Increase in germination and plating efficiency of Neurospora
crassa microconidia by amino acid supplementation
Kandasamy Pitchaimani, Shahana Sultana and Ramesh Maheshwari. Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
A major difficulty that has limited the use of uninucleate microconidia in genetic research is their low and erratic germination. We found that the supplementation of sorbose plating medium by amino acids, notably aspartic acid and methionine, markedly improved germination and plating efficiency of microconidia of mcm and pe fl genotypes of N. crassa. The plating efficiency of mcm microconidia in amino acid supplemented medium was comparable to macroconidia.
N. crassa produces very small numbers of uninucleate microconidia compared to multinucleate macroconidia. If microconidia can be selectively obtained uncontaminated with macroconidia, they would be preferred for use in DNA-mediated transformation, analysis of heterokaryons, and recovery of recessive mutants from wild isolates. This problem is overcome by use of microconidial genotypes or by the use of cellophane culture technique that allows microconidia to be selectively obtained even from wild type strains (Maheshwari 1999 Fungal Genet. Biol. 26: 1-26). However, the problem of erratic or low germination and plating efficiency had remained despite attempts to overcome it (Kalpana et al. 1998 Fungal Genet. Newsl. 45: 19). An observation that macroconidia of a histidine-transformant showed an unusual, non-specific requirement of amino acids for optimal germination led us to examine the effect of amino acid supplementation on plating efficiency of microconidia with surprising results.
The mcm; A genotype (FGSC #7455) produces microconidia by microcycle conidiation when macroconidia from agar grown cultures are incubated in liquid shake cultures for 24 h (Maheshwari 1991 Exp. Mycol. 15: 346-350) whereas pe fl; A (FGSC #3072) and fl;dn; A (FGSC #3517) produces microconidia in agar-grown cultures in 6-8 d. Microconidia (200 or 500, based on haemocytometer counting) were spread on sorbose plating medium (Davis and de Serres 1970 Methods Enzymol. 27A: 79-143). The amino acids (Sigma) were added before autoclaving. The plates were kept at 34C in dark and the colonies were counted on the fourth or the fifth day. The results of mcm and pe fl are from three platings. Microconidia of fl; dn were plated once. Three replicates were used for each plating.
Although we adhered to nearly the same conditions, the plating efficiency of microconidia varied in different platings of the same strain. Our experiments were limited to the strains mentioned above. Addition of 0.1% vitamin-free casamino acids (Difco) markedly improved plating efficiency of both mcm and pe fl microconidia (Table 1); the colonies appeared earlier (day 2) than in control plates (day 4). Plating efficiency of fl; dn microconidia was very low; therefore this strain was not used further. Of the individual amino acid tested, aspartic acid and to a lesser extent, methionine, were most effective. With amino acid supplements, the plating efficiency of mcm microconidia was comparable to macroconidia of different genotypes (55-90%) that have been studied in our laboratory. Alanine, cysteine, glutamic acid, serine and tryptophan improved plating efficiency marginally whereas arginine, glycine, lysine, phenylalanine and histidine had no effect. At the concentrations tested, leucine and threonine were inhibitory. The mechanism of stimulation by amino acid is not known but it is clear that they only triggered germination. The results also show that genotype of strain has a marked influence on microconidial germination.
Table 1. The effect of amino acid supplementation on plating efficiency of microconidiaa
|Amino acid||% colonies ± s.d.|
|mcm; A||pe fl: A||fl; dn; A|
|Control||20 ± 15||9 ± 5||0.6|
|Alanine||46 ± 10||17 ± 7||0.2|
|Arginine||16 ± 9||12 ± 9||0.8|
|Aspartic acid||65 ± 14||55 ± 11||7|
|Casamino acid||76 ± 14||32 ± 9||0.7|
|Cysteine||46 ± 29||44 ± 19||4.5|
|Glutamic acid||45 ± 10||38 ± 18||2.4|
|Glycine||21 ± 19||14 ± 8||n.d.|
|Histidine||32 ± 10||13 ± 7||0.1|
|Leucine||8 ± 5||7 ± 3||2|
|Lysine||23 ± 5||29 ± 7||2|
|Methionine||59 ± 25||55 ± 13||2|
|Phenyl alanine||31 ± 11||16 ± 7||0.06|
|Serine||45 ± 9||20 ± 7||0.1|
|Threonine||2 ± 1||2 ± 2||0.2|
|Tryptophan||48 ± 4||22 ± 5||0.6|
|Tyrosine||56 ± 7||15 ± 7||n.d.|
aAmino acids were used at following concentrations: alanine and glutamic acid at 40 mg/100 ml; arginine, cysteine, glycine, lysine, methionine and serine at 50 mg/100 ml; aspartic acid at 100 mg/100 ml; histidine, leucine, phenylalanine, tyrosine and tryptophan at 20 mg/100 ml; and threonine at 7.5 mg/100 ml. n.d. not determined.
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