Non-embryogenic Callus Research Articles

Changes in biochemistry and histochemical characteristics during somatic embryogenesis in Ormosia henryi Prain

Mature embryos were used as an explant for embryogenic callus (EC) induction, and then EC was further developed to form somatic embryos during somatic embryogenesis (SE) of Ormosia henryi Prain; however, some mature embryos could induced non-embryogenic callus (NEC), browning callus (BC) or snowflake callus (SC). The contents of soluble sugar, starch, soluble protein, H2O2, and endogenous hormones and the activities of polyphenoloxidase (PPO), superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT) were measured at different SE stages, such as EC, globular embryo (GE), and cotyledon embryo (CE), and in abnormal tissue, such as NEC, BC, and SC. The results showed that the contents of soluble sugar and starch; the activities of PPO, SOD, APX and POD; and the ratios of indole-3-acetic acid/abscisic acid (IAA/ABA), IAA/gibberellins (IAA/GAs), auxin /GAs (AUX/GAs), and AUX/ABA decreased gradually at different SE stages. In contrast, the contents of soluble protein, H2O2, all endogenous hormones gradually increased. However, CAT activity and the ratios of IAA/cytokinins (IAA/CKs), AUX/CKs, ABA/CKs, and GAs/CKs first increased and then decreased. The high contents of GAs and ABA, high ratios of ABA/CKs and GAs/CKs and low ratios of IAA/ABA, IAA/GAs, AUX/GAs and AUX/ABA were responsible for the inability of the callus to form EC. The low enzyme activities, low contents of energy substances and H2O2 were related to NEC formation. The high contents of soluble sugar, H2O2, AUX, CKs and PPO activity and the low content of soluble protein were the basic causes of BC formation. The high-energy substances contents and low activities of SOD and POD facilitated SC formation. These results showed that energy substances were the material basis of SE, which affected enzyme activities, regulated reactive oxygen species (ROS) metabolism, and thus regulated the morphogenesis and development of somatic embryos. In addition, the contents and ratios of endogenous hormones affected the dedifferentiation, redifferentiation and embryogenesis of somatic cells. Somatic embryogenesis and abnormal callus tissues formation of Ormosia henryi Prain were associated with energy substances, reactive oxygen species, enzyme activities and endogenous hormones, as well as histochemical characteristics.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Establishment of Pluripotent Cell Cultures to Explore Allelopathic Activity of Coffee Cells by Protoplast Co-Culture Bioassay Method.

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

Polycomb Repressive Complex 2-mediated histone modification H3K27me3 is associated with embryogenic potential in Norway spruce.

Epigenetic reprogramming during germ cell formation is essential to gain pluripotency and thus embryogenic potential. The histone modification H3K27me3, which is catalysed by the Polycomb repressive complex 2 (PRC2), regulates important developmental processes in both plants and animals, and defects in PRC2 components cause pleiotropic developmental abnormalities. Nevertheless, the role of H3K27me3 in determining embryogenic potential in gymnosperms is still elusive. To address this, we generated H3K27me3 profiles of Norway spruce (Picea abies) embryonic callus and non-embryogenic callus using CUT&RUN, which is a powerful method for chromatin profiling. Here, we show that H3K27me3 mainly accumulated in genic regions in the Norway spruce genome, similarly to what is observed in other plant species. Interestingly, H3K27me3 levels in embryonic callus were much lower than those in the other examined tissues, but markedly increased upon embryo induction. These results show that H3K27me3 levels are associated with the embryogenic potential of a given tissue, and that the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Thus, our study provides novel insights into the role of this epigenetic mark in spruce embryogenesis and reinforces the importance of PRC2 as a key regulator of cell fate determination across different plant species.

Early nodulin 93 protein gene: essential for induction of somatic embryogenesis in oil palm.

Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.

TasiR-ARFs Production and Target Regulation during In Vitro Maize Plant Regeneration.

