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As a result of drying, the cytoplasm of desiccation-tolerant organisms, such as seed and pollen, enters into a highly viscous, solid-like, semi-equilibrium state: the glassy state. The work in this dissertation is focussed on the function and characteristics of intracellular glasses in these organisms.It was established that intracellular glasses are formed in both desiccation-tolerant and -intolerant pollen (chapter 1). However, desiccation-intolerant pollen loses its viability during drying before intracellular glasses are formed. This indicates that desiccation tolerance is not related with the formation of glasses during drying. Storage of cattail ( Typha latifolia ) pollen under different water contents and temperatures revealed the existence of an optimum water content for survival at a constant relative humidity (11-15%) (chapter 2). The water content corresponding to this relative humidity shifted to higher values with lower storage temperatures, and was found to be associated with the Brunauer, Emmet, and Teller monolayer value. Drying of the pollen below these water contents had detrimental effects on longevity. The water content-temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. There was a change in ageing kinetics of cattail pollen associated with the melting of the intracellular glass. Above the glass transition temperature (T g ) the activation energy of the ageing rates increased two to three times. This suggests that the presence of glasses in the dry state improves storage stability by decreasing viscosity and, thus, ageing rate. It was concluded that T g curves might be useful for predictions of storage longevity above optimum water contents. However, they cannot be used solely to predict the precise conditions of optimum storage. Subsequently, we sought for a more direct measurement to assess the viscosity of the cytoplasm of tissues.For this purpose, we used electron paramagnetic resonance (EPR) spectroscopy to study the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl (CP) that was incorporated into embryonic axes of pea seeds and cattail pollen. Using the distance between the outer extrema of the EPR spectrum (2 A zz ) as a measure of molecular mobility, a sharp increase in mobility was observed at a definite temperature during heating (chapter 3). This temperature increased with decreasing water content of the samples, and was found to be associated with the melting of the glassy state as measured by differential scanning calorimetry (DSC). Molecular mobility was found to be inversely correlated with storage stability: the higher the molecular mobility, the shorter the longevity: with decreasing water content, the molecular mobility reached a minimum, in concert with ageing rates. At very low water contents, both molecular mobility and ageing rates increased again. Minimum mobility and maximum storage stability occurred at similar water contents, suggesting that storage stability might be partially controlled by molecular mobility. To understand the nature of the changes in 2 A zz in spectra of CP in the tissues, echo detected (ED) EPR spectroscopy was employed (chapter 4). The shape of the ED EPR spectrum revealed the presence of librational motion of the spin probe, a motion typically present in glassy materials. The change in 2 A zz appeared to be the result of librational motion of the spin probe.With the use of saturation transfer (ST) EPR spectroscopy, a more quantitative measure of molecular mobility was acquired: the rotational correlation time (τ R ), which corresponds to the time it takes for the spin probe to rotate a radian around its axis (chapter 5). At room temperature,τ R of CP in pea embryonic axes increased during drying from 10 -11 s in de hydrated state to 10 -4 s in the dry state. At T g ,τ R was constant at10 -4 s for all water contents studied. The temperature dependence ofτ R at all water contents studied followed Arrhenius behaviour with a break at T g . The temperature effect onτ R above T g was much smaller in pea axes as found previously for sugar and polymer glasses. Thus, the melting of the intracellular glass by raising the temperature caused only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.The application of saturation transfer EPR spectroscopy to biological tissues enabled a quantitative comparison between storage stability and molecular mobility in different tissues (section III). The temperature and moisture dependence of ageing rates of seeds and pollen was found to correlate with the rotational motion of CP in the cytoplasm (chapter 6-8). An increase in the temperature resulted in a faster rotational motion in the cytoplasm of cattail pollen, analogous to faster ageing rates (chapter 6). Decreasing the water content of the pollen resulted in a decrease in rotational motion until a minimum was reached, after which rotational motion slightly increased again. The water content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm