Immature Zygotic Embryos Research Articles

Histological Analysis of Somatic Embryogenesis from Immature Zygotic Embryo of Wild Banana Musa acuminata ssp. malaccensis

Somatic embryogenesis, a crucial plant regeneration method, has become indispensable for crop improvement, particularly for species reliant on somatic cell manipulation techniques. Optimization of this process necessitates an understanding of the developmental stages involved. This study investigates the histological aspects of somatic embryogenesis in Musa acuminata ssp. malaccensis derived from immature zygotic embryos. Through detailed histological analysis, we aimed to elucidate the morphological changes and cellular organization occurring during the various stages of somatic embryogenesis, from induction, culture proliferation, and somatic embryo development to plantlet conversion. The initial stages of embryogenesis, characterized by nodules, were primarily composed of meristematic cells with high cell division activity. These cells contained tetrad-like structures that could develop into distinct two- and four-celled proembryoids or proembryogenic aggregates. Our histo-anatomical analysis revealed that embryogenic cultures proliferated through multiple pathways simultaneously: somatic embryo budding, proembryo formation, and pro-embryonic mass formation from both internal and peripheral cells. At the stage of somatic embryo development, embryos with a well-defined protoderm layer, containing cells with prominent nuclei and dense cytoplasm, potentially regenerate into plantlets. Furthermore, histological examination revealed the presence of procambium within mature somatic embryos, which subsequently developed into the vascular system of the complete plantlet

A simple and efficient non-tissue culture method for genetic transformation of grape immature zygotic embryos via VvBBM overexpression

The process of genetic transformation of grapevine has long been hampered by the inefficiency and complexity of tissue culture-based methods, severely limiting cultivar improvement and functional genome research. In this study, we aimed to develop a novel, non-tissue culture-based method for the genetic transformation of grape immature zygotic embryos without relying on tissue culture. This approach involves the overexpression of the Vitis vinifera BABY BOOM (VvBBM) gene, a key member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor family, which is known to enhance plant regeneration and transformation efficiency. Using ‘Cabernet Sauvignon’ immature zygotic embryos, we achieved a transformation efficiency of 42.85 % under non-tissue culture conditions, significantly reducing the cycle duration to approximately two months compared to traditional genetic transformation methods. Phenotypic and molecular analyses of the transformed grapevine plants confirmed the integration and overexpression of the VvBBM gene, resulting in improved growth metrics, including increased plant height, root length, and total root number, in a genotype-independent manner. Our results highlight the potential of VvBBM overexpression to circumvent the limitations imposed by tissue culture dependence, providing a rapid, efficient, and scalable alternative to conventional methods. Our study not only provides a practical approach to overcoming the barriers to genetic transformation in grapevine, but also opens new avenues for the application of this technology in the breeding of other economically important crops.

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.

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.

Plant Regeneration via Adventitious Shoot Formation from Immature Zygotic Embryo Explants of Camelina.

Camelina is an oil seed crop that is enjoying increasing interest because it has a particularly valuable fatty acid profile, is modest regarding its water and nutrient requirements, and is comparatively resilient to abiotic and biotic stress factors. The regeneration of plants from cells accessible to genetic manipulation is an essential prerequisite for the generation of genetically engineered plants, be it by transgenesis or genome editing. Here, immature embryos were used on the assumption that their incomplete differentiation was associated with totipotency. In culture, regenerative structures appeared adventitiously at the embryos' hypocotyls. For this, the application of auxin- or cytokinin-type growth regulators was essential. The formation of regenerative structures was most efficient when indole-3-acetic acid was added to the induction medium at 1 mg/L, zygotic embryos of the medium walking stick stage were used, and their hypocotyls were stimulated by pricking to a wound response. Histological examinations revealed that the formation of adventitious shoots was initiated by locally activated cell division and proliferation in the epidermis and the outer cortex of the hypocotyl. While the regeneration of plants was established in principle using the experimental line Cam139, the method proved to be similarly applicable to the current cultivar Ligena, and hence it constitutes a vital basis for future genetic engineering approaches.

Direct Somatic Embryogenesis in Carica papaya L. Genotypes for Genetic Modification Purposes.

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.

