Embryogenic Cell Research Articles

Identification, Gene Structure, and Expression of BnMicEmUP: A Gene Upregulated in Embryogenic Brassica napus Microspores.

Microspores of Brassica napus can be diverted from normal pollen development into embryogenesis by treating them with a mild heat shock. As microspore embryogenesis closely resembles zygotic embryogenesis, it is used as model for studying the molecular mechanisms controlling embryo formation. A previous study comparing the transcriptomes of three-day-old sorted embryogenic and pollen-like (non-embryogenic) microspores identified a gene homologous to AT1G74730 of unknown function that was upregulated 8-fold in the embryogenic cells. In the current study, the gene was isolated and sequenced from B. napus and named BnMicEmUP (B. napus microspore embryogenesis upregulated gene). Four forms of BnMicEmUP mRNA and three forms of genomic DNA were identified. BnMicEmUP2,3 was upregulated more than 7-fold by day 3 in embryogenic microspore cultures compared to non-induced cultures. BnMicEmUP1,4 was highly expressed in leaves. Transient expression studies of BnMicEmUP3::GFP fusion protein in Nicotiana benthamiana and in stable Arabidopsis transgenics showed that it accumulates in chloroplasts. The features of the BnMicEmUP protein, which include a chloroplast targeting region, a basic region, and a large region containing 11 complete leucine-rich repeats, suggest that it is similar to a bZIP PEND (plastid envelope DNA-binding protein) protein, a DNA binding protein found in the inner envelope membrane of developing chloroplasts. Here, we report that the BnMicEmUP3 overexpression in Arabidopsis increases the sensitivity of seedlings to exogenous abscisic acid (ABA). The BnMicEmUP proteins appear to be transcription factors that are localized in plastids and are involved in plant responses to biotic and abiotic environmental stresses; as well as the results obtained from this study can be used to improve crop yield.

The Effects of Lentivirus-Mediated Gene Silencing of RARβ on the Stemness Capability of Non-Small Cell Lung Cancer.

Retinoic acid receptor beta is a nuclear receptor protein that binds to retinoic acid (RA) to mediate cellular signalling in embryogenic morphogenesis, cell growth, and differentiation. However, the function of RARβ in cancer stem cells (CSCs) has yet to be determined. This study aimed to understand the role of RARβ in regulating cell growth and differentiation of lung cancer stem cells. Based on the clonogenic assay, spheroid assay, mRNA levels of stem cell transcription factors, and cell cycle being arrested at the G0/G1 phase, the suppression of RARβ resulted in significant inhibition of A549 parental cell growth. This finding was contradictory to the results seen in CSCs, where RARβ inhibition enhanced the cell growth of putative and non-putative CSCs. These results suggest that RARβ suppression may act as an essential regulator in A549 parental cells, but not in the CSCs population. The findings in this study demonstrated that the loss of RARβ promotes tumorigenicity in CSCs. Microarray analysis revealed that various cancer pathways were significantly activated following the suppression of RARβ. The changes seen might compensate for the loss of RARβ function, CSCs population's aggressiveness, which led to the CSCs population's aggressiveness. Thus, understanding the role of RARβ in regulating the stemness of CSCs may lead to targeted therapy for lung CSCs.

Integrated proteomics and histochemical analysis of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) in embryogenic suspension culture

t Cell suspension culture is a useful in vitro model-system for both scaling up and conserving the Brazilian conifer Araucaria angustifolia. In the present work, cell suspension of Araucaria was subjected to proteomics, biochemical and histochemical analyses. The results revealed new insights underlying the molecular mechanism of proembryogenic masses transition in cell suspension. Embryogenic cell cultures were cultivated in a basal liquid medium modified in a Steward apparatus (orbital agitator). Cell growth dynamics was evaluated using cell volume after sedimentation, fresh weight, mitotic index, conductivity, pH, and the number of proembryogenic masses (PEMs: I, II, III). Histochemical parameters, cell viability, and cell death analyses were performed to pinpoint growth rates. Proteomics analysis was performed using two-dimensional electrophoresis, and protein identification was carried out by MALDI-TOF-TOF tandem mass spectrometry. Cell growth dynamics showed a predominance of PEM III. Maximum slope of the exponential phase growth in fresh weight occurred at exponential phase after 15 days (optimal cultivation time), after which cell viability and pH decreased, thereby allowing the identification of stressrelated proteins. Several metabolism and growth proteins were abundant, such as: cytoskeletal, WOX1, cytokinin-related, and auxin-related proteins acting on cell wall modification, suspensor cell formation, and PEM I to PEM III transition.

