Summary

The now nearly routine isolation of large numbers of viable sperm cells from pollen or pollen tubes of both dicot and monocot flowering plants has opened up a new field of research in plant biology. These unique plant cells are being studied using techniques that were previously more amenable to cultured animal cells. Investigations of synthetic processes, membrane structure, transport, and fusion and recognition properties are being carried out. When the ability to isolate sperms is equaled by the ability to isolate eggs and/or embryo sacs, biologists studying flowering plants will have powerful tools to probe the molecular aspects of fertilization and early embryogenesis, and the opportunity of attempting to achieve genetic transformation during fertilization.

Introduction

The successful isolation and manipulation of sperm and egg cells of flowering plants has changed the approach to studies of fertilization and embryogenesis from a rather descriptive one to a highly experimental one. The history of flowering plant sperm cell isolation goes back to the 1920s, but there was a considerable gap between the work of Finn (1925) on isolation of sperm cells of Asclepias and that of Cass (1973) on isolation of sperms from pollen of barley for differential interference contrast (DIC) microscopy. The latter work combined DIC microscopy with transmission electron microscopy of sperms in pollen grains. The work of Russell (1984, 1985, 1986) on fertilization and sperm cell dimorphism, including isolation, in Plumbago provided further impetus to others (Dupuis et al. 1987; Matthys-Rochon et al. 1987; Mogensen et al. 1990; Russell et al. 1990; Theunis et al. 1991; Yang and Zhou 1989) to reexamine not only the morphology but also the function of sperms and to consider using them as fusion protoplasts in experimental fusions.

Summary

Chemicals capable of selectively inhibiting pollen development and thus blocking male fertility have been known for some time. Most were identified in screening programs designed to discover chemical hybridizing agents (CHAs) for the large-scale commercial production of hybrid seed, particularly in small grains. Driven by practical requirements, this research has identified newer generations of CHAs with increasingly better selectivity and effectiveness. However, research into the mode of action of these substances has lagged their practical applications in breeding and hybrid production. Compounds are known that cause a range of effects, including feminization of male florets and inhibition of early anther development, interference with tapetal functions and microspore development, and defective germination of the mature pollen. Several CHAs also have demonstrated a stimulatory effect on the induction of androgenic plants from anther cultures derived from treated plants. The strength of this response varies among genotypes. The molecular and physiological mechanisms of these effects are unknown. The purpose of this review is to encourage further research with these substances.

Introduction: Tools and insights into pollen development

The study of complex natural events requires development of tools suitable for their analysis and manipulation. Effective use of such tools then becomes the basis of practical new technologies. So it is with efforts to probe the development and function of the male gametophyte. Descriptive methods have led just so far into understanding the development of pollen and the process of pollination. Beyond description, one must functionally separate its essential from its accidental elements and identify the points of regulation. The classic analytical tools for dissection and disruption of developmental processes are surgical, chemical, and genetic. This discussion considers chemical methods.

Summary

Crossing barriers occur frequently when intra- or interspecific crosses are attempted. These barriers are the result of incompatibility and incongruity. Sexual barriers preventing interspecific hybridization have been distinguished into pre- and postfertilization barriers. The nature of the barrier governs the method to be used to overcome the specific barrier. A range of techniques, such as bud pollination, stump pollination, use of mentor pollen, and grafting of the style, have been applied successfully to overcome prefertilization barriers. In vitro methods in the form of ovary, ovary-slice, ovule, and embryo culture are being used to overcome postfertilization barriers that cause endosperm failure and embryo abortion. An integrated method of in vitro pollination and fertilization followed by embryo rescue has been applied in many crosses. Vital hybrid plants may display lack of flowering or male and female sterility, resulting in failure of sexual reproduction. If sterility is caused by a lack of chromosome pairing during meiosis, fertility may be restored by polyploidization, enabling pairing of homologous chromosomes in the allopolyploid hybrid. Integration of these techniques into the breeding programs would enable the breeder to introgress genes across the species barriers.

Introduction

The phenomena underlying crossing barriers are incompatibility and incongruity. Incompatibility operates in intraspecific crosses and is the result of the activity of S-alleles. Incongruity occurs in interspecific crosses as a result of lack of genetic information in one partner necessary to complete pre- and postpollination processes in the other (Hogenboom 1973). This chapter focuses on different methods used to overcome incongruity.

Interspecific and intergeneric crosses are made to introduce new genetic variation into cultivated plants.In breeding ornamental crops, interspecific hybridization is the most important source of genetic variation.