Somatic embryogenesis from mature sorghum seeds: An underutilized genome editing recipient system

Somatic embryogenesis is a process of cell totipotency in vitro, whereby an embryogenic cell develops from vegetative tissues rather than from zygotes after double fertilization. Sorghum is a recalcitrant crop in genetic transformation; previous recipient systems have usually been from immature zygotic embryos, which needed more time and labors to prepare. Here, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-induced somatic embryogenesis system from mature sorghum seeds was introduced. 2,4-D can induce two types of calli from a plumular axis section. Low-concentration 2,4-D (e.g., 2 mg/L) induces white and loose non-embryogenic calli (type 1), while high-concentration 2,4-D (e.g., 8 mg/L) induces yellow and compact embryogenic calli (type 2), which can be clearly distinguished by Sudan red staining. Germinating seeds have a long 2-day window for SE induction. Somatic embryogenesis can be enhanced by HDAC inhibitor, trichostatin A (TSA), a histone deacetylase treatment, which shows more SE productivity and a bigger size. Importantly, this easily prepared protocol does not show obvious genotype dependency in sorghum hybrids. In this study, a high-concentration 2,4-D-induced SE system was established from mature sorghum seeds. This finding provides a technical option for the genome editing recipient in sorghum.

Collectible Cell Lines of Larix sibirica Obtained by Somatic Embryogenesis and Their Ability to Regenerate

A protocol for the cultivation of Larix sibirica Ledeb. by somatic embryogenesis was developed (RF Patent No. 2456344, 2012). The L. sibirica collection consisted of 22 actively proliferating cell lines (CLs) obtained from immature zygotic embryos. The age of CLs ranged from 1 to 14 years. CLs differed in their growth intensity, embryonic productivity, hormonal balance, and genetic stability, as well as in their regenerative ability. In most proliferating CLs, the formation of globular somatic embryos continued for 2–4 years. Here, a number of CLs actively proliferated for 9–14 years or more. The formation of embryogenic cultures in L. sibirica is associated with the content of phytohormones and their localization in embryo cells. The cytogenetic studies revealed the genetic stability of young CLs (up to 1 year), in which the karyotype consisted of the diploid number of chromosomes (2n = 24). Genomic mutations were observed in the long-term proliferation of CLs. Individual CLs can maintain cytogenetic stability for many years; such CLs can successfully be used to preserve germplasm, obtain planting material, and for plantation reforestation.

Comparative Transcriptome Analysis between Embryogenic and Non-Embryogenic Callus of Davidia involucrata

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata.

Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata

Cunninghamia lanceolata (C. lanceolata) is an important timber tree species in southern China that requires gene function studies to understand its traits. In this study, we investigated the callus induction rates of immature zygotic embryos from reciprocal hybrids between genotypes B46 and B49. With zygotic embryo development, the callus induction rates showed an increasing trend, followed by a decreasing trend. Moreover, the rate of callus induction in genotype B46 × B49 immature zygotic embryos was greater than in genotype B49 × B46. Callus from C. lanceolata with genotype B46 × B49 was selected as the donor material for protoplast isolation. By using an enzymatic digestion solution containing cellulase, macerozyme, and pectinase, combined with an osmotic stabilizer, we obtained 9.76 × 106 protoplasts/mL with 92.7% viability. We subsequently transformed plasmids into C. lanceolata callus protoplasts and observed the location of the H2B-eYFP fusion protein in the nucleus. To achieve transient transfection of C. lanceolata callus protoplasts, we compared transfection efficiencies at different concentrations of PEG4000, PEG6000, or PEG8000 in a modified MMg solution. We found that 20% (w/v) PEG6000 mediated the transient transfection of C. lanceolata callus protoplasts with a 6.70% efficiency. This study provides a technical foundation for future research on transient transfection and functional analysis of C. lanceolata genes.

In Vitro Growth and Regeneration of Brassica oleracea var. gongylodes: A Decade of Research

Kohlrabi (Brassica oleracea var. gongylodes) is a vegetable cultivated for its edible stem tuber. Although valued for its nutritional properties and tolerance to abiotic stress, kohlrabi is one of the least studied brassicas. In this review, we summarize the results of our decade-long research on in vitro morphogenesis of kohlrabi, starting from 2013. Protocols for efficient in vitro regeneration with minimal requirements for external application of plant growth regulators (PGRs) have been developed, both by somatic embryogenesis (SE) and by de novo shoot organogenesis (DNSO). Efficient regeneration by direct SE was achieved using immature zygotic embryos as explants incubated on PGR-free media, and the production process was maintained in culture thanks to highly efficient secondary SE. Conversely, efficient regeneration by indirect DNSO was achieved using entire seedlings as explants and adding only cytokinin (CK) without the need for exogenous auxin. Comprehensive phytohormone analyses revealed that different exogenously applied CKs differentially affected the composition of endogenous phytohormones and induced changes in the expression of cell cycle-related genes and other genes involved in the organogenic response. The addition of high sucrose concentrations to the nutrient media failed to induce the formation of stem tubers in in vitro culture, but revealed a complex interaction with exogenously applied CKs, interfering with both the endogenous phytohormonome and the expression of organogenesis-related genes. Our work has provided substantial biotechnological advances in the field of in vitro regeneration of kohlrabi, as well as in understanding the underlying phytohormonal regulation. The review aims to introduce kohlrabi to the scientific community as a model system for both basic and applied research, while we continue seeking answers to the outstanding questions and trying to pave the way for the development of more resistant varieties.

