Embryogenic Callus Development Research Articles

Enhancing in vitro regeneration via somatic embryogenesis and Fusarium wilt resistance of Egyptian cucumber (Cucumis sativus L.) cultivars

BackgroundSomatic embryogenesis offers a reliable method for cucumber (Cucumis sativus L.) regeneration and genetic enhancement against Fusarium wilt. This study aimed to establish a tailored somatic embryogenesis system for Egyptian cultivars, fostering genetic improvements and Fusarium wilt-resistance lines. ResultsEmploying the Optimal Arbitrary Design (OAD) approach, we optimized the induction medium, initiating prolific embryogenic calli (53.3 %) at 1 mg/L 2,4-D. The cotyledonary leaf (CL) was the preferred explant, showing 60 % embryogenic callus development. Bieth Alpha exhibited higher responsiveness, generating ∼ 18 somatic embryos per explant compared to Prince's ∼ 10. Somatic embryogenesis system validation used quantitative RT-PCR, showing Cucumis sativus splicing factor 3B subunit (CUS1) and an embryogenesis marker gene expression exclusively within embryogenic calli and mainly during embryogenesis initiation. Evaluating fungal toxin filtrate concentrations for selecting embryogenic calli, the S2 selection (25 % filtrate, four subculture cycles) was chosen for somatic embryo development. To gauge the ramifications of selection at the genetic stratum, an in-depth analysis was executed. A cluster analysis grounded in ISSR banding patterns revealed a distinct separation between in vivo-cultivated plants of the two cultivars and regenerated plants devoid of pathogen filtrate treatment or those regenerated post-filtrate treatment. This segregation distinctly underscores the discernible genetic impact of the selection process. ConclusionsThe highest embryogenic capacity (53.3%) was achieved in this study by optimizing the induction stage, which demonstrated the optimal concentrations of BA and 2,4-D for induced proembryonic masses. Moreover, consistent gene expression throughout both stages of embryogenesis suggests that our system unequivocally follows the somatic embryogenesis pathway.

MtWOX2 and MtWOX9-1 Effects on the Embryogenic Callus Transcriptome in Medicago truncatula.

WOX family transcription factors are well-known regulators of plant development, controlling cell proliferation and differentiation in diverse organs and tissues. Several WOX genes have been shown to participate in regeneration processes which take place in plant cell cultures in vitro, but the effects of most of them on tissue culture development have not been discovered yet. In this study, we evaluated the effects of MtWOX2 gene overexpression on the embryogenic callus development and transcriptomic state in Medicago truncatula. According to our results, overexpression of MtWOX2 leads to an increase in callus weight. Furthermore, transcriptomic changes in MtWOX2 overexpressing calli are, to a large extent, opposite to the changes caused by overexpression of MtWOX9-1, a somatic embryogenesis stimulator. These results add new information about the mechanisms of interaction between different WOX genes and can be useful for the search of new regeneration regulators.

Transformation of CRISPR/Cas9 expression construct to enhance saturated fatty acid synthesis in oil palm for biofuel production

New technology innovations and the utilization of elite varieties are necessary for oil palm intensification to improve productivity. In addition to productivity improvement, oil palm intensification is also directed to improve the quality of crude palm oil (CPO) to be used in biofuel production. The modification of the fatty acid biosynthesis pathway through genetic engineering is one of the most promising approaches. The research aims to apply a genetic transformation to oil palm calli and to improve success regeneration of the transformed explants. A CRISPR/Cas9 expression construct to edit the PTE (palmitoyl-acyl carrier protein thioesterase) gene, which protein is involved in the formation of palmitic acid, was generated in the previous research. In this study, the genetic transformation of the construct into 4month-old oil palm calli was conducted via Agrobacterium tumefaciens. The transformed calli were subsequently subcultured into selection media containing 100 ppm of cefotaxime and 50 ppm of kanamycin antibiotics. After several weeks, the transformed calli were transferred into DF10 media without antibiotics. Non-transformed calli were able to grow on the selection media with antibiotics but still in the globular phase. In contrast, the transformed calli on the selection media were able to grow to the scutellar phase with a compact structure in the embryogenic callus development stages. The next step is to optimize the transformation method as well as the regeneration media composition.

