February 1998, Volume 40 Issue 2


          Research Articles
Isolation of Egg Cells and Zygotes in Oryza sativa
Author: HAN Hong-Mei,ZHAO Jie, SHI Hua-Zhong, YANG Hong-Yuan and ZHOU Chang
Journal of Integrative Plant Biology 1998 40(2)
    A method of non-enzymatic, manual microdissection was established to isolate egg cells and zygotes in Oryza sativa L. Generally 5 to 8 protoplasts of egg cells or zygotes could be isolated from 20 ovaries in 2 h. Fluorochromatic reaction proved that the isolated cells were viable and could survive in the isolation medium consisting of 0.5 moL/L mannitol and 5 mmol/L CaC12 for at least 2 h. The egg cells and early zygotes usually turned to spherical shape during the course of isolation, whereas the late zygotes often maintained pear-shaped. The rounded zygotes were proved to be true protoplasts by Calcoflour White fluorescent staining. Chlorotetracycine and fluphenanize fluorescence treatment demonstrated that the isolated zygotes were rich in membrane-bound calcium as well as calmodulin.
Abstract (Browse 1749)  |  Full Text PDF       
Development of Anther- defective Cytoplasmic Male Sterile Line from Interspecific Hybridization Between Dongxiang Wild Rice and Cultivated Rice and Preliminary Observations on Its Derivatives
Author: SONG Dei-Ming, WANG Zhi, LIU Yong-Sheng, WANG Mao-Li, LONG Tai-Kang and SUN Jing-San
Journal of Integrative Plant Biology 1998 40(2)
    Exploring novel source of cytoplasmic male sterility (CMS) is essential to stablize the productivity of hybrid rice. Dongxiang wild rice ( Oryza rufipogon Griff. ) has been recorded as the northest distributed wild rice in China that is resistant to several biotic or abiotic stresses. A male sterile line M01A defective of anther was identified in the F3 population from an interspecific cross between Dongxiang wild rice and cultivated rice ( Oryza sativa L. ssp. indica ). Crosses and successive backcrosses were made between M01A and a variety of breeding materials and 19 progeny families were obtained. Among the families, some were defective of anthers, and some have twisted and degenerated anthers without microsporogenesis or with a few typical aborted pollen. These resuits implicate that the male sterility of M01A was genetically regulated by the interaction between the nucleus and the cytoplasm. Only one cross produced male-fertile hybrid in which the paternal parent contains a part of the genome of Dongxiang wild rice, which implies that Dongxiang wild rice itself could be the source of the fertility restorer to M01A.
Abstract (Browse 1858)  |  Full Text PDF       
X- ray Energy Spectra Analysis of Lanthanum in Tissue Cells of Defferent Parts of Wheat Plant
Author: ZHOU Shi-Gong and LIU Min
Journal of Integrative Plant Biology 1998 40(2)
    Lanthanum entered plants through the root and accumulated mostly in the cell wall of the root tip. Only a few accumulated in the cortex cell of the root elongation zone and in the cell wall of the mesophyllous cells. There was no detectable lanthanum in the cytoplasm. Observation with TEM (transmission electronic microscopy) indicated that lanthanum, which appeared as electron opaque deposits, was present in the cell wall of tissues in every part of the plant. The higher lanthanum contents occurred in the cell wall of root tips caused a dark appearance of the cell wall. The content of lanthanum in the cell wall of the cortex of elongation zone appeared as granules of different size. Those in the mesophyllous cell wall appeared as much less small granules. X-ray energy spectnnn analysis of the deposits confirmed the induction of lanthanum into the plant tissue cells and that its intensity correlated with the density of the deposits.
Abstract (Browse 1729)  |  Full Text PDF       
Identification of Saikosaponin s
Author: TAN Li, ZHAO Yu-Ying, WANG Bin and ZHANG Ru-Yi
Journal of Integrative Plant Biology 1998 40(2)
    Five saikosaponins were isolated from the roots of Bupleurum scorzonerifolium Willd. A new saikosaponin was elucidated as the 3, 16, 23, 28-tetrahydroxy-oleana-ll, 13( 18)-dien-3-O- -D-glucopyranosyl- ( 1 ~ 6 )- [ a-L-rhamnopyranosyl- ( 1 ~ 4 ) ] --D-glucopyranoside on the basis of spectral analysis and named as the saikosaponin s. The other four saikosaponins were determined as the saikosaponin a, saikosaponin c, saikosaponin bi and saikosaponin b2.