Introduction

In this chapter we consider the role of molybdenum (Mo) in the nutrition of legume and grass species cultivated to feed livestock, whether in the form of direct grazing in pastures or as plants cut and removed for use as fodder. It is recognized that crop residues and plant species other than legumes and grasses (e.g., brassicas) are important sources of livestock fodder worldwide and that Mo nutrition can markedly affect their production and quality, but this review is restricted to forage legumes and grasses. We concentrate mainly on legume responses to Mo, because a prime role for this element in plant growth is its involvement in the symbiotic process of nitrogen fixation (Chatt et al., 1969), although Mo imbalances in grasses have also been reported (e.g., Lipsett, 1975).

The probability of Mo deficiency generally increases with increasing soil acidity (Gupta and Lipsett, 1981), and thus we further focus toward legume responses to the Mo-deficiency component of the acid-soil syndrome. It is difficult to diagnose Mo deficiencies, largely because of the relatively small quantities of Mo required for normal plant functions, and thus there is a higher probability than for other elements that Mo deficiencies in legumes will remain undetected. Consequently, legume biomass production and additions of fixed nitrogen (N) to the soil often remain suboptimal. Thus, in this chapter we emphasize the importance of accurate diagnosis of Mo deficiency and suggest appropriate corrective measures for sustaining pastures and forage production.

Introduction

Molybdenum (Mo) is an essential plant nutrient. It acts as a metallic cofactor in plant and animal enzymes. At high concentrations in forages, it can be toxic to ruminants by interfering with assimilation of copper (Cu). The range between toxicity and deficiency in animals is narrow, and therefore careful control of Mo in animal diets is essential.

Bear (1956) reviewed the early literature dealing with Mo in soils and plants and in animal nutrition in a special issue of Soil Science. The agricultural importance of Mo has been discussed (Mortvedt, Giordano, and Lindsay, 1972), and various aspects of the presence of Mo in the environment have been examined (Chappell and Petersen, 1977). Underwood (1977) and Beeson and Matrone (1976) have reviewed the biochemical importance of Mo in animal and human nutrition.

Molybdenum deficiency in tropical and subtropical soils is more widespread than elsewhere because soils in the tropics are highly weathered. In fact, many soils in the tropics are so highly weathered that little remains except sesquioxides and some 1:1 layer silicates (Pasricha and Fox, 1993). The acidic nature of many of these soils with a dominant content of sesquioxides is the major cause of Mo deficiency.

Introduction

The observation that molybdenum (Mo) uptake by plants decreases with increasing concentrations of sulfate was first reported for tomato plants (Lycopersicon esculentum Mill.) in solution culture (Stout and Meagher, 1948). Stout et al. (1951) confirmed that observation with tomato plants grown in tissue culture (Table 14.1) and with tomatoes and peas (Pisum sativum L.) in soil (Tables 14.2 and 14.3). They attributed the action of sulfate ions in suppressing Mo uptake to direct competition between two divalent anions of similar sizes.

Since the report by Stout and co-workers, the effects of sulfur (S) to decrease Mo uptake have been reported in many species grown under a wide range of conditions, including vegetable crops such as beans (Phaseolus vulgaris L.) (Widdowson, 1966), Brussels sprouts (Brassica oleracea L. Gemmifera Group) (Gupta and Cutcliffe, 1968; Gupta, 1969; Gupta and Munro, 1969), cauliflower (Brassica oleracea L. Botrytis Group) (Mulder, 1954), cauliflower and lettuce (Lactuca sativa L.) (Plant, 1956), peas (Reisenauer, 1963; Gupta and Gupta, 1972), and peas and tomatoes (Stout et al. 1951). Similar relations between Mo and S have been found in forages such as berseem (Trifolium alexandrinum L.) (Pasricha and Randhawa, 1972; Shukla and Pathak, 1973; Sisodia, Sawarkar, and Rai, 1975; Pasricha et al., 1977;

Introduction

Molybdenum (Mo) deficiencies have been reported from many countries around the world, mainly in acidic soils. Sandy soils are Mo-deficient more often than are loam or clay soils. Most Mo deficiencies are associated with legume crops, because Mo is an essential constituent of enzymes necessary for fixation of nitrogen (N) by bacteria growing symbiotically with legumes. Molybdenum is also required in other enzyme systems in all plants.

The availability of Mo in soil increases with increasing soil pH. Therefore, liming a soil to the recommended pH range may increase the plant availability of soil Mo sufficiently that Mo fertilization may not be required. This chapter discusses the sources of Mo and the methods of applying Mo fertilizers to those crops that require additional Mo to produce optimum crop yields.

