Production Of Somatic Embryos Research Articles

Temperature and LED lightning affect the regeneration of somatic embryo-derived sporophytes on the internode explants of the tree fern Cyathea delgadii Sternb.

The influence of the temperature and different light qualities emitted by light-emitting diodes (LEDs) and fluorescent lamps (Fl) on the micropropagation of the tree fern Cyathea delgadii was evaluated. The most efficient somatic embryo production was obtained on internode explants at 22 °C. The optimal temperature range for obtaining well-developed plants was 24–28 °C. This stimulated the elongation and development of the first leaf and the formation of the next leaf and roots primordia. Temperatures lower than 24 °C and higher than 28 °C inhibited the formation of young sporophyte organs and delayed their development. The RBUV (35% red, 15% blue, and 50% UV) and B (100% blue) lights and Fl light were beneficial for the sporophyte production on internode explants. However, plants obtained under RBUV light were undeveloped. The white LED light stimulated the number of explants capable of gametophyte production and development. The RB light (70% red and 30% blue) enhanced the number of roots of newly-formed plants. Most of the LED lights tested had a good impact on root elongation compared to Fl light and constant darkness. The R light (100% red) benefits leaf development and elongation. Research shows that temperature and LED lightning play a significant role in the process of morphogenesis in C. delgadii, significantly affecting the embryogenic competence of somatic cells and the development of sporophytes.

History and current status of embryogenic culture-based tissue culture, transformation and gene editing of maize (Zea mays L.).

The production of embryogenic callus and somatic embryos is integral to the genetic improvement of crops via genetic transformation and gene editing. Regenerable embryogenic cultures also form the backbone of many micro-propagation processes for crop species. In many species, including maize, the ability to produce embryogenic cultures is highly genotype dependent. While some modern transformation and genome editing methods reduce genotype dependence, these efforts ultimately fall short of producing truly genotype-independent tissue culture methods. Recalcitrant genotypes are still identified in these genotype-flexible processes, and their presence is magnified by the stark contrast with more amenable lines, which may respond more efficiently by orders of magnitude. This review aims to describe the history of research into somatic embryogenesis, embryogenic tissue cultures, and plant transformation, with particular attention paid to maize. Contemporary research into genotype-flexible morphogenic gene-based transformation and genome engineering is also covered in this review. The rapid evolution of plant biotechnology from nascent technologies in the latter half of the 20th century to well-established, work-horse production processes has, and will continue to, fundamentally changed agriculture and plant genetics research.

Overexpression of HbGRF4 or HbGRF4-HbGIF1 Chimera Improves the Efficiency of Somatic Embryogenesis in Hevea brasiliensis.

Transgenic technology is a crucial tool for gene functional analysis and targeted genetic modification in the para rubber tree (Hevea brasiliensis). However, low efficiency of plant regeneration via somatic embryogenesis remains a bottleneck of successful genetic transformation in H. brasiliensis. Enhancing expression of GROWTH-REGULATING FACTOR 4 (GRF4)-GRF-INTERACTING FACTOR 1 (GIF1) has been reported to significantly improve shoot and embryo regeneration in multiple crops. Here, we identified endogenous HbGRF4 and HbGIF1 from the rubber clone Reyan7-33-97, the expressions of which dramatically increased along with somatic embryo (SE) production. Intriguingly, overexpression of HbGRF4 or HbGRF4-HbGIF1 markedly enhanced the efficiency of embryogenesis in two H. brasiliensis callus lines with contrasting rates of SE production. Transcriptional profiling revealed that the genes involved in jasmonic acid response were up-regulated, whereas those in ethylene biosynthesis and response as well as the S-adenosylmethionine-dependent methyltransferase activity were down-regulated in HbGRF4- and HbGRF4-HbGIF1-overexpressing H. brasiliensis embryos. These findings open up a new avenue for improving SE production in rubber tree, and help to unravel the underlying mechanisms of HbGRF4-enhanced somatic embryogenesis.

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.

