Alleviating Drought Stress in Brassica juncea (L.) Czern & Coss. by Foliar Application of Biostimulants-Orthosilicic Acid and Seaweed Extract.

Agricultural productivity is negatively impacted by drought stress. Brassica is an important oilseed crop, and its productivity is often limited by drought. Biostimulants are known for their role in plant growth promotion, increased yields, and tolerance to environmental stresses. Silicon in its soluble form of orthosilicic acid (OSA) has been established to alleviate deteriorative effects of drought. Seaweed extract (SWE) also positively influence plant survival and provide dehydration tolerance under stressed environments. The present study was conducted to evaluate the efficacy of OSA and SWE on mitigating adverse effects of drought stress on Brassica genotype RH-725. Foliar application of OSA (2 ml/L and 4 ml/L) and SWE of Ascophyllum nodosum (3 ml/L and 4 ml/L) in vegetative stages in Brassica variety RH 725 under irrigated and rainfed condition revealed an increase in photosynthetic rate, stomatal conductance, transpirational rate, relative water content, water potential, osmotic potential, chlorophyll fluorescence, chlorophyll stability index, total soluble sugars, total protein content, and antioxidant enzyme activity; and a decrease in canopy temperature depression, proline, glycine-betaine, H2O2, and MDA content. Application of 2 ml/L OSA and 3 ml/L SWE at vegetative stage presented superior morpho-physiological and biochemical characteristics and higher yields. The findings of the present study will contribute to developing a sustainable cropping system by harnessing the benefits of OSA and seaweed extract as stress mitigators.

Plant age at the time of ozone exposure affects flowering patterns, biotic interactions and reproduction of wild mustard.

Exposure of plants to environmental stressors can modify their metabolism, interactions with other organisms and reproductive success. Tropospheric ozone is a source of plant stress. We investigated how an acute exposure to ozone at different times of plant development affects reproductive performance, as well as the flowering patterns and the interactions with pollinators and herbivores, of wild mustard plants. The number of open flowers was higher on plants exposed to ozone at earlier ages than on the respective controls, while plants exposed at later ages showed a tendency for decreased number of open flowers. The changes in the number of flowers provided a good explanation for the ozone-induced effects on reproductive performance and on pollinator visitation. Ozone exposure at earlier ages also led to either earlier or extended flowering periods. Moreover, ozone tended to increase herbivore abundance, with responses depending on herbivore taxa and the plant age at the time of ozone exposure. These results suggest that the effects of ozone exposure depend on the developmental stage of the plant, affecting the flowering patterns in different directions, with consequences for pollination and reproduction of annual crops and wild species.

Nitric oxide alters nitrogen metabolism and PIN gene expressions by playing protective role in arsenic challenged Brassica juncea L.

Plants have ability to adapt themselves through altering their growth process. In the present study, we examined exogenous application of nitric oxide (NO) on nitrogen metabolism and auxin (PIN) gene expression, and its possible role in alleviation of arsenic (As) toxicity in Brassica juncea seedlings. Seven days old hydroponically grown B. juncea seedlings were exposed to AsIII (150 µM), Sodium nitroprusside (NO donor, 100 µM), AsIII + SNP and control (without metal)for 48 h. Experimental results revealed that AsIII stress: enhanced the level of nitrite, NiR activity, NO3- and NH4+content as well as NADH-GOGAT activity; but GDH level decreased; enhanced content of amino acids; upregulated gene expression level of N metabolism and downregulated polar auxin transporter genes (PIN); inhibited plant growth and morphological parameters; increased MDA, H2O2, cysteine, proline content, enzymatic antioxidants (SOD, CAT, APX; GSH, TT, NPT); and decreased nutrient content. AsIII + SNP combination reduced the accumulation of As; improved growth; chlorophyll, protein and mineral nutrient content by scavenging ROS generation; maintained amino acids content; downregulated expression of N metabolism genes and upregulated expression of auxin transporter (PIN) genes . Additional biochemical data depicts reduction in the level of nitrogen related enzymatic activities, and other stress related parameters. Overall, this study provides an integrated view that exogenous SNP (NO donor) supplementation alleviated the inhibitory role of AsIII in B. juncea seedlings by altering nutrients, amino acids and auxin redistribution via expression of nitrogen and PIN gene profiling.

