Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.     

Handling culture medium composition for optimizing plant cell suspension culture in shake flasks

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L^?1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g^?1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g^?1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g^?1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg^?1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg^?1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens

Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.     

Somatic embryogenesis of Pinus rigida?×?P. taeda and the relationship between the initiation of embryogenic tissue and zygotic embryo development

The pitch-loblolly pine hybrid (Pinus rigida × P. taeda) has useful characteristics of the parents, but its exploitation is hindered by restrictions of conventional breeding and propagation methods. This study was undertaken to establish an effective in vitro system for propagating pitch-loblolly hybrid pine through somatic embryogenesis and to unravel the relationship between the efficiency of embryogenic tissue initiation and zygotic embryo development. To initiate embryogenic tissue, megagametophytes of developing seeds were used as explants. Seeds were collected weekly, examined, and tested during June and July 2004. The medium and seed collection date were the most important factors for the successful somatic embryogenesis of P. rigida × P. taeda. Five embryogenic lines were obtained using a modified P. taeda basal medium, and the highest initiation rate was 0.55%, for seeds collected in 2 weeks, between July 3 and 16. Histological observation revealed that zygotic embryos of those seeds were mostly at the proembryonic stage or in transition to precotyledonary stages. For the successful maturation of somatic embryos, abscisic acid and gellan gum were needed in the medium. The results show that, although further tests and development are required, somatic embryogenesis could provide a viable option for propagating P. rigida × P. taeda hybrids.     

High-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l^?1 of α-naphthaleneacetic acid (NAA) and 1 mg l^?1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Somatic Polyembriogenesis of <Emphasis Type="Italic">Larix sibirica</Emphasis> in Embryogenic in vitro Culture

The initiation of somatic embryos and their propagation in the long-term proliferating embryonal suspensor mass of Larix sibirica were studied. It was found that the increase in the number of somatic embryos in the embryogenic culture occurred as a result of cleavage of the globules of the somatic embryo and the suspensor; it less often occurred as the result of budding of the suspensor and the separation of the embryonal tubes of the suspensor. In the case of long-term proliferating cell lines (more than 8 years), the rate of cleavage did not weaken. A conclusion on the identity of morphogenic processes underlying the development of zygotic and somatic embryos of conifers is made, which is confirmed by the concept of T.B. Batygina (1999) on the parallelism of their development in vivo and in vitro.     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

湿地松体细胞胚胎发生和植株再生

以湿地松的未成熟合子胚为外植体,在附加８ｍｇ／Ｌ,２,４－Ｄ和４ｍｇ／Ｌ ＢＡ的ＬＰ培养基上诱导出胚性愈伤组织。在含１ｍｇ／Ｌ,２,４－Ｄ和０．５ｍｇ／Ｌ ＢＡ的培养基上保持并增殖。提高培养基的渗透压后,愈伤组织内大量的胚性胚柄细胞团和早期原胚。     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1^−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1⁻¹) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N⁶-benzyladenine (BAP, 0.75 mg 1^−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1^−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.     

Effects of cadmium and lead stress on somatic embryogenesis of coniferous species. Part I: Evaluation of the genotype-dependent response

Somatic embryogenesis is an important biotechnological tool that has significant potential to be used in studies related to environmental stress. In this study, embryogenic cell masses of Abies alba and Picea abies were grown on media enriched with 50–500 µM cadmium (Cd²⁺) or lead (Pb²⁺). The effects of cadmium and lead were evaluated during the subsequent stages of somatic embryogenesis: proliferation, maturation, and germination. The following characteristics were evaluated: proliferation potential, cell viability, average number of somatic embryos obtained per 1 g of fresh weight, and morphology of the developed somatic embryos. The tested heavy metals significantly reduced the proliferation rate of A. alba and P. abies embryogenic cell masses. The highest tested cadmium concentration markedly slowed or stopped the growth of embryogenic cell masses in both species. Unexpectedly, the proliferation ratio remained fairly high for the P. abies cell lines treated with lead at all concentrations tested. During the maturation stage, the total number of somatic embryos declined under cadmium exposure. The formation of early precotyledonary and cotyledonary somatic embryos in both species was similarly reduced, although cadmium caused a higher death rate and was more toxic than lead. To the best of our knowledge, this is the first report to study the effects of heavy metals on A. alba embryogenic cell masses during the proliferation stage as well as on the maturation and germination processes of both species. This in vitro system can be used for testing the response of large sets of genotypes, and the best performing lines can be used in the future for in vivo performance tests of heavy metal-polluted soils.     

Regeneration of plant by somatic embryogenesis in <Emphasis Type="Italic">Pinus rigida</Emphasis>×<Emphasis Type="Italic">P. taeda</Emphasis>

Zygotic embryos at different developmental stages were tested for their potential in the initiation of embryogenic suspensor mass (ESM) lines using immature seeds of Pinus rigida × P. taeda. The highest frequency (1.1%) of ESM was obtained with explants from cones collected on July 1. All excised embryos of the July 1 collection were at the early proembryo stage. Two different culture media were compared. Forty-eight ESM lines were initiated on Pinus taeda basal medium (P6) (0.97%) with 13.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA). However, only four ESM were obtained on a modified Murashige and Skoog medium (MSG; 0.55%). Most of the ESM arose from the seeds that were at the stages ranging from late cleavage polyembryony to the early staged proembryo. Out of 52 lines (0.46%) that were produced from 11,388 explants, only two viable lines (0.018%) (PRT11 and PRT28) survived. As for somatic embryo maturation, the highest number (224/g⁻¹ FW) of matured cotyledonary somatic embryos (line PRT 28) was obtained on a medium containing 100 μM abscisic acid (ABA), 0.2 M maltose, and 1.2% gellan gum. For germination of the somatic embryos, the cotyledonary somatic embryos derived from maturation medium were transferred on half-strength Litvay medium (LM) plus 0.4% gellan gum. The germination rates were high (71.4–96.3%) regardless of the concentrations of either ABA or gellan gum in the maturation medium. Approximately 500 somatic plants were recovered from the germination medium and transferred to the green house; finally most of them were transplanted successfully to the experimental field.