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During mustard aphid attack, a huge number of defence proteins’ expression was observed in Indian mustard cultivars cv. Ashirwad and cv. Pusa Mahak. The expression level of those proteins directly correlated with the aphid tolerance level of mustard cultivars. The similar expression of defence proteins was observed in a well-known aphid tolerant wild crucifer Rorippa indica, where a much higher protein expression level was noted. During the study, one Pentatricopeptide repeat containing protein, F26A19.13 was identified as one of the most important defence protein as it directly blocks the aphid salivary amylase’s function in the plant–insect defence cross-talk.

Increasing soil salinity is a mounting concern for agriculture. While economically important Brassica oilseed crops are moderately salt tolerant, there is significant variation at the salt tolerance level among the varieties. Hence analysing the salt tolerance level of individual varieties is important for taking stock of the potential salt tolerant ones besides getting a better understanding about the underlying salt tolerance strategies. In this study we aimed for the first time to estimate the salt tolerance level of B-85 variety of B. juncea by physiological and biochemical analysis in response to salt stress under tissue culture condition. The overall analysis revealed a high strength of 200 mM of NaCl as the lethal dose 50 for B-85 germination and seedling growth. The salt induced increase in Na+/K+ ratio was higher in roots than shoots suggesting the presence of active regulation to protect the sensitive photosynthetic structures which indeed manifested protection well up to 200 mM of NaCl stress, as total chlorophyll content showed significant reduction only at 300 mM NaCl stress level. In this context, significant increase in the content of multipurpose osmolyte glycine betaine at 200 mM of NaCl stress, suggests its probable regulatory role in the maintenance of Na+ and K+ homeostasis, photosynthesis and overall growth of B. juncea B-85 variety under salt stress.

Edible mushrooms are being used as food supplement worldwide for its nutritional attributes. Crop improvement through molecular breeding is adopted in mushroom for different significant targets as well. Thus the molecular genetic analysis is prerequisite and had been carried out for last few decades. Lentinula edodes is a popular shiitake mushroom having medicinal properties and high volume of food value and consume in mainly Asian countries. Genetic studies of L. edodes already been done due to its popularity in breeding programme too. Here, the NADH complex subunit is chosen first time in mushroom to standardize its amplification conditions and sequence analysis. Through the nad1 gene is a conserved region, we tried to give an account that it can be used as genetic marker in various studies in future. However, we have successfully standardized the PCR conditions and showed the nucleotide similarity of L. edodes with its mitochondrial genome. The amplified gene showed 378 bp in size and 99% similarity in this study.

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestion in a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24oC with shaking 60 rpm for 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed 31.60±9.31% regeneration on specific medium and 77.12±2.72% viability by FDA test. Four ISSR primers were used in this study with a total of 27 reproducible bands with mean value 6.75 and they showed similar banding pattern in all with zero percent polymorphism ranged from 280 bp - 2700 bp. The amplified rRNA-ITS gene showed ~ 600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.

Lipaphis erysimi Kaltenbach is one of the economically important pests of Indian mustard Brassica juncea. Plant defense against these pests include impairing digestive processes in the inse...

We have previously established that RiHSPRO2, a nematode resistance protein-like homolog from wild crucifer Rorippa indica (L.) Hiern is a potent candidate to control mustard aphid Lipaphis erysimi. The present study further exploits this protein through structure prediction, biosafety assessment and transgenic study. The RiHSPRO2 protein showed an abundance of alpha helices intervened with loops in the homology-based three-dimensional model. No allergenic moiety was found in its amino acid sequence based on homology search in Protein Data Base. The secondary structure of RiHSPRO2 was unstable at temperatures above 50 °C. In vitro pepsin digestion assay revealed the protein to be digested in pepsin supplemented Simulated Gastric Fluid (SGF) within 2 minutes. The protein is proved to be biologically safe as per the FAO/WHO guidelines. An efficient Agrobacterium-mediated Brassica juncea transformation involving direct organogenesis with mean transformation frequency of 5.06 ± 0.28% is reported. RiHSPRO2, under the influence of constitutive promoter CaMV35S, was transformed into susceptible B. juncea cv. B85. Southern hybridization confirmed stable integration of the transgene and Western blotting confirmed consistent expression of RiHSPRO2 in the genetically modified Brassica lines. The transgene segregated following Mendelian 3:1 ratio in the successive generation. Detached leaf aphid bioassay and in planta aphid bioassay in transgenic B. juncea lines revealed a reduction in aphid survivability by 45% and a decrease in aphid fecundity by 45.6%.

