Optimization of direct somatic embryogenesis from mature zygotic embryos ofPanax ginseng C. A. Meyer

Culture conditions were optimized for somatic embryogenesis ofPanax ginseng. The highest frequency of embryo formation was obtained when tissues were excised from the middle region of the cotyledon segments of zygotic embryos. Only treatment with light could stimulate the formation of single-type somatic embryos, whereas multiple-type somatic embryos and calli were observed under dark conditions. The highest production of somatic embryos was found with an NH₄ ⁺:NO₃ ratio of 21:39. Among the tested media (MS, B5, and SH), maximum formation of somatic embryos was obtained when cotyledon expiants were cultured on an 1% agar MS medium supplemented with 5% sucrose. Regenerated ginseng plantlets were transferred to an autoclaved soil mixture in the greenhouse. These transformants showed no detectable variations in their morphology or growth characteristics compared with the donor plant.     

Induction of adventitious roots and analysis of ginsenoside content and the genes involved in triterpene biosynthesis inPanax ginseng

Adventitious roots were produced directly from root segments ofPanax ginseng seedlings when cultured on an MS solid medium containing 3.0 mg L^-1 IBA. Omitting NH₄NO₃ from this medium greatly enhanced both the frequency of adventitious root formation and the number of roots per expiants. This frequency declined markedly with the age of the root, but could be increased through repeated sub-culturing events. A two-step procedure that included NH₄NO₃ free media for the first two weeks of culture, followed by transfer onto media containing NH₄NO₃ for another four weeks, greatly improved total fresh weights of these adventitious roots compared with a method of continuous culture over six weeks in media that always contained NH₄NO₃. Expression of the genes involved in triterpene biosynthesis was analyzed by RT-PCR. Ginsenoside contents were enhanced by the omission of NH₄NO₃ and were also greatly increased by treatment with methyl jasmonate.     

培养条件对西洋参不定根诱导的影响

为了提高西洋参不定根的诱导率和生长速度,该研究以西洋参鲜根为外植体,在基本培养基的基础上优化IBA、碳源、氮源和磷源等营养成分。结果表明:西洋参不定根诱导过程可以明显分为外植体脱分化(愈伤化)、再分化(根形成)和根伸长等三个阶段; MS基本培养基更有利于西洋参不定根的诱导,可能与MS培养基中矿质元素含量高有关;当培养基中IBA浓度达到2 mg·L~(-1)时,外植体表面上不定根分布密度大,诱导率达到(96±3.5)%;培养基中添加蔗糖到30 g·L~(-1)时,不定根的诱导效果最好,但继续提高浓度后不定根变短、直径变粗;培养基中NO_3~-∶NH_4~+和PO_4~(3-)浓度分别为20∶10(总氮量30 mmol·L~(-1))和25.0mmol·L~(-1)时,西洋参不定根诱导率达到最大。结果提示优化培养条件可以显著改善西洋参不定根的诱导和生长,为后续西洋参不定根规模化培养提供理论支持。     

In vitro propagation of Agave fourcroydes Lem. (Henequen)

In vitro plant regeneration of Agave fourcroydes Lem. (Agavaceae) is described. Results suggest that the NO₃ ^-:NH₄ ⁺ balance in the culture medium is a key factor controlling callus growth and organogenesis in rhizome cultures. Stem callus showed limited organogenic capacity, but high cytokinin concentrations induced adventitious shoot formation on stem explants. When these shoots were excised and subcultured, new callus formed at their base from which new shoots arose. The shoots from stem explants and rhizome callus formed extensive root systems in vitro and were transferred to pot culture with a 90% survival rate.     

Induction,maturation and germination of holm oak (Quercus ilex L.) somatic embryos

Cotyledon explants of Panax ginseng produced somatic embryos directly on solid hormone-free MS medium containing 3% (w/v) sucrose while high concentration of NH₄NO₃ (60 mM) induced embryogenic callus. Ten subcultures of the embryogenic callus on hormone-free MS medium with 40 mM NH₄NO₃ gave hormone-independent proliferation of callus, which exhibited proliferation potential even on MS medium with a standard level of NH₄NO₃ (20 mM). Pulse treatment of callus with exogenous auxin or cytokinin (1.0 mg 1^–1 2,4-D, 1.0 mg 1^–1 kinetin) resulted in the loss of the hormone-independent characteristic and caused the callus to brown. For the suspension culture, embryogenic callus was transferred to MS liquid medium containing 3% (w/v) sucrose in an 500 ml Erlenmyer flask. Embryogenic cell clumps in full-strength MS liquid medium discharged toxic substances, resulting in strong suppression of cell growth. In 1/3-strength MS medium, exudation of toxic material did not occur. Embryogenic cell clumps were mass-grown on a large-scale in a bioreactor (20-1), showing a 7.1 increase of fresh weight in 1/3-strength MS medium with 3% (w/ v) sucrose after 5 weeks of culture. Total ginsenoside content of cultured embryogenic cell clumps was low and 6 times below naturally-cultivated ginseng roots.     

