Embryogenic Callus Proliferation Research Articles

Effect of Media and Plant Growth Regulator on Proliferation of Somatic Embryogenic Callus of Oil Palm (Elaeis gueenesis Jacq.)

Effect of Media and Plant Growth Regulator on Proliferation of Somatic Embryogenic Callus of Oil Palm (Elaeis gueenesis Jacq.)

Physiological, Metabolic, and Transcriptomic Analyses Reveal Mechanisms of Proliferation and Somatic Embryogenesis of Litchi (Litchi chinensis Sonn.) Embryogenic Callus Promoted by D-Arginine Treatment.

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H2O2, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism.

Proper doses of brassinolide enhance somatic embryogenesis in different competent Korean pine cell lines during embryogenic callus differentiation.

Somatic embryogenesis of Korean pine (Pinus koraiensis Sieb. Et Zucc.), an ecologically and econimically very important conifer species, was hindered by the gradually weakens and fast runaway of the embryogenicity and embryo competence of the embryogenic callus. Brassinolide (BL) has shown the enhancing capability of somatic embryo regeneration. For checking the function of BL in this issue, we applied different concentrations of BL to Korean pine callus materials exhibiting different embryogenic capacities and subsequently monitored the physiological alterations and hormone dynamics of the embryogenic callus. Our study revealed that calli with different embryogenic strengths responded differently to different concentrations of BL, but the effect after the addition of BL was very uniform. The addition of BL during the proliferation phase of embryogenic callus may help to stimulate the biological activity of callus during the proliferation process and improve the level of cell metabolism, which is accompanied by a reduction in storage substances. BL could reduce the level of endogenous auxin IAA in embryogenic callus and increase the level of abscisic acid to regulate cell division and differentiation. In addition, the MDA content in the callus was significantly decreased and the activity of antioxidant enzymes was significantly increased after the addition of BL. During the proliferation of embryogenic callus, BL was added to participate in the metabolism of phenylpropane in the cells and to increase the activity of phenylalanine ammonia-lyase and the content of lignin in the cells. We deduced that the proper doses of BL for Korean pine embryogenic callus culture were as follow: calli with low, high and decreasing embryogenicity were subcultured after the addition of 0.75 mg/L, 0.35 mg/L, 2.00 mg/L BL, respectively, during proliferation culture stage.

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.).

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

Reactive oxygen metabolism in the proliferation of Korean pine embryogenic callus cells promoted by exogenous GSH

Exogenous glutathione (GSH) promotes the proliferation of embryogenic callus (EC) cells in Korean pine in the course of somatic embryogenesis, and reactive oxygen species (ROS) may play an important role in regulating the proliferation of EC cells by exogenous GSH. However, the concrete metabolic response of ROS is unclear. In this study, two cell lines of Korean pine with high proliferative potential 001#-001 (F, Fast proliferative potential cell line is abbreviated as F) and low proliferative potential 001#-010 (S, Slow proliferative potential cell line is abbreviated as S) were used as test materials. The responses of ROS-related enzymes and substances to exogenous GSH and L-Buthionine-sulfoximine (BSO) were investigated in EC cells. The results showed that the exogenous addition of GSH increased the number of early somatic embryogenesis (SEs) in EC cells of both F and S cell lines, decreased the amount of cell death in both cell lines. Exogenous addition of GSH promoted cell division in both cell lines, increased intracellular superoxide dismutase (SOD) and catalase (CAT) activities, inhibited intracellular hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) production, and increased NO/ROS ratio. In conclusion, the exogenous GSH promoting the proliferation of Korean pine EC cells, the activity of intracellular antioxidant enzymes was enhanced, the ROS level was reduced, and the resistance of cells to stress was enhanced.

