Introduction

Zygote is a unique cell from which the life cycle of angiosperms begins. It is a product of fertilization between the sperm and the egg cell. Zygote undergoes organized divisions and cell-specifications to give rise to an embryo, a young sporophyte. The process of development of an embryo from a zygote is known as embryogenesis. A particular pattern, form, and polarity exists during the development of embryo which is the outcome of several cellular, molecular and genetic mechanisms. These programmed changes enable the embryo to form the future sporophyte as a unique entity. The present chapter deals with the embryogenesis in angiosperms, encompassing embryo patterning, genetics and physiology involved. Embryos can also develop from somatic cells under in vitro conditions and resulting embryos are known as somatic embryos and the process as somatic embryogenesis. In this chapter, the term embryogenesis will be used for only zygotic embryogenesis.

Structure of the Embryo

Typical mature embryos in both monocots and dicots are similar in the basic design as they share similar embryogeny, at least up to a particular stage (i.e., octant, as will be discussed in Section 10.4); after which ontogenetic differences appear between them. A typical dicot embryo comprises of an embryonal axis with two cotyledons attached to it laterally. The part of embryonal axis above the level of cotyledons is known as the epicotyl; which terminates into the embryonic shoot (also called plumule). The part of embryonal axis below the level of cotyledons is known as the hypocotyl; which gives rise to an embryonic root (also called radicle) (Fig. 10.1 A). A typical monocot embryo differs from a dicot embryo in having only one cotyledon attached to the embryonal axis (Fig. 10.1 B).

The embryos in cereals like Zea mays, Triticum sp., Oryza sp. need special mention because of additional embryonic organs associated with them namely, scutellum, coleoptile and coleorhiza (Fig. 10.1 C). Scutellum is thought to be a seed leaf or a single massive cotyledon in cereals, which fully covers the embryo. Lateral to the scutellum, a short embryonal axis is attached which is divided into an epicotyl (above the level of scutellum) and a hypocotyl (below the level of scutellum). The epicotyl comprises of several young leaves covered by a sheath called coleoptile.

Introduction

In the previous chapter, we learnt that pollination results in transferring of pollen grains from the anther to the stigma of a flower. Such a transfer or landing of pollen grains initiates a series of events that involve a continuous exchange of signals between the haploid pollen and the diploid maternal tissue of the pistil. These events and interactions which include pollen selection or rejection, pollen hydration, pollen tube growth, its nourishment and entry into ovules and embryo sac are recognized as the pollen–pistil interactions, (Herrero & Hormaza 1996; Shivanna 2003; Lora et al. 2016). The pollen–pistil interactions result in screening and selection of conspecific/homospecific pollen (of same species) from the heterospecific pollen (pollen of other species) ensuring fertilization only between the conspecific male and female gametes.

In a pistil, while stigma is the landing platform and recipient of the pollen, the style is the conduit for the transfer of non-motile male gametes to the embryo sac seated in the ovules with the help of pollen tubes. The process of delivery of non-motile sperm to the egg via a pollen tube is known as Siphonogamy. It is regarded as a key innovation in the course of evolution of angiosperms that has allowed flowering plants to carry out sexual reproduction on land without the need for water. Flowering plants have an elaborate screening process for selecting the right pollen and the pollen tubes. This system works at different levels in the pistil. The selection of pollen starts at the stigma itself which allows only the compatible pollen grains to germinate while the incompatible ones are rejected. The selected pollen grains then germinate and put forth pollen tubes which grow in the style, where again a competition takes place to select the best mate. The pollen tubes travel at a rate specific to each species to ultimately reach the ovule. A study by Williams (2008) covering about 130 seed plant families and 717 taxa suggested that the time interval between pollination and fertilization ranges between 15 minutes to >12 months in angiosperms. This time interval is referred to as the fertilization interval.