Two edible mushrooms Calocybe indica and Pleurotus sajor-caju were chosen as parent strains in this study to approach the concept of hybridization through the protoplast fusion technique. Protoplast fusion in presence of polyethylene glycol (PEG) was conducted between the parent strains and by further double selection screening method, six somatic hybrid lines were developed. Those fruit bodies of the hybrid lines showed phenotypic resemblance with Pleurotus sajor-caju when grown on paddy straw under favorable conditions. The hybridity of the newly developed somatic hybrid strains was established by barrage reaction, morphological traits, fruitbody parameter and, inter single sequence repeat (ISSR) profiling. One-way analysis of variance (ANOVA) was used for the analysis of phenotypic data of hybrid lines and parents. Five ISSR primers were used to generate 51 amplified DNA fragments ranged between 250 and 3000 bp in size in six hybrids and two parents with 90.19% polymorphism. Some of the hybrids contain some non-parental bands which indicate that recombination might happen in the hybrid genome hence confirming the hybridity of newly developed strains. The dendrogram was created using the Average Linkage (Between Groups) method based on ISSR profiling and genetic distance between parent-hybrids and hybrid-hybrid was analyzed by Jaccard's proximity matrix. A definite improvement in nutritional properties and biological activity was observed in the study. Due to ease in their cultivation, it can play a significant role in the rural economic development.

Nematode resistance protein HSPRO2 of Arabidopsis thaliana has important roles in defence response to the bacterium (Pseudomonas syringae) and shows response against oxidative stress and salicylic acid. We have explored the gene encoding it from Rorippa indica (L.) Hiern, a wild relative of cultivated crucifers experimentally challenged with mustard aphid Lipaphis erysimi. The gene seems to be a strong candidate for aphid tolerance in cultivated mustards. Presently we are reporting the results of a time-course quantitative relative expression analysis of HSPRO2 of R. indica where we have observed ~ tenfold increase in its expression in R. indica at 12 hours post infestation with L. erysimi. We have also isolated the full-length gene (1314 bp) by both 5′ and 3′ RACE (Rapid Amplification of cDNA Ends). Genome walking experiment identified the promoter sequence. Expression, purification and characterisation of RiHSPRO2 showed that it encodes for a 437 amino acid peptide. The 66 kDa recombinant RiHSPRO2 protein was purified for studying its efficacy against L. erysimi in an artificial diet-based insect bioassay that revealed LC50 (Lethal Concentration 50) values of RiHSPRO2 significantly within limits against L. erysimi. The present study might have a significant implication in future towards aphid management program of Brassica juncea through the development of aphid-tolerant transgenic plants.

Mustard aphids are a serious problem for Brassica oilseed in India causing up to 90% of the crop damage. It was hypothesized that Aphids migrate into the Indo Gangetic plain (IGP) from hilly regions of every year. Exact source and migration pattern of this pest is unknown till date. During their long range migration they infested various places over IGP, which fall on their way of migration. The wind, blown from the hilly regions helps aphids to migrate. Meteorological parameters play a crucial role in this migration of aphids. In this study, we have done the 24 hours air-mass backward trajectory at 100 m above ground level (agl) to detect the source regions of mustard aphids. We have found that mainly Western Himalayan hilly regions act as the source of mustard aphids for IGPs. The dependence upon the micro-meteorological parameters and population dynamics are analyzed and discussed elaborately in this work. In this study, we have proposed the ‘Hop and Fly’ behavior of mustard aphid and further discussed how this migrating behavior could help us to reduce the yield loss of Brassica.

Six fruit body derived lines (pfle FB) generated from six fruiting pfle somatic hybrid mushroom lines showed genetic diversity analysed by fruit body morphology and inter simple sequence repeat (ISSR) markers. Stipe length, pelius diameter and bioefficiency % (BE%) of all the strains showed variations between each other with respect to Pleurotus florida parent. Hybrid pfle 1v and pfle 1q showed the highest value of stipe length and pelius diameter, respectively, compared with parent P. florida. Four ISSR primers amplified a total of 47 reproducible fragments with 82.9% polymorphism in which primer ISSR-03 produced the highest number of amplicons. Unweighted pair group method with arithmetic mean (UPGMA) based dendrogram exhibited two major groups in which hybrids pfle 1r and pfle 1q showed genetical closeness to parents P. florida and Lentinula edodes, respectively. Tissue culture generated line from fruit body of pfle 1r hybrid showed maximum BE% compared with the other hybrids and P. florida parent. For identification of this line, a pair of hybrid strain-specific SCAR marker (RFB2F and RFB2R) was developed based on an unique 813 bp RAPD amplicon.

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestionin a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24ºC, 60 rpm and 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed an average yield of 1.2 × 107 cells/gm tissue with 31.60 ± 9.31% regeneration efficiency on specific medium and 77.12 ± 2.72% viability by FDA test. Four ISSR primers were used in this study resulting a total of 27 reproducible bands with mean value of 6.75. They showed similar banding pattern in all the lines with zero percent polymorphism ranged from 280 bp–2700 bp. The amplified rRNA-ITS gene showed ~600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.

