Investigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic regulation of SE in the context of current molecular understanding of plant development.

Translational genomics is defined as the application of molecular-genetic principles derived from model systems to species of experimental or economic interest. The past 20 years of research in plant model systems such as Arabidopsis thaliana have relinquished vast amounts of information regarding gene function, the integration of genetic components into pathways, and the interrelationships between pathways to control form and function in plants and plant-products alike. At present, the challenge is to relate these paradigms to other species of economic or scientific interest. Apart from being an important and valuable crop, strawberry (Fragaria spp.) is a member of the Rosaceae, a plant family containing fruit, nut, ornamental and wood-bearing species. Strawberry is unique within the Rosaceae in that it is a rapidly growing herbaceous perennial with a small genome and the ability to thrive in a laboratory setting. Strawberry species may also be transformed and regenerated in a time scale of weeks or months instead of years. For these reasons, strawberry has been recognized as the translational genomics model for the Rosaceae family. This review summarizes and synthesizes the technical reports of strawberry regeneration and transformation, consolidating the large body of information regarding genetic modification of this important genus.

Malus micromalus Makino was genetically engineered with the rolC gene via Agrobacterium-mediated transformation. The antibiotics carbenicillin, hygromycin, and kanamycin (Km) inhibited shoot regeneration from in vitro leaf explants. The leaf segments were infected with Agrobacterium tumefaciens strain LBA4404 harboring one of the three different plasmids. Shoots were regenerated only from leaf segments infected with LBA4404 harboring the plasmid with the intron-containing neomycin phosphotransferase II gene as the selectable marker. Preculture of leaf segments on regeneration medium for 2 d before Agrobacterium treatment reduced formation of Km-resistant calluses. Transformation was confirmed by a histochemical β-glucuronidase (GUS) assay, amplification of the rolC gene by polymerase chain reaction (PCR), and by Southern blot analysis. The rolC-transformed M. micromalus shoots showed an increased rooting ability without auxin treatment, and reduced height, internode length and leaf areas. This research shows the potential application of using the rolC gene for developing dwarf apple rootstocks.

In vitro studies related to somatic embryogenesis and organogenesis were performed in different cultivars of cotton. Gossypium hirsutum cultivars SH-131, LH-900, Hybrid H8, Khandwa-2, and Gossypium arboreum cultivars BD-1, BD-6, Sarvottam, and Jawahar Tapti were screened for their ability to regenerate in vitro. Cotyledonary leaves and hypocotyls were used as explants. Vigorous callusing was observed in G. arboreum cultivars. Globular somatic embryos were formed in BD-1, BD-6, Sarvottam, Jawahar Tapti, SH-131, and LH-900. Heart-shaped and torpedo stages were also observed. Embryos of BD-1 and BD-6 germinated and formed distinct shoot and root poles. 2-Isopentenyladenine (2iP) was effective in the induction of somatic embryos. Hybrid H8 and Khandwa-2 regenerated by directly forming shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine and formed roots on hormone-free MS medium.

A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by the enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵–6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6 M mannitol, 2.0 μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0 μM NAA or IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0 μM NAA or 2.5 μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0 μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0 μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.

Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop's (1865) medium supplemented with 20 g l⁻¹ sucrose, 8 g l⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mg l⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mg l⁻¹ 6-(γ,γ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.

Somatic embryos of carob (Ceratonia siliqua L.) were induced from cotyledonary segments excised from immature seeds when cultured on Murashige and Skoog media supplemented with several combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The best frequencies of induction (33.8%) were obtained when 4.4 μM BA and 0.5 μM IBA were used. Shoots were also sporadically formed in the same media. When IBA was replaced by other auxins in the induction media, only α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce somatic embryogenesis, although at lower rates than IBA. 2,4-Dichlorophenoxyacetic acid and 4-amino-3,5,6-trichloropicolinic acid were completely ineffective. Besides culture media composition, the developmental stage of the explants at the time of culture showed a strong influence on somatic embryogenesis induction, with cotyledons from stage II pods providing the highest levels of induction. By contrast, the genotype of the explant did not determine a significant role in the induction process. Attempts to achieve somatic embryo germination were mostly unsuccessful, since only shoot development was observed; the highest frequencies of development occurred on media containing only gibberellic acid (3.0 μM). For plant regeneration, the developed shoots were further rooted on IBA-supplemented media, and the plantlets obtained were transferred to soil, where c. 88% of them survived. Histological observations showed the presence of morphologically normal and abnormal somatic embryos, the latter displaying an abnormal pattern of vascular bundles. Ultrastructural analysis showed that the cells of the globular embryos had a dense cytoplasm, whereas those not involved in somatic embryo formation showed signs of senescence. Histological studies were also used to distinguish between somatic embryos and shoots originated in the same media.

