Open Access Original Research Article

Studies on Calli Mediated Multiple Shoot Regeneration from Rumex vesicarius L.

Lavanya Kakarla, Chakravarthy Rama, Mahendran Botlagunta, M. S. R Krishna

Biotechnology Journal International, Page 520-530
DOI: 10.9734/BBJ/2014/8375

Rumex vesicarius  L  is  a  valuable  potent  medicinal  herb,  which  belongs  to  family Polygonaceae. It possesses antimicrobial, anti- inflammatory, antidiarrhoeal and antioxidant properties. An attempt to study the In vitro callus induction and regeneration of plantlets from calli of leaf and nodal segments as explants has been achieved. Initially mature seeds were excised from plants grown in the departmental garden of KL University. The sterilized seed explants were inoculated aseptically to the solid basal SH media without any growth regulators for seed germination. Effective plantlets observed after 1 week of culture inoculation under maintained controlled conditions. From these In vitro plantlets, leaf and nodal segments were taken as explants for this study. These explants were inoculated on SH medium supplemented with different concentrations of BA (0.5-5.0mg/l) and 2, 4-D (0.5-3.0mg/l) for callus induction and multiple shoot regeneration. 90% of callus induction was observed on media containing BA 4.0mg/l and efficient multiple shoot induction (96.6%) was observed on media containing BA 1.0mg/l from leaf explants. 90% of callus induction and 85% of multiple shoot induction observed on media containing BA 2.0mg/l from nodal explants. Roots were induced from In vitro shoots on SH medium supplemented with 1mg/l IBA after 1 week. Leaf explants were more regenerative with 96.6% response compared to nodal explants 85%. Finally these In vitro regenerated plantlets were hardened, acclimatized and successfully transferred to the field. This protocol will be useful for mass multiplication of plantlets and maintenance of germplasm throughout the year.

Open Access Original Research Article

The Effect of Colchicine on Adventitious Shoot Regeneration from Cultured Leaf Explants of Petunia hybrida

Hassan Abu-Qaoud, Munqez J. Y. Shtaya

Biotechnology Journal International, Page 531-540
DOI: 10.9734/BBJ/2014/8465

Aims: The effect of various concentrations of colchicine on adventitious shoot regeneration of two Petunia hybrid cultivars was studied.

Study Design: All collected data were analyzed using the SAS software. One-way analysis of variance of the treatments was conducted for each experiment followed by means separation using the least significant difference (LSD) at 5% probability level.

Place and Duration of the Study: The study was conducted during January 2012 to December 2012 at the Faculty of Agriculture, An-Najah National University, Palestine.

Methodology: In vitro leaf explants of both ‘Daddy Blue’ and ‘Dreams White’ cultivars were used. Before incubation on the shoot regeneration medium, the leaves were cut transversely into two pieces and were soaked for three hours in different concentrations of colchicine solution (0.025, 0.05 and 0.1% w/v), colchicines application was done at zero incubation time and after 1, 2 or 3 weeks of culture incubation. Wet control was used by soaking explants in sterile distilled water.

Results: The ability to form shoots was highly reduced with colchicine application. Significant reduction in the regeneration frequency was observed with the two cultivars. Shoot regeneration percentage was reduced from 78.3% and 90.0 % without colchicine to 13.3% and 5.2% when colchicine was added after two weeks of incubation at 0.025% in ‘Daddy Blue’ and ‘Dreams White’, respectively. The colchicine treatments at different intervals gave significantly lower regeneration percentages than the control in both cultivars. No regeneration was observed when colchicine was added after three weeks of incubation, and at 0.1% colchicine level at all incubation treatments. A similar trend of average number of shoots produced was observed.

Conclusion: This study showed a high reduction in regeneration percentage due to colchicine application with slight difference in response between the two tested cultivars; therefore, other evaluation methods are needed in the future.

