Ultrastructural Studies of Somatic Embryos of Eucalyptus nitens and Comparisons with Zygotic Embryos Found in Mature Seeds

Although somatic embryogenesis has been observed in tissuesfrom a limited number of Eucalyptus species cultured in vitro,no comparisons have been made of the morphology and structureof eucalypt somatic embryos and zygotic embryos found in matureseeds. We used scanning and transmission electron microscopy,in conjunction with histological analysis, to compare maturezygotic embryos with somatic embryos of the commercially-importanttemperate eucalypt Eucalyptus nitens. Apart from differencesin the nature of the outer coating enclosing both embryo types,somatic embryos of E. nitens were observed to have strong similaritieswith zygotic embryos in seeds in terms of their overall size,morphology and internal cellular organization. Many cells inboth sexually-produced and somatic embryos contained numerouslipid-rich globular bodies. The wider significance of theseobservations is discussed with regard to their potential applicationsin eucalypt plantation biotechnology programmes. Copyright 2000Annals of Botany Company Eucalyptus nitens, shining gum, somatic embryo, tissue culture, ultrastructure, zygotic embryo     

Embryo Rescue in Wide Crosses in Arachis. 2. Embryo Development in Cultured Peg Tips of Arachis hypogaea

Embryo rescue techniques in Arachis are potentially importantfor recovering interspecific hybrids which have the propensityto abort. Pegs are commonly produced in interspecific crosses,but either they fail to reach the soil because growth is arrested,or pods are produced but embryo development is never re-initiated.Peg tips, with the ovule and embryo, of A. hypogaea L. cv. ‘NC6’, were used to determine whether peg tips can be usedas nurse tissue for in vitro culture of embryos. Tissues werecollected 1, 2, 3 and 4 d after self-pollination, after whichpeg meristems were removed from half the pegs, and culturedon five media combinations. Continued reproductive developmentwas observed for embryos cultured at all four collection days;however, the highest frequency of growth was observed in 1-d-oldtissues. Evidence is presented that meristematic activity mayrestrict embryo growth in the 2- to 4-d-old embryos and, oncethe sequence of events is initiated to slow embryo growth, itis not easily reversed in vitro. Achievements of embryo growthto multicellular, globular stages (stages 1–1 or 1–2)encourage the development of methods to recover very young embryosthrough tissue-culture techniques. Embryo culture, morphology, interspecific hybridization, Arachis hypogaea, comparative light and scanning electron microscopy, peanuts, groundnuts     

A Histological Study of Development of Adventive Embryos in Organ Cultures of Phoenix dactylifera L.

Callus cultures from axillary buds of 2–4-year-old offshootsof Phoenix dactyl'fera L have yielded plantlets adventitiouslyAdventive embryos originated from meristematic cytoplasm-richcells located in the epidermal and adjacent sub-epidermal portionsof the periphery of callus on a basal medium supplemented with2, 4-D and isopentenyladenine The formation of adventive embryosis described Morpho-genetically competent cells proliferateinto globular proembryos which differentiate into bipolar structures.Transfer to a medium devoid of hormones enhances embryo developmentand promotes plantlet growth Comparative studies were made betweenzygotic and adventive embryos in various stages of development Phoenix dactylifera L, date palm, Palmae, adventive embryogenesis, histological development     

Ontogeny of Somatic Embryos of Vigna aconitifolia, Vigna mungo and Vigna radiata

Callus cultures were initiated from immature cotyledons of Vignaaconitifolia, V. mungo and V. radiata on MS medium supplementedwith NAA, picloram or 2, 4-D. On transfer to L-6 liquid mediumsupplemented with low concentrations of picloram, GA₃ and cytokinins,large number of somatic embryos differentiated from the callus.The cells destined to become somatic embryos divided to formspherical or filamentous proembryos. From the filamentous proembryo,the embryo proper developed either at single or multiple sites.Development of somatic embryos from multiple sites resultedin several embryos connected by a common suspensor at the radicleend. Continued divisions of the proembryos led to globular,heart shaped and dicotyledonary stages of somatic embryogenesis.The somatic embryos of V. mungo and V. aconitifolia differentiatedinto tiny plantlets at low frequency (1%) in liquid suspensioncultures supplemented with zeatin, picloram and GA₃. Vigna aconitifolia Jacq, Marechal, mothbean, Vigna mungo L. Hepper, urdbean, Vigna radiata L. Wilczk, mungbean, somatic embryo     

Establishment of an in vitro fertilization system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.     