In Vitro Shoot Multiplication and Regeneration of the Recalcitrant Rocket (Eruca sativa Mill.) Variety Domaća Rukola

Eruca sativa is known in traditional medicine for its therapeutic effects, while young plants are used as a salad or green food. Recently, the consumption of rocket has increased considerably, so it has become very important for breeders. Plant tissue culture provides a platform to overcome the problems in improving this species. In the present study, an efficient protocol for in vitro shoot regeneration and propagation of recalcitrant rocket variety Domaća rukola was studied. Murashige and Skoog (MS, 1962) medium containing 0.1 mg L−1 BA and frequent subculture over a period of three weeks proved to be optimal for shoot multiplication with a multiplication index of over 3 and only 8.72% of hyperhydrated shoots without necrosis. Different concentrations of 2,4-D, BA, or TDZ in combination with NAA, with or without the presence of AgNO3, were tested for de novo shoot organogenesis (DNSO) from seedling explants. The hypocotyl explants cultured on MS with a combination of TDZ1.0+NAA0.1+AgNO3 5.0 mg L−1 regenerated viable shoots with the highest rate (25.38%) and an average number of 2.18 shoots per regenerating explant. Somatic embryogenesis from immature zygotic embryos proved to be the best way to regenerate a recalcitrant rocket cultivar. The highest embryogenic efficiency was achieved in explants cultured on MS medium containing 1.0 mg L−1 2,4-D with a frequency of 76.64% and 5.13 mean number of regenerated somatic embryos per explant, which were further converted into normal plants. Additionally, in vitro-produced rocket shoots could serve as a possible promising source for the production of flavonoid kaempferol with proven antioxidant properties.

Establishment of a stable grape immature zygotic embryo-based genetic transformation system

Grapevine, along with other woody perennials, has proven to be a recalcitrant crop to produce transgenic plants, largely restricting its potential for further breed improvement. Here we report the establishment of a stable and reproducible transformation protocol using immature zygotic embryos from 'Chardonnay' seeds. We extensively investigated parameters considered essential for a successful transformation, including antibiotic concentration, pretreatment method, Agrobacterium concentration, concentration of acetosyringone (AS), duration of immature zygotic embryo infection, and the infection method. Our results indicated that transformable immature zygotic embryos may be generated by soaking seeds in a 2.5 g·L−1 gibberellin (GA3) solution for 15 min in a 55 °C constant temperature water bath, followed by 24 h of growth at room temperature before sterilized and inoculated. Furthermore, the transformation rate increased to 12.38% when the immature zygotic embryos of split beak were immersed in the Agrobacterium tumefaciens solution containing 200 μM AS (OD600 = 0.6) for 20 min after 5 days of pre-culture. Transgene integration and expression was further confirmed by the detection of GFP and Western blot in leaves. The newly-developed method took roughly 4 months to obtain the transformation-positive plants, which marked a considerable reduction in the length of time required for the procedure. More importantly, the receptor material is easily accessible, the process unaffected by the changing of the season, and it is straightforward to implement. Our developed transformation protocol on grape immature zygotic embryos offers a tool for gene function analysis and germplasm enhancement via biotechnology.

