Somatic Embryogenesis Protocol Research Articles

Choosing an appropriate somatic embryogenesis medium of carrot (Daucus carota L.) by data mining technology

IntroductionDeveloping somatic embryogenesis is one of the main steps in successful in vitro propagation and gene transformation in the carrot. However, somatic embryogenesis is influenced by different intrinsic (genetics, genotype, and explant) and extrinsic (e.g., plant growth regulators (PGRs), medium composition, and gelling agent) factors which cause challenges in developing the somatic embryogenesis protocol. Therefore, optimizing somatic embryogenesis is a tedious, time-consuming, and costly process. Novel data mining approaches through a hybrid of artificial neural networks (ANNs) and optimization algorithms can facilitate modeling and optimizing in vitro culture processes and thereby reduce large experimental treatments and combinations. Carrot is a model plant in genetic engineering works and recombinant drugs, and therefore it is an important plant in research works. Also, in this research, for the first time, embryogenesis in carrot (Daucus carota L.) using Genetic algorithm (GA) and data mining technology has been reviewed and analyzed.Materials and methodsIn the current study, data mining approach through multilayer perceptron (MLP) and radial basis function (RBF) as two well-known ANNs were employed to model and predict embryogenic callus production in carrot based on eight input variables including carrot cultivars, agar, magnesium sulfate (MgSO4), calcium dichloride (CaCl2), manganese (II) sulfate (MnSO4), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and kinetin (KIN). To confirm the reliability and accuracy of the developed model, the result obtained from RBF-GA model were tested in the laboratory.ResultsThe results showed that RBF had better prediction efficiency than MLP. Then, the developed model was linked to a genetic algorithm (GA) to optimize the system. To confirm the reliability and accuracy of the developed model, the result of RBF-GA was experimentally tested in the lab as a validation experiment. The result showed that there was no significant difference between the predicted optimized result and the experimental result.ConclutionsGenerally, the results of this study suggest that data mining through RBF-GA can be considered as a robust approach, besides experimental methods, to model and optimize in vitro culture systems. According to the RBF-GA result, the highest somatic embryogenesis rate (62.5%) can be obtained from Nantes improved cultivar cultured on medium containing 195.23 mg/l MgSO4, 330.07 mg/l CaCl2, 18.3 mg/l MnSO4, 0.46 mg/l 2,4- D, 0.03 mg/l BAP, and 0.88 mg/l KIN. These results were also confirmed in the laboratory

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.

Exploring somatic embryogenesis in Ajuga multiflora Bunge: Profiling lipophilic metabolites via HPLC, GC-FID, and GCMS– analysis

The present study aims to assess the effect of different concentrations of auxins and combinations of plant growth regulators (PGRs), specifically auxin and cytokinin, on somatic embryogenesis in Ajuga multiflora. We also aim to compare the lipophilic metabolite profiles, such as carotenoids (CAs), fatty acids (FAs), phytosterols (PSs), and tocopherols (TPs), in globular somatic embryos (SEs), germinated SEs, and shoots of SEs derived plantlets. This assessment is carried out utilizing high performance liquid chromatography, gas chromatography-flame-ionization detection, and gas chromatography–mass spectrometry techniques. The results reveal that the application of 2,4-dichlorophenoxyacetic acid (2,4-D) had the most significant impact on somatic embryogenesis, inducing both SE initiation (34.4%) and the number of SEs (3.5) per explant. Among the explants used, leaf explants were found to be the most effective for SE induction (SEI). Additionally, the study explores the combined effect of 2,4-D and cytokinins (N6-benzyladenine, N6-BA, or 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, TDZ) on somatic embryogenesis, highlighting optimal conditions for SEI percentages and the number of SEs per explant. The maximum SE induction rate (100%) and the number of SEs (45.9) per leaf explant were obtained on an SEI medium with 30 µM of 2,4-D and 2.3 µM of TDZ. The maturation and conversion of SEs were also examined, emphasizing the role of GA3 in achieving the highest conversion percentage (94.0 ± 2.7 at 0.8 µM GA3). Furthermore, the study details the biochemical composition during various stages of somatic embryogenesis, including CAs, TPs, PSs, and FAs. Globular SEs displayed comparatively lower concentrations of CAs than their germinated counterparts and shoots from SE-derived plantlets, while the total PSs content remained relatively consistent across all stages. Conversely, fatty acids tended to accumulate more in globular SEs, with the exception of α-Linolenic acid. These findings provide valuable insights into the optimization of somatic embryogenesis protocols in A. multiflora, with implications for plant tissue culture, regeneration and lipophilic metabolites production.

Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

Clonal breeding programs of Pinus maximinoi require the establishment of a robust somatic embryogenesis (SE) protocol to produce enough cell lines to accelerate the effective continuous deployment of elite planting stocks to research and commercial compartments. Somatic embryogenesis was induced from immature zygotic embryo explants enclosed in megagametophytes of P. maximinoi collected from two plantations located in different climatic conditions. Cones were collected during the winter months from July to August and the influence of seed family, cone collection date and culture medium formulation, with emphasis on the organic and inorganic nitrogen supply, were studied. Ammonium to nitrate molar ratios of 1:1 and 1:2 in modified Litvay’s medium (mLV) produced the highest numbers of extrusions, while a 1:4 ratio mostly produced unhealthy, non-embryogenic extrusions. The formation of a tissue showing a rapidly-proliferating, spiky morphotype was produced in a medium supplemented with 1.5 g/L of L-glutamine. Morphologically advanced cultures with nodular structures were produced in megagametophytes from both plantations in a 1:2 NH4+:NO3− medium regardless of L-glutamine supplementation levels. The optimal medium for P. maximinoi SE induction contained a 1:2 NH4+:NO3− molar ratio with 1.5 g/L L-glutamine. The synergy between the molar ratio of NH4+:NO3− and L-glutamine resulted in the highest numbers of extrusions. The overall inductive competence window for somatic embryogenic response in P. maximinoi was determined to be from the second week of July to the first week of August for both plantations. The “peak” period was in the fourth week of July 2022. The success of the SE technology in P. maximinoi seed families is determined by the optimal inductive competence window of the immature megagametophytes enclosing zygotic embryos and the chemical composition of the induction medium in terms of the ammonium to nitrate molar ratio and the concentration of the L-glutamine used.

Effect of Media and Photoperiod on Embryogenic and Organogenic Callus Induction from Mature Seed in Oryza sativa L.

Rice is the staple food for billions of people and a source of income for hundreds of millions. This food security crop production needs to be improved to face the projected demands of the exponentially augmenting world’s population and overcome biotic and abiotic stress in the fields. Optimized in vitro culture protocols are a prerequisite to genetic engineering which appears to be the best potential way to improve the rice plant. In this study, ‘Nerica 3’ and ‘Nerica L36’ seeds were in vitro culture tested into 4 media (m1-4), in dark and photoperiod environments. The percentage of callus induction was calculated and callus weight was recorded. Results show that callus induction was influenced by variety × environment and medium × environment interactions, with a strong influence of the environment used. ‘Nerica 3’ showed the highest mean (88.25%) callus induction after six weeks of incubation on different media. m1 and m2 media showed greater mean callus induction (more than 86%). Higher callus induction came from m1 (82% and 91%) and m2 (81% and 93%) media, while lower rates came from m3 (73%) and m4 (69%) media for embryogenic and organogenic calli respectively. Culturing the explant in dark environment to produce embryogenic callus, resulted in greater callus weight for ‘Nerica 3’ (1.03 g) and ‘Nerica L36’ (0.79 g). This study is a contribution to the rice plant genetic improvement by proposing protocols for somatic embryogenesis of two Nerica rice varieties.

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

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.

In vitro induced cold memory fails to enhance embryogenic cultures development in Araucaria angustifolia.

This research aimed to study how exposing somatic embryos of Araucaria angustifolia to cold temperatures in vitro could enhance somatic embryogenesis. The somatic embryos were induced from immature zygotic embryos, grown for 120 days, and then subjected to 4.5°C for 0, 2, 4, 8, or 16 days. After the cold treatment, the embryogenic cultures were returned to 25±2°C for 20 days and examined using light microscopy and cytochemistry. It was found that the embryogenic cultures at the PEM III stage did not show further development, and the growth of the embryogenic masses was negatively affected in the longer-duration cold treatment. Overcoming these challenges in the somatic embryogenesis protocol for A. angustifolia requires further integrative investigations on epigenetic events driving the zygotic embryo development.

