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Mutation in Neurospora Crassa with Leaf Extract of Citrus Aurantifolia and Their Soluble Protein Content

APURBA LAL RAY
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

MAHBUBA
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

AKHTER JAHAN
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

TAHSINA RAHIM
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

Abstract/ Executive Summary:

Leaf extract of Citrus aurantifolia exhibited remarkable inhibitor effect on the radial mycelial growth of Neurospora crassa. The extract also showed mutagenic effect and atleast six morphological mutants of the fungus were detected including albino (al 243), vigorous (vg 117), fluffy (fl 220), colonial (cl 232), conidial band (con. band 171) and dirty (dir 83). The mutants were used for estimation of soluble protein in comparison with the wild type (Ema). The soluble protein content increased to some extent in case of the mutants con. band 171 (192.86 μg/ml), cl 232 (188.57 μg/ml) and vg 117 (186.43 μg/ml) as compared to the wild type (182.14 μg/ml). On the other hand, the soluble protein content was remarkably decreased in case of the mutant al 243 (94.28 μg/ml), which was about 50% less than the control. This indicates that the leaf extract not only effect colony morphology but possesses profound effect on growth and metabolism of the fungus.

Keywords: Neurospora crassa, Mutation, Leaf extract, Soluble protein

Location: Microbial Genetics Stock, Department. of Botany, University of Dhaka, Dhaka

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Bread

Objectives:

1. Leaf extract of Citrus aurantifolia (Chist) SW was used as natural chemical mutagen for induction of mutation in N. crassa. The mutants were then used for estimation of water soluble protein.

Methodology:

Mutation in Neurospora crassa was induced by using leaf extract of Citrus aurantifolia (Chist) SW. The wild type isolate of N. crassa was obtained from Microbial Genetics Stock, Department. of Botany, University of Dhaka, Dhaka. Vogel’s minimal medium(8) was used for culturing N. crassa in the test tube and also to study the mutagenic effect on culture plate. Sorbose minimal medium (SM) was used for single colony isolation. To test mutagenic effect of Citrus aurantifolia leaf extract on the growth of N. crassa, different concentrations of the extract was prepared with sterilized distilled water. Fungal growth was determined by measuring the radial growth of mycelia of Ema up to 30 hr. For mutation in N. crassa, 4 ml of C. aurantifolia leaf extract was taken into a centrifuge tube and one loop of conidia of Ema was added to it and shaken for homogenous suspension. The tube was kept for 2 hr for mutation and thereafter the suspension was centrifuged and the supernatant above conidia was poured out from the centrifuge tube. The conidial plate was re?suspended with 4 ml of sterile distilled water and centrifuged for 5 minutes. The same procedure was repeated three times. Finally treated conidia were suspended in 1 ml of sterilized distilled water and used as inoculum. One drop of the conidial suspension was mixed with 10 ml of molten SM medium in sterilized Petri dish. The Petri dishes were kept inside the incubator at 25⁰C for 24 hr for growth. Well separated colonies were isolated from each Petri dish by cutting agar blocks from the fungal colonies and were inoculated into small tubes containing VM medium. After five days, all the cultures were observed and classified by comparing their characters with wild type Ema. For estimation of soluble protein content of wild and mutants of N. crassa (Ema), the organisms were grown in VM liquid medium (100 ml) in conical flask for up to 72 hr. After incubation, the cultures were filtered and dried. Dry mycelia of the organisms were crushed with mortar and pastle within ice box. Before that, liquid nitrogen was used to breakdown the cell wall of the organisms. The fine crushed mycelia were taken in Eppendorf tube and centrifuged at 13,000 rpm for 10 minutes to remove cell debris. The supernatant of the samples was taken for estimation of soluble protein by the method of Lowry et al. using bovine serum albumin (BSA) as standard. In each case, 20 μl of sample (supernatant) was taken with 980 μl of sterilized distilled water (total volume 1 ml) for estimation of soluble protein.

Source of Information: Dhaka Univ. J. Biol. Sci. 19(2): 151‐155, 2010 (July)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It is reported that Neurospora has been used in several human societes; either as a food or in processing foods and beverages. It is a major constituent of onchom, a highly nutritious soybean?based presscake produced for human consumption as a cottage industry in West Java. It is also used in preparing oriental foods such as Koji. So, N. crassa and its mutants can also be used in processing valuable foods and beverages in Bangladesh.

Knowledge Management: Journal