Summary

Maize is a flowering plant well suited for biological research on reproduction processes. In addition to interesting sexual traits, there is in this species a large amount of data regarding genetics and cytogenetics. The embryo sac and the female germ unit have been characterized, and precise studies have been developed on male gametes. Finally, the time course of double fertilization has recently been determined. Nevertheless, the intimate mechanisms of double fertilization are still unknown. A new way to approach this phenomenon is to develop systems of “in vitro fertilization.” In this chapter, the technical procedures to isolate and manipulate male and female gametes in maize are described. The embryo sac and egg cells are prepared with the help of an enzymatic treatment. Sperm cells or male nuclei from viable pollen grains are released in an acidic medium and selected from a specific layer of a Percoll gradient after centrifugation steps. With these cellular tools, two in vitro methods of fertilization have been developed: (1) spontaneous fusion of male and female gametes in the presence of 5 mM of calcium and (2) microinjection of male nuclei either into the egg or into the central cell of a mature embryo sac. Culture of the artificial zygotes and microinjected embryo sacs is in progress. All these techniques provide a new window for investigating the first steps of fertilization and early embryogenesis at the cellular and molecular levels.

Introduction

Camerarius (1694, reviewed in Ducker and Knox 1985) was the pioneer in plant reproductive biology.

Summary

The field of self-incompatibility research is at present highly dynamic, and an understanding of the mechanisms of operation of the two most widespread systems is almost within our grasp. This review brings together information on the main types of self-incompatibility systems, outlines the potential benefits of the ability to manipulate self-incompatibility systems to agriculture, and explains how recent breakthroughs made in this field may allow the plant breeders to have this capability within the foreseeable future.

Introduction

Self-incompatibility (SI) is a genetic system possessed by many flowering plants. Indeed, SI has been reported in 66 plant families, representing every major phylogenetic line of the angiosperms (Brewbaker 1957), and it can be defined as “the inability of a fertile hermaphrodite seed plant to produce zygotes after self-pollination” (Lundqvist 1964, p. 222). The significance of SI in the evolutionary context cannot be overstated, for its possession leads to obligate outbreeding and the maintenance of heterozygosity within a species (Stebbins 1950).

Among crop and ornamental plants, most of the perennial grasses (Gramineae), forage legumes (Fabaceae), and members of the Brassicaceae (cabbage, kale, and so forth), Asteraceae (sunflowers, cosmos), Rosaceae (apples, cherries, pears, and so forth) and Solanaceae (petunia, potato, tobacco, and tomato relatives) have SI mechanisms of varying kinds and degrees of effectiveness.

SI presents contrasting prospects to plant breeders. On one hand it will frustrate efforts to produce homozygous lines, but on the other it provides a way to hybridize two lines without emasculation, nuclear or cytoplasmic sterility, or resorting to gametocides.

Summary

There has been long-term interest in cytoplasmic male sterility (CMS) in plants because it provides, at least in theory, a means to produce commercial quantities of hybrid seed for plants where this would otherwise be difficult or impossible. CMS has been observed in at least 150 different plant species. CMS systems have traditionally been characterized by the restorer genes required to overcome the CMS and to provide male-fertile progeny in the male-sterile cytoplasm. More recently, CMS systems have been characterized by DNA analysis techniques applied to cytoplasmic organelles that are able to distinguish genetic differences between normal and male-sterile cytoplasms. CMS systems of pollination control have been developed in several major field and horticultural crops. These CMS systems consist of a malesterile female “A-line,” a male-fertile maintainer “B-line,” and a male-fertility restorer “R-line.” There are a number of limitations associated with the use of CMS in the development of hybrid cultivars. Even with these limitations, CMS is the predominant method of pollination control for hybrid seed production for hybrid cultivars throughout the world. Current research is aimed at enhancing our understanding of the molecular basis of CMS and, through this, enhancing our understanding of improving and extending CMS as a pollination control mechanism. Research on future uses of CMS is focused on the development of cytoplasmic gene transformation systems using CMS components.

Introduction

Plants that do not produce viable, functional pollen grains are male sterile. If such male sterility is exclusively maternally inherited, it is described as cytoplasmic male sterility (CMS). Since the first reference to CMS (Bateson and Gairdner 1921), there have been more than seven hundred papers published on CMS in plants.

Pollen biology

Pollen grains embody the male partners in sexual reproduction. They are generally shed in a desiccated condition and their moisture level is less than 20%. At the time of shedding, pollen grains are either two-celled – a large vegetative cell enclosing a generative cell; or three-celled – a vegetative cell and two sperm cells formed by the division of the generative cell. There is considerable variation in the size and shape of pollen grains (Erdtman 1966; Moore and Webb 1978; Iwanami et al. 1988; Faegri and Iversen 1989; Cresti et al. 1992). Although a majority of pollen grains are spherical, in some marine angiosperms, such as Amphibolis and Zostera, they are filiform (up to 5 mm) (Ducker et al. 1978). The wall of the pollen grain is made up of two layers: an outer, acetolysis-resistant exine composed of sporopollenin and an inner pectocellulosic intine. One of the conspicuous structural features of pollen grains is the ornamentation of the wall formed by the outer part of the exine (Cresti et al. 1992).