Biotechnological and molecular genetic methods in grape breeding

The development of new grape varieties with improved quality and resistance against environmental factors using biotechnological methods is a pressing task in the context of import substitution policy implementation. Due to the high heterogeneity of grape varieties, techniques for regenerating plants from embryogenic callus by somatic embryogenesis have been developed only for a small number of genotypes. This work presents a versatile approach to the development of globular, heart, and torpedo embryos in various grape genotypes. Experiments were carried out using cultures of colchicine embryogenic cell suspensions and somatic globular embryos of the E-342 hybrid form in liquid media. The developed methodology allows the protocols of embryogenesis and polyploidy induction to be effectively improved. The versatility of the presented methodology for polyploidy induction and somatic embryogenesis in grape plants is ensured by a fourfold reduction in the concentration of inorganic nitrogen and vitamins in the NN and PG media and the addition of organic nitrogen and other biological active substances at the stages of embryogenic callus development, colchicination, and development of somatic embryoids in liquid media. The methods of indirect ploidy determination by the properties of the grape leaf epidermis were studied and implemented. Nine promising somaclones were preliminary selected based on a set of four features. It was established that full-fledged transgenic grape plants can only be obtained by the method of transformation, which involves the production of embryogenic callus from a leaf explant, its subsequent passage into a liquid culture to obtain a liquid embryo culture, and the transformation of this culture. In total, 300 leaf explants of the Libya variety and 1 100 globular embryos of the Podarok Magaracha variety were transformed. As a result, six callus kanamycin-resistant lines of the Libya variety and six lines of transgenic plants of the Podarok Magaracha variety were selected. Following genotyping based on nine nuclear microsatellite loci, seven unknown forms with a seedless phenotype were identified. Among the population of hybrid seedlings, hybrids having alleles associated with seedlessness were revealed using the VMC7f2 and p3_VvAGL11 MAS markers.

<i>MtWOX2</i> gene in somatic embryogenesis of <i>Medicago truncatula</i>

Somatic embryogenesis (SE) is one of the plant regeneration pathways. This process is very similar to zygotic embryogenesis, but embryos develop not from zygote, but from vegetative tissues. This process is widely used in biotechnology for plant transformation and propagation. Somatic embryos can derive directly from the vegetative tissues or through the formation of callus. The search for SE stimulators is very important for plant biotechnology.
 Several genes were reported to be the regulators of this process, among them the WOX (WUSCHEL-RELATED HOMEOBOX) family members are presented. These genes encode homeodomain-containing transcription factors, participating in different developmental processes. WOX2 is known for its participation in zygotic embryogenesis. It is expressed in the zygote and later in the apical domain of the embryo. We study the role of this gene in the somatic embryogenesis. Overexpression of MtWOX2 in some cases leads to the development of embryogenic calli with increased size. We performed transcriptome analysis of Medicago truncatula calli with overexpression of this gene compared to the calli overexpressing GUS.
 It was shown that MtWOX2 overexpression led to the changes in expression levels of genes, enriched with several GO pathways, including groups related to oxidative stress and ROS formation, response to toxic substance and auxin-activated signaling pathway. Among differentially expressed genes there are members of several TF families, e.g. MADS-box, BHLH, MYB, bZIP and others. These genes may regulate embryogenic callus development. Together, these results can be used for the search of new morphogenic regulators applicable for plant transformation.
 The research was supported by RFBR grant 20-016-00124.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.).

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

In Vitro Callus Development on Immature Leaf Explants of Liberica Coffee (Coffea liberica L. cv. Liberika Tungkal Komposit) by the Application of 2.4-D and BAP

