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.     

<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.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

A clonal propagation system for Atlantic white cedar (<Emphasis Type="Italic">Chamaecyparis thyoides</Emphasis>) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l^?1 producing more embryos than 0 g l^?1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l^?1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.     

Image Processing and Artificial Neural Network-Based Models to Measure and Predict Physical Properties of Embryogenic Callus and Number of Somatic Embryos in Ajowan (<Emphasis Type="Italic">Trachyspermum ammi</Emphasis> (L.) Sprague)

Trachyspermum ammi (L.) Sprague (Ajowan) is an endangered medicinal plant with useful pharmaceutical properties. Ex situ conservation of this medicinal plant needs the development of an in vitro regeneration protocol using somatic embryogenesis. In the present study, a high-precision image-processing approach was successfully applied to measure physical properties of embryogenic callus. Explant age and the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), and sucrose were used as inputs, and an artificial intelligence technique was applied to predict physical properties of embryogenic callus, and the number of somatic embryos produced. Artificial neural network (ANN) models were tested to find the best combinations of input variables that affected output variables. The lower values of root mean square error, and mean absolute error, and the highest values of determination coefficient, were achieved when all four input variables were applied to predict the number of somatic embryos, the area of the callus, the perimeter of the callus, the Feret diameter of the callus, the roundness of the callus, and the true density of the callus in ANN models. The highest measured and predicted number of somatic embryos were achieved from the interaction of 15-d-old explants?×?1.5 mg L^?1 2,4-D?×?0.5 mg L^?1 Kin?×?2.5% (w/v) sucrose. Based on sensitivity analysis, the 2,4-D concentration was the most important component in the culture medium that affected the number of somatic embryos and physical properties of the embryogenic callus tissue.     

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.     

Somatic embryogenesis and plant regeneration in açaí palm (<Emphasis Type="Italic">Euterpe oleracea</Emphasis>)

An efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from immature zygotic embryos of açaí palm (Euterpe oleracea) has been developed. Embryogenic calli (ECs) were induced from immature zygotic embryos of açaí palm on Murashige and Skoog (MS) modified medium with 2,4-dichlorophenoxyacetic acid and picloram. Embryogenic frequency was dependent on auxin type and concentration. The optimal concentration of picloram for the high-frequency induction of embryogenic calli (72%) was 225 μM. ECs were then subcultured on a differentiation and maturation medium composed of MS modified medium with 2-isopentenyladenine and naphthaleneacetic acid with subcultures at 4-week intervals. SEs were converted to plants on MS modified medium with half-strength macro- and micronutrients, 20 g l^?1 sucrose, and 2.5 g l^?1 activated charcoal and gelled with 2.5 g l^?1 Phytagel. Detailed morpho anatomical changes during the different stages of somatic embryogenesis were characterized. The development of SEs was asynchronous, and ontogenic studies confirmed that the initial cell divisions occur in the epidermal and subepidermal regions of the zygotic embryos. Broad base attachment of SEs to the epidermis indicates the presence of a suspensor.     

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.     

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.     

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.     

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.     

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.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

     

<Emphasis Type="Italic">In vitro</Emphasis> germination of zygotic embryos excised from cryopreserved endocarps of queen palm (<Emphasis Type="Italic">Syagrus romanzoffiana</Emphasis> (Cham.) Glassman)

Queen palm (Syagrus romanzoffiana [Cham.] Glassman) is a palm species best known as an ornamental tree for urban landscaping, but recently, it has been evaluated as a potential crop for biofuel production. The objective of the present work was to establish a cryopreservation technique for queen palm to ensure long-term conservation of this species. The cryopreservation protocol consisted of direct immersion in liquid nitrogen (LN) of whole endocarps with water contents ranging from 5.5 to 10.9%, followed by slow thawing at room temperature (25 ± 2°C) excision and in vitro culture of zygotic embryos. Viability of zygotic embryos isolated from endocarps with different water contents was evaluated before (control) and after freezing in LN using in vitro culture on Woody Plant Medium (WPM) medium. Germination percentages of zygotic embryos isolated from endocarps stored in LN varied from 84 to 93%, whereas those isolated from controls ranged from 55 to 71%. Germination rates were significantly higher for zygotic embryos excised from cryopreserved endocarps. The water content of control or frozen endocarps did not have a significant effect on germination percentages of zygotic embryos. Zygotic embryos excised from endocarps following cryopreservation in liquid nitrogen developed into normal plantlets after in vitro culture. The technique tested is simple, efficient, and can be used in plant gene banks as a routine approach for long-term conservation of queen palm germplasm.