DNA demethylation by transitory 5‐azacytidine treatment improves somatic embryogenesis yield for regeneration and breeding of cork oak

AbstractSomatic embryogenesis (SE) is a crucial biotechnological tool for large‐scale mass propagation of selected material, genetic transformation and breeding, with many advantages for forest tree improvement. However, the application of SE in forest species is limited because of their recalcitrance. SE is also a valuable system for studying cell reprogramming, acquisition of totipotency and embryo development. Increasing evidence reveals that epigenetic reprogramming takes place during SE through DNA methylation, although there is scarce information on forest species. In this work, we have evaluated DNA methylation dynamics during SE and the effects of the DNA demethylating agent 5‐azacytidine (AzaC) in cork oak. After induction and early stages of SE, a reduced DNA methylation level is observed, followed by an increase of methylation during embryo differentiation. These changes in DNA methylation during SE progression were associated with expression profiles of DNA methyltransferase genes QsMET1, QsDRM2 and QsCMT3, and DNA demethylase QsDME‐like. Treatment with AzaC reduced DNA methylation, promoted SE induction rate and proembryogenic masses proliferation, and induced the expression of the SE marker gene QsSERK1‐like. However, continuous AzaC treatment hindered embryo differentiation, suggesting that DNA methylation is needed for further embryo development. Interestingly, AzaC removal from the culture medium of embryogenic masses restored embryo development and led to a significant increase in somatic embryo production compared with untreated cultures. These findings open new possibilities using transitory treatments with small molecule epigenetic modulators, as AzaC, to enhance SE yields for forestry breeding programs.

Somatic embryo production and GFP genetic transformation in elite Ugandan cassava genotypes

Friable embryogenic callus (FEC) production is a prerequisite for the genetic transformation of cassava (Manihot esculenta Crantz). This process necessitates the development of organized embryogenic structures (OES) as a precursor. However, the production of OES and FECs in cassava is dependent on the genotype. This study aimed to optimize somatic embryo production in elite Ugandan cassava genotypes (NASE13, NASE19, and NAROCASS1) and demonstrate genetic transformation using the enhanced green fluorescent protein (eGFP) gene. OES were induced by initiating leaf-lobe explants on Murashige and Skoog (MS) media at various concentrations of 24D and picloram followed by friable embryogenic callus (FEC) production and regeneration. There were highly significant differences among the different media compositions in the percentage response to OES formation (p < 0.001). NASE 13 exhibited the highest regeneration frequency of 38.4%; while NAROCASS1 had the lowest regeneration frequency of 10.5% recorded. The presence of GFP gene in NAROCASS1 was confirmed by polymerase chain reaction (PCR) analysis of the transformed lines. Compared to existing studies, this research pioneers genotype-specific optimization for somatic embryo production and successful genetic transformation in Ugandan cassava variaties. The utilization of these genotypes, already embraced by farmers, presents promising opportunities to enhance them with desirable traits, further augmenting cassava cultivation.

Effects of plant growth regulators on callogenesis and embryogenesis in sarnav and desiree potato (Solanum tuberosum L.) varieties

Somatic embryos play a pivotal role in the production of high-quality potatoes and seed breeding. This study focused on determining the concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzyl amino purine (BAP) in the formation of callus tissue and callus induction. Our goal was to assess the efficiency of potato explants with the highest potential for somatic embryo production. To achieve this, we cultivated Sarnav and Desiree potato varieties under in vitro tissue culture conditions, utilizing the obtained tissue cultures for subsequent experiments. The MS nutrient media were enriched with NAA and BAP at ratios of 1.5: 1, 1: 1.5, and 1: 1 mg/L, along with NAA concentrations of 1.5, 1, or 2 mg/L. Somatic embryogenesis experiments were conducted using various MS nutrient media, enriched with BAP and GA3 at concentrations of 1: 0.5, 0.4: 0.1, 0.5: 0.2, and 0.1: 0.1 mg/L of plant growth regulators. During the course of the study, diverse callus formations were observed in both leaf and internodal stem explants. Among the nutrient media, the M2 medium enriched with 1: 1.5 mg/L of NAA and BAP yielded the highest callus formation rates: 92% for the Desiree variety and 100% for the Sarnav variety, specifically in internodal stem explants. Notably, the index of embryo formation in leaf explants selected for somatic embryogenesis within the SE4 medium was 70% for the Sarnav variety and 65% for the Desiree variety. The inclusion of BAP and GA3 at a ratio of 0.1: 0.1 mg/l in the SE4 nutrient medium resulted in somatic embryogenesis in 80% of calli for the Sarnav variety and 78% for the Desiree variety. These findings underscore the potential for regenerating plants through somatic embryogenesis in the Sarnav potato variety, a significant development with implications for genetic transformation studies involving this particular variety.

