Inter-simple Sequence Repeat Profiles Research Articles

Assessment of genotoxic effects of wastewater of Kitchener pool, Nile Delta Region, North Egypt, using Allium test

BackgroundIn the present study, Allium test bioassay was utilized to evaluate the effects of mixed wastewater of agricultural and sewage effluents at Kitchener pool, Gharbia governate, Middle Delta region, North Egypt. Germination indices, mitotic index and aberrations, α, β-esterase isozymes and inter-simple sequence repeat (ISSR) fingerprinting were tested by different concentrations of the wastewater (tap water as control, 25%, 50% and 100% wastewater).ResultsWater analysis recorded high levels of electrical conductivity, cations and anions compared to control, but were in the permitted limits according to FAO (Food and Agricultural Organization) except Mg2+ and K1+ were above the limits. P, N and heavy metals as Pb, Mn and Ni were also higher than the control. Germination indices showed reduction for all parameters studied (root and shoot lengths, root and shoot fresh and dry weights, and tolerance index). Mitotic index decreased, and the percentage of mitotic aberrations increased as the concentration of treatments increased and the time prolonged. Different types of aberrations were recorded in all treatments and its percentage is time and dose independent. Goat cells are the most common type recorded after different times in all treatments. The expression of α, β-esterase enzymes showed variation in different treatments compared to control and ISSR profiles showed considerable polymorphism. Concentration of 25% mixed water induced different profiles for expression of both α- and β-esterase from other treatments, and the cluster analysis based on polymorphism in ISSR fingerprinting revealed the distinction of plants treated with this concentration and the control plants from those treated with high concentrations.ConclusionIt was suggested that concentration of 25% mixed water may be suitable for growth and act as fertilizer. Mixed water from this pool may be genotoxic for Allium cepa plants at early growth if it is used for irrigation in its present form and usage of this wastewater for agricultural purposes may be harmful and must be partially treated and biologically tested before use.

Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content

Abstract. Jannah N, Pharmawati M, Uslan. 2022. Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content. Biodiversitas 23: 2690-2698. This study investigated the genetic diversity of Sterculia quadrifida R.Br.or faloak based on Inter Simple Sequence Repeats (ISSR) molecular markers and determined stomatal density and chlorophyll content from several localities in Kupang District, East Nusa Tenggara, Indonesia. For genetic diversity analysis, samples were collected from the Oelsonbai forest area of Maulafa Sub-district, Kupang City, whereas for stomatal density and chlorophyll content analysis, samples were collected from areas with various degrees of anthropogenic activity from Kupang city. DNA was isolated using Cetyl Trimethyl Ammonium Bromide (CTAB) method and amplified by three ISSR primers (UBC 811, UBC 812, and UBC 815), whereas stomatal density was observed using a microscope and chlorophyll content was determined using a spectrophotometer. Genetic data were analyzed using the unweighted pair group method with arithmetic mean (UPGMA) method and Nei & Li coefficient similarity in the MVSP software. A total of 78 ISSR bands ranging from 320-3200 bp were obtained, where populations were divided into four main clusters and six sub-clusters, similarly having moderate heterozygosity (He=0.50) not clustered based on their geographical distribution. There was a difference in stomatal density and chlorophyll content, probably influenced by anthropogenic disturbances. A conservation action plan is required to ensure the S. quadrifida's survival in the future.

Genetic diversity of Central Javanese duck (Indonesia) based on Inter Simple Sequence Repeat markers