Hybrid Pine (Pinus attenuata × Pinus radiata) Somatic Embryogenesis: What Do You Prefer, Mother or Nurse?

Development of hybrid pines of Pinus radiata D. Don for commercial forestry presents an opportunity to diversify the current resource of plant material. Climate change and different land uses pose challenges, making alternative species necessary to guarantee wood and non-wood products in the future. Pinus radiata var. cedrosensis × Pinus attenuata hybrid possesses different attributes, such as tolerance to drought conditions, better growth and resistance to snow damage at higher altitudes, and more importantly, different wood quality characteristics. Embryogenic cell lines were successfully initiated reciprocal hybrids using as initial explants megagametophytes, excised zygotic embryos and excised zygotic embryos plus nurse culture. However, the questions raised were: does the initiation environment affect the conversion to somatic plantlets months later? Does the mother tree or the cross have an effect on the conversion to somatic plantlets? In the present work we analysed the maturation rate, number of somatic embryos, germination rate, and the ex-vitro growth in cell lines derived from different initiation treatments, mother tree species, and crosses. Differences were not observed for in vitro parameters such as maturation and germination. However, significant differences were observed due to the mother tree species in relation with the ex-vitro growth rates observed, being higher those in which P. radiata acted as a mother. Moreover, embryogenic cell lines from these hybrids were stored at −80 °C and regenerated after one and five years.

Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures

BackgroundDevelopment of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study.ResultsCitrus sinensis ‘EV2’ embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6–26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1–2 bp), substitution (1 bp), or deletion (1–3 bp) mutations that occurred close to the protospacer adjacent motif.ConclusionsBoth the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.

Retraction Note to: In vitro somatic embryogenesis from immature female flower of Musa AAB cv. Chenichampa and molecular analysis of transcript factors (TFs) during somatic embryogenesis

Somatic embryogenesis (SE) is a process where somatic embryos can form differentiated tissues and regenerate into new plants. Efficient SE and liquid culture system for large-scale production of banana cv. Chenichampa is presented. The embryo maturation media (M3) was modified with different organic additives and high-frequency somatic embryos were observed in M3 media supplemented with CH: Gln (100:150 mg/L) resulting in a tenfold increase in somatic embryo formation as compared to the control. The generated plantlets were hardened and polymerase chain reaction (PCR) with SSR primers were used to confirm the genetic fidelity of the plantlets. To understand the molecular mechanism of SE, expression patterns of transcript factors (TFs) in respect of embryogenic calli (EC) and embryogenic cell suspension (ECS) was also studied. Transcript factor (TF) such as BBM has been reported to play a significant part in converting explants to EC. The higher expression of TFs MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the EC and MaBBM2 and MaWUS2 in ECS, suggested that these genes could play a crucial role in SE. The protocol developed in this commercially important banana cv. Chenichampa could be highly useful for large-scale micropropagation and genetic manipulation studies which can be adapted to other cultivars lacking in male flowers. An efficient micropropagation system using SE for banana cultivar Chenichampa (Musa AAB) is presented. Differential expression profiles of TFs during various stages of SE studied. Organic additives enhanced the SE and this optimized protocol will serve as a platform for developing efficient propagation system for banana cultivars that lack male flowers.

