Genetic diversity and population structure detection in sponge gourd ( Luffa cylindrica ) using ISSR,SCoT and morphological markers

Investigation of genetic diversity is essential for the selection of parents for crop breeding and conservation of genetic resources. To estimate the genetic variability and population structure in the midst of 45 accessions of sponge gourd brought together from different geographical areas of India, morphological traits and two molecular markers, ISSR and SCoT markers were compared. Principal components analysis of 20 morphological traits showed 72.70% variability and significant positive correlations between fruit traits. All three marker techniques clustered all accessions into two groups with few outgroups. High level of polymorphism was observed among ISSR (74.6%) and SCoT (71.5%) primers. The Bayesian model revealed the hidden grouping and showed admixture type of population. The diversity pattern is influenced by genetic marker used, as different molecular markers have different polymorphism evaluation efficiency. This study can be helpful in amplifying the genetic base and selection of specific traits for breeding. Thus, ISSR and SCoT markers are potential marker for identification in sponge gourd and provide valuable data on its genetic correlation and structure.     

Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein^–1 and a molecular mass of 26–28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins.Abbreviations BAP benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - HPLC high pressure liquid chromatography - MS Murashige and Skoog - NAA naphthaleneacetic acid - NCPPB National Collection of Phytopathogenic Bacteria - Ri root-inducing - RIP ribosome-inactivating protein - UV ultra-violet - YMB yeast mannitol broth     

Structural characterization and anti-HIV-1 activities of arginine/glutamate-rich polypeptide Luffin P1 from the seeds of sponge gourd (Luffa cylindrica)

Luffin P1, the smallest ribosome-inactivating peptide from the seeds of Luffa cylindrica was found to have anti-HIV-1 activity in HIV-1 infected C8166 T-cell lines and be able to bind with HIV Rev Response Element. Nuclear magnetic resonance spectroscopy revealed that the Luffin P1 comprises a helix-loop-helix motif, with the two alpha helices tightly associated by two disulfide bonds. Based on our findings, we conclude that unlike the well-studied ribosome-inactivating proteins, which exert their action through N-glycosidase activities, Luffin P1 demonstrates a novel inactivation mechanism probably through the charge complementation with viral or cellular proteins. Our work also provides a new scaffold for the design of novel inhibitors from a simple helical motif.     

Loofa (Luffa cylindrica) sponge: Review of development of the biomatrix as a tool for biotechnological applications

The review discusses the development of loofa sponge (Luffa cylindrica) as a biotechnological tool and the diversity of applications in which it has been successfully used since it was first reported as a matrix for the immobilization of microbiological cells in 1993. The fibro‐vascular reticulated structure, made up of an open network of random lattices of small cross‐sections coupled with very high porosity (79–93%), having very low density (0.02–0.04 g/cm³), and high specific pore volume (21–29 cm³/g), has the characteristics of a carrier/scaffold well‐suited for cell immobilization. This has been confirmed through the immobilization of cells of diverse types, including filamentous and microalgae, fungi, bacteria, yeasts, higher plants, and human and rat hepatocytes. The cells immobilized in loofa sponge have performed well and better than free suspended cells and those immobilized in conventionally used natural and synthetic polymeric materials for the production of ethanol, organic acids, enzymes, and secondary metabolites. The loofa‐immobilized cell systems have been efficiently used for the treatment of wastewaters containing toxic metals, dyes, and chlorinated compounds, and the technology has been used to develop biofilms for the remediation of domestic and industrial wastewaters rich in inorganic and organic matter. In addition, three‐dimensional loofa sponge scaffolds for hepatocyte culture have been suggested to have the potential for development into a bioartificial liver device. Loofa sponge is a cost‐effective, eco‐friendly, and easy to handle matrix that has been used successfully as a biotechnological tool in a variety of systems, purposes, and applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:573–600, 2013     

Inheritance of gynoecism in bitter gourd (Momordica charantia L.)