Abstract (Browse 1741)  |  Full Text PDF       
Study on DNA Diversity of Liaodong Oak Population at Dongling Mountain Region, Beijing
Author: YUN Rui, ZHONG Min, WANG Hong-Xin, WEI Wei, HU Zhi-Ang and QIAN Ying-Qian
Journal of Integrative Plant Biology 1998 40(2)
    Random amplified polymorphic DNA(RAPD) and DNA amplification fingerprinting (DAF) markers were used in detecting genetic structure and DNA diversity of two Liaodong oak ( Quercus liaotungensis Koidz. ) populations at Dongling mountain region, a suburb of Beijing City. Shannon's index of phenotypic diversity was used to partition diversity into components within and between populations. Two hundred and five bands from twelve primers were analyzed. The results showed very high genetic variability within the Liaodong oak populations. The diversity in the central population was higher than that of the marginal one. 95 % of total genetic diversity occurred within populations and the coefficient of gene differentiation was 0.05. Significant difference of gene frequency has been detected in a few loci between populations. The study of different age groups of the oak trees implied that deforestation exerted certain impacts on the genetic structure of the Liaodong oak.
Abstract (Browse 1909)  |  Full Text PDF       
RAPD Analysis of Germplasm in Ancient
Author: ZOU Yu-Ping, CAI Mei-Lin, WANG Xiao-Dong and XU Ben-Mei
Journal of Integrative Plant Biology 1998 40(2)
    By using 40 random primers and 8 anchor-SSR primers, RAPD and microsatellite DNA (SSR) polymorphism were detected in the ancient "Taizi lotus" and the modem Chinese red flower lotus (Nelumbo nucifera Gaertn. ) from Hebei, Harbin (wild population), Jiangxi and Hunan (cultivar). The polymorphism information could be provided with 13 random primers and 2 SSR primers. Total 135 loci were amplified, 71 loci of these were polymorphic (53%). Agarose electrophoresis showed high genetic identity without any genetic variation whithin "Taizi" and Harbin lotus by using the above-mentioned 15 primers. However, there were different extent of differentiation within Hebei, Jiangxi and Hunan lotus. According to the UPGMA analysis of MEGA program, "Taizi", Harbin and Hebei lotus were gathered to a branch in the dendrogram. The genetic distance of "Taizi" and Hebei lotus were very close (0.05). They shared a common ancestry. Comparing with the modem Chinese lotus ( N. nucifera ), the ancient "Taizi" lotus only lacked one locusOPM06-300, so it still belonged to N. nucifera. Hunan and Jiangxi lotus were close to gather and could be grouped in another branch. Their genetic distances from the ancient "Taizi" lotus were farthest (0.67).
Abstract (Browse 2115)  |  Full Text PDF       
Microdissection and PCR Amplification of Single Soybean Chromosome
Author: ZHOU Yi-Hua,DANG Ben-Yuan, HU Zan-Min,CUI Li-Hua, LI Liang-Cai and CHEN Zheng-Hua
Journal of Integrative Plant Biology 1998 40(2)
    Glass needles were successfully used to dissect the soybean (Glycine max L. ) single chromosome under the micromanipulator in this research. Two dissected soybean chromosomes were digested by Sau3A in two 0.5 mL Eppendorf tubes respectively. The two ends of chromosomal fragments were ligated with Sau3A linker adoptor. After two rounds of PCR amplification, smear DNA fragments ranged from 0.3 to 3 kb were acquired. Southern hybridization result showed the PCR products from the two single soybean chromosomes were homogeneous with the soybean genomic DNA, indicating that DNAs from the two single chromosomes have been successfully amplified. At the same time, the amplified products from the two of the distinguished single chromosome appeared somewhat different. The authors dissected the small chromosomes only by a traditional inverted microscope. Therefore, this research provides a plausible chance for amplification and microcloning of single small chromosomes.
Abstract (Browse 1738)  |  Full Text PDF       
Somatic Embryogenic Potential Determined by the Morphological Polarity of the Explant in Tissue Cultures of Freesia refracta
Author: WANG Li, BAO Xiao-Ming, HUANG Bai-Qu and HAO Shui
Journal of Integrative Plant Biology 1998 40(2)
    Inflorescence explants of Freesia refracta Klatt placed on modified N6 medium supplemented with 2 mg/L indole-3-acetic acid (IAA) and 3 mg/L 6-benzyl aminopurine (BAP) might undergo direct somatic embryogenesis leading to plant regeneration. A noticeable phenomenon observed in this morphogenetic process was that all the somatic embryos appeared exclusively at the original morphological lower end of the segments cut from the inflorescence axes (termed the embryogenic end, EE), while no embryos were formed at the morphological upper end (termed the non-em- bryogenic end, NEE), irrespective of the gravity of earth and the position of the explants placed on the medium. Divisions of embryogenic initial cells were observed solely at the EEs of the segments in the early stages of culture. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that two polypeptides appeared specifically at the EEs of the explanted segments after one day in culture, but they were undetectable at the NEEs. Four polypeptides detectable at both ends of the explants at day 1 of culture disappeared from the NEEs at late stages of culture. There was no significant difference in the distribution of the endogenous IAA before culture between the two ends of the segments as measured by high-performance liquid chromatography. However, the level of IAA was remarkably higher at the EE than at the NEE after a certain period of culture. This result showed that the endogenous hormone might play a key role in the process of somatic embryogenesis. The differences in molecular composition and physiological status between the two morphological ends of the explant in relation to the induction of the somatic embryogenesis in F. refracta may be considered as a useful marker in studying the mechanisms of this in vitro morphogenesis.
