Open Access Study Protocol

An Optimised Cetyltrimethylammonium Bromide (CTAB)-Based Protocol for Extracting RNA from Young and Old Cassava Leaves

C. O. Orek

Journal of Advances in Biology & Biotechnology, Page 1-7
DOI: 10.9734/JABB/2018/45370

Ribonucleic acid (RNA) integrity, quality and quantity are critical in most plant molecular studies. Extracting high quality RNA from cassava leaves and other recalcitrant plant tissues are difficult due to the presence of polysaccharides, polyphenols and other secondary metabolites that often co-precipitate with the final RNA extract. This is an optimised a CTAB-based method that suitably extracts RNA from the polysaccharide-rich cassava leaves. The modifications were introduced into a version of the CTAB protocol as described by Gasic and colleagues [1]. The changes included an increased rate or use of Extraction Buffer (EB) for every gram ground leaf tissue (20 ml EB per 1 gram tissue), incubation of the Tissue-EB and Chloroform: Isoamyl alcohol (24:1) mixture at a lower water-bath temperature of 50°C and all centrifugation steps carried out at 4°C. In addition, the EB contained a higher concentration of soluble polyvinylpyrrolidone (PVP-K-30). The pH of sodium acetate was lowered to 5.2 and a final two-step high molarity (10M) Lithium Chloride (LiCl) precipitation was applied. Ethyl alcohol concentration was raised to 100%. The modified CTAB method produced RNA of high concentration (>1.0 μg), high A260:A280 and A260:A230 ratios (> 2.0) and high integrity (distinct and visible 28S rRNA and 18S rRNA bands) from young and old cassava leaves, compared to RNA (from the same leaf tissues) generated by several other published methods or commercial kits. The protocol is efficient, simple, and reproducible and is therefore recommended for RNA extraction from metabolite-rich cassava leaves or plants with similar tissues.

Open Access Original Research Article

Assessment of Genetic Variability Estimates of Selected Traits in Irish Potato Mutants

Emmy Chepkoech, Miriam G. Kinyua, Oliver Kiplagat, Julius Ochuodho, Stephen Kimno, Leah Boit

Journal of Advances in Biology & Biotechnology, Page 1-8
DOI: 10.9734/JABB/2018/44850

Aims: For an effective potato breeding strategy, knowledge of the genetic parameters of traits, such as heritabilities and genetic correlations are essential, hence the need to assess the genetic variability estimates of yield-related traits in Irish potato mutants

Study Design: At M1V1 generation, there was no replication of the mutant minitubers because each does not maintain the same genetical constitution after irradiation.  In M1V2 and M1V3 generation the tubers were replicated 3 times in alpha lattice design.

Place and Duration of Study: Irradiation was done at the Plant Genetics and Breeding Laboratories (PGBL) at IAEA/FAO Seibersdorf, Vienna, Austria. After mutation induction, the mutant microtubers (consisting of Asante, Mpya and Sherekea) were was transported to Kenya, University of Eldoret for establishment between April 2015 and March 2017.

Methodology: A total of 30 tubers each of the three potatoes was sent for irradiation. Two in-vitro radio-sensitivity tests were developed involving different tissues: Irradiation of in vitro nodal cuttings (without leaf) followed by in vitro shoot propagation and irradiation of in vitro nodal cuttings (with leaf) followed by direct in vitro micro-tuber production. After mutation induction, a total of 570 mutant microtubers (Asante 230, Mpya 160, Sherekea 180) were developed from the three potato varieties and was transported to Kenya, University of Eldoret for the establishment. The M1V1 microtubers were established in the greenhouse while M1V2 and M1V3 generations of mutants were planted at the at the University of Eldoret research field.

Results: It showed that the highest positive heritability percentage (H2) estimates in Mpya and Sherekea mutants were in plant height with 81.51% and 87.7% respectively.

Conclusion: Tuber number exhibited high heritability estimates displaying that induced mutation was successful in the development of new potato genotypes which be used in future breeding programs.

Open Access Original Research Article

Effect of Light Quality on the Growth of Microalgae Haematococcus lacustris

L. V. K. Trang, M. Trinh-Dang, T. V. T. Nguyen, N. T. Trang, N. T. Suong

Journal of Advances in Biology & Biotechnology, Page 1-8
DOI: 10.9734/JABB/2018/44933

Haematococcus lacustris (formerly Haematococcus pluvialis) has been widely known as a promising natural source of astaxanthin. To produce a higher amount of astaxanthin content in H. lacustris, two major aspects are considered including increasing biomass production of green cells or enhancing astaxanthin accumulation in red cysts.  The aim of this study was to investigate the effect of light quality of Light emitting diodes (LEDs) on the growth of H. lacustris. In the present research, the effect of different light spectrum, light intensity on the growth of H. lacustris was simultaneously investigated for 11 days of cultivation period. The experiments were performed in Bold’s Basal Medium, illuminated with a circadian light/dark cycle of 16/8 and maintained at 25ºC.  A mixture of red and blue at the light intensity of 30 µmol photons m-2 s-1 was found to be the most effective light quality for growing. The highest cell density obtained with mixed red-blue illumination at the light intensity of 30 µmol photons m-2 s-1 after 9 days of cultivation was 49.57 (105 cells/ml) corresponding to the specific growth rate of 0.509 d-1.