The effectiveness of nitrogen sources in Feijoa somatic embryogenesis

Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH₄ ⁺ and NO₃ ^– ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 M) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH₄ ⁺ and NO₃ ^– concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 M) enhanced the conversion of somatic embryos to plantlets.     

NH4 + and NO3 − requirement for wheat somatic embryogenesis

The influence of three nitrogen salts: NH₄NO₃, KNO₃ and NH₄Cl on wheat in vitro cultures was investigated. Both NO ₃ ⁻ and NH ₄ ⁺ ions were indispensable for proliferation of embryogenic calli and development of wheat somatic embryos. It is possible to obtain wheat somatic embryos when the medium is enriched with NH₄NO₃ only as a source of inorganic nitrogen. The results of the statistical analysis showed that the level of NH₄NO₃ and KNO₃ in the medium had a great influence on the efficiency of somatic embryogenesis. We observed tendency that calli on media containing 50 mM NH₄NO₃ and 0 to 20 mM KNO₃ turned out to be more embryogenic than on control MS medium. High concentrations of KNO₃- 100 mM inhibited somatic embryogenesis, while 100 mM NH₄NO₃ did not. The level of total N did not have significant influence on wheat somatic embryogenesis. Ratio NO ₃ ⁻ :NH ₄ ⁺ also turned out to be not substantial. We observed that mutual connection of concentration levels between NH₄NO₃ and KNO₃ and between NH₄Cl and KNO₃ was more important. The efficiency of somatic embriogenesis obtained in the experiment with NH₄Cl and KNO₃ was significantly lower than in experiment with NH₄NO₃ and KNO₃.     

Adventitious root suspension cultures of <Emphasis Type="BoldItalic">Hypericum perforatum</Emphasis>: effect of nitrogen source on production of biomass and secondary metabolites

The present study investigated the effect of nitrogen source (NH₄⁺; NO₃⁻) at different concentrations on the accumulation of biomass and secondary metabolites in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium with B5 vitamins, 1.0 mg l⁻¹ indole-3-butyric acid, 0.1 mg l⁻¹ kinetin, 3% (w/v) sucrose, and different ratios of ammonium and nitrate (0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM, using NH₄Cl and KNO₃). The cultures were maintained in darkness. The medium supplemented with 5:25 (mM) NH₄⁺/NO₃⁻ resulted in the optimum accumulation of biomass and total phenols and flavonoids. The antioxidant potential of a methanolic extract, measured as the 1, 1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities, of H. perforatum adventitious roots showed that antioxidant activity was high from root extracts that were grown on higher concentrations of NO₃⁻ nitrogen (15, 20, and 25 mM). Further, assessment of hydrogen peroxide (H₂O₂) and malondialdehyde content of the root extracts revealed that cultures supplemented with higher levels of NO₃⁻ nitrogen (15–30 mM) were under oxidative stress, which boosted the levels of secondary metabolites in the adventitious roots. These results suggest that optimal adventitious root biomass could be achieved with the supplementation of cultures with 5:25 ratios of MS nitrogen sources.     

Induction of somatic embryos by macrosalt stress from mature zygotic embryos of Panax ginseng

Mature zygotic embryos of ginseng (Panax ginseng C. A. Meyer) were germinated on a Murashige and Skoog medium lacking growth regulators. However, when the zygotic embryos were cultured on MS medium containing increased levels of macrosalts (NH₄NO₃, KNO₃, KH₂PO₄, MgSO₄, or CaCl₂) to result is a mild salt stress, growth of zygotic embryos was strongly suppressed and eventually browning occurred. Somatic embryos or embryogenic calli were formed directly from these abnormal stressed zygotic embryos. Cotyledons were the most competent tissue for somatic embryo production. The highest frequency of somatic embryo formation (56.3%) was observed on medium containing 61.8 mM of NH₄NO₃. The highest frequency of somatic embryo formation by five different macrosalt treatments occurred in the following order: NH₄NO₃> KNO₃> KH₂PO₄> MgSO₄> CaCl₂. Somatic embryos were regenerated into plants with a shoot and root, and the plants survived on soil in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Combination Effect of NaCl Salinity and Nitrogen Form on Mineral Composition of Sunflower Plants

