Optimized somatic embryogenesis in Pinus strobus L.

Summary Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.     

A clonal propagation system for Atlantic white cedar (<Emphasis Type="Italic">Chamaecyparis thyoides</Emphasis>) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l^?1 producing more embryos than 0 g l^?1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l^?1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.     

The role of activated charcoal in plant tissue culture

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.     

Metabolic evaluation of cellular differentiation of tobacco leaf explants in response to plant growth regulators in tissue cultures using <Superscript>1</Superscript>H NMR spectroscopy and multivariate analysis

To investigate the metabolic changes that precede visible organogenesis in tissue culture, tobacco leaf explants were cultured on media supplemented with various plant growth regulators (PGRs) and analyzed with proton nuclear magnetic resonance (¹H NMR) spectroscopy. Principal component analysis (PCA) of ¹H NMR spectral data was unable to differentiate between leaf explants cultured with α-naphthaleneacetic acid and those cultured with 6-benzyladenine after 4 days of culture; however, a difference was evident after 8 days of culture. A hierarchical dendrogram from PCA analysis could be grouping leaf explants cultured with various auxins separately from those treated by various cytokinins. However, leaf explants cultured with thidiazuron (TDZ) were identified as an outlier group; TDZ appeared to produce pleiotropic metabolic effects that differed from those induced by other PGRs. These results show that dedifferentiation can be initiated by either auxins or cytokinins, which is reflected by similar metabolic changes produced by the two distinct PGRs during the initial incubation period. The subsequent redifferentiation differs according to the PGR treatment, which is reflected by differential metabolic changes, depending on the fate of cells in organogenesis. Glutamine and glutamate levels increased approximately twofold in cytokinin-treated leaf explants compared with auxin-treated explants; however, changes in the levels of sugar compounds did not differ between the two treatments, demonstrating auxin regulation of the carbon/nitrogen ratio in favor of rooting. Taken together, our results suggest that ¹H-NMR spectroscopy combined with multivariate analysis is a promising means for the metabolic evaluation of plant growth and differentiation.     

A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture

This report presents a simple, rapid and accessible validated method for quantification of eight major plant growth regulators (PGR): cytokinins (6-(γ,γ-dimethylallylamino)purine (2-iP), benzylaminopurine (BA) and zeatin), auxin (indole-3-acetic acid; IAA), jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) and abscisic acid (ABA) by liquid chromatography mass spectrometry. This method was tested in eight species including agricultural, ornamental and medicinal species: St. John’s wort, African violet, banana, American elm, tobacco, potato, sweet wormwood, and fennel. The method has good reproducibility and good sensitivity with %RSD (percent relative standard deviation) between 1 and 10% for all matrices and recovery values of 89 to 118% for all analytes. Method detection limits were 50.65 ng/g, 203.4 ng/g, 50.65, ng/g, 50.65 ng/g, 203.4 ng/g, 12.7 ng/g, 193 pg/g and 3.08 ng/g, for SA, IAA, zeatin, JA, GA3, ABA, 2-iP, and BA, respectively. Our results with a range of plant species show that this method represents a simple, low-cost method for analysis of PGRs, and may also serve as an useful starting point for the analysis of other related PGRs, as demonstrated by inclusion of the SA derivative, acetylsalicylic acid, and the JA derivatives: 12-oxo-phytodienoic acid and JA-isoleucine. The efficiency of this method will enable its incorporation into the plant tissue culture work flow and through characterization of endogenous PGR levels, will allow for improved method development for recalcitrant species facilitating fundamental and applied studies in plant morphogenesis, propagation and conservation.     

