Figure 1.
Pollen germination and pollen tube growth of F1 C. crassum × C. chinense on the stigma of F1 C. grandiflorum ‘Zhongshanjingui’ × A. vulgaris ‘Variegata’ plants.
(A) Germinated pollen grains on the stigma 1 hour after pollination (HAP). (B) At 2 HAP, a large number of pollen grains had germinated and many pollen tubes had penetrated the stigma. (C) At 4 HAP, the pollen tubes were growing toward the style (marked by arrows). (D) At 12 HAP, some pollen tubes were growing in the style (indicated by arrow). (E) At 24 HAP, abnormal (twisted and coiled) pollen tubes on the stigma (indicated by arrow). (F) At 48 HAP, swollen pollen tube tip (arrow), twisted tubes and callose deposition (arrow heads) on the stigmatic surface were observed. (G–I) Scanning electron micrographs: (G) pollen grains adhering to the stigma at 1 HAP, (H) short pollen tube penetrating the stigma at 2 HAP, (I) twisted and extended pollen tubes at 24 HAP. Abbreviations: Pg, pollen grain; Pt, pollen tube; St, stigma. Bars: a–f: 50 µm; g: 100 µm; h–i: 10 µm.
Figure 2.
Development of hybrid embryos in the cross between F1 C. grandiflorum ‘Zhongshanjingui’ × A. vulgaris ‘Variegata’ (♀) and F1 C. crassum × C. chinense (♂).
(A) Multicelled proembryo and free nuclei of endosperm at 2 days after pollination (DAP). (B) Globular proembryo and free nuclear endosperm at 4 DAP. (C) Early heart-shaped embryo at 6 DAP; note the crimpled integumentary endothelium and reduced number of endosperm nuclei. (D) Torpedo-shaped embryo at 8 DAP. (E) Cotyledon-shaped embryo at 10 DAP; the endosperm is almost eliminated. (F) An unfertilized embryo sac at 4 DAP. (G) Degenerated multicelled proembryo. (H) Degenerated globular proembryo. (I) Degenerated heart-shaped embryo. (J) Degenerated torpedo-shaped embryo. (K) Degenerated cotyledon-shaped embryo. (I) An abnormal cotyledon-shaped embryo; note the bilateral cotyledons elongated laterally rather than along the longitudinal axis. Abbreviations: Efn: endosperm free nucleus; Em: embryo. Scale bars: a–c and g–j: 40 µm; d–f and k–l: 80 µm.
Table 1.
Development of embryos at 4–12 days after pollination (DAP) in the cross of (Chrysanthemum grandiflorum × Artemisia vulgaris) F1 × (C. crassum × Crossostephium chinense) F1.
Figure 3.
Floral morphology of trigeneric hybrid plants between F1 C. grandiflorum ‘Zhongshanjingui’ × A. vulgaris ‘Variegata’ (♀) and F1 C. crassum × C. chinense (♂).
(A) Standard anemone type and yellow inflorescences of the maternal parent. (B) Non-anemone type and white inflorescences of the paternal parent. (C–K) Putative trigeneric hybrid plants (C: T1, double type and nacarat flower; D: T2, single type and yellow flower; E: T3, standard anemone type and white flower; F: T4, less clear anemone type and white flower; G: T5, least clear anemone type and white flower; H: tubular florets of the female parent (1), male parent (2), and the hybrid lines T3 (3), T4 (4) and T5 (5), respectively; note the pistil in the florets; I: the hybrid line T6, single type and white flower with closely set ray florets; J: the hybrid line T7, single type and white flower with ray florets more widely spaced; K: the hybrid line T8, double type and white flower with 2–3 layers of ray florets). Scale bars: a–g and i–k: 10 cm; h: 10 mm.
Table 2.
Morphological traits of Chrysanthemum grandiflorum × Artemisia vulgaris F1 (CA, ♀), C. crassum × Crossostephium chinense F1 (CC, ♂) and their putative hybrid lines.
Figure 4.
GISH analysis of the trigeneric hybrid line T3.
(A) All 54 chromosomes showing blue fluorescence after staining with DAPI. (B) Among the 54 chromosomes, nine fluoresced green after staining with the probe for Crossostephium chinense genomic DNA, and nine fluoresced red after staining with the probe for Artemisia vulgaris genomic DNA; thus the other 36 chromosomes were obtained from C. grandiflorum ‘Zhongshanjingui’ and/or C. crassum. Bars: 5 µm.
Table 3.
Aphid resistance of Chrysanthemum ‘Zhongshanjingui’ (Zh), C. grandiflorum × Artemisia vulgaris F1 (CA, ♀), C. crassum × Crossostephium chinense F1 (CC, ♂) and their trigeneric hybrid line T3.
Figure 5.
Salinity tolerance test on the trigeneric hybrid and the parents (F1 C. grandiflorum × Artemisia vulgaris, ♀, CA; and F1 C. crassum × Crossostephium chinense, ♂, CC).
Plants were cultured in Hoagland solution supplemented with (A) 0, (B) 100 and (C) 200 mmol L−1 NaCl. Left to right: CA, CC and the hybrid line T3. The arrowheads in (B) and (C) indicate the damaged leaves.
Figure 6.
Ultrastructural observation of mature leaves of the maternal parent F1 C. grandiflorum × A. vulgaris (CA) after one week of NaCl stress.
Leaf ultrastructure of plants cultured in Hoagland solution supplemented with (A–C) 0 (control), (D–F) 100 and (G–I) 200 mmol L−1 NaCl. Ch, chloroplast; CW, cell wall; Mi, mitochondria; OG, osmiophilic globules; Pl, plasmolysis; SG, starch grains. The arrowhead in (F) indicates the vesicles.
Figure 7.
Ultrastructural observation of mature leaves of the paternal parent F1 C. crassum × C. chinense (CC) after one week of NaCl stress.
Leaf ultrastructure of plants cultured in Hoagland solution supplemented with (A–C) 0 (control), (D–F) 100 and (G–I) 200 mmol L−1 NaCl. Ch, chloroplast; CW, cell wall; Mi, mitochondria; OG, osmiophilic globules; Pl, plasmolysis; SG, starch grains.
Figure 8.
Ultrastructural observation of mature leaves of the trigeneric hybrid line T3 CA × CC after one week of NaCl stress.
Leaf ultrastructure of plants cultured in Hoagland solution supplemented with (A–C) 0 (control), (D–F) 100 and (G–I) 200 mmol L−1 NaCl. Ch, chloroplast; CW, cell wall; Mi, mitochondria; OG, osmiophilic globules; Pl, plasmolysis; SG, starch grains. The arrowheads in (F) and (H) indicate the vesicles.
Table 4.
Comparison of Na+ and K+ accumulation in different organs and transport in the parents Chrysanthemum grandiflorum × Artemisia vulgaris F1 (CA, ♀) and C. crassum × Crossostephium chinense F1 (CC, ♂), and their trigeneric hybrid T3 treated with different NaCl concentrations.
Table 5.
Comparison of Na+ and K+ accumulation in different organs and transport in the trigeneric hybrid line T3 treated with different NaCl concentrations.