Temporal dynamics of herbivore‐induced responses in Brassica juncea and their effect on generalist and specialist herbivores

Herbivore feeding may induce an array of responses in plants, and each response may have its own temporal dynamics. Precise timing of these plant responses is vital for them to have optimal effect on the herbivores feeding on the plant. This study measured the temporal dynamics of various systemically induced responses occurring in Brassica juncea (L.) Czern. (Brassicaceae) leaves after insect herbivory in India and The Netherlands. Morphological (trichomes, leaf size) and chemical (glucosinolates, amino acids, sugars) responses were analysed. The effects of systemic responses were assessed using a specialist [Plutella xylostella L. (Lepidoptera: Plutellidae)] and a generalist [Spodoptera litura Fabricius (Lepidoptera: Noctuidae)] herbivore. We tested the hypotheses that morphological responses were slower than chemical responses and that generalist herbivores would be more affected by induced responses than specialists. Glucosinolates and trichomes were found to increase systemically as quickly as 4 and 7 days after herbivore damage, respectively. Amino acids, sugars, and leaf size remained unaffected during this period. The generalist S. litura showed a significant feeding preference for undamaged leaves, whereas the specialist herbivore P. xylostella preferred leaves that were damaged 9 days before. Performance bioassays on generalist S. litura revealed that larvae gained half the weight on leaves from damaged plants as compared to larvae feeding on leaves from undamaged plants. These studies show that although morphological responses are somewhat slower than chemical responses, they also contribute to induced plant resistance in a relatively short time span. We argue that before considering induced responses as resistance factors, their effect should be assessed at various points in time with both generalist and specialist herbivores.     

Floral nectaries and nectar production in brown mustard (Brassica juncea) and white mustard (Sinapis alba) (Brassicaceae)

 The dynamics and abundance of nectar secretion as well as sugar productivity were studied in flowers of brown mustard (Brassica juncea) cv. Małopolska and white mustard (Sinapis alba) cv. Borowska. Moreover, floral nectaries were examined under LM and SEM. In both cultivars lateral and median pairs of nectaries secreted nectar. However, differences were found in morphology and activity of these pairs. The lateral nectaries produced more nectar than the median ones. Nectar secretion started at loose bud and peaked during anther dehiscence. Average amount of nectarsecreted by 100 flowers of cv. Małopolska and cv. Borowska were 119.9 mg and 134 mg. Mean concentration of nectar was 26.7% and 23.4%, respectively. One hundred flowers of cv. Małopolska and cv. Borowska secreted 28.4 mg and 24.9 mg of sugars in nectar. Estimated sugar productivity per 1 ha of crop was 65.5 kg and 71.2 kg, respectively. Received August 28, 2002; accepted November 2, 2002 Published online: June 2, 2003     

Hyperaccumulation of Lead by Roots, Hypocotyls, and Shoots of Brassica juncea

The effects of different concentrations of lead nitrate (10^–5 to 10^–3 M) on root, hypocotyl, and shoot growth of Indian mustard (Brassica juncea L. var. megarrhiza), and the uptake and accumulation of Pb²⁺ by its roots, hypocotyls, and shoots were investigated. Lead had no significant inhibitory effect on the root growth at concentrations of 10^–5 to 10^–4 M during the entire treatment, while at 10^–3 M, Pb slightly inhibited the root and shoot growth. B. juncea has ability to take up Pb from solutions and accumulate it in its roots, and transport and concentrate it. The Pb contents in the parts of plants treated with 10^–3 M Pb were greater than those of untreated plants, by factors of 230 in the roots, 170 in the hypocotyls, and 3 in the shoots.     

The role of extrafloral nectaries in the herbivore defence of Cassia fasiculata

Abstract. 1. An experiment is presented that tests the hypothesis that the extrafloral nectaries of Chssia fasiculata reduce herbivory by attracting pugnacious ants that deter herbivores, and tests the equivalence of two methods of ant exclusion: treatment with Tanglefoot, and nectary excision.
2. The protectionist hypothesis is not supported; treatment plants produced as many pods as did control plants.
3. Treatment with Tanglefoot is equivalent to nectary excision in terms of pod production
4. A graphical model is presented that relates the efficacy of ant protection to plant density and ant activity.     

