Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

Abstract

Preparation and regeneration of mycelial protoplasts from Aliernaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 X 10*/gfresh weight mycelia) was obtained from 24-h-old mycelia digested with Novozym 234 (20 mg/m!) in a stabilized buffer of pH 6.4 and incubated in the dark at 30"C on a rotary shaker (90 r.p.m.) for 6h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration. ZusammenfassuDg HersteOung und Regeneration von Alternaria eicMtorniae-MycelprotoplastenUntersucht wurde die Regeneration von Protoplasten, die aus Alternaria eiehhorniae-Myzel hergestellt worden waren. Um die Protoplasten aus dem Myzel herstellen zu konnen, wurde das kaufliche Enzympraparat Novozym 234 angewandt. Das Alter des Myzels sowie der pH-Wert des stabilisierenden Puifers beeinflufite die Protoplastenbildung. Die maximale Protoplastenproduktion (3,9 X 107g Myzeifrischgewicht) wurde aus 24 h altem Myzel gewonnen, welches mit Novozym 234 (20 mg/ml) in einem stabilisierenden Puffer bei pH 6 verdaut und fur 6h bei 30°C ohne Licht auf einem Kreisschuttler (90 Upm) inkubiert wurde. Die morphologischen Merkmale der Protoplasten waren unterschiedlich und wurden durch das Alter des verwandten Myzels bestimmt. Daruberhinaus hatte das Myzelalter einen sehr signifikanten EinfluB (P = 0,0001) auf die Haufigkeit der Protoplastenregeneration.

Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae

“…For V. dahliae , previous studies have used 24-h-old mycelia for protoplast isolation [ 18 , 19 ], but here we obtained more protoplasts from the 20-h culture than from the 24-h culture. The probable reason for this can be the sensitivity of the younger mycelia to the digesting enzymes, with the increase in growth the cell wall becomes thicker and the mycelia would be digested more difficultly leading to decreased protoplast yield [ 36 , 38 , 39 ]. In addition to the age of mycelia, the protoplast yield also depends on the duration of the enzyme digestion, which has ranged from 3 h to 16 h for maximum protoplast release in other fungal species [ 37 , 40 , 42 ].…”

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33

“…TES and TES1 null mutants were generated by disrupting the targeted gene using homologous recombination, respectively ( Figure 3 A, C). Protoplasts were prepared from A. alternata ZJ33 as described previously with modifications [ 14 , 34 ]. A. alternata ZJ33 was cultured in YPG liquid medium at 28 °C for 48 h in a rotary shaker (180 rpm), and mycelia were harvested.…”

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains

“…The physiological stage of the mycelium plays a significant role in the release of protoplasts [ 62 , 63 ]. Here, the culture medium used had a great influence on the growth of A. niger .…”

“…For instance, A. niger conidia were cultured in 100 mL complete medium (CM) for 12 h (at 100 rpm) to generate protoplasts [ 14 ]. It seems that young mycelia are more susceptible to enzymatic digestion than old mycelia and that the cell wall of old mycelia becomes thicker [ 22 , 63 , 64 , 65 ].…”