The inheritance of sex expression in cucumber (Cucumis sativus) and other cucurbits is well documented; however, the genetics of female sex (gynoecism) expression in bitter gourd (Momordica charantia) has not been described. Inheritance of gynoecism in bitter gourd was studied in a 100% gynoecious line (Gy263B). The F(2) and testcross segregation data revealed that gynoecism in Gy263B is under the control of a single, recessive gene. Following the gene nomenclature of cucurbits, it is proposed that the gene symbol, gy-1, be assigned for the expression of gynoecism in bitter gourd.     

Some competition experiments with alang-alang (Imperata cylindrica (L.) Beauv.) in replacement series

Summary The interaction between alang-alang (Imperata cylindrica) and maize or sorghum was studied in competition experiments according to the replacement principle. Dry matter yield of alang-alang in these experiments appeared hardly affected by the presence of maize or sorghum, while this yield of the latter two was strongly reduced by the presence of alang-alang. The relative yield total (RYT) reached unity except in one experiment in which a value of 0.6 was obtained.The results suggest that the allelopathic activity of alang-alang will find expression in an RYT deviating from one only if alang-alang is not able to utilize all available space.Part of the research supported by the Netherlands Foundation for Advancement of Tropical Research (WOTRO) grant W86-34     

Improved in planta genetic transformation efficiency in bitter gourd (Momordica charantia L.)

In Vitro Cellular & Developmental Biology - Plant - Production of transformed bitter gourd plants through in vitro regeneration is a laborious practice, which may also result in somaclonal...     

Intracellular distribution of the hydrophobic triterpene,bryonolic acid,in cultured cells of Luffa cylindrica L.

Summary Intracellular localization of bryonolic acid, an antiallergic pentacyclic triterpene, in cultured cells of Luffa cylindrica was investigated with reference to the sites of its biosynthesis and accumulation. The results of cell fractionation showed that bryonolic acid was mostly located in the cell wall fraction. The addition of FC-43 emulsion to the culture medium was found to cause the release of bryonolic acid from the cell wall into the medium without affecting cell growth and bryonolic acid production. Under this culture condition, ¹⁴C-labeled sodium acetate administered to the cells was rapidly incorporated into bryonolic acid which was then excreted into the medium within 10 min after administration. Electron microscopic observations suggested that spherical vesicles (ca 0.1 m in diameter) derived from the rough endoplasmic reticulum may be associated with the biosynthesis and excretion of this compound into the cell wall. Furthermore, the activity of 2,3-oxidosqualene cyclase, a key enzyme involved in the biosynthesis of bryonolic acid, was detected in the microsomal fraction containing the endoplasmic reticulum.Abbreviations BA bryonolic acid - ER endoplasmic reticulum - LS Linsmaier-Skoog - NAA 1-naphthaleneacetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PVPP polyvinyl polypyrrolidone     

Sucrose utilization and mineral nutrient uptake during hairy root growth of red beet (Beta vulgaris L.) in liquid culture

Information concerning the sugar status of plant cells is of greatimportance during all stages of the plant life cycle. The aim of this work wasto study primary carbohydrate metabolism in hairy roots of red beet. Growth ofhairy roots of red beet in vitro and changes in concentration of major nutrientsand sugar in the media were measured over a growth cycle of 16 days. We havealso determined the levels of key enzymes in the pathways of sucrose metabolism.Sucrose concentration decreased as hairy root growth proceeded while no changein glucose and fructose levels in the medium was found during the first 3 daysindicating that external sucrose is preferably taken to the cell before it ishydrolyzed by extracellular invertase. The increase in glucose and fructoselevels in the media after 5 days of culture indicates extracellular hydrolysisof sucrose which was further supported by the activity of acid invertaseobserved during that time in the culture medium. The uptake of mineral nutrientsby hairy root of red beet was monitored continuously during the culture cycle.The preferential use of NH₄ ⁺ overNO₃ ^– at the beginning of the culture andacidification of culture media were the two most notable results concerningnitrogen nutrition during hairy root growth of red beet.     