Somatic Embryogenesis of Representative Medicinal Trees in South America—Current Status

Human society is currently facing a growing demand for forest resources, which causes overexploitation and endangers biodiversity. In this regard, it is important to be aware that 10% of trees across the world are used in traditional and modern medicine. South America has the greatest diversity, with 40% of tree species in the world. The aim of our review consists of the assessment of the state of the art of micropropagation through somatic embryogenesis of representative medicinal trees in South America and of targeting the tree species that should be prioritized for conservation plans. From a total of 23,631 tree species from Central and South America, 31 are extinct, 7047 are threatened, and 1434 are possibly threatened. In this sense, in order to conserve them, various strategies are applied both in situ and ex situ. The application of in vitro multiplication protocols represents effective ways both in conservation and in the sustainable use of resources in order to obtain secondary metabolites of interest. Somatic embryogenesis is a well-known method in woody multiplication. According to the VOSviewer analyses, very few studies were available concerning aspects of somatic embryogenesis in medicinal trees. From the 10 representative species selected in our study, somatic embryogenesis protocols were established for 3 species, only for conservation purpose, not for secondary metabolites production. The development of protocols focused on obtaining secondary metabolites of medicinal trees will allow for the obtainment of valuable plant material as a non-invasive alternative.

Somatic Embryogenesis and Plant Regeneration from Stem Explants of Pomegranate

Plant regeneration through somatic embryogenesis provides a solution for maintaining and genetically improving crop or fruit varieties with desirable agronomic traits. For the fruit tree pomegranate (Punica granatum L.), despite some successful applications, the existing somatic embryogenesis protocols are limited by low availability of explants and susceptibility to browning. To address these problems, in this study, we developed an effective system for induction of high-vigor pomegranate somatic embryos derived from stem explants. The usage of stem explants breaks through the difficulty in obtaining material, thus making our system suitable for widespread commercial production. To enhance the performance of our system, we identified the optimal explants, subculture cycles and combination of basal media and plant growth regulators for each step. The results showed that inoculating stem explants onto a Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L 1-naphthaleneacetic acid (NAA) achieved the best induction rate and growth status of pomegranate calli (induction rate = ~72%), and MS medium containing 0.5 mg/L 6-BA and 1.0 mg/L NAA was the optimal condition for the induction of embryogenic calli and somatic embryos (induction rate = ~74% and 79%, respectively). The optimal subculture period for embryogenic calli was found to be 30–35 days. Strong roots were then induced in the developed somatic embryo seedlings, which survived and grew well after transplantation to the natural environment, indicating the good vitality of the induced pomegranate somatic embryos. Together, our system provides a solution to mass somatic embryo induction and plant regeneration of pomegranate and lays a foundation for future genetic transformation and bioengineering improvement of pomegranate with favorable agronomic traits.

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.).

Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance spectroscopy was used to analyze the primary metabolome of these distinct calli to elucidate possible differentiation mechanisms from the common genetic background callus. Standard multivariate analysis methods were then applied, and were complemented by univariate statistical methods to identify differentially expressed primary metabolites and related metabolic pathways. The results showed carbohydrate and lipid metabolism to be the most relevant in all the calli assayed, with most discriminant metabolites being fructose, glucose and to a lesser extent choline. The glycolytic rate was higher in embryogenic calli, which shows, overall, a higher rate of sugar catabolism and a different profile of phospholipids with a choline/ethanolamine analysis. In general, our results show that a distinct primary metabolome between embryogenic and non-embryogenic calli occurs and that intracellular levels of fructose and sucrose and the glucose to sucrose ratio seem to be good candidates as biochemical biomarkers of embryogenic competence.

Establishment of an Efficient Somatic Embryogenesis Protocol for Giant Reed (Arundo donax L.) and Multiplication of Obtained Shoots via Semi-Solid or Liquid Culture

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h.

Recipient Somatic Embryogenesis System Promotes CRISPR/Cas Genome Editing

Somatic embryogenesis (SE) is an important type of plant regeneration, which can be used as a recipient system of genome editing. It is vital to optimize the SE system to improve the mutated somatic cells regenerated into fertile plants after transformation. 2,4-D was used as inducer of somatic embryogenesis from mature seeds. Arabidopsis wild type Col-0 and ABA biosynthesis double mutant <i>nced6 / 9 </i>were grown in 15°C and 25°C respectively, to investigate the effect of sampled plants growth conditions on the SE rate of their seeds and to optimize SE protocol. On the other hand, construct Pbi121-U6-26-Cas9-sgRNA-ELF6-FastR was used for genome editing. The positive seeds after floral dip were separated into two parts: some for sowing and the rest seeds went though SE induction. The efficiency of gene editing was compared by sequencing. Our results show that the SE rate of <i>nced6 / 9</i> seeds from 25°C was significantly lower than that from 15°C. When transgenic positive seeds were sown directly, the target sequence was not found to be edited; However, when seeds went though somatic embryogenesis protocol, gene editing were found around PAM sequence, suggesting that SE system could improve gene editing after transformation. The sampled plants grown at low temperature (15°C) may affect SE ability of their seeds by ABA level. The mature seed somatic embryogenesis system can improve the efficiency of gene editing.

