Acquisition of embryogenic competence during somatic embryogenesis

This review focuses on investigation in acquisition of embryogenic competence during somatic embryogenesis in the last five decades. In tissue culture, differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells during the induction phase. These embryogenic cells are important because they differentiate to form somatic embryos at a later time. Various molecular and structural markers for detecting embryogenic cells or enhancing embryogenic competence are summarized and implications of the findings are discussed.     

Cytological features of developing embryogenic callus and somatic embryos of Iris pumila L. and Iris setosa Pall

Abstract

Cytological examination during somatic embryogenesis in Iris pumila L. and Iris setosa Pall, were performed using light and electron microscopy. The first sign of the cellular differentiation in the initial embryogenic callus (EC; stage 1) of both Iris species was the formation of short and elongated cell types. After the onset of embryogenesis, short cells divided producing a mass of densely packed meristematic cells, closely connected with numerous plasmodesmata. Further differentiation into globular embryos (GE) led to a loss of plasmodesmata and cell separation. In vacuolated elongated cells, cytoplasm was located near the wall and around the nucleus. In both cell types amyloplasts and small mitochondria with poorly developed crystae were abundant.

Cell of GE (stage 2) contained an increased number of mitochondria and plastids comparing to those from stage 1, indicating further differentiation. Thylakoids and starch grains were observed within the plastids, while the number of cristae within the mitochondria was increasing.

In cells of embryos with coleoptile (ECl) (stage 3), plastids differentiated into chloroplasts with thylakoids. In all stages of cell differentiation, short and long cisternae of endoplasmic reticulum with ribosomes were seen. Activity of dictyosomes was increased in stages 1 and 2, then reduced in stage 3.

Ultrastructure of EC cells was identical to that of proembryogenic cells, i.e. of early GE. Ultrastructural appearance of GE cells was identical in both Iris species, but evident, and increasing, differences in mitochondria and plastids were observed between GE and ECl embryos.The presence of bi-, three- and eight- cell proembryos demonstrates that they originate from a single cell in both Iris species.     

Repetitive somatic embryogenesis in carnation on picloram supplemented media

An efficient method of repetitive somatic embryogenesis and plant regeneration of two carnation cultivars (Sagres and Impulse) was established using a two-step protocol. In the first step, embryogenic tissue were induced from petal explants on MS culture medium containing 9% sucrose (w/v), 9 μM 2,4-D and 0.8 μM BA. In the second step, embryogenic tissue transferred onto the MS medium containing 3% sucrose supplemented with different concentrations of picloram (0.8, 2, 4, 8 and 16 μM) to produce primary somatic embryos. Precotyledonary clumps and cotyledonary somatic embryos were then isolated and subcultured onto the same media as the second step where they formed secondary somatic embryos in repetitive cycles. Cotyledony somatic embryos were converted into plantlets when they transferred onto the growth regulator-free half-strength MS medium followed by being acclimated to the greenhouse conditions.     

Plant regeneration of Iris pallida Lam. and Iris germanica L. via somatic embryogenesis from leaves,apices and young flowers

Irones are violet-scented ketonic compounds contained in the rhizome of certain species of iris. As cultivation of the iris tends to decrease, a selection program has been initiated to find the best performing clones in terms of growth and yield. Parallel to this selection, in vitro regeneration studies have been carried out in order to multiply interesting clones. A method of rapid multiplication by somatic embryogenesis associated with multibudding was developed. Callus was obtained from leaf bases, flower pieces or rhizome apices; the best explants were flower pieces. The induction media used to obtain embryogenic callus were Murashige & Skoog (1962) media. Assays with adding of proline in these media have showed that it could double the yield of embryogenic callus. The embryogenic expression medium was the Knudson's orchid agar (Knudson 1946) medium. Conformity of the plants obtained was checked by comparing their chemotypes with those of the mother plants.Abbreviations AIB indolyl butyric acid - BAP 6-benzylaminopurine - Kin 6-furfurylaminopurine - KN Knudson's orchid Agar - MS Murashige & Skoog medium - NAA naphthaleneacetic acid - N6 Chu et al. medium - Pro proline - 2,4-D dichlorophenoxyacetic acid     

Identification of a potential structural marker for embryogenic competency in the Brassica napus spp. oleifera embryogenic tissue

The Brassica napus secondary embryogenesis system requires no exogenous growth regulator to stimulate embryo development. It is stable embryogenically over a long period of culture and has a distinct pre-embryogenic stage. This system was used to investigate the morphological and cellular changes occurring in the embryogenic tissue compared to non-embryogenic tissue using various microscopy techniques. A unique ultrastructural feature designated the extracellular matrix (ECM) was observed on the surface of pre-embryogenic embryoids but not on the non-embryogenic individuals. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. This is a novel aspect of the phenotype previously unreported in the Brassica system. This structure might be linked to acquisition of embryogenic competence.     

On the occurrence of somatic meiosis in embryogenic carrot cell cultures

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.     

Genetic variation in somatic embryogenic response in open-pollinated families of black spruce

Summary Zygotic embryos from open-pollinated seeds of 20 black spruce (Picea mariana) families were used to investigate the proportion of genotypes that would give rise to embryogenic tissue (ET) and mature somatic embryos. Eighty-five percent of the maternal genotypes gave rise to embryogenic tissue. Within-family rates of ET induction ranged from 0 to 17%, with an average of 8%. The largest proportion of variation was among families, indicating the additive nature of the genetic variation. On a medium with 6% sucrose and 3.7 M ABA, 90% of the embryogenic lines gave rise to abundant (>100/100 mg of ET), well-formed, mature somatic embryos. A medium with 2% sucrose, without 2,4-D, was used to germinate the mature somatic embryos. These were grown in the greenhouse and have now been established in field trials.     