Molecular analysis of ERF subfamily genes during coffee somatic embryogenesis

With ample cultivation and economic potential, coffee is one of the main commodities in the world. Despite its vast cultivation, there remain problems related to its productivity as a consequence of pests and diseases, questions regarding its physiology, and difficulty with its propagation. Due to such challenges, the application of biotechnological tools, such as tissue culture and molecular markers, has become essential in the search for improvements to coffee cultivation. Among tissue culture techniques, somatic embryogenesis is considered the best form of micropropagation in vitro since it is possible to produce plants that are identical to the parent plant. It is therefore essential to identify the genes involved in the process of somatic embryogenesis, as well as to understand their functions. Different studies have shown the expression of ethylene response factor (ERF) genes in different tissues and under different conditions, mainly in response to biotic and abiotic stresses. However, little is known about their role during somatic embryogenesis. Thus, this study is aimed to identify and analyze the expression patterns of ERF subfamily genes during Coffea arabica L. somatic embryogenesis. The EST-contig6, 9, and 27 were confirmed to be the ethylene response factor 8, 13, and 12 genes from Coffea arabica L., respectively, and their relative expression analysis suggests that they may be involved in coffee somatic embryogenesis. The expression patterns of ethylene response factor 8 and 13 in embryogenic calluses and embryogenic cell suspensions showed that these genes can potentially act as somatic embryogenic markers.

Genome-wide identification and expression analysis of the SR gene family in longan (Dimocarpus longan Lour.)

Longan (Dimocarpus longan Lour.) is an important commercial fruit tree in southern China. The embryogenesis of longan affects the quality and yield of fruit. A large number of alternative splicing events occurs during somatic embryogenesis (SE), which is regulated by serine/arginine-rich (SR) proteins. However, the functions of SR proteins in longan are poorly understood. In this study, 21 Dlo-SR gene family members belonging to six subfamilies were identified, among which Dlo-RSZ20a, Dlo-SR30, Dlo-SR17, Dlo-SR53 and Dlo-SR32 were localized in the nucleus, Dlo-RSZ20b, Dlo-RSZ20c, Dlo-RSZ20d, Dlo-SC18, Dlo-RS2Z29, Dlo-SCL41, and Dlo-SR33 were localized in chloroplasts, and Dlo-RS43, Dlo-SC33, Dlo-SC37, Dlo-RS2Z33, Dlo-RS2Z16, Dlo-RS2Z24, Dlo-SCL43, Dlo-SR112, and Dlo-SR59 were localized in the nucleus and chloroplasts. The Dlo-SR genes exhibited differential expression patterns in different tissues of longan. The transcript levels of Dlo-RSZ20a, Dlo-SC18, Dlo-RS2Z29, DLo-SR59, Dlo-SR53, and Dlo-SR17 were low in all analyzed tissues, whereas Dlo-RS43, Dlo-RS2Z16, Dlo-RS2Z24, and Dlo-SR30 were highly expressed in all tissues. To clarify their function during SE, the transcript levels of Dlo-SR genes were analyzed at different four stages of SE, comprising non-embryonic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC) and globular embryo (GE). Interestingly, the transcript levels of Dlo-RS2Z29 and Dlo-SR112 were increased in embryogenic cells compared with the NEC stage, whereas transcript levels of Dlo-RSZ20a, Dlo-RS43, Dlo-SC37, and Dlo-RS2Z16 were especially increased at the GE stage compared with the other stages. Alternative splicing events of Dlo-SR mRNA precursors (pre-mRNAs) was detected during SE, with totals of 41, 29, 35, and 44 events detected during NEC, EC, ICpEC, and GE respectively. Protein–protein interaction analysis showed that SR proteins were capable of interaction with each other. The results indicate that the alternative splicing of Dlo-SR pre-mRNAs occurs during SE and that Dlo-SR proteins may interact to regulate embryogenesis of longan.

