Immature Zygotic Embryos Research Articles

Somatic Embryogenesis in Selected Conifer Trees Pinus nigra Arn. and Abies Hybrids.

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.

Transcriptome-wide effect of DE-ETIOLATED1 (DET1) suppression in embryogenic callus of Carica papaya.

Papaya is one of the most nutritional fruits, rich in vitamins, carotenoids, flavonoids and other antioxidants. Previous studies showed phytonutrient improvement without affecting quality in tomato fruit and rapeseed through the suppression of DE-ETIOLATED-1 (DET1), a negative regulator in photomorphogenesis. This study is conducted to study the effects of DET1 gene suppression in papaya embryogenic callus. Immature zygotic embryos were transformed with constitutive expression of a hairpin DET1 construct (hpDET1). PCR screening of transformed calli and reverse transcription quantitative PCR (RT-qPCR) verified that DET1 gene downregulation in two of the positive transformants. High-throughput cDNA 3' ends sequencing on DET1-suppressed and control calli for transcriptomic analysis of global gene expression identified a total of 452 significant (FDR < 0.05) differentially expressed genes (DEGs) upon DET1 suppression. The 123 upregulated DEGs were mainly involved in phenylpropanoid biosynthesis and stress responses, compared to 329 downregulated DEGs involved in developmental processes, lipid metabolism, and response to various stimuli. This is the first study to demonstrate transcriptome-wide relationship between light-regulated pathway and secondary metabolite biosynthetic pathways in papaya. This further supports that the manipulation of regulatory gene involved in light-regulated pathway is possible for phytonutrient improvement of tropical fruit crops.

Assessment of genetic stability in somatic embryo derived plantlets of Pterocarpus marsupium Roxb. using inter-simple sequence repeat analysis.

An efficient plantlet regeneration protocol using immature zygotic embryos (IZEs) via somatic embryogenesis has been developed in Pterocarpus marsupium Roxb. The regenerated plantlets were evaluated for their genetic stability. IZEs were incubated on Murashige and Skoog (MS) media augmented with 1.07-16.11μM naphthalene acetic acid (NAA) or 0.90-13.97μM 2,4-dichlorophenoxyacetic acid. The optimum callus induction (96.6%) was observed on MS medium augmented with 5.37μM NAA. Induction of somatic embryos (SEs) was observed after sub-culture of callion medium with decreased concentrations of NAA (0.54-5.37μM), either alone or 2.69μM NAA in combination with 2.22-8.90μM benzyladenine (BA) or 2.32-9.30μM Kinetin. Maximum number (33.4 ± 0.85) of SEs occurred on MS medium augmented with 2.69μM NAA + 4.40μM BA + 3% sucrose. Highest percentage (67.3 ± 0.37) of SEs matured and developed into cotyledonary stage by subsequent subculture on the same medium. SE formation and maturation decreased when sucrose concentrations were higher than 3%. Seventy percent of mature somatic embryos developed into plantlets on half strength MS medium augmented with 5.80µM gibberellic acid. The various stages of development during somatic embryogenesis includeglobular, heart, torpedo and mature stages as revealed by thestereomicroscopic and histological studies of explants. Plantlets derived from SEs were successfully acclimatized in the greenhouse with a survival rate of 78%. Among the survived plantlets, 9 plantlets were randomly selected for inter-simple sequence repeat (ISSR) analysis. Of the 13 primers used, 8 produced reproducible and monomorphic bands. ISSR analysis revealed a homogenous amplification profile for all regenerated plantlets analyzed validating the genetic stability of somatic embryo derived plantlets.

Embryogenesis followed by enhanced micro-multiplication and eco-restoration of Calamus thwaitesii Becc.: an economic non-wood forest produce for strengthening agroforestry system