Direct somatic embryogenesis in leaves ofCichorium

Summary Leaves from 2-month-old in vitro grown plantlets of a clone ofCichorium placed in agitated liquid induction medium at 35°C in the dark produce embryoids after 5 days of culture, without synchronization. Vascular sheath parenchyma cells react first, but every mesophyll cell is potentially embryogenic. Single cells show an early patchy callosic wall and undergo dedifferentiation. With SEM the cells of those proembryoids just emerging through the epidermis are seen to be linked by a fibrillar network, the nature of which is discussed. Four FITC-labelled lectins were tested; only DBA shows embryogenic specificity.     

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.     

Mitochondrial DMA variation in somatic embryogenic cultures ofLarix

outhern hybridization analysis using wheat mitochondrial gene-specific probes indicates that changes in mitochondrial genomic organization and the relative representation of certain genomic regions occur during in vitro somatic embryogenic cell culture ofLarix species. We observed differences in the mitochondrial (mt)DNA hybridization patterns between somatic embryogenic cell cultures and trees grown from seed forLarix leptolepis,L. decidua, and the reciprocal hybrids of these twoLarix species. This is the first study to describe the correlation of molecular changes in a gymnosperm mitochondrial genome with in vitro somatic embryogenic cell culture. Quantitative differences in mtDNA hybridization signals were also observed among a 4-year-old somatic embryogenic cell culture ofLarix ×eurolepis trees regenerated from this culture, and the seed source tree from which the somatic embryogenic cell cultures were initiated.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Quantitative analysis of ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.)

Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.     

Induction of embryogenic tissue and maturation of somatic embryos in Pinus brutia TEN

Embryogenic tissues (ET) of Turkish red pine (Pinus brutia TEN) were initiated from immature precotyledonary zygotic embryos sampled from 15 different plus trees. Seven collections were made weekly from June 10 to July 22, 2003. DCR basal medium supplemented with 13.6 μM 2,4–dichlorophenoxyacetic acid (2,4-D) and 2.2 μM benzylaminopurine (BAP) was used for initiation and maintenance of ET. Overall initiation frequency of ET in the study was 11.6%, initiation rates ranging between 4.7% and 24.1% per tree. Out of 12,940 explants tested, 3.4% were converted into established cell lines (ECL) following five subcultures. Of the maturation treatments tested, 80 μM ABA, sucrose (3%) and maltose (3% and 6%), and 3.75% PEG combined with 1% gellan gum were the most suitable combination for somatic embryo maturation.     

ISSR marker‐based detection of genomic stability in Cassia occidentalis L. plantlets derived from somatic embryogenesis

An efficient in vitro technology has been designed for mass multiplication of Cassia occidentalis (coffee senna) through somatic embryogenesis. Genetic stability of both regenerants and mother plant was evaluated. Embryogenic calli were produced on Murashige and Skoog (MS) medium supplemented with 20.0 μM 2,4‐dichlorophenoxy acetic acid (2,4‐D). Induction of somatic embryos occurred after transfer of calli to medium with reduced concentration of 2,4‐D (10.0 μM) fortified with 1.0 μM abscisic acid (ABA). Subculturing of these embryos onto the maturation medium (1.5 μM 6‐benzyladenine + 1.0 μM ABA + 0.3 μM α‐naphthalene acetic acid) favored progression of the embryos through torpedo, heart‐shaped, and cotyledonary stages; one‐half MS medium was considered the best for conversion of cotyledonary stage embryos to young plantlets. The plantlets were acclimatized to autoclaved soil rite, after which they were transferred to the green house. Among the survived plantlets, 10 plants for each primer were randomly selected for inter‐simple sequence repeat (ISSR) analysis. Of the 10 primers tested, 5 produced reproducible and monomorphic bands, 2 led to minor variation with the appearance of unique bands, and the remaining 3 did not show any banding pattern. The majority of the regenerants had similar characteristics to the donor plant, suggesting genetic homogeneity of in vitro raised plants.     

Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L.

Daucus carota L. cell lines secrete a characteristic set of arabinogalactan proteins (AGPs) into the medium. The composition of this set of AGPs changes with the age of the culture, as can be determined by crossed electrophoresis with the specific AGP-binding agent, β-glucosyl Yariv reagent. Addition of AGPs isolated from the medium of a non-embryogenic cell line to an expiant culture initiated the development of the culture to a non-embryogenic cell line. Without addition of AGPs or with addition of carrot-seed AGPs an embryogenic cell line was established. Three-month-old embryogenic cell lines usually contain less than 30% of dense, highly cytoplasmic cells, i.e. the embryogenic cells, but when carrot-seed AGPs were added this percentage increased to 80%. Addition of carrot-seed AGPs to a two-year-old, non-embryogenic cell line resulted in the re-induction of embryogenic potential. These results show that specific AGPs are essential in somatic embryogenesis and are able to direct development of cells.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Plant regeneration through somatic embryogenesis in callus culture of green bamboo (Bambusa oldhamii Munro)

Summary Young inflorescence explants of green bamboo (Bambusa oldhamii Munro) in culture show a high capacity for plant regeneration through somatic embryogenesis. Embryogenic callus was initiated from explants maintained on Murashige and Skoog's medium supplemented with 3 mg/l 2,4-D, 2 mg/l kinetin and a high content (60 g/l) of sucrose. Prolonged culture in the embryoid induction medium or transferral of embryonic callus to auxin-free medium resulted in the continued development and eventual germination of embryoids and establishment of rooted plantlets that were successfully transferred to soil.