Floral plasticity: Herbivore-species-specific-induced changes in flower traits with contrasting effects on pollinator visitation.

Plant phenotypic plasticity in response to antagonists can affect other community members such as mutualists, conferring potential ecological costs associated with inducible plant defence. For flowering plants, induction of defences to deal with herbivores can lead to disruption of plant-pollinator interactions. Current knowledge on the full extent of herbivore-induced changes in flower traits is limited, and we know little about specificity of induction of flower traits and specificity of effect on flower visitors. We exposed flowering Brassica nigra plants to six insect herbivore species and recorded changes in flower traits (flower abundance, morphology, colour, volatile emission, nectar quantity, and pollen quantity and size) and the behaviour of two pollinating insects. Our results show that herbivory can affect multiple flower traits and pollinator behaviour. Most plastic floral traits were flower morphology, colour, the composition of the volatile blend, and nectar production. Herbivore-induced changes in flower traits resulted in positive, negative, or neutral effects on pollinator behaviour. Effects on flower traits and pollinator behaviour were herbivore species-specific. Flowers show extensive plasticity in response to antagonist herbivores, with contrasting effects on mutualist pollinators. Antagonists can potentially act as agents of selection on flower traits and plant reproduction via plant-mediated interactions with mutualists.

The effects of iprodione fungicide on survival, behavior, and brood development of honeybees (Apis mellifera L.) after one foliar application during flowering on mustard.

A semifield study to assess the effects of iprodione on honeybees at label use rates was conducted on a bloom mustard crop. The present study followed the Organisation for Economic Co-operation and Development guideline 75 tunnel test and consisted of 3 groups: the iprodione-treated group, the untreated control group, and the toxic reference item group. In addition to the tunnels used for biological assessments, a tunnel was set up in the treatment and control groups to determine the level of residues in flowers, nectar, and pollen. The major endpoints to assess the effects of the application of iprodione were mortality, flight intensity, behavior, condition of the colonies, and development of the brood. Residue analysis showed that honeybees were exposed to significant residues of iprodione. However, no adverse effects were observed on overall mortality, flight intensity, behavior, or brood development of honeybees compared to control. It is concluded that iprodione does not adversely affect the health of honeybees when applied in agriculture at commercially relevant rates in a worst-case exposure scenario. Environ Toxicol Chem 2018;37:3086-3094. © 2018 SETAC.

A quartet pollen phenotype identified in a population of Brassica interspecific hybrids shows incomplete penetrance and variable response to temperature.

Quartet pollen, where pollen grains remain attached to each other post-meiosis, is useful for tetrad analysis, crossover assessment and centromere mapping. We observed the quartet pollen phenotype for the first time in the agriculturally significant Brassica genus, in an experimental population of allohexaploid Brassica hybrids derived from the cross (Brassica napus × B. carinata) × B. juncea followed by two self-pollination generations. Quartet pollen production was assessed in 144 genotypes under glasshouse conditions, following which a set of 16 genotypes were selected to further investigate the effect of environment (warm: 25 °C and cold: 10 °C temperatures) on quartet pollen production in growth cabinets. Under glasshouse phenotyping conditions, only 92 out of 144 genotypes produced enough pollen to score: of these, 30 did not produce any observable quartet pollen, while 62 genotypes produced quartet pollen at varying frequencies. Quartet pollen production appeared quantitative and did not clearly fall into phenotypic or qualitative categories indicative of major gene expression. No consistent effect of temperature on quartet pollen production was identified, with some genotypes producing more and some producing less quartet pollen under different temperature treatments. The genetic heterogeneity and frequent pollen infertility of this population prevents strong conclusions being made. However, it is clear that the quartet phenotype in this Brassica population does not show complete penetrance and shows variable (likely genotype-specific) response to temperature stress. In future, identification of quartet phenotypes in Brassica would perhaps best be carried out via screening of diploid (e.g. B. rapa) TILLING populations.

Selenite-induced nitro-oxidative stress processes in Arabidopsis thaliana and Brassica juncea.