THE EFFECT OF PH AND SUCROSE ON THE EMBRYOGENIC CELLS GROWTH OF SUGAR CANE (Saccharum officinarum) IN LIQUID CULTURE

<p><strong>Background. </strong>Sugarcane (<em>Saccharum officinarum</em> L.) is one of the important commodities in Indonesia as raw material for sugar production. The constraints for sugarcane cultivation are limited availability and poor quality of seedling. The utilization of liquid culture in tissue culture enables to produce good quality seedlings, free of viruses and diseases, and can generate a large quantity in a short time. Liquid culture can produce embryogenic cells faster since embryogenic callus can develop easily in liquid media. <strong>Objective</strong>. This study determined the appropriate pH and concentration of sucrose in the liquid culture of embryogenic cells. <strong>Methodology</strong>. This study was conducted in 3 stages of in vitro culture, which were callus induction, solid media proliferation, and liquid media proliferation that using pH and sucrose treatments. <strong>Result</strong>. The results showed that pH 6.5 + 3% sucrose (A3B1) produced the highest number of callus with the average callus on the first week 29 callus, the second week 8,33 callus and the third week 4,33 callus and the color did not easily turn to brown with color 5Y 8/2. <strong>Implications</strong>. The protocol was developed that allows embryogenic callus of sugarcane plants to be obtained in liquid media that can be used for embryogenic callus proliferation. <strong>Conclusions</strong>. pH 6.5 + sucrose 3% (A3B1) was the best combination treatment of pH and sucrose for sugarcane callus proliferation in liquid culture media, which produced the highest number of calluses and color of callus that not easily turned brown.</p>

IAA combine with kinetin elevates the α-linolenic acid in callus tissues of soybean by stimulating the expression of FAD3 gene

Soybean is a major source of essential fatty acids such as linoleic acid and α-linolenic acid (ALA) where the ALA content is approximately 8.0% which is highly essential for human to synthesize omega 3 fatty acids. To improve the ALA content in soybean, different plant growth plant growth regulators were used in various combinations to induce the callus. In this study, six different combinations of Plant growth Regulators (PGRs) viz, 1) 2,4-D (5 μM) + TDZ (10 μM), 2) 2,4-D (5 μM) + BAP (10 μM), 3) GA3 (5 μM) + Zeatin (1 μM), 4) IAA (5 μM) + Kinetin (10 μM) 5) NAA (10 μM) + IBA (1 μM), 6) 2,4-D (5 μM) + Picloram (1 μM) were evaluated for the gene expression and fatty acid analysis. Within these treatments, IAA (5 μM) + Kinetin (10 μM) showed good proliferation of embryogenic callus with green and friable tissue. Further, mRNA expression analysis showed the varied expression of fatty acid biosynthetic genes like KASI, KASII, SCAPD, FAD2, and FAD3 in callus tissues. The callus tissue regenerated from the IAA (5 μM) + Kinetin (10 μM) treatment was found to be effective in up-regulating the FAD3 gene thereby promoting the production of ALA (up to 20.26%) than the control and any other PGR combinations tested. And hence, it may be concluded that the combination of PGR i.e. IAA (5 μM) with Kinetin (10 μM) enhanced the FAD3 gene expression and thereby improved the ALA production in soybean. Further this PGR combination need to be tested in field condition to validate the ALA content in soybean seeds.

Micropropagation of Tulip via Somatic Embryogenesis

An effective method of tulip regeneration via somatic embryogenesis (SE) was developed. Explants, flower stem slices excised from cooled bulbs were incubated in darkness on MS modified media containing auxins alone (2,4-dichlorophenoxyacetic acid—2,4-D, 1-naphthalene acetic acid—NAA and 4-amino-3,5,6-trichloro-2-pyridine carboxylic acid—picloram) or combined with thidiazuron (TDZ) at 0.1 and 0.5 mg L−1. Yellowish-white callus with a granular structure was developed in the presence of all auxins on the cut surface from the tissues of the vascular bundles. From this, lines of embryogenic calli were derived. The addition of TDZ to the medium with auxins significantly stimulated somatic embryo formation. Cyclic and the most intensive proliferation of embryogenic callus as well as embryo formation was obtained in the presence of 2,4-D at 0.1 mg L−1 combined with TDZ at 0.5 mg L−1. Addition of proline enhanced either callus proliferation rate or frequency of embryo formation. The best quality embryos with cotyledons longer than 10 mm able to form bulbs were recorded when TDZ was replaced with 6-benzylaminopurine (BAP) at the concentration of 0.1 mg L−1. Histomorphology showed that the development of somatic embryos could have either external or internal origins. Embryos of external origin were initiated by cell division on the edge of embryogenic calli. Embryos of internal origin resulted from the division of parenchyma cells inside the tissue. Embryonic cells were characterized by their small volume, regular shape, dense cytoplasm and large nuclei. The globular embryos were covered by a distinct layer of periderm. Then, the embryos developed into structures having leaf-shaped cotyledons with a procambial strand and a sideward-orientated meristem of the vegetative apex (stolon). Cotyledon embryos did not show vascular connections with the parent tissue, and they did not develop embryonic roots.