A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 g l⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.

In oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent.

Cynara cardunculus var. cardunculus L., also known as cardoon, is a perennial weed naturalized in the Pampas region of Argentina. A quantification of cynarine and chlorogenic acid of callus and leaves from cardoon was performed by means of micellar electrokinetic capillary chromatography, showing that the content of cynarine is higher in calluses than in vivo leaves. The scavenging effect of the callus extract, determined by the thiobarbituric acid method, demonstrated its significant antioxidant capacity. The obtained results revealed that in vitro tissue culture is an excellent tool for producing cynarine for therapeutical purposes.

Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.

An efficient seed germination system was developed for an endemic and endangered orchid of the prairies of Bogotá, Colombia. The effects of three culture media [Murashige and Skoog (MS) salts (1962); Knudson C (KC) salts (1946); and Hydro-Coljap^® salts], activated charcoal [0 and 0.5% (w/v)], α-naphthaleneacetic acid (NAA; 0.0, 2.68 and 5.37 μM), and four light regimes (white light, darkness, red light, and far-red light) on asymbiotic germination of Odontoglossum gloriosum, were studied. The best germination percentage and germination time was obtained on agar-solidified medium supplemented with Hydro-Coljap^® salts and 2.68 μM NAA under red light with a 16-h photoperiod. The addition of activated charcoal did not stimulate either germination or the development of O. gloriosum seedlings. This study describes a single medium-based protocol able to achieve more than 330 000 seedlings within 40 wk, starting from a single capsule. This protocol is sufficient for both large-scale propagation and in vitro conservation of this threatened orchid.

An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoot tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3 ± 0.9 and 15.0 ± 0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8 ± 1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4 ± 0.6 shoots per explant) or 10.0 μM kinetin (10.9 ± 0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055 ± 77 mg and 34.2 ± 1.0 mm, respectively) were significantly higher than those derived from full-strength MS semi-solid medium (437.6 ± 17.3 mg and 15.4 ± 0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after hardening.

In vitro plant cultures tend to get contaminated easily with bacteria and fungi because they are grown for long times in sugar-rich media. Contamination of bioreactors is particularly problematic as larger volumes entail larger losses. To study the movement and develop subsequent control of contaminants in the mist bioreactor, the spore-forming microbes Penicillium chrysogenum and Bacillus subtilis were deliberately inoculated into three possible locations in the reactor: the growth chamber (GC), the medium reservoir (R), or the mist-generating chamber (MG). Compared to inoculation into either R or MG regions, the growth of P. chrysogenum inoculated into the GC required 3 more days (c. 60% more time) to move throughout the rest of the reactor. In contrast, regardless of where B. subtilis was inoculated (GC, R, or MG), it took 7 d to contaminate the entire system. The movement of filamentous fungi and bacteria seems to follow the same route of contamination throughout this reactor. Once visibly present in the reactor, neither contaminant was controllable by addition of the biocide, Plant Preservative Mixture (PPM). Both microbes were completely inhibited if PPM was added to the MG at the time of inoculation and then again 2-d post-inoculation of plants. Reactors were run for 3 wk. Plants remained free of contamination. These results will prove useful in the implementation of large-scale in vitro culture systems.