Abstract. Susanti R, Kartikasari AD, Sasi FA, Arlinda DD. 2022. Genetic diversity of Central Javanese duck (Indonesia) based on Inter Simple Sequence Repeat markers. Biodiversitas 23: 1807-1813. Genetic resources of local livestock need to be documented and conserved to prevent genetic erosion of the germplasm. This study aimed to determine the genetic diversity of domestic ducks (Anas platyrhynchos domesticus) in Central Java, Indonesia, based on Inter Simple Sequence Repeat (ISSR) markers. There were seven types of Central Javanese ducks used in this research. DNA was extracted from feathers samples. To give rise to ISSR profile of duck DNA, 5 different ISSR markers were used. Annealing temperature used in PCR was the result of the optimization. The ISSR-PCR results showed that all primers (100%) were polymorphic. From a total of five ISSR primers, resulted in 44 obvious bands in all duck samples, with 41 (91.78%) bands being polymorphic. The quantity of alleles was numerous from 7-14 polymorphic loci, the mean quantity of alleles according to locus was 8.8, and the average PIC score for the Central Javanese duck was 0.777. The relationship investigation showed that the Central Javanese local duck was clustered into two primary offshoots, one heading to Pengging duck breeds and another one consisting of all other Central Javanese local duck. The genetic diversity among Central Javanese local duck based on ISSR primer was high. The ISSR loci were polymorphic for Central Javanese duck and may be useful for study of genetic diversity and genome evolution of Central Javanese ducks in the future.

Ultrastructural and molecular implications of ecofriendly made silver nanoparticles treatments in pea (Pisum sativum L.)

BackgroundSilver nanoparticles (AgNPs) are the most widely used nanomaterial in agricultural and environmental applications. In this study, the impact of AgNPs solutions at 20 mg/L, 40 mg/L, 80 mg/L, and 160 mg/L on cell ultrastructure have been examined in pea (Pisum sativum L) using a transmission electron microscope (TEM). The effect of AgNPs treatments on the α, β esterase (EST), and peroxidase (POX) enzymes expression as well as gain or loss of inter-simple sequence repeats (ISSRs) markers has been described. ResultsDifferent structural malformations in the cell wall and mitochondria, as well as plasmolysis and vacuolation were recorded in root cells. Damaged chloroplast and mitochondria were frequently observed in leaves and the osmiophilic plastoglobuli were more observed as AgNPs concentration increased. Starch grains increased by the treatment with 20 mg/L AgNPs. The expressions of α, β EST, and POX were slightly changed but considerable polymorphism in ISSR profiles, using 17 different primers, were scored indicating gain or loss of gene loci as a result of AgNPs treatments. This indicates considerable variations in genomic DNA and point mutations that may be induced by AgNPs as a genotoxic nanomaterial. ConclusionAgNPs may be used to induce genetic variation at low concentrations. However, considerations should be given to the uncontrolled use of nanoparticles and calls for evaluating their impact on plant growth and potential genotoxicity are justified.

Pathogenic Variation and Molecular Characterization of Macrophomina phaseolina, the Cause of Sesame Charcoal Rot

Macrophomina phaseolina (Tassi) Goid is one of the most important soil-borne fungi that affects sesame and causes charcoal rot disease, with a great economic challenge to sesame growers worldwide. Pathogenic and molecular characterizations of eleven M. phaseolina isolates collected from different geographical regions in Egypt were carried out to determine the pathogenicity and genetic diversity. Pathogenicity tests showed a pathogenic variability among the isolates. The identification of M. phaseolina isolates was confirmed by a specific primer and analyzed for genetic diversity using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The correlation between RAPD analysis of M. phaseolina isolates and their pathogenicity were estimated. Cluster analysis placed the isolates in two distinct clusters and exhibited clear correlation with their pathogenicity. Analysis of ISSR profiles revealed distinct genetic diversity among isolates and showed different clusters according to the geographical regions, in which close geographic origins tend to group nearly. The present study clearly demonstrated that M. phaseolina isolates which were obtained from different geographical regions were highly variable. RAPD and ISSR markers were suitable to reflect the genetic diversity among the studied isolates and could help in DNA finger printing which can be used in future breeding programs of sesame.

Regeneration of Viburnum dentatum L. from Alginate-Encapsulated Shoot Explants after Short-Term Cold Storage and Assessment of Genetic Stability Using ISSR Analysis

The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.

