PMC

Induction of primordium formation and meristem tip growth by auxin and cytokinin. (A–I) Microapplication of auxin and cytokinin to dark-cultured apices. Dissected tomato apices were precultured in the light and transferred to darkness for 6 d. Lanolin containing 1% DMSO (A), 10 mM IAA (B,C), 1 mM zeatin (D–F), or 10 mM IAA plus 1 mM zeatin (G–I) was applied in the dark. These apices were further cultured in the dark for 10 d. (White asterisks) Pre-existing primordia. (F,I) Note that apices treated with 10 mM IAA plus 1 mM zeatin and with 1 mM zeatin alone continued to grow in the dark. (A,B) However, apices treated with 1% DMSO or 10 mM IAA did not grow. (C,E,H) In addition, 10 mM IAA alone, 10 mM IAA plus 1 mM zeatin, and 1 mM zeatin alone promoted the development of pre-existing P1 and I1. The numbers in the bottom left corner show the number of apices that show the displayed phenotype out of the total number of samples. Thus cytokinin induces leaf initiation in the dark, and auxin promotes leaf initiation in the presence of cytokinin. (J–O) Microapplication of auxin and cytokinin to the flank of the meristems of tomato NPA pins. Dissected apices were cultured in the presence of NPA. Resulting pin-shaped apices (NPA pins) were precultured in the light or the dark, and microapplication of IAA and cytokinin was performed. Microapplication of 1% DMSO (mock) lanolin in the light (J), 10 mM IAA lanolin in the light (K), 1% DMSO lanolin in the dark (L), 10 mM IAA lanolin in the dark (M), 10 mM IAA plus 1 mM zeatin lanolin in the dark (N), and 10 mM IAA plus 10 mM zeatin lanolin in the dark (O). (P–S) Microapplication to the flank and the summit of the meristem of NPA pins. Microapplication of 1% DMSO lanolin to the flank and the summit in the light (P), 1% DMSO lanolin to the flank and the summit in the dark (Q), 10 mM IAA lanolin to the flank and 1% DMSO lanolin to the summit in the dark (R), and 1 mM zeatin lanolin to the summit and 1% DMSO lanolin to the flank in the dark (S). (A–I) Scanning electron microscope images. (J–S) Stereomicroscope images. Lanolin dots applied to the flank are colored red, and those applied to the summit are colored blue. Bars, 100 μm.

The leaf length, leaf width and leaf weight of the 1st, 2nd and 3rd leaves after topping of positive transgenic plants/control plants applied with/without 50 µM DEX. P: positive transgenic plants; T: topping; Dex: 50 µM dexamethasone; Y1: the 1st leaves of the application site; Y2: the 2nd leaves; Y3: the 3rd leaves. (a) Comparison of the 1st leaf after topping, from left to right: positive transgenic plants treated with 50 µM dexamethasone, positive transgenic plants treated with lanolin, control plants treated with 50 µM dexamethasone, control plants treated with lanolin; (b) comparison of the 2nd leaf after topping, from left to right: positive transgenic plants treated with 50 µM dexamethasone, positive transgenic plants treated with lanolin, control plants treated with 50 µM dexamethasone, control plants treated with lanolin; (c) comparison of the 3rd leaf after topping, from left to right: positive transgenic plants treated with 50 µM dexamethasone, positive transgenic plants treated with lanolin, control plants treated with 50 µM dexamethasone, control plants treated with lanolin; (d) comparison of the leaf length and leaf width of the 1st, 2nd and 3rd leaves after topping of positive transgenic plants/control plants applied with/without 50 µM DEX; (e): comparison of the leaf weight of the 1st, 2nd and 3rd leaves after topping of positive transgenic plants/control plants applied with/without 50 µM DEX.

Auxin treatment of cotyledons in the isocotylous stage reveals that a higher concentration in one of the cotyledons induces the microcotyledon fate. (A–C) The results of three independent auxin application experiments. “Bigger after treatment” and “Smaller after treatment” indicate individuals in which the cotyledon treated with the lanolin paste grew larger (macrocotyledon) and those that stopped growth (microcotyledon) after treatment, respectively. “One macrocotyledon” indicates that one of the cotyledons differentiated into a macrocotyledon and the other differentiated into a microcotyledon. “Both macrocotyledons” and “Both microcotyledons” indicate individuals that harbored two macrocotyledons and two microcotyledons, respectively. The numbers in the bars indicate the absolute number of individuals in each category. The x-axis shows the types of treatment: “EtOH” indicates the treatment with lanolin only containing ethanol, used to dilute IAAK. “0.2 mM,” “2 mM,” and “20 mM” indicate the concentration of auxin dissolved in the lanolin in the treatment. On the x axis, “both” indicates that the lanolin was applied to both of the cotyledons. (D) The stage at which the lanolin treatment was conducted. Lanolin was applied to the edge of the individual cotyledon, and black carbon particles were stuck on the lanolin to make it easier to show the position of the lanolin. (E) One of the individuals treated with 20 mM IAAK, observed in experiment (A). The arrow indicates the lanolin, which can be seen on the cotyledon on the left (microcotyledon). The hatched white line shows narrower part in the microcotyledon lamina. (F) The individual harboring both microcotyledons in experiment (A). A new organ was produced between the two cotyledons. The hatched white line shows narrower part in the microcotyledon lamina. *p < 0.05, ***p < 0.001 in a binomial test of “Bigger after treatment” and “Smaller after treatment” (The number of occurrences of “Both macrocotyledons” and “Both microcotyledons” is not included because they are very rare). Bar = 0.5 mm in (D) and 1 mm in (E,F).

pOp6/LhGR system in Nicotiana attenuata under glasshouse conditions.
(a) Inverted repeat PDS (irPDS) and PDS transcript level in i-irpds plants 12h after application of DEX-containing lanolin paste to the midvein.
(b) irPDS and PDS transcript levels in the basal and apical parts of the leaf lamina, as well as in the midvein of i-irpds plants three days after application of DEX-containing lanolin paste.
(c) i-irpds plants six days after application of DEX-containing lanolin paste.
(d) IPT transcript and trans-zeatin (ng*gFM⁻¹) levels in i-ovipt plants 12h after application of DEX-containing lanolin paste to the midvein.
(e) IPT transcript and trans-zeatin (ng*gFM⁻¹) levels in the basal and apical parts of the leaf lamina, as well as in the midvein of i-ovipt plants three days after application of DEX-containing lanolin paste.
(f) i-ovipt plants 12d after application of DEX-containing lanolin paste.
(g) trans-Zeatin (tZ), trans-zeatin riboside (tZR), trans-zeatin ribosid O-glycoside (tZROG) and trans-zeatin 7-glycoside (tZ7G) levels in i-ovipt plants after 12 days of treatments with different concentrations of DEX-containing lanolin paste.
Lanolin paste was applied every three days. Asterisks indicate significant differences between DEX treated samples and the corresponding control (0μM) (independent samples t test: * P<0.05, *** P<0.001). irPDS, IPT transcript and trans-zeatin data, 12h after DEX treatment were log₁₀ transformed before t-test analysis. Small letters indicate significant differences between samples (one-way ANOVA, Turkey HSD, P<0.05). Error bars show standard errors (a, b: N=6; d: N=5; e, g: N≥3). FM, Fresh mass.