Showing papers in "In Vitro Cellular & Developmental Biology – Plant in 2013"

Molecular regulation of plant somatic embryogenesis

Abstract: In vitro embryogenesis is an asexual reproduction process by which embryos are produced from either gametophytic (androecium/gynoecium) or sporophytic (somatic) tissues Regardless of the type of explant used, the hallmark of this process is that the explant cells undergo dedifferentiation and acquire meristematic identity The developmental program of such meristematic cells can then be redirected to form somatic embryos, depending on the imposed culture environment Analysis of proteomes and transcriptomes has led to the molecular identification and functional characterization of many genes involved in the initiation and development of somatic embryos These genes can be classified into three categories: embryonic induction, embryonic, and maturation So far, few genes involved in early somatic embryogenesis have been characterized because isolation of early pure embryonic tissue is very difficult This review focuses on genes regulating the induction process Furthermore, we employed bioinformatic tools and pathway databases to identify genes that may play roles in regulating early somatic embryogenesis A total of 51 proteins were identified that may function in early somatic embryogenesis These proteins are predicted to be involved in hormone signal transduction, chromatin remodeling, cell cycle regulation, cellulose biosynthetic and metabolic activity, GTPase signal transduction, transcription regulation, meristem formation and maintenance, and/or apoptosis and microtubule organization This review will help advance knowledge and promote research on molecular regulation of early somatic embryogenesis

Phloroglucinol in plant tissue culture

Abstract: In plant tissue culture research, there is a constant need to search for novel substances that could result in better or more efficient growth in vitro. A relatively unknown compound, phloroglucinol (1,3,5-trihydroxybenzene), which is a degradation product of phloridzin, has growth-promoting properties. Phloroglucinol increases shoot formation and somatic embryogenesis in several horticultural and grain crops. When added to rooting media together with auxin, phloroglucinol further stimulates rooting, most likely because phloroglucinol and its homologues act as auxin synergists or auxin protectors. Of particular interest is the ability of phloroglucinol—a precursor in the lignin biosynthesis pathway—to effectively control hyperhydricity through the process of lignification, thus maximizing the multiplication rate of woody species and other species that are difficult to propagate. Phloroglucinol has also been used to improve the recovery of cryopreserved Dendrobium protocorms, increasing the potential of cryopreservation for application in ornamental biotechnology. Phloroglucinol demonstrates both cytokinin-like and auxin-like activity, much like thidiazuron, and thus has considerable potential for application in a wide range of plant tissue culture studies.

Elevating vitamin C content via overexpression of myo-inositol oxygenase and L-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses

Abstract: l-ascorbic acid (vitamin C) is an abundant metabolite in plant cells and tissues. Ascorbate functions as an antioxidant, as an enzyme cofactor, and plays essential roles in multiple physiological processes including photosynthesis, photoprotection, control of cell cycle and cell elongation, and modulation of flowering time, gene regulation, and senescence. The importance of this key molecule in regulating whole plant morphology, cell structure, and plant development has been clearly established via characterization of low vitamin C mutants of Arabidopsis, potato, tobacco, tomato, and rice. However, the consequences of elevating ascorbate content in plant growth and development are poorly understood. Here, we demonstrate that Arabidopsis lines overexpressing a myo-inositol oxygenase or an l-gulono-1,4-lactone oxidase, containing elevated ascorbate, display enhanced growth and biomass accumulation of both aerial and root tissues. To our knowledge, this is the first study demonstrating such a marked positive effect in plant growth in lines engineered to contain elevated vitamin C content. In addition, we present evidence showing that these lines are tolerant to a wide range of abiotic stresses including salt, cold, and heat. Total ascorbate content of the transgenic lines remained higher than those of controls under the abiotic stresses tested. Interestingly, exposure to pyrene, a polycyclic aromatic hydrocarbon and known inducer of oxidative stress in plants, leads to stunted growth of the aerial tissue, reduction in the number of root hairs, and inhibition of leaf expansion in wild type plants, while these symptoms are less severe in the overexpressers. Our results indicate the potential of this metabolic engineering strategy to develop crops with enhanced biomass, abiotic stress tolerance, and phytoremediation capabilities.