of the pollen.We also investigated the relationship between the longevity of lettuce seeds and the molecular mobility in the cytoplasm of their radicles (chapter 7). Longevity of lettuce seeds was predicted using the viability equation of Ellis and Roberts. Increasing the temperature resulted in faster rotational motion and shorter longevity. There was a linear relationship between the logarithms of rotational motion and estimated longevity for temperatures above 5°C, which is the same temperature range for which experimental data were used to obtain the viability constants of the viability equation. Below 5°C, there was a deviation from linearity, which might stem from inaccurate predictions by the viability equation at low temperatures.Chapter 8 further demonstrates that there is a linear relationship between the logarithms of rotational motion in the cytoplasm of seed and pollen of several plant species and their ageing rates or longevities. This linearity suggests that cytoplasmic mobility might be an important controlling factor of ageing rates. The linear relationship between the two parameters could be used to predict lifespan of germplasm at low temperatures (at which experimental determination of longevity is practically impossible) by simply measuring theτ R values at these low temperatures (chapter 7 and 8). Based on the predictions using the linear regression between ageing rate and rotational motion of CP in pea embryonic axes, an optimum water content of storage was found. This optimum water content shifted to higher values with lowering the storage temperature, as was found previously for cattail pollen based on experimental data (chapter 2). It was predicted that the longevity of seeds at high (0.12 to 0.16 g/g) water content is much higher than previously suggested on the basis of the viability equation. The predictions show that drying germplasm too far leads to decreased longevity compared to storage of germplasm at higher water contents, suggesting that current storage protocols might have to be re-examined.Desiccation-tolerant organisms contain large amounts of soluble sugars. This, and the fact that sugars are excellent glass-formers has led to the suggestion that sugars play an important role in intracellular glass formation. The presence and amounts of oligosaccharides have been found to correlate with longevity. Furthermore, oligosaccharide glasses are known to increase the T g and viscosity of model sucrose glasses. This suggests that oligosaccharides might enhance the stability of intracellular glasses (chapter 9 and 10). Osmo-priming, i.e. pre-imbibition of seeds in an osmotic solution, can result in a decrease in oligosaccharide content and longevity. Priming pea seeds decreased the total oligosaccharide content in the embryonic axes (chapter 9). Despite the change in oligosaccharide:sucrose ratio, no differences in T g values were detected in the dry axes before and after priming as determined by DSC. Also no difference was found between the rotational mobility of CP in dry untreated axes and that of dry primed axes. Chapter 10 demonstrates that osmo-priming of impatiens and bell pepper seeds resulted in considerable decreases in longevity and oligosaccharide contents, while sucrose contents increased. Again, no differences in the T g curves were found between control and primed impatiens seeds. Similarly, there was no difference in rotational motion of CP in the cytoplasm between control and primed impatiens seeds and between control and primed bell pepper embryonic axes. It was concluded that oligosaccharides in seeds do not appear to affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.To understand the nature and composition of biological glasses we investigated the molecular mobility around T g in sugars, poly-L-lysine and dry desiccation-tolerant biological systems, using ST-EPR, 1 H-NMR and FTIR spectroscopy. Two distinct changes in the temperature dependence of molecular mobility were detected in sugars and poly-L-lysine. With heating, the first change was associated with the melting of the glassy state (T g ). The second change, at which the molecular mobility abruptly increased over several orders of magnitude, was found to correspond with a critical temperature (T c ) where the dynamics of the system changed from solid-like to liquid-like. The temperature interval between T g and T c increased with increasing molecular weight of the sugars. The interval between T g and T c in biological tissues was over 50°C, implying that the stability remained high even at temperatures far above T g . A comparably high T c -T g interval was found for the molecular mobility of poly-L-lysine, suggesting that proteins rather than sugars play an important role in the intracellular glass formation. The exceptionally high T c of intracellular glasses is expected to provide excellent long-term stability to dry organisms, maintaining a slow molecular motion in the cytoplasm even at temperatures far above T g .

