杉木未成熟胚胚性愈伤组织诱导影响因素探析

该研究从基因型、6-BA浓度、外植体接种方式和合子胚发育阶段等方面,分析杉木未成熟胚胚性愈伤组织诱导的影响因素。结果表明:基因型、6-BA浓度、外植体接种方式和合子胚发育阶段均对胚性愈伤组织诱导频率有不同程度影响。6种基因型中,有3种基因型诱导出胚性愈伤组织,其中基因型S18胚性愈伤组织诱导频率最高,为11.7%。6-BA浓度在1.0~1.5 mg·L~(-1)范围内时,基因型S18的胚性组织诱导频率较高。以在去皮种子的一端切开一个小口的接种方式为最优,将合子胚剥出的方式易造成合子胚褐化死亡,将未剥皮的种子切开一个小口后直接接入培养基的方式不利于愈伤组织生成。适合胚性愈伤组织诱导的合子胚发育阶段为受精至胚器官分化阶段,合子胚进入成熟阶段后不利于胚性愈伤组织诱导,合子胚易生长成完整植株。     

Evaluation of somatic embryos of interior spruce. Characterization and developmental regulation of storage proteins

Storage proteins of interior spruce ( Picea glauca engelmanii complex) somatic embryos were compared to those of zygotic embryos by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Somatic embryos contain the same storage proteins as zygotic embryos based on similarities of molecular weight, isoelectric variants, solubility characteristics and disulfide linkages. Storage protein levels varied among different somatic embryo genotypes; however, all genotypes tested accumulated significant amounts of storage proteins. Zygotic and somatic embryos display a similar developmental accumulation of storage proteins. The 22, 24, 33 and 35 kDa proteins appear in early stage embryos, while the 41 kDa protein begins to accumulate during mid cotyledon development. The 22, 24 and 41 kDa proteins accumulate continuously during cotyledon development in somatic embryos cultured on abscisic acid. In contrast, zygotic embryos display a more rapid and transient accumulation of these proteins.     

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

Summary An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15–20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4–5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and -glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7–8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.     

Plant regeneration through somatic embryogenesis from callus induced on immature embryos of Alstroemeria spp. L.

Summary The plant regeneration ability of callus obtained from zygotic embryos of the monocot Alstroemeria spp. was studied. The best explants for somatic embryogenesis were immature zygotic embryos in half-ovules when the endosperm was still soft and white. For 2 genotypes embryogenic callus was induced on callus induction medium with a success rate of 54%. The best callus induction period was 10 weeks. The morphology of embryogenic callus was nodular. Somatic embryos were formed after transfer of the callus to regeneration medium. These somatic embryos revealed later on the typical features of zygotic Alstroemeria embryos. The total duration of the plant regeneration protocol, from inoculation till rooted plantlets ready for transfer to the greenhouse, was 28 weeks.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Evolution of Zygotic Linkage Disequilibrium in a Finite Local Population

One crucial feature of zygotic linkage disequilibrium (LD) analysis is its direct use of diploid genotyping data, irrespective of the type of mating system. Previous theories from an evolutionary perspective mainly focus on gametic LD, but the equivalent development for zygotic LD is not available. Here I study the evolution of zygotic LD and the covariances between gametic and zygotic LDs or between distinct zygotic LDs in a finite local population under constant immigration from a continent population. I derive the analytical theory under genetic hitchhiking effects or in a neutral process. Results indicate that zygotic LDs (diploid level) are more informative than gametic LD (haploid level) in indicating the effects of different evolutionary forces. Zygotic LDs may be greater than or comparable to gametic LD under the epistatic selection process, but smaller than gametic LD under the non epistatic selection process. The covariances between gametic and zygotic LDs are strongly affected by the mating system, linkage distance, and genetic drift effects, but weakly affected by seed and pollen flow and natural selection. The covariances between different zygotic LDs are generally robust to the effects of gene flow, selection, and linkage distance, but sensitive to the effects of genetic drift and mating system. Consistent patterns exist for the covariances between the zygotic LDs for the two-locus genotypes with one common genotype at one locus or without any common genotype at each locus. The results highlight that zygotic LDs can be applied to detecting natural population history.     

Selective elimination of chromosomally unbalanced zygotes at the two-cell stage in the Chinese hamster

The gametic and zygotic selection of genome imbalance was investigated in the Chinese hamster by direct chromosome analyses of spermatocytes and preimplantation embryos from crosses between chromosomally normal females and males heterozygous for a reciprocal translocation, T(2;10)3Idr, abbreviated here as T3. The karyotypes and the frequencies of embryos observed at the first cleavage in the cross +/+female X T3/+male were consistent with those expected from MII scoring in male T3 heterozygotes. Therefore, it was concluded that there was neither gametic selection against genome imbalance nor zygotic selection from fertilization until the first cleavage metaphase. However, 9.1-10.8% of embryos were arrested at the two-cell stage, and karyotypes of these embryos were confirmed as 22(2,10,10,10(2)), 21(2,10,10), and 21(2,10,10(2)). The common abnormality of these embryos was partial monosomy of chromosome 2. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of other segments of chromosomes 2 and 10, had fewer blastomeres than chromosomally balanced embryos. This finding suggests that cleavage of these embryos had been retarded by day 4 of gestation.     

