In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

Embryo sac isolation inArabidopsis thaliana: A simple and efficient technique for structure analysis and mutant selection

Arabidopsis thaliana has been widely used as a model plant in gene function analysis. However, its tiny flower and curved embryo sac make it difficult to study gene expression during megagametogenesis, fertilization, and early embryogenesis, especially in the screening of mutants from those developmental processes. The techniques currently available are sectioning and whole-mount clearing of ovules; however, sectioning is time consuming and laborious for quantitative analysis, and whole-mount clearing, makes clear cytological observation impossible. Reported here is a simple and efficient method based on enzymatic isolation of embryo sacs that enables both quantitative analysis and elaborate cytological observation for gene expression investigation and mutant screening.     

Is there a role for auxin in early embryogenesis?

The very young embryo of a flowering plant is not an idealsystem in which to study the effects of auxin. Conversely, auxin isusually not considered as a major component of developmental processesin early embryogenesis. However, recent findings from both experimentalstudies in Brassica and analyses of developmental mutants inArabidopsis make it worthwhile to examine critically thepossibility that auxin may have a role in early embryogenesis. In thisreview, we will focus on specific processes, such as formation of anapical-basal axis of polarity and the initiation of the primary rootmeristem. To provide a conceptual framework in which to discuss possibleeffects of auxin, we will first briefly summarise essential features ofearly embryogenesis in Arabidopsis. This will be followed by anevaluation of relevant data suggesting a role for auxin in axisformation and root meristem initiation. Finally, we will discuss a fewexperimental approaches that we believe are necessary to examine whetheror not auxin plays a role in fundamental processes of earlyembryogenesis.     

Invited review: Genetic regulation of somatic embryogenesis with particular reference to arabidopsis thaliana and Medicago truncatula

Summary Investigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic regulation of SE in the context of current molecular understanding of plant development.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy-brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Leafy cotyledon genes are essential for induction of somatic embryogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.     

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2 gene encoding a signaling peptide promotes cotyledon growth during Arabidopsis thaliana embryogenesis

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

mRNAs for two ribosomal proteins are preferentially translated in the chloroplast of Chlamydomonas reinhardtii under conditions of reduced protein synthesis

Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.     

Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.     

The Arabidopsis APC4 subunit of the anaphase-promoting complex/cyclosome (APC/C) is critical for both female gametogenesis and embryogenesis

The anaphase‐promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that is involved in regulating cell‐cycle progression. It has been widely studied in yeast and animal cells, but the function and regulation of the APC/C in plant cells are largely unknown. The Arabidopsis APC/C comprises at least 11 subunits, only a few of which have been studied in detail. APC4 is proposed to be a connector in the APC/C in yeast and animals. Here, we report the functional characterization of the Arabidopsis APC4 protein. We examined three heterozygous plant lines carrying apc4 alleles. These plants showed pleiotropic developmental defects in reproductive processes, including abnormal nuclear behavior in the developing embryo sac and aberrant cell division in embryos; these phenotypes differ from those reported for mutants of other subunits. Some ovules and embryos of apc4/+ plants also accumulated cyclin B protein, a known substrate of APC/C, suggesting a compromised function of APC/C. Arabidopsis APC4 was expressed in meristematic cells of seedlings, ovules in pistils and embryos in siliques, and was mainly localized in the nucleus. Additionally, the distribution of auxin was distorted in some embryos of apc4/+ plants. Our results indicate that Arabidopsis APC4 plays critical roles in female gametogenesis and embryogenesis, possibly as a connector in APC/C, and that regulation of auxin distribution may be involved in these processes.     

Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

Somatic embryogenesis in Jatropha curcas Linn., an important biofuel plant

Jatropha curcas L. is one potential source of non-edible biofuel-producing energy crop. Its importance also lies in its medicinal properties. The species is primarily propagated through heterozygous seeds, and thus the seed oil content varies from 4 to 40%. Moreover, due to its perennial nature, seed setting requires 2 to 3 years time. The seed viability and rate of germination are low, and quality seed screening is another laborious task; thus, seed propagation alone cannot provide quality planting material for sustainable use. Somatic embryogenesis, a powerful tool of plant biotechnology for faster and quality plant production has been successfully applied to regenerate plants in Jatropha curcas for the first time. Embryogenic calli were obtained from leaf explants on MS basal medium supplemented with only 9.3 μM Kn. Induction of globular somatic embryos from 58% of the cultures was achieved on MS medium with different concentrations of 2.3–4.6 μM Kn and 0.5–4.9 μM IBA; 2.3 μM Kn and 1.0 μM IBA proved to be the most effective combination for somatic embryo induction in Jatropha curcas. Addition of 13.6 μM adenine sulphate stimulated the process of development of somatic embryos. Mature somatic embryos were converted to plantlets on half strength MS basal medium with 90% survival rate in the field condition. The whole process required 12–16 weeks of culture for completion of all steps of plant regeneration. This protocol of somatic embryogenesis in Jatropha curcas may be an ideal system for future transgenic research.