Self‐pollination and parthenocarpic ability in developing ovaries of self‐incompatible Clementine mandarins (Citrus clementina)

This study aimed to determine if self‐pollination is needed to trigger facultative parthenocarpy in self‐incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). ‘Marisol’ and ‘Clemenules’ mandarins were selected, and self‐pollinated and un‐pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self‐pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time‐course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA₁ and GA₄), auxin (IAA) and abscisic acid (ABA) content in the ovary. ‘Clemenules’ showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self‐pollination did not stimulate parthenocarpy, whereas both un‐pollinated and self‐pollinated ovaries set fruit regardless of the cultivar. On the other hand, ‘Marisol’ un‐pollinated flowers showed greater parthenocarpic ovary growth than ‘Clemenules’ un‐pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of ‘Marisol’ paralleled higher levels of GA₁ in the ovary (+34% at anthesis). ‘Marisol’ ovary also showed higher hexoses and starch mobilization, but lower ABA levels (?64% at anthesis). Self‐pollination did not modify carbohydrates or GA content in the ovary compared to un‐pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination‐independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self‐incompatible mandarins is constitutively regulated.     

Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature,polyamines and polyamine synthesis inhibitor

Prunus dulcis L. ‘Mamaei’ is grown widely in souhtwest of Iran. It blooms in early spring when temperatures are still low. Based on our knowledge there are no reports in the literature regarding pollen behavior of this cultivar under specified condition. Thus, the possible factors for low germination percentage in this cultivar have not been reported. The effect of three different temperatures (10, 25, or 35 °C), polyamines (putrescine, spermidine, and spermine) and polyamine synthesis inhibitor, methylglyoxals-bis (guanyl-hydrazone) (MGBG) on in vitro pollen germination and pollen tube growth were investigated in P. dulcis L. ‘Mamaei’. All temperatures and chemicals significantly affected both pollen germination percentage and pollen tube growth. In general, different polyamines stimulated the pollen germination percentage compared to the control at all temperatures, but increasing the temperature, particularly to 35 °C, had demonstrated inhibitory effects on pollen germination. At a concentration of 0.05 mM putrescine and spermidine and 0.005 and 0.025 mM spermine revealed longer pollen tube growth than that of the control at 10 °C, while higher concentrations tended to inhibit pollen tube growth. At 25 °C, most of the treatments had an inhibitory effect on pollen tube growth except for 0.25 mM putrescine and 0.005 mM spermine, which slightly stimulated pollen tube growth. Pollen germination and pollen tube growth were inhibited by MGBG at all temperatures and in all concentrations.     

Pollen tube growth in the self‐compatible sweet cherry genotype, ‘Cristobalina’, is slowed down after self‐pollination

Sweet cherry is a self‐incompatible fruit tree species in the Rosaceae. As other species in the family, sweet cherry exhibits S‐RNase‐based gametophytic self‐incompatibility. This mechanism is genetically determined by the S‐locus that encodes the pollen and pistil determinants, SFB and S‐RNase, respectively. Several self‐compatible sweet cherry genotypes have been described and most of them have mutations at the S‐locus leading to self‐compatibility. However, ‘Cristobalina’ sweet cherry is self‐compatible due to a mutation in a pollen function modifier that is not linked to the S‐locus. To investigate the physiology of self‐compatibility in this cultivar, S‐locus segregation in crosses involving ‘Cristobalina’ pollen, and pollen tube growth in self‐ and cross‐pollinations, were studied. In the crosses with genotypes sharing only one S‐haplotype, the non‐self S‐haplotype was inherited more frequently than the self S‐haplotype. Pollen tube growth studies revealed that the time to travel the whole length of the style was longer for self‐pollen tubes than for cross‐pollen tubes. Together, these results suggest that ‘Cristobalina’ pollen tube growth is slower after self‐pollination than after cross‐pollination. This reproductive strategy would allow self‐fertilisation in the absence of compatible pollen but would promote cross‐fertilisation if cross‐compatible pollen is available, a possible case of cryptic self‐incompatibility. This bet‐hedging strategy might be advantageous for an ecotype that is native to the mountains of the Spanish Mediterranean coast, in the geographical limits of the distribution of this species. ‘Cristobalina’ blooming takes place very early in the season when mating possibilities are scarce and, consequently, self‐compatibility may be the only possibility for this genotype to produce offspring.     

