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.     

Accumulation, detoxification, and genotoxicity of heavy metals in Indian mustard (Brassica juncea L.)

Plants of Indian mustard (Brassica juncea L.) were exposed to different concentrations (15, 30, 60, 120 microM) of (Cd, Cr, Cu, Pb) for 28 and 56 d for accumulation and detoxification studies. Metal accumulation in roots and shoots were analyzed and it was observed that roots accumulated a significant amount of Cd (1980 microg g(-1) dry weight), Cr (1540 microg g(-1) dry weight), Cu (1995 microg g(-1) dry weight), and Pb (2040 microg g(-1) dry weight) after 56 d of exposure, though in shoot this was 1110, 618, 795, and 409 microg g(-1) dry weight of Cd, Cr, Cu, and Pb, respectively. In order to assess detoxification mechanisms, non-protein thiols (NP-SH), glutathione (GSH) and phytochelatins (PCs) were analyzed in plants. An increase in the quantity of NP-SH (9.55), GSH (8.30), and PCs (1.25) micromol g(-1) FW were found at 15 microM of Cd, however, a gradual decline in quantity was observed from 15 microM of Cd onwards, after 56 d of exposure. For genotoxicity in plants, cytogenetic end-points such as mitotic index (MI), micronucleus formation (MN), mitotic aberrations (MA) and chromosome aberrations (CA) were examined in root meristem cells of B. juncea. Exposure of Cd revealed a significant (P < 0.05) inhibition of MI, induction of MA, CA, and MN in the root tips for 24 h. However, cells examined at 24 h post-exposure showed concentration-wise recovery in all the endpoints. The data revealed that Indian mustard could be used as a potential accumulator of Cd, Cr, Cu, and Pb due to a good tolerance mechanisms provided by combined/concerted action of NP-SH, GSH, and PCs. Also, exposure of Cd can cause genotoxic effects in B. juncea L. through chromosomal mutations, MA, and MN formation.     

Phytoextraction of soil cadmium and zinc by microbes-inoculated Indian mustard (Brassica juncea)

Abstract

A pot experiment was carried out to evaluate the effect of Pseudomonas fluorescens and Trichoderma harzianum inoculation on the uptake of zinc (Zn) and cadmium (Cd) by Indian mustard (Brassica juncea) from the soil having three different concentrations of Zn (300, 600, 900 mg/kg) and Cd (5, 10, 15 mg/kg) separately. Microbial inoculation resulted in significantly better plant growth, available metal content and their uptake than control (without microbes). Available Zn was enhanced, ca.1.6- and 1.4-fold and Cd ca. 2.5- and 1.8-fold, by P. fluorescens and T. harzianum, respectively. P. fluorescens resulted in an increase in Zn uptake by 113.9, 51.9 and 58.4% and T. harzianum by 42.6, 32.1 and 33.9% over control from soils having 300, 600 and 900 mg Zn, respectively, while of the corresponding results for Cd were 110.2, 48.9 and 58.1% with P. fluorescens and 42.6, 30.9 and 33.4% with T. harzianum from soil having 5, 10 and 15 mg Cd, respectively, after 90 days of treatment. In general the rate of metal uptake was higher during the initial 30 days and declined later.     

Calcium supplementation modulates arsenic-induced alterations and augments arsenic accumulation in callus cultures of Indian mustard (Brassica juncea (L.) Czern.)

In the present study, the effect of arsenate (AsV) exposure either alone or in combination with calcium (Ca) was investigated in callus cultures of Brassica juncea (L.) Czern. cv. Pusa Bold grown for a period up to 24 h. The AsV?(250 μM) + Ca (10 mM) treatment resulted in a significantly higher level of As (464 μg g^?1 dry weight (DW)) than AsV without Ca (167 μg g^?1 DW) treatment at 24 h. Furthermore, AsV + Ca-treated calli had a higher percent of AsIII (24–47%) than calli subjected to AsV treatment (12–14%). Despite this, AsV + Ca-treated calli did not show any signs of hydrogen peroxide (H₂O₂) accumulation or cell death upon in vivo staining, while AsV-exposed calli had increased H₂O₂, shrinkage of cytoplasmic contents, and cell death. Thus, AsV treatment induced oxidative stress, which in turn elicited a response of antioxidant enzymes and metabolites as compared with control and AsV + Ca treatment. The positive effects of Ca supplementation were also correlated to an increase in thiolic constituents', viz., cysteine, reduced glutathione, and glutathione reductase in AsV + Ca than in AsV treatment. An analysis of selected signaling related genes, e.g., mitogen-activated protein kinases (MAPK3 and MAPK6) and jasmonate ZIM-domain (JAZ3) suggested that AsV and AsV + Ca followed variable pathways to sense and signal the As stress. In AsV-alone treatment, jasmonate signaling was seemingly activated, while MAPK3 was not involved. In contrast, AsV + Ca treatment appeared to specifically inhibit jasmonate signaling and activate MAPK3. In conclusion, Ca supplementation may hold promise for achieving increased As accumulation in plants without compromising their tolerance.     

