Storage of Pollen Grains in Organic Solvents: Effect of Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability

In vitro germinability and membrane integrity (as revealed bythe fluorochromatic reaction (FCR) test) of pollen grains ofCrotalaria retusa L. stored in various organic solvents forsix months at –20±2 °C were studied and correlatedwith leaching of lipids, phospholipids, sugars and free aminoacids from pollen grains into organic solvents during storage.Pollen grains stored in organic solvents with low dielectricconstants (a measure of their non-polar nature), such as hexane,cyclohexane and diethyl ether, showed high scores for germinationand FCR and very little leaching of phospholipids, sugars andamino acids. Pollen grains stored in solvents with high dielectricconstants (a measure of their polar nature) such as isopropanoland methanol did not show germination or positive FCR scores,but showed extensive leaching of phospholipids, sugars and freeamino acids. The viability of pollen grains stored in organicsolvents seems to be determined largely by the effect of theorganic solvents on pollen phospholipid composition, which inturn affects membrane integrity and consequently pollen viability. Crotalaria retusa, organic solvents, pollen storage, viability, phospholipids     

Acceleration of the Growth of Camellia sasanqua Pollen by Soaking in Organic Solvent

Camellia sasanqua pollen that had been soaked in acetone or diethyl ether for only 5 minutes grew three to four times longer pollen tubes than unsoaked pollen. Although the acceleration of pollen tube growth was observed when the pollen had been soaked in cold solvents (5 C, −15 C) for 100 days, soaking in warm solvents (30 C, 24 C) caused it to decrease gradually, and the pollen eventually lost the ability to germinate. The acceleration may be caused by removing inhibitor from the pollen grain by the organic solvents.     

Retaining the viability of Camellia japonica pollen in various organic solvents

Pollen viability in 31 organic solvents was studied. Pollengrains of Camellia japonica which had been soaked in 28 organicsolvents for 3 days retained their viability and grains in 19solvents such as ethyl acetate, n-amyl alcohol and petroleumether grew longer pollen tubes than fresh pollen. (Received July 20, 1972; )     

Role of Steroids and Amino Acids in Pollen Germination of Eichhornia crassipes Solms.

Detection of amino acids from the pollen grains of Eichhornia crassipes together with isolation of β-sitosterol, stigmasterol and free sugars from the pistil were carried out. The role of the growth potential of the pollen and the growth-influencing compounds of the pistil are discussed. Amino acids and free sugars were detected in pollen and in pistils by paper chromatography. The neutral fractions of the alcohol extract (95%) of the pistil on column chromatography [petroleum ether: benzene (2:1)] gave a crystalline compound (m.p. 136-139°C) with positive Liebermann-Burchard test for sterol. A mass spectrum of the compound showed a mixture of β-sitosterol and stigmasterol, which was confirmed by comparative thin-layer chromatography and gas-layer chromatography.     

Characterization of 1,3-regiospecific lipases from new Pseudomonas and Bacillus isolates

Lipase producing ability of 120 bacterial isolates was examined qualitatively, resulting in 32 lipase producers, which were further screened for 1,3-regiospecificity. Three Bacillus (GK-8, GK-31 and GK-42) and one Pseudomonas (GK-80) were found to produce 1,3-regiospecific lipases. These lipases were alkaline in nature as they showed pH optima of 9.0 and high stability in the alkaline pH range of 8.0–11.0. The lipases from three Bacillus isolates, viz. GK-8, GK-31 and GK-42 showed temperature optima of 37 °C, whereas the Pseudomonas (GK-80) lipase showed optimum activity at 50 °C. The lipase of GK-8 was highly stable and showed enhanced activity in different organic solvents like petroleum ether (172%), diethyl ether (143%) and acetone (135%).     

DNA uptake during electroporation of germinating pollen grains

Electroporation of germinating pollen grains of Nicotiana gossei (L.) Domin under a variety of conditions showed that DNA was taken up by the pollen without detrimental effects on the viability of the pollen. By optimizing both the field strength of the electroporation pulse and the DNA concentration in the electroporation medium up to 6% of the donor DNA can be taken up by the germinating pollen while maintaining a pollen viability of 90%. Field strengths as high as 9 kV/cm could be applied to germinating pollen grains without detrimental effects on viability. Southern hybridizations demonstrated that DNA encoding the marker enzyme β-glucuronidase (GUS) was incorporated into electroporated pollen. Germinating pollen, treated in this manner, is capable of producing 300–400 seeds per capsule of viable seed when applied to the stigmas of compatible flowers of N. gossei which has been emasculated 4 days earlier.     