Abstract (Browse 1922)  |  Full Text PDF       
Role of Cell Wall Calcium in Red Light- inhibited Elongation of Hypocotyls of Etiolated Phaseolus radiatus
Author: LONG Cheng, ZHANG Zhi-Liang, SHEN Zeng-You and YAN Ji-Qiong
Journal of Integrative Plant Biology 1998 40(2)
    When the hypocotyl segments of Phaseolus radiatus L. were incubated in CaC12 (1 mmol/L) medium, the cell wall calcium was increased over threefold more than those incubated in a Ca2+ -free medium. However, red light inhibited elongation of the hypocotyl was 20% to 25% both in the medium with or without Ca2 + . The amount of calcium removed from the wall by ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) ( 1 to 10 mmol/L) was 58.13 % to 75.33%, which offset the red light-inhibited elongation of the hypocotyl by 61.29% to 87.1%. Moreover, treatment with the channel blocker, verapamil ( 10 to 100 mol/L), wall calcium was the same as that of the darkness control, by which the red light-inhibited growth was also offset. La3 + ( 100 to 1 000 mol/L) had no effect on wall calcium as compared to hypocotyl segments treated with red light alone, but eliminated the inhibitory effect of red light. Treatment with the calcium ionophore, A23187 (10 to 100 mol/L), red light-inhibited elongation was abolished by 66.67% to 142.45% while wall calcium was reduced by 24.53% to 42.81%. In addition, calmodulin antagonist chlorpromazine (1 to 10 mol/L) also counter acted the red light-induced elongation inhibition. These data indicated that exogenous Ca2+ was involved in the red light-inhibition effect, but that' did not mean that Ca2 + was not required. Perhaps Ca2 + in the wall itself was sufficient for red light-induced inhibition of hypocotyl elongation. The role of wall calcium might be quite complex, it not only acted as a signal of influx Ca2 + from the Ca2 + pool, but also played a regulatory role in the cell wall.
Abstract (Browse 1952)  |  Full Text PDF       
Effect of Osmotic Shock on Protein Phosphorylation in Dunaliella salina Cells
Author: CHEN Si-Xue, LI Lin and JIAO Xin-Zhi
Journal of Integrative Plant Biology 1998 40(2)
    Protein phosphorylation and dephosphorylation are considered as important regulatory mechanisms by which the activity of key enzymes and receptor molecules is altered within cells in response to a wide variety of external stimuli. Previous work is mainly on the purification and characteristics of protein kinase, but the role in stimulus-coupled responses in plants is not very clear. Experiments of in vitro protein phosphorylation demonstrated that in the extract of soluble protein of Dunaliella salina (Dunal) Teed. the activity of some protein kinases was, to some extent, dependent on the calcium concentration. The effects of calcium, verapamil, EGTA and A23187 on the in vivo protein phosphorylation also showed that calcium was important. In comparison, the autoradiograph of the in vivo phosphorylation was different from that of the in vitro phosphorylation. Addition of calcium or molybdate, an inhibitor of phosphatase, increased the extent of protein phesphorylation to a much higher level in hypoosmotic shocked samples (OSS), whereas as in hyperosmotic shocked samples(ESS) ,the extent of protein phosphorylation was lower than the control. In the absence of calcium or molybdate, the stimulation of protein phosphorylation by osmotic shock was hardly observed. The reason for this could be that the osmotic shock inhibited calcium uptake and/or activated protein phosphatase. The response of the intensely labelled 24 kD protein to osmotic shock was further studied. In the absence of calcium, the protein in OSS was more highly phesphorylated than the control and ESS. An increase of the eytosol calcium concentration stimulated phosphorylation of this protein in OSS, but had little effect on ESS. Such differences of calcium effects on protein phosphorylation indicated that the respective mechanisms of signal transduction mediated by protein phosphorylation may not be alike.
