Embryogenesis of European Radish (Raphanus sativus L. subsp. sativus convar. radicula) in Culture of Isolated Microspores In Vitro

The European radish is one of the most unresponsive crops in the Brassicaceae family to embryogenesis in in vitro microspore culture. The aim of this work was to study the process of embryogenesis of European radish and its biological features. In this study, the embryogenesis of European radish is described in detail with illustrative data for the first time. For the first time for the entire family Brassicaceae, the following were found: microspores with intact exines with ordered-like divisions; microspores completely free of exines; and a new scheme of suspensors attachment to the apical parts of embryoids. The morphology of double and triple twin embryoids was described, and new patterns of their attachment to each other were discovered. Uneven maturation of European radish embryoids at all stages of embryogenesis was noted. The period of embryoid maturation to the globular stage of development corresponded, in terms of time, to the culture of B. napus, and into the cotyledonary stage of development, maturation was faster and amounted to 17–23 days. The rate of embryoid development with and without suspensors was the same.

Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata)

Key message Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Abstract Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

Effects of the growth regulators for the induction of somatic embryos from explants in an endemic and threatened Echinocactus parryi Engelm.

Introduction: The list of threatened species is enhancing and needs to be revised by integrating plant tissue culture tools with conventional techniques that support the appropriate management of these species. Objective: To assess the effects of the growth regulators for the induction of somatic embryos from mature seeds, shoots, and compact green callus of Echinocactus parryi Engelm. and the histological analysis of the embryogenic structures. Materials and methods: A completely randomized design was utilized to evaluate three types of explants (apical, medium, and basal) cultured on basal Murashige & Skoog media (MS) with different growth regulators concentrations (2, 4-D [dichlorophenoxy acetic acid], BAP [6-benzylaminopurine] and kinetin, at four levels: 0.5, 1, 1.5, and 2 mg∙L -1 ). Histological analysis of the embryogenic structures was performed. Results and discussion: The 2, 4-D induced both embryogenic and organogenic callus from seeds and shoot explants. The globular stage did not evolve to their maturity, presumably because of 2, 4-D accumulation. The compact callus explants were the more efficient to induce 19.2 somatic embryos per explant when they were cultured in the medium with 0.5 mg∙L -1 kinetin. However, the latest phases did not germinate, probably due to abnormalities generated by genetic and epigenetic changes in the DNA that can cause abnormal somatic embryos. The histology image demonstrated that the globular and torpedo structures were visible under a microscope showing stained nucleus and numerous starch grains. Conclusions: E. parryi is a species that can produce a high number of embryogenic structures, which represents a great potential to grow massive plants.

The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis

Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)—required for purine nucleotide and histidine synthesis—and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.

Selection of salt tolerant embryogenic line in Jatropha curcas L., which has potentiality of biodiesel

The embryogenic calli were grown on MS medium containing NaCl with concentrations from 50 to 300 mM. After 2 weeks of culture, salinity tolerance threshold was identified at 150 mM NaCl. Higher concentrations of NaCl stimulated a significant reduction in the calli survival rate and the highest rate was 78.67% at 50 mM. After subculturing callus to the embryo culture medium con- taining NaCl, the growth and embryogenesis were not affected at the concentrations of 50 – 100 mM. Especially, at 50 mM NaCl the embryogenesis rate reached 83.33%. In contrast, 150 mM NaCl inhibited the somatic embryogenesis. After 4 weeks, culturing somatic embryos on medium MS with addition of 0.07 mg/l spermidin at 50 – 100 mM NaCl, the embryogenesis was considered good and embryos developed through several stages: globular, heart, torpedo and cotyledonary. However, at 150 mM NaCl the globular stage appeared in the culture process. The process of morphohistology and using dye carmine – iod and acridine orange observed the structure of generative callus and embryos at several stages. Mô sẹo có khả năng phát sinh phôi được nuôi cấy trong môi trường có chứa muối NaCl với nồng độ thay đổi từ 50 – 300 mM. Sau 2 tuần nuôi cấy, chúng tôi xác định được ngưỡng chịu mặn của mô sẹo có khả năng sinh phôi cây Cọc rào là 150 mM. Nồng độ muối NaCl càng cao thì tỷ lệ sống của mô sẹo giảm dần và đạt giá trị cao nhất là 78,67% tại nồng độ 50 mM NaCl. Khi chuyển mô sẹo sang môi trường phát sinh phôi có chứa muối NaCl với nồng độ thay đổi, chúng tôi thấy ở nồng độ muối NaCl 50 – 100 mM không ảnh hưởng đến khả năng sinh trưởng và phát sinh phôi, đặc biệt là tại nồng độ 50 mM NaCl giúp kích thích sự hình thành phôi từ mô sẹo với tỷ lệ hình thành phôi đạt 83,33%. Ngược lại, nồng độ từ 150 mM NaCl gây ức chế quá trình hình thành phôi soma từ mô sẹo. Tiếp tục khảo sát ảnh hưởng của muối đến khả năng phát triển và nảy mầm của phôi soma. Ghi nhận kết quả sau 4 tuần nuôi cấy phôi soma trong môi trường MS có bổ sung 0.07 mg/l spermidin, tại nồng độ 50 – 100 mM NaCl khả năng hình thành phôi tốt và phôi phát triển qua các giai đoạn phôi hình cầu, hình tim, hình thủy lôi và hình lá mầm. Đặc biệt ở nồng độ 50 mM số lượng phôi lá mầm đạt giá trị cao với 13,33 phôi. Nồng độ muối NaCl 150 mM chỉ xuất hiện phôi hình cầu trong suốt thời gian nuôi cấy. Quá trình giải phẫu hình thái phôi và sử dụng thuốc nhuộm 2 màu carmin – iod và acridine orange đã cho thấy rõ hơn về cấu trúc mô sẹo có khả năng sinh phôi và phôi hình thái.

