Open Access Original Research Article

Somatic Embryogenesis and Plantlet Regeneration from Protoplast Culture of Stevia rebaudiana

Marisol Lopez-Arellano, Seema Dhir, Nilmarie Colón Albino, Anais Santiago, Terrica Morris, Sarwan K. Dhir

Biotechnology Journal International, Page 1-12
DOI: 10.9734/BBJ/2015/13884

Somatic Embryogenesis and Plantlet Regeneration from Protoplast Culture of Stevia rebaudiana

In order to develop a high-efficiency and reproducible regeneration protocol for Stevia protoplasts, various factors such as type and concentration of enzymes, osmoticum, incubation time, plant material type and age were studied. Protoplasts were successfully isolated from leaves of four-week-old in vitro grown plants using an enzyme mixture comprising of 2% (w/v) Cellulase Onozuka R-10, 1.5% (w/v) Macerozyme Onozuka R-10, 0.2% (w/v) Driselase and 0.1%(w/v) Pectolyase Y-23 in 0.5 M mannitol, 2.5 mM CaCl2.2H2O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH of 5.8. Approximately 8.4±0.40x106 protoplasts g-1fresh weight with 98.8±1.39% viability was obtained after incubating in enzyme solution for 4 hours in dark. Viable protoplasts were collected by centrifugation in the presence of 16% sucrose solution. Protoplasts at density of 5x10mL-1were cultured on modified KM8P medium supplemented with 0.2 mg L-1 2,4-dicholorophenoxyacetic acid (2,4-D), 1 mg L-1 α-naphthalene acetic acid (NAA), 0.5 mg L-1 zeatin, 0.15 M sucrose and 0.3 M mannitol by agarose-bead or thin layer liquid culture technique. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 2-3 days and micro- colonies were formed within 4 weeks. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Compared to liquid culture, agarose bead culture improved division frequency almost 1.5 times effectively and showing a plating efficiency of 13% and 9.1% respectively with survival rate of 23.5% to 14.8%. Upon transfer to Murashige and Skoog’s medium (MS) with 1 mg L-1BA, alone or in combination with NAA or 2, 4-D at 0.1 mg L-1, protoplast-derived calli produced complete plantlets through somatic embryogenesis in 8-weeks. The regenerated plants survived in soil and all were normal with respect to morphology and growth characters. This protocol might lead to the improvement of the Stevia through somatic hybridization, somaclonal variation and genetic engineering by using protoplast based regeneration system.

Open Access Original Research Article

Effect of Arsenic (As) on the Spermatogenesis of Black Bengal Goat (Capra hircus) Reared at the Arsenic Prone Area of Mymensingh District in Bangladesh

M. A. Awal, J. Alam, M. M. Hossain, M. Matsumoto

Biotechnology Journal International, Page 13-23
DOI: 10.9734/BBJ/2015/12325

Effect of Arsenic (As) on the Spermatogenesis of Black Bengal Goat (Capra hircus) Reared at the Arsenic Prone Area of Mymensingh District in Bangladesh

Aim: Arsenic (As) alters the spermatogenic process as well as testicular histology has been reported in different species of domestic, wild, aquatic life, and laboratory animals. The present study was conducted to investigate the impact of toxicity of arsenic poison on the spermatogenesis of the Black Bengal goat at the most arsenic polluted area of Mymensingh district in Bangladesh by using histopathological techniques.

Methods: A total of 12 adult Black Bengal male goats (Capra hircus) were used in this study. Among these, 6 were selected from the arsenic polluted area, and the rest were from the less contaminated area as control. Goats were sacrificed by using conventional animal killing method adopted in the laboratory. Both the right and left testes were collected aseptically. Testicular tissues were cut perpendicular to the long axis of the testis and preserved in Bouin’s solution. Paraffin block was made and tissue sections were cut at 5-μm in thickness. Tissues were processed for routine hematoxylin and eosin and Periodic Acid-Schiff (PAS)-hematoxylin stains. Thickness of tunica albugenia, spermatigenic cell layer, diameters of the seminiferous tubules, number of spermatogenic, sertoli, and leydig cells were counted and tabulated. Apoptotic spermatogenic cells were detected by using Apoptosis Detection Kit. The data collected wastatistically analyzed for any significant differences between the arsenic exposed and control goats.

Results: Our results revealed comparatively increased thickness of the tunica albugenia, wide inter-tubular spaces, low height of the spermatogenic cell layer, decreased diameter of the seminiferous tubules, decreased spermatogenic, sertoli, and leydig cell counts, and marked increased of apoptotic spermatogenic cells in the arsenic affected goats. The data differences between the arsenic affected and control goats were statistically significant (P<0.01).

Conclusion: Our histopathological study revealed alteration of testicular tissues in arsenic affected goats. This morphological changes of testes significantly affected on the spermatogenic processes. But it was not possible to determine the possible stage of the spermatogenesis was interrupted by the arsenic. Decreased number of spermatogenic, sertoli, and leydig cell counts, and distinctly increased number of apoptotic spermatogenic cell indicates high toxic effects of arsenic poisoning on the male gonad. The mechanism of action of toxicity of the arsenic could not be understood clearly. It is suggested here that the Black Bengal goats can be experimentally used as animal model in the laboratory for investigating the role of arsenic on the reproduction of the domestic animals.

Open Access Original Research Article

Effect of kLa and Fed-batch Strategies for Enhanced Production of Xylitol by Debaryomyces nepalensis NCYC 3413

K. Himabindu, Sathyanarayana N. Gummadi

Biotechnology Journal International, Page 24-36
DOI: 10.9734/BBJ/2015/14273

Effect of kLa and Fed-batch Strategies for Enhanced Production of Xylitol by Debaryomyces nepalensis NCYC 3413

Aims: To evaluate the effect of volumetric oxygen transfer coefficient on the production of xylitol by Debaryomyces nepalensis and to enhance the yield and productivity of xylitol by fed-batch fermentation using xylose as substrate.