Temperatures during flower bud development affect pollen germination,self‐incompatibility reaction and early fruit development of clementine (Citrus clementina Hort. ex Tan.)

• One of the key environmental factors affecting plant reproductive systems is temperature. Characterising such effects is especially relevant for some commercially important genera such as Citrus. In this genus, failure of fertilisation results in parthenocarpic fruit development and seedlessness, which is a much‐prized character. Here, we characterise the effects of temperature on flower and ovary development, and on pollen–pistil interactions in ‘Comune’ clementine (Citrus clementina Hort. ex Tan.).
• We examine flower bud development, in vitro pollen germination and pollen–pistil interaction at different temperatures (15, 20, 25 or 30 °C). These temperatures span the range from ‘cold’ to ‘hot’ weather during the flowering season in many citrus‐growing regions.
• Temperature had a strong effect on flower and ovary development, pollen germination, and pollen tube growth kinetics. In particular, parthenocarpic fruit development (indicated by juice vesicle growth) was initiated early if flowers were exposed to warmer temperatures during anthesis.
• Exposure to different temperatures during flower bud development also alters expression of the self‐incompatibility reaction. This affects the point in the pistil at which pollen tube growth is arrested and confirms the role of sub‐ and supra‐optimal temperatures in determining the numbers of pollen tubes reaching the ovary.

     

Post‐pollination process in a partially self‐compatible distylous plant,Primula sieboldii (Primulaceae)

Pollen germination and pollen‐tube growth under natural conditions were observed in a population of a distylous species, Primula sieboldii, in which partial self‐compatibility has been demonstrated in some long‐styled genets. We observed post‐pollination processes microscopically in styles collected after self‐morph and inter‐morph hand pollination (with standardized pollen load on the stigmas) in four genets each from the following three ‘genet types’: self‐incompatible long‐styled (SI), partially self‐compatible long‐styled (SC) and self‐incompatible short‐styled morph genets. Irrespective of the genet type, pollen germination began within 24 h after pollination and tubes of pollen reached to the style base with 48–96 h after inter‐morph pollination. Although pollen tubes germinated after self‐pollination in the SC genets, the number of germinated pollen tubes was significantly lower than in the case of inter‐morph pollination. Few pollen tubes germinated after self‐pollination of the SI or short‐styled genets. In SC genets, the rate of pollen‐tube growth did not differ between self‐morph and inter‐morph pollination (～1.9 mm/day). Therefore, differences in self‐compatibility between SC and SI genets in P. sieboldii are likely to be attributable to differential pollen germination rates rather than to differential pollen‐tube growth rates.     

Reactive oxygen species are involved in regulation of pollen wall cytomechanics

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H₂O₂ and the hydroxyl radical (^?OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H₂O₂, ^?OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn‐5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)21H, 23H‐porphin, or a spin‐trap α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone, which eliminates ^?OH. The inhibiting concentrations of exogenous H₂O₂ and ^?OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo‐osmotic shock. ^?OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H₂O₂, as well as partial removal of endogenous ^?OH, over‐tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H₂O₂‐inducible inter‐polymer cross‐linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross‐links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. ^?OH is involved in local loosening of the intine in the germination pore region, while H₂O₂ is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.     

The optimum sevoflurane concentration for supraglottic airway device Blockbuster™ insertion with spontaneous breathing in obese patients: a prospective observational study

Background

Airway management of the obese patient presenting for surgery is more likely to be a challenging problem. Supraglottic airway device has been adopted as a bridge to connect ventilation and tracheal intubation in obese patients who would be suffered with difficult intubation. The optimum sevoflurane concentration for supraglottic airway device insertion allowing spontaneous breathing in 50% of obese patients (ED₅₀) is not known. The purpose of this study was to determine the ED₅₀ of sevoflurane for supraglottic airway device Blockbuster? insertion with spontaneous breathing in obese patients requiring general anesthesia.

