Comparing responses of generalist and specialist herbivores to various cyanogenic plant features

Plants are obliged to defend themselves against multiple generalist and specialist herbivores. Whereas plant cyanogenesis is considered an efficient defence against generalists, it is thought to affect specialists less. In the present study, we analysed the function of various cyanogenic features of lima bean [Phaseolus lunatus L. (Fabaceae)] during interaction with different herbivores. Three cyanogenic features were analysed, i.e., cyanogenic potential (HCNp; concentration of cyanogenic precursors), β‐glucosidase activity, and cyanogenic capacity (HCNc; release of cyanide per unit time). In no‐choice and free‐choice feeding trials, five lima bean accessions were offered to generalist desert locust [Schistocerca gregaria Forskål (Orthoptera: Acrididae)] and specialist Mexican bean beetle [Epilachna varivestis Mulsant (Coleoptera: Coccinellidae)]. The HCNc was the most important parameter determining host plant selection by generalists, whereas choice behaviour of specialists was strongly affected by HCNp. Although locusts were effectively repelled by high HCNc, this cue was misleading for the detection of suitable host plants, as extensive consumption of low HCNc plant material resulted in strong intoxication of locusts. Balancing cyanide in consumed leaf area, the quantitative release of gaseous cyanide during feeding, and cyanide in faeces suggested that specialists metabolized significantly lower rates of cyanide per consumed leaf material than generalists. We hypothesize that specialists are able to avoid toxic concentrations of cyanide by using HCNp rather than HCNc as a cue for host plant quality, and that they exhibit mechanisms that reduce incorporation of host plant cyanide.     

Cyanogenesis of Wild Lima Bean (Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean (Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans'' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans'' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans'' overall defence in nature. Our results indicate the general importance of analysing ‘multiple defence syndromes’ rather than single defence mechanisms in future functional analyses of plant defences.     

The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10⁻⁶ M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.     

Frequency of cyanogenesis in tropical rainforests of far north Queensland, Australia

BACKGROUND AND AIMS: Plant cyanogenesis is the release of toxic cyanide from endogenous cyanide-containing compounds, typically cyanogenic glycosides. Despite a large body of phytochemical, taxonomic and ecological work on cyanogenic species, little is known of their frequency in natural plant communities. This study aimed to investigate the frequency of cyanogenesis in Australian tropical rainforests. Secondary aims were to quantify the cyanogenic glycoside content of tissues, to investigate intra-plant and intra-population variation in cyanogenic glycoside concentration and to appraise the potential chemotaxonomic significance of any findings in relation to the distribution of cyanogenesis in related taxa. METHODS: All species in six 200 m(2) plots at each of five sites across lowland, upland and highland tropical rainforest were screened for cyanogenesis using Feigl-Anger indicator papers. The concentrations of cyanogenic glycosides were accurately determined for all cyanogenic individuals. KEY RESULTS: Over 400 species from 87 plant families were screened. Overall, 18 species (4.5 %) were cyanogenic, accounting for 7.3 % of total stem basal area. Cyanogenesis has not previously been reported for 17 of the 18 species, 13 of which are endemic to Australia. Several species belong to plant families or orders in which cyanogenesis has been little reported, if at all (e.g. Elaeocarpaceae, Myrsinaceae, Araliaceae and Lamiaceae). A number of species contained concentrations of cyanogenic glycosides among the highest ever reported for mature leaves-up to 5.2 mg CN g(-1) d. wt, for example, in leaves of Elaeocarpus sericopetalus. There was significant variation in cyanogenic glycoside concentration within individuals; young leaves and reproductive tissues typically had higher cyanogen content. In addition, there was substantial variation in cyanogenic glycoside content within populations of single species. CONCLUSIONS: This study expands the limited knowledge of the frequency of cyanogenesis in natural plant communities, includes novel reports of cyanogenesis among a range of taxa and characterizes patterns in intra-plant and intra-population variation of cyanogensis.     

CO2‐mediated changes of plant traits and their effects on herbivores are determined by leaf age

1. Concentration of atmospheric CO₂ is predicted to double during the 21st century. However, quantitative effects of increased CO₂ levels on natural herbivore–plant interactions are still little understood. 2. In this study, we assess whether increased CO₂ quantitatively affects multiple defensive and nutritive traits in different leaf stages of cyanogenic wildtype lima bean plants (Phaseolus lunatus), and whether plant responses influence performance and choice behaviour of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis). 3. We cultivated lima bean plants in climate chambers at ambient, 500, 700, and 1000 ppm CO₂ and analysed cyanogenic precursor concentration (nitrogen‐based defence), total phenolics (carbon‐based defence), leaf mass per area (LMA; physical defence), and soluble proteins (nutritive parameter) of three defined leaf age groups. 4. In young leaves, cyanide concentration was the only parameter that quantitatively decreased in response to CO₂ treatments. In intermediate and mature leaves, cyanide and protein concentrations decreased while total phenolics and LMA increased. 5. Depending on leaf stage, CO₂‐mediated changes in leaf traits significantly affected larval performance and choice behaviour of adult beetles. We observed a complete shift from highest herbivore damage in mature leaves under natural CO₂ to highest damage of young leaves under elevated CO_2. Our study shows that leaf stage is an essential factor when considering CO₂‐mediated changes of plant defences against herbivores. Since in the long run preferred consumption of young leaves can strongly affect plant fitness, variable effects of elevated CO₂ on different leaf stages should receive highlighted attention in future research.     