This work describes an efficient micropropagation protocol of Lippia filifolia. Nodal segments cultivation in MS medium supplemented with 6-benzylaminopurine (4.5 μM)/α-naphthaleneacetic acid (NAA; 54 nM) induced multiple shoots (in average 27 shoots per explant). Elongated shoots were rooted with NAA (0.11 μM) and they maintained ploidy level of the in vitro produced explants. The basic chromosome number were 2n=2x=24. Regenerated rooted shoots were successfully acclimatized under shading house conditions. This is the first report involving the establishment of a protocol for shoot multiplication and rooting for endangered L. filifolia, contributing for germplasm preservation of this species.

In vitro plants in slow-growth storage require routine evaluation for assessment of viability and need for repropagation. Determination of plantlet health by visual assessment is subjective and varies by genus due to variations in growth pattern and plant structure. Developing a standardized plant evaluation system would improve the efficiency of in vitro storage. This study was initiated to develop digital image analysis techniques for plantlets during slow-growth cold storage and to compare that system with visual examinations. Pear (Pyrus communis L.) cultivars were chosen for this initial trial because they have an open structure and clear internode position for image composition. Pear shoots stored at 4°C in tissue culture bags were evaluated monthly by standard visual examination and by digital image analysis. Digital images were evaluated for red, green, blue, modified normalized differences of vegetation index (MNDVI), green/red ratio (G/R), intensity, hue, and saturation at the first two nodes of each plantlet. At 6 mo., the visual ratings had declined steadily for P. communis ‘Luscious’ and ‘Bartlett–Swiss’, while ‘Belle Lucrative’ and ‘Louise Bonne de Jersey’ ratings did not show significant declines until 9 mo. Correlations between visual ratings and G/R and MNDVI values were significant (r²≥0.5) for all cultivars. Regression analysis indicated that the MNDVI and G/R ratios changed significantly over the 15-mo. rating period for most cultivars. Intensity, hue and saturation values were not consistently significant and did not correlate with visual ratings. These results will assist in the development of digital imaging as an alternative technique for evaluation of stored in vitro plantlets.

Dendrobium hybrids have great economic importance in a number of countries. Asymbiotic seed germination and the conventional vegetative method have been commonly used by growers to propagate these plants. To overcome somaclonal variation, which is commonly exhibited by Dendrobium (Nobile group) when micropropagated from protocorm-like bodies, a protocol for propagating Dendrobium Second Love in vitro using axillary buds in the presence of thidiazuron was developed. Random amplified polymorphic DNA analysis was also carried out to check for possible genetic alterations in plants originating from six consecutive subcultures. The results revealed that the established protocol was efficient for the in vitro cloning of this orchid hybrid and the plants obtained from the six subcultures did not exhibit any type of polymorphism.

The photosynthetic and carbohydrate status of an easy-to-acclimatize (EK 16-3) and a difficult-to-acclimatize (EK 11-1) genotype of Uniola paniculata L. (sea oats), a native dune species of the southeastern US, were evaluated during in vitro culture and ex vitro acclimatization. Net photosynthetic rate was eight times greater for EK 16-3 than EK 11-1 plantlets after ex vitro transfer. In vitro-produced leaves were morphologically similar to ex vitro-produced leaves and exhibited similar photosynthetic competence. EK 11-1 plantlets exhibited greater transpiration rates at the time of ex vitro transfer than EK 16-3 plantlets. However, the small magnitude of this difference, although significant, indicated that control of water loss was probably not the main cause for poor acclimatization of EK 11-1 plantlets. Carbohydrate analysis in vitro revealed that EK 16-3 plantlets utilized leaf starch reserves more rapidly than EK 11-1 plantlets. Starch utilization correlated with the development of leaves with expanded leaf blades during in vitro rooting in EK 16-3 plantlets. After ex vitro transfer, both genotypes exhibited significant decreases of starch and soluble sugar content in shoots and roots. However, the higher photosynthetic ability of shoots in EK 16-3 resulted in greater accumulation of shoot soluble sugars than EK 11-1 after 2-wk ex vitro culture. After 6-wk in vitro rooting, there were significantly higher chlorophyll and soluble protein contents, ribulose 1,5-bisphosphate carboxylase (rubisco) and phosphoenolpyruvate carboxylase activities in EK 16-3 than EK 11-1 shoots. These differences also correlated with the development of anatomical and morphological leaf features in EK 16-3 similar to those of greenhouse-produced leaves.

