Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Abstract

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 ,um in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.

Genetic characterization of somatic recombination in Trichoderma pseudokoningii

“…Stasz and Harman (12) studied progenies obtained from many crossings involving different strains from different species of Trichoderma and also observed a very slow development of the recombinant colonies on selective media. Several studies of crossings between strains, within specie or between different species of Trichoderma, through hyphal anastomosis, protoplast fusion or transfer of isolated nuclei into protoplasts, have described the obtainment of unstable recombinants colonies of slow growth on selective media, and with a frequent sector formation (1,3,5,9,10,11,12).…”

Isolation of nuclei from Aspergillus nidulans protoplasts

“…Extractions and colorimetric assays of these macromolecules were carried out as described in MATERIALS AND MEXHODS. Because of the low amount of contamination and the good state of the isolated nuclei, the described protocol is potentially useful for studies on fungal nuclei, including genetic transfer by karyoduction [21,22].…”

Caracterização genética e citológica da recombinação somática em Trichoderma pseudokoningii.

“…interespecíficos (Bagagli et al, 1995;Furlaneto & Pizzirani-Kleiner, 1992;Pecchia & Anné, 1989;Pe'er & Chet, 1990;Silva & Pizzirani-Kleiner, 1999;Sivan et al, 1990;Stasz et al, 1988;Stasz et al, 1989e Stasz & Harman, 1990).…”

“…Alguns autores têm descrito análises de recombinantes instáveis de Trichoderma (Bagagli et al, 1995;Pe`er & Chet, 1990;Sivan et al, 1990;Stasz et al, 1988;Stasz et al, 1989; Figura 12 -Reamplificação de bandas de DNA (setas) isoladas dos géis de agarose das amplificações de RAPD (item 4.4). Com exceção da figura (e), todas as reações de reamplificação foram feitas a partir de bandas isoladas de reações de amplificação do DNA da linhagem parental 12L, no caso da figura (e) foi utilizada uma reação de amplificação do DNA da linhagem recombinante 18R.…”