Influence of plant growth regulators on growth, morpho-physiological characters and yield of summer sesame (Sesamum indicum L. cv. Rama) under moisture stress

Field experiments were conducted with sesame (Sesamum indicum L. cv. Rama) for two years (1997 and 1998) to study the effect of three level of irrigation (F+C, B+C, B+F+C) and two growth regulators (CCC, 200 ppm CCC; 100 ppm and BX-112, 100 ppm; BX-112, 50 ppm) on growth (root and shoot length, average number of primary branches/plant), morpho-physiological growth parameters(LAI, LAD, CGR and NAR), yield attributing parameters(average number of capsule/plant, average number of seeds/capsule) and seed yield. Irrigation at B+F+C stage showed significant effect on these parameters. Among the growth regulators, CCC, 200 ppm showed remarkable results on these parameters and seed yield. Seed yield in CCC, 200 ppm treatment was more than 53% in comparison to water soaked seeds. The interaction between irrigation and PGR showed better seed yield and it was concluded that the growth regulator CCC might be utilized for enhancement of seed yield of summer sesame under field condition.     

Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system

The efficiency of micropropagation of double-node shoots of hop (Humulus lupulus L. cv. Tettnanger) was evaluated using semi-solid and liquid culture medium in RITA® temporary immersion bioreactors. The highest fresh and dry weight of shoots, average number of shoots, and multiplication rate were obtained using the RITA® system, whereas the longest shoots were obtained on semi-solid medium. Moreover, shoot length was affected significantly by the inoculum density of double-node shoots in RITA® vessels. In addition, the RITA® bioreactors were suitable for shoot induction from organogenic calli. The percentage of shoot induction and the shoot fresh and dry weights were significantly higher in the RITA® system than in semi-solid medium. The age of organogenic calli and inoculum density significantly affected the induction of shoots from organogenic calli. The optimum conditions for DNA delivery into hop organogenic calli using the biolistic particle delivery system were also determined. Organogenic calli were bombarded with the plasmid pSR5-2 (gusA and nptII) varying helium pressure (900, 1,100, or 1,350 psi) and target distance (6, 9, or 12 cm). The highest gusA transient activity was obtained using a pressure of 900 psi and a target distance of 6 cm. For stable genetic transformation, 3-wk-old organogenic calli were bombarded with the plasmid pCAMBIA1303 (gusA, mgfp5, and hpt) using these optimum conditions. Stable gusA expression was observed in organogenic calli and shoots after 4 wk of culture on selection medium containing 2.5 mg l^?1 hygromycin. The presence of the mgfp5 gene in the hop genome was confirmed by PCR.     

Identification of sesame (Sesamum indicum L.) chromosomes using the BAC‐FISH system

• Sesame (Sesamum indicum L.; Pedaliaceae) is a commercially valuable oilseed crop with high oil content. Its small genome size favours the genomic analysis of key biological processes, such as oil synthesis and metabolism. However, the 13 chromosome pairs of sesame have not been characterised because of technological limitations and their small size.
• We constructed a BAC library comprising 57,600 BAC clones for sesame. The estimated genome coverage of the sesame BAC library was 13.8×. The successive double colour fluorescence in situ hybridisation (FISH) with bacterial artificial chromosomes (BACs) for sesame was established in this study.
• Subsequently, the 13 sesame chromosome pairs were individually differentiated using 17 specific BACs for the first time. The schematic of the sesame chromosome set was drawn according to the chromosome relative length and relative position of the BAC signal. The cytogenetic characteristics of sesame chromosomes were also explored.
• The results provide the technical background required for further cytogenetic map construction, genome assembly and localisation of the DNA sequence in sesame.

     

Study of genetic variation in sesame (Sesamum indicum L.) using agro-morphological traits and ISSR markers

This research was conducted to study the genetic variation among eighteen genotypes of sesame (Sesamum indicum L.) collected from various agro-climatic regions of Iran along with six exotic genotypes from the Asian countries using both agro-morphological and ISSR marker traits. The results showed significant differences among genotypes for all agro-morphological traits and a relatively high genetic coefficient of variation observed for number of fruiting branches per plant, capsules per plant, plant height and seed yield per plant. Cluster analysis based on these traits grouped the genotypes into five separate clusters. Larger inter- than intra cluster distances implies the presence of higher genetic variability between the genotypes of different groups. Genotypes of two clusters with a good amount of genetic divergence and desirable agronomic traits were detected as promising genotypes for hybridization programs. The 13 ISSR primers chosen for molecular analysis revealed 170 bands, of which 130 (76.47%) were polymorphic. The generated dendrogram based on ISSR profiles divided the genotypes into seven groups. A principal coordinate analysis confirmed the results of clustering. The agro-morphological traits and ISSR markers reflected different aspects of genetic variation among the genotypes as revealed by a non significant cophenetic correlation in the Mantel test. Therefore the complementary application of both types of information is recommended to maximize the efficiency of sesame breeding programs. The discordance among diversity patterns and geographical distribution of genotypes found in this investigation implies that the parental lines for hybridization should be selected based on genetic diversity rather than the geographical distribution.     