Thiourea mediates alleviation of UV-B stress-induced damage in the Indian mustard (Brassica juncea L.)

Abstract

UV-B radiation (280–320 nm) as an environmental stress has damaging effect on plants and its overexposure can potentially interfere with growth and development. The effect of thiourea (TU) on UV-B stress tolerance of 10-day-old Brassica juncea seedlings subjected to supplementary UV-B for 5 days was investigated. An increase in the UV-B absorbing compounds anthocyanin, flavonoids and phenolic compounds was observed in UV-B + TU treated seedlings as compared to that of control. The enhanced synthesis of UV-B screening compounds resulted in lesser damage to chlorophyll and also gain in fresh weight and dry weight in UV-B + TU as compared to UV-B alone treatment. The enzymatic activities of guaiacol peroxidase (GPX) and superoxide dismutase (SOD) also increased in UV-B + TU. The expression profiling of phenylalanine ammonia lyase (pal) and chalcone synthase (chs) indicated an upregulation under UV-B + TU treatment, compared to that of control, suggesting that TU treatment ensured an early and efficient induction of flavonoid biosynthetic pathway. The results indicate that TU helps in ameliorating the damaging effects of UV-B stress by efficiently maintaining the antioxidant status and attenuating the penetration of the UV-B.     

Alternaria incidence in some alloplasmic lines of Indian mustard (Brassica juncea (L.) Coss.)

Summary The cytoplasmic substitution lines of Brassica juncea (L.) Coss were evaluated for their field resistance to Alternaria blight (Alternaria brassicae). The euplasmic B. juncea cv. RLM 198 had a mesothetic reaction while alloplasmic B. juncea lines with cytoplasms of B. campestris, B. chinensis, and B. japonica were highly susceptible. B. nigra cytoplasm did not have any effect on the disease reaction of the B. juncea genome. However, the alloplasmic lines with the cytoplasm of B. napus and B. carinata revealed a comparatively higher degree of resistance. The study underlined the utility of cytoplasmic manipulations in modifying the phenotypic expression of nuclear genes.     

Influence of systemic insecticides on lipid biosynthesis in seeds of Indian mustard (Brassica juncea L.)

The systemic insecticides namely phorate (Thimet 10 G) oxydemeton methyl (metasystox 25 EC) and dimethoate (Rogor 30 EC) decreased oil content in the developing seeds of Indian mustard (Brassica juncea L.) but showed an increase in the mature seeds. The inhibitory effect in the developing seed was accompanied by an increase in soluble sugars and a corresponding decrease in malate dehydrogenase and G6P dehydrogenase activity. The relative proportion of triacylglycerols and glycolipids decreased significantly while that of phospholipids and free fatty acids increased in the developing seeds. In the mature seeds, the proportion of triacylglycerols did not change appreciably from that in control. The erucic acid synthesis which was less at 10 and 20 days after fertilization (DAF) increased at 30 DAF with oxydemeton methyl and dimethoate; phorate was ineffective. In mature seeds, the proportion of erucic acid increased at the cost of linoleic and linolenic acids. All the insecticides appreciably decreased the rate of [1-¹⁴C]acetate incorporation into lipids both in vivo and in vitro experiments. In the in vivo experiment, the synthesis of polar lipids was enhanced at 10 and 20 DAF, and the higher doses of oxydemeton methyl and dimethoate at 30 DAF. On the other hand, the ¹⁴C-incorporation into triacylglycerols showed an opposite trend to that of polar lipids. In the in vitro experiment, oxydemeton methyl and dimethoate enhanced the synthesis of polar lipids at 10 and 20 DAF while these inhibited it at 30 DAF. The synthesis of triacylglycerols was inhibited by the use of these insecticides.     