Development of somatic embryogenesis of mangrove fan palm (Licuala spinosa Thunb.) from culturing immature zygotic embryo

AbstractMangrove fan palm is an endemic plant that grows well and has for a long period been related to traditional ceremonies in southern Thailand. It is an endangered plant with a slow growth and is difficult to propagate in mass production. Tissue culture technique can be used to produce a large number of plants within a short time and to preserve in vitro germplasm resulting in the plant not becoming extinct from natural habitats. Thus, the objectives of this research were to study effects of auxins and embryo preparation on somatic embryogenesis. The sterile immature zygotic embryo was cultured on MS medium with different types and concentrations of auxins. The results showed that 1 mg/l dicamba gave the best results in callus induction at 100% and callus diameter at 4.67 mm after 8 weeks of culture. For callus proliferation, callus chopped at 100 times and transferred to 0.1 mg/l dicamba containing oil palm culture medium gave the highest callus diameter at 10.33 mm after 4 weeks of culture and somatic embryos were also produced. Upon transferring somatic embryos to MS medium with 0.2 M sorbitol, a large number of various developmental stages of secondary somatic embryos were induced within 4 weeks. Therefore, the finding of this study could be highlighted that dicamba and chopping callus have an important role to induce callus and somatic embryo. This is the first report achieved on in vitro propagation of this plant and it will be beneficial for genetic conservation and biotechnological applications in the future.

Fast-track transformation and genome editing in Brachypodium distachyon

BackgroundEven for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets.ResultsWe have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2).ConclusionsWith a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs.

Calcium Dynamics, WUSCHEL Expression and Callose Deposition during Somatic Embryogenesis in Arabidopsis thaliana Immature Zygotic Embryos.

In this work, we studied the induction of somatic embryogenesis in Arabidopsis using IZEs as explants. We characterized the process at the light and scanning electron microscope level and studied several specific aspects such as WUS expression, callose deposition, and principally Ca2+ dynamics during the first stages of the process of embryogenesis induction, by confocal FRET analysis with an Arabidopsis line expressing a cameleon calcium sensor. We also performed a pharmacological study with a series of chemicals know to alter calcium homeostasis (CaCl2, inositol 1,4,5-trisphosphate, ionophore A23187, EGTA), the calcium-calmodulin interaction (chlorpromazine, W-7), and callose deposition (2-deoxy-D-glucose). We showed that, after determination of the cotiledonary protrusions as embryogenic regions, a finger-like appendix may emerge from the shoot apical region and somatic embryos are produced from the WUS-expressing cells of the appendix tip. Ca2+ levels increase and callose is deposited in the cells of the regions where somatic embryos will be formed, thereby constituting early markers of the embryogenic regions. We also found that Ca2+ homeostasis in this system is strictly maintained and cannot be altered to modulate embryo production, as shown for other systems. Together, these results contribute to a better knowledge and understanding of the process of induction of somatic embryos in this system.

Genome editing approaches using reproductive cells/tissues in flowering plants.

Targeted mutagenesis via programmable nucleases including the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been broadly utilized to generate genome-edited organisms including flowering plants. To date, specific expression of Cas9 protein and guide RNA (gRNA) in reproductive cells or tissues is considered one of the most effective genome-editing approaches for heritable targeted mutagenesis. In this report, we review recent advances in genome editing methods for reproductive cells or tissues, which have roles in transmitting genetic material to the next-generation, such as egg cells, pollen grains, zygotes, immature zygotic embryos, and shoot apical meristems (SAMs). Specific expression of Cas9 proteins in initiating cells efficiently induces targeted mutagenesis via Agrobacterium-mediated in planta transformation. In addition, genome editing by direct delivery of CRISPR/Cas9 components into pollen grains, zygotes, cells of embryos and SAMs has been successfully established to generate genome-edited plant lines. Notably, DNA-free genome editing by the delivery of Cas9-gRNA ribonucleoproteins (RNPs) is not associated with any legislative concerns about genetically modified organisms. In summary, the genome editing methods for reproductive cells or tissues have enormous potential for not only basic studies for plant reproduction but also applied sciences toward molecular plant breeding.

In vitro callogenesis essays to induce somatic embryogenesis of the Tunisian chickpea

The present study was the first report on the somatic embryogenesis of the Tunisian chickpea (Cicer arietinum L.) particularly supposed to be recalcitrant and difficult to manipulate in vitro. An efficient protocol has been developed for inducing indirect somatic embryogenesis derived from immature zygotic embryo axis, young leaflet and hypocotylar explants. Callogenesis was achieved on full-strength Murashige and Skoog's (1962) (MS) basal medium supplemented with two auxin/cytokinin combinations; 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylamonopurine (KIN) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to establish the phytohormones requirement to promote the best induction of friable calli. For somatic embryos induction, embryogenic calli developed from zygotic embryo axes, leaflet and hypocotylar explants were cultured on MS full strength and MS/2, half strength, free of phytohormones media. This study found that all explants exhibited high frequency of callus induction. For immature zygotic embryo axes and young leaflet explants, media containing the combination of 2,4-D and KIN were best effective for inducing callogenesis and friable calli. The most important embryogenic callus percentages were obtained on the culture medium containing the highest concentrations of 2,4-D (2 mg L-1) and KIN (0.5 mg L-1). However, media containing the combination NAA and BAP were best effective for inducing embryogenic calli on hypocotylar explants. A maximal rate of embryogenic calli was reached on medium containing 3 mg L-1 NAA and 1 mg L-1 BAP. The highest number of somatic embryos was obtained with embryogenic calli derived from embryo axis explants and cultured on MS half strength free phytohormones medium. About 56% of somatic embryos converted successfully into fertile plantlets.