Key Techniques for Somatic Embryogenesis and Plant Regeneration of Pinus koraiensis

Korean pine is the dominant species of Korean pine forests. It is an economically valuable species that yields oil, high-quality timber and nuts, and it offers great prospects for further development. Complete regenerated plants of Korean pine were obtained via somatic embryogenesis using megagametophytes as the explant. The seeds of 27 families of Korean pine were collected to induce embryogenic lines. We compared the effects of explant collection time, family and medium components (concentrations of sucrose, plant growth regulators and acid-hydrolyzed casein) on embryogenic lines induction. The effects of plant growth regulators and L-glutamine contents on the proliferation and maturation of embryogenic cell lines were studied, and the germinating ability of different cell lines was evaluated. The embryogenic lines induction percentage of Korean pine reached 33.33%. When 4.52 μmol·L−1 2,4-D and 2.2 μmol·L−1 6-BA were added to the medium of embryogenic lines proliferation, the ability of embryo maturation was the best (cell line 001#-100 was 135.71·g−1 fresh weight). Adding 1–1.5g L−1 L-glutamine to the proliferation medium can improve the ability of embryo maturation (cell line 001#-100 was 165.63·g−1 fresh weight). The germination percentage of the three cell lines tested was significant, and the highest was 66%. We report on successful regeneration and cryopreservation methods for somatic embryos of Korean pine. This technology could be used to propagate the excellent germplasm resources of Korean pine and to establish multi-varietal forestry.

Somatic Embryogenesis and Plant Regeneration from Sugi (Japanese Cedar, Cryptomeria japonica D. Don, Cupressaceae) Seed Families by Marker Assisted Selection for the Male Sterility Allele ms1.

One of the possible countermeasures for pollinosis caused by sugi (Cryptomeria japonica), a serious public health problem in Japan, is the use of male sterile plants (MSPs; pollen-free plants). However, the production efficiencies of MSPs raised by conventional methods are extremely poor, time consuming, and resulting in a high seedling cost. Here, we report the development of a novel technique for efficient production of MSPs, which combines marker-assisted selection (MAS) and somatic embryogenesis (SE). SE from four full sib seed families of sugi, carrying the male sterility gene MS1, was initiated using megagametophyte explants that originated from four seed collections taken at one-week intervals during the month of July 2017. Embryogenic cell lines (ECLs) were achieved in all families, with initiation rates varying from 0.6% to 59%. Somatic embryos were produced from genetic marker-selected male sterile ECLs on medium containing maltose, abscisic acid (ABA), polyethylene glycol (PEG), and activated charcoal (AC). Subsequently, high frequencies of germination and plant conversion (≥76%) were obtained on plant growth regulator-free medium. Regenerated plantlets were acclimatized successfully, and the initial growth of male sterile somatic plants was monitored in the field.

Overexpressing rice lesion simulating disease 1-like gene (OsLOL1) in Gossypium hirsutum promotes somatic embryogenesis and plant regeneration

BackgroundCotton somatic embryogenesis is difficult or rarely frequent to present, which has limited gene function identification and biotechnological utility. Here, we employed a rice key somatic embryogenesis-related gene, rice lesion simulating disease 1-like gene (OsLOL1), to develop transgenic cotton callus for evaluating its function in ectopic plants.ResultsOverexpressing OsLOL1 can promote cotton callus to form embryogenic callus, not only shortening time but also increasing transition of somatic callus cells to embryogenic callus cells. And the regenerating plantlets per transgenic OsLOL1 embryogenic callus were significantly higher than those in the control transformed with empty vector. Analysis of physiological and biochemical showed that OsLOL1 can repress cotton superoxide dismutase 1 gene (GhSOD1) expression, possibly resulting in reactive oxidant species (ROS) accumulation in transgenic callus cells. And OsLOL1-overexpressed embryogenic callus exhibited higher α-amylase activity compared with the control, resulting from the promotion of OsLOL1 to cotton amylase 7 gene (GhAmy7) and GhAmy8 expression.ConclusionThe data showed that OsLOL1 could be used as a candidate gene to transform cotton to increase its somatic embryogenesis capacity, facilitating gene function analysis and molecular breeding in cotton.

Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations.

We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.

Induction of Ced9 mediated anti-apoptosis in commercial banana cultivar Rasthali for stable resistance against Fusarium wilt.

Anti-apoptotic gene Ced-9 enhanced resistance against Fusarium oxysporum f. sp. cubense (Foc) in the susceptible banana cultivar Rasthali by arresting tissue necrosis. The embryogenic cell suspension of banana cultivar Rasthali was stably transformed with Ced-9 gene and transformed lines were regenerated independently. The putative transgenic lines were analyzed with PCR using gene primers and further subjected to Southern blot to estimate copy number. The root-challenge bioassay with Foc showed 17-51% Vascular Discoloration Index in independent transformants compared to untransformed banana cv Rasthali (98% VDI). Four transgenic events showed a higher level of resistance over a period of 6months. Overcoming tissue necrosis is the most ideal method to avoid Fusarium multiplication and spread in banana. Oxidative stress-induced cell necrosis is prevented by the activation of antiapoptotic pathways by Ced-9 and is proving to be an effective method to control this dreaded disease. This is the first report from India on the generation of transgenic banana cultivar Rasthali expressing antiapoptotic Ced-9 gene for resistance to Fusarium wilt.

Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells

Key messageIn vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways.In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

Somatic embryogenesis in artificially pollinated seed families of 2nd generation plus trees and cryopreservation of embryogenic tissue in Cryptomeria japonica D. Don (Sugi).

Cryptomeria japonica D. Don (common name is Sugi or Japanese cedar) is the most important forestation tree species in Japan, and 2nd generation plus trees with superior traits have been selected by breeding projects. Biotechnological approaches such as genetic transformation and genome editing are expected to accelerate to add useful traits (e.g., no-pollen traits) to superior trees in short time. To develop a platform for genetic transformation and genome editing of C. japonica superior trees, this study investigated the embryogenic potential of 2nd generation plus trees and obtained good cell lines with high embryogenic potential, which could be useful material for adding new and useful traits to superior trees by genetic transformation. However, the maintenance of embryogenic cell lines is laborious, and prolonged subculture leads to a loss of embryogenesis potential. Therefore, cell lines need to be cryopreserved for long without subculture. Therefore, in this study we made a simple cryopreservation protocol suitable for most C. japonica cell lines. We showed that cryopreserved cells using this protocol formed somatic embryos, which were then converted to plantlets. Transgenic cells produced from cryopreserved cells expressed transgene, gfp. These results indicated that our cryopreservation protocol can be used for prolonged storage of genetic transformation target materials in C. japonica.

Phytochemical Analysis and Establishment of Embryogenic Cell Suspension and Agrobacterium-mediated Transformation for Farmer Preferred Cultivars of West African Plantain (Musa spp.).

Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l’Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l’Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by Agrobacterium-mediated transformation with reporter gene constructs and regenerated into whole plants.

20-Hydroxyecdysone-responsive microRNAs of insects

ABSTRACT 20-Hydroxyecdysone (20-HE) plays essential roles in coordinating developmental transitions of insects through responsive protein-coding genes and microRNAs (miRNAs). The involvement of single miRNAs in the ecdysone-signalling pathways has been extensively explored, but the interplay between ecdysone and the majority of miRNAs still remains largely unknown. Here, by small RNA sequencing, we systematically investigated the genome-wide responses of miRNAs to 20-HE in the embryogenic cell lines of Bombyx mori and Drosophila melanogaster. Over 60 and 70 20-HE-responsive miRNAs were identified in the BmE cell line and S2 cell line, respectively. The response of miRNAs to ecdysone exhibited a time-dependent pattern, and the response intensity increased with extending exposure to 20-HE. The relationship between ecdysone and the miRNAs was further explored through knockdown of ecdysone-signalling pathway genes. Specifically, ecdysone regulated the cluster miR-275 and miR-305 through the coordination of BmEcR-B and downstream BmE75B, and the interaction between BmEcR and miR-275 cluster was strengthened by the feedback regulation of BmE75B. Ecdysone induced miR-275-3p and miR-305-5p through the ecdysone response effectors (EcREs) at the upstream of the pre-miR-275 cluster. Overall, the results might help us further understand the relationship between ecdysone signalling pathways and small RNAs in the development and metamorphosis of insects.

Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm. Callus can be induced from immature embryos of Korean pine (Pinus koraiensis Sieb. et Zucc.; Pinaceae) as explants, but there are problems, such as low proliferation efficiency, loss of embryogenicity, poor vigor; thus, best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear. To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation, we varied hormone concentration, subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages, then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design. The results showed that the family origin and collection date of explants significantly affected callus induction (induction rate reached 1.67%). Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L−1 6-benzyl adenine, 1 mg L−1 naphthaleneacetic acid, 30 g L−1 sucrose, 500 mg L−1, l-glutamine, 500 mg L−1 casein hydrolysis and 6.5 g L−1 agar. In addition, the combination of 2,4-dichlorophenoxyacetic acid + 6-benzyl adenine also had a better proliferative effect on callus. The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different. Transfer to new medium every 13–15 days not only maintained robust callus vigor, but also yielded a larger proliferation coefficient. The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.

Pinus canariensis plant regeneration through somatic embryogenesis

Aim of the study: To develop an efficient method to regenerate plants through somatic embryogenesis of an ecologically relevant tree species such as Pinus canariensis.Area of study: The study was conducted in the research laboratories of Neiker-Tecnalia (Arkaute, Spain).Material and methods: Green cones of Pinus canariensis from two collection dates were processed and the resulting immature zygotic embryos were cultured on three basal media. The initiated embryogenic tissues were proliferated testing two subculture frequencies, and the obtained embryogenic cell lines were subjected to maturation. Germination of the produced somatic embryos was conducted and acclimatization was carried out in a greenhouse under controlled conditions.Main results: Actively proliferating embryogenic cell lines were obtained and well-formed somatic embryos that successfully germinated were acclimatized in the greenhouse showing a proper growth.Research highlights: This is the first report on Pinus canariensis somatic embryogenesis, opening the way for a powerful biotechnological tool for both research purposes and massive vegetative propagation of this species.Keywords: acclimatization; Canary Island pine; micropropagation; embryogenic tissue; somatic embryo.Abbreviations used: embryogenic tissue (ET); established cell line (ECL); somatic embryogenesis (SE); somatic embryos (Se’s).

Highly Efficient Biolistic Transformation of Embryogenic Cell Suspensions of Banana Via a Liquid Medium Selection System

An efficient biolistic transformation system of banana combined with a liquid medium selection system was developed during this study. An embryogenic cell suspension (ECS) of Musa acuminata cv. Baxi (AAA) was bombarded with a particle delivery system. After 7 days of restoring culture in liquid M2 medium, embryogenic cells were transferred to a liquid selection M2 medium supplemented with 10 μg/mL hygromycin for resistance screening. The untransformed cell clusters were inhibited or killed, and a small number of transformants proliferated in the liquid selection medium. After the 0th, first, second, and third generation of antibiotic screening, there were 0, 65, 212, and 320, respectively, vitality-resistant buds obtained from a 0.5-mL packed cell volume (PCV) of embryogenic cell suspension. The β-glucuronidase (GUS) staining, polymerase chain reaction (PCR) analysis, and Southern blot hybridization results all demonstrated a 100% positive rate of regenerated resistant seedlings. Interestingly, the number of buds obtained through third-generation screening was almost equal to that obtained from the original ECS in M2 medium without antibiotics. These results suggested that the liquid medium selection system facilitated the proliferation of a positive transgenic ECS, which significantly improved the regeneration rate of transformants. This protocol is suitable for the genetic transformation of all banana genotypes and is highly advantageous to varieties with low callusing potential.