During in vitro maize plant regeneration somatic cells change their normal fate and undergo restructuring to generate pluripotent cells able to originate new plants. Auxins are essential to achieve such plasticity. Their physiological effects are mediated by auxin response factors (ARFs) that bind auxin responsive elements within gene promoters. Small trans-acting (ta)-siRNAs, originated from miR390-guided TAS3 primary transcript cleavage, target ARF3/4 class (tasiR-ARFs). Here we found that TAS3b precursor as well as derived tasiR-ARFbD5 and tasiR-ARFbD6 display significantly lower levels in non-embryogenic callus (NEC), while TAS3g, miR390 and tasiR-ARFg are more abundant in the same tissue. However, Argonaute (AGO7) and leafbladeless 1 (LBLl) required for tasiR-ARF biogenesis showed significantly higher transcript levels in EC suggesting limited tasiR-ARF biogenesis in NEC. The five maize ARFs targeted by tasiR-ARFs were also significantly enriched in EC and accompanied by higher auxin accumulation with punctuate patterns in this tissue. At hormone half-reduction and photoperiod implementation, plant regeneration initiated from EC with transient TAS3g, miR390 and tasiR-ARFg increase. Upon complete hormone depletion, TAS3b became abundant and derived tasiR-ARFs gradually increased at further regeneration stages. ZmARF transcripts targeted by tasiR-ARFs, as well as AGO7 and LBL1 showed significantly lower levels during regeneration than in EC. These results indicate a dynamic tasiR-ARF mediated regulation throughout maize in vitro plant regeneration.

Comparative transcriptome analysis between embryogenic and nonembryogenic callus of Kalopanax septemlobus

Kalopanax septemlobus is a deciduous medicinal tree species belonging to the family Araliaceae. It contains a number of triterpenoid saponins primarily constituted of hederagenin aglycones. Here, we presented de novo comparative transcriptome analysis using 454 pyrosequencing technology between embryogenic (EC) and nonembryogenic callus (NEC) of K. septemlobus. A total of 372,229 high-quality reads were generated by combining 168,065 EC reads and 204,164 NEC reads, which were then assembled into 80,280 and 63,128 unigenes, respectively. A comparative analysis of transcription profiles showed that 13,766 genes were differentially expressed between EC and NEC. Moreover, functional enrichment analysis revealed that a number of genes involved in the proteolysis and embryogenesis were highly expressed in EC, whereas oxidative stress-responsible genes were overpresented in NEC. Interestingly, the expression levels of genes involved in saponin biosynthesis, such as squalene epoxidase, cytochrome P450s, and glycosyl transferase, were also higher in NEC than in EC, suggesting that their expression is stress-inducible. In this study, we characterized the K. septemlobus transcriptome profiles involved in somatic embryogenesis (SE), which might enhance knowledge of SE process and provide a useful molecular resource for further study of K. septemlobus.

Dynamic Transcriptome Analysis Reveals Uncharacterized Complex Regulatory Pathway Underlying Genotype-Recalcitrant Somatic Embryogenesis Transdifferentiation in Cotton.