Analysis of genetic diversity in oilseed brassica germplasm through ISSR markers and isozyme profiling

Eleven Brassica germplasm were characterized through the application of 12 oligonucleotide Inter Simple Sequence Repeat (ISSR) primers. A total 1248 bands were amplified through polymorphic chain reaction and were visualized by agarose gel electrophoresis. Among the amplified marker bands 71.47% were polymorphic in nature and 352 bands were found to be monomorphic. The polymorphic bands of the amplified DNAs mostly ranged between 110 bp and 3 kb. Genetic distance among the germplasm ranged between 0.0468 and 0.7189. Moreover, three isozyme systems such as esterase, acid phosphatase and peroxidase were analyzed for allozyme variability that detected distinct 93 isozyme loci of which nearly 61.9% were polymorphic. Two dendrograms were constructed based on the ISSR profiling and isozyme data obtained through electrophoresis to find out the relatedness and phylogenetic relationship among the investigating Brassica germplasm. The clusters of the phylogenetic tree revealed 4 distinct groups of Brassica based upon their ISSR banding patterns and isozyme analysis. Nei’s genetic distance analysis provided strong information about the existence of variability among the germplasm of Brassica. All the germplasm was found to be clustered according to their respective species. Brassica carinata (Ethiopian mustard) showed individuality from all the germplasm studied and made a different branch in the phylogenetic tree suggesting its diverse origin. From the clustering pattern and genetic relationship obtained with ISSR markers and isozyme analysis, breeders can successfully identify the diverse germplasm from different cluster and use them in their future breeding program.

Centellaasiatica Morphotypes Differ Genetically as well as Macronutrients Content, Total Phenolic Content and Chemical Fingerprints of Leaves

Purpose: Centellaassiatica is an aromatic herb commonly grown in humid areas of tropical Asia. It has widely been used as a leafy vegetable and for medicinal preparations. Its medicinal and dilatory values have created a high demand for fresh consumption and value added products. Assessing morphological, biochemical and genetic diversity of existing germplasm is the essential first step in cultivation and breeding efforts. Research Method: We analyzed leaf morphological characters, macro nutrient contents, total phenolic content and the high performance liquid chromatography (HPLC) profiles of five most commonly found C. assiatica morphotypes in Sri Lanka. The genetic diversity was assessed using inter simple sequence repeats (ISSR) regions. All morphotypes were collected and grown under controlled environmental conditions. Findings: There were clear morphological differences among different morphotypes. Further, there was a significant difference (P<0.05) among them in leaf macro nutrient content and total phenolic contents. The ISSR profiles and the HPLC profiles of Heengotukola are distinctly different from others. Originality/value: Results show the significance of selecting superior morphotypes and for industrial applications for maintaining the standards.

Studies on single and double layered biocompatible encapsulation of somatic embryos in Albizia lebbeck and genetic homogeneity appraisal among synseed derived lines through ISSR markers

Embryogenic synseeds were prepared in Albizia lebbeck by encapsulating cotyledon stage somatic embryos derived from in vitro maintained embryogenic cultures in different types of Ca-alginate beads. The germination rate of somatic embryos was affected significantly by the bead type, matrix composition and germination substrate. A matrix made of 3% Na2-alginate complexed with 100 mM CaCl2·2H2O for a hardening period of 20 min provided uniform encapsulation of somatic embryo. Among different types of synseeds, type IIA, wherein somatic embryos encapsulated in a single layer of Ca-alginate matrix composed of MS medium supplemented with 2 g L−1 activated charcoal and 1.0 µM gibberellic acid (GA3) as reconstituted endosperm, was found to be the most efficient type having maximum germination rates (88.6 ± 0.51%). Incorporation of GA3 in the alginate beads stimulated greater germination of somatic embryos as against GA3 supplementation in the germination substrate. Further, viability studies on short term cold (4 °C) storage of different types of embryogenic synseeds revealed that double layered synseeds (DLS) were found comparatively more robust to withstand longer storage durations than single layered synseeds as evident by greater germination rates of the former after 4–8 weeks of refrigerated storage. Also, the elevated levels of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and leaf proline content in the plantlets derived from DLS reveals the possible role of alginate coatings in conferring alleviation to low temperature stress generated during different storage durations. Similar Inter simple sequence repeat profiles of embryogenic synseeds derived plantlets and mother tree nullifies the possible occurrence of somaclones, thereby establishing the efficacy of synseed technology for clonal propagation of A. lebbeck germplasm. Encapsulation of cotyledonary stage somatic embryos combined with high in vitro germination rates provides a feasible tool for interim storage and exchange of A. lebbeck germplasm.