A protocol was developed for rapid clonal propagation of the important medicinal climber, Tinospora cordifolia, through in vitro culture of mature nodal explants. Shoots were initiated on both Murashige and Skoog (MS) medium and woody plant medium (WPM) supplemented with 2.32 μM kinetin (KIN). Of the two basal media tested, WPM was found to be superior to MS medium for the induction of multiple shoots. Among the cytokinins tested, N⁶-benzyladenine (BA) was more effective than KIN for axillary shoot proliferation. KIN was superior to BA in terms of shoot elongation. An average multiplication rate of 6.3 shoots per explant was obtained with WPM supplemented with 8.87 μM BA. Shoot clumps harvested from this medium were transferred to WPM supplemented with 2.22 μM BA and 4.65 μM KIN for shoot elongation. Elongated shoots were rooted in half-strength MS medium supplemented with 2.85 μM indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to sand and established with 80% survival.

A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.

Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA:KN (0.57:13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.

Two independent experiments were performed to establish micropropagation of Cleome spinosa from stem segments. In the first experiment, direct shoot organogenesis on hypocotyl explants from 2-mo.-old nursery-grown seedlings was obtained on Murashige and Skoog medium with different combinations of benzyladenine (BA) and 6-furfurylaminopurine, added either individually or in combination. Best proliferation rates occurred in the presence of 2.2 and 4.4 μM BA and the highest mean number of shoots was produced in response to 4.4 μM BA. In the second experiment, regeneration via direct organogenesis was also obtained from nodal and internodal segments of axenic plants cultured in the presence of BA (4.4 and 8.8 μM) in association with indole-3-acetic acid (IAA) (0.57 and 1.14 μM). Internodal explants were the most responsive on all media tested. The best mean number of shoots per explant was achieved on medium with 4.4 μM BA in association with 0.57 μM IAA. Histological studies of the globular structures formed at the apical portion of the explants revealed direct shoot regeneration and adventitious shoot differentiation from meristematic centers around the vascular bundles of the primary regenerants. All shoots elongated and rooted on MS0 medium. The acclimatization rates ranged between 70 and 84%. Plants reached to maturity and flowered 4 mo. after transfer to ex vitro conditions.

The contamination of soils with excess salts is one of the greatest challenges to plant survival, but some unique species have evolved to thrive in highly saline environments. One such species, Alhagi graecorum Boiss., has been previously shown to accumulate high levels of sodium while growing in salt marshes. The aim of this research was to investigate the effects of saline conditions on the growth and the regeneration capacity of this species. Plantlets and explants of A. graecorum were cultured on a medium supplemented with various concentrations of NaCl, where A. graecorum tissues accumulated up to 8% Na . The capacity for regeneration was enhanced by the excess sodium, indicating a requirement of salt for optimal growth and development in this species. Further study of this species may provide new concepts and understanding of the metabolism of sodium in higher plants.

Shoot formation from rhizome explants of Cymbidium kanran was promoted on Murashige and Skoog (MS) medium: (1) with 1 mg l⁻¹ (4.4 μM) 6-benzyladenine (BA) and 0.1 mg l⁻¹ (0.54 μM) α-naphthaleneacetic acid (NAA); (2) with ethylene inhibitor (silver nitrate, AgNO₃); or (3) by reducing ammonium nitrate (NH₄NO₃) and potassium nitrate (KNO₃) to 25 and 50%, respectively, of their original concentrations. Shoot formation by BA and NAA was strongly inhibited with the application of ethephon, an ethylene releaser. The ethylene production from the rhizome explants was reduced 30–55% on low nitrogen medium after 1–3 mo. of culture and 52% on BA and NAA medium after 1 mo. of culture compared with explants on standard MS medium. No difference in endogenous auxin (indole-3-acetic acid, IAA) and cytokinin (isopentenyl adenosine, iPA) contents in the rhizomes was found between the treatments. Low ethylene levels were correlated with higher frequency of shoot formation from the rhizomes.