The present study is focussed on development of a high-frequency micro-multiplication system in Calamus thwaitesii, through somatic embryogenesis from immature zygotic embryos cultured in Murashige and Skoog (MS) medium supplemented with 31.68 µM 2,4-dichlorophenoxyacetic acid (2,4-D). The semi-friable calli when cultured in the same medium augmented with 2.22 µM 6-benzylaminopurine (BAP) and 1.07 µM α-naphthalene acetic acid (NAA) induced ~ 12 discrete globular embryoids in 6 weeks. The isolated embryoids in hormone-free media yielded 65% plantlets. Furthermore, embryoids and axenic shoots exhibited maximum shoot induction in medium supplemented with 0.45 µM Thidiazuron (TDZ). The shoot initials after subculture in media supplemented with 1.78 µM BAP and 0.45 µM TDZ produced shoot proliferation followed by elongation in basal medium. The elongated shoots produced roots in media supplemented with 16.11 µM NAA. With this established protocol, ~ 5940 rooted plantlets could be harvested after 40 weeks from a single embryoid. Genetic stability analysis of the plantlets using inter simple sequence repeat markers recorded only 0.05% genetic polymorphism. The plantlets were hardened in a mist house for 8 weeks, and then to 50% shade house for another 16 weeks, and the well-established 6-month-old nursery plants, reintroduced to selected forest segments, exhibited 86% field establishment even after 3 years of observation. Thus, the mass multiplication system developed could be a breakthrough for large-scale multiplication of C. thwaitesii to ensure continuous supply of quality planting material to the cottage industry through the development of agroforestry systems. Furthermore, the in vitro culture system developed here can be replicated for research activities related to the long-term–short-term conservation, micro-multiplication and sustainable utilization of rare, endangered, endemic, monopodial/single stemmed rattan palms.

Somatic Embryogenesis of Quercus suber L. From Immature Zygotic Embryos.

Quercus suber L., cork oak, is a forest tree of high social and economic value. The cork is traditionally used in the wine industry to produce bottle stoppers, but it is also a very good material for both thermal and acoustic insulation in construction. Since its harvest does not harm the tree, the use of cork in the industry has a positive impact on the environment.Somatic embryogenesis is considered a feasible system for in vitro regeneration procedures, with many advantages in woody species. Classical genetic breeding programs have important limitations in forest trees, like cork oak, due to their long life span and difficulties of seed conservation and vegetative reproduction. Therefore, somatic embryogenesis has a great potential for large-scale propagation and cryopreservation of elite genotypes, as well as for transformation strategies. In the case of Q. suber, several in vitro propagation systems through somatic embryogenesis have been reported, with different efficiency rates.In the present chapter, updated information is reported about an efficient protocol for induction of somatic embryogenesis of Q. suber from immature zygotic embryos, as well as methods for proliferation and maturation of somatic embryos, germination, plantlet regeneration, and acclimatization.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook.

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses.

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

Key messageThis paper describes the micropropagation of Douglas-fir via somatic embryogenesis using media and methods available in the public domain.Douglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programmes is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation is essential biotechnology tool in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on the modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Plant Cell Rep 4(6):325–328, 1985***). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realize increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of Pinus radiata D. don

Pinus radiata is the most important conifer species for commercial forestry in countries such as Australia, New Zeeland and Chile. Nowadays, SE (somatic embryogenesis) is considered the most promising in vitro method for large scale vegetative propagation of woody plants. The understanding of the molecular basis of SE is in its very beginning and a number of embryogenesis-related genes have been identified in conifers. Among the molecular mechanisms involved in regulation of SE, DNA methylation, which is an epigenetic modification associated with transcriptional silencing, has shown to be a pivotal factor controlling gene expression. In this work, we studied the morphological and molecular differences between cell lines previously characterized in terms of their embryogenic potential as embryogenic (E) and non embryogenic (NE), obtained from immature zygotic embryos of P. radiata. In contrast to E lines, NE lines were composed of multicellular aggregates lacking polarity, and they were characterized by the presence of significantly lower transcript levels of embryogenesis-related genes and higher global DNA methylation. Furthermore, the detection of vibrational markers of DNA conformation indicated that DNA samples obtained from E lines presented the common B-DNA conformation, while NE samples presented Z-conformation. Taken together, our results highlight the role of epigenetic mechanisms such as DNA methylation in regulating the expression of embryogenesis- related genes, having impact on the embryo patterning and cell differentiation.

Insights into the early stage of Pinus nigra Arn. somatic embryogenesis using discovery proteomics