Uptake of phosphorus and lead by Brassica juncea and Medicago sativa from chloropyromorphite

In situ remediation of lead (Pb)-contaminated soils via phosphate amendments has been extensively used to immobilize Pb as pyromorphite. However, in phosphorus (P) deficient soils, plants may develop extensive root systems to access P in any P-containing minerals, thereby affecting the stability of Pb5 (PO4)3Cl (Chloropyromorphite; CP). We grew Brassica juncea and Medicago sativa in sand culture to evaluate the stability of CP in the presence or absence of hydroxyapatite (HA) as P source. Treatments (per kilogram of sand) watered with P-nutrient solution were control [PC0, (without CP)], 1, and 5 g Pb as CP [PC1, and PC5] and 0.45 g P as HA (PA), and those of watered with P-free nutrient solution were 1 and 5 g Pb as CP [NC1 and NC5], 5 g Pb as CP plus 0.45 g P as HA [NAC5], and 0.45 g P as HA [NA]. Plants in NC1 and NC5 treatments showed stunted growth and reductions in shoot elongation and leaf size. Among CP treated pots, the highest shoot Pb uptake was observed in NAC5 treatment. The results suggested that Pb accumulation and translocation in the plants was markedly higher in P-sufficient conditions than in P-deficient conditions.     

Production of nutritionally desirable fatty acids in seed oil of Indian mustard (Brassica juncea L.) by metabolic engineering

Development of a designer oilseed crop with improved yield attributes and enhanced nutritional quality for the benefits of mankind and animal husbandry is now achievable with the combination of genetic engineering and plant breeding. In spite of their immense importance, the fatty acid profiles of most oilseed crops are imbalanced that necessitate the use of metabolic engineering strategies to overcome the various shortfalls in order to improve the nutritional quality of these edible oils. Indian mustard (Brassica juncea L.), being one of the important oilseed crops in Indian subcontinent naturally contains ~50 % nutritionally undesirable very long chain unsaturated fatty acids (VLCUFAs), e.g. erucic acid (C_22:1). For the purpose of nutritional improvement of B. juncea seed oil, several metabolic engineering strategies have been employed to divert the carbon flux from the production of VLCUFAs to other important fatty acids. Stearic acid, being a saturated but nutritionally neutral fatty acid, is naturally inadequate in most of the conventional oil seeds. Due to its neutral effect on consumer’s health and as an important industrial ingredient, increased in planta production of stearic acid in the seed oil not only helps in reduction of production cost but also lessens the trans fatty acid production during commercial hydrogenation process. In this review metabolic engineering strategies to minimize the VLCUFAs along with increased production of stearic acid in the seed oil of B. juncea are discussed, so that further breeding attempts can be made to improve the nutritionally desirable fatty acid profile in the suitable cultivars of this important oilseed crop.     

A RFLP-based linkage map of mustard [Brassica juncea (L.) Czern. and Coss.]

 A genetic linkage map of Brassica juncea was constructed based on restriction fragment length polymorphism (RFLP) detected by anonymous cDNA markers from B. napus, using a segregating F₁-derived doubled haploid (DH) progeny from a cross between a canola-quality mustard line (J90-4317) and a high-oil-content mustard line (J90-2733). The RFLP probes consisted of 229 cDNA probes from B. napus and a B. napus tandem repeat sequence, RDA2. The map consisted of 343 marker loci arranged in 18 major linkage groups plus five small segments with two to five marker loci, covering a total map distance of 2073 cM. Twenty-four percent of the markers were dominant in nature. Sixty-two percent of the marker loci were duplicated, and the majority were involved in inter-linkage group duplications, illustrating that complex duplications and subsequent rearrangements occurred after allopolyploidy. Deviation from the Mendelian segregation ratio for a DH population was observed for 27% of the markers. Two-thirds of these markers with a skewed segregation were clustered in 6 linkage groups and two unassigned segments. The overall average marker interval of the B. juncea map reported here was 6.6 cM, which would provide a marker density satisfactory for efficient use of the map in breeding applications, such as tagging of important agronomic traits and marker-assisted selection. Received: 14 May 1996 / Accepted: 11 October 1996     

Mapping of QTLs for oil content and fatty acid composition in Indian mustard [Brassica juncea (L.) Czern. and Coss.]