Loss of viability of tomato pollen during long-term dry storage is associated with reduced capacity for translating polyamine biosynthetic enzyme genes after rehydration

The possibility that a loss of pollen viability during dry storage in a freezer is caused by the reduced pollen capacity to enhance polyamine biosynthetic enzyme activity after rehydration was investigated using pollen grains of tomato (Solanum lycopersicum=Lycopersicon esculentum) stored at -30 degrees C under dry conditions for up to 42 months. Pollen grains showed normal germinability for at least 12 months in storage, but those stored for longer than 24 months exhibited a significant reduction in germinability and fruit-setting ability. This age-dependent reduction in pollen viability coincided with the extent to which the pollen lost the capacity to increase arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) activities and polyamine contents upon rehydration. Immunoblot analysis indicated that the capacity of pollen to translate ADC and SAMDC mRNAs was impaired in accordance with the loss of viability. Also, the capacity to synthesize proteins in general decreased with the increase in storage duration. The addition of 1 mM putrescine, spermidine, or spermine to incubation medium promoted germination, impregnation of pollen grains with 1 mM spermidine restored fertilization ability, and the addition of 1 mM spermidine to incubation medium promoted protein synthesis exclusively in pollen grains which had been stored for a long time. These results indicate that the reduction in viability of tomato pollen during long-term dry storage in a freezer involves a decline in the capacity to enhance gene translation for polyamine biosynthetic enzymes upon rehydration.     

Responses of tobacco pollen to high humidity and heat stress: viability and germinability in vitro and in vivo

Summary Responses of pollen grains of Nicotiana tabacum to high humidity (95% RH, 4 h) and temperature (38°/45° C, 4 h) stresses were investigated. Pollen grains were subjected to only RH or only temperature, or to both of these stresses. Their viability was assessed on the basis of the fluorochromatic reaction (FCR) test, and vigour was assessed on the basis of the time taken for in vitro germination as well as on the emergence of pollen tubes through the cut end of semi-vivo implanted styles. None of the stress conditions affected pollen viability and high RH or high temperature stress did not individually affect pollen vigour. However, pollen vigour was markedly affected when both the stresses were given together. Pollen grains subjected to high RH at 38° C took a longer time to germinate in vitro and the pollen tubes emerged later from the cut end of the semi-vivo styles; division of the generative cell was also delayed. Pollen grains subjected to high RH at 45° C failed to germinate in vitro, but did germinate on the stigma. Many pollen tubes subjected to this treatment showed abnormalities, and the growth of pollen tubes in the pistil was much slower than that observed in other treatments. Pollen samples subjected to all of the stress conditions were able to induce fruit and seed set. The implications of these results on the relationship between the FCR test and viability, and between viability and vigour, especially in stressed pollen, are discussed.     

Effect of Water Loss on Germination Ability of Maize (Zea mays L.) Pollen

The correlation between water content and viability of maizepollen grains was studied on the basis of the germination abilityof pollen from a single cross hybrid. There was found to beclose correlation between viability of the grains and theirtolerance to desiccation. Most of the pollen grains in the hybridexamined survived a reduction by almost 50 per cent of the originalwater content without loss of normal function. With water lossgreater than this, less vigorous pollen grains died or losttheir ability to form pollen tubes. Consequently, when pollinationwas carried out using pollen with a water content reduced bymore than 50 per cent, only the most tolerant pollen grainssurvived to take part in the competition which precedes fertilization.Dry pollen grains required a longer period to establish adhesionto the stigma surface and to initiate pollen tubes than pollengrains with higher water content, but otherwise their behaviourwas normal. If more than 80 per cent of the original water contentwas lost, disturbances occurred in the physiology of the grainssurviving the treatment. This was exhibited as death or a reductionin rate of pollen tube growth. Drying of pollen by an amount which does not irreversibly damagethe more tolerant grains could possibly be used as a means ofpollen selection. Zea mays L., maize, pollen viability, pollen treatment, dehydration, pollen tube     

POLLEN LONGEVITY AND STIGMA PRE-EMPTION IN CLARKIA

The ability of pollen to effect seed set decreases with age when stored at room temperature or on the plant, but not when stored at 5 C. Seed set declines rapidly with pollen stored 10 days or more at room temperature. Seed set declines rapidly with pollen stored 4 days or more on the plant. The degree of pollen germination on the stigma and the number of pollen tubes in the style correlate positively with seed set. Pollen germination on the stigma and pollen tube growth in the style are strongly correlated with seed set for pollen stored at room temperature, but not, or only weakly, correlated for pollen stored at 5 C. In the latter case, seed sets are lower than expected, indicating a possible viability reduction associated with a post-pollination function. Inviable pollen grains temporarily interfere with normal pollen germination on the stigma. Essentially no seeds are produced when in viable pollen, immediately followed by viable pollen, is placed on a fresh stigma. However 27 to 65 seeds are produced when in viable pollen, followed in 1 to 6 days by viable pollen, is placed on fresh stigmas. New stigma surface is exposed at the center as expanding stigma surfaces carry the interfering inviable pollen toward the edge of the stigma.     