Abstract (Browse 1943)  |  Full Text PDF       
Isolation and Preservation of Viable Sperm Cells of Ulmus pumila
Author: CHENG Xiao-Fei and LI Wen-Dian
Journal of Integrative Plant Biology 1998 40(2)
    Viable sperm cells of Ulmus pumila L. were isolated and preserved as follows. Prehydrated pollen were germinated in the 25 % surcose medium solidified with 1% agar for 1.5 to 2 h. The sperm cells were released from the pollen tube by osmotic shock, and purified by surcose density gradient centrifugation. The yield of sperm cells was 2.0 x 106/mL. The effect of bovine serum albumin (BSA) and polyvinylpyrrolidone (PVP) on the viability and longevity of isolated sperm cells were investigated. BSA could increase the viability of isolated sperm cells and also greatly prolonged the life-span of sperm cells at both 0 to 4 and room temperature (23 ) whereas PVP exerted adverse effect on the viability of the sperm cells. The sperm cells could survive for 12 days at 0 to 4 and for 54 hours at room temperature in 25 % surcose medium supplemented with 0.6% BSA.
Abstract (Browse 1851)  |  Full Text PDF       
An Ultrastructural Study on the Development of Egg- apparatus in Rice Embryo Sac
Author: LIU Xiang-Dong, XU Shi-Xiong (S.Y. Zee) and LU Yong Gen
Journal of Integrative Plant Biology 1998 40(2)
    The development of the egg-apparatus (consisted of an egg cell and two synergids) of rice ( Oryza sativa L. ) was studied at the uhrastructural level. The walls of the egg cell and synergids, immediately after their formation, possessed numerous plasmodesmata. Plasmodesmata were also present on walls between the egg cell and synergids. During the enlargement phase of the egg cell and synergids, the walls at the tip region began to loosen and vesiculate. By the time the embryo sac became mature, the part of the wall of the egg cell and synergids, facing the chalaza, disappeared. Consequently, the tip regions of the egg cell and synergids were only protected by a plasma membrane. When the embryo sac reached full maturity, the upper and middle region of the wall of the synergids broke up into pieces. At that time one synergid began to degenerate. Plasmodesmata persisted at the hook region of the wall of both the egg cell and synergids. Most plastids in the egg cell contained starch grains that persisted throughout the period of the embryo sac development. Starch grains in the plastids of the synergids appeared only before the time when the two polar-nuclei moved into the region above the egg-apparatus. They then disappeared and did not appear again until the embryo sac had reached full maturity. The size and location of the vacuoles in the egg cell were different from those in the synergids. The time of formation was also different. Vacuoles in the egg cell formed late in comparison with the synergids. Vacuoles in the chalazal region of the egg cell (especially at the early stage of the embryo sac development) were much larger than those in the micropylar region. Vacuoles in the synergids tended to concentrate mainly in the chalazal region. There was a peak period of lipid formation in the two synergids. The peak appeared when the embryo sac neared maturity. At the early stage of development, the nuclei of the synergids were elliptical in shape and were situated at the central region near the micropyle. The shape of the nuclei at the late stage of development became less regular and tended to move more towards the micropylar region. Changes in the uhrastructure of the egg cell and synergids of rice appeared to be closely related to the metabolic processes controlling the embryo sac formation and development.
Abstract (Browse 1800)  |  Full Text PDF       
Microdissection and PCR Amplification of Rye Chromosome
Author: SONG Wen-Qin, LI Xiu-Lan, XU Wen-Sheng and CHEN Rui-Yang
Journal of Integrative Plant Biology 1998 40(2)
    The 18 ( 14A + 4B) chromosomes of rye ( Secale cereale L. ) were isolated by microdissection technique and the chromosomal DNA was amplified using the artificial synthetic single unique oligonucleotide-primed polymerase chain reaction (SUP-PCR). Southern hybridization demonstrated that the SUP-PCR products have homology with rye genome DNA.