Embryo development and corresponding factors affecting in vitro germination of Cymbidium faberi × C. sinense hybrid seeds

A better understanding of embryo development would provide insights into seed quality and subsequent germination events in the interspecific hybridization of Cymbidium faberi ?Jiepeimei? ? C. sinense ?Qijianheimo?. At the mature stage, 26.1% of the ovules were abnormal. Most of the hybrid embryos could develop normally. Abortions mainly occurred at the zygote (9.5%) and 2-4-celled embryo (15.1%) stages. No germination was observed at 90 and 105 days after pollination (DAP), when the embryo was at the early globular stage, with abundant organelles but no storage materials. During 110-130 DAP, the globular embryo was formed and the starch grains began to accumulate in plastids. The hybrid seeds collected at 120 DAP showed initiation of germination. Germination significantly increased at 135 DAP and was maximal at 150 DAP, during which period the hybrid embryos developed into the late globular stage. The storage materials, i.e. lipid and protein bodies, began to accumulate and the filamentary structures derived from suspensor cells still persisted. After the seeds matured (160 DAP), the germination percentage declined sharply. Safranin staining revealed that the outer seed coat was totally cuticularized and the inner seed coat appeared as a cuticle layer enclosing the embryo proper tightly, which may be the main factor inhibiting the subsequent germination of hybrid seeds. In conclusion, 150 DAP should be the opportune time for the in vitro germination of C. faberi ?Jiepeimei? ? C. sinense ?Qijianheimo? hybrid seeds.

Secondary multiplication of somatic embryos in banana (Musa spp. AAA) in semisolid medium: effect of the culture vessel type and sterilization method

The objective of this work was to evaluate the influence of the type of culture vessel, and sterilization method on secondary multiplication of somatic embryos of the banana cultivar Grande naine (AAA) in semisolid culture medium. From 200 µl of embryogenic cell aggregates, somatic embryos in the globular stage were formed, which were used in the experiments to compare the types of culture vessel (glass and plastic) and the form of sterilization – humid heat (autoclave) and chemical (Vitrofural®). The evaluations of the number of somatic embryos that were multiplied were done at 15 and 20 days of culture. The results obtained with respect to the multiplication phase were highly promising, 40.25 and 80.15 ± 5.0 somatic embryos at 15 and 20 days of culture in glass vessel with plastic cover and using the autoclave as the sterilization method. The results presented in this work open the door for the use of this phase in the mass propagation protocol of this crop via somatic embryogenesis.

A cutin fluorescence pattern in developing embryos of some angiosperms

A cuticle visualized by auramine O fluorescence appears on the developing embryos of 9 species belonging to <em>Cruciferae</em>, <em>Caryophyllaceae</em>, <em>Plantaginaceae</em>, <em>Linaceae</em> and <em>Papilionaceae</em>. In the investigated species the formation and extent of fluorescing and non-fluorescing embryonic areas follow a similar pattern. At first the cutin fluorescing layer is formed on the apical part of the proembryo without delimited protoderm. This layer extends and at the late globular stage envelops the embryo proper, except for a cell adjoining the suspensor. Fluorescing cutin persists during the heart stage but disappears from the torpedo embryo. During these stages there is no cutine fluorescence on suspensorial cells. Continuous cutin fluorescence appears again on the surface of the whole embryo by the late torpedo stage. Then fluorescence disappears from the radicular part of U-shaped embryos, but persists on the shoot apex, cotyledons and at least on the upper part of hypocotyl. It is assumed that polarization and nutrition of the embryo may be influenced by cuticular changes.

Analysis of poly(A) + RNA distribution during maize somatic embryogenesis using digoxigenated oligo-dT probes

The pattern of total transcription activity in terms of steady state levels of poly(A)+ containing mRNA during callus initiation and somatic embryogenesis in a high (A188) and a low (A632) embryogenic line of maize was analyzed using digoxigenin labelled oligo-dT probes. A gradual increase and a preferential accumulation of label was observed in both lines, differing temporally up to 4 days in culture. In the A188 line of maize the callus gave rise to somatic embryos. The globular embryos showed less label than the callus; this labelling was mostly present in the basal part of the embryos. At a later stage upper embryogenic and lower non-embryogenic layers were observed in the A188 callus, showing conspicuous differences in the amount of label. In the late globular stage the poly(A)+ RNA signals were seen all over the embryo but at the junction of the suspensor and the callus tissue no label was observed.