Molybdenum Sources

There are fewer sources of Mo for fertilizers than there are for the other micronutrients (Table 11.1). Ammonium and sodium molybdates and molybdic acid are soluble compounds. These sources of Mo are sometimes applied with other fertilizers or are used as foliar sprays. Both MoO3 a nd Mo frits are insoluble in water, but are effective if applied as fine powders; MoO3 is applied as a seed coating in many cases.

Municipal sewage sludges and fly-ash materials, both waste products, contain Mo that is available to crops. Because these two materials are applied to soils at relatively high rates (5–401 tha–1), their Mo contents should be considered when determining their application rates.

Introduction

Molybdenum (Mo) is an essential element for many plants and animals (Newton and Otsuka, 1980). Because of its chemical properties, Mo readily provides sites for reactions and catalysis in biochemical systems (Haight and Boston, 1973). It is therefore important to understand the processes that control the distribution, speciation, and behavior of Mo in the surficial environment. These processes will affect the bioavalability of Mo and ultimately its passage into the food chain.

In this chapter we discuss the distribution of Mo in the terrestrial environment and examine the factors that control its mobility.

General Chemical Properties of Molybdenum

Molybdenum is a transition element and a member of the 4d series of metals in period 5 of the periodic table. In elemental form, these metals generally are very hard and have high melting temperatures. They exhibit a wide range of oxidation states in their compounds, and they form bonds of high covalent character (Parish, 1977). Other elements that exhibit typical 4d chemistry include zirconium (Zr), niobium (Nb), technetium (Tc), ruthenium (Ru), rhodium (Rh), and palladium (Pd). Molybdenum is also a member of group VIB, along with chromium (Cr) and tungsten (W). There are many chemical similarities between Mo and W, but few similarities between Mo and Cr. The electronic configuration of the free atom of Mo is [Kr]4d55s1 Cotton and Wilkinson (1988) provided an in-depth discussion of the inorganic chemistry of Mo and stated that Mo reactions are among the most complex reactions involving any of the chemical elements.

Introduction

Molybdenum (Mo) is important in ecosystems as a micronutrient for both plants and animals. It can also accumulate in the environment in toxic concentrations. Molybdenum is used widely in industrial societies and is an important fertilizer element in some agricultural systems. Soil Mo averages approximately 1.0–2.3 mg kg–1 as a crustal constituent, making it 53rd in abundance (Krauskopf, 1979), but it can accumulate as a result of biogeochemical cycling to 300mgkg–1 or more in shales rich in organic matter. However, the common range of Mo concentrations in U.S. soils is 0.8–3.3 mg kg–1 (dry weight) (Kubota, 1977). In soils, Mo can be found in four major fractions: (1) dissolved Mo in soil solution (watersoluble), (2) Mo occluded with oxides (e.g., Al, Fe, and Mn oxides), (3) Mo solid phases [e.g., molybdenite (MoS2), powellite (CaMoO4), ferrimolybdite (Fe2(MoO4)3), wulfenite (PbMoO4)], and (4) Mo associated with organic compounds.

Numerous processes take place in soil solution, including plant uptake, ion complexation, adsorption and desorption, and precipitation and dissolution (Figure 2.1). As shown in Figure 2.1, Mo solid phases dissolve upon contact with water and provide dissolved Mo in soil solution. The free molybdate ion reacts with metals to form complexes and ion pairs in soil solution. Plants absorb dissolved Mo, mainly as, from soil solution. Removal of by plants disrupts the electroneutrality of a soil solution.

Introduction

Molybdenum (Mo) is essential for higher plants (Arnon and Stout, 1939). As a constituent of nitrate reductase, sulfite reductase, and, in nodulated legumes, nitrogenase and xanthine oxidase, it is involved in electron-transfer reactions. Nitrate reductase, which catalyzes the conversion of nitrate to nitrite by transfer of electrons from Mo to nitrate, is an inducible enzyme that depends on Mo for its synthesis. There is a close relationship involving Mo supply, nitrate reductase activity, and plant growth. In the nitrogenase of legumes and other plants that fix atmospheric nitrogen, Mo is directly involved in the reduction of N2 (Witt and Jungk, 1977). As a constituent of xanthine oxidase it catalyzes the oxidation of xanthine to uric acid, which is a precursor of allantoin and allantoic acid in most tropical and subtropical legumes. Besides playing a catalytic role, Mo is required for plant reproductive development (Agarwala et al., 1979) and nodulation of leguminous and nonleguminous N-fixing plants (Becking, 1961).