Polymeric derivatives of plant growth regulators: synthesis and properties

The polymeric formulations of plant growth regulators (PGRs) are high molecular weight systems in which the PGR unit is attached to the polymeric chain by a hydrolysable chemical bond. These polymeric derivatives (esters, ethers, or else) of PGRs are characterised by the ability to release the active compound (PGR) from their solutions (mainly aqueous) in certain conditions. The release of the PGR can be controlled by external factors (pH, temperature, enzymes, solution concentration), and inherent properties of the whole macrosystem chemical structure, such as the type of the hydrolysable bond between PGR unit and the main polymeric chain, the structure of the polymer chain (e.g. molecular weight, level of hydrophilicity, and the content of hydrophobic groups, macromolecular conformation in solution etc.). These controlled (slow) release PGRs display certain advantages over conventional PGR formulations due to their prolonged action, improved efficiency (e.g. wide range of effective concentrations) greater safety to non-target organisms and the applicators. In addition the ability of altering the solubility level and modifying the aplication form is of considerable interest. The biological activity efficiency of polymeric PGRs has been documented and the relation of this efficiency to the PGR unit hydrolytic release ability has been mentioned. Slow release polymeric PGRs are considered to solve certain problems in agriculture.Abbreviations PGR plant growth regulator - C(S)RF controlled (slow) release form - PD polymeric derivative - ACC 1-amino-cyclopropane-1-carboxylic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BAP N⁶-benzylaminopurine - ABA abscisic acid - GA gibberellin - LMW low molecular weight - HMW high molecular weight     

Embryo rescue and plant regeneration in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.     

Contribution of the wheat (Triticum aestivum L.) flag leaf to grain yield in response to plant growth regulators

In West-Europe, intensive cereal management uses plant growth regulators (PGRs) especially for wheat. A green-house experiment compared the effects of two PGRs on flag leaf characteristics and yield of winter wheat. Chlormequat chloride + choline chloride (CCC) and chlormequat chloride + choline chloride + imazaquin (CCC+I) were applied to winter wheat at growth stage 5 (Feekes Large scale). CCC and CCC+I significantly increased flag leaf surface area at anthesis. Both treatments also enhanced chlorophyll content of the main stem flag leaf. The grain filling period was extended with PGR application by 2 days. CCC and CCC+I significantly increased net CO₂ assimilation rates during the flag leaf life. No effects of PGR spraying were observed on the pattern of ¹⁴C labelled assimilate distribution. Increased grain yield was due to the increase in average grain weight. The results indicate that PGR treatments increased flag leaf contribution to grain filling. The addition of imazaquin (I) to chlormequat (CCC) improved the effects of CCC.     

Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid

To clarify the causal factors for ploidy variation in plant cell culture, we attempted to alter ploidy distribution in cell cultures of a tetraploid cultivar of Doritaenopsis by changing the plant growth regulators (PGRs) in the culture medium. The original suspension cultured cells, which had been maintained in medium containing 0.1 mg l⁻¹ 1-naphthaleneacetic acid and 1 mg l⁻¹ benzyladenine, were transferred onto various gellan gum solidified media with a single application of PGRs, and the ploidy distributions of the cells were examined using flow cytometry analysis during 3 weeks of culture. Among the PGRs tested, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid caused a drastic reduction in the 4C-cell proportion in cell cultures with an increased cell proportion of 8C or higher C-values. In the case of 2,4-D application, a reduction of cell viability was observed. A decreasing proportion was also observed in the 8C-cell population accumulated by 2,4-D treatment, following transfer back to the medium containing the standard PGR composition. These results suggest that the exogenous application of 2,4-D arrested the cell cycle at G2 phase in the Doritaenopsis cells, and the removal of 2,4-D might induce further endoreduplication or recover the mitotic cycle of the G2-arrested cells.     

