Germination Of Somatic Embryos Research Articles

Endogenous phytohormone analysis during sequential stages of somatic embryogenesis in Musa AAA cv. Grand Naine (subgroup Cavendish)

Abstract This is the first initiative study to provide quantified levels of four endogenous phytohormones during the induction, regeneration, maturation and germination stages of somatic embryogenesis (SE) in banana, using immature male bud explants, in the popular commercial triploid cultivar Grand Naine (AAA). Phytohormones are known to play a major role in the regulation of SE and most protocols have been developed for various cultivars of banana following empirical approaches. Although, several reports are available with regard to the impact of growth regulators exogenously supplemented to the media, the vital role of endogenous growth hormones in various developmental stages of SE remain unclear in banana. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used to analyze endogenous status of indole acetic acid (IAA), zeatin, gibberellic acid (GA) and abscisic acid (ABA). Results from this study revealed that endogenous IAA and GA along with exogenous auxin play a major role in the induction of embryogenic competence of male bud explants. The levels of endogenous IAA and zeatin were found to be higher in embryogenic calli than non-embryogenic calli, although three different types of exogenous auxins were supplemented in callogenesis medium, but not cytokinin. Higher level of ABA and GA was observed in early and late developmental stages of somatic embryo, respectively. GA also played a predominant role in germination of somatic embryos. These findings will be applied for improvement of SE protocol in recalcitrant banana cultivars by suitably altering or supplementing exogenous growth hormones.

Optimization of somatic embryogenesis in Euterpe edulis Martius using auxin analogs and atomic force microscopy

Euterpe edulis Martius is an endangered species of the Atlantic Forest, whose fruits have high antioxidant potential, and propagated exclusively by seeds. The present study assessed the ability of different auxin inducers and picloram analogs to trigger somatic embryogenesis in E. edulis. Immature seeds were harvested, and their zygotic embryos were excised and grown in MS culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram at 150, 300, 450, 600 µM. The activity of picloram analogs triclopyr and clopyralid was evaluated in semisolid MS medium. At maturation and germination, picloram-derived calli and somatic embryos isolated from triclopyr-grown cultures were first transferred to pre-maturation medium and, after 30 days, to basal MS or MS medium supplemented with either 5 µM abscisic acid or 0.53 µM 1-naphthaleneacetic acid plus 12.3 µM 2-isopentenyladenine. Finally, somatic embryos with root protrusions were transferred to MS medium devoid of sucrose for 30 days and then acclimatized ex vitro. Scanning, transmission, and atomic force microscopy revealed that picloram was superior to 2,4-D but less effective than triclopyr (100 µM) in inducing embryogenesis. Maturation and germination of somatic embryos in E. edulis can be maximized by 5 µM abscisic acid, and selecting calli via atomic force microscopy.

Plant Regeneration of Agave cupreata by Somatic Embryogenesis in a Temporary Immersion System with Silver Nanoparticles.

Somatic embryogenesis in Agave genus has been induced; however, it is desirable to increase the rate of growth to get a more efficient propagation system. In this chapter, we present in detailed a protocol for somatic embryogenesis in Agave cupreata and the use of silver nanoparticles in a temporary immersion system. This is an efficient method that can be used commercially to improve the production and germination of somatic embryos.

Suplementação do silício aumenta a regeneração in vitro de embriões somáticos de palma de óleo (Elaeis guineensis Jacq.)

ABSTRACT The use of silicates in plant tissue culture has resulted in positive effects regarding the morphological, anatomical and physiological characteristics of in vitro plants. However, biological responses to this mineral are dose and genotype dependent. The objective of the present study was to evaluate the influence of silicon supplementation on the somatic embryogenesis of oil palm (Elaeis guineensis Jacq.). The study was carried out in three stages of the cloning process, namely: Regeneration (i) and germination (ii) of somatic embryos and during ex vitro acclimatization (iii) of regenerated plantlets. The culture media were supplemented with four doses (0.0, 0.5, 1.0 and 1.5 g L-1) of calcium silicate (CaSiO3). The resulting plantlets were transplanted and acclimatized in a greenhouse. The regression analysis showed that the dose of 0.63g L-1 of CaSiO3 induced a higher rate of regeneration, which would allow a 58% increase in the number of somatic embryos formed. However, the germination of the plantlets, as well as their morphometric characters, were not affected by the different doses of CaSiO3, although the presence of this compound promoted an increase in the thickness of the epidermis and a reduction in the amount of epicuticular wax. Overall, silicon does contribute to the cloning of E. guineensis.

