Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Somatic embryogenesis of a seedless sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbeck)

Somatic embryogenesis is an important in vitro technique used to obtain Citrus sinensis (L.) Osbeck (sweet orange) plantlets for conservation, sanitation, propagation, and breeding. The induction of somatic embryogenesis from adult tissues of sweet orange could be an alternative to in vitro organogenesis from epicotyl segments, especially in seedless cultivars, where the latter is not feasible. The aim of this study was to obtain plantlets from ovary-derived somatic embryos of sweet orange cv. ‘Washington Navel’, an important seedless cultivar for citrus fresh fruit production. The explants used were pistils from flower buds, pre-anthesis, from 20-y-old plants cultivated in the field. Forty plantlets from 47 somatic embryos were obtained, in vitro-grafted, and acclimatized in greenhouse conditions. Ploidy evaluation through flow cytometric analysis, as well as the results of target region amplification polymorphism (TRAP) molecular markers confirmed the somatic origin of embryos as genetically similar to donor plants. This technique could be used for obtaining embryogenic cell suspension cultures or regenerated plants from mature tissues other than seed-derived tissues, especially for seedless genotypes.     

In vitro clonal propagation and stable cryopreservation system for <Emphasis Type="Italic">Platycladus orientalis</Emphasis> via somatic embryogenesis

Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM (P?<?0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l^?1 abscisic acid (ABA), 50 g l^?1 maltose, and 100 g l^?1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells (P?=?0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm.     

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of <Emphasis Type="Italic">Carica papaya</Emphasis>

Efficient protocols for somatic embryogenesis of papaya (Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C. papaya. To study the effects of PEG on somatic embryogenesis of C. papaya, we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG (control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C. papaya somatic embryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.     

Somatic embryos cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> Rupr. in air-lift bioreactors for the production of biomass and resveratrol

Resveratrol are the most important bioactive compounds found in Vitis amurensis. In this study, a somatic embryo induction system for V. amurensis was established in air-lift bioreactors for the production of biomass and resveratrol. The somatic embryos biomass growth was low on solid medium (69.60 g L^?1) compared to in liquid medium in bioreactor (329.45 g L^?1). Bioreactor cultures were found to be superior compared with solid medium culture not only in terms of biomass but also resveratrol productivity. Various culture parameters, including culture method, inoculum density, carbon source, and organic compounds were optimized. An inoculum density of 20 g L^?1 embryogenic calli was found suitable for the accumulation of biomass and resveratrol production, whereas 10 g L^?1 embryogenic calli increased the amount of resveratrol per fresh weight in somatic embryos. For bioreactor culturing, sucrose was an optimum carbon source and 500 mg L^–1 casein hydrolysate acid was conducive to the biomass and resveratrol production. This result indicates that an efficient protocol for the large-scale production of resveratrol can be achieved by bioreactor culturing of V. amurensis somatic embryos and can be used as a source of medicinal raw materials.     

Peculiarities of somatic embryogenesis of long-term proliferating embryogenic cell lines of <Emphasis Type="Italic">Larix sibirica</Emphasis> in vitro

Morphogenesis and maturation of somatic embryos, ploidy, and genotyping of cell lines (CL) of embryogenic cultures of Larix sibirica Ledeb. in vitro were investigated during 2–6 years. It was revealed that from 2000 to 11103 globular somatic embryos were formed in proliferating CL. However, the ability of somatic embryos to the maturation and germination decreased. Cytogenetic study of embryonal-suspensor masses (ESM) of Larix sibirica demonstrated that cells of long-term cultivated cultures remained diploid. According to microsatellite analysis, proliferating CL of Siberian larch were characterized by weak allelic variability, and cell line 6 and cloned seedlings of this line were genetically stable and corresponded to the donor tree. Embryogenic cell lines composed the collection bank, which will be successfully used for plantation forest growing.     

Histological analysis reveals the formation of shoots rather than embryos in regenerating cultures of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

Eucalyptus globulus is an important species in international forestry in regions with Mediterranean climates and comprises 65?% of the plantation hardwood in Australia. Propagation by somatic embryogenesis would offer many advantages and its development has received much attention. Structures regenerating on explants from hypocotyls of mature zygotic embryos of E. globulus cultured on medium with NAA, reported previously to be effective for embryogenic regeneration, were analyzed morphologically and histologically to clarify their pathway of development. Analysis of series of sections revealed organogenic, rather than embryogenic, pathways of regeneration in this system. We show that protocols for propagation of E. globulus must be carefully evaluated by microscopic examination of adequate numbers of serial sections.     