Conventional vegetative propagation is the process in which new plants are grown from a variety of sources, seeds, cuttings and other parts of the plants. Therefore, the conventional vegetative propagation of liberica coffee by cutting or grafting of stems is hampered by the limited number of stem or branches, which can be used as propagating materials. In addition, the tissue culture technique is another method used to propagate liberica coffee. This study aims to investigate an efficient protocol for embryogenic callus development from leaf explants of Coffea liberica cv. Liberika Tungkal Komposit. The explants used are immature leaves of fully opened liberica coffee. The medium used was Murashige and Skoog (MS) composition supplemented with vitamins, 3% sucrose and solidified with 0.7% agar, and the medium pH was adjusted to 5.8 ± 0.1. The experiment was arranged in a factorial randomized block design, and the first factor was 2.4-D (2.0, 3.0, 4.0, and 5.0 ppm) and then the second factor was BAP (0.0, 0.5, and 1.0 ppm). The results showed that the application of 2.4-D and BAP significantly improved the distribution of callus proliferation on cultured explants. The use of 2.0 ppm 2.4-D + 1.0 ppm BAP resulted in the fastest callus proliferation (19 days after culture initiation). In general, the application of different levels of 2.4-D and BAP successfully induced friable with nodular morphology callus on young leaf explant of liberica coffee. It is believe that the callus has embryogenic capacity and will undergo embryogenesis when transferred to a suitable medium composition

Growth and development of young male inflorescences of oil palm (Elaeis guineensis Jacq.) In tissue culture system: The effect of 2, 4-Dichlorophenoxyacetic Acid

An effort in developing a standard protocol for rapid clonal propagation of oil palm through somatic embryogenesis was established with the aim to determine the effect of various concentrations of growth regulators on the development of embryogenic callus from immature inflorescences as explants source. Immature male inflorescences were obtained from 4 – 5 years old oil palm planted in Teaching Farm, Faculty of Agriculture University of Jambi. The inflorescences were taken from leaf axils between 8th and 15th leaf from the top (the upper most exposed leaf). Following surface sterilization with 70% alcohol, the inflorescences were cut into segments of approximately 5 mm long, and cultured on solid MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) at 10, 20, 30, 40, 50, 60, 70 and 80 ppm respectively. Cultures were kept in culture room with photoperiod of 16 hour per day for 8 weeks. The results indicated that the application of 2, 4-D in culture medium was proven to be crucial in accelerating callus formation as well as increasing the percentage of from young male inflorescence of oil palm forming callus. Among the treatments tested, the application of 70 ppm 2, 4-D was the best concentration in initiating callus formation on young male inflorescence explants of oil palm plants.

Functional characterization of a WRKY family gene involved in somatic embryogenesis in Panax ginseng

As a perennial herbaceous species, Panax ginseng is widely cultivated and used as traditional herbal medicine. The root of Panax ginseng commonly remains expensive as conventional breeding of Panax ginseng is difficult. Somatic embryogenesis (S.E.) is a useful tool for plant propagation and optimal model for understanding the mechanisms of plant embryogenesis. In Panax ginseng, increasing studies have been widely performed to optimize the technology of S.E., while the underlying mechanism remains unclear. In this paper, we cloned and identified a WRKY family gene named PgWRKY6 which is upregulated in response to 2,4-D (2,4-dichlorophenoxyacetic acid)-induced embryogenic callus development. The silencing of PgWRKY6 obviously reduces the induction rate of embryogenic callus, indicating its crucial role in S.E. of Panax ginseng hairy root. The expressions of several ROS-scavenging genes are also inducible during embryogenic callus development, and the transcriptions of PgGST, PgAPX1, and PgSOD are demonstrated to be regulated by PgWRKY6. Recombinant PgWRKY6, an approximate 40-KDa protein purified from Escherichia coli, shows a specific DNA-binding activity with a potential recognition site of TTGAC(C/T). This work demonstrated that as a conserved WRKY family transcription factor, PgWRKY6 functions upstream of PgGST, PgAPX1, and PgSOD, and potentially mediated auxins -ROS signaling pathway in the process of S.E. in Panax species.

Explant Type, Culture System, 6-Benzyladenine, Meta-Topolin and Encapsulation Affect Indirect Somatic Embryogenesis and Regeneration in Carica papaya L.