Effect of dark incubation in germination of indirect date palm somatic embryos and conversion into plantlets

Effect of dark incubation in germination of indirect date palm somatic embryos and conversion into plantlets

Tissue Culture of Four Varieties of Date Palm Grown in Indonesia

In vitro propagation of date palms adaptive to Indonesian agroclimate is one of the solutions for providing superior date palm planting material. This study aimed to induce embryogenic calli and regenerate the calli into plantlets of four varieties of date palm grown in Indonesia: three female varieties (Barhee, Ajwa, and Khalas) and one male variety (Jarvis). The materials used were offshoots of the date palm trees that had been flowered at the Jonggol Farm, Bogor, Indonesia. Four layers (layer 1 is the innermost layer and 4 is the outermost layer) of the offshoot were cultured on MMS media added with 100 mg/L 2,4-D combined with 1 and 3 mg/L 2-iP. The results showed that primary callus for var Bahee and Ajwa emerged at 12 weeks after culture. Barhee and Ajwa, and at 18 weeks for Jarvis and Khalas. In var. Jarvis and Khalas, calli were induced only in layer 1 cultured on the media with 1 and 3 mg/L 2-iP. In comparison, callus induction of var. Barhee date palm was successfully performed in layer 1 and 2 on the media with 1 mg/L 2-iP. On the contrary, callus induction in var. Ajwa was successful in layer 1 and layer 2, on the media supplemented both with 1 or 3 mg/L 2-iP. Germination of the date palm somatic embryos into shoots, using non-hormone media occurred successfully in var. Ajwa, Jarvis, and Khalas, but not in Barhee. Regeneration of the shoots into plantlets was carried out successfully only in var. Ajwa and Jarvis. In conclusion, in vitro propagation of date palm was successfully conducted in Jarvis and Ajwa varieties, while var Barhee was the most difficult among the four varieties.

Maduración y germinación de embriones somáticos de guayabo var. Enana Roja Cubana EEA-1840

Somatic embryogenesis is an alternative for the accelerated propagation of promising guava (Psidium guajava L.) materials of agronomic interest. However, low maturation and germination rates of somatic embryos are some of the aspects that limit its application in breeding programs for different purposes. In this sense, the effect of three concentrations (0, 1 and 1.5 mg.L-1) of abscisic acid (ABA) on embryo maturation and two concentrations of sucrose (3 and 5 %) and Murashige and Skoog (MS) macronutrients (50 and 100 %) on the germination of somatic embryos of guava var. Cuban Red Dwarf EEA-1840 were studied. After six weeks of culture, ABA had a negative effect on somatic embryo maturation in culture media supplemented with 1 or 1.5 mg.L-1, whereas 3.95 to 5.49 times more mature embryos were observed in the absence of ABA. Germination of somatic embryos was significantly improved when the concentration of macronutrient in the culture medium was reduced independently of the sucrose concentration [MS 50 % + 3 % of sucrose (73,3 %) and MS 50 % + 5 % de sucrose (55,0 %)]. It is concluded that the simplification of the culture media with reduction of the macroelements MS and sucrose to standard concentration favors the germination of mature embryos of guava var. Cuban Red Dwarf EEA-1840.

EVALUACIÓN DEL EFECTO DE LA PUTRESCINA EXÓGENA DURANTE LA MADURACIÓN Y GERMINACIÓN in vitro DE EMBRIONES SOMÁTICOS EN DOS GENOTIPOS DE AGAVE