Application of Cold Storage and Short In Vitro Germination for Somatic Embryos of Pinus radiata and P. sylvestris.

Somatic embryogenesis (SE) is an advanced vegetative propagation technology that, when used in combination with breeding and cryopreservation, offers the forest industry a powerful tool for the deployment of elite genotypes. Germination and acclimatization are critical and cost-intensive phases in the production of somatic plants. The efficient conversion of somatic embryos into robust plants is a necessity if a propagation protocol is to be successfully adopted by the industry. In this work, these late phases of the SE protocol of two pine species were investigated. A shortened germination protocol and more controlled acclimatization were investigated for Pinus radiata, testing embryos from 18 embryogenic cell lines. A more simplified protocol, including a cold storage phase, was also compared among 10 of these cell lines. A shortened germination period and more controlled protocols significantly improved the acclimatization of somatic embryos directly from the lab to the glasshouse. When results for all cell lines were pooled, there were significant improvements in all growth characteristics (shoot height, root length, root collar diameter, and root quadrant score). When the more simplified protocol involving cold storage was tested, improvements were seen in the root architecture. For Pinus sylvestris, the late phases of somatic embryogenesis were investigated on seven cell lines in a set of two trials (four to seven cell lines per trial). During the germination phase, a shortened and simplified in vitro period, a cold storage option and basal media were explored. Viable plants were obtained from all treatments. However, there is still the need to improve germination and related protocols together with growing regimes for Pinus sylvestris. The improvements to protocols presented here, particularly for Pinus radiata, result in greater survival and quality of somatic emblings, leading to reduced costs and increased confidence in the technology. Simplified protocols using a cold storage option show great promise and, with some further research, could lead to reductions in the cost of the technology.

Propagation of the Colombian genotype of cacao (Theobroma cacao L.) CNCh-12 by somatic embryogenesis

Cocoa production (Theobroma cacao L.) is essential globally and constitutes one of the leading export products for Colombia. Understanding the limitations faced by this crop in Latin American countries, it is required, among other aspects, to contribute to strengthening the first link in the production chain through efficient propagation methods and genetic improvement. Knowing that somatic embryogenesis is an alternative to conventional propagation and constitutes an obligatory step in a breeding platform, the objective of this work was to establish a somatic embryogenesis protocol until the plantlet acclimatization in the nursery for the regional genotype CNCh-12, a promising material with productivities higher than 2,000 kg/ha. Different protocols were evaluated, from callogenesis induction, through the expression of primary somatic embryos (PSE) followed by maturation and subsequent conversion to plantlet two types of explants (petal and staminode) and culture time (according to the stage). Additionally, the induction of secondary somatic embryos (SSE) was evaluated in two culture media (L and F). For CNCh-12, the petal was found as an appropriate explant, with a minimum time of 15 days in induction for PSE formation, without difference between the culture media F and L (22 average embryos). Embryo maturation was achieved in medium F after 30 days, followed by an additional 30 days for conversion to plantlet (52.83%). The concentration of salts to increase the conversion and development of the embryos was 1/5 of that used in F. The highest number of SSE was in the L medium. Finally, the ex-vitro adaptation was achieved when the plants were planted in 50:50 sand-coconut fiber and moistened weekly with Hoagland's solution (1:10). Keywords: Cacao, petals, in vitro propagation, plant growth regulators, somatic embryogenesis.

Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin.

Kinnow (Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I-VII). The ovules of stage III fruits (>21-25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L-1 and malt extract (ME) 1,000 mg L-1. Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA3) 2.0 mg L-1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L-1 + spermidine 100 mg L-1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.

Phytochemical Composition and Biological Activities of Extracts from Early, Mature, and Germinated Somatic Embryos of Cotyledon orbiculata L.