The somatic embryogenesis in conifers represents a suitable model of plant regeneration system facilitating studies of fundamental aspects of an early development as well as in vitro micropropagation. The aim of our study was to deeper understand the somatic embryogenesis in the conifer tree Pinus nigra Arn. Comparative proteomic analysis based on 2D-PAGE in 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) initiated from cotyledons of somatic seedlings of the same genotypes, 3) embryogenic tissues that lost the maturation capacity (E-L) of two cell lines (E362, E366). Investigated pine tissues showed distinct structural features. The 24 protein spots were altered in both cell lines in comparison of embryogenic and non-embryogenic tissues. These proteins are involved in disease and defence mechanism, energy metabolism and biosynthesis of cell wall components. Two of three protein spots detected only in embryogenic form of both cell lines are similar to water deficit inducible protein LP3, the third remains uncharacterised. The loss of the maturation capacity was accompanied by changes in 35 and 38 protein spots in 362 and 366 cell lines, respectively. Only two of them were altered in both cell lines, suggesting non-uniform process of ageing. Biological significanceSomatic embryogenesis in conifers represents an experimental system for the study of early plant development as well as a biotechnological tool for large-scale micropropagation. The obtained results give a new insight into the process of somatic embryogenesis of a conifer Pinus nigra Arn. by revealing differences at proteomic levels among in vitro cultured tissues characterised by different embryogenic potential. Microscopic investigations have also shown differences in the structural organisation of studied tissues.

Callus induction and plant regeneration from immature zygotic embryos of various maize genotypes (Zea mays L.)

옥수수의 최적 조직 배양 조건을 확립하기 위하여 옥수수 국내 5 계통과 국외 11 계통 총 16 계통을 포트와 포장 재배하여 미성숙 배를 분리하여 배발생 캘러스 유도 및 식물체 재분화율을 조사하였다. MS 배지에 auxin으로 1.5 mg/L Dicamba와 0.5 mg/L 2,4-D 가 첨가된 배지에서 캘러스 형성은 본 실험에 사용된 옥수수 ...

Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon.

The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species.

Somatic embryogenesis from immature and mature zygotic embryos of the açaí palm (Euterpe oleracea): Induction of embryogenic cultures, morphoanatomy and its morphological characteristics

A somatic embryogenesis protocol for the açaí palm (Euterpe oleracea Mart.) was developed, based on mature and immature zygotic embryos, to define morphoanatomically the process’s different stages and to analyze the homogeneity of nuclear DNA content by flow cytometry from calli, somatic embryos and regenerated plants. Auxin picloram (4-amino-3,5,6-trichloropicolinic acid) was tested to induce embryogenic calli at 225 and 450μM concentrations, coupled to the physiological maturing stages of zygotic embryos (mature and immature). Murashige and Skoog (MS) medium with 30gl−1 sucrose, 2.5gl−1 Phytagel, 2.5gl−1 activated charcoal and 0.5gl−1l-glutamine was employed for callus induction. Embryogenic calli with somatic embryos in initial differentiation were transferred to a culture medium with 12.3μM of 2iP and 0.6μM of NAA and 300mgl−1 of activated charcoal for differentiating and maturing somatic embryos. Plant regeneration occurred in a medium with 1.0μM BAP (N6-benzylaminopurine) and 0.5μM GA3 (gibberellic acid). The formation of embryogenic calli in all treatments was observed in the induction medium, regardless of the stage of development of the zygotic embryo. Picloram at 450μM concentration provided the best results in forming embryogenic calli (84.7%). In the differentiating and maturing stage, 100% of the explants that had an embryogenic callus formation resulted in somatic embryos. The largest rate of plant regeneration (58.7%) was noted in treatment with an induction medium of 450μM of picloram and somatic embryos obtained from immature zygotic embryos. Morphoanatomical analyses evidenced that induction of somatic embryogenesis reflected stages characteristic of the indirect kind. Regenerated plants showed normal development, with growth of roots and aerial part. Calli, somatic embryos and plants, analyzed by flow cytometry, revealed no significant differences in the estimated rates of DNA content.

Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize ( Zea Mays ) Varieties

In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL-1 L-proline and 0.8 mgL-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties.

De novo comparative transcriptome analysis provides new insights into sucrose induced somatic embryogenesis in camphor tree (Cinnamomum camphora L.).