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

Abstract: An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.

Improving in vitro mineral nutrition for diverse pear germplasm

Abstract: Mineral nutrition in the media used for growth of in vitro plants is often difficult to optimize due to complex chemical interactions of required nutrients. The response of plant tissue to standard growth media varies widely due to the genetic diversity of the plant species studied. This study was designed as the initial step in determining the optimal mineral nutrient requirements for micropropagation of shoot tips from a collection of genetically diverse pear germplasm. Five mineral nutrient factors were defined from Murashige and Skoog salts: NH4NO3, KNO3 ,m esos (CaCl 2·2H20- KH2PO4-MgSO4), micronutrients (B, Cu, Co, I, Mn, Mo, and Zn), and Fe-EDTA. Each factor was varied over a range of concentrations. Treatment combinations were selected using response surface methods. Five pears in three species (Pyrus communis 'Horner 51, '' Old Home×Farmingdale 87, '' Winter Nelis,' Pyrus dimorphophylla ,a ndPyrus ussuriensis 'Hang Pa Li') were grown on each treatment combination, responses were measured, and each response was analyzed by analysis of variance. The analyses resulted in the identification of the following factors with the single largest effects on plant response: shoot quality (mesos), leaf spotting/necrosis (mesos), leaf size (mesos), leaf color (mesos, NH4NO3, and KNO3), shoot number (NH4NO3 and Fe), nodes (NH4NO3 and KNO3), and shoot length (mesos and Fe). Factors with the largest effects (mesos and Fe) were similar among the genotypes. This approach was very successful for defining the appropriate types and con- centrations of mineral nutrients for micropropagation of diverse pear genotypes.

Mineral nutrition influences physiological responses of pear in vitro

Abstract: Abnormal physiological responses of plant cultures such as shoot tip necrosis, callus, and hyperhydricity are some of the most difficult challenges in shoot micropropagation, and their causes are not well understood. Five Murashige and Skoogmineral saltfactors, which influence the growth of pear shoot cultures, were tested in a five-dimensional surface re- sponse experimental design. Pyrus communis 'Old Home × Farmingdale 87, '' Horner 51,' and 'Winter Nelis'; Pyrus dimorphophylla ;a ndPyrus ussuriensis 'Hang Pa Li' shoot cultures were grown on 43 computer-designed treatments to represent the design space of all possible treatment combina- tions.Analysisofshoot responsetothese treatmentsidentified thefactorsthatbothcontributedtophysiologicaldisordersand remedied them. Undesirable callus formation was common for pearshootsculturedonstandardmediumand decreased on formulations with increased NH4NO3, Fe, and mesos (CaCl2, KH2PO4, and MgSO4) for most genotypes. Shoot tip necrosis varied with the genotype, but low mesos or low nitrogen concentrations contributed to the necrosis. Hyperhydricity was more prominent with low mesos or low NH4NO3. Hooked and upwardly curled new leaves were seen in most genotypes and resultedfromuse of low mesos inP. communis and low nitrogen for 'Hang Pa Li' and P. dimorphophylla. Fasciation and hypertrophy were seen infrequently and resulted from wide imbalances in several nutrients simulta- neously. In general, standard concentrations of Murashige and Skoog iron and micros combined with high mesos and mod- erate nitrogen compounds produced normal shoots without physiological disorders.

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

Abstract: We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g L−1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g L−1 sucrose, 2.5 g L−1 activated charcoal, and 2.5 g L−1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.

Antioxidant enzyme activities, malondialdehyde, and total phenolic content of PEG-induced hyperhydric leaves in sugar beet tissue culture

