In vitro induction and identification of polyploid Neolamarckia cadamba plants by colchicine treatment

Polyploidization has played a crucial role in plant breeding and crop improvement. However, studies on the polyploidization of tropical tree species are still very scarce in this region. This paper described the in vitro induction and identification of polyploid plants of Neolamarckia cadamba by colchicine treatment. N. cadamba belongs to the Rubiaceae family is a natural tetraploid plant with 44 chromosomes (2n = 4x = 44). Nodal segments were treated with colchicine (0.1%, 0.3% and 0.5%) for 24 h and 48 h before transferring to shoot regeneration medium. Flow cytometry (FCM) and chromosome count were employed to determine the ploidy level and chromosome number of the regenerants, respectively. Of 180 colchicine-treated nodal segments, 39, 14 and 22 were tetraploids, mixoploids and octoploids, respectively. The highest percentage of polyploidization (20% octoploids; 6.7% mixoploids) was observed after treated with 0.3% colchicine for 48 h. The DNA content of tetraploid (4C) and octoploid (8C) was 2.59 ± 0.09 pg and 5.35 ± 0.24 pg, respectively. Mixoploid plants are made up of mixed tetraploid and octoploid cells. Chromosome count confirmed that tetraploid cell has 44 chromosomes and colchicine-induced octoploid cell has 88 chromosomes. Both octoploids and mixoploids grew slower than tetraploids under in vitro conditions. Morphological characterizations showed that mixoploid and octoploid leaves had thicker leaf blades, thicker midrib, bigger stomata size, lower stomata density, higher SPAD value and smaller pith layer than tetraploids. This indicates that polyploidization has changed and resulted in traits that are predicted to increase photosynthetic capacity of N. cadamba. These novel polyploid plants could be valuable resources for advanced N. cadamba breeding programs to produce improved clones for planted forest development.