AFLP marker polymorphism in cucumber (Cucumis sativus L.) near isogenic lines differing in sex expression

The AFLP technique was used to evaluate the level of polymorphism between two pairs of isogenic cucumber (Cucumis sativus L.) lines (NIL) differing in flower sex expression. The BSA techniques were also applied to find molecular markers linked to sex determination genes (dominant alleles) in those cucumber lines. Sex determination in cucumber is controlled by three main loci F, M and Gy. The interaction of these loci is responsible for the formation of the various phenotypes of flowers in respect to sex in the analyzed lines [corrected]. A female line 2gg with a ff/MM/gygy genotype, isogenic to a monoecious line B10 (genotype ff/MM/GyGy), and a female line Gy3 with a FF/MM/GyGy genotype, isogenic to a hermaphroditic line HGy3 (genotype FF/mm/GyGy). Using 56 combinations of AFLP primers, used for the analysis of lines 2gg and B10, gave 3794 bands, of which 155 (4.1%) were polymorphic. Ten bands distinguished gynoecious and monoecious bulks appearing at the same time in the appropriate parent; they are believed to be linked to the Gy locus. The isogenic lines Gy3 and HGy3 showed a higher level of polymorphism (14.2%). In this case, 55 combinations of primers gave 2996 reaction products, of which 430 showed variation. Twenty bands occurred in one bulk and in one parent, so they are probably associated with the M locus. Using the AFLP technique, the isogenicity of the lines was evaluated. The level of polymorphism (per pair of primer) between lines 2gg and B10 is 0.072% and is four times lower than that between the Gy3 and HGy3 lines (0.27%). The differences in the isogenicity of the lines can result from the degree of their relatedness, which may reflect the way they were derived.     

Nitrogen fertilization and sex expression affect size variability of fibre hemp (Cannabis sativa L.)

Mechanical harvesting and industrial processing of fibre hemp (Cannabis sativa L.) require uniformity of stem length and weight. In 1991 and 1992 we carried out field experiments to investigate the effects of soil nitrogen level (80 and 200 kg ha^-1N) and row width (12.5, 25 and 50 cm) on the variability of weight and height in hemp plants. The crops were sampled 5 times between early June and early September. Row width did not affect size variability. At final harvest coefficients of variation (CV) of both weight and height were about 1.5 times higher at 200 than at 80 kg ha^-1N. Distributions of dry weight were positively skewed at all sampling dates except the first, with skewness larger at 200 than at 80 kg ha^-1N. Distributions of height were negatively skewed at all sampling dates except the first at 80 kg ha^-1N. At 200 kg ha^-1N they changed from negative skewness during the first part of the growing season to negative kurtosis in the second part of the growing season. More suppressed plants were present at 200 than at 80 kg ha^-1N. Contrary to most published results, we did not find a reduction of CV of weight nor of CV of height at the onset of self-thinning. Suppressed hemp plants can survive relatively well in the low-light environment under the canopy. Sexual dimorphism contributed to variability of height and weight, but the effects were smaller than those of nitrogen fertilization. The ratio of female to male plants was higher at 200 than at 80 kg ha^-1N, as a result of a shift in sex-ratio within the population of suppressed plants. As suppressed plants were much more slender than dominating plants, self-thinning eliminated the most slender individuals in a hemp crop. However, the presence of many more heavy individuals of low slenderness at 200 than at 80 kg ha^-1 N was probably the major cause of the difference in slenderness between 200 and 80 kg ha^-1 N.     

Construction of a high-density genetic linkage map and identification of gene controlling resistance to cucumber mosaic virus in Luffa cylindrica (L.) Roem. based on specific length amplified fragment sequencing

Molecular Biology Reports - Luffa cylindrica L. is a cash crop which has important health, medicinal and industrial value, but no high saturation genetic map has been constructed owing to a lack of...     

Molecular cloning and expression analysis of spermidine synthase gene during sex reversal induced by Ethrel in cucumber (Cucumis sativus L.)

A full-length cDNA encoding spermidine synthase CsSPDS of 317 amino acids was isolated from cucumber. CsSPDS showed high similarity to SPDSs of other species, with the greatest similarity to SPDS of Coffea arabica (identity, 87%). Northern blot analysis revealed that CsSPDS was expressed predominantly in rapid growing tissues, such as roots, young leaves and flower buds. The response of CsSPDS to the sex reversal of three-leaf stage androecious cucumber seedlings by Ethrel treatment was also examined. Our results showed that the CsSPDS was up-regulated in shoot apex during sex reversal process, which correlated with increases of spermidine synthase activity and free spermidine (Spd) content, and a decrease of putrescine (Put) content in shoot apices.     