As a result of drying, the cytoplasm of desiccation-tolerant organisms, such as seed and pollen, enters into a highly viscous, solid-like, semi-equilibrium state: the glassy state. The work in this dissertation is focussed on the function and characteristics of intracellular glasses in these organisms.It was established that intracellular glasses are formed in both desiccation-tolerant and -intolerant pollen (chapter 1). However, desiccation-intolerant pollen loses its viability during drying before intracellular glasses are formed. This indicates that desiccation tolerance is not related with the formation of glasses during drying. Storage of cattail ( Typha latifolia ) pollen under different water contents and temperatures revealed the existence of an optimum water content for survival at a constant relative humidity (11-15%) (chapter 2). The water content corresponding to this relative humidity shifted to higher values with lower storage temperatures, and was found to be associated with the Brunauer, Emmet, and Teller monolayer value. Drying of the pollen below these water contents had detrimental effects on longevity. The water content-temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. There was a change in ageing kinetics of cattail pollen associated with the melting of the intracellular glass. Above the glass transition temperature (T g ) the activation energy of the ageing rates increased two to three times. This suggests that the presence of glasses in the dry state improves storage stability by decreasing viscosity and, thus, ageing rate. It was concluded that T g curves might be useful for predictions of storage longevity above optimum water contents. However, they cannot be used solely to predict the precise conditions of optimum storage. Subsequently, we sought for a more direct measurement to assess the viscosity of the cytoplasm of tissues.For this purpose, we used electron paramagnetic resonance (EPR) spectroscopy to study the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl (CP) that was incorporated into embryonic axes of pea seeds and cattail pollen. Using the distance between the outer extrema of the EPR spectrum (2 A zz ) as a measure of molecular mobility, a sharp increase in mobility was observed at a definite temperature during heating (chapter 3). This temperature increased with decreasing water content of the samples, and was found to be associated with the melting of the glassy state as measured by differential scanning calorimetry (DSC). Molecular mobility was found to be inversely correlated with storage stability: the higher the molecular mobility, the shorter the longevity: with decreasing water content, the molecular mobility reached a minimum, in concert with ageing rates. At very low water contents, both molecular mobility and ageing rates increased again. Minimum mobility and maximum storage stability occurred at similar water contents, suggesting that storage stability might be partially controlled by molecular mobility. To understand the nature of the changes in 2 A zz in spectra of CP in the tissues, echo detected (ED) EPR spectroscopy was employed (chapter 4). The shape of the ED EPR spectrum revealed the presence of librational motion of the spin probe, a motion typically present in glassy materials. The change in 2 A zz appeared to be the result of librational motion of the spin probe.With the use of saturation transfer (ST) EPR spectroscopy, a more quantitative measure of molecular mobility was acquired: the rotational correlation time (τ R ), which corresponds to the time it takes for the spin probe to rotate a radian around its axis (chapter 5). At room temperature,τ R of CP in pea embryonic axes increased during drying from 10 -11 s in de hydrated state to 10 -4 s in the dry state. At T g ,τ R was constant at10 -4 s for all water contents studied. The temperature dependence ofτ R at all water contents studied followed Arrhenius behaviour with a break at T g . The temperature effect onτ R above T g was much smaller in pea axes as found previously for sugar and polymer glasses. Thus, the melting of the intracellular glass by raising the temperature caused only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.The application of saturation transfer EPR spectroscopy to biological tissues enabled a quantitative comparison between storage stability and molecular mobility in different tissues (section III). The temperature and moisture dependence of ageing rates of seeds and pollen was found to correlate with the rotational motion of CP in the cytoplasm (chapter 6-8). An increase in the temperature resulted in a faster rotational motion in the cytoplasm of cattail pollen, analogous to faster ageing rates (chapter 6). Decreasing the water content of the pollen resulted in a decrease in rotational motion until a minimum was reached, after which rotational motion slightly increased again. The water content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm of the