Methods

Thirty elective obese patients (body mass index 30-50 kg/m²) undergoing bariatric surgery were recruited in this study. The predetermined target sevoflurane concentration (initiating at 2.5% with 0.5% as a step size) was sustained for >5 min using a modified Dixon’s up-and-down method, and then the supraglottic airway device Blockbuster? was inserted. The patient’s response to supraglottic airway device insertion was classified as either ‘movement’ or ‘no-movement’. The ED₅₀ of sevoflurane were determined by calculating the midpoint concentration of crossover point from ‘movement’ or ‘no-movement’ response.

Results

The ED₅₀ of sevoflurane for supraglottic airway device Blockbuster? insertion in obese patients calculated using up-and-down method were 2.50?±?0.60%. The ED₅₀ and ED₉₅ (95% confidence interval) obtained by probit regression analysis were 2.35 (1.28–3.42) % and 4.03 (3.16–17.83) % for supraglottic airway device Blockbuster? insertion, respectively.

Conclusion

We conclude that the optimum end-tidal sevoflurane concentration required for the supraglottic airway device Blockbuster? insertion allowing spontaneous breathing in 50% of obese patients (ED₅₀) is 2.5?±?0.6%.

Trial registration

Chinese Clinical Trial Registry, ChiCTR-IPR-16009071, Registered on 24 August 2016.

     

Impact of temperature on olive (Olea europaea L.) pollen performance in relation to relative humidity and genotype

Olive varieties ‘Koroneiki’, ‘Kalamata’, ‘Mastoidis’ and ‘Amigdalolia’ were employed in two experiments for 3 years to assess the effect of temperature on olive pollen germination and tube growth in relation to relative humidity and genotype. Pollen samples were subjected to pre-incubation at 10, 20, 30 or 40 °C in combination with decreased air relative humidity – 80, 40, 30 or 20%, respectively – for 24 h to simulate temperature stress that is observed during pollen dispersal; and subsequently in vitro cultured. In the second experiment, pollen was exposed at 15, 20, 25 and 30 °C for 24 h in vitro to evaluate pollen response in conditions of water and nutrients availability and to determine the optimum pollen germination and tube growth temperatures for each cultivar. The highest pre-incubation temperature treatment (40 °C) prevented pollen germination in ‘Koroneiki’ and ‘Mastoidis’, with the less affected varieties (‘Amigdalolia’ and ‘Kalamata’) having average germination percentages of only 7.6 and 2%, respectively. Pre-incubation at 30 °C had a negative impact on pollen germination in ‘Koroneiki’ (?65%), ‘Kalamata’ (?20%) and ‘Amigdalolia’ (?72%) compared to the control (20 °C). Pollen pre-incubation at 40 °C decreased significantly the pollen tube length in ‘Kalamata’ (?50%) and ‘Amigdalolia’ (?52%). In the second experiment, in vitro pollen germination increased after incubation at 25 °C for ‘Koroneiki’ (+6%), ‘Mastoidis’ (+52%), ‘Kalamata’ (+10%) and ‘Amigdalolia’ (+10%) compared to the control (20 °C). At 30 °C germination percentages for ‘Mastoidis’, ‘Kalamata’ and ‘Amigdalolia’ were 8, 6 and 14% higher, respectively, compared to the control (20 °C). Pollen tube length also increased with incubation temperature for all of the studied cultivars. Based on the cumulative stress response index (CSRI) that was calculated for high temperature stress the varieties were classified: ‘Mastoidis’ and ‘Kalamata’ as tolerant and ‘Koroneiki’ and ‘Amigdalolia’ as intermediate at 30 °C while all studied cultivars were sensitive at 40 °C. The observed strong genotype-differentiated response in high and low temperature stress could be exploited by plant breeders towards producing new tolerant olive varieties.     