Evaluation of the genetic diversity and population structure of sesame (Sesamum indicum L.) using microsatellite markers

Sixteen polymorphic microsatellite (SSR) markers, developed from an SSR-enriched genomic DNA library of sesame (Sesamum indicum L.), were used to assess genetic diversity, phylogenetic relationships, and population structure among 150 sesame accessions collected from 22 countries. A total of 121 alleles were detected among the sesame accessions. The number of detected alleles varied from 2 to 18, with an average of 7.6 alleles per locus. Polymorphism information content values ranged from 0.03 to 0.79, with an average of 0.42. These values indicated an excess of heterozygous individuals at 16 loci and an excess of homozygous individuals at three loci. Of these, 32 genotype-specific alleles were identified at 11 of 16 polymorphic SSR markers. Cluster analyses were performed by accession and population, revealing a complex accession distribution pattern with mean genetic similarity coefficient of 0.45 by accession and 0.52 by population. The wide variation in genetic similarity among the accessions revealed by SSRs reflected a high level of polymorphism at the DNA level. Model-based structure analysis revealed the presence of three groups that were basically consistent with the clustering results based on genetic distance. These findings may be used to augment the sesame germplasm and to increase the effectiveness of sesame breeding.     

Aggregation, dissociation and denaturation of sesame (Sesamum indicum L.) alpha-globulin in cetyl trimethyl ammonium bromide solution.

The behaviour of the major protein of sesame seed (Sesamum indicum L.) alpha-globulin has been studied in a cationic detergent, cetyl trimethyl ammonium bromide solution. Up to a critical detergent concentration the protein is precipitated from solution, above which redissolution of the protein is observed. Sedimentation velocity patterns indicate the presence of higher aggregates in the detergent concentration range 5 X 10(-5)--1 X 10(-3) M. These are considered to be the soluble precursors of the insoluble aggregates. Fluorescence measurements show that tryptophanyl groups of the protein which are in contact with the aqueous phase are perturbed by the detergent. The difference spectra of the protein in higher concentration of detergent indicate considerable red shift in the spectrum. Spectrophotometric titration of phenolic groups in 1 X 10(-2) M CTAB indicate that a conformational change in the protein has taken place.     

Purification, physicochemical properties, and subcellular location of alkaline inorganic pyrophosphatase from sesame (Sesamum indicum L.) cotyledons.

Abstract: Changes in the levels of inorganic pyrophosphatases (PPases) were monitored in germinating sesame seeds at regular intervals. Activities of acid and alkaline PPases increased markedly in cotyledons up to day 4, remained at the peak level up to day 7, and then showed a considerable decline thereafter. An alkaline PPase was isolated and purified from 5-day-old sesame cotyledons following acetone precipitation, ammonium sulfate fractionation, and chromatography on DEAE-Sephadex. Current protocol yielded about 20% recovery of total activity with a 6.4-fold purification. The enzyme was a monomer with a molecular mass of 20.8 kDa. Some of the properties of alkaline PPase including stability, substrate specificity, ion requirement, and amino acid composition were studied. Alkaline PPase showed maximum activity at pH 8.6 in the presence of Mg2+ and at 50 degrees C. However, the metal ion could not protect the enzyme against thermal denaturation. Alkaline PPase was highly specific for inorganic pyrophoaphate (PP) as substrate and the Km value was 0.7677 +/- 0.0528 mM. Full activation of the enzyme was achieved with a Mg2+/PPi ratio of 2. Divalent metal ions such as Ca2+, Cu2+, and Zn2+ inhibited PPase activity. Mg2+, partially relieved the inhibition caused by adenosine 5'-triphosphate. Studies related to the localization of alkaline PPase in microbodies revealed that the enzyme was distributed between glyoxysomes and mitochondria, with the former containing more of it.     