Extremes of selenium (Se) exert toxic effects on plants' physiological processes; although plant species tolerate Se differently. This study focuses on the effect of Se (0, 20, 50 or 100µM sodium selenite) on secondary nitro-oxidative stress processes mainly using in situ microscopic methods in non-accumulator Arabidopsis thaliana and secondary Se accumulator Brassica juncea. Relative Se tolerance or sensitivity of the species was evaluated based on growth parameters (fresh and dry weight, root growth) and cell viability. Besides, selenite-triggered cell wall modifications (pectin, callose) and stomatal regulations were determined for the first time. In case of Arabidopsis, relative selenite sensitivity was accompanied by decreased stomatal density and induced stomatal opening, callose accumulation, pronounced oxidative stress and moderate nitrosative modifications. In contrast, the selenite-treated, relatively tolerant Brassica juncea showed larger number of more opened stomata, pectin accumulation, moderate oxidative and intense nitrosative stress. These suggest that selenite tolerance or sensitivity is rather associated with oxidative processes than secondary nitrosative modifications in higher plants.

Castasterone and citric acid treatment restores photosynthetic attributes in Brassica juncea L. under Cd(II) toxicity.

Cadmium(II) toxicity is a serious environmental issue warranting effective measures for its mitigation. In the present study, ameliorative effects of a bioactive brassinosteroid, castasterone (CS) and low molecular weight organic acid, citric acid (CA) against the Cd(II) toxicity to Brassica juncea L. were evaluated. Seeds of B. juncea treated with CS (0, 0.01, 1 and 100nM) were sown in cadmium spiked soils (0 and 0.6mmolkg-1 soil). CA (0.6mmolkg-1soil) was added to soil one week after sowing seeds. Plants were harvested 30 days after sowing. Phytotoxicity induced by Cd(II) was evident from stunted growth of the plants, malondialdehyde accumulation, reduction in chlorophyll and carotenoid contents, and leaf gas exchange parameters. Cd(II) toxicity was effectively alleviated by seed soaking with CS (100nM) and/ or soil amendment with CA (0.6mMkg-1 soil). Relative gene expression of genes encoding for some of the key enzymes of pigment metabolism were also analysed. Expression of chlorophyllase (CHLASE) was reduced, while that of phytoene synthase (PSY), and chalcone synthase (CHS) genes were enhanced with CS and/or CA treatments with respect to plants treated with Cd(II) only. Cd also affected the activities of antioxidative enzymes. Plants responded to Cd(II) by accumulation of total sugars. CS (100nM) and CA treatments further enhanced the activities of these parameters and induced the contents of secondary plant pigments (flavonoids and anthocyanins) and proline. The results imply that seed treatment with CS and soil application with CA can effectively alleviate Cd(II) induced toxicity in B. juncea by strengthening its antioxidative defence system and enhancing compatible solute accumulation.

γ-aminobutyric acid (GABA) confers chromium stress tolerance in Brassica juncea L. by modulating the antioxidant defense and glyoxalase systems.

Chromium (Cr) toxicity is hazardous to the seed germination, growth, and development of plants. γ-aminobutyric acid (GABA) is a non-protein amino acid and is involved in stress tolerance in plants. To investigate the effects of GABA in alleviating Cr toxicity, we treated eight-d-old mustard (Brassica juncea L.) seedlings with Cr (0.15 and 0.3 mM K2CrO4, 5 days) alone and in combination with GABA (125 µM) in a semi-hydroponic medium. The roots and shoots of the seedlings accumulated Cr in a dose-dependent manner, which led to an increase in oxidative damage [lipid peroxidation; hydrogen peroxide (H2O2) content; superoxide (O2•-) generation; lipoxygenase (LOX) activity], methylglyoxal (MG) content, and disrupted antioxidant defense and glyoxalase systems. Chromium stress also reduced growth, leaf relative water content (RWC), and chlorophyll (chl) content but increased phytochelatin (PC) and proline (Pro) content. Furthermore, supplementing the Cr-treated seedlings with GABA reduced Cr uptake and upregulated the non-enzymatic antioxidants (ascorbate, AsA; glutathione, GSH) and the activities of the enzymatic antioxidants including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II), and finally reduced oxidative damage. Adding GABA also increased leaf RWC and chl content, decreased Pro and PC content, and restored plant growth. These findings shed light on the effect of GABA in improving the physiological mechanisms of mustard seedlings in response to Cr stress.