Effects of Long-Term Subculture on Maturation Ability and Plant Conversion in Pinus radiata: Using FT-IR Spectroscopy to Determine Biomarkers of Embryogenic Tissue Aging

The forestry industry has integrated somatic embryogenesis into its clonal programs due to the generation of a high number of plants from selected genotypes at low cost. Somatic embryos are generated in a stressful environment after multiplication of the proembryogenic masses; thus, it is critical to determine the degree of stability of the embryogenic cultures and their potential for mass propagation. Maturation ability in cultures of different ages was evaluated in conjunction with the integrity of the proembryogenic masses, germination rate, hypocotyl and root length, plant conversion, and ex vitro survival. To identify differences in embryogenic tissue from different subcultures, their DNA was analyzed using FT-IR spectroscopy. A significant decrease in the production of somatic embryos was detected from week 15, and some lines even stopped producing embryos. Germination rate, hypocotyl length, and plant conversion were negatively affected by long-term cultivation, while root length and ex vitro survival were not significantly affected. The results obtained from the FT-IR spectroscopy analysis indicate that it is feasible to use mid-infrared spectroscopy to differentiate between embryogenic tissues with different cumulative subculture times based on the spectra obtained from their DNA, which is directly related to maturation ability.

New methods for sorghum transformation in temperate climates.

Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens-mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.

The Arabidopsis Phytoglobin 2 mediates phytochrome B (phyB) light signaling responses during somatic embryogenesis.

During the light induction of somatic embryogenesis, phyB-Pfr suppresses Phytoglobin 2, known to elevate nitric oxide (NO). NO depresses Phytochrome Interacting Factor 4 (PIF4) relieving its inhibition on embryogenesis through auxin. An obligatory step of many in vitro embryogenic systems is the somatic-embryogenic transition culminating with the formation of the embryogenic tissue. In Arabidopsis, this transition requires light and is facilitated by high levels of nitric oxide (NO) generated by either suppression of the NO scavenger Phytoglobin 2 (Pgb2), or its removal from the nucleus. Using a previously characterized induction system regulating the cellular localization of Pgb2, we demonstrated the interplay between phytochrome B (phyB) and Pgb2 during the formation of embryogenic tissue. The deactivation of phyB in the dark coincides with the induction of Pgb2 known to reduce the level of NO; consequently, embryogenesis is inhibited. Under light conditions, the active form of phyB depresses the levels of Pgb2 transcripts, thus expecting an increase in cellular NO. Induction of Pgb2 increases Phytochrome Interacting Factor 4 (PIF4) suggesting that high levels of NO repress PIF4. The PIF4 inhibition is sufficient to induce several auxin biosynthetic (CYP79B2, AMI1, and YUCCA 1, 2, and 6) and response (ARF5, 8, and 16) genes, conducive to the formation of the embryonic tissue and production of somatic embryos. Auxin responses mediated by ARF10 and 17 appear to be regulated by Pgb2, possibly through NO, in a PIF4-independent fashion. Overall, this work provides a new and preliminary model integrating Pgb2 (and NO) with phyB in the light regulation of in vitro embryogenesis.

High throughput in vitro production of somatic embryoswith innovative cryo cell banking system

ArborGen Inc. was originally created as a partnership for forestry research in 2000 by West Rock (MWV), International Paper and Rubicon (Fletcher Challenge). It now produces the largest number of both conventional and advanced genetic tree seedlings in the world. Among its advanced genetics offerings, varietals have the highest level of genetic traits in yield and timber quality such as height, diameter growth, disease resistance and etc. To scale up varietals, in vitro process named somatic embryogenesis (SE) is used as a mean to mass propagate SE seedlings. Over the years of R&amp;D efforts, we recognized that a key challenge for scale-up is the lack of a stable and predictable bioprocess platform. The primary causes that impact stability and predictability include a challenge to receive consistent supplies of good starting cell materials, the risk of somaclonal variation due to long term exposure tomaintenance media containing hormones and risk of contamination or loss from environmental fluctuations. Accordingly our SE R&amp;D program has been targeted to develop a robust bioprocess platform to reduce these variabilities. One of our major achievement is the development of a cell banking (CB) system, which provides high quality starting cell materials for SE production. The cell banking system includes the creations of research cell banks (RCB) and master cell banks (MCB). After the cell banks are created, the banked cells are tested and validated for their qualification for embryo production. Currently, we successfully integrated the cell banking technology into somatic embryo production in a number of economically important tree species and crops, such as pine, spruce, sweetgum, sugarcane, avocado and banana. With the SE&amp;CB platform, the somatic embryo &amp; somatic seedling yields are consistent, predictable and are produced cost-efficiently on a commercial scale.