The effect of two N-forms (NH₄ ⁺ and NO₃ ^–) and NaCl on pattern of accumulation of some essential inorganic nutrients was examined in sunflower (Helianthus annuus L.) cv. Hisun 33. Eight-day-old plants of were subjected for 21 d to Hoagland's nutrient solution containing 8 mM N as NH₄ ⁺ or NO₃ ^·, and salinized with and addition of NaCl to the growth medium had no significant effect on total leaf N. However, root N of NH₄-supplied plants decreased significantly with increase in NaCl concentration, whereas that of NO₃-supplied plants remained unaffected. There was no significant effect of NaCl on leaf or root P, but the NO₃-supplied plants had significa concentration of leaf P than that of NH₄-supplied plants at varying salt treatments. Salinity of the rooting med did not show any significant effect on Na⁺ concentrations of leaves or roots of plants subjected to two differen N. NH₄-treated plants generally had greater concentrations of Cl^– in leaves and roots and lower K⁺ content in leaves than NO₃-supplied plants. Ca²⁺ concentrations of leaves and roots and Mg²⁺ concentrations of leaves decreased in NH₄-supplied plants due to NaCl, but they remained unaffected in NO₃-treated plants.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Morphogenesis in callus tissue ofMedicago sativa: The role of ammonium ion in somatic embryogenesis

Exogenously supplied ammonium ion is critical to alfalfa morphogenesis in vitro. In alfalfa, the ability to induce the formation of either roots or somatic embryos provided an opportunity to examine the effects of ammonium ion on each pattern of morphogenesis. Somatic embryo formation required a minimum of 12.5 mM NH ₄ ⁺ in regeneration medium for optimal expression. Root formation was inhibited by NH ₄ ⁺ levels of 50 mM and above, and occurred in the absence of exogenous NH₄ ⁺. At high levels of NH ₄ ⁺ somatic embryos were formed from cells exposed to the root-inducing combination of hormones. This observation suggests that the growth regulators and exogenously supplied ammonium ion comprise an interactive system controlling the in vitro pattern of alfalfa morphogenesis.     

Genetic engineering of sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) for resistance to necrosis disease through deployment of the TSV coat protein gene

Nitrogen is a major driver of plant growth and the nitrogen source can be critical to good growth in vitro. A response surface methodology mixture-component design and a data mining algorithm were applied to nitrogen (N) nutrition for improving the micropropagation of Prunus armeniaca Lam. Data taken on shoot cultures included a subjective quality rating, shoot number, shoot length, leaf characteristics and physiological disorders. Data were analyzed using the Classification and Regression Tree data mining algorithm. The best overall shoot quality as well as leaf color were on medium with NO₃^??>?25 mM and NH₄⁺/Ca⁺ >?0.8. Improving shoot length to15 mm required 25?<?NO₃^? ≤?35 mM with NH₄⁺/Ca²⁺ ≤?2.33. The most shoots (11.6) were produced with NO₃^? >?25 mM and NH₄⁺/Ca²⁺ ≤ 0.8, but there were 5–10 shoots at other NO₃^? concentrations regardless of NH₄⁺/Ca²⁺ proportion. Leaves increased in size with higher NO₃^? concentrations (>?55 mM). Physiological disorders were also influenced by the nitrogen components. Shoot tip necrosis was rarely present with NO₃^? > 45 mM. Callus production decreased somewhat with NH₄⁺/Ca²⁺ >?2.33. Suggested concentrations for an improved medium considering all of these growth characteristics would be 25?<?NO₃^? ≤?35 mM and NH₄⁺/Ca⁺ ≤ 0.8. Validation experiments comparing WPM and three trial media showed improvements in several shoot growth parameters on medium with optimized mesos and optimized nitrogen components.     

Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.)

The present work deals with optimization of adventitious shoot culture of Bacopa monnieri for the production of biomass and bacoside A and has investigated the effects of macro elements (NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄) and nitrogen source [NH₄ ⁺/NO₃ ⁻] of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) on accumulation of biomass and bacoside A content. Optimum number of adventitious shoots (99.33 shoots explant⁻¹), fresh weight (1.841 g) and dry weight (0.150 g) were obtained in the medium with 2.0× strength of NH₄NO₃. The highest production of bacoside A content was also recorded in the medium of 2.0× NH₄NO₃, which produced 17.935 mg g⁻¹ DW. The number of adventitious shoot biomass and bacoside A content were optimum when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum number of shoots (70.00 shoots explant⁻¹), biomass (fresh weight 1.137 g and dry weight 0.080 g) and also bacoside A content (27.106 mg g⁻¹ DW) were obtained at NH₄ ⁺/NO₃ ⁻ ratio of 14.38/37.60 mM. Overall, MS medium supplemented with 2.0× NH₄NO₃ is recommended for most efficient bacoside A production.     