Abstract (Browse 1724)  |  Full Text PDF       
Physical Localization of Genes for CaM and Ca2+- ATPase in Rice by in situ Hybridization
Author: BI Xue-Zhi, SONG Yun-Chun, REN Nan and LIU Li-Hua
Journal of Integrative Plant Biology 1998 40(2)
    CaM and Ca2 + -ATPase genes are important components of signal transduction chains which affect the regulation of' gene expression and development in plants. These two genes are tunctionally closely related. The rice ( Oryza sativa L. )cDNA probes C419 and SSU304 for these two genes, which are small single-copy ones and 0.8 and 0.3 kb in size respectively, were first physically mapped on rice chromosomes by biotin-labeled in situ hybridization. Both probes were detected on chromosome 5. The detection rate was 6.18 %, and the average chromosome ann ratios and standard deviations of detected chromosomes for probes C419 and SSU304 were 1.79 0.06 and 1.91 0.08 respectively. Probe C419 for CaM gene was located at the end of the long ann, and probe SSU304 for Ca2 + -ATPase gene -- on the short arm near the centromere. As it was reported before, they were closely linked in the high density genetic map. This demonstrated that there was a large discrepancy between the results of genetic and physical mapping of genes, and it indicated that the region between the functionally related genes could be the cold spot. What relationship there is between the region and gene expression is to be shudied further. The nonradioactive in situ hybridization technique about the physical mapping of low/single copy, short DNA fragments is also discussed.
Abstract (Browse 1753)  |  Full Text PDF       
Cytological Studies of the Cytoplasmic Inheritance in Lilium regale and L. davidii
Author: LIU Xing-Liang, HU Shi-Yi, ZHANG Jin-Zhong and LONG Ya-Yi
Journal of Integrative Plant Biology 1998 40(2)
    The distribution and characteristics of plastids and mitochondria in the generative and sperm cells of Lilium regale Wils. and L. davidii Duch. were described. In L. regale there were few plastids and abundant mitochondria in the newly formed generative cell. When the generative cell became free in the vegetative cytoplasm, the plastids degenerated completely within the generative cell. It was further proved by DAPI fluorescent technique that there was no organell DNA in the generative cell within the mature pollen grain or the pollen tube. However, distribution of the plastids was strictly polarizable during the division of the micmspore in L. davidii, resulting the lack of plastids in the newly formed generative cell. Data of RFLP analysis comparable between L. davidii, L. longifiorum and their interspecific hybrid have also proved the plastid inheritance in L. davidii to be of uniparental maternal transmission. Although the mitoehondria were observed both in the generative and sperm cells of L. regale and L. davidii but their DNA was decomposed in the male gametophyte stage. Therefore the mitochondda in the sperm cell could not be transmitted into the offspring. The results provided the detail, cytological evidence that organelles in the microgametophyte are incapable of genetic transmission in the two species of Lilium.
Abstract (Browse 1951)  |  Full Text PDF       
Floral Organogenesis and Its Systematic Significance of the Genus Nandina(Berberidaceae)
Author: FENG Min and LU An-Ming
Journal of Integrative Plant Biology 1998 40(2)
    The initiation of the floral organs of Nandina domestica Thunb. (Berberidaceae) is of the trimemus-whorled pattern. Stamens and petals grew out from the lateral bifurcation of the common stamen-petal primordia; but petals underwent retarded periods of growth in their early development. Carpel initiation belongs to the ascidiate type. Some aspects concerning the trimerous floral organs, the origin of petals, stamen insertion pattem and monocarpellary pistil were discussed. In addition the floral ontogenetic characters among three genera of the Berberidaceae, i.e., Caulophyllum, Podophyllum and Nanclina were compared. It is inferred that the trimerous-whoded arrangement and the diversity of carpel initiation were the two unique characters of Nandina.
Abstract (Browse 1824)  |  Full Text PDF       
Recent Advances in in vitro Fertilization and Zygote Culture of Angiosperms
Author: YANG Hong-Yuan and ZHOU Chang
Journal of Integrative Plant Biology 1998 40(2)
    The successful in vitro fertilization and zygote culture in angiosperms is the most important breakthrough in the experimental researches on sexual plant reproduction during recent years. The present review includes the following parts: 1 ) A brief survey on the historical backgrounds and technical prerequisites of the achievements. 2) Progresses of manipulation techniques, including methods of egg cell isolation, intergametic fusion, microculture of artificial as well as natural zygotes and regeneration of plants, in vitro development from fusion products between gametes of alien species, from unfertilized egg cells and from central cells, etc. 3) Cytobiological and molecular biological studies on in vitro fertilization systems, e. g. time table of developmental events during in vitro fertilization and postfertilization processes, cell wall formation of artificial zygotes in relation to polyspermy preventation, egg cell activation, zygote polarity and asymmetric division of zygotes, construction of cDNA libraries from egg cells, zygotes and early embryos using RT/PCR technique and screening of specific genes, and transient expression of reporter genes introduced into isolated zygotes. 4) Strategies for further broadening and deepening the researches in this area beth in fundamental and applied aspects. It is hopeful that in vitro fertilization and zygote culture will bring new lights in the developmental biology of fertilization and early embryogenesis and open up new prospects for biotechnology in plants.
Abstract (Browse 2085)  |  Full Text PDF       


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