Effects of Colchicine on Populus canescens Ectexine Structure and 2n Pollen Production

Abstract

Triploid breeding is a central way to improve growth traits, timber quality, and stress resistance in Populus. In the present study, the morphology and viability of colchicine-induced 2n pollen, triploid production by crossing induced 2n pollen, and identification of genetic constitution of colchicine-induced 2n pollen were conducted in Populus canescens based on optimizing technology for inducing chromosome doubling in pollen. We found that the meiotic stage, injection time, and the interaction between the meiotic stage and injection time had highly significant effects on the 2n pollen production rate. The most effective treatment for inducing 2n pollen was to give 11 injections of 0.5% colchicine solution when pollen mother cells (PMCs) were at the pachytene stage. The highest 2n pollen production rate was 30.27 ± 8.69%. Colchicine occasionally affected ectexine deposition, and some narrow furrows were detected in the ectexine structure. However, no significant difference was observed in the pollen germination rate between natural 2n pollen and colchicine-induced 2n pollen. Moreover, 5 triploids derived from FDR-type 2n pollen were generated by crossing induced 2n pollen, suggesting that colchicine does not eliminate the function of colchicine-induced 2n pollen. However, slower growth of 2n pollen tubes was responsible for a lower triploid production rate.

Keywords: 2n pollen viability; Populus canescens; chromosome doubling; colchicine; triploid.

FIGURE 1

Cytological observations of PMCs, colchicine-induced 2n pollen (arrow) and naturally occurring 2n pollen in Populus canescens. (A) Leptotene. (B) Pachytene. (C) Diplotene. (D) Diakinesis. (E) Metaphase I. (F) Colchicine-induced 2n pollen derived from the treatment given 11 injections at pachytene in P. canescens (arrow). (G) Natural 2n pollen derived from the control group in P. canescens (arrow). Scale bar = 10.0 μm (A–E) and 20.0 μm (F,G).

FIGURE 2

Scanning electron micrographs of P. canescens pollen grains derived from untreated and treated male flower buds with 0.5% colchicine at pachytene. (A) Morphology of the pollen grains in the control group and natural 2n pollen grains. (B) Morphology of the pollen grains derived from the treatment given 5 injections at pachytene and induced 2n pollen grains. (C) Morphology of the pollen grains derived from the treatment given 11 injections at pachytene and induced 2n pollen grains. (D–F) Ectexine deposition; (D) haploid pollen grains in the control group; (E) haploid pollen grains derived from the 5 injections treatment at pachytene; (F) haploid pollen grains derived from the 11 injections treatment given at pachytene. (G–L) Details of the ectexine structure; (G) haploid pollen grains in the control group; (H) haploid pollen grains derived from the treatment given 5 injections at pachytene; (I) haploid pollen grains derived from the treatment given 11 injections at pachytene; (J) natural 2n pollen grains; (K) 2n pollen grains derived from the treatment given 5 injections at pachytene; (L) 2n pollen grains derived from the treatment given 11 injections at pachytene. Scale bar = 100 μm (A–C), 10 μm (D–F), 5 μm (G–L).

FIGURE 3

Flow cytometric analysis and somatic chromosome counting of the offspring derived from pollen chromosome doubling induced by colchicine in P. canescens (scale bar = 20 μm). (A) Flow cytometric analysis of the mixed simple of a diploid plant and a triploid plant; (B) Chromosome number of the diploid (2n = 2x = 38); (C) Chromosome number of the triploid (2n = 3x = 57).

FIGURE 4

Fresh colchicine-induced pollen of P. canescens derived from the treatment given 11 injection times germinated on media. (A) Germinated fresh colchicine-induced pollen derived from the treatment given 11 injection times. (B) Germinated 2n pollen induced by colchicine (arrow). (C) Germinated natural 2n pollen (arrow). Scale bar = 100.0 (A) and 50.0 μm (B,C).

FIGURE 5

Germination rates of natural 2n pollen derived from the control group and colchicine-induced 2n pollen derived from the treatment given 11 injections with 0.5% colchicine at pachytene in P. canescens.

FIGURE 6

Lengths of the pollen tubes of haploid pollen and colchicine-induced 2n pollen derived from the treatment given 11 injections with 0.5% colchicine at pachytene in P. canescens. Lowercase letters represent significant differences at (P < 0.05).

FIGURE 7

Genetic constitutions of the colchicine-induced 2n pollen in P. canescens were revealed by capillary electrophoresis of the loci of primers GCPM_1158 and GCPM_124. (A) Allelic configuration in triploid progeny derived from P. hopeiensis × P. canescens and their parents. T1 and T2 indicate hybrid triploids. (B) Allelic configuration in triploid progeny derived from (P. alba × P. glandulosa) × P. canescens and their parents. T3, T4, and T5 also indicate three hybrid triploids.

FIGURE 8

Male flower buds of P. canescens after treatment with 0.5% colchicine solution. Scale bar = 2.0 mm.