Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of <Emphasis Type="Italic">Ocotea catharinensis</Emphasis>

In this study we examined the effect of polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) on growth, morphology evolution, endogenous PAs levels and nitric oxide (NO) release in Ocotea catharinensis somatic embryo cultures. We observed that Spd and Spm reduced culture growth, permitted embryo morphogenetic evolution from the earliest to last embryo development stages, increased endogenous PAs levels, and induced NO release in O. catharinensis somatic embryos. On the other hand, Put had little effect on these parameters. Spd and Spm could successfully be used to promote somatic embryo maturation in O. catharinensis. The results suggest that Spd and Spm have an important role during the growth, development and morphogenetic evolution of somatic embryos, through alterations in the endogenous nitric oxide and PAs metabolism in this species.     

Variations in vinblastine production at different stages of somatic embryogenesis,embryo, and field-grown plantlets of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don,as revealed by HPLC

Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anticancer compounds, which are used for the treatment of a wide variety of cancers. We have quantified vinblastine (a major dimeric anticancer compound) in various in vitro raised tissues; embryogenic and nonembryogenic calli, three different embryogenic stages (proliferated, matured, and germinating embryo), somatic embryo derived plantlets and in ex vitro grown plantlets by using high performance liquid chromatography. Of the various obtained callus lines and embryogenesis stages, maximum vinblastine content was found in leaf callus and in germinating embryos. The leaves of somatic embryo-derived plantlets contained more vinblastine than did Catharanthus leaves developed ex vitro. The yield of vinblastine was monitored for 30 wk. The production of vinblastine appeared to be age dependent and tissue specific; the finding of our analyses is discussed in detail.     

Genetic variability analyses of the somatic embryogenesis induction process in Olea spp. using nuclear microsatellites

The crop species Olea europaea L. (olive tree) is of great economic importance in the Mediterranean region. Hence, many efforts have been done in the last decades to propagate this commercially valuable species by in vitro methods. On the other hand, the lesser known Olea maderensis (Lowe) Rivas Mart. & Del Arco which is a native species of the Madeira Archipelago has only been the subject of micropropagation from nodal stem cuttings. Therefore, in this work we analysed the stability of ten nuclear simple sequence repeat (SSR) markers at successive stages of the somatic embryogenesis process in two adult trees belonging to these two species from the Madeira Archipelago. For the induction of somatic embryogenesis, petiole and leaf explants were cultivated on solid Murashige and Skoog medium (MS) with 12.25 μM indole-3-butyric acid (IBA) and 4.56 μM of zeatin, in the dark. After 3 months, different callus tissues (non-embryogenic, pre-embryogenic and embryogenic) developed from leaf explants and petioles were later transferred to MS medium without growth regulators in the dark. All ten SSR markers were able to distinguish between species. However, no mutations were found at the SSR loci at any of the successive developmental stages from PEMs (pre-embryogenic masses) to somatic embryos. This genetic uniformity was observed within material derived from each genotype/species and its respective donor plant. Therefore, we conclude that the genomic integrities of both O. europaea and O. maderensis were maintained throughout the stages of the embryogenic processes in study suggesting the absence of somaclonal variation.     

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.     

Indirect somatic embryogenesis and morphohistological analysis in <Emphasis Type="Italic">Capsicum chinense</Emphasis>

Capsicum chinense is recalcitrant in in vitro morphogenesis. No efficient, reproducible somatic embryogenesis regeneration system exists for this species, impeding regeneration from transformed cells. An indirect somatic embryogenesis protocol is developed using mature C. chinense zygotic embryo segments (ZES). The ZES cultured in semi-solid Murashige-Skoog (MS) medium supplemented with 8.9 μM naphthaleneacetic acid, 11.4 μM indoleacetic acid and 8.9 μM 6-benzylaminopurine, developed an embryogenic callus and 8% of the calli developed somatic embryos. Torpedo-stage somatic embryos were detached from the callus and subcultured in semi-solid MS medium without growth regulators, producing a 75% conversion rate to plantlets with well-formed root tissue. Histological analysis showed the developed structures to have no vascular connection with the callus and to be bipolar, confirming that this protocol induced formation of viable somatic embryos from mature C. chinense ZES. All acclimated plantlets survived under greenhouse conditions. This protocol will facilitate regeneration of genetically transformed plants using either biolistics or Agrobacterium tumefaciens approach.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

Changes in protein pattern during different developmental stages of somatic embryos in chickpea

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg⁻¹(f.m.)] to cotyledonary stage [4.8 μg mg⁻¹(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg⁻¹(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.     

