Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.     

Temperature-induced modifications in the surface features of stamens of a tomato mutant: An SEM study

Summary Stamenless-2 (sl₂/sl₂) is a temperature-sensitive mutant of tomato (Lycopersicon esculentum) which exhibits altered stamen development under different temperatures (Sawhney 1983). By using scanning electron microscopy, this study was conducted to investigate the differentiation of surface features of mutant and normal stamens grown under different temperatures, with the view to further determine the role of temperature in gene expression in stamen development. Mutant stamens grown under intermediate temperatures (23 °C day/18 °C night) differed from the normal in hair production, the shape of epidermal cells and in the pattern of cuticular thickenings. Under low temperatures (18 °C day/15 °C night), all surface features of mutant stamens closely resembled the normal, whereas under high temperatures (28 °C day/23 °C night), the patterns and types of hairs, epidermal cells, stomata, and cuticular thickenings on mutant stamens were similar to that of a gynoecium. The staminal features of normal stamens were not affected by different temperatures. This study shows that the expression of the sl₂/sl₂ allele is influenced by temperature conditions to the extent that the pattern of cellular differentiation characteristic of either the stamens or the carpels can be induced in mutant stamens.     

Comparative analysis of stamen polypeptides of a rapeseed cultivar,Regent, a CMS polima line,and temperature-restored male-fertile polima line

Summary Polypeptides were extracted from stamens of a rapeseed (Brassica napus) cultivar, Regent, a near isogenic male-sterile line, Polima-R7 (Pol-R7), and a high-temperature-restored malefertile Pol-R7 (TR) and subsequently separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis under denaturing conditions. Four variable polypeptides with a pI around 6 were observed. Two stamen polypeptides (40000 Da, 38000 Da) were unique to Regent, and the other two (32000 Da, 30000 Da) were unique to the male-sterile Pol-R7. When the male-sterile Pol-R7 was treated with day/night temperatures of 30°/24° C for 7–10 days prior to flowering, both polypeptides unique to Regent reappeared, while the smaller polypeptides disappeared. Temperature-restored male-fertile Pol-R7 (TR) produced fertile pollen, while its short stamen filaments resembled those of the male-sterile Pol-R7. These changes in protein expression may be causally related to the CMS phenotype.     

Cytogenetic studies in A4 cytoplasmic-nuclear male-sterility system of pigeonpea

Efforts were made to study microsporogenesis and genetics of fertility restoration of A(4) cytoplasmic-nuclear male-sterility (CMS) system in pigeonpea. The process of microsporogenesis in the male-sterile (ICPA 2039) and its maintainer (ICPB 2039) plants was normal up to the tetrad formation stage. The tapetal cells in the male-sterile anthers degenerated soon after tetrad formation, resulting in shriveled and degenerated microspores. In the maintainer plants, the tapetal cells were normal and microspores were functional. The breakdown of the tapetum before the completion of microsporogenesis was the major cause for the expression of male sterility in A(4) CMS system. The studies on the inheritance of fertility restoration showed that in 3 crosses, a single dominant gene; in 1 cross, 2 duplicate genes; and in another cross, 2 complimentary genes governed the fertility restoration.     

Histological aspects of microsporogenesis in fertile,cytoplasmic male sterile and restored fertilePetunia hybrida

Summary A comparative histological study is made of microsporogenesis in fertile, cytoplasmic male sterile and restored fertilePetunia. Microsporogenesis in sterile anthers proceeds normally until leptotene. The development of the restored fertile type at 25°C is normal until the tetrad stage. In both types sporogenesis arrests and the meiocytes, c.q. microspores ultimately degenerate. The first phenomena of deviation are found in the tapetum. The effects of degeneration on cellular structure, vacuolation and cytoplasmic organization of the tapetal and sporogenous cells are variable. The deposition of callose around the meiocytes appears independent of the process of degeneration. The absence of an increase in callase activity possibly explains the remnants of callose found at late stages of development. The failure of callose wall dissolution appears to be the result of metabolic abnormalities in the tapetum and is regarded as an indirect effect of sterility.     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

Temperature effects on endogenous indole-3-acetic acid levels in leaves and stamens of the normal and male sterile 'stamenless-2' mutant of tomato (Lycopersicon esculentum Mill.)