Effects of growth regulator concentrations and explant size on shoot organogenesis from callus derived from zygotic embryos of sunflower (Helianthus annuus L.)

Effects of medium growth regulator composition and embryo size on shoot organogenesis of callus derived from globular- to torpedo-shaped zygotic embryos of five sunflower (Helianthus annuus L.) genotypes were examined. Forty growth regulator combinations composed of 0 to 5 mgl^-1 naphthaleneacetic acid (NAA) and 0 to 1 mgl^-1 6-benzylaminopurine (BA) were tested. The frequency of zygotic embryos forming shoot-regenerating callus was analysed according to categorical data modelling using a maximum-likelihood approach. Both NAA and BA must be present to induce the formation of morphogenic callus from zygotic embryos, but each growth regulator effect varied with the genotype. For four genotypes, NAA and BA effects were neither linear nor quadratic; whereas, they were linear for the fifth one. Most effective concentrations across genotypes were 0.1 mgl^-1 NAA and 0.5 mgl^-1 or 0.2 mgl^-1 BA. However, the optimal growth regulator combination depended on the genotype and an interaction between the two growth regulators. The frequency of shoot-regenerating callus also varied with the size of the embryo explant. For all five genotypes, 0.4 to 1.2 mm long heart-shaped zygotic embryos formed morphogenic callus more frequently than smaller less-developed ones.     

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.     

Development of a cyclic somatic embryogenesis regeneration system for leek (Allium ampeloprasum L.) using zygotic embryos

Summary In leek (Allium ampeloprasum L.) a cyclic system of somatic embryogenesis was developed. Somatic embryos used for cyclic embryogenesis were able to develop the same type of embryogenic callus as zygotic embryos in the primary cycle. For the first time a comparison of the efficiencies of both expiants was made. Ten families were investigated for somatic embryogenesis. There was a genetic relationship with respect to somatic embryo production between the reciprocal crosses. From each family one genotype was selected for investigating cyclic somatic embryogenesis. Different levels of somatic embryo production were found between the expiants of zygotic and somatic embryos. The two best genotypes, 92.001-03 and 92.002-33 produced twice as many somatic embryos as the overall average. On average, 56% of the somatic embryos finally developed into greenhouse plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MS medium Murashige and skoog (1962) basal medium     

Water relations parameters and tissue development in somatic and zygotic embryos of three pinaceous conifers

There is increasing interest in using somatic embryogenesis to meet the demand for high quality seedlings. However, in vitro production of propagules on a large scale depends on the optimization of the maturation and germination steps promoted by desiccation and subsequent imbibition of the embryo, respectively. It is therefore important to characterize zygotic and somatic embryos in terms of their water relations. Bound water, elastic modulus, osmotic potential at full turgor, and relative water content at turgor loss point were determined for somatic and zygotic embryos of western larch and loblolly pine and somatic embryos of white spruce at two developmental stages. These water relations parameters were derived from water-release curves obtained by suspending tissue samples in sealed vials over unsaturated NaCl solutions of known water potential. There was little difference in water relations parameters among species but marked stage dependency for bound water and elastic modulus. The amount of bound water was lowest in western larch somatic embryos (0.02-0.07) and highest in zygotic loblolly pine embryos (0.10-0.18). Elastic modulus ranged from 2.5 to 6.2 MPa in somatic embryos but varied between 1.4 and 1.8 MPa in zygotic embryos. The osmotic potential was lower in somatic embryos than in their zygotic counterparts. Our results show that water relations parameters are remarkably conservative across species but that, within a given species, these parameters are stage specific. It would seem, therefore, that desiccation protocols might be developed for a given developmental stage and applied across a range of species without the confounding effects of differences in water relations parameters.     

Transient gus expression in zygotic embryos ofTaxus Brevifolia

Summary Germinated zygotic embryos of Pacific yew (Taxus brevifolia) were transiently transformed with the expression vector pBl121, as demonstrated by expression of the β-glucuronidase (GUS) gene. Embryos exhibited sectors of blue color following treatment with DNA and dimethyl sulfoxide (optimal concentration of 2.25%). However, transient expression depended on the presence of ions in the buffer, as no GUS activity was observed when deionized water was used in place of other buffers. GUS gene expression was dependent on the developmental stage of the embryo, since the frequency of GUS expression was elevated in embryos incubated with the expression vector during a period from 4 to 8 wk after culture of DCR medium. In addition, the utility of using zygotic embryos from mature seeds for tissue culture and transient expression experiments was also examined. Completed as partial fulfillment of the Masters of Science Degree in the Biological Sciences, Southern Illinois University at Edwardsville. Each author contributed significantly to this work.     