三叶半夏悬浮培养下的体细胞胚胎发生及植株再生

用三叶半夏幼嫩叶片诱导产生的胚性愈伤组织建立了胚性细胞悬浮系,研究了悬浮培养下体细胞胚胎的发生及植株的再生。结果表明,胚性悬浮细胞在附加1．0mg／L2,4-D、0．2 mg／LBA和300mg／L LH的MS液体培养基中产生大量的球形胚,转入液体分化培养基(MS 0．1 mg／LNAA 0．2 mg／L BA 300 mg／L LH)中进一步发育成心形胚、鱼雷形胚和子叶形胚。收集成熟胚转移到MS固体分化培养基上培养获得了再生植株。另外还观察到某些成熟胚上产生了许多次生胚。     

栓皮栎体细胞胚胎发生的细胞组织学观察

以栓皮栎未成熟合子胚为外植体,在添加0.25mg/L 2,4-D和0.5mg/L 6-BA的MS培养基上6周可诱导产生2种类型的胚性愈伤组织,一种表面具光泽、白色;另一种表面光滑湿润具光泽,色泽淡黄或无色透明。组织切片表明,胚性愈伤组织的细胞体积小,细胞核大,细胞质浓,细胞排列紧密;非胚性愈伤组织细胞的体积大,细胞核小,细胞质稀薄。胚性细胞团培养在不含激素的培养基上可诱导产生体细胞胚。体细胞胚直接起源于胚性细胞团表皮或近表皮的单细胞,经历与合子胚相似的球形胚、心形胚、鱼雷胚和子叶胚发育阶段。所有发育时期的体细胞胚的胚轴、子叶均产生次生体胚,它们起源于细胞质较浓的表皮单细胞。     

小麦组织培养中体细胞胚胎发生的细胞胚胎学及淀粉消长动态的研究

小麦幼胚在附加2,4-D 2 mg/L+KT0.5 mg/L+LH 300 mg/L+3%蔗糖的MS 培养基上诱导出愈伤组织,继代2—3次后,降低2,4-D 的浓度,该愈伤组织约以60%的频率转变为胚性愈伤组织,继而形成不同发育期的体细胞胚。这些体细胞胚是起源于愈伤组织表层或近表层的单个胚性细胞,体细胞胚经球形胚、梨形胚、盾片胚、成熟胚而成再生植株。在胚性细胞内淀粉粒大最积累,淀粉颗粒大而密集。随着胚性细胞的分裂,淀粉逐渐被消耗,到多细胞原胚期,胚体细胞内淀粉粒趋于消失。球形胚期淀粉粒又开始积累,但到成熟胚期,淀粉积累只限于生长点区域,尔后随着胚体萌发,淀粉含量又趋于减少。这一消长动态过程与小麦合子胚发生中淀粉代谢动态基本一致。     

Analysis of morphological changes in carrot somatic embryogenesis by serial observation

Carrot somatic embryogenesis was serially observed using a cell cluster immobilizing system with Phytagel. Embryogenic cell clusters ranging in size from 32 to 63 μm were collected by filtration and used for somatic embryo induction. Of the 432 cell clusters, 253 grew, i.e., the size of these cell clusters increased, and 192 developed into globular embryos. Through serial observation, the number of somatic embryos produced from each cell cluster was identified. Cell clusters which developed into two or more embryos grew and developed slowly as compared with cell clusters which developed into single embryos. Serial observation also revealed that some cell clusters consisted of several parts, each of which independently grew as separate units. In cases where two growing parts fused into one embryo, morphological abnormalities such as curvature or lumps in their bodies were occasionally observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Abortion and determination of stages for embryo rescue in crosses between sweet-potato,Ipomoea batatas Lam. (2n=6x=90) and its wild relative,I. trifida (H. B. K.) G. Don. (2n=2x=30)