<p><strong>Background.</strong> Agaves are an important economic source in industrial products, in the food and pharmaceutical industry, steroidal animal feed, ornamental plants and as a material for biofuels. Despite their economic importance, some species present problems in their conventional propagation, which makes their reproduction difficult and has caused some species to be in some risk category. Therefore, there is a need to develop efficient protocols for <em>in vitro</em> micropropagation via somatic embryogenesis. The positive effect of the application of exogenous putrescine on the maturation and germination of somatic embryos is known. <strong>Objective</strong>. To determine the effect of exogenous putrescine on the maturation and germination of somatic embryos of <em>A. cupreata</em> and <em>A. angustifolia</em>.<strong> Methodology</strong>. Two concentrations of putrescine (100 and 150 mg/L) and two controls, one positive and one negative, were evaluated in the somatic embryo maturation medium and later in the germination of somatic embryos. <strong>Results</strong>. Putrescine favors the maturation and germination of somatic embryos in both species. <strong>Implications</strong>. The results of this research contribute to a better understanding of the process of somatic embryogenesis and open the possibility of applying putrescine in the process of somatic embryogenesis at a commercial level. <strong>Conclusion</strong>. Exogenous putrescine in culture medium significantly improves the maturation and germination of somatic embryos in <em>Agave</em>.</p>

Plant-Derived Smoke and Karrikin 1 in Seed Priming and Seed Biotechnology.

Plant-derived smoke and smoke water (SW) can stimulate seed germination in numerous plants from fire-prone and fire-free areas, including cultivated plants and agricultural weeds. Smoke contains thousands of compounds; only several stimulants and inhibitors have been isolated from smoke. Among the six karrikins present in smoke, karrikin 1 (KAR1) seems to be key for the stimulating effect of smoke. The discovery and activity of highly diluted SW and KAR1 at extremely low concentrations (even at ca. 10-9 M) inducing seed germination of a wide array of horticultural and agricultural plants have created tremendous opportunities for the use of these factors in pre-sowing seed treatment through smoke- or KAR1-priming. This review presents examples of effects exerted by the two types of priming on seed germination and seedling emergence, growth, and development, as well as on the content of some compounds and enzyme activity. Seed biotechnology may involve both SW and KAR1. Some examples demonstrate that SW and/or KAR1 increased the efficiency of somatic embryogenesis, somatic embryo germination and conversion to plantlets. It is also possible to stimulate in vitro seed germination by SW, which allows to use in orchid propagation.

Pinus massoniana somatic embryo maturation, mycorrhization of regenerated plantlets and its resistance to Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major quarantine forest disease that poses a threat to various pine species, including Pinus massoniana (masson pine), worldwide. Breeding of PWN-resistant pine trees is an important approach to prevent the disease. To expedite the production of PWN-resistant P. massoniana accessions, we investigated the effects of maturation medium treatments on somatic embryo development, germination, survival, and rooting. Furthermore, we evaluated the mycorrhization and nematode resistance of regenerated plantlets. Abscisic acid was identified as the main factor affecting maturation, germination, and rooting of somatic embryos in P. massoniana, resulting in a maximum of 34.9 ± 9.4 somatic embryos per ml, 87.3 ± 9.1% germination rate, and 55.2 ± 29.3% rooting rate. Polyethylene glycol was identified as the main factor affecting the survival rate of somatic embryo plantlets, with a survival rate of up to 59.6 ± 6.8%, followed by abscisic acid. Ectomycorrhizal fungi inoculation with Pisolithus orientalis enhanced the shoot height of plantlets regenerated from embryogenic cell line (ECL) 20-1-7. Ectomycorrhizal fungi inoculation also improved the survival rate of plantlets during the acclimatization stage, with 85% of mycorrhized plantlets surviving four months after acclimatization in the greenhouse, compared with 37% non-mycorrhized plantlets. Following PWN inoculation, the wilting rate and the number of nematodes recovered from ECL 20-1-7 were lower than those recovered from ECL 20-1-4 and 20-1-16. The wilting ratios of mycorrhizal plantlets from all cell lines were significantly lower than those of non-mycorrhizal regenerated plantlets. This plantlet regeneration system and mycorrhization method could be used in the large-scale production of nematode-resistance plantlets and to study the interaction between nematode, pines, and mycorrhizal fungi.

