Hormone-free Murashige Research Articles

Seed germination and vegetative and in vitro propagation of Hieracium lucidum subsp. lucidum (Asteraceae), a critically endangered endemic taxon of the Sicilian flora.

Hieracium lucidum subsp. lucidum is a critically endangered endemic taxa of the Sicilian flora. It is a relict of the Tertiary period surviving on the cliffs of Monte Gallo (NW-Sicily). This research focused on finding the best protocols for seed germination and vegetative and in vitro propagation to contribute to ex situ conservation. Seed germination tests were carried out using constant temperatures of 15 °C, 20 °C and 25 °C in continuous darkness and an alternating temperature of 30/15 °C (16 h/8 h, light/dark). The seeds had no dormancy, and a high germination capacity (70-95%) was obtained at all tested thermoperiods. The possibility of vegetative propagation of the taxon was evaluated through the rooting capacity of stem cuttings treated or not treated with indole-3-butyric acid (IBA). All cuttings were treated with IBA rooted within 2 months, while only 50% of the untreated cuttings were rooted within a longer time. An efficient protocol for rapid in vitro propagation from leaf portions was developed. The response of explants was tested on hormone-free Murashige and Skoog (MS) basal medium and MS enriched with different types of cytokinins: 6-Benzylaminopurine (BAP) and meta-Topolin (mT) in combination with naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) at the same concentration. The combination of mT (2 mg L-1) and 2,4-D (1 mg L-1) in the medium was the most effective and showed the highest percentage of callus induction and the mean number of regenerated shoots. The maximum rate of root regeneration and the maximum number and length of roots were obtained on hormone-free MS and MS enriched with IBA at concentrations of 1 mg L-1. From the results obtained, it can be concluded that H. lucidum subsp. lucidum can be successfully propagated using one of the tested techniques, subject to the availability of the material for reproduction.

Mg2+ Ions Stimulate both Helical Protoplast-Callose-Fiber Formation and Protoplast Division in a Mangrove Tree, Sonneratia caseolaris: Analysis of Sub-fibril Structures of PCF by Atomic Force Microscopy

The effects of four salts, NaCl, KCl, MgCl2 and CaCl2, on protoplast division and protoplast-callose-fiber (PCF) formation were examined using suspension cultured cells of a mangrove tree, Sonneratia caseolaris. Basal medium was hormone-free Murashige & Skoog’s medium containing 0.8 M sorbitol and 3% sucrose. Addition of 50-100 mM of Mg2+ ions highly stimulated both cell division and PCF formation, while addition of Ca2+ ions was inhibitory. Addition of Na+ ions at 10-25 mM and K+ ions at 50-100 mM stimulated protoplast division but not PCF formation. Helical PCF rapidly elongated from a specific site of cell division plate after 3 days of culture. The β-1,3-glucan (callose) component of the protoplast-fibers was stained with Aniline Blue fluorescent dye and was re-certified enzymatically using laminarinase. After selecting the PCF using a micromanipulator, we analyzed the fibril- and sub-fibril- structures using laser confocal scanning microscopy (LCSM), and atomic force microscopy (AFM). We discuss the uniqueness of the PCF of S. caseolaris as compared to PCFs of several plants.