The indole-3-acetic acid (IAA) concentration in leaves and stamens of the normal and a temperature-sensitive male sterile ‘stamenless-2′ (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.), grown under three temperature conditions, was measured by gas chromatography — mass spectrometry — selected ion monitoring (GC-MS-SIM) and by enzyme-linked immunosorbant assay (ELISA). At low (LTR, 18°C day/15°C night), intermediate (ITR, 23°C day/18°C night), and high temperatures (HTR, 28°C day/23°C night), the mutant leaves had approximately 10 to 20 times higher IAA concentrations, respectively, than the normal leaves under these temperature regimes. Similarly, the stamens of mutant flowers had approximately five and eight times higher IAA concentration at ITR and HTR, respectively, than the normal flowers. In the low temperature reverted mutant stamens, however, the level of IAA was similar to that in normal stamens. Also, with an increase in temperature, there was an increase in the level of IAA in the leaves and stamens of mutant plants. However, different temperatures had no appreciable effect on the IAA content of leaves and stamens of normal plants. It is suggested that the high IAA content in leaves and stamens of the stamenless-2 mutant is one of the factors associated with male sterility and carpellization of stamens in this mutant.     

Thermoperiodic control of stem elongation and endogenous gibberellins in Campanula isophylla

The effect of day/night temperature regimes on stem elongation and on the content of endogenous gibberellins (GAs) in vegetatively propagated plants of Campanula isophylla cv. Hvit have been studied. Compared with a constant temperature regime at 18°C (18/18°C), stem and internode elongation was enhanced significantly by a combination of high day/low night temperature (21/15°C) and inhibited by an opposite regime (15/21°C). Gibberellins A₁, A₁₉, A₄₄, A₅₃, and A₉₇ were identified as endogenous components in Campanula. (GA₉₇ was earlier referred to as 2-OH-GA₅₃.) Quantitative analysis of the endogenous GAs indicates that temperature regimes that stimulate elongation growth are accompanied by an increase in the level of GA₁, GA₁₉, and GA₄₄. On the other hand, in plants grown under conditions that reduced stem elongation growth, there was an increased level of GA₉₇.Abbreviations DIF difference between day temperature and night temperature - GA gibberellin - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass chromatography - SPE solid phase extraction - TMS trimethylsilyl - MSTFA N-methyl-N-TMS-trifluoroacetamide - KRI Kovats retention index - SIM selected ion monitoring - D2 deuterated     

A COMPARATIVE LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF MICROSPOROGENESIS IN MALE-FERTILE AND CYTOPLASMIC MALE-STERILE SUNFLOWER (HELIANTHUS ANNUUS)

Cytoplasmic male sterility (CMS) in sunflower anthers is compared with its normal (N) line by using light and electron microscopy. Degeneration and disintegration of CMS tapetum and microspore tetrads occur after meiosis II, resulting in sterility. At the onset of meiosis, the CMS tapetum enlarges radially and shows signs of disorganization of organelles and walls. The developing CMS meiocytes and tetrads of microspores do not show these abnormalities when compared with their N counterparts. The CMS microspore tetrads remain viable until a rudimentary exine forms around each microspore. At this time, the radially enlarged tapetum disintegrates, followed by disintegration of the tetrads. In N-line microsporogenesis, a peripheral, dense tapetum is present at the tetrad stage, and as each locule enlarges, free spaces occur around the tetrads. After a rudimentary exine with associated spines and colpi is formed around each microspore, the callose holding each tetrad together dissolves, freeing the microspores for further development. Eventually the binucleate tapetum becomes plasmodial, persisting until the vacuolate pollen stage.     

Photosynthetic gas exchange and temperature-induced damage in seedlings of the tropical alpine species Argyroxiphium sandwicense