Establishment of a suitable regeneration protocol is a pre-requisite to carry out transformation study in Sesamum indicum L. (sesame). In this paper, different parameters of regeneration were standardised to develop an efficient protocol for in vitro plant regeneration via direct adventitious shoot organogenesis using de-embryonated cotyledons of sesame as explants. Among the various treatments of MS medium supplemented with 6-benzylaminopurine, thidiazuron and indole-3-acetic acid, maximum regeneration frequency (25.93 ± 2.21%) was obtained in BTI 4 medium (MS supplemented with 33.33 µM BAP with 2.85 µM IAA) within 6 weeks of culture. Regeneration frequency increased further (50.37 ± 2.49%) by fortifying BTI 4 with 29.43 µM silver nitrate (AG 3 medium). Pre-culture of cotyledon explants in AB 3 medium (AG 3 supplemented with 3.78 µM abscisic acid) for 14 days followed by sub-culture in AG 3 medium further improved the regeneration frequency (68.15 ± 2.68%). The highest rate of shoot regeneration (94.82 ± 1.34%) was obtained by pre-culturing 4-day-old cotyledon in a vertical position in AB 3 medium for 14 days and sub-culturing in AG 3 medium for 4 weeks. Regenerated shoots proliferated in MS medium supplemented with 4.44 μM BAP and 1.44 μM gibberelic acid (GA3). The highest frequency (65.33 ± 3.78%) of root induction was achieved by culturing the elongated shoots in MS medium supplemented with 2.69 μM α-naphthalene acetic acid (NAA) for 6 weeks. Rooted plants were acclimatised in soilrite and transferred to soil after 6–8 weeks. The rate of acclimatisation of plants was 76%.

Nine pfle somatic hybrids and their parental strains Pleurotus florida and Lentinula edodes were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of ribosomal RNA internal transcribed spacer (rRNA-ITS) regions. The ITS1-5.8S-ITS2 region was amplified by primer pair ITS1 and ITS4. Restriction digestion of the amplified ITS1-5.8S-ITS2 region with AluI, HinfI, HpaII and HaeIII generated a total of 81 fragments in which HaeIII generated the maximum of 26 restriction fragments and HpaII generated the minimum of 14 fragments for all 11 strains studied. All the pfle hybrids and parents were highly polymorphic in fragment sizes as revealed from the RFLP profiles. The pfle hybrids and parental ITS1-5.8S-ITS2 sequences showed length variations (629–742 bp). Maximum sequence similarity was found in the 5.8S rRNA region followed by ITS1, while maximum polymorphism was identified in the ITS2 region. The nucleotide sequences of the highly conserved 5.8S region of all the pfle hybrids were very similar with minor variations while this variation was greater between the parental strains. The dendrogram generated based on rRNA-ITS sequences of the pfle hybrids and parents by UPGMA method of clustering revealed five distinct groups, where parent P. florida did not group directly with any of the hybrid strains. The cluster of pfle 6w and pfle 6z remained as separate group in the dendrogram, having no intra cluster linkage.

The 12 pfls somatic hybrids and 2 parents of Pleurotus florida and Lentinus squarrosulus were characterized by ISSR and sequencing of rRNA-ITS genes. Five ISSR primers were used and amplified a total of 54 reproducible fragments with 98.14% polymorphism among all the pfls hybrid populations and parental strains. UPGMA-based cluster exhibited a dendrogram with three major groups between the parents and pfls hybrids. Parent P. florida and L. squarrosulus showed different degrees of genetic distance with all the hybrid lines and they showed closeness to hybrid pfls 1m and pfls 1h, respectively. ITS1(F) and ITS4(R) amplified the rRNA-ITS gene with 611–867 bp sequence length. The nucleotide polymorphisms were found in the ITS1, ITS2 and 5.8S rRNA region with different number of bases. Based on rRNA-ITS sequence, UPGMA cluster exhibited three distinct groups between L. squarrosulus and pfls 1p, pfls 1m and pfls 1s, and pfls 1e and P. florida.

Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71–2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest—L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

A new water-soluble heteropolysaccharide (PS-II) with apparent molecular weight ∼1.65 × 10 5 Da, was isolated from the fruiting bodies of hybrid mushroom pfle 1p by hot aqueous extraction . It is composed of d -mannose, d -galactose, and 3- O -Me- d -galactose in a molar ratio of 1.0:0.99:1.1. The structural investigation of PS-II has been carried out using acid hydrolysis, methylation analysis, periodate oxidation study, and 1D/2D NMR experiments. Based on the results of these experiments, it was established that PS-II contained a main chain of (1 → 6) linked α- d -galactopyranosyl residues, one of which was substituted at C-2 by a terminal mannopyranosyl residue and also methylated at C-3 position. This heteropolysaccharide (PS-II) exhibited macrophage activation by NO production as well as in vitro splenocyte and thymocyte stimulation.