Abstract: Hyperhydricity is a physiological abnormality that frequently affects shoots that are vegetatively propagated in vitro. In this study, sugar beet (Beta vulgaris L. cv. Felicita) shoot tip explants were cultured on Murashige and Skoog medium supplemented with different concentrations of polyethylene glycol (PEG) 6000. We observed that higher concentrations of PEG 6000 and longer exposure (up to 4 wk) resulted in increasing levels of hyperhydration as well as browning and/or blackening of tissues in culture. A comparison of hyperhydric shoots with controls on the 28th day showed a marked increase in the content of water, phenolics, and malondialdehyde (MDA), which was positively correlated with an increase in the accumulation of PEG 6000. Selected antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), and polyphenol oxidase (PPO) also increased in hyperhydric shoots, especially at lower concentrations of PEG 6000. Regression analysis indicated that strong linear relationships exist between SOD–APX (R 2 = 0.932), SOD–CAT (R 2 = 0.753), SOD–total phenolic content (R 2 = 0.966), APX–PPO (R 2 = 0.842), APX–total phenolic content (R 2 = 0.904), POX–CAT (R 2 = 0.751), and CAT–total phenolic content (R 2 = 0.806). Despite the correlation between different antioxidant enzymes and between the antioxidant enzymes and antioxidant compounds, was not able to prevent ROS damage in hyperhydric shoots. The negative correlation between SOD–MDA, POX–MDA, CAT–MDA, and MDA–total phenolics also indicated an increase in antioxidant enzyme activities, yet the increase in these antioxidant compound contents did not prevent lipid peroxidation of in vitro propagated beet shoots.

An efficient protocol for the production of chit42 transgenic Furenzhi banana (Musa spp. AA group) resistant to Fusarium oxysporum

Abstract: Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a destructive fungal disease of banana. Transferring antifungal genes into banana provides a feasible way to control fungal disease, but transformation frequencies for banana are often cultivar and cell line dependent. We investigated an efficient liquid medium selection protocol for an Agrobacterium-mediated transformation system for Furenzhi (Musa spp. AA group). Embryogenic cell suspensions (ECS) of Furenzhi were co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring a plasmid containing the endochitinase gene chit42 from Trichoderma harzianum. After co-cultivation, GUS-positive ECS were selected in liquid medium with antibiotics, and compared to semi-solid medium selection. In total, 186 transgenic plantlets were obtained using M2S liquid medium (based on Murashige and Skoog salts) with hygromycin, whereas no transgenic lines were obtained in parallel experiments with semi-solid selection medium. Integration of the transgene was confirmed by PCR, and Southern blots which showed a single copy of the transgene had integrated into the banana genome in three plant lines. Expression of the transgene in regenerated plants was confirmed by s-glucuronidase histochemical assays and real-time PCR. Both in vitro and ex vivo disease assays showed that the majority of transgenic lines (three of seven) expressing chit42 showed a higher level of resistance to Fusarium wilt (Foc race 4), whereas non-transgenic control plants were susceptible. These results imply a relationship between Foc4 disease resistance and the transcription levels of the transgene in the transgenic clones. This study may offer a promising approach to breed bananas resistant to the fungal disease Fusarium wilt.

Organelle antioxidants improve microspore embryogenesis in wheat and triticale

Abstract: Low frequency of green plant production and albinism limits the use of isolated microspore culture (IMC) in cereal breeding programs. The present study was conducted in triticale and bread wheat IMC to increase the production of green plants and minimize albinism. NPB-99 + 10% Ficoll induction medium was supplemented with mitochondrial or plastid antioxidants, in a completely random design, to evaluate their contribution to successful microspore embryogenesis and green plant production. Each group of antioxidants was tested independently: first in triticale and then validated in various spring wheat genotypes. While the response differed by wheat genotype, induction medium supplemented with proline (10 mM) yielded a greater number of embryos/embryo-like structures and green plants in both triticale and wheat. No differences were found with respect to albinism in triticale or wheat except for the cv. Sadash. Among plastid antioxidants tested, glutathione (2 μM) proved to be the best antioxidant to increase embryo and green plant production. Salicylic acid also helped to reduce the number of albino plants in triticale and the wheat genotype SWS366. Overall, induction medium supplemented with proline or glutathione enhanced microspore embryogenesis in both triticale and wheat and increased the number of green plants in the recalcitrant genotypes.

Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

Abstract: Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8–1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH 5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23 ± 2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92–100% and mean number of shoots of 4.7–7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid “Siberia” revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of “Siberia”, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.

Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation

Abstract: Pear accessions and species show a broad response to tissue culture media due to the wide genetic diversity that exists in the available pear germplasm. An initial study of mineral nutrition using a systematic response surface approach with five Murashige and Skoog medium mineral stock solutions indicated that the mesos factor (CaCl2, MgSO4, and KH2PO4) affected most plant responses and genotypes, suggesting that additional studies were needed to further optimize these three mesos components for a wide range of genotypes. Short stature, leaf spots, edge necrosis, and red or yellow coloration were the main symptoms of poor nutrition in shoot cultures of 10 diverse pear genotypes from six species. A surface response experimental design was used to model the optimal factor and factor levels for responses that included overall quality, leaf character, shoot multiplication, and shoot height. The growth morphology, shoot length, and multiplication of these pear shoots could be manipulated by adjusting the mesos components. The highest quality for the majority of genotypes, including five P. communis cultivars, P. koehnei, P. dimorphophylla, and P. pyrifolia ‘Sion Szu Mi’, required higher concentrations (>1.2× to 2.5×) of all the components than are present in Murashige and Skoog medium. ‘Capital’ (P. calleryana) required high CaCl2 and MgSO4 with low KH2PO4; for ‘Hang Pa Li’ (P. ussuriensis), low CaCl2 and moderate to low MgSO4 and KH2PO4 produced high-quality shoots. Suitable combinations of the meso nutrients produced both optimum shoot number and shoot length in addition to general good plant quality.

Genome-wide analysis of key salinity-tolerance transporter (HKT1;5) in wheat and wild wheat relatives (A and D genomes)

Abstract: Exclusion of sodium ions from cells is one of the key salinity tolerance mechanisms in plants. The high-affinity cation transporter (HKT1;5) is located in the plasma membrane of the xylem, excluding Na+ from the parenchyma cells to reduce Na+ concentration. The regulatory mechanism and exact functions of HKT genes from different genotypic backgrounds are relatively obscure. In this study, the expression patterns of HKT1;5 in A and D genomes of wheat were investigated in root and leaf tissues of wild and domesticated genotypes using real-time PCR. In parallel, the K+/Na+ ratio was measured in salt-tolerant and salt-sensitive cultivars. Promoter analysis were applied to shed light on underlying regulatory mechanism of the HKT1;5 expression. Gene isolation and qPCR confirmed the expression of HKT1;5 in the A and D genomes of wheat ancestors (Triticum boeoticum, AbAb and Aegilops crassa, MMDD, respectively). Interestingly, earlier expression of HKT1;5 was detected in leaves compared with roots in response to salt stress. In addition, the salt-tolerant genotypes expressed HKT1;5 before salt-sensitive genotypes. Our results suggest that HKT1;5 expression follows a tissue- and genotype-specific pattern. The highest level of HKT1;5 expression was observed in the leaves of Aegilops, 6 h after being subjected to high salt stress (200 mM). Overall, the D genome allele (HKT1;5-D) showed higher expression than the A genome (HKT1;5-A) allele when subjected to a high NaCl level. We suggest that the D genome is more effective regarding Na+ exclusion. Furthermore, in silico promoter analysis showed that TaHKT1;5 genes harbor jasmonic acid response elements.

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion ( Allium cepa . L.)

Abstract: An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l−1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l−1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l−1 glycine, 690 mg l−1 proline, and 1.0 g l−1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l−1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l−1 6-benzylaminopurine; addition of 2.0 mg l−1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l−1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.

Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions

Abstract: Suboptimal environmental conditions inside closed culture vessels can be detrimental to in vitro growth and survival of plantlets during the acclimatization process. In this study, the environmental factors that affected Doritaenopsis plantlet growth and the relationship between growth and sugar metabolism were investigated. Cultures were maintained under heterotrophic, photoautotrophic, or photomixotrophic conditions under different light intensities and CO2 concentrations. Photoautotrophic growth of Doritaenopsis hybrid plantlets could be promoted significantly by increasing the light intensity and CO2 concentration in the culture vessel. The concentration of different sugars in the leaves of in vitro-grown plantlets varied with different cultural treatments through a 10-wk culture period. Starch, reducing sugars, and nonreducing sugar contents were higher in plantlets grown under photoautotrophic and photomixotrophic conditions than in heterotrophically grown plantlets. Net photosynthesis rates were also higher in photoautotrophically and photomixotrophically grown plantlets. These results support the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high photosynthetic photon flux density. Growth was greatest in plantlets grown under CO2-enriched photoautotrophic and photomixotrophic conditions with high photosynthetic photon flux density. The physiological status of in vitro-grown Crassulacean acid metabolism (CAM)-type Doritaenopsis showed a transition from C3 to CAM prior to acclimatization.

