African Journals On-line
African Journal of Biotechnology

Volume 2 Number 8, August 2003
ABSTRACTS

Stable gene transformation in cowpea (Vigna unguiculata L. walp.) using particle gun method

J. Ikea^1,2*, I. Ingelbrecht², A. Uwaifo1 and G. Thottappilly^2,λ

¹Biochemistry Department, University of Ibadan, Ibadan, Nigeria

²International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria

*Corresponding author, Present address: CSIRO Plant Industry, Black Mountain Laboratories, G.P.O Box 1600, Canberra ACT 2601, Australia. Email: [email protected]

λ Present address: Mahyco Research Foundation, A.G. Heights, Road No. 12,Banjara Hills, Hyderabad- 500 034, India

Abstract

We investigated the possibility of transforming and obtaining transgenic cowpea (Vigna unguiculata L Walp) plants using the particle bombardment process. Meristematic explants that could give rise to whole fertile plants were used in transformation experiments with reporter and selectable marker genes driven by a 35S CaMV promoter. Conditions for optimal delivery of DNA to explants were established based on transient gus expression assays two days after bombardment. The size of microcarriers, microflight distance and helium pressure significantly affected transient expression of reporter genes.  A total of 1692 explants were bombarded with DNA-coated particles and placed on 3 mg/l bialaphos selective medium. Only 12 regenerated shoots produced seeds eventually, and all were Gus negative even though 7 gave positive PCR signals with the bar primer. Eight out of 1400 seeds from T_o plants were GUS positive. DNA from eight of the GUS positive seedlings were amplified with both the gus and bar primers in PCR analysis but only two gave a positive Southern signal. Only two of the 3557 T₂ seedlings obtained were GUS positive. However, 3 seedlings survived Basta spray. The two GUS positive and 3 Basta surviving seedlings gave positive Southern hybridisation signals. Twelve T₃ seedlings from these were GUS positive and also gave positive Southern hybridisation signals. The positive reaction of T₁, T₂ and T₃ seedlings under Southern analysis confirms the stable integration of introduced genes and the transfer of such genes to progenies. However, the level of expression of introduced genes in cowpea cells is very low and this accounted for the high mortality rate of progenies under Basta spray.

Key words: Transformation, particle bombardment, gus assay, transient expression, reporter gene, basta, bar gene.

Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1

S.T. Ogunbanwo*, A.I. Sanni, and A. A. Onilude

Department of Botany and Microbiology University of Ibadan, Nigeria

*Corresponding author: E-mail: [email protected]

Abstract

Lactobacillus plantarum F1 and L. brevis OG1 isolated from Nigerian fermented food products, produced bacteriocins that had broad spectrum of inhibition against both pathogenic, food spoilage organisms and various lactic acid bacteria.  The test organisms exhibited activities of 6400 and 3200 AU/ml respectively against Escherichia coli NCTC10418 and Enterococcus faecalis EF1, but did not inhibit Candida albicans ATCC10231 and Klebsiella sp. UCH15. Comparison of the antimicrobial spectra and characterization of the two bacteriocins were not identical. Bacteriocin produced by L. brevis OG1 was the most heat stable at 121°C for 60 min, while that of L. plantarum F1 was stable at 121°C for 10 min. The bacteriocins produced by the test isolates maintained full stability after storage for 60 days at – 20°C; partial stability after storage for 120 days at 4°C; while activity was not detected after storage for 80 to 120 days at 37°C. Bacteriocin produced by L. brevis OG1 was stable at pH range of 2.0 to 8.0 while, that of L. plantarum F1 was found to be stable at pH 2.0 to 6.0. Their active principle was proteinaceous in nature since the bacteriocins were inactivated by proteolytic enzymes, but not by other non–proteolytic enzymes. mitomycin C and uv light did not affect the activity of the bacteriocins, while chloroform extraction completely destroyed their activity. Exposure to surfactant resulted in an increase in the bacteriocin titre, except Nonidet P-40, which led to total loss of bacteriocin activity. The bacteriocins were able to pass through cellulose membranes with 100,000 KDa and 1,000,000 KDa but could not pass through one with a 10,000 KDa and 1,000 KDa molecular weight cut off. The paper concluded that the ability of bacteriocins produced by the test isolates in inhibiting a wide-range of bacteria, is of potential interest for food safety and may have future applications as food preservative.

