Guidance in Vitro of the Pollen Tube to the Naked Embryo Sac of Torenia fournieri

Abstract

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.

Male and female synchrony and the regulation of mating in flowering plants

“…In all species studied, synergid degeneration occurs on pollen tube penetration ( Jensen 1965;Russell 1996), and recent work with ablation experiments clearly demonstrated that the synergids are required for pollen tube penetration into the embryo sac (Higashiyama et al 2001). Further, embryo sacs with degenerated synergids fail to attract pollen tubes (Higashiyama et al 1998). As the synergid degenerates, an actin corona has been reported to develop in mature embryo sacs (Huang & Russell 1994), which has been shown to play a central part in the delivery of the male gametes to the egg cell and polar nucleus (Russsell 1996;Fu et al 2000) …”

Species Preferentiality of the Pollen Tube Attractant Derived from the Synergid Cell ofTorenia fournieri

“…In Torenia fournieri, pollen tubes are directly attracted to the exserted embryo sac in vitro (Higashiyama et al, 1998). Laser ablation experiments identified the source of the diffusible signal as the two synergid cells (Higashiyama et al, 2001).…”

“…By mixing ovules of two species, one can critically examine whether pollen tubes sense the attraction signal of a different species under conditions in which they can surely respond to the attraction signal of their own species. In addition, the condition of the synergid cell is easily observed in the exserted embryo sac, but the attraction can only be examined in embryo sacs with complete synergid cells (the synergid is a fragile cell; Higashiyama et al, 1998). Ablation experiments using UV lasers are also possible in the exserted embryo sac, as shown in T. fournieri (Higashiyama et al, 2001), and can be used to confirm the role of the synergid cell in pollen tube attraction.…”

Toward in vitro fertilization in Brachiaria spp.

“…The use of a style for pollen germination in Torenia is required to activate targeting of pollen tubes (Higashiyama et al 1998), but in Brachiaria this is not possible because of a barrier mechanism in the style. Potentially, this step may not be required, as in Gasteria (Willemse et al 1995), where free pollen is easy to handle, efficient and fully functional.…”

“…The establishment of in vitro fertilization in general and specifically in Brachiaria can be useful as a tool for plant breeding (Willemse et al 1995;Higashiyama et al 1998). Culture media are used to germinate pollen and to maintain the viability of the buds, or organs.…”