A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

Abstract: The aim of the present study was to evaluate the effects of forced ventilation and CO2 enrichment (360 or 720 μmol mol−1 CO2) on the in vitro growth and development of Pfaffia glomerata, an endangered medicinal species, under photomixotrophic or photoautotrophic conditions. P. glomerata nodal segments showed substantial differences in growth, relative water content and water loss from leaves, photosynthetic pigments, stomatal density, and leaf anatomical characteristics under these different treatments. CO2 enrichment led to increased photosynthetic pigments and reduced stomatal density of in vitro cultivated P. glomerata. A lack of sucrose in the culture medium increased 20-hydroxyecdysone levels, but the increase in CO2 levels did not further elevate the accumulation of 20-hydroxyecdysone. All growth increased in a CO2-enriched atmosphere. In addition, CO2 enrichment, with or without sucrose, gave a lower relative water loss from leaves. This finding indicates that either a photoautotrophic or photomixotrophic system in a CO2-enriched atmosphere may be suitable for large-scale propagation of this species.

Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Abstract: Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg l−1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg l−1 TDZ. The WPM medium supplemented with 2.5 mg l−1 BA + 0.4 mg l−1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg l−1 BA and 3.0 mg 1−1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg l−1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.

Production of biomass and bioactive compounds by adventitious root suspension cultures of Morinda citrifolia (L.) in a liquid-phase airlift balloon-type bioreactor

Abstract: To improve root growth and production of bioactive compounds such as anthraquinones (AQ), phenolics, and flavonoids by adventitious root cultures of Morinda citrifolia, the effects of aeration rate, inoculum density, and Murashige and Skoog (MS) medium salt strengths were investigated using a balloon-type bubble bioreactor. The possible mechanisms underlying changes in activities of enzymic (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase) and nonenzymic (vitamin E) antioxidants, phenylalanine ammonia lyase, and stress levels (accumulation of hydrogen peroxide and proline, peroxidation of lipids) were also studied. Low aeration rate (0.05 vvm [air volume/culture volume/min]) accelerated accumulation of root fresh weight and dry weight (DW). High aeration rates (0.1 to 0.3 vvm) stimulated accumulation of AQ, phenolics, and flavonoids and reduced root growth. Low inoculum densities (5 and 10 g l–1) increased accumulation of those metabolites but inhibited root growth. Culture of adventitious roots with high concentrations of MS salts (1× and 1.5× MS) resulted in induction of oxidative stress that strongly inhibited root growth. Overall, an aeration rate of 0.05 vvm, 15 g l–1 inoculum density, and half-strength (0.5×) MS medium were optimal for enhancing accumulation of root dry biomass (4.38 g l–1), AQ (103.08 mg g–1 DW), phenolics (54.81 mg g–1 DW), and flavonoids (49.27 mg g–1 DW).

Effects of sodium nitroprusside on shoot multiplication and regeneration of Vanilla planifolia Andrews

Abstract: Nitric oxide (NO) plays diverse roles in the growth and development of plants. The effects of a NO donor, sodium nitroprusside (SNP), on shoot multiplication and regeneration of Vanilla planifolia Andrews have been studied. Nodal segments of V. planifolia were used as explants to initiate shoots. The number of shoots per explant showed a significant increase in the presence of SNP and more than 93% of explants formed shoots. Supplementation of 10.0 μM SNP to Murashige and Skoog (MS) basal medium containing 1.0 mg/L 6-benzylaminopurine (BAP) produced the highest number of shoots per explant (10.33) after 60 d of culture. However, in this treatment, shoot length (3.76 cm) was less than in the other treatments, except for the plant growth regulator-free MS medium. MS medium containing only 1.0 mg/L BAP produced the highest shoot length (4.49 cm) with a mean number of 6.26 shoots per explant. These findings indicate that NO stimulated shoot development and may be considered as an intermediary of adventitious shoot regeneration, as has been suggested for other plant species.