In vitro regeneration from adult node explants of Juniperus oxycedrus

In the present study the in vitro propagation from adult native plants of Juniperus oxycedrus was investigated. The effect of seasonal explant collection (spring, summer, winter), in varying media and cytokinin type were examined. The decontamination process of explants taken in the summer was more efficient than that of the other seasons (57.3% of the explants survived). In the spring a low performance in shoot formation took place both on hormone free Murashige and Skoog (MS) media and Rugini Olive Medium (OM) or each containing 6-benzyladenine (BA) at 1.0 mg L-1 by which shoots did not elongate more than 0.5 cm. In the summer OM was used, and a higher in vitro performance was observed on medium containing 0.1 mg L-1 6-(γ,γ-Dimethylallylamino)purine (2iP); the shoot formation percentage was 72.0%, and respectively 3.1 shoots, 1.4 cm in length, were formed. In the winter, the shoot formation percentage was 92.0-100.0%, and respectively 3.9 shoots of 0.4 cm length were formed on medium containing 1.0 mg L-1 2iP. The multiplication phase on cytokinin-containing media led to a satisfactory proliferation rate in terms of shoot formation percentage and shoot number. The higher shoot number (3.3) was recorded on media containing 2iP at 1.0 mg L-1, while the shoot length was longer (2.0) cm, on hormone-free media. Initiation of roots was not observed during the rooting phase on hormone-free half-strength OM, or supplemented with 1.0 and 2.0 mg L-1 IBA. Spontaneous rooting at a low percentage (10.0%) was observed on OM media supplemented with 0.1 mg L-1 2iP and successful acclimatisation took place on a 1:1 (v/v) ratio of peat to perlite substrate.

Regenerative ability and micropropagation of <I>Petunia hybrid</I> in vitro

Scientific relevance. The garden petunia, Petunia hybrida, is a popular and wide spread ornamental crop from the family Solanaceae. It is a promising model plant for molecular and genetic research. In vitro micropropagation plays an important role in the distribution of the garden petunia because the survivability and quality of seed material decreases significantly in every subsequent generation. Besides, micropropagation reduces the cost of production substantially. Considering that very few researchers addressed this question in the Russian Federation, this direction of research is still worthy of attention.Materials and methods. The experiments were conducted by the Laboratory of Breeding and Genetic Research on Field Crops at FSBSI “Federal Scientific Center of Agricultural Biotechnology of the Far East named after A.K. Chaiki”. Seeds of Petunia hybrida (double-flowered) were used as primary explants. Liquid bleacher ACE diluted with distilled water in the proportion 1:9 was used as a sterilizing agent (the working solution contained 0.50% NaOCl). The total time of exposure was 15 minutes. The primary explants were subcultured onto a hormone-free Murashige and Skoog basal medium containing 20 g/L sucrose and 6 g/L agar. Isolated in vitro objects were cultured in test tubes with cotton-gauze plugs at an illuminance of 4000 lx, a temperature of 22–25 °C, and a 16h photoperiod in a culture room. The duration of one passage was 60 days. Micropropagation was carried out using 7- 10 mm cuttings with one or two nodes. The pot culture of the regenerants was established under controlled conditions in a light room (photoperiod was 16 hours, temperature was 23°С).Results. The optimal method for introducing Petunia hybrida into cell culture is the use of seeds treated with the solution of bleacher ACE that was diluted with distilled water in the proportion 1:9. The optimal time of exposure is 15 minutes. Petunia hybrida demonstrated a high regeneration rate on the hormone-free MS medium – it had a fast growth and development rate, and good rhizogenesis; the reproductive rate was 8.77. For the micropropagation of the garden petunia, it is advisable to use cuttings of test tube plants, which should be placed onto a hormone-free MS medium. The test tube plants of Petunia hybrida acclimatized successfully on a soil substrate. This shows the high plasticity of the culture.

Assessment of different kenaf (Hibiscus cannabinus L.) accessions in hormone-free plant growth medium and identification of the associated fungal contaminants

