Isozyme Profile During Somatic Embryogenesis and In Vitro Flowering of Bambusa vulgaris

Peroxidase and esterase isozymes were investigated during plant regeneration via somatic embryogenesis in Bambusa vulgaris, The transition of non-embryogenic calli to embryogenic calli, somatic embryo development, germination and subsequent flowering of somatic embryo derived shoots were associated with selective expression or repression of isoforms of peroxidase and esterase. Non-embryogenic callus showed six peroxidase and four esterase bands. During somatic embryogenesis and germination of somatic embryos, some bands were suppressed and new isoforms of peroxidase and esterase appeared. During flowering, in addition to four peroxidase bands, a new unique esterase band ‘a’ appeared. Each developmental stage was thus associated with a definite isozyme profile.     

Large impact of the apoplast on somatic embryogenesis in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> offers possibilities for improved developmental control <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Clonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop Cyclamen persicum by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.     

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L^?1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L^?1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

落叶松体胚发育中5个miRNA前体与成熟体的表达

利用同源比对或RACE克隆了5个落叶松(Larix leptolepis) miRNA前体。结果显示, 在各物种miRNA前体间, 成熟序列高度相似, 但其它序列相似度差异大, 序列相似度与亲缘关系有关。采用qRT-PCR分析了5个miRNA、前体和靶基因在落叶松体胚8个发育阶段的表达变化。结果显示, miRNA表达最高峰出现在后期子叶胚, 暗示与促进胚胎休眠有关; 表达次高峰出现时期不同, 表现为miR397和miR408在PEMIII, miR398在早期单胚, miR156和miR166在早期子叶胚, 表明其与保持薄细胞壁、质子传递、顶端分生组织形成等调控有关。miRNA成熟体表达与前体含量不呈线性相关, 可能受多重调控。研究结果对于阐明MIR基因进化、表达调控及在体胚发育中的调控功能具有重要理论意义。     

Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest

Pinus halepensis Mill. is a common forest species in the Mediterranean area and it is important for environmental conservation. This study established a method of regenerating Pinus halepensis Mill. through somatic embryogenesis. The effect of culture medium (mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was analysed during the first steps of embryogenesis process. This study showed a marked effect of the culture medium tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture medium and the proliferation stage was significantly affected by the combination of plant growth regulators. At the end of the maturation phase the presence of activated charcoal was also evaluated. Finally, maturation of embryogenic tissue was affected by the nitrogen source in the culture medium and these results were different for high and low mature embryo producing cell lines. To the best of our knowledge, this is the first report on Aleppo pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.     

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes     

Rice embryogenic calli express a unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic development

In vitro cultured embryogenic callus was employed as a model to investigate microRNAs (miRNAs) associated with embryogenesis and post-embryonic development. Thirty-one miRNAs including 16 novel species were identified from a large number of small RNAs which were cloned from both differentiated and undifferentiated rice embryogenic calli. Four target genes of the miRNAs were further validated. A set of the miRNAs, including miR397 and miR156, exhibited intriguing expression patterns during the transition from undifferentiated to differentiated calli. By exploiting the correlations between the differential expression patterns of these miRNAs and their targets, the regulatory roles of the miRNAs on meristem maintenance and embryogenesis were indicated.     

Physiological and biochemical features of embryogenic and non-embryogenic peach palm (<Emphasis Type="Italic">Bactris gasipaes</Emphasis> Kunth) cultures

Both embryogenic and non-embryogenic peach palm (Bactris gasipaes Kunth) cultures arise during somatic embryogenesis induction, and both tissue types are often observed growing side-by-side from the same explant. To better understand why this occurs, samples from each tissue type were analyzed for their endogenous concentrations of indole-3-acetic acid (IAA), abscisic acid (ABA), polyamines, and amino acids with high-performance liquid chromatography and for total phenolics with spectrophotometry. Embryogenic cultures contained significantly higher concentrations of IAA, ABA, and total amino acids, whereas non-embryogenic tissue contained more total polyamines and phenolics. The greater IAA concentrations in embryogenic cultures supported the role of that hormone as a marker of embryogenic potential. Putrescine was especially prevalent in non-embryogenic cultures; however, the decreased putrescine/spermine + spermidine ratio in embryogenic cultures added support to the conclusions of previous studies in other species that this can serve as a marker of embryogenic competence. Though embryogenic cultures contained higher total amino acids, each culture type had different concentrations of specific amino acids.     

Embryogeny of gymnosperms: advances in synthetic seed technology of conifers

Synthetic seed technology requires the inexpensive production of large numbers of high-quality somatic embryos. Proliferating embryogenic cultures from conifers consist of immature embryos, which undergo synchronous maturation in the presence of abscisic acid and elevated osmoticum. Improvements in conifer somatic embryo quality have been achieved by identifying the conditions in vitro that resemble the conditions during in ovulo development of zygotic embryos. One normal aspect of zygotic embryo development for conifers is maturation drying, which allows seeds to be stored and promotes normal germination. Conditions of culture are described that yield mature conifer somatic embryos that possess normal storage proteins and fatty acids and which survive either partial drying, or full drying to moisture contents similar to those achieved by mature dehydrated zygotic embryos. Large numbers of quiescent somatic embryos can be produced throughout the year and stored for germination in the spring, which simplifies production and provides plants of uniform size. This review focuses on recent advances in conifer somatic embryogenesis and synthetic seed technology, particularly in areas of embryo development, maturation drying, encapsulation and germination. Comparisons of conifer embryogeny are made with other gymnosperms and angiosperms.Abbreviations ABA abscisic acid - LEA late embryogenesis abundant - PEG polyethylene glycol - PGR plant growth regulator - RH relative humidity - TAG triacylglycerol