Growth response modulation by putrescine in Indian mustard Brassica juncea L. under multiple stress.

Plants, in general, are put to various kinds of stress, biotic and abiotic, both natural and manmade. Infestation by insect pests and diseases, and extreme conditions such as salinity, temperature, etc., as well as heavy metal contamination affect their growth performance. Here, we studied the impact of salinity and heavy metal pollution on the growth performance of Indian Mustard Brassica juncea L. and its amelioration by the diamine, putrescine, a known media supplement. We evaluated the putrescine (Put) modulation potential on multiple stress effect in 7-day old Indian mustard. The germination, seedlings length and photosynthetic pigments decline under salinity and metal (Cd/Pb) stress condition, alone or in combination, were checked by putrescine. The stress induced increase in root-shoot ratio, RNA and total amino acids content, as well as Na⁺/K⁺ ratio in leaf tissues were also comparatively less. The increased endogenous Cd/Pb accumulation in plants exposed to either metal further elevated under salinity was also found decelerated. However, the multiple stressed seedlings showed increase in glutathione content, which was further elevated with putrescine application. The increase in protein contents in leaf under single or combined stresses in the presence of putrescine could be a qualitative change. The differential changes in parameters examined here resulted in improved growth (> 10%) suggests stress mitigation by the putrescine up to an extent.

Difference in root K+ retention ability and reduced sensitivity of K+-permeable channels to reactive oxygen species confer differential salt tolerance in three Brassica species.

Brassica species are known to possess significant inter and intraspecies variability in salinity stress tolerance, but the cell-specific mechanisms conferring this difference remain elusive. In this work, the role and relative contribution of several key plasma membrane transporters to salinity stress tolerance were evaluated in three Brassica species (B. napus, B. juncea, and B. oleracea) using a range of electrophysiological assays. Initial root growth assay and viability staining revealed that B. napus was most tolerant amongst the three species, followed by B. juncea and B. oleracea At the mechanistic level, this difference was conferred by at least three complementary physiological mechanisms: (i) higher Na(+) extrusion ability from roots resulting from increased expression and activity of plasma membrane SOS1-like Na(+)/H(+) exchangers; (ii) better root K(+) retention ability resulting from stress-inducible activation of H(+)-ATPase and ability to maintain more negative membrane potential under saline conditions; and (iii) reduced sensitivity of B. napus root K(+)-permeable channels to reactive oxygen species (ROS). The last two mechanisms played the dominant role and conferred most of the differential salt sensitivity between species. Brassica napus plants were also more efficient in preventing the stress-induced increase in GORK transcript levels and up-regulation of expression of AKT1, HAK5, and HKT1 transporter genes. Taken together, our data provide the mechanistic explanation for differential salt stress sensitivity amongst these species and shed light on transcriptional and post-translational regulation of key ion transport systems involved in the maintenance of the root plasma membrane potential and cytosolic K/Na ratio as a key attribute for salt tolerance in Brassica species.

Alleviation of selenium toxicity in Brassica juncea L.: salicylic acid-mediated modulation in toxicity indicators, stress modulators, and sulfur-related gene transcripts.

The present work reveals the response of different doses of selenium (Se) and alleviating effect of salicylic acid (SA) on Se-stressed Brassica juncea seedlings. Selenium, a micronutrient, is essential for both humans and animals but is toxic at higher doses. Its beneficial role for the survival of plants, however, is still debatable. On the other hand, SA, a phenolic compound, is known to have specific responses under environmental stresses. Experiments were conducted using leaves of hydroponically grown seedlings of Pusa bold (PB) variety of B. juncea, treated with different concentrations of Se (50, 150, 300 µM) for 24- and 96-h exposure times. Increasing Se concentrations inhibited growth and, caused lipid peroxidation, concomitantly increased stress modulators (proline, cysteine, SOD, CAT) along with sulfur-related gene transcripts (LAST, APS, APR, GR, OASL, MT-2, PCS) in Brassica seedlings. On the basis of the above studied parameters, maximum inhibition in growth was observed at 300 µM Se after 96-h exposure time. Further, co-application of SA along with 300 µM Se helped to mitigate Se stress, as shown by improved levels of growth parameters, toxicity indicators (chlorophyll, protein, MDA), stress modulators (proline, cysteine, SOD, and CAT), and expression of sulfur-related genes as compared to Se-treated seedlings alone. Altogether, this study revealed that Se + SA combinations improved seedling morphology and were effective in alleviation of Se stress in PB variety of B. juncea.