An AFLP linkage map of Brassica juncea (L.) Czern and Coss was constructed using 88 recombinant inbred lines (RILs) from a cross between an Indian cultivar ‘Varuna’ and an accession from Poland ‘BEC-144’. The map included 91 AFLP markers organized on 19 linkage groups covering a total map distance of 1679.1 cM. A total of 14 QTLs were detected for oil content (2 QTLs), erucic acid (2 QTLs), eicosenoic acid (2 QTLs), linolenic acid (3 QTLs), linoleic acid (3 QTLs) and palmitic acid (2 QTLs). A specific genomic region on LG2 was associated with contents of three fatty acids: erucic acid, eicosenoic acid and linoleic acid. Some of the markers showed absolute linkage with the QTLs associated with the levels of linolenic acid, linoleic acid and oil content. These markers may be used for improvement of fatty acid profile of B. juncea.     

Linked epistasis for six quantitative traits in Indian mustard (Brassica juncea (L.) Czern & Coss)

Summary Gene effects, and interactions, and associations between days-to-flower initiation and maturity, number of secondary branches and siliquae per plant, and 1,000-seed weight and yield per plant were studied in a cross of Indian mustard (Brassica juncea (L.) Czern & Coss) using the parents and F₁, F₂, F₃, B₁, B₂, B₁₁, B₁₂, B₂₁, B₂₂, B_1S, B_2S, B₁F₁, B₂F₁, B_1bip, B_2bip, F₂P₁, F₂F₁, and F_2bip generations. A linked digenic model was adequate for all characters studied. According to this model, the main effects, additive and interactions between linked pairs of genes, were present in varying proportions for days-to-flower initiation and maturity and number of siliquae per plant. The contribution of linked epistatic effects, however, was much greater than that of additive effects. Dominance effects contributed significantly to the inheritance of days-to-flower initiation. Duplicate epistasis was observed for all traits except 1,000-seed weight where epistasis was of the complementary type. A complete association among the genes of similar effect (increasing or decreasing) was observed for number of secondary branches and siliquae, and yield per plant. Coupling phase linkage was observed for days-to-flower initiation whereas repulsion phase linkage was observed for daysto-maturity and 1,000-seed weight.     

Molecular cloning and characterization of five annexin genes from Indian mustard (Brassica juncea L. Czern and Coss)

Plant annexins constitute a multigene family having suggested roles in a variety of cellular processes including stress responses. We have isolated and characterized five different cDNAs of mustard, Brassica juncea (AnnBj1, AnnBj2, AnnBj3, AnnBj6 and AnnBj7) encoding annexin proteins using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins are ～36.0 kDa with acidic pIs. At the amino acid level, they share high sequence similarity with each other and with annexins from higher plants. Phylogenetic analysis revealed their evolutionary relationship with corresponding orthologous sequences in Arabidopsis and deduced proteins in various plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. The expression patterns of these genes also showed regulation by various stress conditions such as exposure to signaling molecules, salinity and oxidative stress and wounding. Additionally, the in silico promoter analysis (of AnnBj1, AnnBj2 and AnnBj3) showed the presence of different cis-responsive elements that could respond to various stress conditions. These results indicate that AnnBj genes may play important roles in adaptation of plants to various environmental stresses.     

Selection of genetically transformed Brassica juncea L. cv. Varuna (Indian mustard) based on Positech system

The use of antibiotic and herbicide resistance based negative selection in plant transformation experiments remains a major impediment in the acceptance of transgenic crops. To overcome this, Positech selection system involving the use of phosphomannose isomerase (pmi) gene from Escherichia coli and mannose as selection agent was exploited for the selection of transgenic Brassica juncea L. cv. Varuna. The transgenic plants were generated by transformation with Agrobacterium tumefaciens harbouring the pmi gene driven by a constitutive Cestrum leaf curling viral promoter. Supplementing the selection medium with 0.09 gl^-1 mannose and 5 gl^-1 glucose provided the optimal condition for the selection of transformed explants. Stable integration and expression of pmi gene was confirmed by Southern and northern blot analysis, respectively. Our results show that the pmi gene driven by the constitutive Cestrum leaf curling viral promoter can be successfully used for positive selection in transformation of B.juncea, an important agronomic oil-seed crop, and that a combination of mannose and glucose rather than mannose alone is more suitable for the selection. To the best of our knowledge, Positech system has not been used so far in transformation of Brassica juncea.     