Kenaf is an industrial crop, which serves as income to small-holder farmers in Sub-Saharan Africa. Unfortunately, the oily and recalcitrant nature of its seeds makes seed germination during conventional propagation almost impossible. Plant tissue culture is a viable alternative to conventional propagation of kenaf seeds, however, it requires the use of expensive plant growth hormones and it is prone to fungal contaminations. This study therefore aimed at comparing the seedling growth of four different accessions of kenaf seeds (Tianung 2, Ifeken 100, Ifeken 400, and Ifeken D1 400) in half-strength growth hormone-free Murashige and Skoog (MS) medium as well as to identify the associated fungal contaminants in this medium. Data were obtained on plant length (cm), plant width (mm), number of leaves, leaf length (cm), number of roots, and root length (cm) at 14 days after culture (DAC). There were significant differences in all the growth parameters observed (p<0.05) except the plant width (mm) [p>0.05] at 14 DAC. Ifeken 400 had the highest average plant length (14.5 cm) followed by Ifeken 100 (10.5 cm) at p<0.05. On the other hand, the least average plant length (4.16 cm) was observed from both Tianung 2 and Ifeken D1 400 at p<0.05. The highest average root length (5.3 cm) was produced by Ifeken 400 while Ifeken DI 400 had the least average root length (2.6 cm) [p<0.05]. The fungal contaminants associated with the kenaf cultures in the half-strength growth hormone-free MS media were Aspergillus spp., Fusarium spp. and Penicillium spp. Therefore, half-strength growth hormone-free MS medium could be considered as a low cost alternative method of propagating kenaf seeds in order to prevent poor seed germination and allow for improvement of kenaf as an industrial crop. Keywords: Aspergillus spp ., Fungal contamination, Hibiscus cannabinus L., Plant growth hormones, Plant tissue culture DOI: 10.7176/JBAH/12-10-05 Publication date: May 31 st 2022

Abietane Diterpenoids from the Hairy Roots of Salvia corrugata.

Salvia corrugata Vahl. is an interesting source of abietane and abeo-abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of S. corrugata and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 Agrobacterium rhizogenes onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of rolC and virC1 genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA®. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7-O-acetylhorminone and 7-O-methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of S. corrugata can be considered a source of these compounds.

In Vitro Preservation of Potato Plants

Abstract Potato plants free of viruses, bacteria and viroid could be maintained in vitro for a long time. Proper preparation of potato plants for in vitro culture provide its long-term storage in good condition. First step is to establish in vitro culture from young greenhouse grown plants in early developmental stage. Explants are maintained and propagated by nodal subculture on hormone-free Murashige and Skoog (MS) medium at temperature 20-22°C. After rooting, for longer preservation plants are maintained at temperature 8-10°C.

High Concentration of Benzyladenine Solution Stimulates Anthers for Inducing Callus in Ricinus Communis L.

An high-frequency protocol for induction of callus from anther explants of Ricinus communis was described. When anther explants of R. communis was cultured directly onto medium containing 6-benzylaminopurine (BA) induced formation of only poor quality callus that had a low induction frequency of anther callus (10.67%). However, treating the anther explants with high concentrations (7.5-120 mg/L) of BA solution for short time periods (5-80 min) helped to improve the induction frequency and enhance the quality of the callus formation significantly. The best callus induction (41.25%) was observed when anther explants were treated with 15 mg/L BA solution for 10 min before being inoculated onto hormone-free Murashige and Skoog (MS) medium for 30 days. In order to further optimize the culture system, after treated with 15 mg/L BA for 10 min, anther explants were inoculated on the hormone-free MS medium contained concentrations of sodium nitroprusside (SNP). The results showed that SNP significantly promoted the response of callus induction, especially when 8 mg/L SNP was applied, the the highest percentage of callus induction (60.37%) were gained.

In Vitro Propagation and Cryopreservation by V-Cryoplate Technique of Foeniculum Vulgare Mill = الإكثار الدقيق والحفظ باستخدام تقنية صفائح الألمنيوم لنبات الشومر البري

In this study, mass production of Foeniculum vulgare Mill was successfully achieved from seeds using in vitro propagation technique. Seeds were surface-sterilized and inoculated on the surface of hormone-free Murashige and Skoog (MS) medium until full germination. For shoot proliferation, different concentrations (0.0, 0.1, 0.6, 1.0, 1.6, 2.0) of (BA, kinetin, 2ip, and TDZ ) were used. The best results were obtained on MS medium supplemented with BA (1.6 mg/L) which gave (7.4 shoot with 3.9 cm height). Rooting was also experimented at different levels of (IBA, IAA or NAA). The highest root number (8.0) and length (3.6 cm) revealed on MS medium supplemented with 1.0 mg/L NAA. For cryopreservation of Foeniculum vulgare Mill shoot tips; a regrowth percentage up to (90 %) was obtained when shoot tips were precultured on 0.3 M sucrose for 7 days. These results pave the way to conserve and propagate this valuable medicinal plant.