Low level of selenium increases the efficacy of 24-epibrassinolide through altered physiological and biochemical traits of Brassica juncea plants.

This study was conducted to provide an insight into the effect of Se (through soil) induced changes in Brassica juncea plants in the presence and absence of 24-epibrassinolide (EBL; foliar). The Se treatments showed dual response, 10 µM of Se significantly increased growth, water relations, photosynthetic attributes along with carbonic anhydrase activity whereas its higher concentrations proved inhibitory in concentration dependent manner. The follow-up application of EBL to the Se stressed plants improved growth, water relations, photosynthesis and simultaneously enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase with the excess accumulation of proline. In addition to this, 10 µM Se increases the efficacy of 10(-8) M of EBL and both in combination showed maximum increase for the growth and photosynthetic traits of plants. On the other hand, the elevated level of antioxidant enzymes as well as proline could have conferred tolerance to the Se-stressed plants resulting in improved growth, water relations and photosynthesis.

Optimization of chelators to enhance uranium uptake from tailings for phytoremediation.

A greenhouse experiment was set up to investigate the ability of citric acid (CA), oxalic acid (OA), nitrilotriacetic acid (NTA) and EDTA for phytoremediation of uranium tailings by Indian mustard [Brassica juncea (L.) Czern. et Coss]. Uranium tailings were collected from Umra mining region and mixed with 75% of garden soil which yielded a 25:75 mixture. Prepared pots were divided into four sets and treated with following different concentrations - 0.1, 0.5, 2.5 and 12.5 mmol kg(-1) soil additions for each of the four chelators. Control pots which were not treated with chelators. Experiments were conducted in completely randomized block design with triplicates. The optimum concentrations of these chelators were found on the basis of biomass production, tolerance and accumulation potential. The data collected were expressed statistically. EDTA produced maximum growth depression whereas, minimum occurred in the case of NTA. Maximum U uptake (3.5-fold) in the roots occurred at 2.5 mmol of CA, while NTA proved to be the weakest for the same purpose. Severe toxicity in the form of reduced growth and plant death was recorded at 12.5 mmol of each chelator. Minimum growth inhibition produced by chelators occurred in NTA which was followed by OA, moderate in CA and maximum was traced in EDTA applications. Chelator strengthened U uptake in the present study follows the order: CA>EDTA>OA>NTA.

Reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in cytoplasmic male-sterile Brassica juncea.

The signal from organelle to nucleus, namely retrograde regulation of nuclear gene expression, was largely unknown. Due to the nuclear-cytoplasmic incompatibility in cytoplasmic male-sterile (CMS) plants, we employed CMS Brassica juncea to investigate the retrograde regulation of nuclear gene expression in this study. We studied how reduced BjRCE1 gene expression caused by the nuclear-cytoplasmic incompatibility altered the auxin response in CMS of B. juncea. We isolated the BjRCE1 gene that was located in the nucleus from B. juncea. Over-expression of BjRCE1 enhanced auxin response in transgenic Arabidopsis. The expression of BjRCE1 was significantly reduced in CMS compared with its maintainer fertile (MF) line of B. juncea. There were fewer lateral roots in CMS than MF under normal and treatment of indole-3-acetic acid (IAA) conditions. Expression patterns of several auxin-related genes together with their phenotypes indicated a reduced auxin response in CMS compared to MF. The phenotypes of auxin response and auxin-related gene expression pattern could be mimicked by inhibiting mitochondrial function in MF. Taken together, we proposed reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in CMS B. juncea. This may be an important mechanism of retrograde regulation of nuclear gene expression in plants.

Assessment of the interactive effects of ambient O3 and NPK levels on two tropical mustard varieties (Brassica campestris L.) using open-top chambers.