Plant morphology and root hydraulics are altered by nutrient deficiency in Pistacia lentiscus (L.)

The plants in arid and semiarid areas are often limited by water and nutrients. Morpho-functional adjustments to improve nutrient capture may have important implications on plant water balance, and on plant capacity to withstand drought. Several studies have shown that N and P deficiencies may decrease plant hydraulic conductance. Surprisingly, studies on the implications of nutrient limitations on water use in xerophytes are scarce. We have evaluated the effects of strong reductions in nitrogen and phosphorus availability on morphological traits and hydraulic conductance in seedlings of a common Mediterranean shrub, Pistacia lentiscus L.. Nitrogen deficiency resulted in a decrease in aboveground biomass accumulation, but it did not affect belowground biomass accumulation or root morphology. Phosphorus-deficient plants showed a decrease in leaf area, but no changes in aboveground biomass. Root length, root surface area, and specific root length were higher in phosphorus-deficient plants than in control plants. Nitrogen and phosphorus deficiency reduced both root hydraulic conductance and root hydraulic conductance scaled by total root surface area. On the other hand, nutrient limitations did not significantly affect root conductance per unit of foliar surface area. Thus, adaptation to low nutrient availability did not affect seedling capacity for maintaining water supply to leaves. The implications for drought resistance and survival during seedling establishment in semi-arid environments are discussed.     

Silicon in Imperata cylindrica (L.) P. Beauv: content,distribution, and ultrastructure

Silicon concentration, distribution, and ultrastructure of silicon deposits in the Poaceae Imperata cylindrica (L.) P. Beauv. have been studied. This grass, known for its medicinal uses and also for Fe hyperaccumulation and biomineralization capacities, showed a concentration of silicon of 13,705?±?9,607 mg/kg dry weight. Silicon was found as an important constituent of cell walls of the epidermis of the whole plant. Silica deposits were found in silica bodies, endodermis, and different cells with silicon-collapsed lumen as bulliforms, cortical, and sclerenchyma cells. Transmission electron microscope observations of these deposits revealed an amorphous material of an ultrastructure similar to that previously reported in silica bodies of other Poaceae.     

Production of recombinant human deoxyribonuclease I in Luffa cylindrica L. and Nicotiana tabacum L.: evidence for protein secretion to the leaf intercellular space

Plant Cell, Tissue and Organ Culture (PCTOC) - The sequence encoding hDNase I with its native N-terminal signal peptide was codon-optimized for heterological expression in cucurbit plants. The...     

In vitro conservation of Bacopa monnieri (L.) using mineral oil

Bacopa monnieri (L.) Wettst., a traditional Indian medicinal plant with high commercial potential, is used as a potent nervine tonic. A slow growth protocol was developed for medium-term conservation using mineral oil (MO) overlay. Nodal segments of B. monnieri (two genotypes; IC249250, IC468878) were conserved using MO for 24?months. Single node explants were implanted on MS medium supplemented with 0.2?mg?l^?1 BA and were covered with MO. Subculture duration could be significantly enhanced from 6 to 24?months, on the above medium. Normal plants regenerated from conserved cultures were successfully established in soil. On the basis of 20 random amplified polymorphic DNA and 5 inter-simple sequence repeat primers analyses and bacoside A content using HPLC, no significant reproducible variation was observed between the controls and in vitro-conserved plants. The results demonstrate the feasibility of using MO for medium-term conservation of B. monnieri germplasm without any adverse genetical and biochemical effects.     

Analysis of secondary sex ratio in the fox (Vulpes vulpes L.)

Secondary sex ratio and its variability in relation to some paratypic and genetic factors were studied in the silver fox by analysis of data obtained at the Pushkinskii fur farm (Moscow oblast) in 1980-1989. A total of 17285 whelps were examined. The mean proportion of males over the ten years of observation was 0.536+/-0.004. No effect of parent age or litter size on this proportion was found. Individual analysis of the progeny of a single parent revealed 44 males and 49 females showing significant deviations from the expected sex ratio (l male : 1 female). These results can be used for reconstruction of pedigrees whose progeny yields regular deviations from the expected sex ratio.