Somatic embryogenesis from zygotic embryos and shoot-tips of the Chilean tree <Emphasis Type="Italic">Gomortega keule</Emphasis>

The endangered Chilean tree Gomortega keule (Mol.) Baillon produces edible fruit, making it a potential crop. However, its cultivation from seed or cuttings is extremely difficult. This paper reports the induction of somatic embryogenesis and the initiation of liquid cultures in this species. Callus was induced from zygotic embryos and field-collected shoots. Somatic embryogenesis on zygotic embryos occurred at a low frequency. Induction of somatic embryogenesis was accomplished on micropropagated shoots after 6.5 months on semi-solid Murashige and Skoog (MS) medium with 30 g/l sucrose, 1.0 mg/l 2,4-dichlorophenoxyacetic acid and 1.0 mg/l 6-(γ,γ-dimethylallylamino) purine (2iP). Liquid cultures of compact callus and small aggregates were obtained and showed optimum proliferation in MS medium with 20 g/l sucrose, 0.01 mg/l α-naphthaleneacetic acid and 0.1 mg/l 2iP. The proliferation of friable embryogenic callus was observed in liquid medium and will allow the propagation of selected genotypes of this tree on a large scale. Genetic variation in two embryogenic genotypes cultured in vitro was not detected in an assessment using microsatellites; this approach is suitable for tracing genotypes.     

Diphenylurea Derivatives Induce Somatic Embryogenesis in Citrus

The present research investigates the possibility that three diphenylurea (DPU) derivatives, N-phenyl-N′-benzothiazol-6-ylurea (PBU), N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methilendioxyphenyl)urea (3,4-MDPU), stimulate the induction of somatic embryogenesis in three Citrus species. The hypothetical embryogenic activity was assessed using stigma and styles of Citrus myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three compounds influenced the production of somatic embryos differently as regards the concentrations tested and the citrus species. PBU was able to induce somatic embryogenesis at all the concentrations tested and in all the three species with percentages that ranged from 44 (C. limon) to 85% (C. myrtifolia). 2,3-MDPU and 3,4-MDPU were completely unable to induce the production of somatic embryos in C. myrtifolia while both the compounds at the higher concentration (12 μM) acted positively in both C. madurensis and C. limon (68% of embryogenic explants). The phenylurea derivatives, used for the first time in this study to induce somatic embryogenesis in plant, showed a higher embriogenic performance when compared with 6-benzylaminopurine (BAP), a classical adenine-cytokinin, and with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), a classical DPU derivative.     

Somatic embryogenesis from immature zygotic embryos of annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl⁻¹ activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.     

Cyclic secondary somatic embryogenesis and efficient plant regeneration in camphor tree (<Emphasis Type="Italic">Cinnamomum camphora</Emphasis> L.)

An efficient protocol for secondary somatic embryogenesis in camphor tree is reported. Secondary somatic embryos (SSEs), initially obtained from the primary embryos of a nascent embryogenic culture in 2002, were proliferated and maintained for more than 4 yr via cyclic secondary somatic embryogenesis. Throughout this period, the embryo populations retained a high level of competence for plant regeneration. SSEs were produced on the surfaces of the cotyledons and radicular ends of maternal somatic embryos (MSEs). Histological observations of the various stages of secondary embryo development revealed four typical stages, namely, globular, heart-shaped, torpedo, and cotyledonary. The process of secondary embryogenesis continued in a cyclic way, with each newly formed embryo producing a subsequent generation of secondary embryos. In order to progress developmentally beyond proliferation cycles, cotyledonary embryos from one of embryogenic lines (L14) were cultured on Murashige and Skoog (MS) medium with 0.1–3.0 mg l⁻¹ abscisic acid (ABA) or 0.05–1.0 mg l⁻¹ thidiazuron (TDZ) in darkness for 2 mo to achieve maturation. Matured embryos were then transferred to MS-based germination medium containing either 0.1 mg l⁻¹ TDZ, 0.2 mg l⁻¹ indole-3-butyric acid (IBA), and 0.5 mg l⁻¹ 6-benzylaminopurine (BA) or 0.1 mg l⁻¹ TDZ and 0.2 mg l⁻¹ IBA and were cultured in light for germination. Over 50% of embryos matured in the presence of 0.5 mg l⁻¹ ABA were able to germinate with shoots and poor root system. Frequencies of embryos germinating normal shoots among different genotypes did not change significantly. A total of 93% of the shoots from the germinated embryos converted to plantlets on half strength MS medium with 0.5 mg l⁻¹ IBA by 3 wk. Plantlets acclimatized successfully to ex vitro conditions and developed as field-grown plants with normal appearance.     