Key words: Bacteriocins, lactic acid bacteria, indicator organisms, fermented foods, antagonistic activity. 

Protein enrichment of solid waste from cocoyam (Xanthosoma sagittifolium (L.) Schott) cormel processing using Aspergillus oryzae obtained from cormel flour

Duru, C. Chidi* and  Uma, Ngwanma U.

Department of Botany and Microbiology, University of Lagos, Akoka-Yaba, Lagos, Nigeria.

*Corresponding author; Email: [email protected],  tel: +234 802 314 1252

Abstract 

Aspergillus oryzae obtained from spoilt cormel flour was subjected to mutation treatments using X-rays, solar radiation and bleach. Following selection and screening of viable colonies on a medium containing Xanthosoma cormel solid process waste as the only carbon source, A. oryzae A7 which significantly (p < 0.05) produced more biomass at a higher growth rate than the wild parent, was chosen for protein enrichment. Protein content of substrate enriched with the mutant fungal strain was higher than that enriched with the wild strain. Addition of (NH₄) ₂SO₄, NH₄NO₃, NH₄Cl, and urea to Xanthosoma solid process waste increased the growth rate of mutant, with the highest  increase observed with urea. Medium amended with urea also had the highest protein level of 26.23% strain compared to a protein yield of 17.41% obtained in the control with no added nitrogen. The optimal temperature for protein enrichment was found to be 35°C.

Key words: Aspergillus oryzae, cormel, protein enrichment, waste, Xanthosoma.

Constructional features of a 15-litre home-made bioreactor for fed-batch fermentations

Gueguim Kana, E.B.¹*, Oloke, J.K. ¹, Lateef, A. ¹ And Zebaze Kana, M.G. ²

¹Biotechnology Centre, Ladoke Akintola University of Technology, PMB 4000, Ogbomoso, Nigeria.

²Advanced Physics Laboratory, Sheda Science and Technology Complex, Abuja, Nigeria.

*Corresponding author; E-mail: [email protected].

Abstract

A 15-litre bench-top multipurpose bioreactor was designed and constructed. The vessel is a glass type with a stainless flat headplate incorporating 9 access ports allowing for a variety of interchangeable probes and actuators. The stirring speed ranges between 0 and 250 rpm, the aeration rate (0-2 l/m), the pH control loop uses HI 1131 probe, two 100 ml bottles of HCl and NaOH solutions and operates a close feedback system. The temperature control module is a close loop using a PT 100 RTD   thermocouple and an auxiliary vessel containing a cooling solution. The aeration and feed flow rates are open loops. The system incorporates attributes of a good bioreactor design as discussed by Naraendranathan (1998). Sterility is achieved by autoclaving different units of the system. This machine has been tested on an array of local standard fermentation processes.

Key words: Fermentation, bioreactor, control, sensors, actuators.

Comparison of an African herbal formula with commercially available haematinics

Okochi Vi¹, Okpuzor J²*, Alli La¹

¹Department of Biochemistry, University of Lagos, Lagos, Nigeria.

²Department of Cell Biology and Genetics, University of Lagos, Lagos, Nigeria.

*Corresponding author; Present Address: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, West Bank, Sheffield S10 2TN, UK; Email: [email protected]. 

Abstract 

The haematological changes observed with commercially available haematinics (Fagon 9® and Chemiron®) were compared with those of a local haematinic referred to as African Herbal Formula (AHF). Results showed that AHF produced effects in haemoglobin (Hb) and packed cell volume (PCV) levels, which are reasonably comparable with the reference commercial and chemically defined haematinics.

Key words: Haematological changes, haematinics, African Herbal Formula, Trypanosome brucei brucei.

Rhesus negative pregnant women in a traditional birth home in Abeokuta, Nigeria

Idowu, O.A. ¹*, Mafiana, C.F. ¹ and Sotiloye, Dapo²

¹Department of Biological Sciences, University of Agriculture,Abeokuta, Nigeria

²Department of Obstetrics and Gyneacology Federal Medical Centre, Abeokuta, Nigeria

*Corresponding author; E-mail: [email protected]