Genetic transformation of Eucalyptus globulus through biolistics: complementary development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue

Stable transformation of Eucalyptus globulus after biolisticDNA delivery was investigated using zygotic embryos as targetmaterial. Conditions for plant regeneration were first investigated.Six-day-old cultured embryos, which had shown to be a good targetfor DNA transient expression, appeared to be suitable for regeneration.Whole plants were recovered through organogenesis after particlegun bombardment. Transformation experiments were performed witha linear T-DNA fragment harbouring GUS and NPTII genes, usingthe biological and physical conditions defined for optimum transientexpression. After 2 months on a culture medium, neoformed GUS-positivecalli were obtained from the ‘T-DNA’ bombarded embryos.GUS-expressing calli were also recovered after selection withkanamycin following bombardment. The integration of both GUSand NPTII genes into the Eucalyptus genome was confirmed bySouthern blot analysis, demonstrating stable transformationof Eucalyptus globulus cells for the first time. Key words: Eucalyptus, zygotic embryos, biolistics, stable transformation, regeneration     

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.)

Summary Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.     

Production ofAgrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos ofDatura innoxia

This work describes a new method to obtain transgenic somatic embryos fromAgrobacterium-infected immature zygotic embryos ofDatura innoxia. It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency. Critical steps for optimal transformation were the embryo stage and a short preculture treatment. The marker gene -glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development. The transgenes were transmitted to the progeny in a Mendelian fashion. The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform. We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency forAgrobacterium infection was developmental stage-specific. This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.     

Differentiation of isolated wheat zygotes into embryos and normal plants

Efficient and reproducible embryo development has been obtained from fertilized wheat (Triticum aestivum L.) egg cells isolated 3–6 h after hand-pollination of emasculated spikes. It is possible to routinely isolate viable zygotes from about 75% of the excised ovaries from cultivars of both winter and spring types. Co-culture with barley microspores which had been stimulated to sporophytic development resulted in embryonic development of the cultivated wheat zygotes. Within 23 h of pollination; the zygotes underwent their first cell division. They proceeded to develop into club-shaped embryos, most of which turned subsequently to dorsiventral differentiation. The morphological patterns of in-vitro-grown embryos were in accordance with those of normal zygotic embryos growing in planta. The formation of twin or multiple embryos originating from a single zygote was dependent on genotype and exogeneously supplied auxin. Upon transfer onto a suitable solidified medium, zygote-derived embryos usually germinated and developed into plants. After optimizing the feeder system, the nutrient medium and the concentration of 2,4-dichloro phenoxyacetic acid (2,4-D), more than 80 and 90% of the zygotes eventually developed into plants in genotypes Florida and Veery #5, respectively. All regenerated plants were morphologically normal and fertile. The in-vitro development from isolated zygotes of a higher-plant species into typically patterned zygotic embryos is shown here for the first time. Since the entire process, including early zygotic development, is now freely accessible to observation and micromanipulation, the method presented opens up new approaches in fundamental as well as applied fields of reproductive biology. Received: 4 September 1997 / Accepted: 28 November 1997     

Artificial endosperm of Cleopatra tangerine zygotic embryos: a model for somatic embryo encapsulation

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype-derived plants in a short time. In this work, a range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. Different complexing ions in the form of alginate capsules, zeolite as an ion exchanger and the relationship between capsule-nutrient gel on germination of zygotic embryos, were evaluated. Artificial endosperm assays showed that abscisic acid (1 μM) and mannitol (0.25 M) delayed germination and conversion of zygotic embryos, whereas amino acid supplements (proline, glutamic acid and arginine) accelerated the conversion process. An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency

Summary Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos. In an effort to improve the transformation protocol, the effects of gold particle size and callus morphology on transformation efficiency were investigated. Reducing gold particle size from 1.0 μm or 0.6 μm resulted in a significant increase in the rate of recovery of bialaphos-resistant clones from Type II callus. The average transformation efficiency of pre-embryogenic, early embryogenic and late embryogenic callus did not vary significantly. Rates of transformation, regeneration and fertility achieved for Type II callus are summarized and compared to those achieved for greenhouse- and field-derived immature zygotic embryos.     

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA₃ (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.     

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos. This revised version was published online in June 2006 with corrections to the Cover Date.