English

Being a tropical country, Vietnam has an exceptional diversity of medicinal plants, particularly in the highland provinces (for example, Lam Dong, Daklak and Kontum provinces). However, a list of many rare medicinal plants are recorded in the Vietnamese Red Data Book and most of them are globally threatened. In this study, the effect of the type of culture media and the concentration of phytohormones on germination, shoot height and shoot number derived from Codonopsis javanica (Blume) Hook f. & Thomson synthetic seeds were evaluated under laboratory conditions. The germination rate and shoot-formatting potential of C. javanica artificial seeds were greatly affected by the components of plant tissue culture media after 40 days of sowing. Particularly, germination rate of Codonopsis artificial seeds was maximum when they were sown in hormone-free Murashige and Skoog (MS) medium in comparison with other culture media (1/2 MS, 1/2 Schenk and Hide-brandt (SH), and SH). Addition of 1.0 mg l-1 N-6-benzyladenine (BA) and 0.1 mg l-1 naphthaleneacetic (NAA) into MS medium led to increased shoot height, shoot number and germination percentage of the synthetic seeds compared to other concentrations of these phytohormones. The height of shoots derived from the synthetic seeds was inhibited according to the increase of supplemented abscisic acid (ABA) concentration in MS medium. The best planting medium to use to germinate the artificial seeds of C. javanica and to increase their shoot-forming potential was the MS supplemented with 1 mg l-1 BA and 0.1 mg l-1 NAA. The study is expected to provide new insights into the application of synthetic seeds in rapid multiplication of Vietnamese C. javanica, establishing the traditional herbal medicine resource for human demands. Key words: Medicinal plant, Codonopsis javanica, artificial seed, plant tissue culture medium.

Micropropagation of Pluchea sagittalis (Lam.) Cabrera

ABSTRACT:The objective of this study was to develop an in vitro protocol for the micropropagation of Pluchea sagittalis (Lam.) Cabrera. Plants were regenerated in vitro from stem segments. The procedure employed includes: 1) surface sterilization of shoots by immersion in 70% ethanol for 10 s followed by 1.0% NaOCl for 10 min, and subsequent immersion in 0.05% HgCl2 for 3 min and two washes with sterile distilled water; 2) induction of root and shoot by culture on hormone-free Murashige and Skoog medium (MS); 3) acclimatization of 60 day-old-plantlets in soil under ex vitro conditions. Minimum contamination was observed for apical shoot explants (10%). However, independently of the explant position in the stem, all explants regenerated new shoots. Various successive cultivations from stem explants every 60 days during more than 1 year have been shown to be a suitable method to propagate P. sagittalis in vitro. Low salt concentration (25% of the normal concentration) in the medium promoted greater growth of plantlets because the plants had a higher number of roots and longer roots in such an environment. Our protocol for the micropropagation of P. sagittalis can be accomplished as a two-step procedure within a short period of time (two months) before transplanting.

Efficient culture protocol for plant regeneration from petiole explants of physiologically mature trees of Jatropha curcas L.