Bacterial cyanogenesis: impact on biotic interactions

The ability of bacteria to influence organisms that they associate with via metabolite production is one of the hallmarks of microbial interactions. One metabolite of interest is the metabolic poison cyanide. Production of this metabolite is an unique characteristic of certain bacteria that inhabit a wide array of habitats ranging from the human body to the rhizosphere. This review focuses on four targets of cyanogenic bacteria: the human lung, plant pathogens, plants and invertebrates. For a number of cyanogenic bacteria, the contribution of cyanide to the interaction has been rigorously tested using mutants altered in cyanide production. Both deleterious and stimulatory effects of cyanogenic bacteria on other organisms have been documented. In addition, the HCN synthase‐encoding gene cluster hcnABC has served as a marker of cyanogenic capability in the soil environment revealing both genetic diversity at this locus and regulatory influences by other organisms. The pervasive nature of cyanogenesis in a number of different ecological contexts encourages exploration of this bacterial ability and its possible optimization for improving human health, crop production and pest control.     

Pollen feeding,resource allocation and the evolution of chemical defence in passion vine butterflies

Evolution of pollen feeding in Heliconius has allowed exploitation of rich amino acid sources and dramatically reorganized life‐history traits. In Heliconius, eggs are produced mainly from adult‐acquired resources, leaving somatic development and maintenance to larva effort. This innovation may also have spurred evolution of chemical defence via amino acid‐derived cyanogenic glycosides. In contrast, nonpollen‐feeding heliconiines must rely almost exclusively on larval‐acquired resources for both reproduction and defence. We tested whether adult amino acid intake has an immediate influence on cyanogenesis in Heliconius. Because Heliconius are more distasteful to bird predators than close relatives that do not utilize pollen, we also compared cyanogenesis due to larval input across Heliconius species and nonpollen‐feeding relatives. Except for one species, we found that varying the amino acid diet of an adult Heliconius has negligible effect on its cyanide concentration. Adults denied amino acids showed no decrease in cyanide and no adults showed cyanide increase when fed amino acids. Yet, pollen‐feeding butterflies were capable of producing more defence than nonpollen‐feeding relatives and differences were detectable in freshly emerged adults, before input of adult resources. Our data points to a larger role of larval input in adult chemical defence. This coupled with the compartmentalization of adult nutrition to reproduction and longevity suggests that one evolutionary consequence of pollen feeding, shifting the burden of reproduction to adults, is to allow the evolution of greater allocation of host plant amino acids to defensive compounds by larvae.     

Adaptive cyanogenesis clines evolve recurrently through geographical sorting of existing gene deletions

Identifying the genetic basis of parallel phenotypic evolution provides insight into the process of adaptation and evolutionary constraint. White clover (Trifolium repens) has evolved climate‐associated adaptive clines in cyanogenesis (the ability to produce hydrogen cyanide upon tissue damage) in several world regions where it has been introduced. Gene‐deletion polymorphisms at the CYP79D15 and Li loci underlie the presence/absence of the cyanogenic phenotype. Both loci have undergone multiple independent gene‐deletion events, which are identifiable through molecular signatures in flanking regions. To investigate whether cyanogenesis clines in introduced populations have evolved through the sorting of standing genetic variation or de novo gene deletions, we examined cyanogenesis gene‐flanking regions in three world regions. In comparison with native Eurasian populations, we find no evidence for novel gene deletion events in any introduced region, which suggests that these adaptive clines have evolved through the geographical sorting of pre‐existing genetic variation.     