Compared with those for other micronutrients, the physiological requirements for Mo are very low (Stout and Meagher, 1948; Hewitt, 1963). The adequate concentrations of Mo in most crops range between 0.1 and 1. Omgkg–1 (Bergmann, 1988), but the threshold for Mo toxicity in plants is much higher than that for any other micronutrient. Some plants can accumulate 1,000 times their adequate concentrations of Mo without exhibiting any visible signs of toxicity: cotton (Gossypium spp.), tomato (Lycopersicon esculentum Mill.), and Phaseolus beans (Joham, 1953; Johnson, 1966; Widdowson, 1966).

Introduction

A number of factors can affect the availability of molybdenum (Mo) to crops. The most important ones include the nature of the parent rock, soil pH, the organic matter in the soil, drainage, interactions with other nutrients, and plant species, plant part, and stage of plant growth at sampling. This chapter attempts to review the information available on the soil and plant factors that affect Mo uptake by plants in different parts of the world.

Soil Factors

Molybdenum Occurrences in Parent Rocks

The concentrations and forms of Mo in rocks and soils tend to vary according to the particular origins and conditions of formation. Molybdenum is a versatile element insofar as valence is concerned, and it can precipitate under either oxidizing (Mo6+ predominant) or reducing (Mo4+) conditions (Manheim and Landergren, 1978). Consequently, there may be local enrichments or depletions, and recent work has largely been concerned with elucidating the sequences of occurrence, mobilization, and deposition in particular situations.

Occurrences in Igneous and Metamorphic Rocks

Igneous rocks make up some 95% of the crust of the earth (Mitchell, 1964), and Mo occurs in both acid and basic igneous rocks. Manheim and Landergren (1978) suggested an overall Mo content of nearly 2.0 ppm for granitic rocks and somewhat lower values for basalts.

Although the occurrences of Mo in metamorphic rocks have not been widely studied, metamorphism would be expected to alter the form and site of occurrence rather than the amount of Mo present.

The chief purpose of preparing this book was to condense all the information available on the subject of molybdenum (Mo) as it relates to soils, crops, and livestock. Because the problems related to the requirements for Mo in soil and in crop production differ considerably from one part of the world to another, I attempted to solicit the assistance of experts from the different regions of the world who were best suited to write about the topics of the various chapters. These contributions by authors from different geographical areas have helped to provide a broader viewpoint of the subject matter than would have been the case if only a single author had prepared the book in its entirety.

Molybdenum deficiencies in field-grown plants were first recorded in Australia more than 55 years ago. This book contains a chapter authored by two Australian scientists, Drs. Chris Johansen and Peter Kerridge, who have advanced our understanding of the responses of agricultural plants to Mo in Australia and elsewhere, particularly in tropical regions. Currently, they are senior research managers at international agricultural research institutes: ICRISAT in India and CIAT in Colombia, respectively.

This book reviews our current knowledge of the following topics:

Introduction

The symptoms of molybdenum (Mo) deficiency are common in certain crops under certain soil and climatic conditions. However, Mo toxicity is uncommon and is found only when unusually high concentrations of Mo are present.

Deficiency symptoms for most micronutrients appear on the young leaves at the top of the plant, because most micronutrients are not readily translocated. Molybdenum is an exception in that it is readily translocated, and its deficiency symptoms generally appear on the whole plant.

The symptoms associated with deficiency of Mo are closely related to nitrogen (N) metabolism. Because Mo is needed for nitrogenase activity, Mo deficiency prevents the fixation of N2. This process involves higher plants and symbiotic organisms such as Rhizobium, and so Mo deficiency can produce symptoms associated with a deficiency of nitrogen. If the Mo is needed directly by the plant for nitrate reductase, then symptoms peculiar to Mo occur, although essentially the plant can be considered to be suffering from a shortage of protein due to failure of the initial processes of reduction.

The first type of symptom can be relieved by supplying fixed nitrogen. Even may serve the purpose, because the Mo requirement for nitrate reductase is lower than that for nitrogenase.

Introduction

The fact that higher plants need molybdenum (Mo) was recognized as early as 1930s, as described in many early review articles (Hewitt, 1956; Anderson, 1956; Stout and Johnson, 1956; Rubins, 1956; Evans, 1956; Davies, 1956; Purvis and Peterson, 1956; Reisenauer, 1956). A later review, by Gupta and Lipsett (1981), covered every aspect of soil fertility: Mo fertilizers and their application, the physiological roles of Mo, determinations of Mo in plants and soils, factors affecting plant uptake of Mo, and the problems of toxicity and deficiency of Mo. The importance of Mo was first noted in legumes, because clovers (Trifolium spp.) were extensively used in mixed pastures in Australia. Since that time, Mo deficiencies have been identified in many other legumes, such as soybeans, alfalfa, peas, and various beans. Molybdenum deficiencies have also been reported in nonlegume crops. The early experiments revealed that Mo not only is required for nitrogen (N) fixation by rhizobia for use by legumes but also is required for nitrate reductase utilization by legumes and nonlegumes. Field data on yield responses to applied Mo are more limited than for other essential elements; because of the low requirements for Mo, it has not been as thoroughly studied as other elements. Because Mo deficiencies are found in acid soils, other infertility factors have tended to mask Mo deficiencies, and thus we have fewer data on crop yields in response to Mo in field and horticultural crops. The plant part and age at testing are important in diagnosing Mo deficiencies.

