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Characteristics of
protoplast
inter, intra-fusant
and regeneration of antagonistic fungi
Trichoderma
harzianum
and Trichoderma viride
Natesan Balasubramanian1*
and Damodaran Lalithakumari2
1CIRN
and Department of Biology, University of Azores,
9501-801
Ponta Delgada, Azores,
Portugal.
2Centre
for Advanced Studies in Botany, University of Madras, Guindy
campus Chennai-600 025, India.
*Corresponding author. E-mail:
yenbala2007@gmail.com.
Tel: +351- 296 650 000- Ext1470.
Fax:+351-296650100.
Accepted 3 September, 2008 |
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A standard method for isolation, fusion and regeneration of
protoplasts from Trichoderma harzianum and
Trichoderma viride was developed. The protoplasts from
T. harzianum and T. viride were isolated using
Novozym 234 as lytic enzyme and potassium chloride as
osmotic stabilizer. The maximum number of protoplasts (9.37
× 105/ml) was obtained from 16 h old mycelium of
T. harzianum and 11.1 × 105/ml for T.
viride from 14 h old mycelium at pH 5.5, 28ºC for 3 h.
The interspecific and intraspecific fusion frequency was
determined using 40% polyethylene glycol (PEG) as fusogen.
The intrafusants were selected based on their growth,
sporulation, pigmentation on chitin and cellulose amended
media, where as the interfusants were selected on fungicide
resistance as a marker. The protoplast fusion frequency was
found to be 1.92% for interspecific fusion. In the case of
intraspecific fusion it was about 6.2 and 7.2%,
respectively, for T. harzianum and T. viride.
The protoplast regeneration frequency of intrafusant was 17%
for T. harzianum on chitin medium and 19.2% for T.
viride on cellulose medium after two days. The
regeneration frequency of 11% for interfusants on fungicide
amended medium was observed after three days. The
regenerated fusants morphology, growth, sporulation and
pigmentation were compared with parental strains.
Keywords:
Fusion, isolation, regeneration, Trichoderma harzianum,
Trichoderm viride. |