Nine inter-generic somatic hybrids named as pfle were produced through PEG-mediated protoplast fusion between Pleurotus florida and Lentinula edodes using double selection method. Hybridity of the newly developed strains was established on the basis of colony morphology, mycelial growth, hyphal traits, fruit-body productivity and inter single sequence repeat (ISSR) marker profiling. Hybrid population was assessed with different phenotypic variables by one-way analysis of variance. Principal component matrices were analyzed for the six phenotypic variables in scatter plot showing maximum positive correlation between each variable for all strains examined. Six ISSR primers generated 66 reproducible fragments with 98.48 % polymorphism. The dendrogram thus created based on unweighted pair-group method with mathematic averages method of clustering and Euclidean distance which exhibited three major groups between the parents and pfle hybrids. Though P. florida parent remained in one group but it showed different degrees of genetic distance with all the hybrid lines belonging to the other two groups while L. edodes was most distantly related to all the hybrid lines. L. edodes specific sequence-rich ISSR amplicon was recorded in all the hybrid lines and in L. edodes but not in P. florida. All the fruit body generating pfle hybrid lines could produce basidiocarp on paddy straw in sub-tropical climate and showed phenotypic resemblance to the P. florida parent.

A water soluble glucan isolated from hot aqueous extract of the fruit bodies of an edible hybrid mushroom pfle1v of Pleurotus florida and Lentinula edodes , consisting of terminal, (1→3,6)-, two (1→6)- and two (1→3)-linked β -D-glucopyranosyl moieties in a molar ratio of nearly 1:1:2:2 showed macrophages, splenocytes, and thymocytes activation. The structure of the repeating unit of the glucan was established on the basis of chemical analysis (acid hydrolysis, methylation, periodate oxidation study, and Smith degradation) and NMR studies ( 1 H, 13 C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC). This glucan contained a backbone of β -(1→3)-,(1→6)-glucose residues with branching at O-6 position of one of the (1→3)-linked residues.

Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) dramatically improved Agrobacterium infection efficiency in Sesamum indicum (sesame) cotyledon. Different parameters of this method such as the duration of sonication, Agrobacterium concentration and acetosyringone concentration during co-cultivation was standardized to develop a balanced protocol that gave optimum β-glucuronidase (GUS) expression with minimal tissue damage. Nine seconds was identified as the optimum duration for sonication. Agrobacterium concentration having OD600 nm 1.0 was found not to induce tissue damage. Usage of 100 μM acetosyringone, which facilitates T-DNA transfer by activating the vir gene in Agrobacterium, increased the transient GUS activity. The optimized protocol facilitated 85.56 ± 0.35% transient GUS activity. Mimicking SAAT by increasing the concentration of acetosyringone during the co-cultivation period to 400 μM in the conventional Agrobacterium-mediated transformation gave only 32.22 ± 1.53% GUS activity. The results suggest that not only acetosyringone but also other compounds were released during formation of micro-wound by sonication that increased the infection efficiency.

An immunostimulating water-insoluble β-glucan isolated from hot alkaline extract of the fruiting bodies of an edible somatic hybrid mushroom of Pleurotus florida and Calocybe indica var. APK2 showed significant macrophage, splenocyte, and thymocyte activations. On the basis of total hydrolysis, methylation analysis, and NMR experiments (1H, 13C, DQF-COSY, TOCSY, NOESY, DEPT-135, and HSQC), the repeating unit of the polysaccharide is established.

The water-soluble polysaccharide isolated from hot aqueous extract of the fruit bodies of somatic hybrid mushroom ( pfle 1q ) obtained through intergenic protoplast fusion between Pleurotus florida and Lentinula edodes consists of d -galactose and d -mannose in a molar ratio of nearly 2:1. The polysaccharide showed immunoenhancing activity by stimulating the macrophage, splenocyte, and thymocyte. Based on the results of acid hydrolysis, methylation analysis, periodate oxidation, and NMR experiments ( 1 H, 13 C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC and HMBC) the repeating unit of the purified polysaccharide was established as

Rorippa indica, a wild crucifer, has been previously reported as the first identified plant in the germplasm of Brassicaceae known to be tolerant towards the mustard aphid Lipaphis erysimi Kaltenbach. We herein report the full-length cloning, expression, purification and characterization of a novel R. indica defensin (RiD) and its efficacy against L. erysimi. Structural analysis through homology modeling of RiD showed longer α-helix and 3rd β-sheet as compared to Brassica juncea defensin (BjD). Recombinant RiD and BjD was purified for studying its efficacy against L. erysimi. In the artificial diet based insect bioassay, the LC50 value of RiD against L. erysimi was found to be 9.099 ± 0.621 µg/mL which is far lower than that of BjD (43.51 ± 0.526 µg/mL). This indicates the possibility of RiD having different interacting partner and having better efficacy against L. erysimi over BjD. In the transient localization studies, RiD signal peptide directed the RiD: yellow fluorescent protein (YFP) fusion protein to the apoplastic regions which indicates that it might play a very important role in inhibiting nutrient uptake by aphids which follow mainly extracellular route to pierce through the cells. Hence, the present study has a significant implication for the future pest management program of B. juncea through the development of aphid tolerant transgenic plants. Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-2144-2) contains supplementary material, which is available to authorized users.