Open Access Original Research Article

Evaluation of Halobacterial Extracellular Hydrolytic Activities in Several Natural Saline and Hypersaline Lakes from Romania

Ioan Păceşilă, Roxana Cojoc, Mădălin Enache

Biotechnology Journal International, Page 541-550
DOI: 10.9734/BBJ/2014/10239

This work presents the evaluation of extracellular hydrolytic activities involved in the degrading of organic compounds from several natural salt lakes located in the southern part of Romania. The paper approaches the extracellular enzymatic decomposition activity in accordance with several physico-chemical parameters and microbial abundance. The chloride content was 13 g/l in Balta Albă salt lake, around 15 – 17 g/l in Amara salt lake, 80 g/l in Movila Miresei hypersaline lake and close to saturation, 252 g/l respectively in the case of Ocnele Mari hypersaline sample. The chemical composition revealed that salinity is due to compounds of sulphur, chloride and potassium in lakes Balta Alba and Amara. In Movila Miresei, potassium is replaced by sodium and in Ocnele Mari the salinity is due mainly to chloride and sodium. The enzyme α –glucosidase showed high values in the hypersaline lakes Movila Miresei and Ocnele Mari and a similar profile was recorded also for β-glucosidase. The activity of the enzyme alkaline phosphatase is directly correlated with the chloride content. The enzyme aminopeptidase showed high values if compared with previously mentioned enzymes.

Open Access Original Research Article

Molecular Relatedness and Morpho-Agronomic Characteristics of Congolese Accessions of Cassava (Manihot esculenta Crantz) for Breeding Purposes

G. Mamba-Mbayi, K. K. Nkongolo, R. Narendrula, P. Tshilenge Djim, A. Kalonji-Mbuyi

Biotechnology Journal International, Page 551-565
DOI: 10.9734/BBJ/2014/7423

Cassava serves as primary staple food of millions of people in the tropics and subtropics, and is used as a carbohydrate source in animal feed. Knowledge of agro-morphological characteristics and genetic relatedness is essential for an efficient recombination of varieties in a breeding program. The objective of the present study was to determine genetic relatedness and morpho-agronomic differentiation among Congolese cassava collection for breeding purposes. The morphological and agronomic characters were highly variable among accessions. Every accession could be differentiated from any other one. There were significant genotypes x location interactions for storage root yields. Root weights were positively correlated with the number of roots per plant. In general, all the improved varieties were tolerant or resistant to the Cassava Mosaic Virus (CMV) while the local (non-improved) varieties were susceptible. But the reaction to Cassava Bacterial Blight (CBB) confirmed that genetically improved accessions are susceptible and local varieties are resistant. Molecular analysis revealed that the accessions analyzed were genetically distant with 80% of genetic distance values estimated above 0.5. One local accession was an out-group that was separated from the main groupings with 100% degree of confidence. More importantly, there were no associations between genetic relationships and morphological similarities based on lobe shape, leaf colour, petiole colour, petiole orientation, and stem colour. Although the Congolese cassava genepool is small, there is enough variability to sustain a breeding program without new introductions of germplasms.

Open Access Original Research Article

Stability Evaluation and Degradation Kinetics of Ascorbic Acid in Baobab Fruit Pulp Formulated with the Seed Oil

Addai- Mensah Donkor, Matthew Tei, Jennifer Suurbaar, Abdallah Yakubu, Daniel Addae

Biotechnology Journal International, Page 566-578
DOI: 10.9734/BBJ/2014/9837

Aims: Baobab (Adansonia digitata) fruit pulp and the seed oil contain appreciable amount of vitamins and nutrients which help fight off diseases and afford commendable source of nourishment. It is essential to screen for the stability of the high vitamin C contents and validate the mechanism of its kinetic degradation in the fruit pulp with and without the oil extracted from seeds, during heat treatment. Experiments were planned according to standard methods and practices.

Methodology: Ascorbic acid degradation in both raw baobab fruit pulp and the pulp formulated with baobab seed oil were investigated at varying temperatures (25–80ºC) and at different time intervals. Kinetic data analysis was then conducted by utilizing the absorbance data collected and the validated calibration curve of standard method using DCPIP to determine the ascorbic acid contents.