Open Access Original Research Article

Bioremediation Potential of Aspergillus clavatus and Pichia spp. on Oil Spill Dispersant Polluted Marshland

Janet Olufunmilayo Williams, Barisi Samuel-Penu

Journal of Advances in Biology & Biotechnology, Page 1-14
DOI: 10.9734/JABB/2018/46012

Aims: The aim of the study is to identify the bioremediation potential of Aspergillus clavatus and Pichia spp. of oil spill dispersant on polluted marshland in Kegbara-Dere community in Gokana Local Government Area of Rivers State.

Study Design: The study employs experimental assay and statistical analysis of the data and interpretation.

Place and Duration of Study: Polluted marshland were collected from Kegbara-Dere community in Gokana Local Government Area of Rivers State with sterile spade from three different spot at the same location and put in black polythene bag and transported to the microbiological laboratory within 24 hours for physicochemical and microbiological analyses.

Methodology: Standard microbiological techniques were used to enumerate, isolate and identify the fungi. Aspergillus clavatus and Pichia spp. in contaminated soil samples with oil spill dispersants were observed for bioremediation potential for a period of 1day, 7days, 14days, 21days and 28days respectively.

Results: The results indicate that the total hydrocarbon (THC) content of soil samples at day one was 8006.58 mg/kg but reduces at day 28. Thus THC was: marshland polluted with Seacare (CTRL and CTRL 2b), 2988.49 mg/kg and 3453.95 mg/kg> polluted marshland and Aspergillus clavatus, 2942.11 mg/kg> polluted marshland and Pichia, 2973.68 mg/kg> polluted marshland and consortium, 1473.68 mg/kg. The bioremediation potential of the fungi expressed in percentage was: marshland and Seacare (control), Aspergillus clavatus, Pichia and consortium, 56.86%<64.50%>62.86%<81.59%.

Conclusion: These results show that bioremediation of dispersant pollutants by activation of naturally occurring microorganisms such as Aspergillus clavatus and Pichia spp. will be cost effective in cleaning up the environment.

Open Access Original Research Article

Nematicidal Properties of Endophytic Fungi Isolated from Some Musa Species in Cameroon, for the Management of Radopholus similis and Platylenchus coffeae

Eneke Esoeyang Tambe Bechem, Stéphane Fadanka Wapouo, Pierre Michel Loubana

Journal of Advances in Biology & Biotechnology, Page 1-19
DOI: 10.9734/JABB/2018/45952

Aims: To evaluate the effectiveness of endophytic fungi isolated from banana and plantain cultivars, in the control of Radopholus similis and Pratylenchus coffeae in vitro.

Place and Duration of Study: African Centre for Research on Bananas and Plantain (CARBAP) Njombé and New Biotechnology Laboratory, Faculty of Science, University of Buea, between June 2017 and May 2018.

Methodology: Endophytic fungi were isolated from banana and plantain roots and corms using Potatoes Dextrose agar. The isolates were initially identified based on their cultural and micro-morphological characteristics. They were then screened for nematicidal activity against R. similis and P. coffeae in vitro.  DNA was extracted from each isolate which showed nematicidal activity and the 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) of the rDNA were amplified and sequenced in order to confirm their identity.

Results: A total of eighteen endophytic fungal isolates were obtained from the plant materials. All isolates demonstrated nematicidal activity on both nematodes with the mortality ranging from 5-100%. Filtrate from three isolates: MB2, GN4 and BT1 were the most effective and were able to kill 100% of R. similis and P. coffeae after 12 hours of incubation. Filtrate from isolate GN4 was still very effective (100%) after 12 hours when diluted to 1/100. Solutions of broken mycelia from most of the isolates killed the nematodes, but the nematicidal activity was lower than that for culture filtrates. Isolates were identified to belong to five genera: Fusarium, Aspergillus, Trichoderma, Ceratobasidium and Mucor.

Conclusion: The findings here clearly demonstrated the potential of these fungi as biological control agents against R. similis and P. coffeae. However, more work is required to help identify the active ingredients exuded by these fungi in culture which has been demonstrated to have nematicidal effects.