A protocol to propagate papaya hybrid plants through indirect somatic embryogenesis was developed considering the effect of explant type, culture system, particular cytokinins and encapsulation, in different stages of the process. Optimal 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations for non-embryogenic callus formation ranged between 9.0 and 27.1 μM in half-cut seeds, while higher concentrations were harmful. Non-embryogenic callus was also obtained with 22–158 μM 2,4-D from hypocotyl segments. Callus with embryogenic structures was only obtained in half-cut seeds cultured in the darkness on half-strength Murashige and Skoog culture medium supplemented with 2,4-D, while hypocotyl segments and isolated zygotic embryos failed to produce this type of callus regardless of the 2,4-D and sucrose (30 and 70 g l-1) concentrations tested in this study. Both, embryogenic callus development and quantity of somatic embryos formed per embryogenic callus, which ranged between 11 and 31 units after 14 months, required 2,4-D, but without any effect of the concentration. Histological studies confirmed the multicellular origin of the somatic embryos. In further steps, liquid medium induced over four times more somatic embryos than agar-gelled medium and showed significantly higher production of globular somatic embryos (85 vs. 57%). Both, 6-benzyladenine (BA) and meta-topolin (Mtop) stimulated sprouting (40–45%) of the somatic embryos (development of shoots only) in concentrations of up to 2.7 and 10 μM, respectively. Sprouting probability showed a 2nd order polynomial trend despite the range of concentration used for each cytokinin. This is the first report about the positive effect of Mtop on the apical shoot development of Carica papaya somatic embryos known to the authors. Radicle growth was observed in 5% or less of the cultivated embryos, regardless of the BA concentration. Finally, all encapsulation conditions tested (2.5, 3.5, and 4.5% sodium alginate, combined with 50 and 100 mM CaCl2) reduced sprouting of somatic embryos when compared to the non-encapsulated ones, whereas capsule hardness showed low correlation with embryo sprouting. Embryos were further cultivated until they became plantlets approximately 5 cm long. They were acclimatized and afterward planted in the field, where they flowered and produced fruit.

PENGARUH JENIS DAN KONSENTRASI ZAT PENGATUR TUMBUH PADA INDUKSI KALUS EMBRIOGENIK KLON CENDANA (Santalum album Linn.)

Sandalwood (Santalum album Linn.) is a tree which has a high rate of natural sprouting ability. Eventhough the propagation by the conventional techniques (shoot and root cuttings) and by the tissue culture have been reported, the percentage of plants regeneration is still low. The propagation using somatic embryogenesis was reported as better result than using shoots multiplication technique or organogenesis. The objective of this research is to examine the effect of clones, type and concentration of plant growth regulator on the development embryogenic callus of sandalwood. The three tested clones are C1, C2, and C3. The plant growth regulators are 2.4-D, Dicamba, and Picloram with the three level of concentrations:1 mg/L, 3 mg/L, and 5 mg/L. The result of study showed that the clone of C3 performed best on embryogenic callus development. It was observed through morphological analysis that 58.12% of explants performed embryogenic callus with friable texture and white, yellowish in colours. However, there were not significant differences between the types of plant growth regulator, the level of concentrations and their interactions on embryogenic callus development of sandalwood

Polyamine Plays a Role in Subculture Growth of in Vitro Callus of Indica Rice

AbstractIn vitroembryogenic callus is a critical factor for genetic transformation of rice, especially for indica varieties. In this study, we investigated the relationship between polyamines, including putrescine (Put), spermidine (Spd) and spermine (Spm), and callus browning, and we studied the effect of exogenous Put on callus regeneration and on the content of endogenous polyamines. In addition, the expression levels of arginine decarboxylase gene (Adc1) and S-adenosylmethionine decarboxylase gene (Samdc) in embryogenic callus were studied by quantitative Real-time PCR analysis. The results showed that the contents of endogenous Put and Spd in the browning callus were significantly lower than those in normal callus. Exogenous Put could effectively improve the growing state of callus of indica rice and enhance the development of embryogenic callus. The content of endogenous polyamines in embryogenic callus, especially Spd and Spm, was increased after addition of exogenous Put. Additionally, exogenous Put also had an obvious impact on the expression levels ofAdc1but partial effect on the expression levels ofSamdcgene. This study could increase the knowledge of both embryogenic callus induction and polyamine catabolism in callus in indica rice.

Identification and Comparative Analysis of microRNA in Wheat (Triticum aestivum L.) Callus Derived from Mature and Immature Embryos during In vitro Culture.