Results: The results showed that reaction kinetics through heat treatments of the fruit pulp were well characterized by zero-order reactions. The activation energy (Ea) for the ascorbic acid degradation in the raw fruit pulp and the pulp treated with the seed oil were 0.000274 and 0.001903 Kcal/mol respectively. The shelf life of ascorbic acid in the formulated fruit pulp at 25ºC was approximately seven times that of the raw fruit pulp. Conclusion: The results indicate that the baobab seed oil exhibits both antioxidant enrichment and preservative properties.

Open Access Original Research Article

Molecular Identification of Cassava Mosaic Begomoviruses Associated with Cassava Mosaic Disease in the DR-Congo Using Primer Pairs

Marcel Muengula-Manyi, Claude Bragard, Stephan Winter, Adrien Kalonji-Mbuyi, Kabwe K. C. Nkongolo, Djim Tshilenge-Kanana

Biotechnology Journal International, Page 579-588
DOI: 10.9734/BBJ/2014/8112

Molecular analysis of cassava leaf samples from different regions of the Democratic Republic of Congo (DR-Congo) was conducted to identify cassava mosaic begomoviruses (CMBs)  associated with cassava infections in farmers' fields in Gandajika (Eastern Kasai), Bas-Congo and Kinshasa. Four specific primer pairs M1F/M1R, M2F/M2R, Begomo 146/Begomo 672, and EAC4F/EAC4R were used to detect East African cassava mosaic virus (EACMV) AC2, African Cassava Mosaic Virus (ACMV) AC2, ACMV DNA-A, and EACMV-UG DNA-A AC4, respectively. Based on PCR analysis, only African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV-UG) were identified in the cassava growing regions. Overall, 67% of cassava samples were infected with ACMV, 10% with EACMV-UG, and 10% with both, ACMV and EACMV-UG. No virus was detected in 13% of symptomatic samples. Thus, although EACMV-UGG is present in targeted areas, ACMV remained the most common CMB in the DR-Congo. 

Open Access Original Research Article

Study of Genetic Fidelity of Somatic Embryos - Derived Plants of Gentiana kurroo Royle from Western Himalayas - Plant of Great Medicinal Value

Anshu Sharma, Rajinder Kaur, Ravinder Raina

Biotechnology Journal International, Page 589-611
DOI: 10.9734/BBJ/2014/8653

Aim: To study the effect of plant growth regulators on direct and indirect somatic embryogenesis on Gentiana kurroo Royle – an endangered medicinal plant.

Place and Duration of Study: Department of Biotechnology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan-173230, Himachal Pradesh, India, 2010-2013.

Methodology: For induction of direct and indirect somatic embryogenesis petiole, leaf and petiole derived callus (after incubation in activated charcoal) were cultured on MS medium supplemented with a range of combinations of auxins (2,4-D, picloram, dicamba) and cytokinins (zeatin, kinetin).

Results: Somatic embryos were observed from both direct and indirect method. The best performance was observed on MS basal medium supplemented 1.0mg/l dicamba. Development of somatic embryos was observed on MS basal+0.50mg/l GA3+1.0 mg/lKn and the maximum plantlets formation was achieved when somatic embryos (directly and indirectly induced) were shifted to half strength MS basal medium.

Conclusion: In direct pathway somatic embryos were in contact with maternal tissue in a suspensor like structure. In indirect pathway, the explants first proliferated to give rise to callus before embryoids were induced. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.

Open Access Original Research Article

Molecular Identification of Biodiversity of Fusarium species Isolated from Wilted Oil Palm and Date Palm in Nigeria