Patent portfolio-based indicators to evaluate the commercial benefits of national plant genetic resources

According to property rights theory, national plant genetic resources (PGRs) are sovereign properties rather than resources belonging to the common heritage of humankind. Consequently, provider states can claim compensation from users of their national PGRs, leading to the need for bilateral or multilateral agreements to share national PGRs' commercial benefits. However, as benefit-sharing agreements are made exante, estimating the potential profit is difficult. Thus, issues around asymmetric information about the commercial value of such resources have emerged. In this paper, we use a patent portfolio as a proxy to estimate the potential commercial benefits of national PGRs and propose new evaluation indicators. We propose a comprehensive evaluation process that covers constructing a patent portfolio for each PGR, establishing indicators in terms of marketability, technology, and exclusiveness, and assigning weights to the indicators using fuzzy analytic hierarchy. In addition, we illustrate this process using a case of Korean national PGRs based on the opinions of stakeholders and experts. This research is expected to help promote national PGR transactions with equitable access and benefit sharing agreements.     

Fulvic Acid,Brassinolide, and Uniconazole Mediated Regulation of Morphological and Physiological Traits in Maize Seedlings Under Water Stress

Water stress is one of the most important factors limiting sustainable crop production. Therefore, the effects of the plant growth regulators (PGRs) fulvic acid (FA), brassinolide (BR), and uniconazole (Uni) on seedling growth and physiology of two maize (Zea mays L.) varieties were evaluated under???0.7 MPa water stress induced by polyethylene glycol-6000. Under drought stress, the PGRs promoted seedling growth, altered the root-to-shoot ratio, and significantly increased root biomass, length, surface area, diameter, and volume. In addition, depending on the PGR, net photosynthesis rate, SPAD value (indicating chlorophyll content), and water use efficiency increased significantly, under drought stress, whereas transpiration rate decreased. The PGRs also significantly increased antioxidant enzyme activities and significantly decreased malondialdehyde accumulation in leaves and roots under drought stress. Zhengdan958 showed greater variation in physiological responses and stronger drought resistance than Xundan20. In alleviating drought stress in maize seedlings, FA had the greatest effects on shoot growth and leaf physiology; Uni exerted its effects by regulating root structure, and BR effects were intermediate. Under drought stress, the three PGRs increased maize seedling growth, which reduced drought stress-induced damage and improved plant ability to resist the adversity. Based on a comprehensive analysis of physiological indices of drought resistance, Uni is recommended as the best PGR to improve maize seedlings resistance to drought.

     

4. The role of plant growth regulators in the germination of forest tree seeds

To establish a role for PGRs in the germination of forest tree seeds, it must be demonstrated that PGRs are involved in the mechanisms that control the induction of and release from dormancy, and also that these mechanisms are operative in the seeds of woody plants. Four currently known concepts of seed dormancy are reviewed, citing the published evidence relating PGRs to tree seed germination. The germination of tree seeds appears to be controlled by a variety of external and internal factors. PGRs figure prominently among these factors, but the range of mechanisms by which such control is mediated may vary considerably—from physical to metabolic. Although significant support can be found for PGR involvement in the regulation of angiosperm seed dormancy, the evidence for gymnosperms is still inconclusive. This is primarily because our understanding of the control of germination—by PGRs or other means—is generally unsatisfactory. However, recent advancements in PGR technology, together with a more holistic concept of PGR action in biological systems, signal a more favourable climate and a revitalized approach to the investigation of the role of PGRs in plant growth and development.     

In vitro micro-tuber initiation and dormancy in yam

Dormancy is a mechanism that regulates the timing of sprouting (germination) of affected plant parts as well as ensures that the food quality of edible parts is maintained in storage until the following growing season. In yam, however, little is known about the control of tuber initiation or tuber dormancy. The objective of this study was to determine the effects of selected plant growth regulators (PGRs) on tuber initiation and dormancy, using an in vitro system. In two replicated experiments, 2-chloroethylphosphonic acid (ethephon, an ethylene source), abscisic acid (ABA) and gibberellin (GA₃) – and their inhibitors silver nitrate, fluridone and 2-chloroethyl-trimethylammonium chloride, respectively – were added at two concentrations to the culture medium prior to explant culture. Dates of micro-tuber initiation and sprouting (end of dormancy) and tuber number were recorded. In the control (no PGR) in Experiment 1, micro-tubers were initiated at the base of the stem after 176 days and sprouted 235 days later, that is 411 days after culturing. Most PGR treatments had only small effects (±30 days) on the duration of dormancy and the time of micro-tuber initiation. However, in GA₃ micro-tuber initiation occurred after 76 days, about 100 days earlier than in the control, whereas fluridone affected the position of micro-tubers and duration of dormancy. With fluridone treatments, tubers were found at the base of the stem (normal position) and on lower and upper nodes. Lower node tubers sprouted within 225 days of culturing compared with about 420 days after culturing at other nodal positions and in other PGR treatments. These data suggest an important role for ABA and gibberellic acid in yam micro-tuber initiation and the induction of dormancy.     