A water soluble heteroglycan, isolated from the alkaline extract of the fruit bodies of the somatic hybrid mushroom (PfloVv1aFB), raised through protoplast fusion between the strains of Pleorutus florida and Volverilla volvacea, was found to consist of (1→3)-, (1→6)-, (1→3,4)-linked, and terminal β-D-Glcp along with (1→2,6)-α-D-Galp and terminal α-D-Manp in a relative proportion of approximately 1:1:1:1:1:1. This polysaccharide exhibited strong immunostimulating activity of macrophages as well as splenocytes and thymocytes. Structural investigation was carried out using sugar analysis, methylation analysis; periodate oxidation study, and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above mentioned experiments, the structure of the repeating unit of the polysaccharide was established as: [formula see text].

Two different glucans (PS-I and PS-II) were isolated from the alkaline extract of the fruiting bodies of an edible mushroom, Pleurotus florida blue variant and the PS-I showed macrophage, splenocyte and thymocyte activations. On the basis of sugar analysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC and HMBC), the structure of the repeating unit of these polysaccharides were established.

A water-soluble polysaccharide of an edible mushroom Calocybe indica var. APK2 showed immunoenhancing (macrophage, splenocyte, thymocyte, and bone marrow activation) and cytotoxic activity toward HeLa cell lines and found to consist of d -glucose, d -galactose, and l -fucose in a molar ratio of nearly 3:1:1. On the basis of acid hydrolysis, methylation analysis, and NMR studies ( 1 H, 13 C, DEPT-135, TOCSY, DQF–COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of the fuco-galacto-glucan was established as: Download full-size image

A water-soluble glucan, isolated from the alkaline extract of the fruit bodies of an edible mushroom, Lentinus squarrosulus (Mont.) Singer was found to consist of (1→3,6)-linked, (1→3)-linked, (1→6)-linked, and terminal β- d -glucopyranosyl moieties in a relative proportion of approximately 1:2:1:1. This polysaccharide showed optimum activation of macrophages as well as splenocytes and thymocytes at 10 μg/mL. Structural investigation was carried out using sugar analysis, methylation analysis, periodate oxidation study, and NMR experiments ( 1 H, 13 C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments, the structure of the repeating unit of the polysaccharide was established as

A water soluble polysaccharide isolated from the hot aqueous extract of the fruit bodies of the somatic hybrid mushroom (PfloVv1aFB), raised through protoplast fusion between the strains of Pleurotus florida and Volvariella volvacea was found to consist of d-glucose, d-galactose, and d-mannose in a molar ratio of nearly 4:1:1 and showed macrophage, splenocyte, and thymocyte activation. On the basis of sugar analysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC and HMBC), the structure of the repeating unit of the polysaccharide was established as: [structure: see text].

A water-soluble polysaccharide was isolated from the aqueous extract of the fruit bodies of somatic hybrid PCH9FB, obtained through intergeneric protoplast fusion between the strains Pleurotus florida and Calocybe indica var. On the basis of total acid hydrolysis, the polysaccharide was found to contain galactose, fucose, and glucose in a molar ratio of nearly 2:1:2. Methylation analysis and NMR experiments (1H, 13C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) showed that the structure of the repeating unit present in the polysaccharide was Download : Download full-size image This molecule showed macrophage, splenocyte, thymocyte activation as well as antioxidant property.

An immunoenhancing polysaccharide isolated from the aqueous extract of the fruit bodies of the mushroom, Pleurotus florida blue variant, was found to consist of d-glucose and d-galactose in a molar ratio of nearly 5:1. On the basis of sugar analysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of the polysaccharide was established as: →6)-β-D-Glcp-(1→3)-α-D-glcp-(1→[6)-β-D-Glcp-(1]3→ α-D=Glcp. The molecule activated macrophages, splenocytes, and thymocytes.

A water-soluble polysaccharide, isolated from the hot water extract of the fruiting bodies of an edible mushroom, Lentinus squarrosulus (Mont.) Singer was found to contain d -galactose, l -fucose, and d -glucose in a molar ratio of nearly 1:1:5. Structural investigation of the polysaccharide was carried out using acid hydrolysis, methylation analysis, periodate oxidation study, and NMR experiments (1H, 13C, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments, the structure of the repeating unit of the polysaccharide was established as: Download : Download full-size image This polysaccharide showed macrophage as well as splenocyte and thymocyte activation.