Summary The frequency of aborted fruits and the changes and abnormalities that occur during the embryo development in intraspecific crosses of sweet-potato Ipomoea batatas (2n=6x=90) and interspecific crosses between I. batatas and I. trifida (2n=2x=30) were investigated in order to study the causes of the low seed production. Three genotypes of I. batatas and 18 genotypes of I. trifida were intermated. The frequency of aborted fruits was below 25% in the intraspecific crosses and over 90% in the interspecific crosses. Paraffin sections were used to examine the developmental stages of fruits and seeds. Embryos in different developmental stages were observed to determine the stage of abortion. These observations permitted the identification of developmental stages of embryo rescue in interspecific crosses. There were no significant differences in the frequency of embryo abortion before the early globular stage among female sweet-potato progenitors for the intraspecific and interspecific crosses. The frequency of the late occurrence of embryo abortion (when embryo abortion occurs after the pre-globular stage) was higher in interspecific crosses (19.1%) than in intraspecific crosses (5.5%). The frequency of the late occurrence of embryo abortion in interspecific crosses was higher at the globular stage (9.6%) than at the heart stage (4.3%). Providing that embryo rescue is conducted in interspecific crosses, the estimated number of potentially viable embryos could be increased: 30 times with embryos at the globular stage; 20 times with embryos at the heart stage; and 11 times if embryos at the torpedo stage were used for the rescue with respect to the seed set. The results suggested that the appropriate time for embryo rescue in interspecific crosses is at the globular stage. If embryos could be rescued at the globular stage, it would be possible to increase the number of surviving embryos up to 30 times in interspecific crosses and 0.02 times in intraspecific crosses with respect to natural conditions without embryo rescue.This research was initiated during sabbatical of M.I. at the Asian Vegetable Research and Development Center (AVRDC) in Taiwan     

陕西宁强早寒武世磷酸盐化具星状表饰胚胎化石研究

后生动物胚胎化石研究对了解寒武纪大爆发中生命的个体及系统发育具有十分重要的意义。陕南早寒武世灯影组宽川铺段发现大量磷酸盐化的球状化石标本,其中的一些为保存有十分精细的星状表饰的胚胎化石,目前发现至少3种具星状纹饰的胚胎化石:圆球状胚胎、具中央收缩带的球状胚胎和表面具“拉伸构造”的花生状胚胎,其中后者为新类型。研究表明,此新类型在演化序列上处于圆球状胚胎、具中央收缩带的球状胚胎发育阶段之后,是胚胎向成体演化的一个重要中间过渡环节,这一发现为研究早寒武世早期后生动物胚胎及个体发育提供了新线索。     

Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius) embryos

This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n=12: six Day 6 and six Day 7 embryos) or vitrification (n=12: four Day 6 and eight Day 7). The remaining 8 'control' embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4% paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity. Vitrified-warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P<0.05). Although the proportion of dead cells in conventionally frozen embryos tended to be higher than in controls, the difference was not significant (P> or =0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified-warmed embryos >300 microm in diameter had a significantly higher percentage of dead cells than embryos < or =300 microm (P=0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3%) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.     

Photosynthesis in the Chloroembryo of Cyamopsis tetragonaloba Taub

The intraseminal embryo of Cyamopsis tetragonaloba is greenfrom the globular stage onwards. The chloroembryos do not photosynthesizein vivo but are capable of in vitro photosynthesis at a ratewhich is similar to that of leaf tissue and they utilize bicarbonateions in this process. Both ribulose bisphosphate (RuBP) andphospho-enol-pyruvate (PEP) carboxylases are functional in CO₂fixation by the embryos. Application of aminotriazole does notinhibit the development of chlorophyll in chloroembryos Cvamopsis tetragonaloba Taub, cluster bean, chloroembryo, photosynthesis     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

Embryos and Plantlets from Cultured Anthers of Hybrid Grapevines

Embryos and plantlets were produced in large numbers from callusformed by cultured anthers of hybrid grapevines (Vitis viniferax Vitis rupestris). Anthers of Vitis vinifera produced smallamounts of callus or failed to grow in vitro. For embryo formationanthers containing uninucleate microspores were chilled (4 °C)for 72 h before culture with Nitsch medium containing 2, 4-D(5µM) and benzyladenine (1 µM). Highest yields ofembryos were with anthers cultured in darkness. For productionof normal plantlets embryos required chilling (4 °C) for2 weeks. Unchilled embryos produced mainly abnormal plantlets.Chilling was effective in promoting plantlet growth when appliedat any stage of embryogeny. In grapes ability to produce plantlets from cultured anthersis a genetically-determined trait and maleness, as distinctfrom hermaphroditism, may be a predisposing factor. Callus derivedfrom anthers contained both haploid and diploid cells but allplantlets produced so far are diploid. The genetic constitutionof plantlets, whether they are diploids of somatic origin ordiploids from spontaneously doubled haploid cells, is not yetknown and is being determined by standard genetic methods.     