pollen.We also investigated the relationship between the longevity of lettuce seeds and the molecular mobility in the cytoplasm of their radicles (chapter 7). Longevity of lettuce seeds was predicted using the viability equation of Ellis and Roberts. Increasing the temperature resulted in faster rotational motion and shorter longevity. There was a linear relationship between the logarithms of rotational motion and estimated longevity for temperatures above 5°C, which is the same temperature range for which experimental data were used to obtain the viability constants of the viability equation. Below 5°C, there was a deviation from linearity, which might stem from inaccurate predictions by the viability equation at low temperatures.Chapter 8 further demonstrates that there is a linear relationship between the logarithms of rotational motion in the cytoplasm of seed and pollen of several plant species and their ageing rates or longevities. This linearity suggests that cytoplasmic mobility might be an important controlling factor of ageing rates. The linear relationship between the two parameters could be used to predict lifespan of germplasm at low temperatures (at which experimental determination of longevity is practically impossible) by simply measuring theτ R values at these low temperatures (chapter 7 and 8). Based on the predictions using the linear regression between ageing rate and rotational motion of CP in pea embryonic axes, an optimum water content of storage was found. This optimum water content shifted to higher values with lowering the storage temperature, as was found previously for cattail pollen based on experimental data (chapter 2). It was predicted that the longevity of seeds at high (0.12 to 0.16 g/g) water content is much higher than previously suggested on the basis of the viability equation. The predictions show that drying germplasm too far leads to decreased longevity compared to storage of germplasm at higher water contents, suggesting that current storage protocols might have to be re-examined.Desiccation-tolerant organisms contain large amounts of soluble sugars. This, and the fact that sugars are excellent glass-formers has led to the suggestion that sugars play an important role in intracellular glass formation. The presence and amounts of oligosaccharides have been found to correlate with longevity. Furthermore, oligosaccharide glasses are known to increase the T g and viscosity of model sucrose glasses. This suggests that oligosaccharides might enhance the stability of intracellular glasses (chapter 9 and 10). Osmo-priming, i.e. pre-imbibition of seeds in an osmotic solution, can result in a decrease in oligosaccharide content and longevity. Priming pea seeds decreased the total oligosaccharide content in the embryonic axes (chapter 9). Despite the change in oligosaccharide:sucrose ratio, no differences in T g values were detected in the dry axes before and after priming as determined by DSC. Also no difference was found between the rotational mobility of CP in dry untreated axes and that of dry primed axes. Chapter 10 demonstrates that osmo-priming of impatiens and bell pepper seeds resulted in considerable decreases in longevity and oligosaccharide contents, while sucrose contents increased. Again, no differences in the T g curves were found between control and primed impatiens seeds. Similarly, there was no difference in rotational motion of CP in the cytoplasm between control and primed impatiens seeds and between control and primed bell pepper embryonic axes. It was concluded that oligosaccharides in seeds do not appear to affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.To understand the nature and composition of biological glasses we investigated the molecular mobility around T g in sugars, poly-L-lysine and dry desiccation-tolerant biological systems, using ST-EPR, 1 H-NMR and FTIR spectroscopy. Two distinct changes in the temperature dependence of molecular mobility were detected in sugars and poly-L-lysine. With heating, the first change was associated with the melting of the glassy state (T g ). The second change, at which the molecular mobility abruptly increased over several orders of magnitude, was found to correspond with a critical temperature (T c ) where the dynamics of the system changed from solid-like to liquid-like. The temperature interval between T g and T c increased with increasing molecular weight of the sugars. The interval between T g and T c in biological tissues was over 50°C, implying that the stability remained high even at temperatures far above T g . A comparably high T c -T g interval was found for the molecular mobility of poly-L-lysine, suggesting that proteins rather than sugars play an important role in the intracellular glass formation. The exceptionally high T c of intracellular glasses is expected to provide excellent long-term stability to dry organisms, maintaining a slow molecular motion in the cytoplasm even at temperatures far above T g .