In vitro conservation of chestnut ( Castanea sativa ) by slow growth

Abstract: Slow growth storage has been achieved for Castanea sativa (cv. ‘Montemarano’) shoot cultures over a duration of 48 mo at a temperature of 8°C, where 82% of explants survived and were able to resume normal growth after transfer to standard culture conditions at 23°C. The evaluation of the chlorophyll content of leaves also showed no differences between material stored for 48 mo and control material subcultured at 23°C. With a storage temperature of 4°C, the survival of shoots was significantly lower at approximately 56% after 12 mo, and no plants recovered after 24-mo storage. The presence of 6-benzyladenine 0.44 μM in the culture medium proved to be necessary for the recovery of healthy shoots, while pre-treatments with different concentrations of abscisic acid did not significantly influence the survival of shoots following storage conditions. A low level of light during slow growth storage resulted in positive effects on the rate of shoot survival over the longest preservation periods.

The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity

Abstract: Panax quinquefolium, American ginseng, is valued for its triterpene saponins, known as ginsenosides. These constituents possess a number of pharmacological properties and hairy root cultures can synthesize similar saponins to those of field-cultivated roots. The antibacterial activity of extracts from three hairy root clones of P. quinquefolium L. was tested against a range of standard bacterial and yeast strains. The agar diffusion method was used to evaluate inhibition of microbial growth at various extract concentrations. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains. Susceptibility testing to antibiotics was also tested using the disk diffusion method. The minimal inhibitory concentration values of the extracts, obtained by agar diffusion, ranged from 0.8 to 1.4 mg/ml. The results showed that extracts from hairy root cultures inhibited the growth of bacteria and yeast strains and suggest that they may be useful in the treatment of infections caused by pathogenic microorganisms.

Somatic embryogenesis from stamen filaments in grapevine (Vitis vinifera L. cv. Mencía): changes in ploidy level and nuclear DNA content

Abstract: Somatic embryogenesis was induced from stamen filaments and an embryogenic suspension culture was established in the grapevine cultivar Mencia using thidiazuron and 2,4-dichlorophenoxyacetic acid. Four combinations of each growth regulator were assessed for somatic embryo induction in a basal medium containing Nitsch and Nitsch salts and Murashige and Skoog vitamins, and an embryogenic suspension was established in liquid medium containing 1 μM 2,4-dichlorophenoxyacetic acid plus 4.5 μM thidiazuron. By using thidiazuron instead of benzyladenine, induction rates were improved over those previously reported for this cultivar and were relatively high compared with previous results in other cultivars. Three combinations of indole-3-acetic acid and benzyladenine and two inoculum levels were tested in a differentiation medium containing activated charcoal. The size of the inoculum affected the developmental stage of the somatic embryos, whereas the type of growth regulator did not. Both the germination and plant conversion rates were high (87.8% and 88.2%, respectively). An analysis of plant ploidy levels by flow cytometry revealed that 5.6% of the somatic embryo-derived plants were tetraploid. The mean nuclear DNA content of the diploid somatic embryo-derived plants was, on average, 6.7% lower than that of diploid field-grown plants, indicating that this protocol produces low levels of somaclonal variation. The results obtained here indicate that such variations in grapevine can occur both through changes in the ploidy level and by loss of genetic material during somatic embryogenesis.

Direct somatic embryogenesis of Agave fourcroydes Lem. through thin cell layer culture