As a notable illustration of totipotency and plant regeneration, somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway, the key step for genetic engineering. Investigations examining the totipotency process are of great fundamental and practical importance in crop biotechnology. However, high-frequency regeneration of cotton via SE has been limited due to genotype-dependent response. The molecular basis deciphering SE genotype recalcitrance remains largely unexplored in cotton. In the current study, to comprehensively investigate the dynamic transcriptional profiling and gene regulatory patterns involved in SE process, a genome-wide RNA sequencing analysis was performed in two cotton genotypes with distinct embryogenic abilities, the highly embryogenic genotype Yuzao 1 (YZ) and the recalcitrant genotype Lumian 1 (LM). Three typical developmental staged cultures of early SE—hypocotyls (HY), nonembryogenic calli (NEC) and primary embryogenic calli (PEC)—were selected to establish the transcriptional profiles. Our data revealed that a total of 62,562 transcripts were present amongst different developmental stages in the two genotypes. Of these, 18,394 and 26,514 differentially expressed genes (DEGs) were identified during callus dedifferentiation (NEC-VS-HY) and embryogenic transdifferentiation (PEC-VS-NEC), respectively in the recalcitrant genotype, 21,842 and 22,343 DEGs in the highly embryogenic genotype. Furthermore, DEGs were clustered into six expression patterns during cotton SE process in the two genotypes. Moreover, functional enrichment analysis revealed that DEGs were significantly enriched in fatty acid, tryptophan and pyruvate metabolism in the highly embryogenic genotype and in DNA conformation change otherwise in the recalcitrant genotype. In addition, critical SE-associated expressed transcription factors, as well as alternative splicing events, were notably and preferentially activated during embryogenic transdifferentiation in the highly embryogenic genotype compared with the recalcitrant genotype. Taken together, by systematically comparing two genotypes with distinct embryogenic abilities, the findings in our study revealed a comprehensive overview of the dynamic gene regulatory patterns and uncharacterized complex regulatory pathways during cotton SE genotype-dependent response. Our work provides insights into the molecular basis and important gene resources for understanding the underlying genotype recalcitrance during SE process and plant regeneration, thereby holding great promise for accelerating the application of biotechnology to cotton for improving its breeding efficiency.

Molecular cloning and characterization of five genes from embryogenic callus in Miscanthus lutarioriparius

The regeneration from embryogenic callus of higher plants in tissue culture is regulated by explants types and developmental stage and also regulated by some genes. In Miscanthus lutarioriparius, five candidate genes were selected to decide the differential expression between embryogenic and non-embryogenic calli, including MlARF-GEP (guanine nucleotide-exchange protein of ADP ribosylation factor), MlKHCP (kinesin heavy chain like protein), MlSERK1 (somatic embryogenesis receptor-like kinases 1), MlSERK2 (somatic embryogenesis reportor-like kinases 2), and MlTypA (tyrosine phosphorylation protein A) with Genbank accession numbers KU640196–KU640200. Multiple sequence alignment analysis showed that five genes were highly conserved among members of their gene families respectively. Phylogenetic relationship analysis showed that five genes were closest with homologous genes of Zea mays and Sorghum. The qRT-PCR results showed significant differences of five genes expression pattern between two different callus types, the relative expression in embryogenic callus was detected to exceed in non-embryogenic callus. Furthermore, simple sequence repeats (SSR) marker statistics results via Chi-square showed a significant correlation between MlSERK1 genotype and induction of embryogenic callus in M. lutarioriparius. This study may lay the foundation of the molecular mechanism on the embryogenic callus induction of M. lutarioriparius and perhaps provide some gist for further study on genetic manipulation.

Genome-wide identification and expression analyses of Sm genes reveal their involvement in early somatic embryogenesis in Dimocarpus longan Lour.

The Sm proteins are a conserved protein family with Sm motifs. The family includes Sm and Sm-like proteins, which play important roles in pre-mRNA splicing. Most research on the Sm proteins have been conducted in herbaceous plants, and less in woody plants such as Dimocarpus longan (longan). And the embryo development status significantly affects the quality and yield of longan. In this study, we conducted a genome-wide analysis of longan Sm genes (DlSm) to clarify their roles during somatic embryogenesis (SE) and identified 29 Sm genes. Phylogenetic analysis deduced longan Sm proteins clustered into 17 phylogenetic groups with the homologous Sm proteins of Arabidopsis thaliana. We also analyzed the gene structures, motif compositions, and conserved domains of the longan Sm proteins. The promoter sequences of the DlSm genes contained many light, endosperm development, hormone, and temperature response elements, which suggested their possible functions. In the non-embryogenic callus(NEC) and during early SE in longan, the alternative splicing(AS) events of DlSm genes indicated that these genes may influence SE development by changing gene structures and sequences. The kinetin(KT) hormone, and blue and white light treatments affected the differentiation and growth of longan embryonic callus(EC) probably by affecting the AS events of DlSm genes. Expression profiles showed the possible functional divergence among Sm genes, and different hormones and light qualities affected their expression levels. The expression trends of the DlSm genes determined by RNA sequencing as fragments per kilobase of exon model per million mapped reads (FPKM) and by real-time quantitative PCR(qRT-PCR) during early SE in longan showed that the expression of the DlSm genes was affected by the growth and differentiation of longan SE, and decreased as the somatic embryo differentiation progressed. The results will contributed to understanding the longan Sm gene family and provide a basis for future functional validation studies.