Phytoremediation of arsenic and lead in contaminated soil using Chinese brake ferns (Pteris vittata) and Indian mustard (Brassica juncea)

Field and greenhouse experiments were performed to assess the performance of phytoremediation of arsenic and lead from contaminated soil at an EPA Superfund site (Barber Orchard). Chinese Brake ferns (Pteris vittata) were used to extract arsenic. On average, fern shoot arsenic concentrations were as high as 20 times the soil arsenic concentrations under field conditions. It was estimated that 8 years would be required to reduce the acid-extractable portion of soil arsenic to safe levels (40 mg/kg). The effect of soil pH on arsenic extraction was also investigated. Results indicate that increasing soil pH may improve arsenic removal. Indian mustard plants (Brassica juncea) were used under greenhouse conditions to phytoextract soil lead. EDTA was applied to soil and was found to improve lead extraction. When the EDTA concentration was 10 mmol EDTA/kg soil in soil containing 338 mg Pb/kg soil, mustard plants extracted approximately 32 mg of lead. In conclusion, phytoremediation would be a suitable alternative to conventional remediation techniques, especially for soils that do not require immediate remediation.     

Distinct uptake of tellurate from selenate in a selenium accumulator, Indian mustard (Brassica juncea)

Tellurium (Te) is widely used in industry because of its unique chemical and physical properties, and has recently become a part of everyday life as a component of phase-change optical magnetic disks. However, the recovery of Te from the environment has not been discussed yet. In this regard, we evaluated the potential use of Indian mustard (Brassica juncea), a selenium (Se) accumulator, for the phytoremediation of Te. The Indian mustard plant was exposed to selenate and tellurate and the concentrations of Se and Te and the chemical species in the plant were determined. The Indian mustard plant accumulated less Te than Se, and the amount of Te accumulated in the plant was approximately 1/69 of that of Se. Although the incorporation of selenate was reduced by increasing sulfate concentration in the medium, the incorporation of Te was not affected by it, suggesting that this plant was able to discriminate tellurate from selenate in the roots. Three Te species were detected in the plant. The major species was tellurate. The other two species were not identical to available Te standards and thus could not be identified. Consequently, the Indian mustard plant is inappropriate for the phytoremediation of Te because it can strictly distinguish tellurate from selenate.     

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.     

The value of certain plant parameters as an index for salt tolerance in Indian mustard (Brassica juncea L.)

In a micro-plot study of 6 cultivars of Indian mustard (Brassica juncea L.) it was observed that germination of seeds and seed yield per plot decreased linearly with rising salinity levels. On the contrary, seed yield per plant increased with the salinity until at a critical salinity level it fell abruptly. Compared to the cultivars of the sensitive group the tolerant ones exhibited a higher magnitude of reduction in number of leaf stomata per unit area (stomatal frequency of leaf) at the critical salinities (12 mmhos/cm and above) of irrigation water, whereas the decrease in leaf water potential (-leaf) was not so marked at the same salinity levels. Tolerant cultivars were also notable for having a wider K/Na ratio (1.63 and 1.28) and a higher accumulation of free proline (2.10 and 2.30 mole/g) at EC irrigation water of 12 and 16 mmhos/cm respectively. Reductions in yield at these salinities were rather lower being 27.0 and 79.8% respectively in tolerant cultivars compared with 59.0 and 94.2% respectively in sensitive ones.     

Nickel accumulation in rape shoots (Brassica napus L.) increased by putrescine

Application of exogenous putrescine (Put) increases nickel accumulation in rape shoots (Brassica napus), improving potential for phytoremediation of contaminated soils. Plants were grown within a growth chamber in water culture for five weeks, then 250-500 microM NiCl2 was added to rooting media. Within 5 days of treatment, damaging effects of nickel manifested as a reduction of root system size and a decrease of Cu and especially Fe content in young leaves. Spraying leaves of adult plants with Put markedly reduced toxic effects of Ni on root growth, enhanced leaf supply with Fe, and increased Ni content in young leaves by 2.5 times. Plant growth in medium with elevated levels of Ni stimulated accumulation of endogenous spermidine (Spd), spermine (Spm), and especially Put (by 4 times as compared with the control). Results suggest that Ni-induced accumulation of endogenous polyamines in rape leaves is caused by activation of long-distance metal transport within the plant and reduction of its toxicity due to Put chelating action.