Abstract: Here, we describe a new protocol for the induction of direct somatic embryogenesis of Agave fourcroydes through thin cell layer (TCL) culture technology. The protocol was optimized for the main factors known to affect the process, including the type of explant (stem or leaf tissue), type and concentration of exogenous growth regulators (α-naphthalene acetic acid [NAA], 2,4-diclorophenoxyacetic acid [2,4-D], 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid [picloram], and 3,6-dichloro-2-methoxybenzoic acid [dicamba]), and the influence of plant genotype. Thin tissue segments cut transversally (tTCLs) from stems of in vitro-cultured plants gave the best embryogenic response when cultured with 2.26 μM dicamba (92.22 embryos/explant) or 2.07 μM picloram (81.72 embryos/explant). It was interesting to observe that the embryogenic capacity of these tissues was affected by the presence of 6-benzylaminopurine (BA) in the culture medium in which the explant donor plantlets were maintained. Thirteen clonal lines (each derived from a different parental plant), compared for their embryogenic competence under the same culture conditions, produced very different embryogenic responses that varied from very high (117 embryos/explant) to null. The histological analysis revealed that the amount of meristematic tissue present in the tTCLs varied according to the region of the stem (apical, middle, or basal) from which they originated. The cells of the vascular procambium became competent and developed into cell lines that formed embryos, either by a unicellular or a multicellular pathway. Mature embryos germinated in half-strength Murashige and Skoog medium without growth regulators and 85% of regenerated plants was successfully acclimatized in a greenhouse.

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Abstract: Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

Abstract: We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD600 ≅ 0.6 and diluted to 109 cells ml−1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg ml−1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T0). Among T1 seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ 2) test. Southern hybridization of genomic DNA from kanamycin-resistant T1 transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T1 transgenic plants, thereby confirming stable expression of the nptII gene.

Improvement of peanut (Arachis hypogaea L.) transformation efficiency and determination of transgene copy number by relative quantitative real-time PCR

Abstract: The biolistic method is reliable for delivering genes of interest into various species, but low transformation efficiency can be a limiting factor in its application. To test various conditions that could improve peanut transformation via particle bombardment, embryogenic tissues of the peanut cultivar Georgia Green were co-bombarded with two plasmids: one containing a green fluorescent protein gene and one containing a gene of interest plus a selectable marker. The fluorescence in bombarded embryogenic tissues was measured to evaluate transformation efficiency. A 4.6-fold improvement of transformation efficiency was achieved in stably transformed peanut lines by introducing protamine instead of conventional spermidine in a bombardment mixture with 70 ng/shot plasmid DNA and 50 μg/shot gold. Unexpectedly, the reduction of plasmid DNA from 700 to 70 ng/shot produced transgenic lines with significantly increased numbers of transgene copies. To determine the transgene copy number during plantlet regeneration, relative quantitative real-time polymerase chain reaction (qPCR) was established using fluorescently labeled universal library probes. A correlation of 95% was found for estimation of copy number between Southern blot and qPCR data. Given its speed and high-throughput nature, qPCR can be employed as an effective screening tool to separate high copy number events from low copy number events as early as the shoot formation stage of regeneration.

Callus induction and plant regeneration from anthers of Dendrocalamus latiflorus Munro

Abstract: Bamboo varieties are very difficult to improve by traditional breeding methods. Here, we established an efficient plant-regeneration system for Dendrocalamus latiflorus (tropical giant bamboo) by anther culture. Culture conditions, especially the plant growth regulators required for callus induction and shoot differentiation, were optimized by orthogonal design. M8 medium supplemented with 5.37 μΜ α-naphthaleneacetic acid (NAA), 1.33 μM N 6 -benzyladenine (BA), 110.17 μM phenylacetic acid (PAA), and a pretreatment time of 3 d produced the highest rate (5.08 ± 0.61%) of callus induction. The maximum shoot differentiation rate reached 28.3 ± 4.29% in M8 medium supplemented with 2.32 μM kinetin (KT), 8.89 μM BA, 1.08 μM NAA, and 110.17 μM PAA. The results of the ploidy level test showed that most of the regenerated plants were dodecaploid (96/100), a few were hexaploid (3/100), and one was triploid (1/100). The average chlorophyll content of dodecaploid lines was significantly higher than that of hexaploid lines. The present study provides an innovative method for bamboo ploidy breeding and a useful method for genetic improvement.

