Open Access Original Research Article

Investigation of Differential Genes Expression in the Genome of Epipactis flava Seidenf. (Orchidaceae) under Flooded Condition Using cDNA-SRAP Analysis

Waroon Suwankitti, Surin Peyachoknagul, Somjit Homchan, Pattamon Sang-In, Anupan Kongbangkerd, Maliwan Nakkuntod

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BJI/2018/43501

Epipactis flava Seidenf. is classified into a rare type of orchid species, a rheophyte. The specific habitat for its life cycle requires a strong current and flooding time. In this study, differential gene expression between submerged and non-submerged conditions was studied using the cDNA-SRAP method. RNAs were extracted from rhizomes at 0, 6, 12, 24 and 48 hours after submergence. Seven candidate genes were selected to study the relative gene expression by real-time RT-PCR and found to be upregulated in submergence condition. Six of these, RNA polymerase sigma factor gene, ATPase gene, Plant homeodomain finger Alfin-like genes, DNA-3-methyladenine glycosylase gene, and Allene oxide synthase gene were induced to the highest level within 12 hrs. Subsequently, the expression of five genes decreased at 24 and 48 hrs of treatment except for UDP-glucosyl transferase gene which showed stable expression at all time. Apart from others, dolichyl-diphosphooligosaccharide glycosyl transferase gene was induced to the highest level at 24 hrs and was stable until 48 hrs. This suggested that the induction of genes expression by flooding occurred within 12-24 hrs and most genes in this finding involved in stress responses which were easily upregulated in rheophytic plants during submergence. The more differentially expressed genes should be analysed for a further understanding of the rheophytic habit of this plant.

Open Access Original Research Article

Use of Palm Male Inflorescence and River-Sand as Acclimatization Substrate for Plantain (Musa sp.) Cultivars

Ekwa Yawa Monono, Jemimah Evenye Ngale, Levai Lewis Dopgima, Akongte Peter Njukang

Biotechnology Journal International, Page 1-10
DOI: 10.9734/BJI/2018/43879

The objective of this work was to investigate the use of Palm Male Inflorescence (PMI) and river-sand as substrate for the acclimatization of plantain. Plantlets from three plantain cultivars (Batard, Ebanga and French Clair) were obtained after 16 weeks of tissue cultures and the plantlets were subjected to routine acclimatization under screen house conditions using two different substrates mixed in different ratios (100% Sand, 100% PMI, 75% PMI, 60% PMI and 50% PMI). The experiment was arranged in a completely randomized design with ten (10) replications; each replicate consisting of one micro-pot. The different substrates used significantly influenced the performance of the cultivars. The best medium for acclimatization for French Clair was 60% PMI in terms of percentage survival of plantlets (96.88%), plantlet height (6.03 cm), diameter (0.60 cm), number of leaves (4.42 leaves), leaf area (20.23 cm2), leaf emergence rate (1.64), number of roots (7.70 roots), and root length (18.86 cm). Ebanga plantlets had the  best results with 75% PMI in terms of percentage survival of plantlets (96.88%), plantlet height (6.18 cm), diameter (0.62 cm), number of leaves (4.39 leaves), leaf area (20.48 cm2), leaf emergence rate (1.76), and total fresh weight (10.05 g). Meanwhile with Batard cultivar, 50% PMI  was the best substrate in terms of percentage survival of plantlets (96.88%), plantlet height (4.41 cm), diameter (0.55 cm), number of leaves (4.55 leaves), leaf area (12.96 cm2), leaf emergence rate (1.55), and number of roots (5.73 roots). This study clearly show that PMI can be a viable substrate to use with sand in plantlet acclimatization; however, the different plant cultivars had optimal result at different proportions of PMI.