The capacity of Argyroxiphium sandwicense (silverword) seedlings to acclimate photosynthetic processes to different growing temperatures, as well as the tolerance of A. sandwicense to temperatures ranging from –15 to 60° C, were analyzed in a combination of field and laboratory studies. Altitudinal changes in temperature were also analyzed in order to explain the observed spatial distribution of A. sandwicense. A. sandwicense (Asteraceae) is a giant rosette plant that grows at high elevation on two Hawaiian volcanoes, where nocturnal subzero temperatures frequently occur. In addition, the soil temperatures at midday in the open alpine vegetation can exceed 60° C. In marked contrast to this large diurnal temperature variation, the seasonal variation in temperature is very small due to the tropical maritime location of the Hawaiian archipelago. Diurnal changes of soil and air temperature as well as photosynthetic photon flux density were measured on Haleakala volcano during four months. Seedlings were grown in the laboratory, from seeds collected in ten different A. sandwicense populations on Haleakala volcano, and maintained in growth chambers at 15/5, 25/15, and 30/25° C day/night temperatures. Irreversible tissue damage was determined by measuring electrolyte leakage of leaf samples. For seedlings maintained at each of the three different day/night temperatures, tissue damage occurred at –10° C due to freezing and at about 50° C due to high temperatures. Tissue damage occurred immediately after ice nucleation suggesting that A. sandwicense seedlings tend to avoid ice formation by permanent supercooling. Seedlings maintained at different day/night temperatures had similar maximum photosynthetic rates (5 mol m^–2 s^–1) and similar optimum temperatures for photosynthesis (about 16° C). Leaf dark respiration rates compared at identical temperatures, however, were substantially higher for seedlings maintained at low temperatures, but almost perfect homeostasis is observed when compared at their respective growing conditions. The lack of acclimation in terms of frost resistance and tolerance to high temperatures, as well as in terms of the optimum temperature for photosynthesis, may contribute to the restricted altitudinal range of A. sandwicense. The small seasonal temperature variations in the tropical environment where this species grows may have prevented the development of mechanisms for acclimation to longterm temperature changes.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

HEAT INJURY DURING FLORAL DEVELOPMENT IN COWPEA (VIGNA UNGUICULATA,FABACEAE)

High night temperatures during floral development induce male sterility in cowpea (Vigna unguiculata [L.] Walp.). The objectives of this study were to determine: the possible causes of the male sterility; the stage of floral development when damage due to heat stress occurs; and whether specific tissues are damaged during the period of sensitivity to heat. Plants were grown under controlled temperatures in both greenhouses and growth chambers in separate experiments. Floral development was normal under a night temperature of 20 C, whereas flowers developed under high night temperature (30 C) set no pods due to low pollen viability and anther indehiscence. Anthers developed under 33/30 C day/night temperatures did not exhibit endothecial formation, whereas anthers developed under 33/20 C day/night temperatures exhibited normal development of the endothecial layer. Reciprocal transfers of plants between chambers with high or optimum night temperature demonstrated that the stage of floral development most sensitive to heat stress occurs 9 to 7 d before anthesis. Anthers developed under either optimal or high night temperatures were compared cytologically. Development was similar through meiosis, but after tetrad release, which occurred 8 d before anthesis, the tapetal layer degenerated prematurely under high night temperature. Premature degeneration of the tapetal layer and lack of endothecial development may be responsible for the low pollen viability, low anther dehiscence, and low pod set under high night temperatures.     

A laboratory study on the life cycle of Sericostoma personatum (Kirby & Spence), and light dark-dependent food consumption

The life cycle of Sericostoma personatum (Spence) was studied at 6 °C, 10 °C and 14 °C and at each temperature at 8 and 14 hrs daylength. Embryogenesis was not temperature dependent in the 12°–18°C range. Only 7 of 38 (app. 18%) had a direct development, the rest remained in diapause with partly developed larvae. Hatching success of single egg masses was over 95%. At 6 °C at both LDs, about 452 days are required for larval development. At 10 °C 370 days (LD 8/16), or 320 days (LD 14/10) and at 14° C 319 days (LD 8/16) and 295 days (LD 14/10) were required. Duration of instars III and IV was longer at 6 °C (both LDs), compared with all other groups. Vth instar larvae of the 14 °C (LD 14/10) group grew fastest. Instar VI larvae of the 10 °C short day group developed faster than all others. Instar VII larvae of both 14 °C groups and of the 10 °C long day group develop faster than the rest. Duration of pupal instar is only temperature dependent, regardless of light regime. The field life cycle of S. personatum may require 2–5 years. Larvae are night active. They feed on Coarse Particular Organic Material (CPOM) on the sediment surface at night. They release faeces (Fine Particular Organic Material, FPOM) into the sediment where they rest by day at a few cm depth. Their burrowing behavior thus contributes to the retention of FPOM in the stream channel. Daily food consumption at constant 10 °C is significantly dependent on night length (r ² = 0.979, p < 0.05). Two factors thus may limit food consumption: in winter, low temperatures, and in summer short nights. The species thus avoids competition by day-active shredders and predation by day-active predators.     

