Observation of Pollen Tube Behaviour and Early Embryogenesis Following Interspecies Pollination Between Actinidia deliciosa and A. arguta

The pollen tube behaviour in the style and early embryogenesis following interspecies pollination between Actinidia deliciosa No. 26 and A. arguta were observed by means of fluorescence and light microscopy. Pollen grains germinated on the papillate stigma and pollen tubes grew along the V-shaped open-type style. Pollen tubes showed slower growth and reached the ovules 50--60 hours later than those of the control. Several abnormalities of pollen tubes have been observed at the base of the style, including wave-like pollen tubes, pollen tubes with swollen or pointed tips, with variable diameters, and a few with irregular growth. Random deposition of callose along pollen tube wall and even the whole wall was observed. About 26.74 % of the ovules were successfully fertilized and developed into seeds, among them 68.50% of the seeds were normal and 31.50% were abortive. About 11.41% were empty seeds without embryo and endosperm. Unfertilized small ovule was 61.45 %. Normal seed and its embryo were smaller than those of the control. The development of embryo was of the Soland type. The endosperm was cellular. The zygote remained quiescent for about 12-15 days before it started to divide, eventually forming a cotyledonary embryo 50 days after pollination.     

Changes of Microtubular Skeleton in the Generative Cell in Pollen Tube During Mitosis of Lilium davidii

Previous observations indicated that division of the generative cell (GC) in some plant genura such as Lilium and Tradescantia is characterized by several unusual features, including persistence of surrounding microtubule (MT) bundles during mitosis, lacking a matephase plate, the cytokinesis is completed with furrow. The authors have further studied the changes of MT organizations and the chromosome (CHs) behavior in the GC during mitosis using electron microscopy and method of tubulin localizations. No MTs in the GC before GC division and during prophase was seen under electron microscopy. However, there was tubulin in the GC with antitubulin staining. During promatephase to matephase, the CHs appeared and arranged in a complexed tangled pattern lengthwise along the cell. Correspond- ingly, transverse pairs of kinetochores were located along the length and depth of the cell. They stacked successively like the rungs of a ladder. In this phase, a large mount of MTs appeared in the GC, which distributed in the cortex of the cell and among the CHs and along the whole length of the CHs. In the beginning, one or two kinetochore pairs changed from transversely to longitudinally situated in each cell. MTs ended on the kinetochore to form kinetochore MTs (KMTs). With the electron microscopy, authors did not find the image of lateral connection between the MTs and the kinetochores as previous reported with immunofluorescent method. As karyokinesis proceeded, more transverse kinetochore pairs gradually became longitudinal, and KMTs gradually increased in number. However, a distinct spindle was not evidenced. During anaphase, CHs seperation started at various positions along the length of the cell. The distribution of MTs in the GC was similar to that of promatephase to matephase. In late anaphase, the CHs segregated as two groups. Most MTs disappeared but only some remained in the polar regions and the interzone. Authors also measured and compared the lengths of the CHs and indirectly identified the existing anaphase B. During late tolephase, the MTs increased in number gradually in the region between the two newly formed sperm nuclei. The region might be the MT interdigitating zone visualized with antitubulin localization. The MTs disappeared after the cell plate (CP) appeared.     

Numerical Characteristics of Pollen Assemblages of Surface Samples from the West Kunlun Mountains

A total of 31 suface sediment samples were collected from West Kunlun Mountain in south Xinjiang Autonomous Region in northwest China. These samples are from seven types of vegetation: Picea schrenkiana Fisch. et Mey. forest, Sabina Spach. woodland, sub-alpine steppe, alpine meadow, desert vegetion, cushion-vegetation and vegetation adjancent to glaciers. Pollen percentages and pollen concentrations were calculated in all samples. The dominant pollen types in the region are Chenopodiaceae, Artemisia, Picea, Ephedra, Gramineae, Cyperaceae, Rosaceae, Leguminosae, Compositae etc. In order to reveal the relationship between pollen composition and the vegetation type from which the soil sample was collected, principal component analysis and group average cluster analysis were employed on the pollen data. The results revealed that the major vegetation types in this region could be distinguished by pollen composition: a. Samples from desert vegetation were dominated by pollen of Chenopodiaceae (about 60195%). The percentages of all other pollen types were low. b. Picea forest samples were rich in Picea pollen (about 20%) Sabina forest had more Sabina pollen grains than other vegetation types (about 5%, others <1%). Pollen percentages of Artemisia, Chenopodiaceae and Ephedra were comparatively higher (each about 20%) in these samples from the two types of vegetations. C. Pollen percentages of Artemisia, Cyperaceae, Gramineae and Chenopodiaceae were high in both sub-alpine steppe and alpine meadow. But steppe containal more Artemisia and Chenopodiaceae (steppe 33.75% and 32.30%, meadow 15.57% and 19.48% in average), less Cyperaceae and Gramineae (steppe 2.58% and 7.60%, meadow 22.35% and 12.93% in average) than meadow. d. Samples from cushion-vegetation and vegetation adjacent to glaciers were mainly composed of pollen grains transported from other sites. It was not easy to distinguish them from other vegetation types. Principal component analysis and cluster analysis distinguish samples from Picea forest, Sabina woodland, sub-alpine steppe, alpine meadow and desert vegetation. Therefore we think it will be possible to apply the module to reconstruct past vegetation in this region and other similar regions. Regression analysis was also applied to reveal the relationships between pollen and plant percentages of Artemisia, Chenopodiaceae, Cyperaceae and Gramineae. The results indicated that a linear relationship existed between pollen and plant percentages for Artemisia, Chenopodiaceae and Cyperaeeae.     