Introduction

The testing of soils for molybdenum (Mo) and other micronutrients has been reviewed extensively in recent publications (Gupta and Lipsett, 1981; Anderson and Mortvedt, 1982; Cox, 1987; Johnson and Fixen, 1990; Sims and Johnson, 1991; Sims, 1996). The general objectives for testing soils for any nutrient have been to assess the soil's capacity to supply plant-available nutrient during the growth of crops and to gather data that can guide producers in obtaining the best economic response to fertilizer application. Fitts and Nelson (1956) suggested that soil testing can be divided into four phases: (1) sampling the soil, (2) conducting tests to determine nutrient availability, (3) calibrating test findings with crop responses, and (4) interpreting the findings and making recommendations.

Because of the relatively small amounts of Mo in soils (0.1–30mg kg–1) (Kubota, 1976), the importance of seed Mo reserves in supplying crop needs (Peterson and Purvis, 1961; Harris, Parker, and Johnson, 1965; Gurley and Giddens, 1969), the importance of soil properties that affect Mo availability (Lowe and Massey, 1965; Massey, Lowe, and Bailey, 1967; Karimian and Cox, 1978,1979; Burmester, Adams, and Odom, 1988), and the low requirements of most crops for Mo (0.1–0.5 mg kg–1 tissue), the testing of soils for Mo in the classic sense is rendered difficult.

Introduction

Many early studies reported the molybdenum (Mo) requirements of bacteria (Bortels, 1930; Horner et al., 1942), fungi (Steinberg, 1936, 1937), green algae (Arnon et al., 1955), and higher plants (Arnon and Stout, 1939) long before recognition of its various biochemical and physiological functions. Because of subsequent advances in biochemical and physiological research techniques and instrumentation, Mo has been found to be an essential constituent of several important enzymes, besides having a range of nonspecific roles in plant metabolism. This chapter reviews the information available on the biochemical and physiological roles of Mo in crop plants.

Chemical Basis for the Roles of Molybdenum in Biochemical Reactions

It is worthwhile to examine the chemistry of Mo to emphasize what makes it suitable for catalyzing many unique biochemical reactions. Molybdenum is a transition element with an atomic number of 42 and electronic configurations of Is2, 2s22p6, 3s23p63d10, 4s24p64d5, and 5s1. The outermost 4d electrons can be easily removed to yield several oxidation states of Mo, ranging from zero to VI. However, the oxidation states IV, V, and VI are more common and involve only low potentials. Higher oxidation states of Mo have a tendency to form oxocomplexes. As Mo(VI) has no electron in its d orbital, it accepts two e– from an O2– anion to form a sigma (σ) bond. As the Mo—O bond length is short, the d orbital of Mo suffers sidewise weak overlap with the p orbitals of an O atom, to yield a slightly double-bond (Mo=O°) character.

Introduction

Among the micronutrients essential for plant growth, molybdenum (Mo) is required in the smallest amounts. In most soils, the Mo requirements of plants can be met by liming the soil. Because of its low requirement, the deficiency and sufficiency concentrations of Mo in most plants are extremely small. Molybdenum toxicity to plants under field conditions seldom occurs, and usually it can be induced only under extreme experimental conditions (Johnson, 1966). Therefore, this chapter does not place great emphasis on the toxic concentrations of Mo in plants. However, plants can, under certain conditions, accumulate large concentrations of Mo and induce molybdenosis in ruminants that eat such material. That will be dealt with in Chapter 15.

The precision of modern analytical methods is such that even microquantities of Mo in plants can be detected accurately, and considerable data have accumulated over the past 40 years regarding Mo concentrations in a number of plant species. The purpose of this chapter is to report the sufficient, deficient, and toxic concentrations of Mo in a number of cultivated crop species as found by workers around the world.

Usually when one talks about the deficient, sufficient, and toxic concentrations of nutrients in crops, there is a range of values, rather than one definite number that can be considered as critical. Therefore, use of the term “critical” in crops is somewhat misleading. A nutrient concentration considered critical by workers in some areas may not be critical under conditions in other areas.