Insect visitors to extrafloral nectaries of Byttneria aculeata (Sterculiaceae): relative importance and roles

Abstract. 1. Ants, parasitoids and flies are about equally frequent at foliar nectaries of Byttneria aculeata (Sterculiaceae) in lowland Costa Rica during the dry season, a pattern previously unreported but also observed at other plants in the area.
2. Species of Ectatomma, Crematogaster and Camponotus were the most frequent of twenty-four ant species on Byttneria, eight of which nested in the hollow stems. Ants spent most time at nectaries and little in patrolling.
3. Collections at nectaries yielded large numbers of species of parasitoid Hymenoptera with few individuals of each. Rearing studies of leaf-feeding herbivores yielded several species of parasitoids, including one species taken at a nectary and two others congeneric or closely related.
4. Flies appear to be nectar thieves, in the same sense as non-pollinating floral visitors, despite close association with Byttneria.
5. Ant-plants may be poor models for the larger number of species of less specialized plants with extrafloral nectaries. Plants which have only extrafloral nectaries may better suit the needs of parasitoids than ants, and plants such as Byttneria may benefit as much from parasitoids as from ants.     

Understanding ontogenetic trajectories of indirect defence: ecological and anatomical constraints in the production of extrafloral nectaries

Background and Aims

Early ontogenetic stages of myrmecophytic plants are infrequently associated with ants, probably due to constraints on the production of rewards. This study reports for the first time the anatomical and histological limitations constraining the production of extrafloral nectar in young plants, and the implications that the absence of protective ants imposes for plants early during their ontogeny are discussed.

Methods

Juvenile, pre-reproductive and reproductive plants of Turnera velutina were selected in a natural population and their extrafloral nectaries (EFNs) per leaf were quantified. The anatomical and morphological changes in EFNs during plant ontogeny were studied using scanning electron and light microscopy. Extrafloral nectar volume and sugar concentration were determined as well as the number of patrolling ants.

Key Results

Juvenile plants were unable to secrete or contain nectar. Pre-reproductive plants secreted and contained nectar drops, but the highest production was achieved at the reproductive stage when the gland is fully cup-shaped and the secretory epidermis duplicates. No ants were observed in juvenile plants, and reproductive individuals received greater ant patrolling than pre-reproductive individuals. The issue of the mechanism of extrafloral nectar release in T. velutina was solved given that we found an anatomical, transcuticular pore that forms a channel-like structure and allows nectar to flow outward from the gland.

Conclusions

Juvenile stages had no ant protection against herbivores probably due to resource limitation but also due to anatomical constraints. The results are consistent with the growth-differentiation balance hypothesis. As plants age, they increase in size and have larger nutrient-acquiring, photosynthetic and storage capacity, so they are able to invest in defence via specialized organs, such as EFNs. Hence, the more vulnerable juvenile stage should rely on other defensive strategies to reduce the negative impacts of herbivory.     

Isozyme studies in Indian mustard (Brassica juncea L.)

Summary A comparative study of peroxidase and esterase isozymes was carried out at five developmental stages of siliqua in order to characterize twelve genotypes of Indian mustard. The studies showed nearly the same number of isozyme bands at every stage for peroxidase and a varying number of isozyme bands for esterase. The appearance and disappearance of bands, along with their intensity scores, indicated the role of different isozymes at different stages of siliqua development. It has been ascertained that these patterns, especially the intensity scores, can be successfully used to characterize different Indian mustard genotypes.     

Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea

Transgenic Indian mustard (Brassica juncea) overexpressing y-glutamylcysteine synthetase (ECS) or glutathione synthetase (GS) were shown previously to have two-fold higher levels of glutathione and total nonprotein thiols, as well as enhanced cadmium tolerance and accumulation. Here, the hypothesis was tested that these transgenics have enhanced tolerance to organic pollutants, based on the reasoning that many organic xenobiotics are detoxified via conjugation to glutathione. Both the ECS and GS transgenics showed enhanced tolerance to atrazine: while root growth of wildtype seedlings was inhibited 50% by 100 mg L(-1) atrazine, ECS and GS root growth was inhibited 20-30% (P < 0.05). The tolerance of the transgenics to CDNB (1-chloro-2,4-dinitrobenzene). metolachlor, and phenanthrene was also somewhat higher than wild type, but these differences were not as pronounced. Each of the organics treatments significantly enhanced total nonprotein thiol levels in all plant types (2 to 12-fold). Overall, these results suggest that GSH biosynthesis is limiting for atrazine detoxification in Indian mustard and that overexpression of enzymes involved in GSH biosynthesis offers a promising approach to create plants with the enhanced capacity to tolerate not only heavy metals, but also certain organics.     

Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea)

Chimeric constructs with the hydrophilic octapeptide FLAG epitope (DYKDDDDK) have been widely used as multipurpose tags for identification, detection, and purification of FLAG fusion proteins. Constructs consisting of C-terminal FLAG-tagged genomic and cDNA clones of anArabidopsis phytochelatin synthase gene,AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression ofAtPCS1 in transgenic lines were confirmed by using PCR and Northern blot analyses. However, immunoblot analysis revealed strong nonspecific binding of a monoclonal anti-FLAG M2 antibody to an endogenous protein in both shoot and leaf tissues of wild-type Indian mustard (85-kDa) that masked presence of the phytochelatin synthase (PCS) protein of interest (55-kDa). Further analysis revealed absence of a nonspecific protein in root tissues of transgenic plants, thus allowing detection of the FLAG-tagged PCS protein.     

Sixteen new simple sequence repeat markers from Brassica juncea expressed sequences and their cross‐species amplification

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 16 expressed sequence tags (EST)‐SSR markers from Brassica juncea and their cross‐amplification across Brassica species. Sixteen primer pairs were assessed for polymorphism in all genomes of the diploid and amphidiploid Brassica species. The markers show reliable amplification, considerable polymorphism and high transferability across species, demonstrating the utility of EST‐SSRs for genetic analysis of brassicas.     

An ecogenomic analysis of herbivore‐induced plant volatiles in Brassica juncea

Upon herbivore feeding, plants emit complex bouquets of induced volatiles that may repel insect herbivores as well as attract parasitoids or predators. Due to differences in the temporal dynamics of individual components, the composition of the herbivore‐induced plant volatile (HIPV) blend changes with time. Consequently, the response of insects associated with plants is not constant either. Using Brassica juncea as the model plant and generalist Spodoptera spp. larvae as the inducing herbivore, we investigated herbivore and parasitoid preference as well as the molecular mechanisms behind the temporal dynamics in HIPV emissions at 24, 48 and 72 h after damage. In choice tests, Spodoptera litura moth preferred undamaged plants, whereas its parasitoid Cotesia marginiventris favoured plants induced for 48 h. In contrast, the specialist Plutella xylostella and its parasitoid C. vestalis preferred plants induced for 72 h. These preferences matched the dynamic changes in HIPV blends over time. Gene expression analysis suggested that the induced response after Spodoptera feeding is mainly controlled by the jasmonic acid pathway in both damaged and systemic leaves. Several genes involved in sulphide and green leaf volatile synthesis were clearly up‐regulated. This study thus shows that HIPV blends vary considerably over a short period of time, and these changes are actively regulated at the gene expression level. Moreover, temporal changes in HIPVs elicit differential preferences of herbivores and their natural enemies. We argue that the temporal dynamics of HIPVs may play a key role in shaping the response of insects associated with plants.     

Pot experiment to study the uptake of Cd and Pb by three Indian mustards (Brassica juncea) grown in artificially contaminated soils

Most of the metals-contaminated and fallow lands in Taiwan are a result of irrigation with illegal effluent of factories. Phytoextraction methods can be applied to reach the target of fallow-lands reuse and earn more incomes for farmers. In many studies, Indian mustards (Brassica juncea) were planted in the metal-contaminated soils to study their suitability in phytoextraction. However, the total removal of metals by plants was quite different between accessions. In this pot study, three accessions of B. juncea (cv. 182921, cv. 211000, and cv. 426308) were planted in artificially Cd- or Pb-contaminated soils to investigate the differences between them. EDTA was applied to study its effect in increasing the bioavailability of Cd and Pb and their uptake by these Indian mustards. Experimental result showed that three accessions of Indian mustard can accumulate a high concentration of Cd and Pb when growing in the artificially Cd- and Pb-contaminated soils. Their shoot Cd or Pb concentrations were significantly enhanced, resulting from the application of EDTA. Among the three accessions, B. juncea cv. 211000 accumulated the highest concentrations of Cd and Pb in their shoots compared with B. juncea cv. 182921 and cv. 426308, but its total removal was the lowest due to its lower biomass.     