N. I. Chidi, A. A. Adekunle, E. I. Eziashi, I. B. Omamor, E. E. Odigie, J. Osagie

Biotechnology Journal International, Page 612-621
DOI: 10.9734/BBJ/2014/3473

Biodiversity of sixteen Fusarium isolates, isolated from the roots of oil palm and date palm in Nigeria was studied. Ten Fusarium isolates out of the sixteen were obtained from the oil palm while six isolates were from date palm. Random Amplified Polymorphic DNA (RAPD) was used to detect the phylogenetic similarity between them. The unweighted Pair Group Method with Arithmetic average (UPGMA) dendrogram clearly separated these sixteen Fusarium isolates into five groups (clusters). The first at SC values of 100 grouped six Fusarium isolates (5, 2, 13, 9, 8 and 14,) of both oil and date palms. They belonged to F. oxysporum and F. chlamydosporum. The second at SC values of 100 had one Fusarium isolate (16) of date palm. It belonged to F. solani. The third at DC values of 71-100 grouped six isolates (6, 4, 3, 7, 11 and 10) of oil and date palms. They all belonged to F. solani. The fourth at DC value of 76 had one Fusarium isolate (1) of oil palm, it belonged to F. nelsonii. The fifth at SC value of 100 grouped two Fusarium isolates (15,12) of date palm. They belonged to F. nelsonii and chlamydosporum. The sequence alignment technique showed there were similarities between these sixteen Fusarium isolates. The analysis of RAPD showed that the sixteen Fusarium isolates were five groups while sequence alignment technique grouped them into five Fusarium isolates. The slight difference in similarity may be attributed to ecological origin and zone of the Fusarium isolates.

Open Access Original Research Article

Allelopathic Effect of Cell-Free Medium of Microcystis aeruginosa Kützing on the Chromosomal Changes in Allium cepa Root Tips and Plumule Formation of Zea mays Seedling

M. M. EL-Sheekh, H. M. Khairy, R. EL-Shenody

Biotechnology Journal International, Page 622-629
DOI: 10.9734/BBJ/2014/8414

This work confirm the allelopathic toxic effect of both cell-free medium in log and death phase of growth of Microcystis aeruginosa on chromosomal changes of Allium cepa root tips and the plumule formation of Zea mays seedlings. The results showed high significant inhibition of mitotic index in Allium cepa root tips in addition, chromosomal abnormalities were also detected. There were morphological change occurred in the plumule formation of Zea mays seedlings. The results revealed that, seed soaked in log phase cell-free medium had less inhibitory effect on plumule formation than that the seed soaked in death phase cell-free medium of M. aeruginosa. This was confirmed by the allelopathic effect of toxins released during death phase of growth was more than log phase.

Open Access Original Research Article

Analysis of Bio-gas Production from Cow Dung by Indigenous Microbial Consortia

V. O. Ifeanyi, I. W. Ossai

Biotechnology Journal International, Page 630-639
DOI: 10.9734/BBJ/2014/5834

The study analyzed quantitatively and qualitatively the biogas produced from cowdung by indigenous microbial consortia. Four 20L bioreactors were used for the study. The bioreactors were constructed to imitate the fixed batch prototype. The study lasted for six months and it was carried out at the Microbiology Laboratory of Anambra State University, Anambra State, Nigeria. Slurry was prepared in bioreactors. The substrates in the bioreactors were water and manure (WM), rumen fluid and manure (RM-1 and RM-2), medium and manure (MM). The pH, the total solids (TS), volatile solids (VS) and total volatile fatty acid (VFA) characteristics of the substrate before and after digestion were determined using standard method. Quantification and qualitative analysis of biogas production was by liquid displacement and gas chromatography methods respectively. The microbial analysis of the substrate was carried out using spread plate method. The results of the TS, VS and VFA were 400 mg/l, 92mg/l and 16.7 mg/l respectively in the predigested samples and 92 mg/l, 17.4mg/l and 28.3mg/l respectively in the post digested samples. The quantity of biogas produced at fourth month was 60 ml, 128ml and 220ml from WM, MM and RM-1 respectively. The qualitative analysis showed that the prominent biogas produced was methane. The cultural morphology revealed Gram positive rods with creamy irregular edges. The average heterotrophic counts at the end of each month for a period of four months were 18.5x105cfu/ml, 21.1 x105cfu/ml and 26.7x105cfu/ml for WM, MM and RM respectively. The results of the research concluded that high quantity of biogas can be produced using cow dung. Approaches and technology for more efficient biogas producing consortia are proposed.