Effective Induction of Fertile Gametes in Oriental × Trumpet (OT) Lily by High Temperatures

Abstract

Lily is a very important bulb crop, and interspecific distant hybridization is a crucial method of lily breeding. However, F₁ interspecific hybrids tend to be highly sterile due to low levels of genetic homozygosity. This can be addressed by meiotic polyploidization, which has the advantage of reducing breeding time and being able to promote genetic recombination resulting in many variant progenies. High temperatures have been proven to induce 2n gametes via hindering a spindle formation in several plants, but little has been reported in lilies. In the present study, after observing the correlation between the development of the pollen mother cells (PMCs) and the length of the buds, 28-31 mm long buds were selected as the experimental material, which were at the stage of prophase I-metaphase I. Individual buds were induced at different temperatures (40 °C, 42 °C, and 44 °C) and durations (4 h and 6 h) using self-made multiwire heating equipment, and successfully induced fertile male gametes.. The best results were achieved with treatment of 42 °C for 4 h, reaching a maximum fertile pollen induction rate of 36.64%, while bud mortality was 40%. Two chemicals, colchicine and oryzalin, were also used by injection, and only the treatment with oryzalin obtained fertile gametes, with the highest fertile gamete rate of 15.39% at a concentration of 0.005%, while the bud mortality was 36.67%. This suggests that high temperatures have a superior effect on lily 2n gamete induction. In addition, the pollen obtained from the 6 h induction of high temperature was significantly larger than that from the 4 h induction, with an average diameter of 138.64 μm and 107.88 μm, respectively, 2.35 and 1.84 times wider than haploid pollen. The fertile pollen was crossed with four cultivars and two species, and a total of 267 embryonic seeds were obtained, with the highest embryonic rate of 4.52% in OT lily 'Mister Cas' as the parent, which had a germination rate of 26.27%. This suggests that the method of high-temperature induction for fertile gametes probably has important significance for ploidy and distant hybrid breeding in lilies.

Keywords: Lilium; fertility restore; high temperature; meiosis.

Figure 1

Meiotic processes of PMCs of ‘Black Beauty’. (a) Interphase. (b) Prophase I. (c) Metaphase I. (d) Anaphase I. (e) Telophase I. (f) Prophase II. (g) Metaphase II. (h) Anaphase II. (i) Telophase II. (j) Tetrad.

Figure 2

A mixture of meiotic stages in one flower bud. Arrows show M I (metaphase I), P II (Prophase II), and T II (Telophase II). (Bar = 100 μm).

Figure 3

Flower buds and their corresponding anther morphology on the day of flowering for the different treatments and controls. (a,f) Control flower bud and anthers without any treatment. (b,g) Flower bud and anthers treated at a high temperature of 40 °C for 4 h. (c,h) Flower bud and anthers treated at a high temperature of 42 °C for 6 h (Reduced or no pollen where anthers turn white). (d,i) Flower bud and anthers treated with 0.01% concentration of oryzalin. (e,j) Flower bud and anthers treated with 0.01% concentration of colchicine (outer petals deciduous) (Bar = 10 mm).

Figure 4

Comparison of sterile and fertile pollen from ‘Black Beauty’. (a) Control group. (b) Pollen grains obtained after 4 h treatment at 42 °C. (c) Pollen grains obtained after 6 h treatment at 42 °C. (Nonfertile pollen indicated by green arrows, and fertile pollen indicated by red arrows).