Developmental Stages Of Somatic Embryos Research Articles

Scanning Electron Microscopy Analysis of Tea’s Embryo Axis Explant Cultured on Murashige and Skoog Medium Containing 2,4-Dichlorophenoxyacetic acid

Camellia sinensis L. is an important crop in Indonesia as healthy beverage that contains several secondary metabolism compounds, such as polyphenols and catechins. Tissue culture including somatic embryogenesis and organogenesis has been used for propagating plant for various needs. In this present short-communication, scanning electron microscopic (SEM) analysis of tea was conducted and discussed. This study aimed to investigate surface ultrastructure of TRI2025 embryo axis tea clone cultured on Murashige and Skoog (MS) medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D). The results revealed two different forms of explant’s development, i.e. somatic embryo and transitional form between somatic embryogenesis and organogenesis; or called by “Globular-like Structure” (GLS). Surface ultrastructure analysis of somatic embryo and GLS revealed respectively many stages of somatic embryo development i.e. globular, torpedo, and cotyledon stage, and leaf development form GLS regeneration.

Genome-Wide Identification and Expression Analysis of GATA Family Genes in Dimocarpus longan Lour.

GATA transcription factors, which are DNA-binding proteins with type IV zinc finger binding domains, have a role in transcriptional regulation in biological organisms. They have an indispensable role in the growth and development of plants, as well as in improvements in their ability to face various environmental stresses. To date, GATAs have been identified in many gene families, but the GATA gene in longan (Dimocarpus longan Lour) has not been studied in previous explorations. Various aspects of genes in the longan GATA family, including their identification and classification, the distribution of their positions on chromosomes, their exon/intron structures, a synteny analysis, their expression at different temperatures, concentration of PEG, early developmental stages of somatic embryos and their expression levels in different tissues, and concentrations of exogenous hormones, were investigated in this study. This study showed that the 22 DlGATAs could be divided into four subfamilies. There were 10 pairs of homologous GATA genes in the synteny analysis of DlGATA and AtGATA. Four segmental replication motifs and one pair of tandem duplication events were present among the DlGATA family members. The cis-acting elements located in promoter regions were also found to be enriched with light-responsive elements, which contained related hormone-responsive elements. In somatic embryos, DlGATA4 is upregulated for expression at the globular embryo (GE) stage. We also found that DlGATA expression was strongly up-regulated in roots and stems. The study demonstrated the expression of DlGATA under hormone (ABA and IAA) treatments in embryogenic callus of longan. Under ABA treatment, DlGATA4 was up-regulated and the other DlGATA genes did not respond significantly. Moreover, as demonstrated with qRT-PCR, the expression of DlGATA genes showed strong up-regulated expression levels under 100 μmol·L-1 concentration IAA treatment. This experiment further studied these and simulated their possible connections with a drought response mechanism, while correlating them with their expression under PEG treatment. Overall, this experiment explored the GATA genes and dug into their evolution, structure, function, and expression profile, thus providing more information for a more in-depth study of the characteristics of the GATA family of genes.

Cloning, Characterization, and Expression Pattern Analysis of the BBM Gene in Tree Peony (Paeonia ostii)

BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.

Production of Black Cumin via Somatic Embryogenesis, Chemical Profile of Active Compounds in Callus Cultures and Somatic Embryos at Different Auxin Supplementations

