Efficient induction of tetraploids via adventitious bud regeneration and subsequent phenotypic variation in Acacia melanoxylon

Abstract

BACKGROUND ACACIA MELANOXYLON: is an important species for establishing pulpwood plantations due to its high application value in engineered wood products. However, the lack of a well-established in vitro regeneration system has severely constrained its industrial-scale propagation and the induction of tetraploids. RESULTS: In this study, using the superior A. melanoxylon clone SR3, an in vitro regeneration system using a bud-bearing stem segment was established. A DKW medium supplemented with 0.5 mg/L 6-BA, 0.1 mg/L IAA, and 0.2 mg/L NAA was determined as the optimal differentiation medium. Adding 0.5 mg/L IBA and 0.25 mg/L NAA to the 1/2 MS medium produced a higher rooting percentage and root number. To determine the optimal timing for tetraploid induction in A. melanoxylon, morphological, cytological, and flow cytometric analyses were conducted on the swollen tissue at the base of the bud-bearing stem segment. On the 5th day of preculture, white callus tissue was observed, characterized by vigorous cell division and the highest G₂/M-phase cell content in the adventitious bud primordia. After colchicine treatment, the tetraploid induction efficiency on the 5th day of preculture was significantly higher compared to the 4th or 6th day. The highest induction rate of 12.26 ± 0.80% was achieved with 100 mg/L colchicine for 72 h on the 5th day of preculture. Furthermore, tetraploid A. melanoxylon exhibited morphological traits such as reduced plant height, leaf number, and stomatal density. CONCLUSIONS: This study establishes a stable and effective method for in vitro tetraploid induction in A. melanoxylon, providing theoretical and technical support for polyploid breeding and laying the groundwork for subsequent triploid development.

Keywords: Acacia tree; Adventitious bud; Bud-bearing stem segment; Developmental stage observation; Morphological variation; Tetraploid induction.

Fig. 1

Establishment of an in vitro regeneration system from bud-bearing stem segment of A. melanoxylon. (a) Bud-bearing stem segment inoculated on differentiation medium. (b) Swelling initiated at the basal region of the explants. (c) Continued expansion of the swollen tissue with the emergence of several adventitious buds. (d) Numerous adventitious buds formed within the swollen tissue. (e) Aerial part of a rooted plantlet. (f) Root system of the rooted plantlet

Fig. 2

Anatomical observation of adventitious bud regeneration at the basal part of A. melanoxylon bud-bearing stem segment. (a) Transverse section of the stem segment before culture. (b) Dedifferentiation of cortical parenchyma cells into bud primordium cells. (c) Proliferation and organization of bud primordium cells. (d) Development of bud primordium into adventitious buds. Note: Xy - xylem; Ve - vessel; Pe - parenchyma cell; En - enlarged tissue; Mc - meristematic cell; Mn - meristematic nodule; Lp - leaf primordium; Ab - adventitious bud

Fig. 3

Observation of developmental status and detection of cell cycle proportions of tissues cultured in vitro. (a-g) Morphological characterization of the basal region of bud-bearing stem segment of 0-6d. (h-n) Cytological observation of the basal region of bud-bearing stem segment of 0-6d. (o-u) Flow cytometric analysis of the proportion of G₁, S, and G₂/M phase cells in adventitious bud primordia of 0-6d

Fig. 4

Ploidy identification of regenerated A. melanoxylon plantlets. (a) Flow cytometry result of diploid plantlets. (b) Flow cytometry result of tetraploid plantlets. (c) Chromosome number of diploids (2n = 2x = 26). (d) Chromosome number of tetraploids (2n = 4x = 52)

Fig. 5

Morphological comparison of diploid and tetraploid A. melanoxylon plantlets at 80 days after transplantation. (a) Comparison of plant height between diploid and tetraploid plants. (b) Comparison of leaf morphology between diploid and tetraploid plants. (c) Growth curve of diploid and tetraploid plants over 80 days. (d) Stem diameter at the 5th internode in diploid and tetraploid plants. (e) Number of leaves per plant in diploid and tetraploid plants. (f) Phyllode area of diploid and tetraploid plants. (g) Phyllode thickness of diploid and tetraploid plants. All values represent the mean ± SD. Asterisks denote significant differences, determined by Student’s t-test: *, P < 0.05; ***, P < 0.001

Fig. 6

Stomatal phenotypic characteristics of diploid and tetraploid A. melanoxylon at 80 days after transplantation. (a) Stomatal morphology of diploid leaves under 100× magnification. (b) Stomatal morphology of diploid leaves under 40× magnification. (c) Stomatal morphology of diploid leaves under 20× magnification. (d) Stomatal morphology of tetraploid leaves under 100× magnification. (e) Stomatal morphology of tetraploid leaves under 40× magnification. (f) Stomatal morphology of tetraploid leaves under 20× magnification. (g) Stomatal length in diploid and tetraploid plants. (h) Stomatal width in diploid and tetraploid plants. (i) Stomatal density in diploid and tetraploid plants. (j) Stomatal index in diploid and tetraploid plants. All values represent the mean ± SD. Asterisks denote significant differences, determined by Student’s t-test: **, P < 0.01; ****, P < 0.0001