Investigation of the genetic variation of anise (Pimpinella anisum L.) using RAPD and ISSR markers

Anise (Pimpinella anisum L.) is an annual spice and medicinal plant belonging to the family Apiaceae. Only a limited number of anise breeding studies are available. Therefore, genetic variation among anise landraces has not been extensively examined with molecular markers. In this study, the molecular diversity of 15 Turkish anise landraces collected from different regions of Turkey and four foreign landraces obtained from Cyprus, Syria and France were analyzed using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. The discriminatory power of these markers was assessed by calculating various marker parameters, namely percent polymorphism, polymorphism information content (PIC), resolving power (RP), and marker index (MI). The genomic DNA of 19 anise landraces was amplified with nine RAPD primers that generated 71 polymorphic bands and five ISSRs primers that produced 45 polymorphic bands. The percentage of polymorphic loci was 59.8% in RAPD profiling and 51.5% in ISSR profiling. The average PIC values obtained using RAPD and ISSR markers were 0.40 and 0.38, respectively. The mean RP (2.90 and 2.67) and MI values (1.23 and 1.05) of the RAPD assay were found to be somewhat higher than those of the ISSR assay, but ISSR was more favorable than the RAPD techniques in terms of presenting the phylogenetic relationships in anise landraces. A UPGMA cluster grouped the 19 anise landraces into two major clusters based on both markers. The RAPD and ISSR marker systems were found to be useful for determining genetic diversity in P. anisum L. and identifying variation among landraces.

Germination of Persian Walnut Somatic Embryos and Evaluation of their Genetic Stability by ISSR Fingerprinting and Flow Cytometry

Somatic embryos (SEs) can play important roles in genetic manipulation and breeding. They can be used as targets for induced mutagenesis, as material for cryopreservation and germplasm conservation, and for transformation or gene editing in support of plant improvement and proof of gene function. However, germination rates of walnut (Juglans regia) SEs are low, and the genetic stability of plantlets regenerated from them has not been explored. Here, we studied first the effects of gibberellic acid (GA3) and low temperature storage (LTS) on germination of walnut somatic embryos. Second, we assessed the genetic fidelity of plantlets regenerated from these SEs by comparing them to each other and to their cultivar of origin. Results showed that GA3 and LTS increased the walnut SE germination rate. The best rate was observed when SEs were subjected to LTS for 60 d followed by culture on a medium with either 1 or 3 mg·L−1 GA3 (56.6% and 46.6% germination respectively). Genetic stability was evaluated, using flow cytometry and 15 sets of ISSR primers. Flow cytometry indicated that all samples (i.e., regenerated and parental counterpart) showed the same peak. Amplified fragments of inter simple sequence repeats (ISSR) primers ranged in size from ≈200 to 1800 bp. All ISSR profiles of regenerants were monomorphic. Results did not show any genetic differences among plantlets regenerated from SEs or from their parental counterpart. Due to this apparent genetic stability, walnut SEs can be useful for genetic transformation and germplasm conservation.