Artificial Seed Production and Cryopreservation by Encapsulation Dehydration for Medicinal Herb of Himalayan Region, Swertia Chirayita

BACKGROUND: Cryopreservation of germplasm in liquid nitrogen is an ideal technique for the longer term storage of plant genetic material, including medicinal species. OBJECTIVE: To develop a somatic embryo production system for the medicinal species Swertia chirayita and to evaluate their potential for storage in liquid nitrogen (- 196˚C). MATERIALS AND METHODS: An efficient protocol of somatic embryogenesis was developed for the first time using leaves of in-vitro grown shoots of S. chirayita . Somatic embryos were then encapsulated in 3% sodium alginate, 0.85 M sucrose and 100 mM calcium chloride for synthetic seed production and subjected to cryopreservation. Marker medicinal compounds were determined by RP-HPLC analysis. RESULTS: A medium containing 1 mg/L 2,4-D+ 0.5 mg/L BAP+ 0.5 mg/L TDZ was found to stimulate the highest callus induction. Somatic embryos were recovered after 5 weeks, when cultured on the same media. Synthetic seeds were dehydrated and immersed in liquid nitrogen for 1 h. Cryopreserved synthetic seeds were successfully revived and germinated on MS media supplemented with 1 mg/L IBA+ 2 mg/L KN + 3 mg/L GA3 in which 93.3% somatic embryos differentiated into shoots. One month old in-vitro grown shoots from cryopreserved somatic embryos had similar marker medicinal compounds, such as amarogentin (4.72 ± 0.11 μg/mg) and mangiferin (14.54 ± 0.05 μg/mg), as control material. CONCLUSION: This protocol offers vast scope for multiplying material of an endangered medicinal herb and subsequent cryopreservation.

Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.

We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant's genome. We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.

Somatic Embryogenesis in Citrus (Citrus spp.), var. Valencia.

Somatic embryogenesis has been obtained in many citrus cultivars; however, the efficiency of the system is genotype dependent and culture synchronization is important to reach more efficient systems. In this chapter we present a detailed protocol of somatic embryogenesis induction from nucellar tissue and the use of an alternative method of callus sieving for culture synchronization and embryo production. This is a simple method which can also be evaluated for other species aiming at better culture efficiency and somatic embryo production.

Evaluation of somatic embryo production during embryogenic tissue proliferation stage using morphology, maternal genotype, proliferation rate and tissue age of Pinus thunbergii Parl

To determine the optimal embryogenic capacity (somatic embryo production) of the selected elite nematode-resistant genotypes of Pinus thunbergii, variables such as embryogenic tissue (ET) morphology, maternal genotype, proliferation rate and tissue age were analyzed. ET morphology and histological evaluation of the proliferation stage showed a decrease in filamentous clump and protuberant surfaces and a decline in the acetocarmine-staining area, which indicates a decrease in somatic embryo production (SEP). Variations in cell physiology during the proliferation stage showed that SEP was positively correlated with soluble sugars and proteins, but negatively correlated with starch, peroxidase, and superoxidase. In addition, SEP was significantly (p < 0.001) affected by maternal genotype, tissue age and proliferation rate. Moreover, SEP was positively correlated with proliferation rate (r = 0.98, p < 0.001), but negatively correlated with tissue age (r = − 0.95, p < 0.001). In general, the results suggest that SEP could be assessed in ET proliferation stages by the apparent cell morphology, histology, proliferation rate and tissue age, which provides novel insights for evaluating the ET maturation capacity (number of somatic embryos) during the proliferation stage of P. thunbergii somatic embryogenesis.

Proteome-Wide Analysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a Combined Response of Sugar Metabolism and Translational Regulation Mechanisms.

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.

Telomere Length in Norway Spruce during Somatic Embryogenesis and Cryopreservation

Telomeres i.e., termini of the eukaryotic chromosomes protect chromosomes during DNA replication. Shortening of telomeres, either due to stress or ageing is related to replicative cellular senescence. There is little information on the effect of biotechnological methods, such as tissue culture via somatic embryogenesis (SE) or cryopreservation on plant telomeres, even if these techniques are widely applied. The aim of the present study was to examine telomeres of Norway spruce (Picea abies (L.) Karst.) during SE initiation, proliferation, embryo maturation, and cryopreservation to reveal potential ageing or stress-related effects that could explain variation observed at SE process. Altogether, 33 genotypes from 25 families were studied. SE initiation containing several stress factors cause telomere shortening in Norway spruce. Following initiation, the telomere length of the embryogenic tissues (ETs) and embryos produced remains unchanged up to one year of culture, with remarkable genotypic variation. Being prolonged in vitro culture can, however, shorten the telomeres and should be avoided. This is achieved by successful cryopreservation treatment preserving telomere length. Somatic embryo production capacity of the ETs was observed to vary a lot not only among the genotypes, but also from one timepoint to another. No connection between embryo production and telomere length was found, so this variation remains unexplained.