Retaining the viability of the resting egg of brine-shrimp (Artemia salina) in organic solvent

The resting egg of brine-shrimp (Artemia salina) which had been soaked in various organic solvents such as acetone, acetonitril, benzol, chloroform, ethyl ether, petroleum benzene, pyridine, and xylol for 10 days hatched and grew in artificial sea water in just the same way as did unsoaked eggs.     

野菊与菊花杂交中花粉活力和柱头可授性及胚胎发育研究

应用石蜡制片、活体压片、光学显微镜及扫描电子显微镜观察等方法,研究了四倍体河南云台山野菊(Dendranthema indicum)与栽培菊花'钟山金山'(D.grandiflorum 'Zhongshanjinshan')种间杂交中父本花粉活力、花粉在柱头萌发、花粉管生长及胚胎发育情况等.结果发现,父本云台山野菊的花粉活力在授粉时为12%左右.人工授粉后的不同时间,在柱头上都观察到正常萌发的花粉粒,且花粉管都能进入柱头,其中,在授粉后0.5 h时,平均每柱头有5.9粒花粉萌发;12 h时,为59.9粒;而24和48 h时,则分别降为47.1和35.7粒.此外,在授粉后8、10、12和15 d时,分别在49.1%、40.8%、39.7%和38.5%子房内观察到正常发育的胚胎,最终杂交结实率为44.8%,而母本自然开放结实率为52.3%.研究表明,授粉前其多数母本雌蕊发育良好、授粉后多数花粉能在柱头正常萌发和花粉管正常生长,在受精后大部分胚胎发育正常是野菊与栽培菊种间杂交较高结实率的重要保证,而授粉前父本较低的花粉活力对杂交结实率影响不大.     

漆酶催化苯甲醇氧化制备苯甲醛

以自制的高活性漆酶为催化剂,考察漆酶催化苯甲醇制备苯甲醛的工艺条件(底物浓度、介质体系、溶剂体系、氢受体、酶的用量、通氧方式等)对氧化反应的影响。结果发现:优化反应条件为以2,2,6,6-四甲基哌啶-1-氧基(TEMPO)为介质体系且TEMPO与苯甲醇的摩尔比为1∶4、60 mmol/L的丙酮为氢受体、漆酶比酶活80 U/mL、60mmol/L的苯甲醇,反应体系通O20.5 h后密闭反应36 h,苯甲醛的产率达98%。     

Molecular evidence that most RNAs required for germination and pollen tube growth are stored in the mature pollen grain in petunia

After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for up-regulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.     

Applications of Fluorochjromes to Pollen Biology. Ii. The Dna Probes Ethidium Bromide and Hoechst 33258 in Conjunction with the Callose-Specific Aniline Blue Fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

甜瓜细菌性斑点病拮抗菌P-13的鉴定及其抑菌物质的初步研究

从新疆甜瓜根际土壤中分离到1株甜瓜细菌性斑点病拮抗放线菌P-13, 根据其形态学、生理特征和16S rRNA序列分析, 鉴定该菌株为娄彻氏链霉菌(Streptomyces rochei)。琼脂扩散法生物活性研究表明, 其发酵液对细菌性果腐病菌(Acidovorax avenae subsp. citrull)BFB、细菌性角斑病菌(Pseudomonas syringae pv. Lachrymans)P4的抑菌圈直径分别为19 mm和17 mm以上; 发酵液中抑菌物质主要为胞外代谢物, 不溶于石油醚, 乙醚, 乙酸乙酯等有机溶剂。在100°C处理10 min、pH 6处理6 h或紫外线照射7 h, 该物质抑菌活性不变。纸层析结果表明, 该物质主要为碱性水溶性物质。     

Involvement of the ubiquitin/proteasome pathway in the organisation and polarised growth of kiwifruit pollen tubes

We recently reported the involvement of the ubiquitin pathway in microgametophyte development, and a direct role for the 26S proteasome in regulating pollen tube emergence in kiwifruit. Here we show that the ubiquitin/proteasome proteolytic pathway is involved not only in early kiwifruit pollen tube organisation, but also in maintaining polarised growth of tubes. By immunofluorescence analysis we show that ubiquitin and ubiquitin-protein conjugates are distributed mainly at the apex of emerging tubes, in both untreated pollen grains and pollen grains treated with MG132, an inhibitor of proteasome function. In the latter case, polysiphonous germination occurred and all the emerging areas were highly fluorescent. By adding MG132 to pollen when normal tube growth had already been established, accumulation of ubiquitin-protein conjugates, as well as a drastic reduction in tube growth and dramatic modifications of tube tip morphology were observed. Significantly, differential interference contrast microscopy analysis demonstrated that the clear zone was largely reduced or absent, and the nuclei were disconnected in their movements, reaching, in some cases, the extreme apex of the tip. These findings provide evidence that the ubiquitin- and proteasome-dependent proteolytic system could modulate the abundance and/or activity of key regulatory proteins involved in pollen tube emergence and polarised growth.     