Pollen Cryopreservation and Pollen Protoplast Isolation in Brassica carnpestris var.purpurea

The authors have investigated the factors affecting pollen cryopreservation in Brassica campestris var. purpurea, such as pollen development stages cryoprotectant and the process of freezing. A suitable procedure was established as follows :Pollen grains suspended in B5 medium containing 10X DMSO and 1SM sucrose were frozen by a three- –1℃/min – 1 ℃/min step method(0℃———→–10 ℃ ,standing for 15 min———→–40 ℃ , standing for 1 hr→liquid nitrogen)and later thawed in 40℃ water bath. During a period of 60, 90 days′preservation, the relative survival percentage of mature (at the day of anthesis)and nearly mature(2 days before anthesis, trinucleate stage)pollens maintained at ca. 91% that of young pollens(7-8 days before anthesis, late uninucleate stage to early binucleate stage)slightly declined from the original 91.6% to 84. 3%. Culture. experiment showed that the cryopreserved young pollen could be induced to cell division just as well as the fresh pollen. The method of isolating protoplasts from fresh mature pollen developed previously was improved and simplified. As a result, protoplasts were isolated more conveniently from mature pollen and young pollen for the first time. The protoplasts from cryopreserved mature and young pollen could be obtained as well with an isolation rate of 77.4% and 35.9% respectively. However, for isolation of protoplasts from preserved young pollen, an incubation in NLN medium at 35℃ after thawing was necessary.     

On the Ultrastructure of Pollen Exine in Metasequoia Hu and Cheng

Metasequoia is endemic to China. Present study deals with ultrastructure of pollen exine of M. glyptostroboides Hu et Cheng, and in comparision with other genera of the family. Pollen grains of Metasequoia are spheroidal or subsphoroidal and 27.8(24.3–32.3) μm in diameter. There is a papilla in the distal face. The papilla is wide at the base, 3.5–5.2 μm high, with pointed and circular end and the base crooked toward one side. Exine is about L5 μm thick, layers distinct, Nexine is as thick as sexine. Surface weakly granulate. According to observation by SEM, exine is covered with fine granules and rather coarse tuberculae. The former can be easily separated from the latter. The loose and uneven tuberculae are provided with minute spinules on the surface and generally fall off after acetolysis. The fine and dense granulae, however, remain intact after acetolysis. The study by TEM shows that ektexine is made of granules densely arranged and connected with each other. In addition, sparse Ubisch bodies are unevenly distributed on granular layer with geminate surface. The thick endexine, is composed of 10–15 lamellae. It is worthy to note that all lamellae possess tripartite structure. But lamellae of endexine in other genera of Taxodiaceae have no tripartite structure except the lamella near ektexine. Number of lamella and thickness of endexine in Metasequoia differ from those of other genera in Taxodiaceae; for example endexine with 8–10 lamellae in Taxodium, 8–9 lamellae in Sequoia, 6–7 lamellae in Glyptostrobus, 6–8 lamellae in Cunninghamia, about 16 lamellae in Cryptomeria etc.     

A Fluorescence Staining-Methyl Salicylate Clearing Technique for Demonstrating Pollen Nuclei

Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization.     

Studies on Pollen Morphology and Taxonomy of Lespedeza and Its Allied Genera from NE China