Pollen biology involves a comprehensive understanding of the structural and functional aspects of pollen grains. The main function of the pollen is to discharge male gametes in the embryo sac for fertilization and for subsequent seed and fruit development. This function depends on the successful completion of a number of sequential events. The following are considered the major events in pollen biology:

• Pollen development

• Free dispersed phase

• Pollination

• Pollen–pistil interaction

• Fertilization

Pollen grains develop inside the anther and are dispersed by dehiscence of the anther. After dispersal, pollen grains remain as independent functional units and are exposed to the prevailing environmental conditions for varying periods.

Pollen biotechnology offers a powerful tool for (a) the optimization of crop production, (b) exploitation of hybrid vigor, and (c) crop improvement through breeding. A large number of papers on various aspects of pollen biology and biotechnology are being published in a variety of journals. There is no book that brings together various aspects related to pollen biotechnology. Because most researchers in pollen biology and biotechnology work in specialized areas, their interests tend to be confined to journals in their fields. This has resulted in a wide gap between biologists interested in fundamental knowledge on pollen biology and those interested in the application of this knowledge to crop production and improvement.

This volume aims to provide a comprehensive account, written by international authorities, of different aspects of pollen biotechnology. We hope that this volume will help bridge the gap between pollen biologists, and agri-horticulturists, foresters, and, particularly, plant breeders. The information contained should also help pollen biologists focus their research in areas directly relevant to crop production and improvement.

The introductory chapter gives a general account of the scope of pollen biology and biotechnology. The remaining chapters are grouped under four sections. The first section gives an overview of pollen biology and includes pollen development, pollination, and pollen–pistil interaction. This is intended to provide, particularly for nonspecialists, the basic information on pollen biology necessary for a better understanding of different aspects of pollen biotechnology covered in other sections. The remaining three sections include chapters pertaining to the role of pollen biotechnology in (a) optimization of crop production, (b) hybrid seed production, and (c) plant breeding.

Summary

Structural details of pollen development are quite uniform in most of the species studied. The main structural events associated with pollen development are (i) the formation of a syncytium of microspore mother cells (MMCs), also referred to as pollen mother cells (PMCs) or meiocytes, in each anther locule, followed by the isolation of each MMC and the resulting microspores encased in a callose wall; (ii) cytoplasmic reorganization resulting in the breakdown of most of the RNA and ribosomes of MMCs, and de-differentiation of plastids and mitochondria; (iii) release of microspores by the activation of callase; (iv) development of microspores accompanied by the synthesis and buildup of RNA, ribosomes, and proteins, and redifferentiation of plastids and mitochondria; (v) asymmetric division of the microspore; and (vi) desiccation and dispersal of pollen grains. The tapetum undergoes several changes and plays a crucial role in pollen development. Although the pistil shows tremendous morphological diversity, the surface of the stigma and the path of pollen tube growth in the pistil invariably contain extracellular components that come into contact with the pollen grain and the pollen tube. Pollination initiates a series of events leading to the discharge of sperm cells in the embryo sac and double fertilization. There is a close interaction between pollen and pistil throughout the postpollination period, and so far only a beginning has been made in understanding the details of these interactions.

Introduction

The development of normal, viable pollen grains, their transfer to the stigma, and pollen germination and successful completion of pollen–pistil interaction are prerequisites for fruit and seed development. Since the beginning of this century, extensive studies have been carried out on various aspects of pollen biology.

Summary

Enhancing insect pollination makes a significant contribution to the value of many commercially important crops by decreasing the time to crop maturity and by increasing crop uniformity, quantity, and quality. There are still many crops, however, that suffer yield losses in situations where adequate pollinator populations cannot be maintained. Plant and pollinator components of cropping systems can be managed to enhance pollinator visitation, increase the rate and quality of pollen transfer, or both. This chapter discusses the efficiency, costs, and benefits of different management strategies that have been developed to maintain and enhance pollinator populations on crops and to increase the rate and quality of pollen transfer.

Introduction

Insect pollinators are a crucial component in the production of many commercially important crops. Enhancing the amount or quality of insect pollination can lead to increases in crop value by decreasing the time to crop maturity and by increasing crop uniformity, quantity, and quality. The diverse group of insect species that play a role in crop pollination range from those that are incidental flower visitors to those that are superbly adapted for pollinating flowers on which they forage exclusively for their food.