In this thesis membrane lipid composition is studied in relation to pollen viability during storage. Chapter 1 reviews pollen viability, membranes in the dry state and membrane changes associated with cellular aging. This chapter is followed by a study of age-related changes in phospholipid composition in Typha latifolia L. pollen.Typha pollen was stored at room temperature, with internal moisture contents of respectively 0.06 and 0.15 g H 2 O g -1dry weight. In the course of the storage period, imbibition was accompanied by increased leakage of endogenous K +and germination declined. Simultaneously, phospholipids were deesterified in situ, leading to the accumulation of lysophospholipids and free fatty acids in the membranes. Viability declined much faster at the higher internal moisture content. An analysis of the fatty acid composition of the pollen phospholipids after aging indicated that the membrane degradation was not mediated by phospholipase A2. When stored with 0.06 g H 2 O g -1dry weight, the level of polyunsaturated fatty acids remained constant, at the higher internal moisture content a slight decline in the level of linolenic acid content was found. The observed phospholipid degradation could be mediated by an unspecific lipid acyl hydrolase, but free-radical activity is more likely because of the low metabolic activity at the studied moisture levels. The pollen lipids were purified for liposome studies. These revealed that the lysophospholipids and the free fatty acids, accumulated during aging, enhanced the leakage of entrapped solutes from the liposomes (Chapter 2).The phase behaviour of liposomal membranes containing lysophospholipids was studied after drying, to investigate the effect of these compounds in dried and in reimbibed phospholipid bilayers. Liposome studies, using Differential scanning calorimetry and Fourier transform infrared spectroscopy, showed that the lysophospholipids caused a dehydration dependent lateral phase separation. Membrane phase behaviour was also studied in Typha pollen and isolated pollen membranes. After aging the pollen membranes also exhibited lateral phase separation. The phase separation clearly coincided with the above described deesterification of membrane lipids in situ after aging, and caused an extremely fast leakage of endogenous K +upon imbibition (Chapter 3).Two other pollen species were studied to assess whether the observed phospholipid deesterification is a general characteristic of pollen aging. In the course of storage with an internal moisture level 0.06 g H 2 O g -1dry weight, germination declined and the leakage of endogenous K +upon imbibition was increased in pollen from Papaver rhoeas L. and Narcissus poeticus L. At the same time phospholipid deesterification was observed in these species and lysophospholipids and free fatty acids accumulated. The degradation was again accelerated when the pollen was stored with an internal moisture content of 0.15 g H 2 O g -1dry weight. In pollen species with high levels of linolenic acid such as Papaver and Narcissus the deesterification occurred at a higher rate than in pollen species such as Typha in which linoleic acid is predominant. However, no selective loss of polyunsaturated fatty acids was observed. The degradation of the phospholipids in the pollen during dry storage was again most likely free radical-mediated (Chapter 4).Although there was no large preferential degradation of linolenic acid, longevity was somehow correlated with the linolenic acid content. To study whether membrane fluidity in general determines longevity, attempts were undertaken to manipulate the level of linolenic acid in situ . Unfortunately catalytic hydrogenation, using Pd-alizarine as the catalyst, failed to result in saturation of the phospholipids in intact pollen, owing to an inhibition of the activity of the catalyst in viable pollen (Chapter 5).Because catalytic hydrogenation was unsuccessful, an in situ modification of the level of linolenic acid during the germination process was not possible. So, to study the role of linolenic acid in relation to pollen germination, Arabidopsis thaliana L. Heynh. mutants exhibiting desaturase-deficiencies were used. Pollen tube growth rate was severely decreased in pollen from the mutant fad2, in which the activity of the 16:0/18:1 desaturase located in the endoplasmatic reticulum is suppressed. In pollen from this mutant line the level of polyunsaturated fatty acids was decreased, confirming the correlation between membrane fluidity and pollen tube growth rate. In the other mutants in which chloroplast located enzymes were affected, pollen tube growth rate was unaffected (Chapter 6).The results from these studies show that with respect to aging and storage of pollen, the key factor in controling viability is the fluidity of the phospholipid bilayer. When the pollen membranes are in the high fluidity liquid crystalline phase during storage, aging is much more rapid than when the membranes are in the low fluidity gel phase. So for storage gel phase is preferable, but for rapid tube growth, on the contrary the liquid crystalline phase is required. Thus for both proper storage and germination conditions, membrane fluidity has to be carefully monitored, for instance with the aid of Fourier transform infrared spectroscopy (Chapter 7).