Physiological parameters and differential expression analysis of N-phenyl-N'-[6-(2-chlorobenzothiazol)-yl] urea-induced callus of Eucalyptus urophylla × Eucalyptus grandis.

In this study, we analyzed differences in the enzyme activities and transcriptomes of embryogenic and non-embryogenic calli to gain insights for improving the success of tissue culture-based breeding. A total of 2,856 differentially expressed genes (DEGs; 1,632 up-regulated and 1,224 down-regulated) were identified based on RNA sequencing and verified by reverse transcription quantitative polymerase chain reaction. Gene set enrichment analysis revealed that many of the up-regulated DEGs in embryogenic callus were enriched in the photosynthesis processes. Furthermore, the enzyme activity, hormone content, and cytokinin oxidase/dehydrogenase (CKX) gene expression analyses were found to be consistent with the transcriptome results. Cytokinin biosynthesis in N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU)-induced embryogenic callus increased owing to CKX repression. Measurement of endogenous hormones by high-performance liquid chromatography revealed that, compared with non-embryogenic callus, in embryogenic callus, the indole-3-acetic acid, abscisic acid and trans-zeatin riboside content had significantly higher values of 129.7, 127.8 and 78.9 ng/g, respectively. Collectively, the findings of this study will provide a foundation for elucidating the molecular mechanisms underlying embryogenic callus differentiation and can potentially contribute to developing procedures aimed at enhancing the success of callus-based plant regeneration.

Genome-wide identification of miRNAs and their targets during early somatic embryogenesis in Dimocarpus longan Lour.

miRNAs are endogenous regulatory factors that play pivotal roles in post-transcriptional regulation. However, their specific roles in early somatic embryogenesis (SE) remain unclear. Study of the SE system is fundamental for clarifying the molecular mechanisms in Dimocarpus longan. We identified 289 known miRNAs from 106 different miRNA families and 1087 novel miRNAs during early longan SE, including embryogenic callus (EC), incomplete pro-embryogenic culture (ICpEC), globular embryo (GE), and non-embryogenic callus (NEC). The abundances of known miRNAs were concentrated in GE. The differentially expression (DE) miRNAs showed five expression patterns during early SE. Largely miRNAs were expressed highly and specially in EC, ICpEC, and GE, respectively. Some miRNAs and putative target genes were enriched in lignin metabolism. Most potential targets were related to the pathways of plant hormone signal transduction, alternative splicing, tyrosine metabolism and sulfur metabolism in early longan SE. The regulatory relationships between dlo-miR166a-3p and DlHD-zip8, dlo-miR397a and DlLAC7, dlo-miR408-3p and DlLAC12 were confirmed by RNA ligase-mediated rapid amplification of cDNA ends. The expression patterns of eight DE miRNAs detected by qRT-PCR were consistent with RNA-seq. Finally, the miRNA regulatory network in early SE was constructed, which provided new insight into molecular mechanism of early SE in longan.

Dynamic changes in the expression pattern of miRNAs and associated target genes during coconut somatic embryogenesis.