Male gametophyte defective 4 encodes a rhamnogalacturonan II xylosyltransferase and is important for growth of pollen tubes and roots in Arabidopsis

In flowering plants, the growth of pollen tubes is essential for the delivery of sperm to the egg cells. Although many factors (including cell‐wall properties) are involved in this process, little is known about the underlying molecular mechanisms that regulate the growth of pollen tubes. We report here the characterization of an Arabidopsis mutant male gametophyte defective 4 (mgp4) that is severely defective in pollen tube growth. The mgp4 mutation also impairs root growth of pollen‐rescued mgp4 mutant plants generated by expressing MGP4 cDNA under the control of a pollen grain/tube‐specific promoter. The MGP4 gene encodes a putative xylosyltransferase and is expressed in many organs/tissues, including pollen tubes and roots. MGP4 protein expressed in Pichia pastoris exhibited xylosyltransferase activity and transferred d ‐xylose onto l ‐fucose. The pectic polysaccharide rhamnogalacturonan II (RG‐II), isolated from 7‐day‐old pollen‐rescued mutant seedlings, exhibited a 30% reduction in 2‐O‐methyl d ‐xylose residues. Furthermore, an exogenous supply of boric acid enhanced RG‐II dimer formation and partially restored the root growth of the pollen‐rescued mutant seedlings. Taken together, these results suggest that MGP4 plays important roles in pollen tube and root growth by acting as a xylosyltransferase involved in the biosynthesis of pectic RG‐II.     

Monomeric G‐actin is uniformly distributed in pollen tubes and is rapidly redistributed via cytoplasmic streaming during pollen tube growth

Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G‐actin) and filamentous actin (F‐actin) is crucial for actin assembly and array construction, and the local concentration of G‐actin thus directly impacts actin assembly. The localization and dynamics of G‐actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I‐binding loop of ACT11 (GFP_Met49–ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFP_Met49–ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)‐treated pollen tubes. Careful observations showed that G‐actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G‐actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.     

Response of in vitro pollen germination and pollen tube growth of groundnut (Arachis hypogaea L.) genotypes to temperature

Air temperatures of greater than 35 °C are frequently encountered in groundnut‐growing regions, especially in the semi‐arid tropics. Such extreme temperatures are likely to increase in frequency under future predicted climates. High air temperatures result in failure of peg and pod set due to lower pollen viability. The response of pollen germination and pollen tube growth to temperature was quantified in order to identify differences in pollen tolerance to temperature among 21 groundnut genotypes. Plants were grown from sowing to harvest in a poly‐tunnel under an optimum temperature of 28/22 °C (day/night). Pollen was collected at anther dehiscence and was exposed to temperatures from 10° to 47·5 °C at 2·5 °C intervals. The results showed that a modified bilinear model most accurately described the response to temperature of percentage pollen germination and maximum pollen tube length. Genotypes were found to range from most tolerant to most susceptible based on both pollen characters and membrane thermostability. Mean cardinal temperatures (T_min, T_opt and T_max) averaged over 21 genotypes were 14·1, 30·1 and 43·0 °C for percentage pollen germination and 14·6, 34·4 and 43·4 °C for maximum pollen tube length. The genotypes 55‐437, ICG 1236, TMV 2 and ICGS 11 can be grouped as tolerant to high temperature and genotypes Kadiri 3, ICGV 92116 and ICGV 92118 as susceptible genotypes, based on the cardinal temperatures. The principal component analysis identified maximum percentage pollen germination and pollen tube length of the genotypes, and T_max for the two processes as the most important pollen parameters in describing a genotypic tolerance to high temperature. The T_min and T_opt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating genotypes for high temperature tolerance. Genotypic differences in heat tolerance‐based on pollen response were poorly related (R² = 0·334, P = 0·006) to relative injury as determined by membrane thermostability.     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

Spermidine oxidase‐derived H2O2 regulates pollen plasma membrane hyperpolarization‐activated Ca2+‐permeable channels and pollen tube growth

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca²⁺ concentration that accompanies pollen tube growth. Using the whole‐cell patch clamp and outside‐out single‐channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization‐activated Ca²⁺ current: the addition of Spd cannot induce the channel open probability increase in excised outside‐out patches, indicating that the effect of Spd in the induction of Ca²⁺ currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization‐activated Ca²⁺‐permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca²⁺‐permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal‐encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca²⁺‐permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca²⁺ influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd‐derived H₂O₂ signals Ca²⁺ influx, thereby regulating pollen tube growth.     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.     