In this thesis membrane lipid composition is studied in relation to pollen viability during storage. Chapter 1 reviews pollen viability, membranes in the dry state and membrane changes associated with cellular aging. This chapter is followed by a study of age-related changes in phospholipid composition in Typha latifolia L. pollen.Typha pollen was stored at room temperature, with internal moisture contents of respectively 0.06 and 0.15 g H 2 O g -1dry weight. In the course of the storage period, imbibition was accompanied by increased leakage of endogenous K +and germination declined. Simultaneously, phospholipids were deesterified in situ, leading to the accumulation of lysophospholipids and free fatty acids in the membranes. Viability declined much faster at the higher internal moisture content. An analysis of the fatty acid composition of the pollen phospholipids after aging indicated that the membrane degradation was not mediated by phospholipase A2. When stored with 0.06 g H 2 O g -1dry weight, the level of polyunsaturated fatty acids remained constant, at the higher internal moisture content a slight decline in the level of linolenic acid content was found. The observed phospholipid degradation could be mediated by an unspecific lipid acyl hydrolase, but free-radical activity is more likely because of the low metabolic activity at the studied moisture levels. The pollen lipids were purified for liposome studies. These revealed that the lysophospholipids and the free fatty acids, accumulated during aging, enhanced the leakage of entrapped solutes from the liposomes (Chapter 2).The phase behaviour of liposomal membranes containing lysophospholipids was studied after drying, to investigate the effect of these compounds in dried and in reimbibed phospholipid bilayers. Liposome studies, using Differential scanning calorimetry and Fourier transform infrared spectroscopy, showed that the lysophospholipids caused a dehydration dependent lateral phase separation. Membrane phase behaviour was also studied in Typha pollen and isolated pollen membranes. After aging the pollen membranes also exhibited lateral phase separation. The phase separation clearly coincided with the above described deesterification of membrane lipids in situ after aging, and caused an extremely fast leakage of endogenous K +upon imbibition (Chapter 3).Two other pollen species were studied to assess whether the observed phospholipid deesterification is a general characteristic of pollen aging. In the course of storage with an internal moisture level 0.06 g H 2 O g -1dry weight, germination declined and the leakage of endogenous K +upon imbibition was increased in pollen from Papaver rhoeas L. and Narcissus poeticus L. At the same time phospholipid deesterification was observed in these species and lysophospholipids and free fatty acids accumulated. The degradation was again accelerated when the pollen was stored with an internal moisture content of 0.15 g H 2 O g -1dry weight. In pollen species with high levels of linolenic acid such as Papaver and Narcissus the deesterification occurred at a higher rate than in pollen species such as Typha in which linoleic acid is predominant. However, no selective loss of polyunsaturated fatty acids was observed. The degradation of the phospholipids in the pollen during dry storage was again most likely free radical-mediated (Chapter 4).Although there was no large preferential degradation of linolenic acid, longevity was somehow correlated with the linolenic acid content. To study whether membrane fluidity in general determines longevity, attempts were undertaken to manipulate the level of linolenic acid in situ . Unfortunately catalytic hydrogenation, using Pd-alizarine as the catalyst, failed to result in saturation of the phospholipids in intact pollen, owing to an inhibition of the activity of the catalyst in viable pollen (Chapter 5).Because catalytic hydrogenation was unsuccessful, an in situ modification of the level of linolenic acid during the germination process was not possible. So, to study the role of linolenic acid in relation to pollen germination, Arabidopsis thaliana L. Heynh. mutants exhibiting desaturase-deficiencies were used. Pollen tube growth rate was severely decreased in pollen from the mutant fad2, in which the activity of the 16:0/18:1 desaturase located in the endoplasmatic reticulum is suppressed. In pollen from this mutant line the level of polyunsaturated fatty acids was decreased, confirming the correlation between membrane fluidity and pollen tube growth rate. In the other mutants in which chloroplast located enzymes were affected, pollen tube growth rate was unaffected (Chapter 6).The results from these studies show that with respect to aging and storage of pollen, the key factor in controling viability is the fluidity of the phospholipid bilayer. When the pollen membranes are in the high fluidity liquid crystalline phase during storage, aging is much more rapid than when the membranes are in the low fluidity gel phase. So for storage gel phase is preferable, but for rapid tube growth, on the contrary the liquid crystalline phase is required. Thus for both proper storage and germination conditions, membrane fluidity has to be carefully monitored, for instance with the aid of Fourier transform infrared spectroscopy (Chapter 7).