Stage-specific nitrogen metabolism in developing carrot somatic embryos

The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and ¹⁴C-labeling. ¹⁵N NMR spectroscopy was used to examine the uptake and incorporation of ¹⁵N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α-amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non-embryogenic cells and germinating embryos were glutamine and α-amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y-aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon-14 labeling indicated that labeled glutamine pools in non-embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non-embryogenic cells and germinating stage embryos. Furthermore, during early to mid-embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

Interspecific Hybridization of Trifolium repens with T. hybridum Using In Ovulo Embryo and Embryo Culture

In ovulo embryo culture followed by culture of excised immatureembryos produced interspecific hybrids between Trifolium repensL. (white clover) and autotetraploid T. hybridum L. (alsikeclover). Ovules containing hybrid embryos were excised 12–14 dafter pollination and cultured on Nitsch (1951) medium supplementedwith 15% young cucumber juice for 5–6 d. Embryos weresubsequently excised and transferred to hormone-free EG medium,a medium suitable for the culture of immature embryos. A total of 118 hybrid seedlings were obtained from 1978 reciprocalpollinations. All seedlings produced showed various chlorophylldeficiencies, either totally albino or albino with green sectors.Transmission electron microscope studies were carried out toinvestigate plastid development in embryos and seedlings. Someembryos produced only callus. Plants were regenerated from sevencalli. Two semi-albino plants survived transfer to soil, andone plant produced flowers. Backcrosses to T. repens producedone green plant. Hybridity is supported by analysis of morphological characters,karyotype and the gel electrophoretic separation of leaf isozymes. Pollen irradiated with 40 Gy of gamma rays was also used forpollinations. Results indicate that in certain cases ionizingradiation might be useful in overcoming hybrid inviability. Trifolium repens, Trifolium hybridum, clover, interspecific hybridization, in ovulo embryo culture, irradiation     

The role of the peanut (Araschis hypogaea) ovular tissue in the photo-morphogenetic response of the embryo

Correlative interactions between the embryo and the ovular tissue in peanut, were studied in vitro. Growth arrest of the embryo in the light was controlled by the ovule. Light perceived by the ovular tissue inhibited embryo growth and the inhibitory effect was diminished by decreasing the amount of ovular tissue remaining attached to the embryo. The suspensor played a major role in the development in vitro of isolated globular and heart-stage but not in the cotyledonary stage embryos. Injury to the suspensor in isolated pro-embryos, inhibited their growth in vitro. Electron microscopy showed disintegrated cells, devoid of starch grains and protein bodies, in nuclear cells surrounding the embryo sac at the micropylar, but not at the chalazal end. This may indicate a role of the suspensor in metabolite absorption by the young embryo. Pro-embryos cultured in a medium with abscisic acid in the dark exhibited a mode of growth arrest similar to that by light. The results suggest that light perceived by the ovular tissue induces the production of substances which diffuse to the embryos and arrest their development.     

Abscisic acid and ancymidol promote conversion of somatic embryos to plantlets and secondary embryogenesis inAsparagus officinalis L.

Summary The effects of abscisic acid (ABA) (0, 0.09 μM, 0.19 μM, 0.28 μM, and 0.38 μM) or ancymidol (0, 0.98 μM, 1.95 μM, 2.93 μM, 3.90 μM) in embryo germination medium on the conversion of primary embryos to plantlets and secondary embryogenesis were evaluated for asparagus. ABA and ancymidol each significantly enhanced both responses. ABA was more effective than ancymidol in promoting the conversion of primary embryos to plantlets, while the converse was true for the production of secondary embryos. The most effective treatments for embryo conversion were 0.19 and 0.28 μM ABA; 75–77% bipolar and 55–57% globular embryos converted to plantlets. For secondary embryogenesis, the most effective treatments were 1.95 and 2.93 μM ancymidol; 99–101 and 84–86 somatic embryos were produced from 10 globular and 10 bipolar embryos, respectively. Bipolar embryos generally converted to plantlets better than globular embryos, but more secondary embryos were produced from globular embryos than from bipolar embryos in all treatments. ABA and ancymidol also affected the morphology of the plantlets produced. The plantlets from the embryos incubated on the medium with ancymidol had strong and thick shoots and roots, while those on the medium with ABA had long, thin shoots and short thin roots.