Net fluxes of ammonium and nitrate in association with H+ fluxes in fine roots of Populus popularis

Poplar plants are cultivated as woody crops, which are often fertilized by addition of ammonium (NH₄ ⁺) and/or nitrate (NO₃ ^?) to improve yields. However, little is known about net NH₄ ⁺/NO₃ ^? fluxes and their relation with H⁺ fluxes in poplar roots. In this study, net NH₄ ⁺/NO₃ ^? fluxes in association with H⁺ fluxes were measured non-invasively using scanning ion-selective electrode technique in fine roots of Populus popularis. Spatial variability of NH₄ ⁺ and NO₃ ^? fluxes was found along root tips of P. popularis. The maximal net uptake of NH₄ ⁺ and NO₃ ^? occurred, respectively, at 10 and 15 mm from poplar root tips. Net NH₄ ⁺ uptake was induced by ca. 48 % with provision of NO₃ ^? together, but net NO₃ ^? uptake was inhibited by ca. 39 % with the presence of NH₄ ⁺ in poplar roots. Furthermore, inactivation of plasma membrane (PM) H⁺-ATPases by orthovanadate markedly inhibited net NH₄ ⁺/NO₃ ^? uptake and even led to net NH₄ ⁺ release with NO₃ ^? co-provision. Linear correlations were observed between net NH₄ ⁺/NO₃ ^? and H⁺ fluxes in poplar roots except that no correlation was found between net NH₄ ⁺ and H⁺ fluxes in roots exposed to NH₄Cl and 0 mM vanadate. These results indicate that root tips play a key role in NH₄ ⁺/NO₃ ^? uptake and that net NH₄ ⁺/NO₃ ^? fluxes and the interaction of net fluxes of both ions are tightly associated with H⁺ fluxes in poplar roots.     

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH₄⁺) as the sole source of nitrogen. Growth of NH₄⁺-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH₄⁺ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH₄⁺ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH₄⁺ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH₄⁺ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.     

Influence of inorganic nitrogen sources on K+/Na+ homeostasis and salt tolerance in sorghum plants

This work aimed to study the regulation of K⁺/Na⁺ homeostasis and the physiological responses of salt-treated sorghum plants [Sorghum bicolor (L.) Moench] grown with different inorganic nitrogen (N) sources. Four days after sowing (DAS), the plants were transferred to complete nutrient solutions containing 0.75 mM K⁺ and 5 mM N, supplied as either NO₃ ^? or NH₄ ⁺. Twelve DAS, the plants were subjected to salt stress with 75 mM NaCl, which was applied in two doses of 37.5 mM. The plants were harvested on the third and seventh days after the exposure to NaCl. Under the salt stress conditions, the reduction of K⁺ concentrations in the shoot and roots was higher in the culture with NO₃ ^? than with NH₄ ⁺. However, the more conspicuous effect of N was on the Na⁺ accumulation, which was severely limited in the presence of NH₄ ⁺. This ionic regulation had a positive influence on the K⁺/Na⁺ ratio and the selective absorption and transport of K⁺ in the plants grown with NH₄ ⁺. Under control and salt stress conditions, higher accumulation of free amino acids and soluble proteins was promoted in NH₄ ⁺ grown roots than NO₃ ^? grown roots at both harvesting time, whereas higher accumulation of soluble sugars was observed only at 7 days of salt stress exposure. Unlike the NH₄ ⁺ grown plants, the gas exchanges of the NO₃ ^? grown plants were reduced after 7 days of salt stress. These results suggest that external NH₄ ⁺ may limit Na⁺ accumulation in sorghum, which could contribute to improving its physiological and metabolic responses to salt stress.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

The effects of KNO3 concentration in callus induction medium for wheat anther culture

KNO₃ concentration was found to significantly affect the anther culture of wheat (Triticum aestivum L.). When KNO₃ was increased from 0 to 15 mM (in cultivar Jinghua 1) or from 10 to 15 mM (in cultivars 2531-10, Xiaoyan 759 and Norin 10), the callus induction frequency increased significantly. When KNO₃ was increased further above 20 mM, the callus induction frequency decreased significantly in all the tested cultivars. The subsequent frequency of green plantlet regeneration increased significantly, and the ratio of green to albino regenerants increased sharply when KNO₃ concentration increased. Further experiments found that the decrease of callus induction frequency in the medium with too much KNO₃ might be caused by NO₃ ^- ion alone, while the effect of KNO₃ on green plantlet regeneration might be caused by both K⁺ and NO₃ ^- ions, and that the effects of NO₃ ^- concentration were independent of NH₄ ⁺ concentration in the medium.