NEUROSPORA LIFE CYCLE AND SINGLE GENE INHERITANCE

JACK KENNELL

INTRODUCTION

Fungi display a wide variety of lifecycles which alternate between haploid and diploid generations. Among the fungi one may find those which propagate asexually, and have no diploid phase in their lifecycle. There are those which exist mainly in the haploid phase, but can enter a sexual cycle resulting in a brief diploid phase. Still others exist in the diploid phase for an extended period during their lifecycle. In this experiment we will observe the lifecycle of a filamentous fungi called Neurospora crassa. This fungus, which incidentally is responsible for some bread molds, is useful for genetic research because its dominant phase is haploid. Genetic traits can be most easily studied in haploid phases of organisms, since a recessive trait cannot be obscured by a second, dominant copy, as may be present in diploid organisms.

OBJECTIVES

A. Demonstrate the sexual and asexual cycle of a filamentous fungi.

B. Follow the inheritance of a single genetic trait.

PROCEDURE

A. Propagation by Asexual Conidiaspores - Week 1

1. Obtain two test tubes, one containing the Neurospora with conidia, and one which contains normal agar media, only.

2. Using your best sterile techniques, draw approximately 1 ml of sterile water into a sterile pipet and add the water to the Neurospora test tube. Mix conidia and water with the tip of the pipet, then draw up the conidia suspension.

3. Add one drop of the suspension to the test tube containing normal agar media, and allow to grow at room temperature until next week.

Neurospora Conidia Color

Transfered to

Resulting growth

B. Inducing the Sexual Phase of the Lifecycle - Week 1

1. Obtain two test tubes, one containing the same Neurospora used in part A, and a larger one which contains nitrogen-deficient agar media.

2. Add one drop of the suspension of Neurospora conidia to the large test tube containing nitrogen-deficient agar media, and allow to grow at room temperature until next week.

Neurospora Conidia Color

Transfered to

Resulting growth

C. Sexual Reproduction and Resulting Ascospores - Week 1

1. Obtain two test tubes, one containing the same Neurospora used in part A, and another which contains Neurospora of the opposite mating type that has been grown in a nitrogen-deficient agar media. These are identical to the ones which you prepared in part B., but have had a week to mature.

2. Add 5-6 drops of the same conidia suspension prepared from Neurospora in part A, to the large test tube containing Neurospora protoperithecium, and allow to grow at room temperature until next week.

Neurospora Conidia Color

Transfered to

Resulting growth

D. Germination of Ascospores - Week 1

1. Obtain the test tube containing mature, black perithecia with ascospores. This growth, prepared several weeks ago, resulted from the same cross that you performed in part C.

2. Using your best sterile techniques, draw approximately 1 ml of sterile water into a sterile Pasteur pipette and add the water to the test tube containing parithecia with ascospores. Mix ascospores and water with the tip of the pipette, then draw up the suspension.

3. Add the suspension to a small eppendorf tube. Cap the tube and incubate at 60 C for 45 minutes.

4. After 45 minutes, use a pipet to remove the suspended liquid from the tube, and place it onto the sorbose and agar media in a petri plate. Spread the liquid over the agar. Allow to grow at room temperature until next week.

Ascospores from

Transfered to

Resulting growth

LIFE CYCLE OF NEUROSPORA

CONCLUSION — Week 2

1. When placing conidia onto normal agar, what type of propagation occurs? How do the conidia produced by the new cultures compare in color those of the culture from which they were started?

2. What factor induces the Neurospora to begin the sexual phase of the lifecycle?

3. What change in appearance do you notice when the Neurospora are growing in the sexual phase of the lifecycle?

4. What new structures are produced during the sexual phase of the lifecycle?

5. Sexual reproduction results in the production of a diploid cell, which quickly undergo meiosis to produce the haploid ascospores. In what structure does this occur?

6. Can ascospores be produced by combining conidia and protoperithecium from the same mating strain? How do you know?

7. If you cross a white conidia- producing Neurospora with an orange conidia- producing Neurospora, can you predict how many of the offspring will produce orange conidia? Do your results in part D. agree with your assumption?

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