Root Rot Resistance Locus PaLAR3 Is Delivered by Somatic Embryogenesis (SE) Pipeline in Norway Spruce (Picea abies (L.) Karst.)

Research Highlights: The Norway spruce somatic embryogenesis (SE) pipeline is suitable for multiplication of material with root rot resistance traits. Background and Objectives: Heterobasidion root rot is the economically most severe forest pathogen in Europe, reducing the benefit of planting elite forest material. In this study, the SE-propagation ability of elite Norway spruce material carrying root rot resistance traits was studied. Materials and Methods: We analyzed the presence of the root rot resistance locus PaLAR3B among 80 Finnish progeny-tested Norway spruce plus-trees used for SE-plant production as well as in 241 SE lines (genotypes) derived from them. Seven full-sib families with lines having either AA, AB, or BB genotype for PaLAR3 locus were further studied for their SE-plant propagation ability. Results: The results indicate that 47.5% of the studied elite trees carry the PaLAR3B allele (45% are heterozygous and 2.5% homozygous). The resistance allele was present among the SE lines as expected based on Mendelian segregation and did not interfere with somatic embryo production capacity. All embryos from PaLAR3 genotypes germinated well and emblings were viable in the end of first growing season. However, in three families, PaLAR3B homo- or heterozygotes had 23.2% to 32.1% lower viability compared to their respective hetero- or PaLAR3A homozygotes. Conclusions: There is no trade-off between root rot resistance locus PaLAR3B and somatic embryo production ability, but the allele may interfere with Norway spruce embling establishment.

The induction and development of somatic embryos from the in vitro culture of Catharanthus roseus (L.) G. Don

Catharanthus roseus is containing anticancer alkaloids vincristine and vinblastine as is an important medicinal plant. Several studies have conducted on in-vitro culture of this plant. To optimize the somatic embryogenesis, a factorial based on CRD experiment with 10 replications was conducted. Root, hypocotyl and leaf explants grown in-vitro were transferred and cultured on MS media containing different combinations of 2,4-D, NAA and 2,4-D×BAP. The results revealed that in callogenesis, the interaction effects of root and hypocotyl explants×2,4-D and NAA as well as hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was superior than other treatments (p≤0.01). For calli fresh weight, hypocotyl×NAA and hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was the treatment of choice (p≤0.01). The calli produced were sub-cultured to attain the pre-embryos and somatic embryos. For the number of pre-embryos and somatic embryos; the interaction of hypocotyl×2,4-D was the best and most efficient treatment. Seemingly, the production of somatic embryos is accessible in this plant by the logical management of growth regulator combinations. Furthermore, the production and genetic engineering of the somatic embryos could be a promising trend in the subsequent production of high-valued metabolites from this plant.

English

Some improved genotypes of cocoa tree (Theobroma cacao L.), have shown in standard study conditions of absence or a very low response to the induction of somatic embryos. This is the case of the recalcitrant genotypes C8 and C15, partially recalcitrant genotypes C14 and C16. This study aims to improve the production of somatic embryos of recalcitrant genotypes. Staminodes and petals excised from the immature buds of the five genotypes C1, C8, C14, C15 and C16 were used as plant material. These floral explants were cultured on different media containing two type of mineral salts such as potassium sulphate (K2SO4) at concentrations 18, 27, 36 and 45 mM and magnesium sulphate (MgSO4) whose concentrations used are 5, 10, 15 and 20 mM. Calli induction was obtained in the five (05) genotypes at percentages ranging from 50 to 100% with staminodes and from 61 to 100% with petals on all media. The transfer of the callogenic explants on the developmental medium allowed the induction of embryogenic calli and somatic embryos after 84 days only with petal explants. The most important PEC and NSE were obtained with two mineral salts concentrations, 27 mM K2SO4 and 15 mM MgSO4 for all genotypes and varied to 20 to 40%. Potassium sulphate (K2SO4) at 27 and magnesium sulphate (MgSO4) at 15 mM are the concentrations of the most suitable mineral salts to overcome recalcitrance of cocoa genotypes. &nbsp; Key words: Recalcitrant, Theobrama cacao, improved genotypes, mineral salts, somatic embryogenesis.