Introduction

Angiosperms possess a vast diversity of flowers which serve various purposes for the different groups of living beings, including humans. Due to their color, fragrance, and beauty flowers have always occupied a special place in human lives. Flowers are considered sacred across most cultures and have inspired much artistic expression. Apart from their aesthetic value, flowers possess myriad medicinal properties that further enhance their value to humans. Describing from a botanist's perspective though, the flower is a unit of reproduction in angiosperms. A flower may be defined as a modified determinate shoot system with four distinct whorls, viz. calyx, corolla, androecium and gynoecium arranged on a receptacle. Outer whorls, calyx and corolla are leaf-like structures which are not directly involved in reproduction. The two inner whorls, the androecium and the gynoecium harbor the reproductive organs of the flower and are the ones involved in reproduction. Flowering plants exhibit enormous diversity in size, shape, color, symmetry and the other morphological features (Fig. 2.1). This diversity in floral forms plays a huge role in ensuring pollinator services by different groups of pollinators. The diverse forms of flowers are accompanied by an array of mating strategies and sexual systems in angiosperms.

The timing of flowering in plants is critical for their reproductive success as both late and premature flowering can limit proper seed development. Plants also attempt to realize their reproductive potential by synchronizing their flowering to match pollinator availability. Floral induction is promoted by distinct environmental cues such as photo-period, vernalization and endogenous regulators like phytohormones. These signalling cues are perceived in the leaves and the shoot apical meristem (SAM) for induction of flowering. Plants use genetic machinery to control all events starting from induction of flower to development of different whorls. Research in the last few decades has identified numerous genes which are involved in floral induction, floral meristem formation, and floral organ development. Genes which control floral organ development are called floral organ identity genes. These genes belong to the MADS box gene family and are also known as homeotic genes. The functioning of these genes is explained by the ABCDE model of flower development. This chapter gives an outline of the organization of a flower, sexual system seen in angiosperms and a summary of the components that play important role in the floral induction and floral organ development.

Introduction

Sexual reproduction involves delivery of sperm cells, via the pollen tube, to the egg cell present in the embryo sac, where fertilization occurs and the new sporophyte is formed (Dumas & Mogensen 1993). While the formation of male gametophyte (pollen grains) takes place within the anther, the female gametophyte (embryo sac or megagametophyte) develops within the ovule. Thus, the ovule can be defined as a specialized sporophytic structure within which development of female gametophyte or mega-gametophyte takes place. Ovule is the site for delimitation of megasporocyte, production of a functional megaspore (megasporogenesis) and eventually formation of embryo sac (megagametogenesis). The embryo sac harbors the female gamete or the egg, which subsequently gets fertilized by the male gamete to form an embryo. In angiosperms, apart from the female gametophyte and egg cell development, important reproductive events such as pollen tube attraction and guidance, double fertilization, and embryo and endosperm development all occur within the ovule. The ultimate result of all these events is the formation of seed and therefore, the ovule is also considered the developmental precursor or progenitor of the seed.

Among angiosperms, different modes of female gametophyte ontogeny are seen, leading to different types of female gametophytes. The cells of female gametophyte are very peculiar in their ultrastructure and with the help of electron microscopy, great details of these cells are known. In various species, besides the typical parts of ovule, there are many specialized structures associated with the ovules which aid in pollen tube guidance and facilitate fertilization. All these aspects of structure and development of the angiosperm ovule, female gametophyte and their types have been discussed in the present chapter. The chapter also includes exceptions to these developmental patterns and details of extra ovular structures.

Basic Structure of Ovule

In general, ovules among angiosperms are fundamentally similar in their basic structure, consisting of three major tissues: a nucellus, protective coat(s) or integument(s), and a funiculus. Besides these, a typical ovule also consists of a micropyle, a chalaza and its vascular supply (Fig. 5.1). In angiosperms, the ovules remain enclosed in the ovary, and a stalk like structure through which ovules remain attached to the ovary wall or placenta is known as the funiculus.

The inception of interest in plant reproduction is as old as the inception of interest in biology. The science of sexual plant reproduction is more than 400 years old. During all these years there has been an accumulation of information which has greatly enriched our understanding of plant reproduction. Our current knowledge of plant reproduction is a result of continuous efforts of scientists world-wide that transformed it from an observational investigation to an important field of experimental science. This chapter provides a summary of important mile stones in the history of reproductive biology of flowering plants. To maintain conciseness, only significant contributors across the world including India have been mentioned in the chapter without undermining the importance of others whose ideas and concepts have shaped the science of plant reproduction today.