Induction of cytomixis affects microsporogenesis in Sesamum indicum L. (Pedaliaceae)

Cytomixis was recorded during microsporogenesis in sesame (Sesamum indicum L.), a member of the family Pedaliaceae. The phenomenon of cytomixis was observed at various stages of meiosis in 0.5% sodium azide (SA) treated populations of Sesamum indicum L. Cytomixis was observed to occur through various methods, i.e. by forming cytoplasmic channels and direct fusion of pollen mother cells (PMCs), the former was more frequent than the latter. The migration of nuclear content involved all the chromatin/chromosomes or part of it from donor to recipient cell/cells. Some completely empty meiocytes were also observed. Stickiness, precocious movement, laggards, unorientation and micronuclei were observed in almost all the sets treated with various doses of SA. Increase in the doses of SA had a positive effect on the percentage of PMCs showing cytomixis and chromosomal abnormalities. The impact of cytomixis on meiotic behaviour, reduced pollen viability and heterogeneous sized pollen grains were observed.     

Genetic transformation of chickpea (Cicer arietinum L.) with insecticidal crystal protein gene using particle gun bombardment

Here, we report the establishment of an efficient particle gun bombardment mediated genetic transformation in chickpea (Cicer arietinum L.) using cryIAc gene of Bacillus thuringiensis. Explants were bombarded with recombinant plasmids engineered for the expression of cryIAc transgene in plants and stable transformants regenerated in presence of benzyladenine, kinetin and kanamycin. Transformation frequency showed dependence on explant type, cultivars, plasmids, helium pressure and microcarrier type used. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization approaches in T ₀ plants. The expression of CryIA(c) delta-endotoxin and GUS enzyme was ascertained by enzyme linked immunosorbent assay and histochemical assays, respectively. These transgenic plants (T ₀) showed more protection and high mortality for Heliothis armigera and Spodoptera litura larvae as compared to control plants. The results of the present study indicate that highest transformation frequency (18%) could be achieved by use of gold as a microcarrier in combination with helium pressure of 900 psi. Among the other factors tested, plasmid pHS 102 was the most efficient plasmid, while epicotyl explant was the best explant source for particle gun bombardment. Among the different cultivars of chickpea tested, cultivar ICCC37 and PG-12 produced higher frequency of transformation frequency compared to others.     

Comparative analysis of genetic diversity of sesame (Sesamum indicum L.) from Vietnam and Cambodia using agro-morphological and molecular markers

The purpose of this study was to comparatively analyze the genetic diversity of sesame (Sesamum indicum L.) using agro-morphological and molecular markers. Twelve sesame populations collected from three regions in Cambodia and Vietnam were used in this study. A high genetic variation was revealed both by agro-morphological and RAPD markers within and among the 12 sesame populations. The range of agro-morphological trait based average taxonomic distance among populations (0.02 to 0.47) was wider than that of RAPD based genetic distance (0.06 to 0.27). The mean distance revealed by agro-morphological markers (0.23) and RAPD markers (0.22) was similar. RAPD based analysis revealed a relatively higher genetic diversity in populations from South Vietnam as compared to the other two regions. Interestingly, populations from this region also had higher values for yield related traits such as number of capsules per plant, number of seeds per capsule, and seed yield per plant suggesting positive correlation between the extent of genetic variation within population and yield related traits in sesame. A highly significant positive correlation (r = 0.88, P < 0.001) was found between agro-morphological and RAPD markers in estimating the genetic distance between populations. Both methods suggested the existence of a substantial amount of genetic diversity both in the Vietnamese and Cambodian populations. Although both agro-morphological and RAPD markers were found to be useful in genetic diversity analysis in sesame, their combined use would give superior results.     

Achieving improved control of foliar diseases of sesame (Sesamum indicum L.) intercropped with maize (Zea mays L.) through foliar spray of plant extracts

Field trials were conducted to evaluate the effect of foliar spray of aqueous extracts of Tithonia diversifolia or Ocimum gratissimum on Cercospora leaf spot (CLS) and Alternaria leaf blight (ALB) diseases of sesame intercropped with maize. Spraying regime was at 2 weeks interval from 3 weeks after planting (WAP) until 12 WAP. Extracts of T. diversifolia or O. gratissimum reduced the incidence and severity of both diseases. CLS incidence and severity as well as defoliation was significantly (p < 0.05) reduced below what obtained in the unsprayed intercrop. ALB lesion size was significantly (p < 0.05) reduced by T. diversifolia extract at 8.0% (w/v) from 154.7 mm² (sole crop) or 13.4 mm² (unsprayed intercrop) to 4.9 mm² (sprayed intercrop). T. diversifolia extract at 8.0% (w/v) enhanced higher values of grain yield/plant and incidence of normal seeds, and lower incidence of fungal infection of seeds than in the unsprayed intercrop.     

