Yeung Bot Stud (2017) 58:33 DOI 10.1186/s40529-017-0188-4 REVIEW Open Access A perspective on orchid seed and protocorm development Edward C. Yeung* Abstract This perspective draws attention to the functional organization of orchid seed and protocorm during the course of development. The orchid embryos have a well-organized developmental plan generating a blue-print of a protocorm as they mature. The diferent phases of embryo development in orchids, i.e. histodiferentiation, storage product syn- thesis and accumulation, and maturation are essentially similar to other fowering plants. The protocorm is considered as a unique structure designed to establish symbiotic association with mycorrhizal fungi and with the primary goal to form a shoot apical meristem. This perspective brings forth arguments that the processes of embryo and proto- corm development are highly programmed events, enhancing survival of orchid seeds and plantlets in their natural habitats. Furthermore, the ability of protocorm cells to divide, makes them ideal explants for micropropagation and transformation studies. Through seed germination and micropropagation using protocorms as explants, orchid con- servation eforts are greatly enhanced. Keywords: Embryo, Suspensor, Endosperm, Seed coat, Protocorm, Phytohormones, Shoot apical meristem, Regeneration, Protocorm-like bodies, Orchid mycorrhizae, Micropropagation Background embryo development in orchids, i.e. histodiferentiation, An understanding of plant reproductive biology is essen- storage product accumulation, and maturation are essen- tial to plant conservation eforts. In orchids, after a suc- tially similar to other fowering plants. Te protocorm cessful pollination event, numerous seeds are produced formed at the time of seed germination is considered as within a single capsule. Utilizing seeds through asym- a unique structure designed to establish symbiotic asso- biotic and symbiotic seed germination for plant main- ciation with mycorrhizal fungi and with the primary goal tenance and propagation are key approaches in orchid to form a shoot apical meristem. Tis perspective brings conservation eforts. In order to further optimize cur- forth arguments that the process of embryo and proto- rent methodologies on seed germination and plantlet corm formation involves highly programmed events, establishment, we need to have a proper understanding enhancing survival of orchid seeds and plantlets in their of seed biology, as well as the structure and function of natural habitats. Te intent of this article is to generate protocorm. additional interests, discussions, and further studies on Key features of orchid embryos, seeds, and protocorms orchid seed biology in the near future. are summarized in this article. Te globular-shape and small sizes of orchid embryos imply that the embryos Embryo development are simple and not as well developed as in other fower- The zygote and the proembryo ing plants. On the contrary, the author sees that orchid In fowering plants, a successful fertilization event results embryos have developed a plan, generating a blue-print in seed formation in which an ovule becomes a seed, of a protocorm as they mature. Te diferent phases of the egg cell becomes a zygote, the central cell forms the endosperm, and the integuments develop into the seed coat. Detailed accounts on changes of the egg cell before *Correspondence: [email protected] Department of Biological Sciences, University of Calgary, Calgary, and after fertilization about orchids are few. In Epiden- AB T2N 1N4, Canada drum scutella, after fertilization, the size of the zygote is © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Yeung Bot Stud (2017) 58:33 Page 2 of 14 similar to the egg cell and there is little change to the vac- the varied patterns of suspensor formation, and limited uolar system within its cytoplasm (Cocucci and Jensen number of cells in the embryo proper, suggest new regu- 1969). Rough endoplasmic reticulum and polysomes latory controls in proembryo establishment in orchid. become more abundant, indicating an increase in meta- Based on the cell division pattern in the zygote and bolic activities within the cell. It is interesting to note proembryo, as well as the varied forms of suspensor that dictyosomes could not be detected in the zygote morphologies, diferent embryo types were proposed after fertilization (Cocucci and Jensen 1969). At the light (Swamy 1949; Johansen 1950). Moreover, the suggested microscopic level, the zygote maintains a uniform dense embryological system of classifcation sees limited use by cytoplasm, e.g. Cymbidium sinense (Yeung et al. 1996) embryologists. More recently, Clements (1999) provided and Calypso bulbosa (Yeung and Law 1992), or it can a more extensive description of embryo developmental have a polarized appearance with a distinct vacuole(s) types, incorporating