Histological and Micro-CT Evidence of Stigmatic Rostellum Receptivity Promoting Auto-Pollination in the Madagascan Orchid Bulbophyllum bicoloratum

Abstract

BackgroundThe rostellum, a projecting part of the gynostemium in orchid flowers, separates the anther(s) from the stigma and thus commonly prevents auto-pollination. Nonetheless, as a modified (usually distal) portion of the median stigma lobe, the rostellum has been frequently invoked of having re-gained a stigmatic function in rare cases of orchid auto-pollination. Here it is shown that a newly discovered selfing variant of Madagascan Bulbophyllum bicoloratum has evolved a modified rostellum allowing the penetration of pollen tubes from in situ pollinia.MethodsGynostemium micro-morphology and anatomy of selfing and outcrossing variants of B . bicoloratum was studied by using light and scanning electron microscopy and histological sections. Pollen tube growth in the selfing variant was further observed via X-ray computed microtomography (micro-CT), providing 3D reconstructions of floral tissues at a micron scale.FindingsSelfing variants possess a suberect (‘displaced’) rostellum rather than the conventional, erect type. Very early in anthesis, the pollinia of selfers are released from the anther and slide down onto the suberect rostellum, where pollen tube growth preferentially occurs through the non-vascularized, i.e. rear (adaxial) and (semi-) lateral parts. This penetrated tissue is comprised of a thin layer of elongate and loosely arranged cells, embedded in stigmatic exudates, as also observed in the stigmatic cavity of both selfing and outcrossing variants.ConclusionsOur results provide the first solid evidence of a stigmatic function for the rostellum in orchid flowers, thereby demonstrating for the first time the feasibility of the micro-CT technique for accurately visualizing pollen tube growth in flowering plants. Rostellum receptivity in B . bicoloratum probably uniquely evolved as an adaptation for reproductive assurance from an outcrossing ancestor possessing an erect (non-receptive) rostellum. These findings open up new avenues in the investigation of an organ that apparently re-gained its ‘primordial function’ of being penetrated by pollen tubes.

Plant Tissues in 3D via X-Ray Tomography: Simple Contrasting Methods Allow High Resolution Imaging

“…Although our results allow the straightforward use of CT for qualitative morphological work [43], quantitative applications are now more straightforward as well. Our results allow for the streamlined acquisition of high-resolution phenotypic data, which can be correlated with genomic, transcriptomic, or metabolomic data via the setting of morphometric landmarks, in order to advance our understanding of phenotypic evolution and developmental trajectories [1].…”

Evaluation of X‐ray tomography contrast agents: A review of production, protocols, and biological applications

“…Madagascan orchid was reported by Gamisch, Staedler, Schönenberger, Fischer, and Comes (), where the CT technique was applied to plants to evaluate the pollen tube structure. Researchers applied various visualization methods such as electron microscopy, optical microscopy, and micro‐CT technique.…”

<p align="left">Gastrodia flexistyloides (Orchidaceae), a new mycoheterotrophic plant with complete cleistogamy from Japan

“…The usual mechanism of autonomous self-pollination involves the pollinia falling onto the stigma surface, allowing contact between the pollinia and stigma (e.g. Liu et al 2006, Chen et al 2012, Suetsugu 2013a, b, c, 2014, but also see Gamisch et al 2013). However, the autogamous system of G. flexistyloides is notable because the central lobe of the column, which is strongly incurved, also contributes to the pollination process, allowing the anther to contact with stigma directly and thus facilitate selfpollination.…”