Each species of insect has specific morphological, physiological, behavioral, and life history constraints that affect its abundance, effectiveness, and distribution on pollinated crops. These constraints have a direct bearing on the ways in which insects can be most effectively and economically manipulated for the purpose of enhancing crop pollination.

Summary

We review here the results on gene delivery into pollen grains and transformation by particle bombardment. Most of the studies that have been done up to date on bombardment-mediated transformation of pollen are still confined to transient expression of several pollen- or anther-specific promoters. However, these studies do provide evidence that pollen grains can be transformed at least transiently, and suggest the potential use of these “transformed” pollen grains or those bearing foreign DNA for direct pollination to obtain transgenic seeds via natural reproduction system. The alternative way of using pollen transformation for crop improvement is to develop haploid plants via in vitro culture of bombarded immature pollen grains. Included here are the authors' own recent results on successful production of transgenic haploid plants derived from in vitro culture of bombarded pollen. These results provide a basis for a discussion of the usefulness of pollen transformation for crop improvement.

Significance of pollen transformation for crop improvement

Pollen transformation is an attractive approach for plant breeding and crop improvement. There have been reports of various techniques for gene delivery into pollen or microspores, including imbibition of pollen with DNA (Hess 1980), Agrobacterium-mediated transformation (Hess 1987; Pechan 1989), electroporation of pollen (Matthews et al. 1990; Fennell and Hauptmann 1992; Jardinaud et al. 1993), and polyethylene glycol-mediated transformation (Fennell and Hauptmann 1992).

However, all these techniques are problematic in their applicability or reproducibility. Electroporation is useful for introducing foreign DNA into germinating pollen (Matthews et al. 1990), but it is not applicable to plant species in which in vitro pollen germination is difficult.

Summary

Incompatibility barriers are major impediments in crop improvement programs. Nonsynchronous flowering and/or geographical isolation of parental species is common, particularly in wide crosses, and is critical in tree species. Postpollination barriers may operate before and/or after fertilization. Prefertilization barriers act either on the surface of the stigma (by inhibiting pollen germination or pollen tube entry into the stigma) or in the transmitting tissue of the stigma and style. Occasionally, pollen tubes may be inhibited in the ovary or in the ovule. More often, prefertilization barriers are not restricted to a particular level but may be active at all levels. The proportion of pollen grains that complete sequential postpollination events is reduced at each level, with the result that very few or no pollen tubes reach the ovule. In most of the interspecific crosses, pollen inhibition is passive (not as a result of active recognition of the pollen) because of the lack of co-adaptation between the pollen and the pistil. The most common postfertilization barrier is the abortion of the hybrid embryo at different developmental stages. In many of the crosses, this is a result of the lack of endosperm development or of its early breakdown. An understanding of the details of barriers at different levels is important for the application of effective techniques to overcome such barriers.

Introduction

Hybridization is one of the most effective methods of crop improvement programs. Most of the hybridization work carried out so far has used genetic variability within the species, and thus crossability barriers were not the main constraints in breeding programs.

Summary

Pollen tube growth is the result of rapid polar extension of a single cell and is fundamentally different from the standard model of cellular growth in plants. The pollen tube emerges from the germpore, penetrates the stigma, and grows within the intercellular matrix of the style. As the pollen tube elongates, the living cytoplasm is confined within the tip of the pollen tube, isolated by callose plugs. The process concludes when the tube tip reaches the micropyle and discharges the sperm nuclei into the embryo sac. This chapter reviews the information available concerning the genetic control of these processes, the role of the pistil in supporting and modulating pollen function, and the genetic basis of the interaction between pollen and pistil. As well, since a positive correlation between pollen and sporophytic responses to certain traits has been demonstrated in many systems, the use of pollen assays and pollen selection for the identification of plants with desirable traits is discussed. These technologies, when integrated into the conventional breeding program, form powerful breeding tools.

Introduction

The male gametophyte has a very short life-span in the biological cycle of higher plants. Nevertheless, it is able to exist as a free organism and to express a large portion of the plant genome, including many specific genes. These genes control the development and function of a highly specialized structure, the pollen tube, which can grow at a very fast rate and respond to a sophisticated cell-cell communication system. Pollen tube growth is a unique model of plant cell growth, which more resembles fungal growth than the cell growth in a higher organism.

Summary

Hybrid varieties of crop plants are grown when the increased productivity gained from heterosis offsets the extra cost of their development and seed production. One important factor in economically viable hybrid seed production is the availability of a practical and effective pollination control system, which is employed to prevent sib- or self-pollination of the female parent. Such a system is not available in many important crops and, in others, difficult and costly manual emasculation of the male flower or flower parts from the female parent plants is the only possibility. Alternative methods of pollination control have long been desired in these crops. The development of recombinant DNA technologies has opened new possibilities for creating and manipulating male sterility for pollination control. These possibilities are the subject of this chapter. Recombinant DNA concepts devised only in the last five years have already developed into practical tools for hybrid seed production and will be one of the first and most important contributions of biotechnology to plant agriculture.