Molecular genetic studies on abiotic stress resistance in sorghum entries through using half diallel analysis and inter-simple sequence repeat (ISSR) markers

BackgroundWater deficit stress is considered as one of the most important environmental stresses which is more harmful to strategic crops, as it reduces the final crop yield by up to 40%. Therefore, the aim of this research is to evaluate some promising and superior sorghum entries for water stress tolerance and determine the most agro-morphological parameters and reasons responsible for drought tolerance in this regard.ResultsFifteenth sorghum genotypes (five parents and their ten F1 crosses resulting from half diallel analysis) were used in this investigation under two levels of irrigation (normal and drought experiment). The recent genotypes were estimated through some physiological parameters related to water stress tolerance in sorghum; besides that, eight inter-simple sequence repeat (ISSR) primers were used to identify among the five sorghum parents and the highest five crosses resistance to water deficit conditions depending on the data calculated from all studied traits under both conditions. The following genotypes P1, P2, P3, P1 × P2, P1 × P3, P2 × P3, P2 × P4, and P3 × P4 confirmed high resistance to water deficit conditions under the drought treatment compared with the control. This high resistance was affirmed through the calculated data for all studied traits. The ISSR profile analysis showed 151 fragments as taxonomic divisions among the ten sorghum genotypes (38 of them were monomorphic and 113 polymorphic with 74.83% polymorphism).ConclusionThe entries (P1, P2, P3, P1 × P2, P1 × P3, P2 × P3, P2 × P4, and P3 × P4) were succeeded in achieving the highest concept of water deficit resistance under both conditions. Therefore, this work will be the nucleus for producing resistant sorghum varieties for drought stress in the future.

Characterization of the Genetic Diversity of Acid Lime (Citrus aurantifolia (Christm.) Swingle) Cultivars of Eastern Nepal Using Inter-Simple Sequence Repeat Markers.

Acid lime (Citrus aurantifolia (Christm.) Swingle) is an important fruit crop, which has high commercial value and is cultivated in 60 out of the 77 districts representing all geographical landscapes of Nepal. A lack of improved high-yielding varieties, infestation with various diseases, and pests, as well as poor management practices might have contributed to its extremely reduced productivity, which necessitates a reliable understanding of genetic diversity in existing cultivars. Hereby, we aim to characterize the genetic diversity of acid lime cultivars cultivated at three different agro-ecological gradients of eastern Nepal, employing PCR-based inter-simple sequence repeat (ISSR) markers. Altogether, 21 polymorphic ISSR markers were used to assess the genetic diversity in 60 acid lime cultivars sampled from different geographical locations. Analysis of binary data matrix was performed on the basis of bands obtained, and principal coordinate analysis and phenogram construction were performed using different computer algorithms. ISSR profiling yielded 234 amplicons, of which 87.18% were polymorphic. The number of amplified fragments ranged from 7–18, with amplicon size ranging from ca. 250–3200 bp. The Numerical Taxonomy and Multivariate System (NTSYS)-based cluster analysis using the unweighted pair group method of arithmetic averages (UPGMA) algorithm and Dice similarity coefficient separated 60 cultivars into two major and three minor clusters. Genetic diversity analysis using Popgene ver. 1.32 revealed the highest percentage of polymorphic bands (PPB), Nei’s genetic diversity (H), and Shannon’s information index (I) for the Terai zone (PPB = 69.66%; H = 0.215; I = 0.325), and the lowest of all three for the high hill zone (PPB = 55.13%; H = 0.173; I = 0.262). Thus, our data indicate that the ISSR marker has been successfully employed for evaluating the genetic diversity of Nepalese acid lime cultivars and has furnished valuable information on intrinsic genetic diversity and the relationship between cultivars that might be useful in acid lime breeding and conservation programs in Nepal.