Genome-wide analysis of small RNAs identifies somatic embryogenesis- specific miRNAs and their targets and provides novel insights into the mechanisms governing somatic embryogenesis in coconut, a highly in vitro recalcitrant species. Coconut, a major plantation crop of the tropics is recalcitrant to in vitro culture with a very low rate of somatic embryo turnover. Clonal propagation to enhance the production of high yielding, disease-free planting material in coconut has remained a distant reality. To better understand the molecular basis of this recalcitrance and to throw light on the complex regulatory network involved in the transition of coconut somatic cells to embryogenic calli, genome-wide profiling of small RNAs from embryogenic (EC) and non-embryogenic calli (NEC) was undertaken using Illumina Hiseq 2000 platform. We have identified a total of 110 conserved miRNAs (representing 46 known miRNA families) in both types of calli. In addition, 97 novel miRNAs (48 specific to EC, 21 specific to NEC and 28 common to both the libraries) were also identified. Among the conserved miRNAs, 10 were found to be differentially expressed between NEC and EC libraries with a log2 fold change > 2 following RPM-based normalization. miR156f, miR167c, miR169a, miR319a, miR535a, and miR5179 are upregulated and miR160a, miR166a, miR171a, and miR319b are down-regulated in NEC. To confirm the differential expression pattern and their regulatory role in SE, the expression patterns of miRNAs and their putative targets were analyzed using qRT- PCR and most of the analyzed miRNA-target pairs showed inverse correlation during somatic embryogenesis. Selected targets were further validated by RNA ligase mediated rapid amplification of 5' cDNA ends (5'RLM-RACE). Our data suggest that a few conserved miRNAs and species-specific miRNAs act in concert to regulate the process of somatic embryogenesis in coconut. The results of this study provide the first overview into the regulatory landscape of somatic embryogenesis in coconut and possible strategies for fine-tuning or reprogramming to enhance somatic embryo turn over in coconut.

Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar

Optimization of tissue culture and regeneration conditions of eggplant is necessary for achieving different goals such as gene transformation and the development of somaclonal variations. In this study, hypocotyl explants ware used to produce callus in a medium containing different concentrations of NAA and BAP. Moreover, the concentration of the elements Ca, Mn, Mg, Fe and K were measured and analysed between embryogenic and non-embryogenic calli. For shoot elongation, embryogenic calli were transferred to a new culture medium containing 3.5, 4 and 4.5 mg l-1 BAP plus 2 mg l-1 GA3. Finally, produced shoots were rooted in a culture medium containing 1, 1.5 and 2 mg l-1 NAA. Results showed that the best treatment for the embryogenic callus induction was MS medium containing 0.5 mg l-1 BAP plus 0.25 mg l-1 NAA. Two elements, Fe and K, had the highest amount in non-embryogenic calli compare to the embryogenic one. For plant regeneration, MS medium containing 4.5 mg l-1 BAP plus 2 mg l-1 GA3 and 2 mg l-1 NAA were the best treatments for shooting and rooting, respectively. In this study, the best treatments for plant regeneration produced 35 shoots from an explant with 92 % shooting. This regeneration protocol could be useful for gene transformation and micro-propagation studies.

Antioxidant enzyme activities during somatic embryogenesis in Musa acuminata Colla (AAA group) ‘Grand Naine’ and Musa spp. (AAB group) ‘Rasthali’

Antioxidant enzymes play a major role in scavenging reactive oxygen species that arise during somatic embryogenesis (SE). Activity levels of antioxidant enzymes such as peroxidase (POX), catalase (CAT), and superoxide dismutase (SOD) were investigated during developmental stages of SE of Musa acuminata Colla (AAA group) ‘Grand Naine’ and Musa spp. (AAB group) ‘Rasthali’. Using native polyacrylamide gel electrophoresis, five POX isoforms were detected in ‘Rasthali’, and four POX isoforms were observed in ‘Grand Naine’. The POX1 isoform was highly expressed in non-embryogenic callus (NEC) of both cultivars, and the POX2 isoform was present in somatic embryos (se) at 0, 30, 45, and 60 d of development of ‘Rasthali’, but not ‘Grand Naine’. Among two CAT isoforms, the CAT2 isoform showed higher expression in embryonic stages than in vegetative stages in explants and NEC of both cultivars. A total of five SOD isoforms detected in different developmental stages of both cultivars, SOD4 and SOD5, were detected only during the developmental stages of se. The enzyme assay showed that the highest CAT and SOD activity was in embryogenic callus (EC) of both cultivars, whereas activity of POX was reduced in EC. During somatic embryo development, the activity of SOD, POX, and CAT was found to be higher in the early globular stage than in the later coleoptilar stage embryos. During germination, the activity of SOD and POX was found to be higher in germinating se of ‘Grand Naine’. The specifically detected isoforms can be used as biochemical markers.