Place and Duration of Study: All experiments were performed at the Applied and Industrial Microbiology Laboratory, Indian Institute of Technology Madras, from March 2013 to May 2014.

Methodology: Batch cultivations were carried out in a 7.5 L fermentor under various oxygen transfer coefficients in the range 12 to 39.6 h-1 in order to understand the effect of oxygen on xylitol production. Fed-batch studies were performed in 2.5 L bioreactor with a working volume of 1 L. The cultures were initially grown as batch cultures.  Feed containing xylose and nitrogen source was added to the medium intermittently.

Samples were periodically collected at regular intervals of time and the concentrations of xylose, xylitol and glycerol were determined by HPLC.

Results: Maximal xylitol yield (0.64 g/g) and productivity (0.43 g/L·h) were obtained at kLa 13.68    h-1. The effect of pH was also studied at this kLa. A pH value of 6.0 was found to be favorable for xylitol accumulation. Fed-batch fermentation involving feeding of xylose and nitrogen source was used for xylitol production by D. nepalensis. Within the fed-batch phase, the yield of xylitol was 0.83 g/g and the productivity was increased to 0.83 g/L.h with a final product concentration of 90 g/L.

Conclusion: Higher kLa favors biomass production whereas product formation was favored at lower kLa. Fed-batch process resulted in enhancement of final product concentration by 73%.

Open Access Original Research Article

Effect of Process Variables on Survival of Bacteria in Probiotics Enriched Pomegranate Juice

Mina Khanbagi Dogahe, Kianoush Khosravi-Darani, Azadeh Tofighi, Mehrnaz Dadgar, Amir Mohammad Mortazavian

Biotechnology Journal International, Page 37-50
DOI: 10.9734/BBJ/2015/12114

Effect of Process Variables on Survival of Bacteria in Probiotics Enriched Pomegranate Juice

Aims: The consumption of foods and beverages containing probiotic microorganisms is a growing, global consumer trend. In this research, production of probiotic pomegranate juice containing Lactobacillus plantarum and Lactobacillus delbrueckii was studied.

Study Design: Plackett-Burman statistical design was used to evaluate the impact of eleven process variables on the viability of both probiotics. Impact of incorporation of grape juice, tomato juice and pomegranate peel extract as well as phenolic compounds and vitamins have been investigated.

Place and Duration of Study: Department of Food Science and Technology, Varamin Branch, Islamic Azad University, Varamin, Tehran, Iran, between Sep 2012 and July 2013.

Methodology: Pomegranate juices were inoculated with probiotic bacteria and their survival was evaluated every week by pure plate method. The effect of 11 variables (in two levels) on survival of bacteria by the statistical design of Plackett-Burman was evaluated. For this purpose, 12 treatments in triplicate by the Minitab (version = 11.0) software at significant levels α= .01 were analyzed.

Results: The highest survival rate of L. plantarum (4.74 ×106 CFU/mL) and L. delbrueckii (4 ×106 CFU/mL) was obtained by 10% v/v inoculation of a 48 h inoculum culture in MRS broth medium to enriched pomegranate juice (10% v/v Grape juice, 5% v/v tomato juice, 0.1% v/v pomegranate peel extract and 2.0 g.L glucose) which was inoculated in anaerobic condition for 72 h at 37ºC and kept for 2 weeks at environment temperature. Sensory evaluation shows the probiotic juice was accepted by consumers with no significant difference in comparison to control in terms of taste, odour and overall acceptability (P>.05).

Conclusion: The results of this study suggest that grape, tomato juices and pomegranate peel extract exert a protective effect on L. plantarum and L. delbrueckii viability under acidic condition of pomegranate juice and storage time, which was associated with the chemical composition of them. This study indicates that develop of probiotic pomegranate juices with acceptable viability and stability of the probiotic is possible.

Open Access Original Research Article

Biochemical Characterization, 16S rRNA Sequence Analysis and Multiple Sequence Alignment of Bacteria Isolated from Fermented and Unfermented Coconut

Hailu Weldekiros Hailu, Viktor Abdallah, Ari Susilowati, Saleh Muhammed Raqib, Ali Ramadan Ali ALatawi

Biotechnology Journal International, Page 51-61
DOI: 10.9734/BBJ/2015/14523

Biochemical Characterization, 16S rRNA Sequence Analysis and Multiple Sequence Alignment of Bacteria Isolated from Fermented and Unfermented Coconut

Coconut (Cocos nucifera L.) is a tropical and subtropical plant which has great versatility due to many benefits of its different parts. Appropriate methods such as biochemical, morphological, serological, physiological and molecular techniques are required to clearly differentiate beneficial from harmful bacteria that are found in coconut. The objectives of this research were to characterize bacterial diversity in coconut based on biochemical tests and molecular techniques, to study the evolutionary relationship among the bacteria isolates based on 16S rRNA BLAST analysis and Multiple Sequence Alignment. Eight bacteria isolates, 3 from fermented and 5 from non-fermented coconut were identified. Biochemical characterization, polymerase chain reaction amplification of 16S rRNA using universal primers (63f-1387r), BLAST analysis and multiple sequence analysis were performed. The result indicated that the bacteria were detected as Klebsiella pneumonia, Enterobacter aggromerans, Pseudomonas, Ralstonia pickettii and Burkholderia. The first two were carbohydrate fermenting while the last three were non-fermenting bacteria. All these species are related to human pathogenicity. This can be related to hygiene in food process and handling procedures. Therefore, to favour the selective growth of beneficial bacteria, appropriate environmental hygiene is required.