Triploid cultivars of Cymbidium act as a bridge in the formation of polyploid plants

Abstract

Triploid is considered a reproductive barrier and also a bridge in the formation of polyploids. However, few reports are available in Cymbidium. In this study, diploid 'Xiaofeng', sexual triploid 'Yuchan' and 'Huanghe' of Cymbidium were used to evaluate hybridization compatibility of the triploids. Results showed that the sexual triploids were fertile whether they were used as male or female parents. 'Yuchan' produced male gametes of 1x, 1x~2x, 2x, 2x~3x, and 3x at frequencies of 8.89%, 77.78%, 6.67%, 3.33%, and 3.33%, respectively; while 'Huanghe' produced 3.33% 1x, 80.00% 1x~2x, 8.89% 2x, 5.56% 2x~3x, and 2.22% 3x male gametes. The cross of 'Xiaofeng' with 'Yuchan' produced progenies with a wide range of ploidy levels, including one diploid, 34 2×~3× aneuploids, 12 triploids, and one tetraploid, indicating that male gametes produced by sexual triploid were fertile and could be transmitted and fused with egg cells. On the other hand, 10 progenies obtained from the cross of 'Yuchan' × 'Xiaofeng' were all aneuploids. The cross of 'Yuchan' with 'Huanghe' produced 40 progenies including three 2×~3× aneuploids, nine 3×~4× aneuploids, 21 tetraploids, six 4×~5× aneuploids, and one pentaploid, suggesting that 2x gametes, instead of the unreduced ones played a more important role in the formation of tetraploids. The survival rates of the hybrids were all above 80.00%, with the tetraploids at 96.67%. Cytological analysis revealed that during meiosis of sexual polyploids, two chromosome sets of the 2n gamete were inclined to enter into the same daughter cell, resulting in the production of 2x gametes. Our results indicate that the triploid cymbidiums are not reproductive barrier but serve as a bridge in the formation of polyploid plants.

Keywords: Cymbidium; cross compatibility; polyploidy; triploid bridge; unreduced gamete.

Figure 1

Ploidy identification of hybrid progenies. 1. In vitro cultured seedlings; 2. Chromosome numbers in a root tip cell; 3. Flow cytometry histogram of leaf tissue (arrow represents plant ploidy). (A) ‘Xiaofeng’ (diploid, 2n = 2× = 40). (B, C) Hybrid progenies of ‘Yuchan’ × ‘Xiaofeng’: (B) ‘18-21-8’ (aneuploidy, 2n = 50), (C) ‘18-21-10’ (aneuploidy, 2n = 75). (D–G) Hybrid progenies of ‘Xiaofeng’ × ‘Yuchan’ where (D) ‘18-50-1’ (diploid, 2n = 2× = 40), (E) ‘18-50-86’ (aneuploidy, 2n = 48), (F) ‘18-50-125’ (triploid, 2n = 3× = 60), and (G) ‘18-50-140’ (tetraploid, 2n = 4× = 80). (H–L) Hybrid progenies of ‘Yuchan’ × ‘Huanghe’ where (H) ‘18-24-50’ (aneuploidy, 2n = 56), (I) ‘18-24-33’ (aneuploidy, 2n = 72), (J) ‘18-24-15’ (tetraploid, 2n = 4× = 80), (K) ‘18-24-1’ (aneuploidy, 2n = 93), and (L) ‘18-24-172’ (pentaploid, 2n = 5× = 100).

Figure 2

Types of male gametes in diploid and triploid cymbidiums. (A–F) represent early microspore stage where (A) The aneuploidy gamete of ‘Xiaofeng’: x-2 = 18 (arrow) and (B–F) The gamete of ‘Yuchan’: (B) 1x gamete: x = 20 (arrow), (C) Aneuploidy gamete: x+10 = 30 (arrow), (D) 2x gamete: 2x = 40 (arrow), (E) Aneuploidy gamete: 2x +2 = 42 (arrow), and (F) Unreduced gamete: 3x = 60 (arrow). Bar = 10 μm. Additionally, (G–L) represent mature pollens stained with 4, 6-diamidino-2-phenylindole (DAPI): (G) Dyad (arrow), (H) Triad (arrow), (I) Tetrad with the same size of nuclei (arrow), (J) Tetrad with two large (arrow) and two small nuclei (arrowhead), (K) Tetrad with one large (arrow) and three small nuclei (arrowhead), and (L) Tetrad with three large (arrow) and one small nuclei (arrowhead). Bar = 50 µm.

Figure 3

Pollen fertility of ‘Xiaofeng’ and ‘Yuchan’. (A) Pollen stained with 2, 3, 5-triphenyltetrazolium chloride: Pollen grains of (1) ‘Xiaofeng’ and (2) ‘Yuchan’, (3) ‘Yuchan’ pollens showed dyad (arrow) and tetrad with two small nuclei (small arrowhead) and two big nuclei (big arrowhead), and (4) ‘Yuchan’ pollens with triad (arrow). Bar=50 μm. (B) The percentage of stained pollens of ‘Xiaofeng’ and ‘Yuchan’, the same letter above the bars indicates no significant difference between cultivars analyzed by Duncan’s multiple range test at P< 0.05 level.

Figure 4

Meiotic abnormalities of sexual triploid ‘Yuchan’. (A) Univalent (big arrow), bivalent (small arrow), trivalent (big arrowhead), and multivalent (small arrowhead) observed at diakinesis. (B) Metaphase I, microspore mother cells at pachyten (arrow) or diakinesis (arrowhead) stage were observed. (C) Telophase I: microsporocyte failed to carry out meiosis I (arrow). (D) Telophase II: microsporocyte that missed meiosis I but proceeded with normal meiosis II (arrow), which resulted in the formation of dyad (arrow) (E). (F–I) Lagging chromosomes (arrow) at metaphase I, anaphase I, telophase I, and metaphase II. (J) Chromosome bridge (arrow) at anaphase (I). (K) Lagging chromosomes (arrow) and a chromosome bridge (arrowhead) at anaphase II. (L) Lagging chromosomes (arrow) and a chromosome bridge (arrowhead) at telophase II. (M) Tetrad stage: indicating micronucleus (arrow). (N) Metaphase II: tripolar spindles (arrow). (O) Metaphase II: fused spindles (arrow). (P) Tetrad stage, indicating triad (arrow). Bar = 20 μm.

Figure 5

Survival rates of hybrid seedlings with different ploidy levels after being transplanted to plastic bags containing a substrate and grown in a shaded greenhouse. Bars represent standard error, and different letters on the top of bars indicate significant difference in survival rates among hybrid progenies analyzed by Duncan’s multiple range test at P< 0.05 level.

Figure 6

Possible pathways for producing tetraploid through ‘triploid bridge’. (A) and (B) represent possible pathways in the cross of 2××3×. (C-G) represent possible pathways in the cross of 3××3×. The size of ellipse represents ploidy level of gamete, the larger the size is, the higher the ploidy level is.