Black cumin or Nigella sativa L. is a medicinal plant of the Ranunculaceae family that has enormous importance. It has traditionally been used to cure a lot of diseases since ancient times. In the current study, the effects of different auxins on callus induction and subsequent somatic embryo formation of N. sativa L. cv. Black Diamond were examined. The best result of callus induction was observed when cotyledon explants were incubated in a Murashige and Skoog (MS) medium supplemented with 1.0 mg L−1 α-naphthaleneacetic acid (NAA). The formation of somatic embryos was achieved efficiently from cotyledon-derived calli cultured on a 2 mg L−1 Indole-3-butyric acid (IBA)-containing medium. Furthermore, histological analysis of embryogenic calli was used to detect the presence of different developmental stages of somatic embryos. In contrast to the calli and embryos of N. sativa ‘Black Diamond’, which initiated in the dark, light was necessary for the complete differentiation of callus and embryo cultures into shoots/developed plants. Hypocotyl-derived calli and embryos were successfully differentiated on IBA at 2.0, 1.0 mg L−1, and NAA at 2.0 mg L−1. To the best of our knowledge, this work can be considered the first report on the differentiation of N. sativa ‘Black Diamond’ somatic embryos into developed plants. Moreover, the metabolic profiles of secondary products of N. sativa ‘Black Diamond’ callus and embryo cultures originated from the best auxin treatments identified and were compared with that of intact seeds. Callus cultures of N. sativa ‘Black Diamond’ contained thymoquinone (TQ) in a significant percentage of the peak area (2.76%). Therefore, callus cultures could be used as a perfect alternative source of TQ for pharmaceutical and therapeutic purposes. In addition, fatty acids and/or their esters were recorded as the major components in callus and embryo cultures. These vital compounds could be isolated and used for numerous industrial applications.

Dynamics of Endogenous Auxin and Its Role in Somatic Embryogenesis Induction and Progression in Cork Oak

Somatic embryogenesis (SE) is a feasible in vitro regeneration system with biotechnological applications in breeding programs, although, in many forest species, SE is highly inefficient, mainly due to their recalcitrance. On the other hand, SE represents a valuable model system for studies on cell reprogramming, totipotency acquisition, and embryogenic development. The molecular mechanisms that govern the transition of plant somatic cells to embryogenic cells are largely unknown. There is increasing evidence that auxins mediate this transition and play a key role in somatic embryo development, although data on woody species are very limited. In this study, we analyzed the dynamics and possible role of endogenous auxin during SE in cork oak (Quercus suber L.). The auxin content was low in somatic cells before cell reprogramming, while it increased after induction of embryogenesis, as revealed by immunofluorescence assays. Cellular accumulation of endogenous auxin was also detected at the later stages of somatic embryo development. These changes in auxin levels correlated with the expression patterns of the auxin biosynthesis (QsTAR2) and signaling (QsARF5) genes, which were upregulated after SE induction. Treatments with the inhibitor of auxin biosynthesis, kynurenine, reduced the proliferation of proembryogenic masses and impaired further embryo development. QsTAR2 and QsARF5 were downregulated after kynurenine treatment. Our findings indicate a key role of endogenous auxin biosynthesis and signaling in SE induction and multiplication, as well as somatic embryo development of cork oak.

Construction of ceRNA Networks at Different Stages of Somatic Embryogenesis in Garlic.

LncRNA (long non-coding RNA) and mRNA form a competitive endogenous RNA (ceRNA) network by competitively binding to common miRNAs. This network regulates various processes of plant growth and development at the post-transcriptional level. Somatic embryogenesis is an effective means of plant virus-free rapid propagation, germplasm conservation, and genetic improvement, which is also a typical process to study the ceRNA regulatory network during cell development. Garlic is a typical asexual reproductive vegetable. Somatic cell culture is an effective means of virus-free rapid propagation in garlic. However, the ceRNA regulatory network of somatic embryogenesis remains unclear in garlic. In order to clarify the regulatory role of the ceRNA network in garlic somatic embryogenesis, we constructed lncRNA and miRNA libraries of four important stages (explant stage: EX; callus stage: AC; embryogenic callus stage: EC; globular embryo stage: GE) in the somatic embryogenesis of garlic. It was found that 44 lncRNAs could be used as precursors of 34 miRNAs, 1511 lncRNAs were predicted to be potential targets of 144 miRNAs, and 45 lncRNAs could be used as eTMs of 29 miRNAs. By constructing a ceRNA network with miRNA as the core, 144 miRNAs may bind to 1511 lncRNAs and 12,208 mRNAs. In the DE lncRNA-DE miRNA-DE mRNA network of adjacent stages of somatic embryo development (EX-VS-CA, CA-VS-EC, EC-VS-GE), by KEGG enrichment of adjacent stage DE mRNA, plant hormone signal transduction, butyric acid metabolism, and C5-branched dibasic acid metabolism were significantly enriched during somatic embryogenesis. Since plant hormones play an important role in somatic embryogenesis, further analysis of plant hormone signal transduction pathways revealed that the auxin pathway-related ceRNA network (lncRNAs-miR393s-TIR) may play a role in the whole stage of somatic embryogenesis. Further verification by RT-qPCR revealed that the lncRNA125175-miR393h-TIR2 network plays a major role in the network and may affect the occurrence of somatic embryos by regulating the auxin signaling pathway and changing the sensitivity of cells to auxin. Our results lay the foundation for studying the role of the ceRNA network in the somatic embryogenesis of garlic.