Early Discoveries

The long and venerable history of studies in plant reproduction dates back to seventeenth century with the ideas of European naturalists Rudolf Jacob Camerarius in Germany and Nehemiah Grew in England. Though, Grew first proposed the idea of sexual processes occurring in plants for generation of seeds, to Camerarius must go the principal credit for experimentally establishing the existence of plant sexuality. N. Grew, in an address to the Royal Society of London in 1676, had expressed the view that the stamens are the male organs of a flower and the pollen act as vegetable sperm. He is credited for documenting stamens as the male sex organ of plants in his book The Anatomy of Plants (1682). The experiments of Camemarius were primarily based on the removal of stamens and styles along with isolation of female plants in dioecious species. He provided evidence for the inevitability of both sex organs in seed formation. His publications On the Sex of Plants (1694) and Botanical Works (1697); are landmarks in the history of botany.

In the history of plant reproduction, Adam Zalužanský is a little-known botanist. He was a professor at the University of Prague and his book Methodi herbariae libri tres was published in 1592 and 1604. This book includes a chapter De sexu plantarum in which, almost a century before the work of Camerarius, sexuality of plants was suggested.

Introduction

Strasburger's work in Monotropa identified the embryo as the product of fertilization of egg cell by the one of the male gametes. The mystery around the fate of the second male gamete discharged by the pollen tube was resolved through the discovery of double fertilization by Nawaschin (1898). It is a common knowledge now, that one of the male gametes undergoes fusion with the nucleus of the egg cell and the other fuses with the two polar nuclei of the central cell. The fusion of three nuclei in the latter is known as triple fusion which was also referred to as vegetative fertilization by Strasburger in 1900. Triple fusion results in a triploid nucleus known as Primary Endosperm Nucleus (PEN), which divides and forms the endosperm. Thus, double fertilization initiates development of embryo and endosperm. Discovery of triple fusion led to questions like what is endosperm and what role does it play? The term endosperm means ‘with-in the seed,’ i.e., a tissue that develops inside a seed. A plethora of studies have established that endosperm is the nutritive tissue for a growing embryo inside a seed. The two tissues are closely connected in their growth within a seed, reflecting the importance of the embryo-endosperm relationship. Recent investigations show that failure of endosperm formation leads to the abortion of the developing embryo which establishes that embryo development is regulated by endosperm.

Formation of the PEN is a well-organized event which is preceded by several ultrastructural changes in the central cell. The PEN follows different developmental pathways forming the basis for classifying the types of endosperm. The PEN and the endosperm cells are mostly triploid but the ploidy level may vary with the type of female gametophyte from which a central cell develops. In most angiosperm families, the endosperm is short-lived and the developing embryos consume the endosperm completely before germination. This leaves mature seeds without any endosperm and such type of seeds are known as non-endospermous or ex-albuminous seeds, e.g., Cucurbita, pea, and beans. In other angiosperms, endosperms act as a storage tissue and persist in mature seeds. Such seeds where endosperm is present at maturity are known as endospermous or albuminous seeds, e.g., cereals, coconut, and castor bean.

Introduction

Life cycle of an angiosperm is characterized by alternation of generation between a diploid sporophyte and a haploid gametophyte. Unlike lower plants, gametophytic generation in angiosperms is much shorter and dependent on sporophytic generation for its development. Gametophyte develops from the cells of a sporophyte in preparation for reproduction. The gametophytic cells undergo meiotic division and produce haploid gametes within the specialized structures of a flower. While the male gametophyte develops within the anther, the female gametophyte develops within the ovule. Pollen grain is the male gametophyte in flowering plants and contains the two male gametes (also called the sperm cells). Pollen grains are also involved in the formation of pollen tubes to facilitate the movement of sperm cells for fertilization with female gametes.