Feasible and efficient tissue culture plays an important role in plant genetic engineering. Wheat (Triticum aestivum L.) immature embryos (IMEs) are preferred for tissue culture to mature embryos (MEs) because IMEs easily generate embryogenic callus, producing large number of plants. The molecular mechanisms of regulation and the biological pathways involved in embryogenic callus formation in wheat remain unclear. Here, microRNAs (miRNAs) potentially involved in embryogenic callus formation and somatic embryogenesis were identified through deep sequencing of small RNAs (sRNAs) and analyzed with bioinformatics tools. Six sRNA libraries derived from calli of IMEs and MEs after 3, 6, or 15 d of culture (DC) were constructed and sequenced. A total of 85 known miRNAs were identified, of which 30, 33, and 18 were differentially expressed (P < 0.05) between the IME and ME libraries at 3, 6, and 15 DC, respectively. Additionally, 171 novel and 41 candidate miRNAs were also identified, of the novel miRNA, 69, 67, and 37 were differentially expressed (P < 0.05) between the two types of libraries at 3, 6, and 15 DC, respectively. The expression patterns of eight known and eight novel miRNAs were validated using quantitative real-time polymerase chain reaction. Gene ontology annotation of differentially expressed miRNA targets provided information regarding the underlying molecular functions, biological processes, and cellular components involved in embryogenic callus development. Functional miRNAs, such as miR156, miR164, miR1432, miR398, and miR397, differentially expressed in IMEs and MEs might be related to embryogenic callus formation and somatic embryogenesis. This study suggests that miRNA plays an important role in embryogenic callus formation and somatic embryogenesis in wheat, and our data provide a useful resource for further research.

In vitro plant regeneration from mature seeds of finger millet (Eleusine coracana) through somatic embryogenesis

Mature seeds of two genotypes of finger millet, viz., PR 202 and PES 400, were cultured on 20 different callus induction media containing four levels of 2,4-d (1, 1.5, 2.0 and 2.5 mg l−1) alone or in combination with two levels of cytokinin (0.5 and 1.0 mg l−1 kinetin or BAP). 2,4-d was found to be most effective for callus induction and development when combined with kinetin. Genotypic response for embryogenic callus development ranged from 29.5 to 78.2 % for PR 202 and 10 to 35.7 % for PES 400. After 45–50 days of culture, calli were transferred in light to develop into green nodular calli. Plantlets were obtained by transferring the green nodular embryogenic calli onto regeneration media containing different concentrations and combinations of GA3, kinetin and BAP. Kinetin was found to be more effective for shoot regeneration as compared to BAP. When Callus derived from BAP containing media were transferred on regeneration media containing GA3 alone or with BAP, developed shoots were either devoid of roots or had few thin roots. These shoots were rooted on MS basal media and MS media supplemented with IBA (0.5 or 1.0 mg l−1), NAA (1 mg l−1) and IBA + NAA (0.5 mg l−1 each). Rooting media containing 0.5 mg l−1 IBA and 0.5 mg l−1 NAA showed remarkable rooting response, inducing a thick bunch of 11.5–12 healthy, white and long roots per shoot. Regenerated plants were transferred to pots containing sterilized soil:sand:vermiculite:FYM (2:2:2:1) and were irrigated with Hoagland solution. Regenerated plants grew well and were successfully established in soil.

Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan

RAN (Ras-related nuclear protein) plays crucial roles in multiple cellular processes in yeast, animals and plants. Here we present a DlRan gene and its alternative splicing transcripts containing premature terminator codons (PTCs), identified from embryogenic cultures in longan. Multiple alignment and splicing pattern analyses indicated that DlRan-1 transcript harboring PTC was the consequence of alternative splicing. The accumulation of DlRan PTC-containing transcripts increased significantly when the embryogenic calli were treated with the translation inhibitor, cycloheximide, indicating that DlRan-1 may be targeted by NMD. The analysis of expression profiles of DlRan transcripts revealed that differential expression levels of the alternative spliced DlRan transcripts occurred during the development of embryogenic callus, globular-shaped embryos, and cotyledon-shaped embryos, respectively, in the longan somatic embryogenesis, and were in consistent with the embryo development in corresponding wild-type transcripts. The present work offers evidence to speculate that the alternatively spliced PTC-containing transcripts can be functional and may shed light on expression regulation of DlRan during development of the longan somatic embryos.