Induction of somatic embryogenesis in different explants from Ormosia henryi Prain

An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain, which is an important precious tree in the timber industry. Five types of explants, six types of basic media and different plant growth regulators (PGRs) were used for embryogenic callus (EC) initiation, EC proliferation, somatic embryo (SE) induction and SE germination. The EC and SE induction rates from mature zygotic embryos (MZE) were higher than those from stem segments, cotyledons, leaves and hypocotyls. MZE were used as explants, and the EC induction rate was greater than 30% on B5 medium supplemented with 1.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l naphthaleneacetic acid (NAA) or 0.2 mg/l BA and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The highest induction rate (22.83%) for SE was obtained with B5 medium supplemented with 0.5 mg/l kinetin (KT) and 1.0 mg/l 2,4-D. The EC proliferation rate and SE number in the suspension culture were increased compared to those in the solid culture. Fifty-four percent of SE developed to the cotyledonary stage on B5 medium supplemented with 0.5 mg/l BA and 0.2 mg/l NAA. The number of cotyledon embryos was the highest on medium supplemented with 0.5 mg/l thidiazuron (TDZ) and 0.2 mg/l NAA. Histological analysis confirmed the bipolar organization of the somatic embryos, which were comprised of shoot and root meristems, and showed that SE was characterized by globular, heart-shaped, torpedo-shaped and cotyledon stages in O. henryi, the developmental process of which was similar to that of zygotic embryos. An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain using mature zygotic embryos as explant.

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

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

Somatic embryogenesis and plant regeneration in Betula platyphalla

Betula platyphylla is a native tree species in northern China that has high economic and medicinal value. We developed an efficient protocol for the induction of somatic embryogenesis in B. platyphalla from immature zygotic embryos and assessed the effects of explant type, genotype, and plant growth regulators (PGRs) on embryogenic callus induction. Among the various explants evaluated, embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid (2,4-D). Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction. On the 20 days after pollination, immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency (16.8%) when cultured on half-strength MS medium supplemented with 2.0 mg L−1 2,4-D and 0.2 mg L−1 6-benzylaminopurine (6-BA). The process of proliferation of embryogenic callus, somatic embryo formation, and subsequent plantlet conversion occurred under optimal culture conditions. When regenerated plants were transplanted to soil, 95% of them developed normally and grew vigorously. This somatic embryogenesis system required 3–4 months for the regeneration of B. platyphalla plantlets from immature zygotic embryos.

Enhancement of embryogenic callus proliferation in Chinese pine ( Pinus tabuliformis ) by airlift bioreactor

Enhancement of embryogenic callus proliferation in Chinese pine ( Pinus tabuliformis ) by airlift bioreactor

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application.

The family Gentianaceae consists of 1736 species, which play an important role in human being existence due to their pharmacological and horticultural values. Many species accumulate bitter iridoid substances used medicinally and in flavorings, while others are cultivated because of beauty of their flowers showing a wide range of colors and patterns. Out of 99 genera belonging to the gentian family, process of somatic embryogenesis (SE) was reported for 5. The first reports, aimed at micropropagation of ornamental cultivars and production of secondary metabolites, concerned Centaurium erythraea Rafn., Eustoma russellianum Grieseb. and Exacum affine Balf. Somatic embryos were induced on different explants cultured in the liquid Murashige and Skoog medium supplemented with auxins and cytokinins. In the 1990s of the last century, significant progress in the exploration of the phenomenon of SE and its biotechnological application was made for the genus Gentiana. The process was induced on various explants and studied at the structural and ultrastructural levels. Regenerated plants were screened for genetic stability using flow cytometry, chromosome counting, and molecular markers. Besides typical indirect SE, the use of leaf fragments enabled to obtain single-cell origin of somatic embryos. On the other hand, proliferation of embryogenic callus in liquid medium resulted in the establishment of long-term embryogenic cell suspension cultures, paving the way not only to study the formation of somatic embryos and the development of regenerants but also to preserve the morphogenic potential of cell aggregates by cryopreservation. Cell suspensions re-established after storage in liquid nitrogen maintained their embryogenic character and allowed to obtain somatic embryo-derived regenerants that were true-to-type at both genetic and epigenetic levels. Another application of SE was related to genetic manipulation purposes. Efficient protocols of plant regeneration from callus-, cell suspension-, or leaf mesophyll-derived protoplasts allowed engaging procedures of somatic hybridization or protoplast electroporation for gentian genome modifications. Also, high embryogenic potential existing in the numerous gentian species enabled successful Agrobacterium-mediated transformation of G. cruciata L. and G. dahurica Fisch.