Genetic transformation of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' via microprojectile bombardment

 An effective method has been developed for the stable transformation and regeneration of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' by microprojectile bombardment. Embryogenic cell suspensions were initiated using immature male flowers as the explant. Cells were co-bombarded with the neomycin phosphotransferase (nptII) selectable marker gene under the control of a banana bunchy top virus (BBTV) promoter or the CaMV 35S promoter, and either the β-glucuronidase (uidA) reporter gene or BBTV genes under the control of the maize polyubiquitin promoter. Plants were regenerated, under selection with kanamycin, that were co-transformed with nptII and either the uidA or BBTV genes. Molecular characterisation of transformants demonstrated that the transgenes had been stably integrated into the banana genome. Received: 22 June 1998 / Revision received: 29 March 1999 / Accepted 1 May 1999     

A survey of sesamin and composition of tocopherol variability from seeds of eleven diverse sesame (Sesamum indicum L.) genotypes using HPLC-PAD-ECD

The objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as alpha-tocopherol (alphaT), delta-tocopherol (deltaT) and gamma-tocopherol (gammaT) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, alphaT, deltaT and gammaT levels were 0.67-6.35 mg/g, 0.034-0.175 microg/g, 0.44-3.05 microg/g and 56.9-99.3 microg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and gammaT compared with alphaT and deltaT. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content.     

Growth,photosynthesis and ion balance of sesame (Sesamum indicum L.) genotypes in response to NaCl concentration in hydroponic solutions

A hydroponic, greenhouse experiment was conducted to assess the effects of NaCl on growth, gas-exchange parameters, chlorophyll (Chl) content, and ion distribution in seven sesame (Sesamum indicum L.) genotypes (Ardestan, Varamin, Naz-Takshakhe, Naz-Chandshakhe, Oltan, Yekta, Darab). The plants were grown in 4-L containers and subjected to varying levels of salinity (0, 30, and 60 mM NaCl). After 42 days, salt treatments induced decreases of plant fresh and dry mass, total leaf area, and plant height in all genotypes. Increasing NaCl concentration caused significant, genotypedependent decrease in the net photosynthetic rate, stomatal conductance, Chl content, and maximum quantum efficiency of photosystem II, while it increased the intercellular CO₂ concentration. Based on the dry matter accumulation under salinity, the genotypes were categorized in two groups, i.e., salt-tolerant and salt-sensitive. The impact of salt on plant ion concentrations differed significantly among the sesame genotypes and between both two groups. The plant Na⁺ concentrations were significantly lower in Ardestan, Darab, and Varamin genotypes than those found in the remaining genotypes. The highest plant K⁺ and Ca²⁺ concentrations together with the lowest Na⁺/K⁺ and Na⁺/Ca²⁺ ratios were observed in Ardestan, Varamin, and Darab genotypes. Our results indicated the presence of differences in salt response among seven sesame genotypes. It suggested that growth and photosynthesis could depend on ion concentrations and ratios in sesame.     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus

Commercial barley cultivars are difficult to transform because of the lack of an efficient regeneration system. By modifying certain components in the standard culture medium, we have developed a reproducible and more efficient regeneration system. Herbicide-resistant transgenic plants from barley (Hordeum vulgare L. cv. Conlon) were obtained using this medium. Embryo-derived callus was bombarded with pAHC25, which contains the screenable marker gus (#-glucuronidase) and the selectable marker bar (bialaphos resistance gene), both driven by the maize ubiquitin promoter (Ubi1) and followed by the nos terminator. Following bombardment, callus was transferred to callus-induction medium supplemented with 5 mg/l bialaphos for selection. Resistant calli were subsequently transferred to maintenance medium containing 5 mg/l bialaphos for further selection and finally transferred to regeneration medium with 5 mg/l bialaphos. Green shoots that developed on the regeneration medium were transferred to rooting medium containing 3 mg/l bialaphos. Eighty-five transgenic plants were obtained from 13 independent transformation events. Progeny tests showed Mendelian inheritance for the transgenes. This is the first report of the production of large numbers of transgenic plants from a commercial cultivar adapted to Midwestern US barley production.     

Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens

The leaf disc transformation/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced efficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.Abbreviations BA benzyl adenine - IAA indole acetic acid - LB Luria Broth