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro.

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don)

Vanda tessellata (Roxb.) Hook. ex G.Don (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. The present study investigates asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos (SEs), and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months. Seeds cultured on full strength Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP) had the highest frequency of asymbiotic seed germination (94%) as compared with half- and one-fourth strength MS media supplemented with various concentrations and combinations of 6-furfurylaminopurine (kinetin) and BAP. The protocorm like bodies (PLBs) gained dedifferentiation and proliferated into embryogenic calli on MS medium containing 2.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid (IAA). Somatic embryos (SEs) were differentiated from the callus when cultured on MS medium in combination with 1.0 mg L−1 BAP and 0.5 mg L−1 IAA. The morpho-anatomical elucidations authenticated somatic embryogenesis and various developmental stages of SEs from embryogenic calli. Short-term cold storage of SEs was carried out by encapsulating with 2% sodium alginate and 100 mM calcium chloride and stored under dark at − 4 °C for 12 months to conserve this valuable germplasm. The stored artificial seeds were germinated (91%) on MS medium incorporated with additives and 0.5 mg L−1 each of BAP, kinetin, and IAA. Complete retrieval of plantlets was achieved in 8 week with formation of shoots (4.2 cm average length) and roots (2.5 cm). The well developed plantlets were acclimatized in the greenhouse with 90% survival rate. Vanda tessellata (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. A reproducible protocol has been successfully established for asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos, and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months.

Proteomic analysis of somatic embryo development in Musa spp. cv. Grand Naine (AAA)

Somatic embryos are comparable to their zygotic counterparts for morphological traits but are derived from somatic cells through various metabolic regulations, collectively referred as somatic embryogenesis (SE). It has been well exploited for germplasm conservation, genetic engineering, mutation breeding, for artificial seed technology and as a tool for mass multiplication. Though somatic embryo development is an important area of interest in growth, and developmental studies, the underlying molecular mechanism remains unclear. Therefore, understanding the molecular basis behind somatic embryo development can provide insight into the signaling pathways integrating this process. Proteomic analysis of somatic embryo development in cv. Grand Naine (AAA) was carried out to identify the differentially expressed protein during somatic embryo development stages, using two dimensional gel electrophoresis together with mass spectrometry. In total, 25 protein spots were differentially expressed during sequential developmental stages of somatic embryos. Among these, three proteins were uniquely present in 30 days globular stage and six proteins in 60 days old mature somatic embryo. Functional annotation of identified spots showed that major proteins are involved in growth and developmental process (17%) followed by defense response (12%) and signal transportation events (12%). In the early stage, cell division and growth related proteins are involved in the induction of somatic embryos whereas in the late developmental stage, cell wall associated proteins along with stress related proteins played a defensive role against dehydration and osmotic stress and resulted in the maturation of somatic embryo. The identified stage specific proteins are valuable indicators and genetic markers for screening and for media manipulation to improve SE efficiency in recalcitrant crops and varieties.

Identification of an Embryonic Cell-Specific Region within the Pineapple SERK1 Promoter.