A Stable and Efficient Genetic Transformation Method for Embryogenic Callus of Fraxinus mandshurica

Fraxinus mandshurica is a widely used greening and ornamental tree species. However, its genetic transformation system has been hampered by problems such as low transformation efficiency, among others, which can hinder research related to molecular breeding and the analysis of functional genes. Thus, in this study, a novel genetic transformation method for efficient transformation of the embryonic callus of Fraxinus mandshurica was investigated. The method was optimized in terms of factors such as antibiotics, infection solution concentrations, co-culture time, and somatic embryo maturation. The results indicated that the optimal antibiotic concentration was 10 mg·L−1 of hygromycin (Hyg). At this point, the callus proliferation multiple was only 0.12. The highest transformation efficiency was found to be 93.93% when the absorbance of the infection solution concentration at OD600 was 0.4. Interestingly, transformation efficiency was found to be highest (77.9%) at 48 h of co-culture, with a GUS staining rate of 88.23%. The medium for somatic embryo maturation of transformed callus was half-strength MS medium (MS 1/2) containing 60 g·L−1 polyethylene glycol, 1 mg·L−1 abscisic acid, 400 mg·L−1 casein enzymatic hydrolysate (CH), 20 g·L−1 sucrose, 1 g·L−1 activated charcoal, and 5 g·L−1 gellan gum. The medium for somatic embryo germination was MS ½, containing 0.2 mg·L−1 of N-(Phenylmethyl)-9H-purin-6-amine(6-BA) and 5.0 mg·L−1 of gibberellin (GA). These results are of significance for the verification of the gene function and future genetic improvement of Fraxinus mandshurica.

Efficient Micropropagation of Genetically Stable Panax ginseng Meyer by Somatic Embryogenesis

Panax ginseng Meyer is a valuable medicinal crop. However, the species’ propagation is limited by its long reproductive cycle and low seed yield. The present study focused on P. ginseng plant regeneration via somatic embryogenesis and evaluated the genetic stability of regenerated plantlets. We assessed the effects of carbon source type and concentration on somatic embryo induction, maturation, and germination. Somatic embryogenesis was optimal in Murashige and Skoog (MS) medium supplemented with 5% sucrose; however, maturation peaked in 1/2 MS containing low concentrations of sucrose ranging from 1 to 2%. Germination and plant regeneration were optimal in germination medium supplemented with 2% sucrose based on high germination rates, efficient plantlet production, and balanced growth characteristics. Molecular marker analysis suggested that the genetic fidelity of the regenerated plants was comparable with that of the control. High-performance liquid chromatography (HPLC) analysis showed that in vitro-grown roots (IGRs) accumulated more ginsenoside than those of the control, but the ginsenoside content of 2 year old IGRs was similar to that of the controls after acclimatization. Our study provides valuable insights into the optimization of genetically stable micropropagation and could promote the distribution of superior P. ginseng cultivars with high product yields and quality.

Multi-strategy approach towards optimization of maturation and germination in radiata pine somatic embryogenesis

Somatic embryogenesis is a powerful technique for forestry improvement programs when implementing multi-varietal forestry. However, it still faces some bottlenecks to be efficient in many forestry species. In this work we have studied the effect of some physico-chemical modifications at proliferation, maturation and germination stages of Pinus radiata D. Don somatic embryogenesis, as well as the impact of this modifications on plant conversion, survival, and on the morphology and biochemical profile (carbohydrate and amino acid analysis) of the resulting plantlets. Given the long culture period needed for maturation and germination in radiata pine, an increase of the plant yield could be very beneficial for its commercial application. Following these premises and based on the experiments performed, a preculture of 14 days without plant growth regulators before maturation seems to be beneficial for the development and germination of somatic embryos. Before germination, a cold storage at 4 °C had no detrimental effect and even increased plant conversion in some embryogenic cell lines (85% of germination and 64.6% of planted explants). During germination, supplementation of the culture medium with glutamine and a reduction of the sucrose content had a significant effect on germination (88%) and doubled the percentage of planted explants. Similarly, germinants were demonstrated to be influenced by the light source: fluorescent light enhanced root formation, whereas blue LED light increased the shoot height of somatic plants. Moreover, the highest plants showed lower contents of arginine, asparagine and total carbohydrates. Modification of the physico-chemical environment before or during somatic embryo maturation and germination improves the success of the process.

