Livro das Actas - advid

Livro Actas
Other important factors, determining the efficiency of ATMT, are the co-cultivation conditions.
These include the concentration of A. tumefaciens and fungal recipient, temperature,
pH, length of the co-cultivation time and filters type. During the design of our
experimental setup, we optimized the concentration of the fungus as well as filters type.
The concentration of the fungus is important because the addition of too many fungal
recipients can result in too much fungal growth during co-cultivation, which makes the
subsequent isolation of single transformants difficult (Covert et al. 2001). Based on these
previous findings, conidia of each strain were collected and resuspended in water and
their concentration adjusted to 103 – 108 conidia ml-1 . The conidial suspension was mixed
with an equal volume of A. tumefaciens culture, prepared as described by Covert et al.
(2001). A 200µl aliquot of this mixture was plated on induction medium (IM) amended
with 200µM acetosyringone. The greatest number of transformants was collected from
experiments in which the number of conidia co-cultured with A. tumefaciens was 104 or
105 /plate. Other concentration of conidia either yielded no transformant (i. e. 103 / plate)
or too much fungal growth to identify transformant colonies (i.e. 106 /plate or higher).
As for the type of filter that can be used in the co-cultivation step, previous studies have
employed nitrocellulose, Hybond N + , filter paper, cellophane sheets polyvinylidene
difluoride (Michielse et al. 2005). The experiments were carried out by testing the efficiency
of transformation by co-incubation on nitrocellulose filter with 0.45µm pores
(Whatman) or Hybond N + (Amersham International). It was found that both types of filters
resulted in equally high transformation rates.
The last parameter that it was necessary to optimize was the level of sensitivity of the
fungi to hygromycin B. B. cinerea B0510, A. alternata Aa1 and A. carbonarius
CECT2086 strains were tested for hygromycin B sensitivity using different concentrations
from 50 – 500 µg/ml. Freshly grown mycelial edges were transferred to potato
dextrose agar amended with hygromycin B, and the optimal concentration was determined
by each fungal species.
Once optimized main parameters that can affect the efficiency of transformation, we
have carried out the Agrobacterium tumefaciens- mediated transformation of B. cinerea,
A. alternate and A. carbonarius using the protocol described by de Yan et al. (2010)
with the subsequently modifications. A. tumefaciens strain AGL-1 was grown at 28 ºC
for 2 days in minimal medium (MM) supplemented with kanamycin (100µg/ml). The
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