Loss of secreted antibody from transgenic plant tissue cultures due to surface adsorption

The role of surface adsorption in the disappearance of secreted foreign proteins from the medium of transgenic plant cell and organ cultures was investigated. When mouse monoclonal IgG1 was added to sterile plant culture media in glass shake flasks, the antibody concentration declined rapidly demonstrating that antibody was labile in the plant culture environment even in the absence of biomass and proteases. Elution of bound antibody from the surfaces of the flasks indicated that adsorption had contributed to the observed loss of antibody from solution. Antibody retention in sterile plant culture media was improved significantly when protein-resistant polymer coatings were applied to the glass vessels containing the antibody solutions. Pluronic F127 applied at a concentration of 1 mg mL(-1) to a primary dimethyldichlorosilane layer on glass yielded the best results in sterile Murashige and Skoog medium. When this coating was used in shake flasks for culture of transgenic tobacco hairy roots, there was a significant improvement in the accumulation of secreted recombinant antibody in the medium consistent with a reduction in antibody adsorption. Medium antibody levels eventually declined, however, as medium protease concentrations rose rapidly towards the end of the culture period. This work demonstrates that surface adsorption reduces the medium antibody titre observed in transgenic plant tissue cultures.     

The combined impact of pH and activated carbon on the elemental composition of a liquid conifer embryogenic tissue initiation medium

This study considered the elemental composition of plant tissue culture media in response to pH and two different types of activated C (AC; tissue culture and non acid-washed grades) in liquid media. When tissue culture medium is supplemented with AC the method of AC addition and pH adjustment can greatly impact the final medium pH, in turn, altering mineral availability. Over the pH range of 4–7, Cu and Zn adsorbed (95% and 50%) onto the two physically different ACs to the same extent. As the pH exceeded 5.8, precipitation became pronounced, resulting in 50% reductions in Mn and Fe and smaller reductions in Ca (20%), and P (15%), independent of AC. Non acid-washed AC released significant levels of Mg (65% increase) and Ca (10% increase) at pH 5.8 compared to the no-AC control. No adsorption was indicated for inorganic anions. Low levels for Cu and Zn are a concern when using AC, and low levels of Fe and Mn are a concern when the pH of the medium exceeds 5.8. Due to its impurity content and difficulty associated with its neutralization, non-acid-washed AC may be a poor choice for use in tissue culture medium.Communicated by W.A. Parrott     

Direct measurement of water availability in gelled plant tissue culture media

Summary Water constitutes nearly 100% of the volume and 95% of the mass of gelled plant tissue culture media. Even so, plant growth and development responses observed to occur with relatively small changes in gelling agent concentration (0.1% of media total mass) have been attributed to changes in media water availability. Measurements with three alternative direct techniques, specific for measuring physiochemical water availability indicated the effects of a change of this magnitude in gelling agent concentration negligibly affected the media water potential and water conductivity. Sensitive pressure membrane measurements indicated that incremental gelling agent concentration increases of 0.1% (of media total mass) within the range normally used for plant tissue culture media, depressed water matric potential only 1–2cm H₂O (1–2×10⁻⁴ MPa (mega pascal.)); these values were confirmed with equally sensitive tensiometer measurements. Moreover, no effect of concentration on water movement could be detected with a precise constant-head permeameter over a broader range of gelling agent concentrations. These results indicate that eitherin vitro plants are extremely sensitive to subtle shifts in water status, or other physiochemical factors that also change with gelling agent concentration are contributing to the reported effects on plant growth and development.     

