Tetraploidization promotes radial stem growth in poplars

Abstract

Somatic polyploidization often increases cell and organ size, thereby contributing to plant biomass production. However, as most woody plants do not undergo polyploidization, explaining the polyploidization effect on organ growth in trees remains difficult. Here we developed a new method to generate tetraploid lines in poplars through colchicine treatment of lateral buds. We found that tetraploidization induced cell enlargement in the stem, suggesting that polyploidization can increase cell size in woody plants that cannot induce polyploidization in normal development. Greenhouse growth analysis revealed that radial growth was enhanced in the basal stem of tetraploids, whereas longitudinal growth was retarded, producing the same amount of stem biomass as diploids. Woody biomass characteristics were also comparable in terms of wood substance density, saccharification efficiency, and cell wall profiling. Our results reveal tetraploidization as an effective strategy for improving woody biomass production when combined with technologies that promote longitudinal stem growth by enhancing metabolite production and/or transport.

Keywords: polyploidization; poplar; tetraploid; woody biomass.

Figure 1. Tetraploid poplars have larger cells in the stem. (A) Flow cytometric analysis of the diploid (2n) and tetraploid (4n) lines. Plants were grown in a medium for four weeks, and nuclei were isolated from leaves. (B–E) Stem sections of diploids (2n) and tetraploids (4n). The 10th internode of plants cultured in vitro for 50 days was used for sectioning. Magnified images of the xylem tissue surrounded by white squares in (B) and (C) are shown in (D) and (E), respectively. Scale bars: 100 µm (B, C) and 25 µm (D, E). (F, G) Cell number and cell area in the xylem. The 10th internode of plants cultured in vitro for 50 days was sectioned and used for measurements. The cell number was counted in each radial cell file of the secondary xylem (F), and the cell area was measured for xylem fiber cells (G). Data are presented as mean±SD [n≥150 for (F), and n≥300 for (G)]. Significant differences from the diploid were determined using Student’s t-tests: ** p<0.01.

Figure 2. Growth analysis of tetraploid poplars. (A) Diploid (2n) and tetraploid (4n) lines of poplars grown for four weeks in a greenhouse. Scale bar, 10 cm. (B–F) Stem and leaf growth in a greenhouse. Plants were acclimated for 10 days and grown for 17 weeks to measure stem height (B), basal stem diameter (C), and leaf number (E) every week. The relationship between stem height and basal stem diameter is shown in (D). After 17-week cultivation, the sizes of the 6–12th leaves were measured (F). (G, H) Fresh and dry weights of the stem. After 17-week cultivation, the whole stem was measured for fresh weight (G) and dry weight (H). Data are presented as mean±SD [n≥7 for (B, C, E), and n=6 for (F, G, H)]. Significant differences from the diploid were determined using Student’s t-tests: * p<0.05; ** p<0.01.

Figure 3. Assessment of woody biomass. (A, B) Dry weight per 5-cm stem section (A) and wood substance density (B) of diploid (2n) and tetraploid (4n). Part 1, 0–15 cm from the ground; part 2, 46–61 cm from the ground; and part 3, 92–107 cm from the ground. (C) Enzymatic saccharification efficiency. The amounts of released glucose are shown for samples of whole shoots grown in an agar medium for four weeks. Each sample was saccharified by incubation with cellulase and cellobiase for 0, 5, or 24 h. Data are presented as mean±SD (n=6). Significant differences from the diploid were determined using Student’s t-tests: ** p<0.01. (D, E) Cell wall profiling by NMR analysis. Plants were grown for 19 weeks in a greenhouse, and 20-cm stem sections from the basal part were subjected to NMR analysis. Fifty-one descriptors were used for PCA (D). The ratio of lignin monomer units—syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H)—was calculated from the NMR data (E).