BackgroundSomatic embryogenesis is a notable illustration of cell totipotency, by which somatic cells undergo dedifferentiation and then differentiate into somatic embryos. Our previous work demonstrated that pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in camphor tree. To better understand the molecular basis of somatic embryogenesis induced by osmotic stress, de novo transcriptome sequencing of three tissues of camphor tree (immature zygotic embryos, sucrose-pretreated immature zygotic embryos, and somatic embryos induced from sucrose-pretreated zygotic embryos) were conducted using Illumina Hiseq 2000 platform.ResultsA total of 30.70 G high quality clean reads were obtained from cDNA libraries of the three samples. The overall de novo assembly of cDNA sequence data generated 205592 transcripts, with an average length of 998 bp. 114229 unigenes (55.56 % of all transcripts) with an average length of 680 bp were annotated with gene descriptions, gene ontology terms or metabolic pathways based on Blastx search against Nr, Nt, Swissprot, GO, COG/KOG, and KEGG databases. CEGMA software identified 237 out of 248 ultra-conserved core proteins as ‘complete’ in the transcriptome assembly, showing a completeness of 95.6 %. A total of 897 genes previously annotated to be potentially involved in somatic embryogenesis were identified. Comparative transcriptome analysis showed that a total of 3335 genes were differentially expressed in the three samples. The differentially expressed genes were divided into six groups based on K-means clustering. Expression level analysis of 52 somatic embryogenesis-related genes indicated a high correlation between RNA-seq and qRT-PCR data. Gene enrichment analysis showed significantly differential expression of genes responding to stress and stimulus.ConclusionsThe present work reported a de novo transcriptome assembly and global analysis focused on gene expression changes during initiation and formation of somatic embryos in camphor tree. Differential expression of somatic embryogenesis-related genes indicates that sucrose induced somatic embryogenesis may share or partly share the mechanisms of somatic embryogenesis induced by plant hormones. This study provides comprehensive transcript information and gene expression data for camphor tree. It could also serve as an important platform resource for further functional studies in plant embryogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2357-8) contains supplementary material, which is available to authorized users.

Recurrent Somatic Embryogenesis and Plantlet Regeneration in Psidium guajava L.

A simple and efficient protocol for recurrent somatic embryogenesis and plant regeneration is one of the prerequisites for genetic improvement of guava. An efficient reproducible regeneration somatic embryogenesis protocol was developed in four genotypes of Psidium guajava L. using immature zygotic embryo as starter explant. Somatic embryogenesis induction was obtained on MS basal medium supplemented with 2.0 mgL-1 2, 4-D, 400 mgL-1 L-glutamine, 6% sucrose and 500 mgL-1 Malt extract. Following SE induction different developmental stages of somatic embryos (Globular, heart-shaped, torpedo, cotyledonary) was directly obtained and further recurrent embryogenesis also obtained upon prolonged incubation in induction media. Addition of polyethylene glycol (50 mgL-1) significantly improved the embryos maturation in MS supplemented with and 3% sucrose. The regeneration in MS medium supplemented with BAP (0.5 mgL-1), NAA (0.2 mgL-1), casein hydrolysate (100 mgL-1) and 3% sucrose. High plant regeneration frequency and intensity of somatic embryos (58.5%) obtained. Plant maturation on MS medium supplemented with BAP 2.0 mgL-1 with 2% sucrose. The rooted plants was successfully acclimatize in the greenhouse with a survival rate of 85%. This somatic embryogenesis protocol developed would be helpful in establishment of genetically modified guava aimed at seedlessness, increased shelf life and wilt disease.

Somatic Embryogenesis in Olive (Olea europaea L. subsp. europaea var. sativa and var. sylvestris).

Protocols for olive somatic embryogenesis from zygotic embryos and mature tissues have been described for both Olea europaea sub. europaea var. sativa and var. sylvestris. Immature zygotic embryos (no more than 75 days old), used after fruit collection or stored at 12-14 °C for 2-3 months, are the best responsive explants and very slightly genotype dependent, and one single protocol can be effective for a wide range of genotypes. On the contrary, protocols for mature zygotic embryos and for mature tissue of cultivars are often genotype specific, so that they may require many adjustments according to genotypes. The use of thidiazuron and cefotaxime seems to be an important trigger for induction phase particularly for tissues derived from cultivars. Up to now, however, the application of this technique for large-scale propagation is hampered also by the low rate of embryo germination; it proves nonetheless very useful for genetic improvement.

Somatic Embryogenesis and Plant Regeneration from Zygotic Embryo in &amp;lt;i&amp;gt;Carica papaya&amp;lt;/i&amp;gt; L., cv. Red-Lady