Plant tissue culture methods, such as somatic embryogenesis, are attractive alternatives to traditional breeding methods for plant propagation. However, they often suffer from limited efficiency. Somatic embryogenesis receptor kinase (SERK)1 is a marker gene of early somatic embryogenesis in several plants, including pineapple. It can be selectively induced and promotes a key step in somatic embryogenesis. We investigated the embryonic cell-specific transcriptional regulation of AcSERK1 by constructing a series of vectors carrying the GUS (Beta-glucuronidase) reporter gene under the control of different candidate cis-regulatory sequences. These vectors were transfected into both embryonic and non-embryonic callus, and three immature embryo stages and the embryonic-specific activity of the promoter fragments was analyzed. We found that the activity of the regulatory sequence of AcSERK1 lacking −983 nt ~−880 nt, which included the transcription initiation site, was significantly reduced in the embryonic callus of pineapple, accompanied by the loss of embryonic cell-specific promoter activity. Thus, this fragment is an essential functional segment with highly specific promoter activity for embryonic cells, and it is active only from the early stages of somatic embryo development to the globular embryo stage. This study lays the foundation for identifying mechanisms that enhance the efficiency of somatic embryogenesis in pineapple and other plants.

Indirect somatic embryogenesis and Agrobacterium-mediated transient transformation of ginger (Zingiber officinale Rosc.) using leaf sheath explants

ABSTRACTThis study has established an efficient and simple Agrobacterium-mediated transient transformation method for a valuable spice crop ‘ginger’ via indirect somatic embryogenesis. Leaf sheath explants were transformed using Agrobacterium tumefaciens strains EHA105 and LBA4404 harbouring vector pGFPGUSPlus containing β-glucuronidase (gus) reporter and hygromycin phosphotransferase (hptII) selection marker. Different parameters enhancing agro-infection were investigated to optimise transformation efficiency. High transformation frequency was achieved when explants were co-cultivated with bacterial cell density of 0.6 OD600 on medium containing 150 µM acetosyringone for 2 days, followed by selection regime of 40 mg/l hygromycin. Histochemical GUS analysis confirmed the transient expression of gus gene, which was further confirmed by PCR analysis of hptII and nptII genes specific primers. The origin and developmental stages of somatic embryos from leaf sheath explants were investigated with the help of scanning electron micrographs. This newly developed transient transformation system provides a basis for quick expression of marker genes in regenerated plantlets.Abbreviation: GUS - β-glucuronidase; PCR - Polymerase chain reaction; LB - Luria broth; BA - 6- Benzylaminopurine; 2,4-D - 2,4-Dichlorophenoxyacetic acid; TDZ - Thidiazuron; NAA - 1-Naphthaleneacetic acid

Somatic embryogenesis-related gene expression and functional genomics in mangosteen

The atypical somatic embryogenesis of mangosteen leads us to enquire what is happening at each stage of somatic embryogenesis (SE) at the level of the molecular regulation controlling transition between embryogenic stages. The expression of selected genes somatic embryogenesis receptor-like kinase 1 (SERK1), brassinosteroid insensitive 1-associated kinase 1 (BAK1)/SERK3, brassinosteroid insensitive 1 (BRI1), Leafy cotyledon 1 (LEC1) and Wuschel (WUS) were examined during the stages of SE in mangosteen. Somatic embryos of mangosteen were induced on Murashige and Skoog medium with 3.08 μM 6-benzylaminopurine, 3.18 μM thidiazurone and 60 mg/L glucose. Selected genes were isolated from early, mid and late globular stages of somatic embryo development. Quantitative PCR analyses showed that the early globular stage is characterised by low level of expression of SERK1, BRI1 and WUS and no expression of LEC1. The mid globular stage is characterised by a higher level of expression of the SERK1 and BRI1 genes, while the late globular stage is characterised by a high level of expression of LEC1 and WUS genes with low expression of the BRI1 gene. This demonstrates a clear match between globular stages and the expression of key genes at each stage. Transcriptome data from embryogenic structures showed differentially expressed genes involved in hypothetical signal transduction (brassinosteroid, auxin, cytokinin, jasmonic acid, abscisic acid and glucose) and biosynthetic (glucose and polyamine) pathways. The five selected genes showed interconnection with differentially expressed unigenes (up-regulated) indicating molecular interaction involved in somatic embryogenesis of mangosteen.

Histological Evidence of Indirect Somatic Embryogenesis from Immature Female Date Palm Inflorescences.