Optimization of culturing conditions for production of somatic embryos and lignins of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill

Schisandra chinensis (Turcz.) Baill. is a valuable medicinal plant species increasingly used in phytotherapy worldwide. This study systematically detected the lignin content and production during somatic embryogenesis of S. chinensis. The effect of various culture parameters on biomass accumulation and lignin production were also examined to optimize the accumulation of lignins in SEs in bioreactors, including the culture method, inoculum density, aeration volume and photoperiod. An inoculum density of 20 g L^??1 embryogenic calli enhanced production of lignin, while 30 g L^??1 embryogenic calli increased the biomass of somatic embryos. During somatic embryo induction, an aeration volume of 0.2 vvm and photoperiod of 16 h day^??1 were found to be optimal for biomass accumulation and lignin production. An approximately threefold increase in the biomass production rate and a fourfold increase in the total lignin production rate in SEs were achieved in bioreactors than on solid medium. The present study indicated, therefore, that the culturing of S. chinensis somatic embryos in bioreactors is an effective method for the industrialized production of lignin in vitro.     

Developmental expression of the cucumber <Emphasis Type="Italic">Cs-XTH1</Emphasis> and <Emphasis Type="Italic">Cs-XTH3</Emphasis> genes,encoding xyloglucan endotransglucosylase/hydrolases,can be influenced by mechanical stimuli

The expression of two genes encoding xyloglucan endotransglucosylase/hydrolases (XTHs), Cs-XTH1 and Cs-XTH3, was upregulated during the onset of cucumber somatic embryogenesis. As a means of characterising the developmental regulation of these genes, the activity of the respective upstream regulatory regions was investigated in seedlings and somatic embryos of Arabidopsis thaliana and Cucumis sativus. GUS assays revealed that both genes are under developmental control. In addition, elevated promoter activity was found in the tension-bearing regions of the plant and in response to touch and wounding, which is consistent with the existence of numerous stress-related cis elements in the 5′-regulatory regions. In vivo xyloglucan endotransglucosylase (XET) action assays were performed to gain an overview on the role of XTHs during somatic embryogenesis. The highest XET action was observed in the external cell layers of somatic embryos in the cotyledonary region and in the presumptive region of peg formation. Based on the results, we propose a dual mechanism (one developmental and the second adaptive) for the regulation of Cs-XTH1 and Cs-XTH3 activity wherein the developmental pattern can be modified by mechanical stimuli.     

MIR166a Affects the Germination of Somatic Embryos in <Emphasis Type="Italic">Larixleptolepis</Emphasis> by Modulating IAA Biosynthesis and Signaling Genes

The somatic embryogenic regeneration system is an ideal model system to study the regulation of early developmental processes and morphogenesis in gymnosperms. We have previously generated five larch (Larix leptolepis) LaMIR166a overexpression cell lines. The germination rates of mature somatic embryos in transgenic and wild-type (WT) lines were calculated and the results showed that overexpression of the miR166a precursor (LaMIR166a) markedly enhanced germination, especially in the a-3, a-4, and a-5 lines. The relative expression of LaMIR166a and miR166a in the LaMIR166a overexpression lines was higher than in the WT control line during the germination process, whereas the expression levels of LaHDZ31–34 increased markedly throughout germination, potentially as a result of feedback regulation of miR166. The effect of miR166a on auxin biosynthesis and signaling genes was also studied. During germination, mRNA levels of Nitrilase (LaNIT), Auxin response factor1 (LaARF1), and LaARF2 were markedly higher in LaMIR166a overexpressing lines. These results indicated that indole-3-acetic acid (IAA) synthesis is required for germination in L. leptolepis. Further exogenous application of IAA at different concentrations showed that 2 mg L^?1 IAA clearly promoted germination, resulting in a 56% germination rate for L. leptolepis somatic embryos. This shows that IAA plays a vital role in controlling the germination ability of someatic embryos in L. leptolepis. Our results suggest that miR166a and LaHDZ31–34 have important roles in auxin biosynthesis and signaling during the germination of somatic embryos in L. leptolepis.     

Establishment of neomycin phosphotransferase II (<Emphasis Type="Italic">nptII</Emphasis>) selection for transformation of soybean somatic embryogenic cultures

Soybean transformation is limited by the lack of multiple efficient selectable marker systems. Biolistic transformation of somatic proliferative embryogenic cultures, one of the commonly used soybean transformation methods, relies largely on hygromycin phosphotransferase II (hptII) selection. The purpose of the present study was to establish another efficient selectable marker system to facilitate multiple gene transformations of soybean. We tested neomycin phosphotransferase II (nptII) that has been used successfully in cotyledonary node transformation, but with limited success in transformation of embryogenic cultures. Transgenic events were obtained using nptII with improved G418 selection without generating escapes. G418 selection required longer recovery and selection periods, and resulted in a lower efficiency of initial transformants compared to hygromycin selection. Six independent fertile transgenic plants were recovered using nptII and G418, a frequency similar to that obtained with hygromycin selection. Soybean embryogenic cultures co-transformed with the hptII and nptII markers showed resistance to both hygromycin B and G418, while regeneration and plant fertility were not adversely affected. The nptII will be useful as a second selectable marker for multiple gene transformations in basic and applied soybean research.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.