An efficient and reproducible protocol for induction of adventitious shoot buds and plant regeneration from petiole explant cultures of Jatropha curcas, an important biofuel crop, is described. Physiologically mature trees of three J. curcas genotypes were selected and explants were prepared from young petioles. Treating the explants with high concentrations (5 to 120 mg/L) of thidiazuron (TDZ) solution for short time periods (5 to 80 min) helped increase the regeneration frequency and improved the quality of the regenerated buds significantly. The age of the petioles and inoculation methods were found to influence the culture results. The best shoot buds induction (65.78%) and number of buds (6.77) per explant was seen in the second petiole explants of genotype M-1 treated with 20 mg/L TDZ solution for 20 min, followed by 35-day culture on hormone-free Murashige and Skoog medium. The regenerated buds could elongate to become shoots in a medium containing gibberellic acid. The elongated shoots initiated roots to become intact plantlets in rooting medium containing indole-3-butyric acid and L-glutamine (Gln), and supplementing 16 mg/L Gln into the rooting medium effectively stimulated the initiation and growth of roots, with the best rooting rate (51.72%). After acclimatization, these plantlets were transplanted to soil wherein normal growth was observed. Therefore, an intact plantlet could usually be obtained at 60 days of culture by using the culture protocol described in this study. This protocol can be used for mass production of true-to-type plants and the production of transgenic plants through Agrobacterium/biolistic-mediated transformation.

Differing Sucrose Requirements for In-vitro Conservation of Cassava Genotypes

Field conservation of vegetative propagated crops poses a major problem to curators of germplasm, especially in developing countries. An alternative method to ensure security of germplasm is the use of tissue culture techniques in media formulated for slow growth. However, tissues of plant species may require different nutrients for optimum growth. The objectives of this study were to a) assess the effects of sucrose on the performance of different cassava genotypes and b) recommend sucrose levels for in-vitro conservation of the genotypes. Four sucrose levels (0, 10, 20, and 30 g l -1 ) and apical meristems of five cassava genotypes (Bankye Hemaah, Bankye Botan, Tek Bankye, Doku Duade, and Essam Bankye) cultured in-vitro was studied at the Kwame Nkrumah University of Science and Technology’s plant biotechnology laboratory. Growth media were prepared using hormone-free Murashige and Skoog (MS) basal media formulation. Inoculated cultures were exposed to 16 hours of light and 8 hours of darkness with light illuminance of 3500 lux and also maintained at 24±2°C temperature and relative humidity of 70%. All genotypes Original Research Article Sintim and Akromah; IJPSS, 7(1): 45-54, 2015; Article no.IJPSS.2015.130 46 showed a direct regeneration without callus formation. Generally, sucrose enhanced the growth performance of plantlets; however, the genotypes responded differently to sucrose in leaf formation, plant height, and rooting ability with time. For long term conservation, growth medium must sustain the health of plantlets with infrequent need for sub-culturing. As such, sucrose levels of 10 g l for Essam Bankye, 20 g l for Doku Duade and Tek Bankye, and 30 g l for Bankye Hemaa and Bankye Botan were the recommended rates for in-vitro conservation.

Molecular, chemical and biological stability of plants derived from artificial seeds of Centella asiatica (L.) Urban—An industrially important medicinal herb

Synthetic seed technology offers a viable option for large scale multiplication and in vitro conservation of endangered and elite plant genotypes with economy of space, time, and operational cost. In the present study an efficient method for the preparation, short-term conservation, and plantlet regeneration from synthetic seeds of Centella asiatica (L.) Urban, is described for the first time. Axillary buds/nodal segments excised from multiple shoot cultures were encapsulated in 4% (w/v) sodium alginate beads following complexation in 75mM calcium chloride solution. The seeds were kept on moist filter paper in sealed petri plates and stored at 25±3°C temperature for 200 days. The seeds thus conserved recorded more than 85% germination and plantlet conversion frequency upon shifting onto a hormone-free Murashige and Skoog medium. The rooted plantlets recorded 85-90% establishment in soil and did not show any morphological variation when compared with mother plants. The genetic fidelity of the regenerated plants was further confirmed using random amplified polymorphic DNA (RAPD) analysis wherein 19 decamer primers produced a total of 60 distinct monomorphic bands. The HPLC analysis of leaf extracts of synthetic seed-derived plant progeny and the mother plant showed qualitative and quantitative uniformity in terms of four major bioactive centellosides namely, asiaticoside, madecassoside, asiatic acid, and madecassic acid. The leaf extracts of the regenerated plants depicted the same magnitude of wound healing activity in excision wound model of mice as that of the mother plant. The relevance of the developed synseed technology for short-term in vitro conservation, micro-cloning and exchange of selected germplasm of C. asiatica without affecting the chemical and biological activities is discussed.