Cotyledon orbiculata L. (Crassulaceae)-round-leafed navelwort-is used worldwide as a potted ornamental plant, and it is also used in South African traditional medicine. The current work aims to assess the influence of plant growth regulators (PGR) on somatic embryogenesis (SE) in C. orbiculata; compare the metabolite profile in early, mature, and germinated somatic embryos (SoEs) by utilizing ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); and determine the antioxidant and enzyme inhibitory potentials of SoEs. A maximum SoE induction rate of 97.2% and a mean number of SoEs per C. orbiculata leaf explant of 35.8 were achieved on Murashige and Skoog (MS) medium with 25 µM 2,4-Dichlorophenoxyacetic acid and 2.2 µM 1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea. The globular SoEs were found to mature and germinate best on MS medium with gibberellic acid (4 µM). The germinated SoE extract had the highest amounts of both total phenolics (32.90 mg gallic acid equivalent/g extract) and flavonoids (1.45 mg rutin equivalent/g extract). Phytochemical evaluation of SoE extracts by UHPLC-MS/MS reveals the presence of three new compounds in mature and germinated SoEs. Among the SoE extracts tested, germinated SoE extract exhibited the most potent antioxidant activity, followed by early and mature somatic embryos. The mature SoE extract showed the best acetylcholinesterase inhibitory activity. The SE protocol established for C. orbiculata can be used for the production of biologically active compounds, mass multiplication, and conservation of this important species.

In Vitro Propagation by Axillary Shoot Culture and Somatic Embryogenesis of Daucus carota L. subsp. sativus, ‘Polignano’ Landrace, for Biodiversity Conservation Purposes

Carrots are a multi-purpose vegetable; hence, they have become a very popular consumers’ choice, also thanks to their nutritional value with health benefits. Several varieties and landraces exist of carrot (Daucus carota L. ssp. sativus). In Apulia (Italy), for example, a multicolored carrot landrace is present, the Polignano carrot (PC), threatened by the risk of genetic erosion. With the purpose of verifying the possibility of using in vitro culture for ex situ conservation purposes of PC, a rapid and reliable protocol for micropropagation and a highly efficient somatic embryogenesis protocol have been set up. For micropropagation studies, axillary buds from taproot were used as initial explants. Supplementation on basal medium (BM, a Murashige–Skoog modified medium) with low cytokinine (6-benzyladenine, BA, 0.22 μM) dosage was enough to obtain a good multiplication rate, also depending by the PC color. Rooting of proliferated shoots occurred easily in hormone-free medium and acclimatization was successfully reached in 6 weeks. Somatic embryos were formed from embryogenic callus induced by taproot sections placed on 2,4-dichlorophenoxyacetic acid (2,4-D)-added medium. After in vitro conversion in hormone-free medium, somatic embryos were also successfully acclimatized. Both microplants and somatic embryos produced true-to-type taproots (multicolored carrots), demonstrating the suitability of in vitro techniques for biodiversity conservation purposes.

Somatic embryogenesis in Abies nebrodensis, an endangered Sicilian fir

Somatic embryogenesis, as a promising biotechnological tool for many conifer trees, has never been applied for the Abies nebrodensis species. Although all the encouraging results previously obtained by the EU LIFE (European LIFE program) funded projects in over ten years, the critically endangered Sicilian fir remains alarmingly close to extinction. In this study, we reported the first protocol of somatic embryogenesis obtained from mature zygotic embryos of the Abies nebrodensis. Seeds from Abies adult trees with specific identification numbers (IN) were collected and full seeds were identified by X-ray. Different experiments were carried out for callus initiation, from both zygotic immature and mature embryos, testing different culture media. The immature embryos did not give embryogenic tissue (ET). Embryogenic callus (EC) was successfully induced from mature embryos with variable frequencies (0–40%). Schenk and Hilderbrandt (SH) was the most suitable initiation medium where the obtained callus initiation rate reached up to 40% for IN7 (first experiment). 6-benzylaminopurine (BAP) showed to be essential to induce EC (second experiment). IN8 presented the highest callus initiation rate (40%) among all tested donor trees, whereas IN13 recorded the lowest rate with 4% (third experiment). ET maturation from each singular embryo of IN7, IN8, IN10 and IN21 was successfully achieved in SH medium containing 37,83 µM abscisic acid (ABA), 8% of polyethylene glycol (PEG-4000) and 4% maltose. The encapsulation technology was assessed on the obtained ET and its proliferation was observed after encapsulation.