Rapid production of somatic embryogenesis and date palm regeneration is achieved by culturing immature female inflorescence explants. Inflorescence explants are soft, creamy in color, average 6-7cm in length, and cultured on Murashige and Skoog (MS) medium containing 1mg/L thidiazuron (TDZ). Callus induction occurs after 4-5weeks of culture on the callus induction medium. Subsequently, callus develops embryogenic calli on MS medium supplemented with 0.1mg/L naphthalene acetic acid (NAA). Histological samples were collected successively at the culturing time and during morphogenetic changes throughout the developmental stages of somatic embryos. Initiation of callus and different successive developmental stages for somatic embryos including two-celled, four-celled, globular, bipolar, and fully developed cotyledonary somatic embryos were observed. Mature somatic embryos develop within 10-12weeks after culture establishment.

Chemical Compositions, Somatic Embryogenesis, and Somaclonal Variation in Cumin.

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis of the content of catalase, ascorbate peroxidase, proline, protein, terpenic compounds, alcohol/phenols, aldehydes, and epoxides) and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan). Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 0.1 mg/L kinetin proved to be the best primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet. Results indicated that different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potential. The high content of catalase, ascorbate peroxidase, proline, and terpenic hydrocarbons and low content of alcoholic and phenolic compositions had a stimulatory effect on somatic embryogenesis. Band patterns of RAPD markers in regenerated plants were different from those of the mother plants. This may be related to somaclonal variations or pollination system of cumin. Generally, measurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cumin. Also, somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs.

Enhancing plant regeneration of Lachenalia viridiflora, a critically endangered ornamental geophyte with high floricultural potential

Lachenalia viridiflora is a critically endangered bulbous plant with high potential on the international floriculture market. In the present study, an efficient protocol for in vitro plantlet regeneration through somatic embryogenesis was developed. Embryogenic callus was established from leaf explants cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations and combinations of picloram and thidiazuron (TDZ). A high number of somatic embryos (28.5±1.49) at different developmental stages of somatic embryos (SEs: globular embryos, juvenile embryo and cotyledon with bipolar characteristics) was obtained on Murashige and Skoog (1962) medium with 2.5μM picloram, and 1.0μM TDZ. Histological and scanning electron microscope (SEM) analyses confirmed the presence of somatic embryos. Mature somatic embryos germinated and developed into plantlets after 6 weeks on full strength MS medium. High plant regeneration frequency (91.1%) was achieved on full-strength MS medium supplemented with 5μM phloroglucinol (PG). Well-developed healthy plantlets were successfully acclimatized in the greenhouse with a survival rate of 90%. The result of this study is beneficial in the mass propagation of high-quality L. viridiflora plants for the commercial horticultural market and also provides a platform for future genetic transformation studies.

Somatic embryogenesis in Hypoxis hemerocallidea: An important African medicinal plant

An efficient in vitro plant regeneration system was established via somatic embryogenesis of Hypoxis hemerocallidea, an extensively utilized medicinal plant in South African traditional medicine. Embryogenic callus was obtained on Murashige and Skoog (MS) basal medium supplemented with 15μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5μM N6-benzyladenine (BA). Different developmental stages of somatic embryos (SEs: globular embryos, torpedo-shaped embryos and cotyledonary embryos) were obtained and further matured from the embryogenic callus by subsequent subculture on the same medium. Highest frequency of somatic embryos (30.56±1.15) was recovered after 6weeks. Scanning electron micrographs revealed the presence of somatic embryos. Mature somatic embryos germinated and developed into plantlets after 4weeks on half strength MS basal agar medium. The germination frequency reached 88.9% when using half strength MS basal agar medium supplemented with 1.44μM gibberellic acid (GA3). Rooted plantlets were successfully acclimatized in the greenhouse with a survival rate of 90%. This is the first report on the establishment of H. hemerocallidea somatic embryogenesis. The protocol developed would be helpful in reducing stress on natural populations, provide a system for germplasm conservation, regeneration of large numbers of high value clonal plants for commercial production, genetic transformational studies and provides a system for analysis of bioactive compounds.