Somatic embryogenesis creates a number of opportunities to facilitate large-scale propagation, synthetic seed production, genetic improvement through somaclonal variation, in vitro mutagenesis, protoplast fusion and genetic transformation. Induction of somatic embryogenesis from the vegetative parts of papaya plant was met with low success rates and a slow process of regeneration. Success depends on the choice of explants, the species being used and on various methods of embryogenesis will study. The most suitable explant for somatic embryogenesis is a large portion of either meristematic tissue or cell that retain an ability to express totipotency. Various investigations were made on somatic embryo induction of Carica spp. such as ovules of immature fruits, zygotic embryos from immature fruits, axillary buds, peduncles of immature fruits etc. In this study, somatic embryo induction from zygotic embryo of an immature fruit was examined. Various concentrations of 2,4-D and two levels of sucrose concentrations with or without addition of glutamine were assessed to determine the best response for induction of somatic embryogenesis from immature zygotic embryo of immature fruits in papaya cv. Red-lady (Carica papaya L.). The results showed that MS medium supplemented with 5.0 mg/l 2,4-D + 400 mg/l glutamine + 60 g/l sucrose promoted the formation of highest (70%) percentage of somatic embryo and also the average maximum number (35.80±4.40) of somatic embryo per explant. The highest (44%) percentage of somatic embryo germination into complete plantlets were obtained on MS medium devoid of plant growth regulators. The embryo developed only shoots of 22% and only roots of 10% instead of complete plantlet formation in this medium of MS0. The average highest (4.25 ± 0.32 cm) shoot length per germinated somatic embryo derived complete plantlets were also achieved on MS0 medium. The maximum (90%) percentage of root induction and average highest (7.50 ± 0.75) number of root per somatic embryo derived shoot and also the average maximum (5.20 ± 0.40 cm) root length were obtained on MS medium without plant growth regulators. The survival rate of somatic embryo derived plantlets in the field was about 10% to 50% depending on management practices.

Plant regeneration through somatic embryogenesis and shoot organogenesis from immature zygotic embryos of Sapium sebiferum Roxb.

Sapium sebiferium Roxb. is a highly valued ornamental plant species in tropical and subtropical regions. Here we present an efficient protocol for plant regeneration from immature zygotic embryos (IZEs) of S. sebiferium through direct somatic embryogenesis and shoot organogenesis for the first time. The results showed that mechanical damage, plant growth regulators (PGRs) and light regimes had great influence on in vitro morphogenesis. High frequency of somatic embryogenesis was obtained when the mechanical damaged IZEs were cultured on the Murashige and Skoog (MS) medium containing 1.0mgl−1 indole-3-acetic acid (IAA) under continuous darkness for two weeks followed by a light/dark photoperiod cycle for four weeks. The induction frequency of somatic embryogenesis was up to 90.0%. Somatic embryos (SEs) at early cotyledonary stage showed highest proliferation frequency (81.7%) and 80.0% of secondary somatic embryos (SSEs) germinated on plant growth regulators (PGRs)-free MS medium. For direct shoot induction pathway, the best shoot regeneration rate (63.3%) was obtained on the medium supplemented with 1.0mgl−1 6-benzyladenine (6-BA). The plantlets derived from two ways were successfully acclimatized in the greenhouse with more than 90% survival rate. This study will be beneficial for the rescue of immature embryo, inheritance of heterosis as well as the genetic improvement of S. sebiferium.

Maize miRNA and target regulation in response to hormone depletion and light exposure during somatic embryogenesis.

Maize somatic embryogenesis (SE) is induced from the immature zygotic embryo in darkness and under the appropriate hormones' levels. Small RNA expression is reprogrammed and certain miRNAs become particularly enriched during induction while others, characteristic to the zygotic embryo, decrease. To explore the impact of different environmental cues on miRNA regulation in maize SE, we tested specific miRNA abundance and their target gene expression in response to photoperiod and hormone depletion for two different maize cultivars (VS-535 and H-565). The expression levels of miR156, miR159, miR164, miR168, miR397, miR398, miR408, miR528, and some predicted targets (SBP23, GA-MYB, CUC2, AGO1c, LAC2, SOD9, GR1, SOD1A, PLC) were examined upon staged hormone depletion in the presence of light photoperiod or darkness. Almost all examined miRNA, except miR159, increased upon hormone depletion, regardless photoperiod absence/presence. miR528, miR408, and miR398 changed the most. On the other hand, expression of miRNA target genes was strongly regulated by the photoperiod exposure. Stress-related miRNA targets showed greater differences between cultivars than development-related targets. miRNA/target inverse relationship was more frequently observed in darkness than light. Interestingly, miR528, but not miR159, miR168 or miR398, was located on polyribosome fractions suggesting a role for this miRNA at the level of translation. Overall our results demonstrate that hormone depletion exerts a great influence on specific miRNA expression during plant regeneration independently of light. However, their targets are additionally influenced by the presence of photoperiod. The reproducibility or differences observed for particular miRNA-target regulation between two different highly embryogenic genotypes provide clues for conserved miRNA roles within the SE process.