Ploidy Level Influences Pollen Tube Growth and Seed Viability in Interploidy Crosses of Hydrangea macrophylla

Abstract

All Hydrangea macrophylla cultivars tested to date are diploid or triploid and triploid H. macrophylla have thicker stems, larger flowers, and larger stoma compared to related diploids. It is unknown whether interploidy crosses between diploid and triploid hydrangeas can be used to develop triploid varieties. The objective of this study was to compare pollen tube development, fruit formation, and seed viability among intra- and interploidy pollinations of H. macrophylla and evaluate the genome size and pollen viability of resultant progeny. By 24 h post-pollination, pollen tubes had reached the ovaries of diploid flowers in 48.7% of samples while pollen tubes reached the ovaries in only 8.7% of triploid flowers (χ ² = 30.6, p < 0.001). By 48 h post-pollination pollen tubes reached the ovaries of diploid and triploid flowers in 72.5% and 53.8% of samples, respectively (χ ² = 26.5, p = 0.001). There was no difference in percentage of flowers with pollen tubes reaching the ovaries in diploid and triploid flowers at 72 h after pollination (χ ² = 7.5, p = 0.60). Analysis of covariance showed that pollen tube length at 24 and 48 h post-pollination was significantly influenced by ploidy and flower length of the female parent. Progeny of interploidy crosses was diploid and aneuploid; no triploid progeny were recovered from crosses using triploid parents. Mean genome sizes of offspring from each cross type ranged from 4.56 pg for 2x × 2x offspring to 5.17 pg for 3x × 3x offspring. Estimated ploidy of offspring ranged from 2x for 2x × 2x crosses to 2.4x for 3x × 3x crosses. Pollen stainability rates of flowering offspring using a modified Alexander's stain ranged from 69.6% to 76.4%.

Keywords: hydrangea; mating barrier; ornamental plant breeding; pollen tube; polyploid; triploid.

Figure 1

Stained pollen of Hydrangea macrophylla diploid cultivars Decatur Blue (A), Oakhill (B), and Zaunkoenig (C), and triploid cultivars Blaumeise (D), Nachtigall (E), and Taube (F). Fresh pollen was stained with modified Alexander's stain and observed after 30 min at 10× magnification using an Olympus BX-60 compound microscope with an Olympus Q Color 5 digital camera for image capture.

Figure 2

Stained pistils showing pollen tube growth from (A) 2x × 2x, (B) 2x × 3x, (C) 3x × 2x, and (D) 3x × 3x controlled crosses of Hydrangea macrophylla collected 24 h (left), 48 h (center), and 72 h (right) post-pollination. Crosses shown from top to bottom are: “Decatur Blue” x “Oakhill”, “Decatur Blue” x “Kardinal”, “Kardinal” x “Decatur Blue”, and “Taube” x “Kardinal”. Pistils were fixed, rinsed, hydrolyzed, stained with decolorized aniline blue, and placed on microscope slides. Slides were observed after 1 h using an Olympus BX-60 compound microscope with an Olympus Q Color 5 digital camera for image capture.

Figure 3

Mean percentage of Hydrangea macrophylla flowers with pollen tube reaching the ovaries at 24, 48, and 72 h post-pollination. 2 = 2x = diploid; 3 = 3x = triploid. Means are based on six crosses per cross type per time period. Error bars represent standard error of the mean. Pearson's chi-square values for association between cross type and percentage of flowers with pollen tubes reaching the ovaries were χ ² = 30.6, p < 0.001 at 24 h, χ ² = 26.5, p = 0.001 at 48 h, and χ ² = 7.5, p = 0.60 at 72 h post-pollination.

Figure 4

Hydrangea macrophylla seedlings from interploidy and interploidy crosses after five months of growth in a greenhouse under 56% shade at 24°C/20°C day/night and natural light. Photo taken 11 July 2018 in McMinnville, TN, USA.