Global scale transcriptome analysis reveals differentially expressed genes involve in early somatic embryogenesis in Dimocarpus longan Lour

BackgroundSomatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Longan SE has been established and wildly used as model system for studying embryogenesis in woody plants, SE-related genes had been characterized. In spite of that, a comprehensive overview of SE at a molecular level is still absent. To understand the molecular mechanisms during longan SE, we examined the transcriptome changes by using Illumina HiSeq from the four distinct developmental stages, including non-embryogenic callus (NEC), embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE).ResultsRNA-seq of the four samples generated a total of 243.78 million high quality reads, approximately 81.5% of the data were mapped to longan genome. The cDNA libraries of NEC, EC, ICpEC and GE, generated 22,743, 19,745, 21,144, 21,102 expressed transcripts, 1935, 1710, 1816, 1732 novel transcripts, 2645, 366, 505, 588 unique genes, respectively. Comparative transcriptome analysis showed that a total of 10,642, 4180, 5846 and 1785 genes were differentially expressed in the pairwise comparisons of NEC_vs_EC, EC_vs_ICpEC, EC_vs_GE, ICpEC_vs_GE, respectively. Among them, plant hormones signalling related genes were significantly enriched, especially the auxin and cytokinin signalling components. The transcripts of flavonoid biosynthesis related genes were mainly expressed in NEC, while fatty acid biosynthesis related genes mainly accumulated in early SE. In addition, the extracelluar protein encoding genes LTP, CHI, GLP, AGP, EP1 were related to longan SE. Combined with the FPKM value of longan nine tissues transcription, 27 SE specific or preferential genes (LEC1, LEC1-like, PDF1.3, GH3.6, AGL80, PIN1, BBM, WOX9, WOX2, ABI3, et al.) and 28 NEC preferential genes (LEA5, CNOT3, DC2.15, PR1–1, NsLTP2, DIR1, PIP1, PIP2.1, TIP2–1, POD-P7 and POD5 et al.) were characterized as molecular markers for longan early SE. qRT-PCR validation of SE-related genes showed a high correlation between RNA-seq and qRT-PCR data.ConclusionThis study provides new insights into the role of the transcriptome during early SE in longan. Differentially expressed genes reveal that plant hormones signalling, flavonoid and fatty acid biosynthesis, and extracelluar protein related genes were involved in longan early SE. It could serve as a valuable platform resource for further functional studies addressing embryogenesis in woody plants.

Essential oil yield estimation by Gas chromatography-mass spectrometry (GC-MS) after Methyl jasmonate (MeJA) elicitation in in vitro cultivated tissues of Coriandrum sativum L.