The male reproductive organ in flowering plants is the stamen. It consists of two morphologically distinct parts, the anther and the filament (Fig. 4.1 A). Filament is an entirely sporophytic structure which anchors the stamen to the flower. It also contains vascular tissue for transporting water and nutrients. The anther on the other hand contains both sporophytic and gametophytic tissues that are responsible for producing and releasing pollen grains. Anther development is a perfectly timed and orchestrated event which follows different pathways in different groups of angiosperms. Development of pollen grains (male gametophyte) takes place within the anther and is divided into two phases. It begins with the meiosis in the microspore mother cells to produce four haploid microspores, each of which later develops into a pollen grain and the process is called as microsporogenesis. This is followed by a second phase of pollen development where the formation of two sperm cells takes place and the process is known as microgametogenesis.

Pollen development includes participation of various sporophytic cells of the anther and the associated molecules. Pollen grains vary immensely in size, shape and surface characteristics among different plant species. At maturity, the pollen grains are surrounded by an elaborate cell wall which consists of a thin inner wall known as the intine, and an outer thicker wall called the exine. The shape and the external features of the exine are highly variable, and often used to distinguish pollen grains produced by different species.

A community has a diverse suite of definitions, as many as there are scientific fields and sociological units. Among humans, there is a place-based variant such as a city defined by political boundaries, or an interest-based variant focused on a group of people defined by their interactions. The same issues plague the field of community ecology, especially when we address a topic such as a mycorrhiza, which is a functional relationship. Early measurements of communities looked for spatial boundaries, the edge of a meadow; a shift in forest type with a physical edge such as a shift in topography; an obvious shift in plant types such as a forest edge. Theophrastus (~44 BCE) noted that most mushrooms were found in forests (which included EM species) but not grasslands (which were almost exclusively AM). Alexander von Humbolt drove some of the earliest developments in community and ecosystem ecology by showing relationships between climate and vegetation both up elevation gradients, such as his beautiful and accurate 1807 Tableau Physique, drawings of vegetation up Mount Chimborazo in Ecuador, and his Geographical Distribution of Plants – climate consortia of plants – in relation to global climate patterns (371).

The definition of symbiosis is two organisms living intimately together, and this chapter examines the physiological basis of the interaction. A mycorrhiza is comprised of two distinctly different organisms, a plant and a fungus, that interface down to the molecular level. Because of this intimate physical closeness, the biochemistry, physiology, and ecology become highly intertwined. At the most basic definitional level, the fungus picks up nutrients and water in the soil, transfers those resources to the host, in exchange for carbon fixed by the plant from the atmosphere. This physical dimension means that resources available to one partner are less available to the other. But both sets of resources are essential to both organisms.

The study of population ecology in plants is as old as the field of ecology but is more complex for fungi. Due to their microscopic morphology, identifying individuals for measuring and modeling is challenging. We have examined the general morphology of fungi and plants comprising the mycorrhizal symbiosis, and we have looked at the larger-scale evolutionary patterns that resulted in the mycorrhizae that we observe and study today. However, selection acts on the individual organism (472). An organism survives to reproduce offspring that in turn reproduce, or it does not: a binary outcome. And, an organism is comprised of a complete genetic code that allows it to survive to reproduction (or not).

Probably no research topic in mycorrhizae has undergone as much change over the past few decades as the evolution of the symbiosis. The rapid development of techniques and reduction in costs of sequencing, increase in databases and new approaches to sequence database management, data mining, and sequencing analyses has generated a plethora of new phylogenic reorganization, molecular clocks, and theory. Newer sequencing concepts often readily integrate with the fossil record as the field of paleoecology itself rapidly evolves. But for understanding the mechanisms of evolution in a symbiosis, we need to go beyond phylogenetic relationships to understanding both the role of and the shifts in environments that determine how mycorrhizae develop, adapt, and diversify. Here I will summarize the key topic areas relating to mycorrhizal symbiosis, recognizing that there are likely many ideas that will change in the near future. Specifically, I address the hypotheses that: (1) mycorrhizae were crucial to the invasion of land and related to the regulation of atmospheric CO2, (2) mycorrhizal symbioses are fundamentally stable, and (3) there are both genetic and ecological underpinnings supporting the mycorrhizal symbiosis. Here I explore the four lines of evidence of how evolution has played a key role in the ecology of modern mycorrhizae (36), including (1) paleobiology evidence, (2) extant plant mycorrhizal status, (3) the molecular basis of interaction, and (4) models of mutualism.