the classifcation system proposed located at the micropylar end such as in Cypripedium by Dressler (1993). passerinum (Law and Yeung 1993) and Phaius tanker- villiae (Ye et al. 1997). Te phenomenon of ‘zygote The suspensor shrinkage’ (see Natesh and Rau 1984) prior to the frst Te diferent morphologies of the suspensor within zygote mitosis appears to be absent. Changes in micro- Orchidaceae generate a lot of interest in the study of tubule cytoskeleton have been noted, refecting dynamic embryo development (see Yam et al. 2002). Te suspen- changes within the zygote prior to its frst mitotic divi- sor can appear as a single cell, highly elongated hausto- sion. Soon after zygote formation, the microtubules are rial structures, or multicellular swollen forms that fll the randomly arranged. As the zygote increases in size, the embryo cavity (Swamy 1949). What directs the growth microtubules organize into a meshwork in the cytoplasm and morphogenesis of the varied suspensor forms? In and a dense array of microtubules begins to appear in the the study of the nun orchid suspensor development, cortical region near the chalazal pole (Huang et al. 1998). cytoskeletal elements, i.e. microtubules and microfla- Moreover, additional studies are needed to decipher cel- ments, play an important role in suspensor morpho- lular events leading to the establishment of cell polarity genesis (Ye et al. 1997), especially in the elongation of within the zygote at this critical stage of development. suspensor cells. Dynamic changes in the microtubule In the zygote, varying degree of vacuolation appears cytoskeleton are also observed in C. sinense during the within its cytoplasm. In C. bulbosa (Yeung and Law course of suspensor formation and elongation (Huang 1992) and C. sinense (Yeung et al. 1996) the cytoplasm et al. 1998). However, other extrinsic and intrinsic sig- of the zygote remains dense through the frst mitotic nals regulating suspensor development are not known at division. In these examples, the frst zygote division is present. usually symmetrical or slightly asymmetrical in appear- With the small size of the embryo proper and the lack ance. For orchids having a polarized zygote with large of an endosperm, it is logical to speculate that the varied vacuole(s) located at the micropylar end of the cell, such forms of suspensors can play important roles in nutri- as those found in C. passerinum (Law and Yeung 1993) ent uptake. Histochemical staining of cell walls indirectly and Phaius tankervilliae (Ye et al. 1997), the frst division supports the notion that orchid suspensor can serve as a is asymmetrical giving rise to two cells of unequal sizes. nutrient uptake site. Te polychromatic stain toluidine Changes to the orientation and distribution of the micro- blue O (TBO) can distinguish lignin, cellulose, and pec- tubule cytoskeleton are observed at this time (Huang tic substances on the basis of color diferences (O’Brien et al. 1998). Irrespective of the cell division symmetry, et al. 1964; Feder and O’Brien 1968; O’Brien and McCully cell division results in the formation of a terminal and a 1981). Te fact that the suspensor cell wall stains pink- basal cell, each having a distinct fate. Te basal cell can ish-purple with no detectable lignin and cutin deposits, diferentiate into a short-lived embryonic organ, known suggests that water and water soluble substances should as the suspensor, and the terminal cell will give rise to the be able to move apoplastically from the seed coat to the embryo proper. developing embryo proper via suspensor cells (Lee and One of the key events occurring after zygote division is Yeung 2010). Te pinkish-purple color of the suspensor the establishment of a polarized embryo axis leading to wall after TBO stain also suggests the presence of nega- cell fate determination and the formation of a body plan tively charged groups which can increase cation-binding (Ueda and Laux 2012; Wabnik et al. 2013). Such infor- capacity of a wall (Smith 1972), and this can aid in solute mation similar to the study of Arabidopsis is lacking in uptake. A simple plasmolysis experiment indicates that orchids. Te oblique divisions observed in the formation the suspensor cell in the nun orchid has a more negative of the proembryo in C. sinense (Yeung et al. 1996), the osmotic potential than the surrounding seed coat cells changes in microtubule organization (Huang et al. 1998), (Lee and Yeung 2010). Te negative osmotic potential Yeung Bot Stud (2017) 58:33 Page 3 of 14 provides a driving force for water uptake by the suspen- has a patchy appearance and it is initially absent from sor. In order to visualize the uptake pathway, a fuores- the walls at the chalazal end (Yang and Lee 2014). Tese cent tracer 6-carboxyfuorescein diacetate (CFD) was structural features indicate that the C. javanica embryo is applied to developing seeds.