Open Access Original Research Article

DNA Analysis of Ralstonia solanacearum Based on 270-bp PCR-amplified Fragment from the Lowlands and Highlands of Kenya

E. K. Kago, Z. M. Kinyua, J. M. Maingi, P. O. Okemo

Biotechnology Journal International, Page 1-15
DOI: 10.9734/BJI/2018/43906

Aims: Bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease resulting in tremendous losses of economic crops such as plants in the Solanaceae. Studies have shown that R. solanacearum is spreading from the lowlands to the highlands. This has seen increased need for understanding the genetic diversity of R. solanacerum strains common in these areas as a basis for better strategies in their control.

Methodology: Sixty-nine bacteria isolates obtained from various wilting plant hosts (Tomato, capsicum and potato) from 11 different sites in Nyeri, Nyahururu, Kirinyaga, Kiambu, Nakuru Murang’a and Embu were subjected to molecular analysis.

Results: All the bacteria isolates were confirmed to be R. solacearum following PCR amplification of about 270-bp fragment using specific primers OLI 1-F and Y2-R. Based on the targeted 16 S rDNA sequences using primers OLI 1 and Y2, the 69 bacteria isolates had 98 to 100% identity with other DNA sequences for R. solanacearum isolates deposited in the NCBI database. Analysis of genetic differentiation showed there were total of 26 haplotypes from the 11 studied populations. The total number of segregating sites in all populations was 225.

Conclusion: Through this study, it was realized that the main causative agent of bacterial wilt in potatoes, tomatoes and capsicum grown in the lowland and highland regions in Kenya is R. solanacearum. The isolates are in two main groups (Cluster A and B) that represent mainly the phylotypes I and II respectively.

Open Access Original Research Article

α-L- rhamnosidases Produced under Solid State Fermentation by Few Aspergillus Strains

Sarita Yadav, Dhirendra Kumar, K. D. S. Yadav

Biotechnology Journal International, Page 1-8
DOI: 10.9734/BJI/2018/43518

The production of α-L-rhamnosidase  from Aspergillus ochraceous MTCC -1810, A. wentii MTCC- 1901, A. sydowii MTCC- 635, A. foetidus MTCC-508 under solid- state fermentation using easily available agro- industrial residues such as corn cob, rice bran, sugarcane bagasse, wheat bran and citrus peel as substrate. Among these, sugarcane bagasse in combination with naringin and sucrose were found to be the best substrate. The α-L-rhamnosidase production was highest after the 4th day of incubation at 30ºC and a substrate to moisture ratio of 1:1 w/v. Supplementation of the medium with 10% naringin caused the maximum production of the enzyme. The temperature optima and pH optima of α-L-rhamnosidases were determined in the range of 50-60ºC and 4.0-5.0 respectively. The α-L-rhamnosidases secreted from the above fungal strains is suitable for the debittering of orange fruit juice.

Open Access Original Research Article

Cost Effective Pilot-scale Ajmalicine Production by Catharanthus roseus Cell Suspension Cultures in a 100 Lit Bioreactor

Devanand P. Fulzele, Ajay G. Namdeo

Biotechnology Journal International, Page 1-15
DOI: 10.9734/BJI/2018/43510

Aims: The cosmic production of biomass and bioactive compounds at pilot scale with minimum production costs is an important task to achieve feasible production process of corresponding secondary metabolites at a commercial level.

Materials and Methods: The cell suspension cultures of Catharanthus roseus in MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (9.05 µM), kinetin (4.52 µM) were scaled up in a pilot plant bioreactor (100 lit). The cost of production was reduced by addition of substitute carbon source in a basal medium which hardly costs 30% in the medium. Preliminary studies were performed in the 7-lit bioreactor. A 100 lit stainless steel bioreactor equipped with helical impeller top mounted was used for scale-up of C. roseus suspension cultures and ajmalicine production.

Results: The culture medium reduced the cost by 36% by addition of commercial grade sugar whereas medium consist of tissue culture grade sucrose costs 53 USD per 100 lit. The suspension cultures were cultivated in a 100 lit bioreactor containing MS medium fortified with cost-effective carbon source produced ajmalicine 73.18 mg/l DW and achieved 36 kg of fresh biomass on day 20.

Conclusion: The results of the present finding demonstrated the feasible and cost-effective production process of ajmalicine at pilot scale.