In vitro somatic embryo productions from Curculigo orchioides derived gold nanoparticles: Synthesis, characterization, its biomedical applications, and their eco-friendly approaches to degradation of methylene blue under solar light irradiations

Curculigo orchioides rhizome explants were employed to develop a rapid and effective strategy for increased plant regeneration using somatic embryogenesis. Direct somatic embryo development was shown on rhizome explants cultivated on Murashige and Skoog (MS) making with 2, 4-D (1.0–3.0 mg/L). Rhizome explants cultivated on MS media supplemented with 2.0 mg/L 2, 4-D yielded the highest frequency of embryogenesis (87.5%) and the maximum number of somatic embryos (1596.7/explant). Somatic embryo germination was accomplished using MS media with 2.0 mg/L 6-benzylaminopurine (BAP). With an 80% survival rate, the germination plantlets were acclimated in the greenhouse. The current study is the first evidence of the efficacy of in vitro-produced plants and C. orchioides somatic embryo callus cultures of stable gold nanoparticles. The UV–Vis spectrophotometric absorbance, at 510 nm, revealed the absorption spectra of the AuNPs. The FT-IR revealed functional groups and reaction processes in green AuNP formation. High-resolution transmission electron microscopy (HR-TEM) was used to assess the surface morphology and structure of the AuNPs after their elemental composition was determined using a dispersive energy X-ray (EDAX) spectrum. The average size of AuNPs was around 35 nm in diameter. The crystalline nature of the AuNPs was investigated by X-ray diffraction (XRD). The highest growth inhibition was found for C. orcthioides against Klebsiella pneumoniae (17.5 mm) and Serratia marcescens (16.5 mm). The AuNPs exhibited antioxidant activity against free radicals such as DPPH and ABTS. Furthermore, the cytotoxicity of AuNPs was assessed, and inhibitory concentration (IC50) was 20 μg/mL and 80 μg/mL for breast carcinoma (MDA-MB-231) and Vero cell lines. The degradation of methylene blue measures the photocatalytic activity of the manufactured AuNPs when subjected to visible sunlight (MB). Thus, the result showed a maximum degradation efficiency of MB (84%).

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60days after being transferred to auxins and cytokinin (1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg Kinetin L-1) along with 200mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC–MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g−1 dry wt) and lupeol (1.232 µg g−1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13353-022-00727-7.

Impact of cultural conditions on germination of olive (Olea europaea L.) somatic embryos and plantlets development from the Algerian cultivar Chemlal

The in vitro propagation techniques are currently a commercial alternative for the production of plants with good quality in several plant species, including the olive tree (Olea europaea L.). Somatic embryogenesis is the process practically used for the application of several biotechnological tools of improvement and in vitro plant regeneration via the germination of somatic embryos. Our work aims to evaluate the effect of the chemical and hormonal composition of the culture medium on the germination of olive somatic embryos (cv. Chemlal) as well as the micropropagation of the obtained plantlets before their acclimatization to natural conditions. The results indicated that the production of olive plants by somatic embryogenesis depends strongly on the genotype of the somatic embryos (cell line) and more on the culture conditions, particularly the presence of growth regulators. Indeed, a solid OM medium supplemented with hormones (BA and IBA) permitted an advanced root emergence and germination allowing the production of well-developed plants with several leaves. In addition, an OM medium supplemented with Zeatin and IBA allowed better reactivity of micro-cuttings producing well-developed shoots with several emitted roots which facilitates their further acclimatization to natural conditions.

Integrated Lipidomic and Transcriptomic Analysis Reveals Phospholipid Changes in Somatic Embryos of Picea asperata in Response to Partial Desiccation.

Partial desiccation treatment (PDT) is an effective technology for promoting the germination and conversion of conifer somatic embryos (SEs). PDT, as a drought stress, induces intensive physiological responses in phospholipid metabolism, which are not well understood in the conifer SEs. Here, we integrated lipidomics, transcriptomics and proteomics analyses to reveal the molecular basis of lipid remodeling under PDT in Picea asperata SEs. Among the 82 lipid molecular species determined by mass spectrometry, phosphatidic acid (PA) had a significant effect after PDT and was the most critical lipid in the response to PDT. The transcriptomics results showed that multiple transcripts in the glycerolipid and glycerophospholipid metabolism pathways were differentially expressed, and these included five PLDα1 transcripts that catalyze the conversion of phosphatidylcholine (PC) to PA. Furthermore, the enzyme activity of this phospholipase D (PLD) was significantly enhanced in response to PDT, and PDT also significantly increased the protein level of PLDα1 (MA_10436582g0020). In addition, PA is a key factor in gibberellin, abscisic acid and ethylene signal transduction. One GDI1, one DELLA, three ABI1s, two SnRK2s, one CTR and 12 ERFs showed significantly differential expression between SEs before and after PDT in this study. Our data suggest that the observed increases in the PA contents might result from the activation of PLDα by PDT. PA not only affects the physical and chemical properties of the cell membrane but also participates in plant hormone signal transduction. Our work provides novel insight into the molecular mechanism through which PDT promotes the germination of SEs of coniferous tree species and fills the gap in the understanding of the mechanism of somatic embryo lipid remodeling in response to PDT.