Influence of temperature on the in vitro pollen germination and pollen tube growth of various native Iranian almonds (Prunus L. spp.) species

Pollen germination and pollen tube growth was quantified among various native Iranian wild almonds (P. dulcis (Mill.) D. A. Webb, P. eleaegnifolia Mill., P. orientalis Mill., P. lycioides Spach, P. reuteri Bioss. et Bushe, P. arabica Olivier, P. glauca Browick and P. scoparia Spach in order to identify differences in the tolerance of pollen to temperature variations. Pollen germination and pollen tube growth were observed after incubation in darkness in a germination medium for 24?h at 10?C50°C at 5°C intervals. Maximum pollen germination of the wild almond species and specify that 60% was obtained for P. orientalis pollen and 98% for P. scoparia. Pollen tube length ranged from 860???m was obtained in P. lycioides and 1490???m in P. scoparia. A modified bilinear model best described the response to temperature of pollen germination and pollen tube length. Almond species variation was found for cardinal temperatures (T _min, T _opt and T _max) of pollen germination percentage and pollen tube growth. Mean cardinal temperatures averaged over eight almond species were 14.7, 24.2, and 43.7°C for maximum percentage pollen germination and 14.48, 25.3, and 44.4 °C for maximum pollen tube length. The principal component analysis (PCA) identified maximum percentage pollen germination and pollen tube length of the species, and T _max for the two processes as the most important pollen parameters in describing a species tolerance to high temperature. PCA also classified Prunus L. spp. into four groups according to the tolerance of pollen to temperature variations. The T _min and T _opt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating species for high temperature tolerance.     

Stigma development and receptivity of two Kalanchoë blossfeldiana cultivars

Several members of the Kalanchoë genus are popular as ornamental plants. Cross-breeding and wide hybridisation are essential to continuously introduce novel traits into cultivated plant material. This study aimed to identify the major factors related to the stigma affecting cross-pollination in the Kalanchoë blossfeldiana. Pollen tube growth after pollination of K. blossfeldiana ‘Jackie’ and ‘Reese’ was examined at different stigma developmental stages. Five distinct developmental stages were identified based on changes in morphology and activity of stigmatic peroxidase. After reciprocal pollination at the five stigma developmental stages, fluorescence microscopy was used to estimate the number of pollen tubes in situ. Both cultivars had receptive stigmas from stage I to IV, which concurred with the continuous expansion of the stigma covered with exudates. No pollen tube growth was observed at stage V for both cultivars. The number of pollen tubes was significantly higher in carpels pollinated at stage III, characterized by loose arrangement of the papillae and maximal amount of exudates, compared to all other developmental stages. Stigmas showing drying exudates and absence of peroxidase exhibited a relatively decreased number of pollen tubes in situ. No pollen tubes germinated on wilting stigmas. The arrangement of the papillae, the presence of exudates and peroxidase activity affected the number of pollen tubes in cross-pollination of K. blossfeldiana cultivars ‘Jackie’ and ‘Reese’. These results will help breeders to better select the optimal time for effective pollination. The findings may be applicable for other cultivars of K. blossfeldiana and relevant for different species of Kalanchoë.     

A metal‐chelate affinity reverse micellar system for protein extraction

A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X‐45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X‐45 less than 0.6, thermodynamically stable reverse micelles of water content (W₀) up to 30 are formed. Di(2‐ethylhexyl) phosphoric acid (HDEHP; 1–2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine‐rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper‐chelate reverse micelles (Cu‐RM) are characterized for their W₀, hydrodynamic radius (R_h), and aggregation number (N_ag). Similar with reverse micelles of bis‐2‐ethylhexyl sodium sulfosuccinate (AOT), R_h of the Cu‐RM is also linearly related to W₀. However, N_ag is determined to be 30–90 at W₀ of 5–30, only quarter to half of the AOT reverse micelles. Then, selective metal‐chelate extraction of histidine‐rich protein (myoglobin) by the Cu‐RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine‐tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.