch, chloroplast; Mi, mitochondrion; nu, nucleus; SG, starch granule; Va, vacuole; T; thylakoid; O, osmiophilic droplet; Me: nuclear membrane; Chr, chromatin. Scale bars: (a), (b) 50 µm, (e)−(i) 2 µm, (j)−(n) 500 nm, (o)−(s) 1 µm.

    • Figure 5. 

      Relative expression of genes and microRNAs in the plantlets/seedlings and mother trees. (a) miR156, (b) miR172, (c) RpVAL, (d) RpSPL6, (e) RpSPL9, and (f) RpSPL10. Asterisks indicate statistically significant differences compared to the seed-derived seedlings (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant difference). Abbreviations: SS, seed-derived seedlings; RS, root-sprout plantlets; RC, root-cutting plantlets; SC, shoot-cutting plantlets; MT, mother trees.

    • miRNA/GeneSpeciesJuvenileAdultFunctionReferences
      miR156A. thalianaHighly expressed−Prolongs juvenility and delays the onset of the adult phase.[30,36]
      miR172A. thaliana−Highly expressedIts expression is regulated by miR156 via SPL, thereby regulating plant vegetative phase change.[36,37]
      SPL3/4/5A. thaliana−Highly expressedRegulates flowering time and phase change[38,39]
      SPL9/15A. thaliana−Highly expressedPromotes juvenile-to-adult phase transition and flowering; directly regulates genes involved in trichome formation.[36,40]
      SPL2/10/11A. thaliana−Highly expressedRegulates leaf shape and trichome distribution with shoot maturation in the reproductive phase.[36,39]
      SPL6/13A. thaliana−Highly expressedRegulates the switch from cotyledon to vegetative leaf stage.[36]
      TOE1/2A. thalianaHighly expressed−Represses flowering[39]
      VAL1/2A. thaliana−Highly expressedBoth VAL1 and VAL2 regulate vegetative phase change by constitutively decreasing miR156 levels and indirectly promoting SPL expression[27]
      SPL, SQUAMOSA PROMOTER BINDINGPROTEIN-LIKE; TOE, TARGET OF EAT; VAL, VIVIPAROUS/ABI3-LIKE.

      Table 1. 

      Key miRNAs and genes known to function in plant vegetative phase change.

    • MTMT Age (y)SS (N)SC (N)RC (N)RS (N)
      MT118158714
      MT21915157
      MT3211531013
      MT417153016
      MT524158621
      Total75232971
      MT616152018
      MT719152017
      MT817158510
      MT922154108
      MT1018154012
      Total75201565
      MT1120151307
      MT1219156618
      MT132215539
      MT1424151455
      MT1517156112
      Total75441551
      Grand total2258759187
      MT, mother tree; SS, seed-derived seedling; SC, shoot-cutting plantlet; RC, root-cutting plantlet; RS, root-sprouting plantlet.

      Table 2. 

      Ages of mother trees and numbers of seedlings and plantlets obtained from each mother tree.