Influence of two selective factors on cyanogenesis polymorphism ofTrifolium repens L. in Darjeeling Himalaya

Cyanogenesis-the production of toxic hydrogen cyanide (HCN) by damaged tissue-inTrifolium repens L. (white clover), a type of most important pasture legume, has been studied at different elevations of Darjeeling Himalaya (latitude-27° 2′ 57″ N, longitude-88° 15′ 45″ E). Release of HCN takes place due to reaction between cyanogenic glucosides stored in vacuoles of the leaf cell and the corresponding enzyme β-glucosidase present in another compartment, often cell wall. Cyanogenesis, a defense system in plant, protects the clover from herbivore and inhibits grazing. Biochemical analysis showed the presence and absence of the cyanogenesis trait within the population in different proportions at different elevations. Acyanogenic individuals also showed variations with respect to presence or absence of either cyanogenic glucosides or β-glucosidase enzyme or both. The distribution of cyanogenic and acyanogenic plants was found in all places, but at lower altitudes (2084–2094 m) the dominating plants were cyanogenic whereas in higher altitude (2560 m) the dominating plants were acyanogenic. It was observed that blister beetle (Mylabris pustalata Thunb.) and the mollusc (Macrochlamys tusgurium Benson.) were the most common consumer of leaflets ofT. repens. Six categories of damage on white clover leaf by these animals were recorded. Our results suggest that the two selective factors or forces i.e. very cold temperature (harmful to cyanogenic plants) at higher altitude as well as indiscriminate but preferential predation (harmful to acyanogenic plants) interact to affect the system of cyanogenesis and also to cause the stable and protective polymorphism inT. repens rather than genotypic differences present among the plants.     

Age versus stage: does ontogeny modify the effect of phosphorus and arbuscular mycorrhizas on above‐ and below‐ground defence in forage sorghum?

Arbuscular mycorrhizas (AM) can increase plant acquisition of P and N. No published studies have investigated the impact of P and AM on the allocation of N to the plant defence, cyanogenic glucosides. We investigated the effects of soil P and AM on cyanogenic glucoside (dhurrin) concentration in roots and shoots of two forage sorghum lines differing in cyanogenic potential (HCNp). Two harvest times allowed plants grown at high and low P to be compared at the same age and the same size, to take account of known ontogenetic changes in shoot HCNp. P responses were dependent on ontogeny and tissue type. At the same age, P‐limited plants were smaller and had higher shoot HCNp but lower root HCNp. Ontogenetically controlled comparisons showed a P effect of lesser magnitude, and that there was also an increase in the allocation of N to dhurrin in shoots of P‐limited plants. Colonization by AM had little effect on shoot HCNp, but increased root HCNp and the allocation of N to dhurrin in roots. Divergent responses of roots and shoots to P, AM and with ontogeny demonstrate the importance of broadening the predominantly foliar focus of plant defence studies/theory, and of ontogenetically controlled comparisons.     

Semi‐quantitative tests of cyanide in foods and excreta of Three Hapalemur species in Madagascar

Three sympatric Hapalemur species (H. g. griseus, H. aureus, and H. (Prolemur) simus) in Ranomafana National Park, Madagascar are known to eat bamboo food parts that contain cyanide. How these lemurs avoid cyanide poisoning remains unknown. In this study, we tested for the presence/absence of cyanide in bamboo lemur foods and excreta to (1) document patterns of cyanide consumption among species with respect to diet, (2) identify routes of elimination of cyanide from the gastrointestinal tract, and (3) determine whether cyanide is absorbed from the diet. We tested 102 food, urine, and fecal samples for hydrogen cyanide (HCN) during two “pre‐dry” seasons (April 2006, May 2007) using commercially available Cyantesmo test strips. The test strips changed color in the presence of HCN, and we recorded color change on a scale of 0 (no change) to 5 (cobalt) at preset intervals with a final score taken at 24 hr. We detected cyanide in bamboo food parts and urine of all three Hapalemur species. Time to color change of the test strips ranged from almost instantaneous to >12 hr incubation. Of the foods tested, only bamboo contained cyanide, but results differed among bamboo species and plant parts of the same species. Specifically, branch shoot and culm pith of the giant bamboo produced strong, immediate reactions to the test paper, whereas parts of liana bamboos produced either weak or no color change. Cyanide was present in almost all urine samples but rarely in fecal samples. This suggests that dietary cyanide is absorbed in the gastrointestinal tract of the Hapalemur species and excreted, at least in part, by the kidneys. Samples from H. griseus exhibited lower, though still detectable, cyanide levels compared with H. simus and H. aureus. Differences among lemur species appear to be related to the specific bamboo parts consumed. Am. J. Primatol. 72:56–61, 2010. © 2009 Wiley‐Liss, Inc.     

Cyanogenesis in plants

Several thousand plant species, including many economically important food plants, synthesize cyanogenic glycosides and cyanolipids. Upon tissue disruption, these natural products are hydrolyzed liberating the respiratory poison hydrogen cyanide. This phenomenon of cyanogenesis accounts for numerous cases of acute and chronic cyanide poisoning of animals including man. This article reviews information gathered during the past decade about the enzymology and molecular biology of cyanogenesis in higher plants. How compartmentation normally prevents the large-scale, suicidal release of HCN within the intact plant is discussed. A renewed interest in the physiology of these cyanogenic compounds has revealed that, in addition to providing protection for some species against herbivory, they may also serve as storage forms for reduced nitrogen.     