Recurrent Somatic Embryogenesis and Plantlet Regeneration in Psidium guajava L.

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

Ultrastructural observations in somatic embryogenesis ofnatural tetraploid Trifolium pratense L.

Previous reports of plant regeneration of natural tetraploid T. pratense L. 'Elci' could be realized only through the apical meristem calli. In order to proceed to the production stage, other regeneration methods need to be tried. Aseptic seedlings were used for the production of somatic embryos through various 2,4-D and kinetin trials. Nonuniform external callus cells with translucent cytoplasm were observed in various developmental stages of somatic embryos. Beneath these cells, there were uniformly aligned, dark- stained embryo cells with dense cytoplasm. Despite the similar developmental stages and cell characteristics of zygotic and somatic embryos, the walls of somatic embryo cells revealed a highly wavy pattern. The nucleus generally contained only one nucleolus, which was spherical, dark stained, and electron-dense. Electron-dense droplets were seen in vacuoles. The cytoplasm consisted of starch- containing amyloplasts, mitochondria, plastids, ribosomes, endoplasmic reticulum, dictyosomes, lipid, and protein bodies. In some of the somatic embryos at the globular and heart stages, vacuole or electron-translucent zones were observed in the nucleolus. Additionally, a few embryo degenerations were recorded during developmental stages of the zygotic embryo. For the first time, the somatic embryos of natural tetraploid T. pratense were produced from hypocotyl (85%), cotyledon (75%), and apical meristem (60%) explants in 0.3 mg/L 2,4-D and 2 mg/L kinetin-containing MS medium. Our study developed an effective and efficient in vitro production method for using natural tetraploid T. pratense in biotechnological studies.

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in Butea monosperma (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L−1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L−1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L−1 2,4-d with 0.1 mg L−1 Kn, and 0.5 mg L−1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L−1 thidiazuron with 0.5 mg L−1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L−1 AA with 3.0 mg L−1 Kn and 0.5 mg L−1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L−1 Kn, 0.3 mg L−1 indole-3-butyric acid, and 0.25 mg L−1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.

In vitro propagation and biochemical changes in Aloe pruinosa

Aloe pruinosa is a vulnerable traditional medicinal plant extensively used in South Africa. Efficient in vitro mass propagation and analysis of biochemical changes of the species was established to counter extinction threats, for commercial cultivation and to understand the effect of oxidative enzymes and phenolic metabolism on oxidative browning in vitro from shoot-tip and leaf explants using various concentrations and combinations of plant growth regulators (PGRs), sucrose and phloroglucinol (PG). The highest number of regenerated shoots per shoot-tip explant (15.4±0.9) was obtained on solid Murashige and Skoog (MSS) medium supplemented with a combination of 5μM benzyladenine (BA) and 4μM indole-3-acetic acid (IAA). Shoots developed significantly more roots (8.6±0.7 per shoot) on ½-MSS medium containing 2μM indole-3-butyric acid (IBA) and 40μM PG. The plantlets were successfully acclimatized (100%) ex vitro in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Somatic embryogenesis with all developmental stages of somatic embryos (SEs: 38–26) were obtained from friable embryogenic callus (FEC) of leaf explants in liquid MS (MSL) containing 0.5μM picloram (Pic), 2μM BA or 1μM thidiazuron (TDZ) and 10μM PG. However, germination and conversion of plantlets from SEs were inhibited as a result of oxidative browning. Polyphenolic enzymes (polyphenol oxidase (PPO) and peroxidase (POD)) activity and total phenolics of different in vitro growth phases (adventitious shoots, regenerated plantlets and calli) with different PGR treatments were determined. The application of PGRs and PG resulted in the control of morphogenic properties, oxidative exudates and browning in A. pruinosa. The study highlights the role of natural PGR (meta-topolin riboside) and PG in reducing the impact of polyphenolic exudation and can be used to improve future in vitro programmes. In addition, micropropagation protocols facilitate commercial and rapid propagation of A. pruinosa for conservation and could satisfy pharmaceutical, cosmetic and food industry demands.