Perbanyakan In Vitro Dendrobium Indonesia Raya ‘Ina’ melalui Embriogenesis Somatik Berbasis Sistem Bioreaktor

<pre><em></em><em>ABSTRACT<br /><br />An effective and efficient in vitro propagation system has important roles in preparing and producing high quality-seedlings of Dendrobium for commercial scale. The objective of this research was to establish an effective and efficient embryogenic callus (EC) proliferation method using bioreactor system and regeneration EC into plantlet for producing high quality seedlings of Dendrobium Indonesia Raya ‘Ina’. Differences in callus densities (5, 10, 15, and 20 g callus in 250 mL medium), aeration levels (2.5, 5.0, and 10.0 O2 volume per medium volume per minute; vvm), and regeneration media half-strength MS and 2 g L-1 NPK (32:10:10) combinated by 0.00, 0.05 mg L-1 BA, 150 mL L-1 coconut water and their combinations were tested in this experiment. The experiments were arranged using randomized completely block design (RCBD) with three replications for EC proliferation and randomized completely desaign (RBD) for EC regeneration. The results showed that combination of aeration at 2.5 vvm and 10 g of EC was the most suitable aeration level and callus density for proliferation of EC in the 500 ml airlift bioreactor with 6.85 multiplication rate, 92.5% EC formation, and malformed callus morphology as low as 6.1%. The highest somatic embryos (SEs) formation was 87.7% with 44.5 SEs per clump and 92.1% SEs germination with 41.0 germinated-SEs per clump, 85.1% normal germinated-SEs, and whereas the best performance of plantlet was obtained from 1/2 MS + 0.05 mg L-1 BA semi solid medium. Plantlets were successfully acclimatized using Cycas rumphii medium with high survival rate (91.6%). <br /><br />Keywords: aerations, callus densities, germination, media, somatic embryos<br /><br /></em></pre>

Induksi dan Proliferasi Embriogenesis Somatik In Vitro pada Lima Genotipe Kedelai

ABSTRACT<br /><br />Somatic embryo induction medium was reported to be genotype dependent for soybean. This study was aimed to obtain the optimum medium for embryo somatic induction and proliferation, and to regenerate somatic embryo of five soybean genotypes. Five soybean genotypes (Tanggamus, Anjasmoro, Yellow Biloxi, CG-22-10, and SP-10-4) were used in this study. The research was divided into four steps: (1) embryogenic callus induction of five soybean genotypes, (2) embryogenic callus proliferation of five soybean genotypes, (3) optimation of embryo somatic induction on five soybean genotypes and (4) embryo somatic regeneration of five soybean genotypes. The induction experiment showed that based on number of embryogenic callus, the best somatic embryo-induction medium was 3% sucrose+ NAA 5 mg L-1+2,4-D 5 mg L-1+ Vitamin B5. Embryogenic callus number for each genotype tested was increased on proliferation media of 3% sukrosa + 2,4-D 5 mg L-1 + NAA 5 mg L-1+ Vit B5, and Yellow Biloxi gave the highest number of proliferated somatic embryos compared to other genotypes. Increasing number of globular somatic embryo of all genotypes was obtained from the optimation of somatic embryo induction media being used, and Tanggamus genotype gave the highest number of globular somatic embryo which followed by Yellow Biloxi genotype. Tanggamus and Yellow Biloxi genotypes were also successfully formed the four steps of somatic embryos (globular, heart, torpedo, and cotyledonary stages), but in regeneration medium of MS0 and media MS + sukrosa 10 g L-1 + GA3 2 mg L-1 + BAP 4 mg L-1 + Vit B5 only Tanggamus genotype was regenerated into plantlet. <br /><br />Keywords: 2,4-D, NAA, somatic embryos, induction, proliferation<br /><br />

Proliferation of embryogenic callus of Satoimo taro (Colocasia esculenta var. antiquorum) in culture media with various levels of sucrose and gelling agent