A water-soluble immunoenhancing polysaccharide was isolated from the aqueous extract of fruit bodies of somatic hybrid (Pflo Vv5 FB), obtained through protoplast fusion between Pleurotus florida and Volvariella volvacea strains. On the basis of acid hydrolysis, the polysaccharide was found to contain glucose only. Methylation analysis, periodate oxidation along with 1H, DEPT-135, and 13C NMR spectroscopy, including two-dimensional TOCSY, DQF-COSY, NOESY, ROESY, 1H,13C-HMQC, and HMBC experiments showed that the polysaccharide was a (1→6)-β- d -glucan, which was not a constituent of any of the parent mushrooms previously reported. This glucan stimulated the macrophages, splenocytes, and thymocytes.

This article primarily focuses on the production of somatic hybrid sporophores through PEG-mediated protoplast fusion between Calocybe indica var. APK2 and Pleurotus florida. To screen the hybrid strains a selection strategy was followed based on the differential tolerance of NaCl level by the two parental genera. Basidiocarps could be successfully generated from eight out of fourteen hybrid lines that were maintained in culture. Hybridity of the fusant lines was established on the basis of their colony morphology, mycelial growth rate and hyphal traits, while the fruit-body-generating lines were demarcated on the basis of nature of sporophores, isozyme and RAPD markers. The degree to which the hybrid population differed among themselves and from their parents was assessed by analysing each of these morphological variables by one-way analysis of variance (ANOVA). RAPD bands of the fusant lines were similar to either parental bands or were new non-parental bands, which classified them into the microgenome and macrogenome insertion types. A dendrogram created with the help of UPGMA method of clustering and Euclidean distance exhibited three major clusters, in which the Pleurotus–Calocybe hybrids showed intra-cluster variations. Notably, P. florida was genetically distant from the hybrid lines, while C. indica was phylogenetically the dominant parent. Significant increase in bio-efficiency and γ-linoleic acid content in these hybrid lines indicated quantitative as well as qualitative improvement of the newly developed somatic hybrids.

Twelve somatic hybrid lines were raised through polyethylene glycol-mediated intergeneric protoplast fusion between Volvariella volvacea and Pleurotus florida using a double selection method. Four hybrid lines belonging to two distinct colony morphology types could be maintained in culture. Basidiocarps could be generated from two hybrid lines, one of which showed resemblance to the P. florida parent while the other showed intermediate parental morphology. Hybridity of the fusant lines was established on the basis of colony morphology, mycelial growth rate, hyphal traits, fruit body morphology, isozyme and RAPD analysis. Four isozyme activities were analyzed to provide biochemical criteria for detection of polymorphism among the hybrids and their parents. Using 20 random decamers a total of 203 RAPD bands ranging from 0.09 to 1.6 kb were observed. The UPGMA method of clustering exhibited two major phylogenetic clusters, the first of which comprised of the parents, two fruit body generating hybrid lines and their subsequent fruit body derived lines, while the two non-fruit body generating hybrid lines formed the out group of the first major cluster. Screening of a Pleurotus type hybrid line having high biological efficiency and generation of a temperature tolerant Pleurotus type line through backcross mating between a non-fruit body generating somatic hybrid and V. volvacea parent are the key achievements of this fusion programme.

The twenty-first century human society is dealing with many difficulties besides climate challenge, and most of them are though diverse but highly interconnected. Transformation in agriculture and rural development are demanding urgent attention in this scenario. Human civilization is basically an agrarian one, and rural people who practice agriculture are literally the real bread earners. Neglecting agriculture and these endogenous people has visibly started to affect our ecosystem, environment, economy, and civilization. Sustainable solutions to most of these problems can be created only by combining endogenous knowledge reserve and the modern biotechnological know-how. In order to successfully carry forward this process, we first need to ensure endogenous development. Rural development is inevitably connected to several key endeavors having global impacts like food security, protection of environment, and conservation of natural resource base including local landraces, germplasm, and biodiversity. Successful application of rurally relevant biotechnology will be a necessity in transforming agriculture besides enhancing and protecting rural livelihoods as well as rural integrity.

A water-soluble heteropolysaccharide (PS-I) having molecular weight ∼2.1 × 105 Da was isolated from hot aqueous extract of the fruit bodies of hybrid mushroom pfle 1p. The hybrid mushroom pfle 1p was obtained through intergenic protoplast fusion between Pleurotus florida and Lentinula edodes. The heteropolysaccharide contained d-glucose, d-galactose, and d-mannose in a molar ratio of nearly 4:2:1. The structural investigation of PS-I has been carried out using sugar and methylation analyses as well as 1D/2D NMR experiments (1H, 13C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). Based on the results of these experiments, the repeating unit of the PS-I was established as: Figure options Download full-size image Download as PowerPoint slide PS-I showed in vitro macrophage activation by NO production and also stimulated splenocytes and thymocytes.

