Unreduced Male Gamete Formation in Cymbidium and Its Use for Developing Sexual Polyploid Cultivars

Abstract

Polyploidy plays an important role in crop improvement. Polyploid plants, particularly those produced through unreduced gametes (2n gametes), show increased organ size, improved buffering capacity for deleterious mutations, and enhanced heterozygosity and heterosis. Induced polyploidy has been widely used for improving floriculture crops, however, there are few reported sexual polyploid plants in the floriculture industry. This study evaluated nine cultivars of Cymbidium Swartz and discovered that 2n male gametes occurred in this important orchid. Depending on cultivars, 2n male gamete formation frequencies varied from 0.15 to 4.03%. Interspecific hybrids generally produced more 2n male gametes than traditional cultivars. To generate sexual polyploid plants, seven pairs of crosses were made, which produced five triploid and two tetraploid hybrids. Two triploid hybrids were evaluated for in vitro regeneration and growth characteristics. Compared to the diploid parents, the triploids were more easily regenerated through rhizomes or protocorms, and regenerated plants had improved survival rates after transplanting to the greenhouse. Furthermore, the sexual polyploid plants had more compact growth style, produced fragrant flowers, and demonstrated heterosis in plant growth. Through this study, a reliable protocol for selection of appropriate parents for 2n gamete production, ploidy level evaluation, in vitro culture of polyploid progenies, and development of new polyploid cultivars was established. Our study with Cymbidium suggests that the use of 2n gametes is a viable approach for improving floriculture crops.

Keywords: 2n gametes; Cymbidium; floriculture crops; micropropagation; sexual polyploidization.

FIGURE 1

Analysis of unreduced male gametes in Cymbidium cultivars using the squash method. (A) Mature pollens were stained with carbolfuchsin, and dyad (a), triad (b), and tetrad (c) gametes were identified by the squash method. Bar = 10 μm. The dyad with 2n gametes (arrow) (d), triads with one unreduced gamete (arrow) and two reduced gametes (e), and tetrad with four reduced gametes (f) were stained with 4’,6-diamidino-2-phenylindole (DAPI). Bar = 50 μm. (B) 2n gamete formation frequencies (means of 3-year data, Supplementary Table S1) among nine cultivars, bars represent standard error (n = 30). Different letters on the top of bars indicate significant cultivar difference in 2n gamete formation frequencies analyzed by Duncan’s multiple range test at P < 0.05 level.

FIGURE 2

Evaluation of ploidy levels of Cymbidium hybrids of “DH” and “Huanghe” resulted from cross of “Dafeng” × “Hezhihua.” (A) Morphology of “DH” (left) and “Huanghe” (right). (B) Flow cytometric DNA histograms of “DH” and (C) chromosome numbers of root tip cell of “DH” (2n = 2x = 40). (D) Flow cytometric DNA histograms of “Huanghe” and (E) chromosome numbers of root tip cell of “Huanghe” (2n = 3x = 60).

FIGURE 3

Evaluation of ploidy levels of Cymbidium hybrids of “XY” and “Yutao” developed from cross of “Yunv” × “Xiaoxiang.” (A) Morphology of “YX” (left) and “Yutao” (right). (B) Flow cytometric DNA histograms of “YX” and (C) chromosome numbers of root tip cell of “YX” (2n = 2x = 40). (D) Flow cytometric DNA histograms of “Yutao” and (E) chromosome numbers of root tip cell of “Yutao” (2n = 3x = 60).

FIGURE 4

Morphological characteristics of polyploid Cymbidium cultivars with their diploid counterparts. (A) Mature plants of “DH” (left) and “Huanghe” (right). (B) Flowers of “DH” (left) and “Huanghe” (right). (C) Sepals (left), petals (middle), and lips (right) of “DH” (above) and “Huanghe” (below). (D) Mature plants of “XY” (left) and “Yutao” (right); (E) Flowers of “YX” (left) and “Yutao” (right); (F) Sepals (left), petals (middle), and lips (right) of “YX” (above) and “Yutao” (below).

FIGURE 5

Regeneration ability of polyploid Cymbidium cultivars developed from sexual polyploidization. (A) Induction of rhizomes from shoot tips: ① Shoot tip of “Huanghe” on day one; ② Shoot tip of “Yutao” on day one; ③ Shoot tip of “DH” on day one; ④ Shoot tip of “Huanghe” on day 40; ⑤. Shoot tip of “Yutao” on day 40; ⑥ Shoot tip of “DH” on day 40. (B) Rhizome induction frequencies (%) of the three cultivars. (C) Rhizome proliferation: ① Rhizomes of “Huanghe” on day one; ② Rhizomes of “Yutao” on day one; ③ Rhizomes of “DH” on day one; ④ Rhizomes of “Huanghe” on day 40; ⑤ Rhizomes of “Yutao” on day 40; ⑥ Rhizomes of “DH” on day 40. (D) Rhizome proliferation coefficient of three cultivars. (E) Shoot regeneration from rhizomes: ① Shoot induction of “Huanghe” on day one; ② shoot induction of “Yutao” on day one; ③ shoot induction of “DH” on day one; ④ Shoot induction of “Huanghe” on day 40; ⑤ Shoot induction of “Yutao” on day 40; ⑥ Shoot induction of “DH” on day 40. (F) Shoot production rate of three cultivars from cultured rhizomes. Bars represent standard error, and different letters on the top of bars indicate significant cultivar difference for individual traits analyzed by Duncan’s multiple range test at P < 0.05 level.

FIGURE 6

The survival rate of “Huanghe,” “Yutao,” and “DH” plantlets after transplanting into soilless substrate grown in a shaded greenhouse. Bars represent standard error, and different letters on the top of bars indicate significant cultivar difference in survival rates analyzed by Duncan’s multiple range test at P < 0.05 level.