Abstract. Fitriani H, Aryaningrum PD, Hartati NS. 2016. Proliferation of embryogenic callus of Satoimo taro (Colocasia esculenta var. antiquorum) in culture media with various level of sucrose and gelling agent. Nusantara Bioscience 8: 316-320. Satoimo taro (Colocasia esculenta var. antiquorum) is one of the promising varieties of taro which have potentially high value to cultivate. Tissue culture or in-vitro culture considers an economically useful technique to mass propagates this variety of taro. The research was conducted to figuring out the optimum media for proliferation success of embryogenic callus of Satoimo. The explants used embryogenic callus of 12-months-old storage materials obtained from the previous study. Proliferation media include a basal half-strength MS salt (Murashige and Skoog) enriched with of 0.1 mg/L TDZ, 0.05 mg/L 2.4 D, and 100 mg/L L-glutamine. Adding to the basal media, we set the treatments by combining two factors i.e. Sucrose with three levels of concentration i.e. 3, 4, and 5% which cross-tabulated with three gelling agents, i.e., Agarose, Phytagel, and Gerlite. The research parameters included the percentage of callus diameter growth, the color and callus discoloration, and the structure of callus. The data recorded periodically every 7 d in one month. Data were analyzed using analysis of variance and followed by Duncan Multiple Range Test (DMRT) to determine the significant difference (p<0.05) among the treatments. The result revealed that the callus diameter growth has the greatest percentage at medium with a combination of Phytagel and sucrose 4%. The Micro-agar enhanced the presence of callus with yellow color. Phytagel combined with a high percentage of sucrose (5%) indicated browning properties of callus. Gelrite causes the callus with discoloration appearance (white).

De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

Plant regeneration via somatic embryogenesis (SE) is the key step for genetic improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, but the molecular mechanisms underlying SE in cotton is still unclear. Here, RNA-Sequencing was used to analyze the genes expressed during SE and their expression dynamics using RNAs isolated from non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryos (SEs). A total of 101, 670 unigenes were de novo assembled. The genes differentially expressed (DEGs) amongst NEC, EC and SEs were identified, annotated and classified. More DEGs were found between SEs and EC than between EC and NEC. A significant number of DEGs were related to hormone homeostasis, stress and ROS responses, and metabolism of polyamines. To confirm the expression dynamics of selected DEGs involved in various pathways, experiments were set up to investigate the effects of hormones (Indole-3-butytric acid, IBA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results showed that exogenous application of IBA and KT positively regulated the development of EC and SEs, and that polyamines and H2O2 promoted the conversion of EC into SEs. Furthermore, we found that low and moderate stress is beneficial for proliferation of EC and SEs formation. Together, our global analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress response synergistically regulating SE in cotton.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-016-0511-6) contains supplementary material, which is available to authorized users.

Plantlet regeneration through somatic embryogenesis in Nordmann’s fir (Abies nordmanniana)

I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana (Steven) Spach. Additionally, the effect of ABA, PEG-4000 and different wavelengths on the maturation of somatic embryos was tested. The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of micropropagation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nordmanniana can be carried out, including the preceding 24-h seed disinfection with NaOCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.

Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs.

THE USE OF SOMATIC EMBRYOGENESIS IN ARTIFICIAL SEEDS PRODUCTION IN CAULIFLOWER (BRASSICA OLERACEAE VAR. BOTRYTIS)

A reliable method was investigated for high production of cauliflower artificial seeds through somatic embryogenesis. Optimum Embryogenic callus proliferation was obtained using friable callus derived from hypocotyls and root explants on semi-solidified MS (Murashige and Skoog, 1962) media containing 0.15, 0.5 mg/L of 2,4-D and 0.5, 1.0, 2.0 mg/L kinetin. This callus induction medium (CIM) was used to maintain callus tissue by subculturing every 21 days. However, the initiation of liquid cultures from chopped embryogenic clusters and the high production of somatic embryos SEs were established on somatic induction medium (SIM) consisting of MS agitated liquid medium supplemented with 0.05 mg/L IAA, 0.5 mg/L kinetin and 20% sucrose after 20 days. The highest germination and conversion rate 60 and 100%, respectively, was achieved after one month when somatic embryos cultivated on MS medium without hormones. Mature somatic embryos were encapsulated in Ca-alginate beads to produce artificial seeds. The ability of cauliflower SEs to regenerate complete plantlets was discovered.