PEMBENTUKAN KALUS REMAH DARI EKSPLAN DAUN RAMIN (Gonystylus bancanus (Miq) Kurz.)

Ramin (Gonystylus bancanus (Miq.) Kurz) is the most favourite and the most over exploited woody species. This species has been listed in CITES APPENDIX II from 2004. The purpose of this experiment is to obtain the best treatment for friable and embryogenic callus formation that can develop to somatic embryo. Basal Murashige and Skoog (MS) media was used as growth medium. The experiment was conducted in three stages : callus induction and propagation stage, friable callus induction stage and embryogenic callus induction stage. The treatment of 3.0 – 5.0 mg/l 2,4-D was used for callus induction. The best of callus was propagated in 5.0 mg/l 2,4-D + 1.0 – 2.0 mg/l thidiazuron. The best of callus was subcultured for callus friable induction used 6.0 mg/l 2,4-D + 1.0 – 2.0 mg/l thidiazuron + 1.0 – 2.0 mg/l biotin. The best friable callus was subcultured for embryogenic callus induction used 7.0 – 8.0 mg/l 2,4-D + 1.0 – 2.0 mg/l biotin. The observation was made on texture, percentages, performance and color of friable callus. The results showed that callus can be induced on 5.0 mg/l 2,4-D. The treatment of 6.0 mg/l 2,4-D + thidiazuron produced friable callus yellowish on color. 2,4-D + biotin treatment produced very friable callus with yellowish on color and with no embryogenic callus development.

Plant tissue culture studies in Sorghum bicolor: immature embryo explants as the source material

Sorghum is a wonder crop from physiological point of view. It is the most important cereal crop, after rice and wheat. The number of reports describing the use of transgenic Sorghum for basic studies in Biotechnology is still limited when compared to other crops. In one hand, the success of the transformation techniques is mainly dependent upon the availability of optimal protocols for highly efficient tissue culture techniques. On the other hand, regeneration in Sorghum is difficult. Hence, in this study an efficient and reproducible method for in vitro development of embryogenic callus and regeneration in Sorghum bicolor was developed. Immature embryo explants of Sorghum bicolor were cultured on MS nutrient medium supplemented with different concentrations and combinations of auxins and cytokinins. Embryogenic callus was initiated on MS medium supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l Kinetin (KN). Addition of KN to MS medium containing 2,4-D resulted in a significant enhancement on embryogenesis of the embryos. Amino acids also supported an improved frequency of embryogenesis. Therefore induction of a high frequency of somatic embryogenesis in immature embryos on MS medium is possible. Development of embryogenic callus, induction of somatic embryo and subsequent shoot regeneration was proficient at a concentration of 2 mg/l BAP. The regenerated shoots readily rooted on half strength MS medium supplemented with 1 mg/l naphthalene acetic acid (NAA). The regenerated plantlets were successfully transferred to soil and subsequently plants produced seeds. There was no difference between the acclimatized plants in comparison with in vivo plants in the respect of morphological characters.

Regeneration in sugarcane via somatic embryogenesis and genomic instability in regenerated plants

In the present study, embryogenic calli of sugarcane variety BL4 were induced using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in different concentrations and combinations. In contrast to earlier studies, embryogenic callus sectors were identified and isolated microscopically within 1–2 weeks. Subsequently, 51 media formulations were used for regeneration of proliferated embryogenic callus, using MS medium supplemented with three different cytokinins [kinetin, 6-Benzylamino purine (BAP), and thidiazuron (TDZ)] and auxins (IAA/NAA and IBA) in different combination and concentrations. After acclimatization, the genomic DNA of regenerated plants was studied to explore the insertion polymorphism of transposable elements in order to ascertain the variation among somaclones. Though low concentration of kinetin with 2,4-D was found supportive to embryogenic callus development, the highest number of regenerated plantlets was observed using BAP (1 μM), however the plantlets had very low fresh weight (2.2 g). Conversely, TDZ alone supported a significant increase in the number of plantlets regenerated (38–40) with higher fresh weight. The somaclones generated during this study showed considerable positional polymorphism of activator-like transposable elements possibly due to the stress associated with in vitro culture. This study provides a procedure to produce regenerated sugarcane plants from embryogenic callus in a relatively short time.

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula. Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression. Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature. The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.