Shoot regeneration from spinach hypocotyl segments by short term treatment with 5,6-dichloro-indole-3-acetic acid

Factors affecting shoot regeneration from hypocotyl segments of spinach (Spinacia oleracea L.) were investigated. When expiants were cultured on medium containing 10 mg/l IAA for 7 weeks, 3 out of 9 cultivars showed relatively high shoot regeneration response (15 – 35%). The other PGRs tested had no effect on shoot regeneration. However, the transfer of explants from auxin-containing medium to auxin-free medium 20 d after culture induced shoot formation from expiants cultured on media containing each of the auxin sources tested individually. By applying this short term auxin treatment, more than 80% shoot regeneration was obtained on medium containing 5–20 mg/l 5,6-Cl₂-IAA, compared to less than 30% with 10–20 mg/l IAA treatment.Abbreviations BAP 6-benzylaminopurine - NAA 1naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - 5,6-Cl₂-IAA 5,6dichloro-indole-3-acetic acid - PGR plant growth regulator - A-PGR auxin-like plant growth regulator     

Plant growth regulators and the orchid cut-flower industry

Research involving plant growth regulators (PGRs) and orchids in areas of orchid growth and development are reviewed. For all areas covered in the review — seed germination and seedling growth, lateral shoot production, root production, flower initiation and development, postharvest physiology, and photosynthate partitioning — it was concluded that further studies would assist in clarifying potential uses for PGRs in the orchid cut-flower industry. It is suggested that extra PGR research on orchids is justified at the present time because of favourable prospects facing the orchid cut-flower industry.     

Induction of water deficit tolerance in wheat due to exogenous application of plant growth regulators: membrane stability,water relations and photosynthesis

Our experiment was carried out in order to explore effects of plant growth regulators (PGR; thidiazuron, paclobutrazol, and ascorbic acid) on physiological traits of wheat genotypes under water surplus and deficit conditions. Study revealed that relative water content, membrane stability index, chlorophyll content, photosynthetic rate (P_N), and maximal quantum yield of PSII improved with PGRs application across the genotypes both under irrigation and water stress. The response of HD 2733 genotype was more positive toward PGRs treatment as compared to other genotypes under water stress. Higher P_N and chlorophyll contents were observed in HD 2987 followed by C 306 genotype under water-stress conditions. Moreover, Rubisco small subunit (SSU) expression was lower in wheat genotypes under water stress as compared to irrigated conditions. Application of PGRs led to upregulation of SSU under water stress, while no significant change was found in Rubisco level and activity under irrigated condition in dependence on PGRs treatments. Yield-related traits showed also significant reduction under water-stress conditions, while application of PGRs enhanced the yield and its components. Results indicated that the PGRs exhibited a positive interaction and synergetic effect on water stressed wheat plants in terms of photosynthetic machinery and yield.     

西双版纳傣族曼仑村植物遗传资源研究:现状与保护策略

本文采用民族植物学和文化人类学的方法, 对地处我国西南边陲西双版纳勐仑镇的傣族村寨曼仑村的植物遗传资源进行了调查和研究, 结果表明: 当地仍然保存有十分丰富的植物遗传资源, 栽培植物有68个科, 136个属, 204个种, 地方品种也较丰富, 集中分布在葫芦科、天南星科、禾本科等8个科; 当地人与植物遗传资源的形成和发展关系密切, 随着社会变迁、经济发展和人口膨胀等各种因素的影响, 原本丰富的植物遗传资源锐减, 从而使与之共存的传统知识也面临消亡的危机。作者认为只有充分调动当地人的积极性, 主动参与到植物遗传资源的保护领域, 才能进行有效保护。