An immunostimulating water-soluble heteroglycan (PS-II) was isolated from an aqueous extract of the fruit bodies of a hybrid mushroom, pfls1h produced by intergeneric protoplast fusion between Pleurotus florida and Lentinus squarrosulus (Mont.) Singer. Structural characterization of PS-II was carried out using sugar analysis, methylation experiment, periodate oxidation and 1D/2D NMR studies. Sugar analysis indicated the presence of glucose, mannose and galactose in a molar ratio of 1:1:2. Methylation analysis revealed that PS-II was composed of (1→6)- and (1→2,4,6)-α-d-galactopyranosyl, terminal β-d-mannopyranosyl and terminal β-d-glucopyranosyl residues in a relative proportion of approximately 1:1:1:1. On the basis of chemical analysis and NMR studies, the structure of the repeating unit of the heteroglycan was established to consist of a backbone chain of two (1→6)-α-d-galactopyranosyl residues, one of which is branched at O-2 with terminal β-d-Manp and at O-4 with terminal β-d-Glcp. This heteroglycan (PS-II) showed in vitro splenocyte, thymocyte and macrophage activations.

Mustard aphid, Lipaphis erysimi (L.) Kaltenbach is a perpetual annual threat in the cultivation of rapeseed- mustard (Brassica spp.) crop in tropical and sub-tropical climate. Cultivated Brassica germplasm has failed so far to provide any source of resistance. Wild germplasm is a potential source of resistance against many threatening herbivores. On wild germplasm screening, we noted that the wild crucifer Rorippa indica (L.) Hiern confers resistance against L. erysimi. In the present study L. erysimi challenged transcriptome of R. indica was compared to un-infested R. indica sample to get a molecular insight about the aphid resistance mechanism and identify the candidate defense response genes. Cloning, sequencing and in silico sequence analysis of complimentary DNA amplified fragment length polymorphism identified 116 differentially expressed transcript derived fragments revealed thirty candidates which are from different functional categories including redox regulation, signalling, photosynthesis, structure, metabolism, defense response as well as a few of unknown function. Twenty four identifications were then studied by quantitative real time RT PCR analysis at 6, 12, 24 and 48 hour time point post infestation to understand the early-to-late defense response through their relative gene expression profiles. Seventeen fragments showed significant up or down regulation at p

An attempt to transfer genes from droughttolerant Diplotaxis harra, a wild relative of Brassica species, to an elite oil-yielding cultivar, B-85, of mustard (Brassica juncea) was made through protoplast fusion, as the two plant systems are sexually incompatible. By following the standard protocol for PEG-mediated protoplast fusion followed by high pH, high Ca(++), DMSO treatment and appropriate cell-culture technique, 16 presumptive somatic hybrid plants could be regenerated. Chromosomal analysis of four such somatic hybrids revealed that three of them were asymmetric. Analysis of morphological characters, meiotic chromosomes, and esterase isoenzyme pattern revealed that all the somatic hybrids were different from each other. Furthermore four chromosomes of each genome could undergo homoeologous pairing at meiosis indicating the possibilities for genetic recombination and chromosomal rearrangements. Irregular distribution of chromosomes at anaphase-II at meiosis has been a consistent feature of these plants. Eventually, pollen of all the somatic hybrids showed complete infertility preventing the recovery of any selfed seed. Nevertheless, ovule fertility of one somatic hybrid was not totally impaired as it had set some seeds upon backcrossing with the B. juncea parent. The esterase isoenzyme banding pattern of 24 individual progeny plants of this backcross provided evidence for their recombinant nature. It was thus confirmed that a transfer of genetic traits from Diplotaxis harra to B. juncea had indeed taken place. Furthermore, it was conceptualised that a transfer of alien genes through the protoplast-fusion technique is primarily possible in situations where meiotic pairing of the chromosomes of the two participating genomes generates recombinant gametocytes which can pass through subsequent filial generations.

A water soluble polysaccharide isolated from the alkaline extract of the somatic hybrid mushroom ( PfloVv5FB ), obtained through protoplast fusion between Pleurotus florida and Volvariella volvacea strains was found to contain d -glucose only. Structural investigation was carried out using acid hydrolysis, methylation analysis; periodate oxidation, and NMR studies ( 1 H, 13 C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC). On the basis of the above mentioned experiments the structure of the repeating unit of the polysaccharide was established as: Download full-size image This polysaccharide exhibited strong immunoactivation of macrophages, splenocytes as well as thymocytes.