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

The β‐cyanoalanine synthase pathway: beyond cyanide detoxification

Production of cyanide through biological and environmental processes requires the detoxification of this metabolic poison. In the 1960s, discovery of the β ‐cyanoalanine synthase ( β ‐CAS) pathway in cyanogenic plants provided the first insight on cyanide detoxification in nature. Fifty years of investigations firmly established the protective role of the β ‐CAS pathway in cyanogenic plants and its role in the removal of cyanide produced from ethylene synthesis in plants, but also revealed the importance of this pathway for plant growth and development and the integration of nitrogen and sulfur metabolism. This review describes the β ‐CAS pathway, its distribution across and within higher plants, and the diverse biological functions of the pathway in cyanide assimilation, plant growth and development, stress tolerance, regulation of cyanide and sulfide signalling, and nitrogen and sulfur metabolism. The collective roles of the β ‐CAS pathway highlight its potential evolutionary and ecological importance in plants.     

Relationship between cyanogenesis and latex stability on tapping panel dryness in rubber trunk girth

The presence of high cyanogenic glycoside concentrations may predispose plant to the tapping panel dryness (TPD). This study aimed to verify the involvement of cyanogenesis in the reduction of latex stability and in the establishment of TPD. The following parameters were evaluated in rubber tree trunk bark: concentration of cyanogenic glycosides with determination of cyanogenic potential (HCNp) and latex stability with lutoid bursting index (LBI). The study of the relationship between cyanogenesis and TPD was performed by semiquantitative comparison of hydrogen cyanide (HCN) gas released from the trunk bark under the following conditions: without (0%) and with (100%) TPD. The positive correlations between HCNp values and LBI indicate that cyanogenic glycosides present in the bark reduce latex stability, resulting in low yield due to the short duration of flow during tapping. The largest amount of HCN released by trunk bark tissues when the plant exhibits TPD symptoms strengthens the evidence of the involvement of this compound in the establishment of this condition.     

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.     

In vitro shoot regeneration from leaf explants of Echinops kebericho: an endangered endemic medicinal plant

Echinops kebericho is a critically endangered endemic medicinal plant of Ethiopia. It is threatened due to over harvesting of its roots for medicinal purposes and from poor seed viability. This study aimed to develop a protocol for in vitro shoot regeneration from leaf explants of E. kebericho. The seeds were sterilized using ethanol followed by Clorox or calcium hypochlorite. Shoots from the germinated seeds were cultured on Murashige and Skoog (MS) medium containing different concentrations of α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP). Young leaves were cultured on MS medium containing different concentrations of BAP and NAA for shoot regeneration. For shoot multiplication, shoots were excised and cultured on MS medium containing different concentrations of BAP or kinetin (KIN) and NAA. The highest mean number of initiated shoots (4.00 ± 0.57) with 100% shoot induction was obtained on medium containing 1.0 mg/L BAP and 0.2 mg/L NAA. The highest shoot regeneration (33%) and shoot number (2.13 ± 0.06) were obtained on MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. Medium containing 1.0 mg/L KIN and 0.2 mg/L NAA produced the highest number of shoots (4.67 ± 0.33) per explant. This protocol can be used for genetic improvement and conservation of this endangered species.     

Antiherbivore defenses alter natural selection on plant reproductive traits

While many studies demonstrate that herbivores alter selection on plant reproductive traits, little is known about whether antiherbivore defenses affect selection on these traits. We hypothesized that antiherbivore defenses could alter selection on reproductive traits by altering trait expression through allocation trade‐offs, or by altering interactions with mutualists and/or antagonists. To test our hypothesis, we used white clover, Trifolium repens, which has a Mendelian polymorphism for the production of hydrogen cyanide—a potent antiherbivore defense. We conducted a common garden experiment with 185 clonal families of T. repens that included cyanogenic and acyanogenic genotypes. We quantified resistance to herbivores, and selection on six floral traits and phenology via male and female fitness. Cyanogenesis reduced herbivory but did not alter the expression of reproductive traits through allocation trade‐offs. However, the presence of cyanogenic defenses altered natural selection on petal morphology and the number of flowers within inflorescences via female fitness. Herbivory influenced selection on flowers and phenology via female fitness independently of cyanogenesis. Our results demonstrate that both herbivory and antiherbivore defenses alter natural selection on plant reproductive traits. We discuss the significance of these results for understanding how antiherbivore defenses interact with herbivores and pollinators to shape floral evolution.     

The evolutionary appearance of non‐cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous β–glucosidases resulting from a crucial amino acid substitution

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α–hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β–glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non‐cyanogenic γ‐ and β–hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β–glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site‐directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non‐flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β–glucosidase, resembling BGD2, and required no more than a single amino acid substitution.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

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