UV TREATMENT IN MONUSCUS SP.

Presented by Steve Irwin

INTRODUCTION Monuscus sp is a fungus that belong to class

ascomycota and family Monasaceae. Under microscope Monuscus purpureus appear filamentous fungi. Can be obtained from tofu and rice.

Some special characteristics of this fungi are : This fungus bears asexual reproducing spores called conidia and bears sexual reproducing ascus. Monuscus purpurea produces red and yellow pigment.

Monuscus sp have many commercial and medicinal use.

COMMERCIAL AND MEDICINAL USAGE1. Pigment is used as food dyes, preservative,

and food condiments.2. Ingredient in making rice wine, tofu, etc. 3. Red rice is used as medicine to treat

dysentery, external and internal trauma4. Fermentate of this fungus is used to bring

down cholesterol level and triglyceride level.

5. This fungus also contain antibiotic property.

OBJECTIVE OF THE EXPERIMENT The main objective of the lab was to produce

colored mutant and albino variety of Monuscus sp. by UV treatment and observe the morphology of the various mutants.

Ultraviolet light causes DNA mutation in some cases by forming pyrimidine dimers ‘T-T’ (Klug et al. 2007).

Prediction was to observe change in colony morphology, or reduced pigmentation in the Monuscus sp.

GROWTH MEDIUMS FOR THE FUNGUS PDA – Potato Dextrose Agar. 39 grams of PDA

powder in 1000 ml of distilled water and autoclave.

MYS- Malt Yeast Starch. 30 grams cassava starch, 3 gram malt extract, 3 gram yeast extract, 5 gram peptone, 15 gram agar in 1000 ml distilled water and autoclave.

White Rice.

FUNGAL CULTURE Thai Vietnam

Isolate fungus PDA

Store Grow on PDA. 2 Weeks incubation.

Glycerol Suspension

Isolate single spore conidia.3 days incubation on PDA

Single spore clones

UV treatment Grow on MYS to see Mutation

Store

UV TREATMENT In order to do UV treatment fungus must be

cloned from conidia and grown in PDA. Glycerol suspension was made with

monoclonal fungus with 1% glycerol. The UV treatment can be done by taking 1 ml

glycerol suspension into empty Petri dishes and treat it in 20 cm, 40 cm, 60 cm in different times such as 20 min, 40 min, 60 min.

Sample Time Distance 1

Distance 2

Vietnam 10 single spore # 1 (V10 SS 1)

20 minutes 20 cm 40 cm40 minutes 20 cm 40 cm60 minutes 20 cm 40 cm

Sample Time Distance 1

Distance 2

Vietnam 6 single spore # 2 (V10 SS2)

20 minutes

20 cm 40 cm

40 minutes

20 cm 40 cm

60 minutes

20 cm 40 cm

RESULTS OF ULTRAVIOLET TREATMENT

Sample (Monuscus purpurea.)

UV Treatment

TimeDistance Observations

Vietnam 10 (V10 SS 1 )

20 Minutes

20 cm

Negative

40 Minutes Negative

60 Minutes Negative

Vietnam 10 (V10 SS 1)

20 Minutes

40 cm

Negative

40 Minutes Negative

60 Minutes Negative

Sample (Monuscus purpurea.)

UV Treatment Time Distance Observations

Vietnam 6 Single Spore 2 (V6 SS 2 )

20 Minutes

20 cm

Negative

40 Minutes

White sector was observed in UV treated colonies

compared to normal.

60 Minutes

White colonies compared to

Normal colony. Sectors of different

morphology.

Vietnam 6 single spore 2 (V6 SS 2)

20 Minutes

40 cm

Negative

40 Minutes

Mutation was observed and

white colony of fungus was seen

without pigments.60 Minutes Negative

SUB CULTURING MUTANT V6 SS 2 mutant was sub-cultured into PDA and

MYS if more than 2 times in presence of light will reverse pigmentation in the Monuscus sp.

Wong, C., and Lin, Y., et al. reported that slightly colored and albino mutants of Monuscus purpureus produced pigments after having been exposed to UV for 5 minutes.