Rising O(3) concentrations in agricultural areas have been identified as a significant threat to crop production in Asia including India. The present work reports the results of a field study conducted to assess the usefulness of higher than recommended NPK dose in modifying the physiological, growth, yield, and seed quality responses of two mustard (Brassica campestris L. var. Vardan and Aashirwad) varieties under ambient ozone level at a rural site of India, using open-top chambers. Twelve hourly mean O(3) concentrations ranged between 27.7 and 59.04 ppb during the growth period. Plants in nonfiltered chambers (NFCs) showed reductions in photosynthetic rate, stomatal conductance, and growth parameters compared to the plants in filtered chambers (FCs), but reductions were of lower magnitude at 1.5 times recommended dose of NPK (1.5 RNPK) compared to recommended (RNPK). Yield and seed quality reduced significantly in plants of NFCs compared to FCs at RNPK, but no significant differences were recorded at 1.5 RNPK. There were higher N uptake and N uptake efficiency of plants in FCs compared to NFCs. Nitrogen utilization efficiency increased in Vardan, but decreased in Aashirwad in NFCs compared to FCs suggesting higher capability of N acquisition and utilization under ambient O(3), which led to a less pronounced reduction in the yield of the former than the latter variety. The differential nitrogen utilization efficiency in these varieties may be potentially used as measure of sensitivity characteristics in breeding programs for yield improvement in mustard under the present trend of increase in O(3) concentrations.

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development.

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.

Effects of selenium accumulation on reproductive functions in Brassica juncea and Stanleya pinnata.

Selenium (Se) is an essential micronutrient for many organisms, but is also a toxin and environmental pollutant at elevated levels. Due to its chemical similarity to sulphur, most plants readily take up and assimilate Se. Se accumulators such as Brassica juncea can accumulate Se between 0.01% and 0.1% of dry weight (DW), and Se hyperaccumulators such as Stanleya pinnata (Brassicaeae) contain between 0.1% and 1.5% DW of Se. While Se accumulation offers the plant a variety of ecological benefits, particularly protection from herbivory, its potential costs are still unexplored. This study examines the effects of plant Se levels on reproductive functions. In B. juncea, Se concentrations >0.05-0.1% caused decreases in biomass, pollen germination, individual seed and total seed weight, number of seeds produced, and seed germination. In S. pinnata there was no negative effect of increased Se concentration on pollen germination. In cross-pollination of B. juncea plants with different Se levels, both the maternal and paternal Se level affected reproduction, but the maternal Se concentration had the most pronounced effect. Interestingly, high-Se maternal plants were most efficiently pollinated by Se-treated paternal plants. These data provide novel insights into the potential reproductive costs of Se accumulation, interactive effects of Se in pollen grains and in the pistil, and the apparent evolution of physiological tolerance mechanisms in hyperaccumulators to avoid reproductive repercussions.

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials.

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC(3)) and selfed (S(3)) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC(3) plants (originated from somatic hybrids 3, 4, 10) with 2-8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.

Analysis of genetic diversity in cytoplasmic male sterility, and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee).

In our previous study, we bred a stable cytoplasmic male sterility (CMS) line of tuber mustard by using distant hybridization and subsequent backcrosses. In this CMS plants, all floral organs are normal except the anthers, which are transformed into petals or tubular structures. Recently, 2 mitochondrial genes-atpA and orf220-that are distinctively present in the CMS line of tuber mustard were cloned and partially characterized. In our study of genetic diversity analysis of CMS, 7 species of Brassica and Raphanus crops, which included 5 CMS lines and their respective maintainer lines, were used to compare the constitution of protein-coding genes in the mitochondrial genomes. In 4 of the 43 mitochondrial genes, namely, atpA, orf220, orf256, and orf305/orf324, polymorphisms were detected among the tuber mustard CMS line and its maintainer line. The results of a cluster analysis indicate that petaloid CMS phenotype of tuber mustard is a novel CMS type and is nearer to the nap CMS in Brassica napus at the phylogenetic level. The results of individual amplifications of these genes indicate the presence of 4 sequence-characterized amplified region (SCAR) markers, which enable rapid and reliable identification of this CMS. Expressions of the orf220 and orf256 genes were detected only in the CMS line, while expression of the orf305 gene was detected in the maintainer line. The different expression patterns of different mitochondrial-specific marker genes indicate that the quantity of mitochondrial proteins is differentially regulated during organ/tissue development in tuber mustard. The results of this study suggest that the above mentioned 4 mitochondrial genes are associated with the petaloid CMS phenotype in tuber mustard.