Agro-Biochemical Based Characterization and Molecular Profiling of Linseed (Linum usitattissimum L.) Cultivars

Nineteen commercial cultivars of flax (Linum usitatissimum L.) were subjected to genetic diversity analysis, fingerprinting and identification of informative marker allele(s) for seed purity assessment based on agro-morphological and biochemical quality traits and inter simple sequence repeat (ISSR) marker. A total of 74 reproducible bands ranged from 120 to 1200 bp were amplified out of which 68 were polymorphic among the cultivars. Dendrogram constructed showed no clear grouping of cultivars with regard to the parentage/pedigree record and breeding centres. The 2D plot obtained with PCA analysis clearly supported the results obtained by cluster analysis. Further, ISSR profiling helped in the identification of 15 genotypes by cultivar specific bands. The findings of this study would provide a useful guide for selecting specific cultivar with a desirable agronomic trait with distinct genetic background for linseed breeding program in India.

Volatile Metabolomic Composition of Vitex Species: Chemodiversity Insights and Acaricidal Activity.

The Vitex genus (Lamiaceae) produces a plethora of metabolites that include ecdysteroids and terpenoids, some of which have demonstrated insect repellent properties. The volatile composition of several members of this genus has not been chemically defined, as many taxa are endemic to remote ecosystems. In this study, leaves were collected from the northeast of Brazil from Vitex capitata, V. megapotamica, V. gardneriana, and V. rufescens plants and examined for their chemical profile via GC-MS/FID of essential oil extracts. The analyses showed a diversity of terpenoids. Of particular note were seven-member ring sesquiterpenes which were present in great abundance; a dendrogram showed clades separating by the production of bicyclogermacrene, aromadendrane and 5,10-cycloaromadendrane sesquiterpenoids for the four species. Comparison of volatile metabolite profiles to 13 other Vitex species showed strong similarities in the production of some monoterpenes, but varied by their production of larger terpenes, especially those with gem-dimethylcyclopropyl subunits on seven-member ring compounds. From this work, we suggest that the sesquiterpene skeleton with seven member rings is a good chemosystematic biomarker candidate for the Vitex genus. Separation using this biomarker was then validated using Inter-Simple Sequence Repeat profiling. Lastly, experiments examining the toxicity of these four oils against the coconut mite Aceria guerreronis showed that only the oil of V. gardneriana had strong acaricidal activity, with an LC50 of 0.85 mg/mL, thus demonstrating its potential for use as a natural pesticide.

In vitro propagation and assessment of genetic stability of acclimated plantlets of Cornus alba L. using RAPD and ISSR markers.

Cornus alba L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegetative propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of C. alba cultivars ‘Aurea’ and ‘Elegantissima’ were cultured on media enriched with six different sources of macronutrients. Media were supplemented with either N6-benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L−1 BA, 0.1 mg L−1 NAA, and 20–30 g L−1 sucrose. Rooting of regenerated shoots was achieved by an in vitro method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L−1 NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for ‘Aurea’ and 90% for ‘Elegantissima’. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymorphic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197–199 and 184–187 distinct and reproducible band classes, respectively, in ‘Aurea’ and ‘Elegantissima’ plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant.

Pittosporum eriocarpum Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. In vitro regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine in vitro raised plantlets with mother plant. DNA fingerprints of in vitro regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of P. eriocarpum. The protocol would be useful for the conservation and large scale production of P. eriocarpum to meet the demand for medicinal formulations and also for the re-introduction of in vitro grown plants in the suitable natural habitats to restore the populations.

In vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants

The present study describes a simple, efficient and one step regeneration system for rapid shoot proliferation and in vitro rooting of Cannabis sativa nodal explants using meta-topolin (mT), an aromatic natural cytokinin. The best response in terms of explants producing maximum number of shoots with maximum shoot length and percent explants producing shoots was recorded on Murashige and Skoog (MS) medium supplemented with 2μM mT. Shoots multiplied on the same medium for two sub-cultures were able to induce healthy roots within 4–6 weeks. A separate medium containing auxin was not required for root induction. Regenerated plantlets were successfully acclimatized and hardened off in the climatic controlled grow room with 100% survival rate. Genetic fidelity of in vitro propagated plants was tested using inter simple sequence repeat (ISSR) markers. Our results show that all the ISSR profiles from in vitro propagated plants were monomorphic and comparable to that of the mother plant, thereby confirming the genetic fidelity. Qualitatively and quantitatively, cannabinoid profiles and the content, using gas chromatography-flame ionization detector (GC–FID), in mother plant and in vitro propagated plants were found to be similar to each other. Furthermore, regenerated plants were eco-physiologically and functionally comparable to that of the mother plant. The maximized regeneration protocol using mT is thus effective and safe for large scale production of true to type C. sativa plants.