Factors influencing direct shoot regeneration from mature leaves of Jatropha curcas, an important biofuel plant

Abstract: In order to establish a highly efficient and sustainable regeneration system, we systematically researched the key factors affecting direct shoot regeneration from Jatropha curcas leaves that were collected from Hainan (HN1-1), Lijiang (LJ3-1), and Yuxi (YX2-12) provinces in China. The L9(34) orthogonal test of thidiazuron (TDZ), kinetin (Kn), and gibberellic acid (GA3) were studied, and the explant type, growth age, and cultivar of leaves were subsequently investigated. Simultaneously, the combinations of plant growth regulators (PGRs) promoting shoot bud proliferation, elongation, and root establishment were examined. The results showed that the best medium for shoot bud induction was Murashige and Skoog (MS) medium supplemented with 1.0 mg/L TDZ, 0.5 mg/L Kn, and 0.5 mg/L GA3. TDZ was the key PGR, while Kn and GA3 played an important role in shoot bud elongation and the number of shoots per leaf disk, respectively. The induced shoot buds proliferated and readily elongated in MS medium with 0.3 mg/L 6-benzylaminopurine and 0.01 mg/L indole-3-butyric acid (IBA) and established roots in half-strength MS medium supplemented with 2.0 mg/L IBA. Using the previously described methods, the third to fifth leaves were found to be the best explant source for shoot bud induction, with a high induction rate, large shoot numbers per disk, excellent proliferation, and consistent rooting. With the use of this regeneration system, the shoot bud induction rate increased from the reported rate of 53.5% to more than 90% using different explants and cultivars, and the shoot number per leaf disk (shoot length ≥ 0.5 cm) increased from 1.6 to 3.5. Thus, this optimized regeneration system will effectively promote the propagation and genetic transformation of J. curcas.

Shoot regeneration and ploidy variation in tissue culture of honeydew melon (Cucumis melo L. inodorus)

Abstract: The Cucumis melo L. inodorus honeydew melon variant is one of the most consumed melons in the USA, and has important commercial and nutritional value. There is a need for improvement of several genetic traits in the US honeydew melon, such as nutrition, drought tolerance, and disease resistance. We investigated the existing regeneration media and optimized the medium composition for an elite honeydew diploid breeding line, “150”, using cotyledonary explants. Four combinations of three different plant growth regulators, 6-benzyladenine, abscisic acid, and indole-3-acetic acid (IAA), were tested in the shoot regeneration media. The presence of IAA in the medium caused the cotyledon explants to curl away from the medium, which made antibiotic selection problematic in our previous study. Omission of IAA from the culture media eliminated this problem and did not impact the shoot regeneration capacity of the cotyledonary explants. We also estimated the ploidy of regenerated plants using flow cytometry, and 50–60% were found to be polyploid (tetraploid or mixoploid). However, contrary to other studies, these polyploid plants did not show major morphological differences compared to the diploid plants.

Salt tolerant screening in eucalypt genotypes ( Eucalyptus spp.) using photosynthetic abilities, proline accumulation, and growth characteristics as effective indices

Abstract: Eucalypts are highly regarded plantation trees due to their fast growing nature, high water consumption, tolerance to abiotic stresses, and ease of conversion to pulp and paper We screened five genotypes of Eucalyptus camaldulensis (T5, BD4, 1-7-1, H1, and SH4) and three genotypes of the E camaldulensis × Eucalyptus urophylla hybrid (H4, 58H2, and 27A2) for salt tolerance Fresh weight and leaf area in hybrid genotypes (H4, 58H2, and 27A2) were greater than those in the E camaldulensis population after plantlets were subjected to 200 mM NaCl for 14 d The chlorophyll a content in hybrid genotypes decreased by 1971–3711% compared to 5145–6600% decline in E camaldulensis Similarly, total chlorophyll content was retained at a high level in the hybrid population, leading to stabilization of the net photosynthetic rate The amount of proline, an osmolyte, was significantly increased in all Eucalyptus genotypes when exposed to 200 mM NaCl Multivariate analyses of proline accumulation, photosynthetic pigment degradation, diminishing chlorophyll fluorescence, Pn reduction, and growth inhibition in salt-stressed plantlets of Eucalyptus genotypes were performed to classify salt-tolerant- and salt-sensitive groupings The hybrid eucalypt genotypes H4, 58H2, and 27A2 were identified as salt tolerant while the selection genotypes of E camaldulensis, T5, BD4, 1-7-1, H1, and SH4, were classified as salt susceptible