Abstract 

In a survey of 200 pregnant women (mean age 24 years) attending a traditional birth home (TBH) in Abeokuta, Nigeria, 19 (9.5%) were found to be rhesus negative, 8 (42.1%) of which were primigravidae while 11 (57.9%) were multigravidae. 87.5% of the Rhesus negative primigravidae delivered at the TBH without being given the post partum injection of anti-D-gama globulin within 72 h of delivery, thereby having their systems likely sensitized (if baby is rhesus positive) against subsequent pregnancies involving Rhesus positive fetuses. Of the multigravidae involved in this study 27.3% women delivered live babies at the traditional birth home while the remaining 72.7% women were not seen again at the TBH.  One woman who has had an induced abortion and was carrying a second pregnancy lost the pregnancy in the course of this study. The knowledge of these women (who were mostly without formal education) on their haematological status is nil. The need to educate the public, especially women patronizing TBH, on the rhesus problem is recommended.

Key words: Pregnancy, Rhesus factor, traditional birth home.

Chloroquine-resistant Plasmodium falciparum in Sokoto, North Western Nigeria

K. Abdullahi¹, S. Muhammad¹*, S. B. Manga¹ and I. M. Tunau²

¹ Department of Biological Sciences Usmanu Danfodiyo University, P.M.B. 2346, Sokoto-Nigeria.

² Sokoto Specialist Hospital, Sokoto-Nigeria

*Corresponding author; E-mail: [email protected]

Abstract 

Three patients, 30, 2 and one and a half years, were diagnosed as having falciparum malaria and were placed on chloroquine therapy which failed. They were then placed on quinine therapy that then cleared the parasitaemia. This case report seeks to draw the attention of the presence of possible chloroquine-resistant falciparum malaria in Sokoto, North Western Nigeria.

Key words: Chloroquine-resistant malaria, Plasmodium falciparum, haemoglobin level, packed cell volume.

Genetic fingerprinting and phylogenetic diversity of Staphylococcus aureus isolates from Nigeria

Onasanya A.^1,2*, Mignouna H.D.^2,α and Thottappilly G.^2,۸

¹Department of Microbiology, Federal University of Technology Akure, Akure, Nigeria.

²International Institute of Tropical Agriculture, PMB 5320, Ibadan, Nigeria.

*Corresponding author; Present address: West Africa Rice Development Association, BP 320, Bamako, Mali. Tel.: 223 222 33 75. Fax: 223 222 86 83, E-mail address: [email protected]. 

^αPresent address: Virginia State University Agricultural Research Station Box 9061 Petersburg, VA 23806, USA.

^۸Present address: Mahyco Research Foundation, Kamalapuri colony, Hyderabad- 500073, India.

Abstract

Genetic fingerprinting of 18 different isolates of Staphylococcus aureus from Nigeria using random amplified polymorphic DNA (RAPD) was carried out. Ten out of 100 Operon primers showed polymorphism among the isolates tested generating 88 bands, 51 of which were polymorphic with sizes ranging between 200 and 3,000 bp. All the isolates were classified completely into two major groups (Sa-1 and Sa-2) with twelve different subgroups. Sa-1 group originated from human while isolates from plant and animal origins formed the Sa-2 group. The twelve different subgroups suggest adaptation of S. aureus in the different host cells. This indicates possible relationship between host origin and genetic variation among S. aureus isolates. The DNA fingerprint defined for each race of S. aureus could be useful in epidemiological studies, medical diagnosis and the identification of new strains and their origins.

Keywords: Staphylococcus aureus, foodborne-acquired infections, genetic fingerprinting; phylogenetic diversity, RAPD, polymorphism.

Isolation of total DNA from bacteria and yeast

Marco Ligozzi* and Roberta Fontana

Dipartimento di Patologia, Sezione di Microbiologia Università degli studi di Verona

Corresponding author; Phone: 0039-45-8027601, fax: 0039-45-584606, e-mail: [email protected]

Abstract 

Many procedures in molecular biology require the isolation of high quality genomic DNA. This study investigated a new method to extract DNA from Gram-negative, Gram-positive bacteria, Mycobacteria and yeasts. Guanidine thioisocyanate present in DNAzol is capable of binding DNA to silica particle column. Subsequently the silica with adsorbed DNA is washed to remove impurities and the clean DNA eluted in appropriate buffer. Results indicated that the new extraction method is simple and reproducible. This isolation technique is faster and easier to perform than the other conventional extraction methods. Finally the recovered DNA is of high quality and suitable for downstream applications.

Key words: DNAzol, DNA isolation, bacteria, yeast.