Integrated transcriptomic and metabolic analyses provide insights into the maintenance of embryogenic potential and the biosynthesis of phenolic acids and flavonoids involving transcription factors in Larix kaempferi (Lamb.) Carr.

Somatic embryogenesis (SE) techniques have been established for micropropagation or basic research related to plant development in many conifer species. The frequent occurrence of non-embryogenic callus (NEC) during SE has impose constraints on the application of somatic embryogenesis SE in Larix kaempferi (Lamb.) Carr, but the potential regulatory mechanisms are poorly understood. In this study, integrated transcriptomic and metabolomic analyses were performed in embryogenic callus (EC) and NEC originating from a single immature zygotic embryo to better decipher the key molecular and metabolic mechanisms required for embryogenic potential maintenance. The results showed that a total of 13,842 differentially expressed genes (DEGs) were found in EC and NEC, among which many were enriched in plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and the biosynthesis of amino acids pathways. Metabolite profiling showed that 441 differentially accumulated metabolites (DAMs) were identified in EC and NEC. Both EC and NEC had vigorous primary metabolic activities, while most secondary metabolites were upregulated in NEC. Many totipotency-related transcription factor (TF) genes such as BBMs, WUSs, and LEC1 showed higher expression levels in EC compared with NEC, which may result in the higher accumulation of indole 3-acetic acid (IAA) in EC. NEC was characterized by upregulation of genes and metabolites associated with stress responses, such as DEGs involved in jasmonic acid (JA) and ethylene (ETH) biosynthesis and signal transduction pathways, and DEGs and DAMs related to phenylpropanoid and flavonoid biosynthesis. We predicted and analyzed TFs that could target several key co-expressed structural DEGs including two C4H genes, two CcoAOMT genes and three HCT genes involved in phenylpropanoid and flavonoid biosynthesis. Based on the targeted relationship and the co-expression network, two ERFs (Lk23436 and Lk458687), one MYB (Lk34626) and one C2C2-dof (Lk37167) may play an important role in regulating phenolic acid and flavonoid biosynthesis by transcriptionally regulating the expression of these structural genes. This study shows an approach involving integrated transcriptomic and metabolic analyses to obtain insights into molecular events underlying embryogenic potential maintenance and the biosynthesis mechanisms of key metabolites involving TF regulation, which provides valuable information for the improvement of SE efficiency in L. kaempferi.

Comparison of tissue culture-induced variation in triticale regenerants obtained by androgenesis and somatic embryogenesis

Triticale is becoming an increasingly important livestock crop production. This is evidenced by increasing triticale-producing areas and by improved yields. In addition, meeting the increasing demand for cereals involves the introduction of high-yielding and stress-resistant varieties into breeding. In vitro culture techniques can accelerate the development of new varieties. Therefore, it seems extremely important to develop efficient plant regeneration methods through in vitro cultures and to understand the mechanisms involved in gaining regenerants. Obtaining regenerants of triticale through somatic embryogenesis and androgenesis may lead to tissue culture-induced variation. In the present study, we compared regenerants obtained in both regeneration systems (anther and immature zygotic embryo cultures), considering the level of genetic and epigenetic changes observed in different DNA sequence contexts for methylated cytosine (CG, CHG, CHH). The changes concerning the DNA sequence (so-called sequence variation) and the changes concerning the DNA methylation patterns, i.e., the removal of methylated cytosine (DNA demethylation) and the introduction of methylation to cytosine (de novo DNA methylation), were analyzed. We observed that regenerants derived via somatic embryogenesis and androgenesis differ notably for demethylation in the symmetrical CG sequence context and de novo methylation in the asymmetrical CHH context. These changes may be related to the reprogramming of microspore development from gametophytic to sporophytic and lack of such process in zygotic embryos.