B) Vietnam 6 single spore 2 colonyA) Vietnam 6 single

spore 2 Mutant 3 colony

Figure 1.1

Figure 1.3:- V6 SS2 M1a Pigment less single spore colony of Monuscus purpureus on MYS.Date of Sub culturing: April 2, 2009.Date of Snap Shot: April 6, 2009.

Figure 1.2

A) B)

Figure 1.3:- V6 SS2 M3b comparison between albino and normal V6 SS2 pigment production on PDA. Date of Sub culturing: April 2, 2009.Date of snap shot: April 6, 2009.

Figure 1.3

A) B)

Figure 1.4:- V6 SS2 M3b comparison between albino and normal V6 SS2 pigment production on PDA. Date of Sub culturing: April 2, 2009.Date of Snap Shot: April 9, 2009.

Figure 1. 4

A) B)

Figure 1.5: - Comparison between normal pigment producing V6 SS2 in rice medium and pigment less mutant V6 SS2 M3 in rice medium. Date of inoculation: - April 13, 2009Date of snap shot: - April 24, 2009

Figure 1. 5

DISCUSSIONUV treatment produced mutant variety at

distance 20 cm and 40 cm, in 40 minutes and 60 minutes.

Reverse in pigment production in albino

mutant was observed after sub-culturing the mutant more than two times in presence of light.

Albino mutant can be retained in dark in MYS medium.

CONCLUSIONIt was possible to produce mutant variety of

Monuscus sp with Ultra Violet light. It can be predicted that Ultra violet light

interrupt with red pigment producing gene.

For further studies, molecular techniques can be used to isolate red pigment gene.

ACKNOWLEDGEMENT I would like to thank Dr. Kung for guiding me

through out the project. I would like to think Ms. Sonya and Isha also for helping me when I needed their help.

REFERENCE Kranz, C. Pantiz, C. and Kunz, B. (1992). Biotransformation of Free Fatty Acids

in Mixtures to Methyl Ketones by Monuscus Purpureus. Applied Microbiology and Biotechnology, 36, pp. 436-439.  

Lin, Ching-Fwu. (1973). Isolation and Cultural Conditions of Monuscus sp. for the Production of pigment in a Submerged Culture. Journal of Fermentation and Technology, 51 (6), pp. 407-414.

Lotong, N. and Suwanarit, P. (1989). Fermentation of ang-kak in plastic bags and regulation of pigmentation by initial moisture content. Journal of Bacteriology, 68, pp. 567-570. 

Omamor, B.I. Eziashi, E. I. and Adekunle, A.A. (2008). Carbon Nutrition in Relation to Growth of Three Monuscus sp. isolated from decaying date fruits. African Journal of Microbiological Research, 2, pp. 153-155.

Wong, Hin-Chung and Bau, Yun-Shen. (1978). Morphology and Photo responses of Fast-Neutron and X-Ray-Induced Strains of Monuscus Purpureus. Mycologia, 70, pp. 645-648.  

Wong, Hin-Chun and Koehler, P. (1981). Production and Isolation of an Antibiotic from Monuscus Purpureus and its relationship to pigment production. Journal of Food Science, 46 (2), pp. 589-592. 

Wong, H., C. Lin, Y., C. and Koehler, P. E. (1981). Regulation of Growth and Pigmentation of Monuscus purpureus By Carbon and Nitrogen Concentrations. Mycologia, 73, pp. 649-654.

Klun, William, S. Cummings, Michael, R. and Spencer, Charlotte, A. (2007). Gene Mutation, DNA Repair and Transcription. In: Klun, William, S. Cummings, Michael, R. and Spencer, Charlotte, A. Essentials of Genetics. 5th Edition. USA: William S. Klug and Michael R. Cummings, pp. 314-315.

Anonymous. (2001). Red Yeast Rice (Monuscus purpureus). [Online] Penton Media. Available from: http://www.healthwell.com/healthnotes/Herb/Red_Rice.cfm?path=hw [25 September 2001]