Application of somatic embryogenesis for development of emerald ash borer-resistant white ash and green ash varietals.

Emerald ash borer (Agrilus planipennis; EAB) has devastated populations of ash (Fraxinus spp.) trees in dozens of U.S. states and Canada over the past few decades. The continued survival of scattered ash trees known as “lingering ash” in heavily infested natural stands, however, offers evidence of genetic resistance or tolerance to EAB. These surviving or “lingering” ash individuals may form the basis for reforestation programs in EAB-impacted areas, and clonal mass-propagation of these genotypes can help accelerate these efforts. Between 2013 and 2018, we initiated embryogenic cultures by culturing immature zygotic embryos from open-pollinated (OP) seeds collected from several surviving white ash and green ash trees in Michigan and Pennsylvania. In addition, in 2018, we initiated cultures from crosses made between lingering green ash parents from the USDA Forest Service ash breeding program in Ohio. Somatic embryos were produced by growing cultures in liquid suspension, followed by fractionation and plating on semisolid medium to produce developmentally synchronous populations of somatic embryos. Somatic embryo germination and conversion were enhanced by a combination of pre-germination cold treatment and inclusion of activated charcoal and gibberellic acid in the germination medium. Ash somatic seedlings derived from OP explants grew rapidly following transfer to potting mix and somatic seedlings representing nine ash clones were acclimatized, grown in the greenhouse and planted in a preliminary field test, along with EAB-resistant Manchurian ash (F. mandshurica) and EAB-susceptible control seedlings. Somatic seedlings have now been produced from cultures that originated from seeds derived from the progeny of lingering green ash parents and an ex vitro germination protocol has shown some promise for accelerating early somatic seedling growth. Results of this research could provide the basis for scaled-up production of EAB-resistant ash varieties for seed orchard production for forest restoration and cultivar development for urban tree restoration.

An Improved and Simplified Propagation System for Pollen-Free Sugi (Cryptomeria japonica) via Somatic Embryogenesis.

Sugi (Japanese cedar, Cryptomeria japonica) is the most important forestry tree species in Japan, covering 44% of the total artificial forest area. Large amounts of pollen released from these forests each spring cause allergic reactions in approximately 40% of the population, which are a serious social and public health problem in Japan. As a countermeasure, there is an urgent need to reforest using male-sterile plants (MSPs; pollen-free plants); however, the production of MSPs via conventional methods is inefficient, time consuming, and requires considerable resources in terms of labor and space. In the present paper, we described an improved and simplified methodology for the efficient propagation of pollen-free Japanese cedar, combining the use of genetic markers (marker-assisted selection or marker-aided selection) for the early selection of male-sterile genotypes and the use of somatic embryogenesis (SE) for the clonal mass propagation of seedlings. We describe all the stages involved in the production process of somatic seedlings. Our results demonstrated that this methodology easily and efficiently produces MSPs with a discrimination rate of 100% in a short period of time. Production of 243.6 ± 163.6 cotyledonary embryos per plate, somatic embryo germination, and plantlet conversion frequencies of 87.1 ± 11.9% and 84.8 ± 12.6%, respectively, and a 77.6 ± 12.1% survival rate after ex vitro acclimatization was achieved. Moreover, we also describe an easy method for the collection of somatic embryos prior to germination, as well as an efficient and practical method for their storage at 5°C. Finally, a representative schedule for the propagation of pollen-free sugi somatic seedlings is presented as a reference for practical uses. This methodology will definitively help to accelerate the production of C. japonica MSPs across Japan.