Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea

Abstract

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry of pollen tubes at different developmental stages that were cultured in vitro revealed changes in the nuclear phase at 9 h and 18 h after culture. Sperm cell formation occurred at 6-9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cell showed a tendency to increase. The association was suggested as male germ unit. When sperm cells, vegetative nuclei, and partial male germ units were separately collected from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial male germ units was counted as 2C. Therefore, the second change in the nuclear phase, which resulted in an increase in 2C nuclei, was possibly related to the formation of male germ units.4

Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii

“…That the proportion of 3C DNA did not change (Table 1) suggests that no changes in MGU were detected by flow cytometer from 12 to 36 h of culture. Isolated paired sperm cells from pollen grains often separate in buffer solution (Zhang et al 1998), and a similar phenomenon occurred in our previous study on FCM analysis of pollen tubes (Hirano and Hoshino 2009). Therefore, some MGUs appear to dissociate into individual cells and nuclei, or into vegetative nucleus and pair of sperm cells during isolation or in the extraction buffer used for FCM.…”

“…Since the FCM-based method for analyzing pollen tubes could be applied to A. aurea (Hirano and Hoshino 2009) and C. mackenii, we believe that this method may be applicable for the analysis of pollen tubes of other species, as well, and has the potential to be used as a high throughput technique for preparing MGU material for molecular approaches in combination with a cell-sorting system. In the present study, we detected two kinds of sperm dimorphisms in terms of nuclear shape and microtubule accumulation and prior work has indicated that microtubules play a role in cell shaping, cell polarity determination, and vesicular transport (Southworth and Cresti 1997).…”

“…f. (Amaryllidaceae) plants used in this study were greenhouse grown with anthers were collected from the flowers after dehiscence and maintained at -20°C. Pollen grains from two anthers were sown in 2 ml of liquid pollen culture medium (Hirano and Hoshino 2009) and cultured at 25°C in the dark. For microtubule depolymerization, oryzalin (final concentration, 5 μM) was added to culture medium after 12-h incubation, and the pollen grains were cultured for an additional 12 h. During 6-36-h culture, pollen tubes were collected at appropriate intervals from the culture medium using a 77-μm nylon mesh and were used for the subsequent male gamete analysis.…”

“…Pollen tubes and isolated cells and nuclei were analyzed by FCM according to the method described by Hirano and Hoshino (2009). To determine nuclear ploidy level in pollen tubes, we collected pollen tubes as noted above and transferred them to 200 μl of nuclear extraction buffer (CyStain UV Precise P kit; Partec, Münster, Germany), chopped tubes with a sharp razor blade, and incubated then with 800 μl of staining buffer from the CyStain UV Precise P kit.…”

“…We previously reported that flow cytometry (FCM) analysis combined with micromanipulation is useful for a rapid evaluation of the general behavior of male gametes during pollen tube growth in Alstroemeria aurea Graham (Hirano and Hoshino 2009). However, the applicability of this method to other species has not yet been examined.…”