Mechanism of male sterility in Petunia

Summary The free amino acid contents in the anthers of male fertile, cytoplasmic male sterile (cms) and genic male sterile (gms) petunia lines were compared at different developmental stages of the male gametophyte. Quantitative differences in the amounts of free amino acids were found between the fertile and male sterile lines and between the cms and gms lines. The differences between the sterile lines were correlated with the different developmental stages at which the breakdown in microsporogenesis occurred. In the Rosy Morn (RM) cms line, where breakdown of microsporogenesis occurred at the end of prophase 1, there was an associated increase in asparagine and decrease in the other amino acids. In the RM gms line, in which breakdown occurred at the tetrad stage, an accumulation of asparagine in the anthers corresponded with an accumulation of glutamine beginning at prophase 1. Compared with fertile anthers, the sterile anthers accumulated much proline at the early meiotic stages, but no -aminobutyric acid. Comparison of the free amino acids of the fertile and the male sterile lines indicates that certain biochemical events leading to breakdown of microsporogenesis precede the observed cytological breakdown. The results from adding asparagine and glutamine to extracts of anthers at different developmental stages suggest that the amino acid balance may contribute to the changes in pH in the fertile and male sterile anthers which we observed previously.Contribution from the Volcani Center, Agricultural Research Organization Bet Dagan, Israel. 1972 Series, No. 2083 E.     

Effects of Chilling Temperature on the Activity of Enzymes of Sucrose Synthesis and the Accumulation of Saccharides in Leaves of Three Sugarcane Cultivars Differing in Cold Sensitivity

The effects of short-term exposure to chilling temperature (10 °C) on sucrose synthesis in leaves of the cold-tolerant sugarcane cultivars Saccharum sinense R. cv. Yomitanzan and Saccharum sp. cv. NiF4, and the cold-sensitive cultivar S. officinarum L. cv. Badila were studied. Plants were grown at day/night temperatures of 30/25 °C, and then shifted to a constant day/night temperature of 10 °C. After 52-h exposure to the chilling temperature, sucrose content in the leaves of NiF4 and Yomitanzan showed a 2.5- to 3.5-fold increase relative to that of the control plants that had been left on day/night temperatures of 30/25 °C. No such increase was observed in Badila leaves. Similarly, starch content in the leaves of NiF4 and Yomitanzan was maintained high, but starch was depleted in Badila leaves after the 52-h exposure. During the chilling temperature, sucrose phosphate synthase (SPS; E.C.2.4.1.14) activity was relatively stable in the leaves of NiF4 and Yomitanzan, whereas in Badila leaves SPS activity significantly decreased. There was no significant change in cytosolic fructose-1,6-bisphosphatase activity for the three cultivars at the chilling temperature. This supports the hypothesis that: (1) on exposure to chilling temperature, sucrose content in sugarcane leaves is determined by the photosynthetic rate in the leaves, and is not related to SPS activity; (2) SPS activity in sugarcane leaves at chilling temperature is to be determined by sugar concentration in the leaves.     

Effects of high-temperature stress on microsporogenesis in heat-sensitive and heat-tolerant genotypes of Phaseolus vulgaris

High ambient temperature (32/27 °C, day/night, 12 h photoperiod) applied prior to anthesis to Phaseolus vulgaris plants results in abnormal pollen and anther development during microsporogenesis. Scanning and transmission electron microscopy were used to examine anther and pollen morphology and pollen wall architecture after heat stress was applied to two genotypes that differ with respect to yield potential under high‐temperature field conditions: one, a heat‐sensitive, Mesoamerican genotype, A55, the second, a heat‐tolerant, Andean genotype, G122. High‐temperature treatment of both genotypes was applied 1–13 d before anthesis. Under heat stress, the heat‐tolerant genotype showed anther and pollen characteristics that were generally similar to the low temperature controls. In contrast, after 9 d of heat treatment before anthesis, the anthers of the heat‐sensitive genotype were indehiscent and contained abnormal pollen. Pollen wall architecture was also affected in the 12 and 13 d treatments. In addition to the morphological changes, the heat‐sensitive genotype also experienced reduced pollen viability and reduced yield in high‐temperature experiments conducted in both the greenhouse and field.     

Diel activity and thermoregulatory behavior of a fully aquatic frog: Xenopus laevis

Ten adult Xenopus laevis were tested individually for 48-hr periods, following an initial 24-hr introductory period, in electronic shuttleboxes which allowed them to control water temperatures without operant conditioning. Locomotor activity was recorded via photocell-monitored light beams. The frogs were nocturnal, being nearly twice as active at night as during the day. The mean preferred temperature was 22.4°C, with no significant difference between night (22.5°C) and day (22.3°C), although the modal preferendum shifted from 24°C by day to 22°C at night, with a corresponding change in skewness. The range of voluntarily occupied temperatures was 14–32°C by day and 14–29°C at night. The median thermal preferendum was 22°C both day and night.