Influence of paclobutrazol on growth and development of fruit in Brassica juncea (L.) Czern and Coss

Foliar spraying of paclobutrazol(5, 10 and 20 g mL^–1) on Brassica juncea (cv. RLM 514) plants, at the green floral bud stage of the terminal inflorescence, exerted a profound effect on growth and metabolism of fruits (siliquae). Paclobutrazol caused a decrease in siliqua length but enhanced significantly siliqua width, seed size (diameter) and seed number per siliqua. Paclobutrazol increased dry matter production and also hastened the relative growth rate of the siliqua wall and seed. In paclobutrazol-treated plants the siliquae were darker green, and during active phases of growth both siliqua wall and seed exhibited higher total chlorophyll (Chl) content, decreased chl a/b ratios, greater hill reaction activity of chloroplasts and increased activities of -amylase, -amylase and invertase than controls. Paclobutrazol enhanced the levels of total soluble sugars, starch, protein and free amino acids in mature seeds. The seed oil content increased by 2 to 3 per cent with a treatment of 10 g mL^–1 pacolobutrazol.     

The function of extrafloral nectaries in the pollination and reproduction of Sambucus javanica

Sambucus javanica is a perennial herb with extrafloral nectaries (EFNs) on its inflorescences. To explore the ecological functions of EFNs, a factorial combination experiment of ant (access or exclusion) and EFNs (with or without) at the plant level was created in two populations. The role of EFNs in the attraction of ants and flying pollinators, the defensive role of ants against foliar herbivores, the effects of ants on pollinator visitation and the effects of ant–pollinator interactions on fruit production in one or both populations were assessed. Ants were common on the ant-access plants with EFNs, but absent from the ant-access plants without EFNs. Foliar herbivory was independent of ant and EFN treatments and their interactions. The visitation frequency of flying pollinators (honeybees and syrphid flies) and fruit set were significantly higher for plants with EFNs than plants without EFNs, but were not affected by ant treatments or their interactions with EFN treatments. These results suggest that EFNs in S. javanica attracted both ants and flying pollinators, but ants did not present a defensive role against herbivores, did not deter flying pollinators from visiting inflorescences and had no effects on fruit production. In addition, ants were not significant pollen vectors.     

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose.     

Effect of ‘Mixtalol’ on growth,yield and yield components of Indian mustard (Brassica juncea)

Mixtalol (a mixture of long chain aliphatic alcohols varying in chain length from C₂₄ to C₃₂) applied to Brassica juncea plants as foliar spray caused an increase in secondary and tertiary branching with consequent enhancement in seed yield through increased number of inflorescences and siliquae per plant. The percentage of immature siliquae and shattering of siliquae decreased with this treatment. Mixtalol increased total dry matter of plants, partitioning coefficient and harvest index. The contents of starch, protein and oil were also higher in seeds from Mixtalol treated plants.     

The occurrence and abundance of plants with extrafloral nectaries, the basis for antiherbivore defensive mutualisms, along a latitudinal gradient in east Asia

Abstract. The occurrence and abundance of indigenous plants with extrafloral nectaries was evaluated within local communities and regional floras along a north to south gradient from tundra in northeastern Russia (64–70°N) through temperate types in eastern Russia and Korea to subtropical vegetation in the Bonin Islands (26–27°N) south of Japan. Moving from tundra to subtropical vegetation, there is a pattern of increasing abundance of extrafloral bearing plants as a function of total plant cover (from 10.25 to 40.18%), number of species per sampled area (from 0.11 to 1.13/100 m), and proportion of species within regional floras (from 0.32 to 7.46%). There were some plants with extrafloral nectaries in all communities but their abundance varied greatly, c. 1–25% in the four northern latitudes and c. 7–70% in the subtropical region. Ants, the primary mutualists associated with plants bearing extrafloral nectaries, have a similar pattern of increasing abundance (species richness, nest density, and colony size) along the same north–south latitudinal gradient.     

提高榨菜离体培养植株再生频率

采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg／L6．BA 0．2mg／L2,4-D的组合较为适宜,其不定芽再生频率可达50％,且外植体以下胚轴为好：而CPPU和2,4-D的适宜组合为1．5mg／L 0．2mg／L,其不定芽再生频率高达66．67％,最适外植体为带柄子叶。同时,研究结果显示,添加0．25～1mg／L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。