A water soluble heteroglycan isolated from hot aqueous extract of fruit bodies of an edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula edodes consists of d-glucose, d-mannose, and d-galactose residues in a molar ratio of nearly 1:1:1. This polysaccharide showed macrophage, splenocyte, and thymocyte activation. On the basis of sugar hydrolysis, methylation, periodate oxidation study, and NMR studies (1H, 13C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC), the structure of the repeating unit of the polysaccharide was established as: --6)-α-D-Galp-(1--6)-α-D-Glcp-(1--2--1β-D-Manp.

A water soluble polysaccharide was isolated from the aqueous extract of the fruit bodies of somatic hybrid mushroom PCH3FB, obtained through protoplast fusion between the strains Pleurotus florida and Calocybe indica var. APK2. On the basis of sugar analysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC and HMBC), the structure of the repeating unit of the polysaccharide was established as: [formula, see text] This molecule showed macrophage, splenocyte and thymocyte activations.

A water-soluble glucan (PS-I) was isolated from the aqueous extract of the fruit bodies of a hybrid mushroom, pfls1h of Pleurotus florida and Lentinus squarrosulus (Mont.) Singer. Structural characterization of PS-I was carried out using total hydrolysis, methylation analysis, periodate oxidation, and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). Methylation analysis revealed that PS-I was composed of (1→3, 6), (1→3), (1→6)-linked and terminal β-d-glucopyranosyl residues in a relative proportion of approximately 1:1:1:1. The repeating unit of the glucan consists of a backbone chain of two (1→6)-β-d-glucopyranosyl residues, one of which is branched at O-3 position with (1→3)-β-d-glucopyranosyl and terminated with a β-d-glucopyranosyl residue. Study of immunological activity revealed that PS-I stimulates the splenocytes, thymocytes and macrophages.

An immunostimulating water-soluble glucan was isolated from hot aqueous extract of fruit bodies of an edible mushroom Calocybe indica. Structural investigation of the glucan was carried out using acid hydrolysis, methylation analysis, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments, the structure of the repeating unit of the polysaccharide was established as [see figure in text]. This glucan stimulated the splenocytes and thymocytes.

Two different glucans (water-soluble PS-I, water-insoluble PS-II) were isolated from the alkaline extract of the fruit bodies of hybrid mushroom. PS-I was found to consist of only (1→6)-linked β- d -glucopyranose. PS-II was composed of terminal, (1→3,4)-linked, and (1→3)-linked β- d -glucopyranosyl moieties in a molar ratio of nearly 1:1:1. PS-I showed macrophages, splenocytes, and thymocytes activation as well as antioxidant property. On the basis of sugar analysis, methylation analysis, and NMR studies (1H, 13C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these glucans were established as: Download : Download full-size image

Three polysaccharide fractions (PS-I, PS-II, and PS-III) were isolated from the aqueous extract of a hybrid mushroom obtained through backcross mating of a somatic hybrid mushroom PfloVv12 (Sterile line) with Volvariella volvacea. PfloVv12 was obtained through protoplast fusion of Pleurotus florida and V. volvacea. PS-I was identified as 1,6-β glucan. PS-II and PS-III were identified as mannoglucogalactan but differing in molecular weights only. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and NMR experiment ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) the structures of these polysaccharides were established as

A water-soluble glucan isolated from an alkaline extract of fruit bodies of a somatic hybrid mushroom PCH9FB of Pleurotus florida and Calocybe indica var. APK2 strains showed antioxidant properties with immune activation of macrophage, splenocyte, and thymocyte. On the basis of acid hydrolysis, methylation, periodate oxidation and NMR studies ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) the structure of the repeating unit of the glucan was established as: [structure: see text].

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2-7.1×10(5) protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS2 medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.

Carisoprodol, a centrally acting muscle relaxant, has recently been noticed to be abused. A series of 16 cases with carisoprodol abuse or dependence is described. Carisoprodol was started by the majority of patients as a substitute for opiates, although its independent distinct effects, similar to the features of hypomania, were recognized and described by most users. The drug is currently available freely over the counter and is a much cheaper substitute for the legally prohibited "harder" drugs. Unless awareness spreads and cautionary measures are taken, carisoprodol abuse might become a great menace in the near future.

The need to transfer genetic traits from Diplotaxis muralis (2n=42) to Brassica juncea (2n=36), a major oil seed crop of the tropical world, was realised. Since the two plant types are sexually incompatible, attempts were made to evolve parasexual hybrids as the result of protoplast fusion. Protoplasts of hypocotyl-derived calli of two cultivars of B. juncea were fused with normal and γ-irradiated mesophyll protoplasts of Diplotaxis muralis. Regeneration of 110 plants from the fused products was successfully achieved. Upon analysis of some of them, we realised that true somatic hybrids and partial somatic hybrids had been generated. Thus the primary goal of evolving intergeneric hybridisation products between these two plant types was fulfilled.