Introduction: The importance and production of F1 hybrid varieties

The exploitation of heterosis through the use of hybrid varieties is arguably the single most important contribution of genetic research to agriculture (Peacock 1992). The term “heterosis,” or hybrid vigor, was defined by Shull (1952) as the increase in vigor and productivity resulting from the differences in parental gametes. Thus, in contrast to the open-pollinated or inbred varieties they replace, hybrid (F1) varieties are derived from controlled crossings between two genetically distinct groups of parents, usually inbred lines. An added premium in the case of hybrids of inbred lines is uniformity, which is often a major parameter of quality and can also facilitate mechanical harvesting.

Our knowledge of pollen, the gold dust that carries the male germ line of flowering plants and is vital for sexual reproduction and seed formation, has “come of age” with the publication of this book. Here, for the first time in a single volume, are all the ideas and techniques developed in the last two decades concerning the manipulation of pollen and pollen tubes in plant breeding and biotechnology. Pollen has never been an easy topic to come to grips with, with its variable and often inexplicable terminology that has made it a difficult field in which to work. This book will remedy that, with its overview of pollen biology and pollen–pistil interactions that explains terms and concepts of the male function of pollen in a way that is readily understandable.

This new biotechnology of pollen had its origins 40 years ago in developments in plant tissue culture, physiology, and electron microscopy, and, more recently, with the advent of molecular genetics. In reviewing pollen developmental processes and genetic defects in the early 1970s, Professor Jack Heslop-Harrison FRS showed how the opportunities for manipulation might be achieved:

Summary

Nuclear encoded, genie male sterility (GMS) is a common occurrence in angiosperms and is reported in nearly every major crop species. GMS can result from mutations in any one of a number of genes controlling pollen and/or stamen development and, accordingly, the phenotypes of GMS mutants vary. There are a number of cytological, physiological, and biochemical processes affected in GMS mutants, but the causative mechanisms of GMS are not clearly understood. Although GMS is not commonly used in hybrid seed production, primarily because of the maintenance of pure male-sterile lines, there are several proposals put forward to circumvent these problems. One promising approach is to select for chemical- or environment-sensitive GMS lines in which fertility can be restored by appropriate treatments. Such systems are potentially useful in hybrid seed production.

Introduction

Normal development of the male reproductive organ (stamen) and male gametophyte (pollen grain) is essential for the successful completion of sexual reproduction in angiosperms. Abnormalities at any stage of stamen and pollen development can result in male sterility.

Male sterility is a wide occurrence in flowering plants and has been of interest to various plant biologists and plant breeders. Male-sterile mutants serve as useful tools for investigations into the genetic, molecular, physiological, and developmental processes involved in stamen and pollen development. The sterility of the male reproductive organ is also considered a major mechanism by which gynodioecy is believed to have originated (Bawa 1980). For plant breeders, male-sterile plants are useful systems for interspecific hybridization and for performing backcrosses.Perhaps the most widely accepted use of male sterility is in the production of F1 hybrid seed in monoecious and hermaphrodite crops (Frankel and Galun 1977; Kaul 1988).

Summary

Insects visiting flowers to collect nectar, pollen, and other rewards often serve as important incidental agents of pollination. The efficiency of insect species as pollinators varies greatly. In this chapter, emphasis is placed on (a) the interactions between foraging insects, (b) comparison of pollinating abilities of insect sexes and castes, (c) comparisons of nectar and pollen foragers, (d) insect tongue length and its influence on flower visitation patterns, (e) characteristics of pollen grains and insect body hairs and their influence on pollen movement, and (f) pollen removal, and various other pollen losses, during insect visits. To date, most of our knowledge on insect pollination efficiency is derived from plant species growing in natural settings. Additional research is required for agricultural and horticultural crops. Some recommendations are given for future studies aimed at identifying the relative importance of different insect species as pollinators in agriculture. The importance of obtaining data within a plant species, for both insect removal and deposition of pollen, is emphasized.

Introduction

Insects are important vectors of pollen for many agricultural crops, and this chapter focuses on various characteristics that influence their efficiency as pollinators. Although some comparisons to non-insect agents of pollination are referred to herein, readers seeking information on pollen dynamics in other pollination syndromes may refer to the following: anemophily (wind pollination)–Di-Giovanni and Kevan (1991), McCartney (1994), and Niklas (1985); zoophily (mammal and bird pollination) – Fleming and Sosa (1994), and Chapter 4 of this volume.

Perhaps no other term in pollination biology has carried such ambiguity as “pollination efficiency.”