High Frequency of Plant Regeneration through Cyclic Secondary Somatic Embryogenesis in Panax ginseng.

Somatic embryogenesis is one of good examples of the basic research for plant embryo development as well as an important technique for plant biotechnology such as medicinally important plants. Single embryos develop into normal plantlets with shoots and roots. Therefore, direct single embryogenesis derived from single cells is highly important for normal plant regeneration. Here we demonstrate that the cyclic secondary somatic embryogenesis in Panax ginseng Meyer is a permanent source of embryogenic material that can be used for genetic manipulations. Secondary somatic embryos were originated directly from the primary somatic embryos on hormone-free Murashige and Skoog medium, and proliferated further in a cyclic manner. EM medium (one third of modified MS medium [MS medium containing half amount of NH4NO3 and KNO3] with 2% to 3% sucrose) favored further development of proliferated secondary somatic embryos into plantlets with root system. The plantlets developed into plants with well-developed taproots in half-strength Schenk and Hildebrandt basal medium supplemented with 0.5% activated charcoal.

Cryopreservation of Teucrium polium L. shoot-tips by vitrification and encapsulation-dehydration

Teucrium polium L. with the common name of Felty Germander is one of the plants flora that is widely used in folk medicine in many Middle East countries, it is an endangered plant species and must be highly considered for preservation. Cryopreservation of T. polium by vitrification and encapsulation-dehydration was successfully achieved in this study. Shoot-tips were excised aseptically from in vitro grown plants and incubated for 3 days on solid hormone free-Murashige and Skoog (HF-MS) media supplemented with 0.3 M sucrose under complete darkness at 24 ± 1 °C. In vitrification, shoot-tips were loaded in 0.4 M sucrose and 2 M glycerol for 20 min followed by desiccation with different combinations and concentrations of plant vittrification solution 2 (PVS2), before immersion in Liquid Nitrogen (LN). Whereas for the encapsulation-dehydration; shoot-tips were encapsulated in calcium alginate and dehydrated under laminar air flow cabinet for 0, 3, 6, or 9 h. A total of 60 % of the cryopreserved vitrified shoot-tips survived when desiccated in concentrated PVS2 solution for 20 min, whereas, 28 % of the cryopreserved vitrified shoot-tips were regrown after 20 min of desiccation by two step increase in PVS2 concentration. Complete survival were obtained for the non-cryopreserved encapsulated shoot-tips treated for 3 days in 0.5 M sucrose with MS media without or with 3 h of dehydration, whereas, only 20 % of the cryopreserved encapsulated shoot-tips were regrown. The procedures developed in this study are easy to handle and produced a high levels of shoot formation.

Enhanced accumulation of flavonolignans in Silybum marianum cultured roots by methyl jasmonate

Root cultures of Silybum marianum (L.) Gaertn. (Asteraceae) were established from in vitro germinated sterile plantlets. The cultures grew in hormone-free Murashige and Skoog medium. The flavonolignan content in the cultured roots was determined by HPLC using 30% acetonitrile in acidified water (0.5% phosphoric acid). The major flavonolignans produced by the cultured roots were silychristin (74.2μgg−1 fresh weight (FW)) and silydianin (8.1μgg−1 FW). The flavonolignan precursor taxifolin was also detected in the cultured roots (40.8μgg−1 FW). Addition of methyl jasmonate to 7-days-old root cultures for 48h increased the content of the produced flavonolignans and taxifolin to approximately 300% of the control cultures. Methyl jasmonate also enhanced about sixfold the accumulation of a compound identified as 3,3′,5,5′,7-pentahydroxyflavanone.