Overzicht van de pollenontwikkeling m.b.t. meiose, calloseontwikkeling, incompatibiliteit, organeleliminatie en mannelijke steriliteit. Bespreking van manipulatiemogelijkheden zoals m.b.v. androgenese in vitro

Overzicht van de mogelijkheden voor het ontwikkelen van nieuwe selectie- en transformatiemethoden op het niveau van pollen. Selectiemethoden met pollen vinden plaats via in vivo- en in vitrotechnieken. Selectiemethoden met microsporen worden bijv. toegepast bij koolzaad. Recent is het gelukt tarwe te transformeren door kort na bestuiving een oplossing met DNA op de stempel te brengen

Overzicht van de mogelijkheden voor het ontwikkelen van nieuwe selectie- en transformatiemethoden op het niveau van pollen. Selectiemethoden met pollen vinden plaats via in vivo- en in vitrotechnieken. Selectiemethoden met microsporen worden bijv. toegepast bij koolzaad. Recent is het gelukt tarwe te transformeren door kort na bestuiving een oplossing met DNA op de stempel te brengen

Er zijn een aantal methoden om de levensvatbaarheid van pollen te kunnen vaststellen. Vitaliteitstoetsen worden uitgevoerd m.b.v. kleuringsmethoden, kieming in vitro en in vivo. Bij bewaringstoetsen wordt de uitdroogtolerantie bepaald. Bij de kiemingstoetsen wordt de levensduur van uitdroogtolerante pollen nagegaan en wordt onderzocht hoe imbibitie-problemen kunnen worden voorkomen

Overzicht van de pollenontwikkeling m.b.t. meiose, calloseontwikkeling, incompatibiliteit, organeleliminatie en mannelijke steriliteit. Bespreking van manipulatiemogelijkheden zoals m.b.v. androgenese in vitro

Chapter 1Effects of various components upon germination in vitro were studied in order to develop an optimal germination medium for Compositae pollen. Equilibration of pollen in humid air, preceding germination, improved the reliability of results considerably.Irregular germination ability of pollen samples, originating from different collections, was studied by exposing flowering plants to different climatic conditions. High relative humidity and temperature at dehiscence cause a rapid decrease in pollen vitality. Data for an optimal germination medium and for acquisition of good pollen quality are presented.Chapter 2The respiration and vitality of ungerminated bi- and trinucleate pollen were studied in order to determine the influence of relative humidity and temperature on metabolic activity. The gas exchange, gerniination capacity and staining with tetrazolium bromide were followed under standardized conditions.A constant respiration rate occurred under conditions of high relative humidity (97 %). Per mg pollen, the trinucleate grains of Compositae and Gramineae respired 2 to 3 times as intense as 6 species of binucleate grains. Per unit of pollen protein the differences were even larger. In contrast to binucleate pollen, the longevity of trinucleate pollen was very short and the ability to germinate was lost twice as fast as the respiration capacity. This limits the use of tetrazolium bromide as an indicator of viability.At reduced relative humidities respiration was strongly restricted, but the longevity of bi- and trinucleate pollen considerably increased.Pollen of Gramineae, however, was very sensitive to changes in relative humidity; short exposure to low relative humidity decreased both the vitality and the capacity to respire.Chapter 3Bi- and trinucleate pollen generally differ in the extent of their mitochondrial development at anther dehiscence and in the rate of their attainment of maximum-phosphorylative capacity during germination in vitro, as judged from experiments with representatives of both groups.The typically trinucleate pollen of Aster tripolium L. immediately respired at a high rate, maintaining a high energy charge. Mitochondria attained maximum electron-transducing capacity within 2 min of incubation, while tube growth started within 3 min. In contrast, the binucleate pollen of Typha latifolia L. only gradually reached a relatively low rate of respiration, concomitant with a