The pollen morphology of 11 species and 1 variety in the genus Lespedeza and its allied genera (Campylotropis, Kummerowia) from NE China was examined under light and scanning electron microscopes. 1. Lespedeza Michx. (plate 1:1-6; 2:1-6; 3:1-6; 4:1-2) Pollen grains prolate, rarely subprolate or spheroidal, elliptic or rarely suborbicular in equatorial view, 3-lobed-rounded in polar view, tricolporate, colpus margins smooth or jagged. Polar axis 20.7-33.1μm long, equatorial axis 15.4-20.9 μm long. Exine reticulate or foveolate, lumina verrucose or smooth under SEM. 2. Campylotropis Bge. (plate 4:3-4) One species in NE China, C. macrocarpa (Bge.) Rehd. Pollen grains prolate, elliptic in equatorial view, 3-lobed-rounded in polar view, 3-colporate, colpus linear, 25.1μm long, 1.79μm broad, colpus margins jagged, with a series of verrucae equal in size along one side visible under SEM. Polar axis 19.7μm long, equatorial axis 14.6μm long. Exine reticulate, lumina nearly rounded, verrucose at periphery under SEM. 3. Kummerowia Schindl. (plate 4:5-6) Pollen grains spheroidal, oblate or prolate, elliptic in equatorial view, obtuse-triangular in polar view, tricolporate, colups linear, 25.1μm long, 2.01μm broad, colpus margins sinuate. Polar axis 24.7-27.9μm long, equatorial axis 19.7-26.6μm long. Exine reticulate or subreticulate, lumina nearly rounded, with verrocae visible under SEM. According to the pollen morphology of Lespedeza and its allied genera, the division of Lespedeza (s. lat.) into Lespedeza (S. str.), Campylotropis and Kummarowia by Schindler (1912) is reasonable. The subdivision of Lespedeza (s. str.) into Sect. Macrolespedeza and Sect. Lespedeza by many botanists, and the treatment of Lespedeze juncea (L. f.) Pers. var. inschanica Maxim. as an independent species (i.e. Lespedeza inschanics (Maxim). Schindl.) are also supported by the pollen morphology shown in the present work.     

A Study on Pollen Morphology of Eucommia ulmoides Oliver

Eucommia ulmoides Oliver is endemic to China. The pollen morphology and exine ultrastructure were examined under LM, SEM and TEM. Pollen grains are prolate, polar axis 30.5-54.8 μm long, with the average 32.7 μm, equatorial axis 27.8-31.3 μm long, with the average 29.2 μm, tricolporoidate, but sometimes the outline of ora could be observed and elliptic in shape. Colpi are narrow, uequal in length, often two long and one short or two short and one long, sometimes rather irregularly arranged, with indistinct and thin colpus membrance. Exine psilate under LM, granulate under SEM, and shortly baculate under TEM. Tectum is thin with dense and small granules, columellae layer consists of short bacules, and foot layer very thick. Some taxonomists (Cronquist, 1968) consider that Eucommiaceae is related to Hamamelidales, but others (Takhtajan, 1969) to Urticales. The Urticales is of the porate type of pollen grains, while Eucommiaceae of tricolporoidate type, and thus the former is more advanced than the latter. Pollen grains of some members of Hamamelidales, tricolporoidate, are similar to those of Eucommiaceae. We therefore consider that Eucommiaceae is related to Hama-melidales.     

Studies on Pollen Morphology of Taxaceae of China

The present study deals with pollen morphology of 4 genera and l0 species of Taxaceae in gymnosperms. Pollen grains of the family are spheroidal or subspheroidal, 20.8μm in diameter and with laptoma or papilla in the distal face. Exine is two-layered, with sexine equal to nexine in thickness, but sometimes the stratification is indistinct. The surface is scabrous or slightly granular under LM. Coarse verrucae and fine tuberculae on pollen surface are observed under SEM. From thin section, endexine is shown to have lamellate structure, and ectexine is made of verrucate elements. In Amentotaxus argotaenia, some pollen grains show remnant saccate. According to pollen morphology, this family may be divided into two tribes: 1, Pseudotaxeae (including Pseudotaxus only), and 2, Taxeae (including Taxus and Torreya). Owirg to the special feature of pollen grains in Amentotaxus the present author suggests that the genus be separated from Taxaceae and raised to the level of family, Amentotaxaceae.     

Studies on the Free Amino Acid Content in Anthers of Malesterile and Fertile Plants of Taigu Wheat,with Special Reference to Free Proline Content

Biochemical analyses were made on anthers and pistils at various developmental stages of both male-sterne and fertile plants of Taigu wheat. Analyses ineluded total free amino aeids and free proline. The following results were obtained: 1. There was no significant difference between the content of free proline in anthers of male-sterile and fertile plants at reduction division of mierospore mother cells. 2. In anthers with early uninucleate miorospores, the content of free proline of fertile plants was remarkably higher than that of male-sterile plants. It is interesting to note that at this stage the content of free proline in fertile plants rose to 1.65% of the dry weight of the anther, constituting 50% of the total free amino acids, and amounted to 7-fold of that in male-sterile plants. This result is in line with the results obtained with most cytoplasmic malesterile plants reported by other workers, although malesterility in Taigu wheat is controlled by the nueleus. 3. In pistils, at the stages eorresponding to the early uninneleate and the binueleate stages of the pollen, the free proline content of fertile plants was twice as much as that of the male-sterile plants. This differenee disappeared gradually after fertilization. 4. Tile content of total free amino aoids did not fluetuate as much as the free proline content. There was no differenee in anthers of both types of plants during reduction division of mierospore mother cells. In anthers with early uninueleate pollen grains, total free amino acid content of fertile plants exceeded that of male-sterile plant, the difference levelled off at latter stages. In pistils, before fertilization, the content of total free amino acids of the fertile phmts was slightly higher than that of the male-sterile plants. After fertilization t}fis difference was no nmre noticeable.