Establishment of plant regeneration of Michelia champaca L. through cell suspension culture technique

Michelia champaca L. is a woody tree species, which has high economical value as a basic material for medicinal and fragrance products. However, there are several problems encountered in conventional propagation of M. champaca, which are generally low through seed germination, while vegetative layering technique produces limited planting materials. This experiment was conducted to establish high frequency M. champaca regeneration through cell suspension culture technique. Liquid Murashige and Skoog medium containing 2 mgL-1 α-naphthalene acetic acids induced high frequency of somatic embryos formation (47.67 ± 4.53 per ml). Meanwhile for germination of somatic embryos, the highest total number of somatic embryos germinated into plantlets obtained was 34% in hormone free Murashige and Skoog medium. Plant regeneration of M. champaca via cell suspension culture technique which was successfully established in this study could be used for mass production of planting materials of this tree. Key words: Michelia champaca, somatic embryogenesis, cell suspension culture technique.

Establishment of plant regeneration system in Erianthus arundinaceus (Retz.) Jeswiet, a potential biomass crop

Erianthus arundinaceus (Retz.) Jeswiet is regarded as one of the most promising biomass crops because of its high-yield biomass and resistance to biotic and abiotic stresses. We investigated the frequencies of callus formation from seeds and leaf sheaths of E. arundinaceus under 25 culture conditions containing various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Callus formation from seeds was significantly affected by 2,4-D but not by BA. BA markedly inhibited callus formation from leaf sheaths, indicating that the pattern of the callus formation frequency in the tested media differed depending on the explant types. We showed that media containing 4.0 or 6.0 mg L−1 2,4-D effectively produced callus from both explants; medium containing 2.0 mg L−1 2,4-D was shown to be preferred for callus maintenance among the five tested subculture media. Plants were successfully regenerated on a hormone-free Murashige and Skoog medium, subsequently acclimatized and transplanted to an experimental field. To evaluate the genetic variability of the regenerated plants, random amplified polymorphic DNA (RAPD) analysis was performed in 31 regenerated plants with 17 primers. The result showed very few genetic changes among the regenerated plants in RAPD analysis. The present study demonstrated the establishment of the callus induction and plant regeneration system using seeds and leaf sheaths in E. arundinaceus, which contributed to the development of genetic engineering techniques for crop improvement.

Somatic embryogenesis and organogenesis from mature caryopses of North African barley accession “Kerkena” (Hordeum vulgare L.)

The regeneration ability of a Tunisian barley accession originated from Kerkena islands was monitored through somatic embryogenesis and organogenesis. To prevent or to reduce normal germination, longitudinally bisected as well as base-wounded mature caryopses were cultured on a modified Chee and Pool-based medium (CP) enriched with different phytohormonal combinations. The greatest embryogenesis response was obtained when base-wounded caryopses were cultured on CP enriched with 2 mg/l chlorophenoxyacetic acid + 2.5 mg/l kinetin (76.85%). The same combination coupled to longitudinally bisected caryopses led to the embryogenic induction at the hypocotyl base of the germinated caryopses (61.9%). Embryogenic calluses differentiated into globular, heart-shaped, torpedo, and fully differentiated stages of somatic embryos on hormone-free Murashige and Skoog-based medium. Rooted plantlets were successfully transferred to soil and grown to maturity in the greenhouse and produced fertile seeds within 3 mo. On the other hand, organogenesis was achieved on CP enriched with 2 mg/l 2,4-dichlorophenoxyacetic acid + 2.5 mg/l kinetin. Histological aspects and scanning electron microscopy of both regeneration methods confirmed further the embryogenic and organogenic nature of the established processes. This efficient plant regeneration system provides a foundation for generating transgenic plants and germplasm preservation of “Kerkena” barley accession.