temporary decrease in energy charge, upon immersion in the germination medium. Development of the mitochondrial, electron-transducing system occurred in about 75 min, after which the first pollen tubes emerged. Starting from a poor differentiation, mitochondria became increasingly normal in appearance as germination proceeded.The binucleate pollen of Nicotiana alata Link et Otto and Tradescantia paludosa Anders. et Woods. showed intermediate characteristics: Nicotiana resembled Typha but mitochondria developed at a higher rate; Tradescantia germinated more rapidly and resembled the trinucleate pollen of Aster.Inhibitors of mitochondrial or cytoplasmic protein synthesis failed to affect the development of the mitochondrial, respiratory capacities during pollen germination. It is concluded that the duration of the lag period is determined by the level and rate of mitochondrial development and not by the division of the generative cell.Chapter 4The equal rates of water vapour absorption by both bi- and trinucleate pollen indicate that their widely-differing rates of respiration have an intrinsic, biochemical basis. This was investigated with various metabolic inhibitors that were previously introduced into dry pollen via anhydrous acetoneThe uncoupler, CCCP, inhibited the O 2 uptake of rapidly respiring pollen and stimulated that of slowly respiring types to similar absolute values, that probably reflect the rates of substrate transport across the mitochondrial membranes.The extent of inhibition of the O 2 uptake by oligomycin, DCCD, antimycin A, and SHAM, alone and in combinations, indicates that hardly any oxidative phosphorylation and anabolic activities occur in slowly respiring, binucleate pollen species, having low-developed mitochondria and high EC values. The presence of the alternative pathway was insignificant.In other binucleate pollen species, characterized by recognizable mitochondria and low EC values, a limited ATP synthesis was established. The low EC values point to imbalance between phosphorylative and anabolic activities.In rapidly respiring, trinucleate pollen, containing well- developed mitochondria, a significant activity of the alternative oxidase was found. The EC values were high notwithstanding the large demand for ATP, mounting to 1.7μmol h -1mg pollen -1.In some pollen species, oligomycin highly stimulated the flow of electrons through the cytochrome pathway, which made an estimation of the ATP synthesis impossible.Chapter 5Under humid conditions both bi- and trinucleate pollen species incorporate very low amounts of leucine, 0.4 pmol min -1mg pollen -1on an average. During germination in vitro , however, the two types of pollen greatly differ in their capacity for protein synthesis.Binucleate pollen species such as Typha , which are characterized by slow respiration in humid air and prolonged lag periods during germination in vitro , contain large amounts of monoribosomes at dehiscence. Polyribosomes are formed soon after the pollen is wetted in the germination medium and a considerable incorporation of leucine is initiated after 10-15 min.More rapidly respiring, binucleate pollen, such as Tradescantia , showing a short lag period, may contain many polysomes at dehiscence already and incorporates leucine within 2 min of incubation.On the contrary, rapidly respiring, trinucleate Compositae pollen contains very limited amounts of ribosomal material and never attains any substantial level of incorporation.Cycloheximide completely inhibited both protein synthesis and tube emergence and growth in the slowly respiring binucleate pollen species. The more rapidly respiring types are less dependent on protein synthesis, while germination of the phylogenetically advanced, trinucleate Compositae pollen proceeds completely independently.It is concluded that the level of phylogenetic advancement of the male gametophyte is characterized by its overall state of metabolic development at dehiscence rather than by the number of its generative cells.