Cost-effective tissue culture media for large-scale propagation of three commercial banana (Musa spp.) varieties

ABSTRACTCost-effective tissue culture protocols have been established for the commercial multiplication of three banana varieties, ‘Rasthali’ (AAB – Silk), ‘Grand Naine’ (AAA – Cavendish), and ‘Udhayam’ (ABB – Pisang Awak). Reverse osmosis water and 3% (w/v) table sugar were used as the low-cost water and carbon source, respectively. Six different gelling agent treatments were tested: sago alone (T1), Isabgol alone (T2), sago + agar (T3), Isabgol + agar (T4), sago + Isabgol (T5), and agar alone as a control (T6). Full-strength Murashige and Skoog (MS) medium supplemented with 3 mg l–1 6-benzylaminopurine (BAP) and 1 mg l–1 indole-3-acetic acid (IAA) were used for culture initiation and subculturing. Rooting was accomplished on low-cost MS medium containing 1.0 mg l–1 α-napthaleneacetic acid (NAA), 1.0 mg l–1 indole-3-butyric acid (IBA), and 250 mg l–1 activated charcoal. Statistical analysis indicated that sago + Isabgol (T5) produced the maximum number of shoots (10 per explant) in ‘Udhayam’ and ‘Rasthali’, while sago alone (T1) produced the maximum number of shoots (6 per explant) in ‘Grand Naine’. The genetic stability of tissue-cultured banana plantlets produced using these low-cost substitutes was assessed using inter-simple sequence repeat (ISSR) markers. The results indicated that the ISSR profiles of the five treatments and the control (T6) were similar, indicating genetic stability using these cost-effective tissue culture protocols. Reductions in cost over the control (l–1 of MS medium) ranged from 65% to 86%, while the per plant production cost was reduced by 12.5%–20.0%. Adoption of these treatments (T1–T5) as low-cost tissue culture protocols for in vitro propagation would reduce production costs significantly, leading to an expansion of the area planted with tissue-cultured banana, thereby increasing productivity.

Evaluation of genotoxicity of Artemisia herba-alba and Jasmina montana extracts against Zucchini Yellow Mosaic Virus in Cucurbita pepo

Zucchini yellow mosaic virus (ZYMV) presents a critical problem in cucurbit production in Egypt and worldwide due to generation of aggressive viral strains. Therefore, novel approaches must be used for control of viral infections. In this study the effects of extracts from Artemisia herba-alba and Jasmina montana on ZYMV multiplication in Cucurbita pepo plants were evaluated. For this purpose, SDS-PAGE protein profiles and enzyme activities were examined and induction of resistance by plant elicitors against ZYMV was studied. The results showed that application of the aqueous extracts of A. herba-alba or J. montana extracts prior to ZYMV inoculation gave 100% inhibition of virus infection. The C. pepo plants treated with mixture of A. herba-alba extract and virus inoculum were 100% resistant. Treatment of squash plants with extracts resulted in production of proteins and phenolic compounds and activated various defense-related enzymes in the squash plants. Genetic variability in DNA genomes of treated and untreated plants was compared. Random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) profiles for DNA samples differed among treatments and control plants with visible change as indicated by the appearance or disappearance of DNA amplicons. The changes in RAPD and ISSR profiles of representative squash plants can be exploited for an understanding of induced systemic resistance. These elicitors, when activated, induced resistance in squash plants. Consequently, elicitors should be considered in the breeding programs for protection against ZYMV.