红松配子体的发育过程

红松是我国主要用材树种,材质优良。我国主要分布在长白山和小兴安岭,随着森林工业的发展,大面积的原始森林迅速减少,人工更新日趋重要。在生产中采用种子育苗,因此对红松种子的发育规律应全面掌握,以便为种子园建立、良种选育采取有效措施提供理论依据。根据国内外有关报导,红松雌雄配子的形成、受精作用已有一定的研究^[14、15],但这些研究只侧重在小孢子母细胞的减数分裂和受精作用。故对雌雄配子体的发育过程仍需深入研究,尤其是授粉后的雄配子体发育和精子进入颈卵器的过程,均未见详细报导。     

葎草不同溶剂提取物和萃取物的杀虫活性

对律草[Humulus scandens(Lour.) Merr.]全草干粉的不同溶剂提取物以及丙酮提取物的不同溶剂萃取物的杀虫活性进行了研究.在葎草的石油醚、苯、三氯甲烷、乙酸乙酯、丙酮和乙醇提取物(333 g·L-1)中,丙酮和乙醇提取物对小菜蛾(Plutella xylostella L.)的触杀作用较强,其中丙酮提取物的触杀作用最强,试虫的48h校正死亡率达到86.67％.6种溶剂提取物(100g·L-1)对小菜蛾的拒食活性均较弱,24和48 h拒食率仅为9.65％～ 20.45％.6种溶剂提取物对棉蚜(Aphis gossypii Glover)均有较强的触杀效果.其中丙酮提取物的触杀作用最强,经50g·L-1丙酮提取物处理后棉蚜24和48 h的校正死亡率分别为76.78％和85.64％,而经100g·L-1丙酮提取物处理后棉蚜24和48 h的校正死亡率分别为82.63％和92.53％.丙酮提取物的石油醚、三氯甲烷和乙酸乙酯萃取物对小菜蛾均有一定的触杀活性,且随萃取物浓度提高及处理时间的延长触杀活性增强;其中石油醚萃取物的触杀作用最强,经25.0g·L-1石油醚萃取物处理后小菜蛾24和48 h校正死亡率分别达到80.00％和96.67％ 研究结果表明:葎草丙酮提取物对小菜蛾和棉蚜的杀虫活性均最强,其主要有效杀虫活性成分存在于丙酮提取物的石油醚萃取物中.     

Polyester and nylon powders used as pollen diluents preserve pollen germination and tube growth in controlled pollinations

Pollen acquisition for seed production, breeding programs and supplemental pollination can be costly and difficult. The identification of dry particulates for use as pollen diluents would facilitate the use of limited amounts of pollen and aid in accurate pollen application and dispersion. Four powders - Rilsan ES, polyester, wheat flour, and Lycopodium spores - were evaluated as pollen diluents using petunia as a model system. Diluents were combined with petunia pollen at a 5:1 (v/v) ratio. Two types of studies were conducted: (1) storage studies evaluated the viability of pollen combined and held with diluent for different durations; and (2) in vivo studies evaluated pollen tube growth in the styles of flowers pollinated with pollen-diluent mixtures. Pollen germination was not affected when stored as pollen-diluent mixtures for 4 days. Slight detrimental effects on pollen germination were observed after 6 days storage with Rilsan ES powders. Pollination with all the pollen-diluent mixtures resulted in fewer pollen tubes growing in the style compared to controls diluted with heat-killed pollen instead of diluent powders. Lycopodium-pollen mixtures were the most inhibitory, providing only 8% of the tube numbers observed in controls. Pollen mixed with polyester powders, Rilsan ES powders or wheat flour had tube numbers ranging from 47 to 61% of the control, but still had 175 or more pollen tubes per style, which would be sufficient for high rates of seed set in petunia. Wheat flour-pollen mixtures tended to clump and degrade pollen flow. Rilsan ES and polyester were identified as two promising pollen diluent powders that can facilitate accurate metering and distribution of pollen, produce large numbers of pollen tubes, and maintain pollen viability under storage.