Coriandrum sativum is an important spice plant known for its unique fragrance. Coriander oil is also one of the major essential oils in world global market. The oil yield varies with different coriander varieties; and the content and quality of oil is governed by several factors. In recent times, a variety of technologies have been exploited to improve phyto-compounds including essential oils. In this present study, Methyl jasmonate (MeJA) was amended in medium and the yield of essential oil was measured and compared in different cultivating tissues. The cultured tissues were nonembryogenic callus and embryogenic tissues (induction, proliferation and maturation stages of embryos). MeJA acts as a signaling molecule in accumulating secondary metabolites. Four different MeJA treatments i.e. T1 = 50, T2 = 100, T3 = 150 and T4 = 200μM, along with a control (T0) were used and the yield of coriander essential oil was estimated in different in vitro cultivating tissues by using Gas chromatography-mass spectrometry (GC-MS). The addition of MEJA enriched essential oil yield, maximum oil being in maturation stage of embryos at T3 (150μM). Other added treatments also had varied stimulatory role. The addition of MeJA induced stress as the stress marker enzymes like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) content were high compared to non treated tissue (T0). In T4, the CAT activity was maximum i.e. 5.83 and 6.28mg-1 protein min-1 in Co-1 and RS respectively in matured somatic embryos. The SOD activity was also high at maturation stage of embryos at T4 (5.3mg-1 protein min-1 in RS). The APX activity on the other, was high (3.32mg-1 protein min-1) in induction stage of embryogenesis at T3. The comparative biochemical (sugar, protein and proline) analyses of tissues were performed and presented that had high and low essential oil. MeJA induced stress may help in accumulating essential oils in C. sativum.

Study on the effect of stress treatment on embryogenic callus of raspberry

In order to obtain the embryogenic callus of raspberry, the friable calluses of raspberry were used as materials to be treated by different time of starvation and desiccation, and were further cultivated and induced embryoid. By use of embryogenic observation, the embryogenic degree of callus could be determined. The results showed that the embryogenic degree of calluses which were treated by starvation for 6 days was the highest, and the desiccation treatment had no effect on embryogenic degree of calluses. Therefore, appropriate starvation treatment can facilitate the transformation of non-embryogenic callus into embryogenic callus. The results of this experiment could provide reference for callus embryogenesis induction of other plants.

Genome-Wide Analysis of Cotton Auxin Early Response Gene Families and Their Roles in Somatic Embryogenesis.

Auxin is well known to regulate growth and development processes. Auxin early response genes serve as a critical component of auxin signaling and mediate auxin regulation of diverse physiological processes. In the present study, a genome-wide identification and comprehensive analysis of auxin early response genes were conducted in upland cotton. A total of 71 auxin response factor (ARF), 86 Auxin/Indole-3-Acetic Acid (Aux/IAA), 63 Gretchen Hagen3 (GH3), and 194 small auxin upregulated RNA (SAUR) genes were identified in upland cotton, respectively. Phylogenetic analysis revealed that the ARF, GH3, and SAUR families were likely subject to extensive evolutionary divergence between Arabidopsis and upland cotton, while the Aux/IAA family was evolutionary conserved. Expression profiles showed that the ARF, Aux/IAA, GH3, and SAUR family genes were extensively involved in embryogenic competence acquisition of upland cotton callus. The Aux/IAA family genes generally showed a higher expression level in the non-embryogenic callus (NEC) of highly embryogenic cultivar CCRI24 than that of recalcitrant cultivar CCRI12, which may be conducive to initializing the embryogenic transformation. Auxin early response genes were tightly co-expressed with most of the known somatic embryogenesis (SE) related genes, indicating that these genes may regulate upland cotton SE by interacting with auxin early response genes.

Early detection of somaclonal variation in oil palm callus culture through cytological and SDS-PAGE protein analysis

The major problem of oil palm in vitro propagation is the occurrence of somaclonal variation. This study aimed to obtain somaclonal variation analytical method based on chromosome activities and protein molecular weight level using SDS-PAGE technique on callus culture. Generally, three types of callus were observed, i.e. rooty, nodular friable, and nodular aggregate callus. During mitotic analyses, normal and abnormal cell division of each callus was successfully observed in several phases of mitotic. Rooty callus showed the highest rate of abnormal mitotic divisions up to 7.0 %. In contrast, nodular friable resulted in 3.2 % abnormality rate which is even less than normal plant cell division that was 3.7 %. The most mitotic abnormality type was a reduction-grouping mechanism of the chromosome during methaphase and prophase. SDS-PAGE revealed different protein molecular weight between nodular friable, nodular aggregate, and rooty callus. Protein 20 kDa is a specific protein found in nodular friable callus; this protein was known as a biochemical marker to distinguish embryogenic and non-embryogenic callus.