In vitro somatic embryogenesis in two major rattan species: Calamus merrillii and Calamus subinermis

Summary Occurrence of somatic embryogenesis in in vitro cultures of Calamus merrillii and Calamus subinermis, two major largecaned rattan species, was scientifically demonstrated for the first time. Tissue responsiveness varied markedly according to the species and the type of primary explants used when initiated on 10.4–31.2 μM picloram-enriched Murashige and Skoog callus induction media. In C. merrillii, within 6 wk after inoculation, 84% of the leaf and 90% of the zygotic embryo explants produced friable embryogenic calluses, by contrast with those formed by 74% of the root explants. In C. subinermis, callogenesis was observed only 6 mo. after inoculation in 68% of root and 48% of zygotic explants. Leaf explants did not respond at all. Only root-derived calluses developed into nodular embryogenic structures. Irrespective of these initial differences, the further steps of the somatic embryogenesis developmental pattern was similar for both species. Histological analyses established that callus formation took place in the perivascular zones, and could give rise to embryogenic isolated cells from which the proembryos were derived. Reducing the picloram concentration stimulated the maturation process resulting ultimately in the germination of somatic embryos that exhibited bipolar development, despite an apparent lack of starch and protein reserves. The somatic embryo-derived plantlets of C. merrillii, overall more prone to somatic embryogenesis than C. subinermis in the given conditions, were successfully acclimatized to outdoor conditions.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Establishment and Growth of Embryogenic Suspension Cultures of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was to test the effects of 2,4-D, sucrose, culture media and initial inocula on the development of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Suspension cultures were established in half-strength MS medium supplemented with 2% (w/v) sucrose either in the absence or in the presence of 2.2 μM 2,4-D, when higher cell viability was achieved. Under this culture condition the maximum fresh weight increase occurred in the fourth week. The cultures were yellow and consisted of a mixture of highly cytoplasmic single cells and small cell aggregates (<0.25 mm). The best proportion of inoculum per volume of medium for suspension culture development was 5% (w/w). Suspension cultures consisting of somatic embryos at the globular and cotyledonary stages (structures ranging from 1 to 3 mm) were successfully established on half-strength MS supplemented with 2% (w/w) sucrose through repetitive embryogenesis from the desiccated mature somatic embryos used as initial inoculum. The failure to initiate liquid cultures from non-desiccated mature somatic embryos was overcome by pre-treatment with air desiccation and reduction of the water content to 6.1 g H₂O g⁻¹ dry weight.     

Somatic embryogenesis in <Emphasis Type="Italic">Buchanania lanzan</Emphasis> spreng

Summary We report a protocol for somatic embryogenesis and plantlet regeneration of Buchanania lanzan Spreng (Family—Anacardiaceae), which is a tropical fruit tree widely distributed in the dry forests of India. Calluses were initiated from immature zygotic embryos cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) and/or 1-naphthaleneacetic acid (NAA). The highest frequency (60%) of somatic embryo induction was obtained in cultures grown on MS medium fortified with 4.53 μM 2,4-D, 5.32 μM NAA and 4.48 μM BA. The medium supplemented with 15 μM abscisic acid (ABA) was most effective for maturation and germination of somatic embryos. This is the first report on somatic embryogenesis in B. lanzan, which may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of this species.     

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l⁻¹) and BA (1.5 mg l⁻¹). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.