Bt Research 2012, Vol.3, No.3, 11
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improved. It may thus lead to the control of
agricultural pests that live within plants of major
economic importance to Brazil and other countries
worldwide.
3
Materials and Methods
The experiments were carried out in the Laboratories
of Entomopathogenic Bacteria, Apomixes and Electronic
Microscopy at Embrapa Genetic Resources and
Biotechnology, Brasília, Federal District of Brazil.
3.1
Strains of
B. thuringiensis
Four strains were used:
S1450–
B. thuringiensis
subspecies
kurstaki
HD
-
1 (
Btk) a standard obtained
from the Collection of
B. thuringiensis
and
Lysinibacillus
sphaericus
at the Pasteur Institute, France, and S1905,
S2122 and S2124 from the Bank of Bacteria of
Invertebrates at Embrapa Genetic Resources and
Biotechnology.
3.2
Plant material
Seeds of hybrid cabbage Astro Plus were used in
assays to evaluate colonization by the strains of
B.
thuringiensis
in cabbage seedlings grown
in vitro
.
3.3
Bacterial growth
The strains were cultivated in Embrapa medium
(
Monnerat et al., 2007) in a rotating incubator at
28
℃
,
for 72 hours and at 200 rpm. The lots of each
strain were viewed under a phase control optical
microscope with 1000× magnification to observe
spores and crystals.
3.4
Inoculation of
B. thuringiensis
strains in
cabbage seeds
Cabbage seeds were superficially disinfected in
ethanol 70% for 5 minutes, and in sodium hypochlorite
2 %
with Tween 20 added (0.01%) for 30 minutes.
After this period the seeds were washed three times in
sterile distilled water and were transferred to autoclaved
filter paper to remove the excess of water. All
procedures with seeds, bacteria and
in vitro
culture
were carried out in a laminar flow chamber. After
disinfecting, each lot of 18 seeds was treated with one
of the four strains of
B. thuringiensis
.
Sterile distilled
water and Embrapa medium were applied as negative
controls. The seeds were immersed in 10 ml of each of
the treatments for 5 minutes, and transferred to dry on
Petri dishes with filter paper. Once dry, the seeds were
inoculated into Petri dishes containing MS medium
(
Murashige and Skoog, 1962) solidified with 0.7% of
agar and at pH 5.8. For seed germination, the dishes
were incubated in a culture room at (25±2)
℃
in the
dark. The treatments were arranged completely
randomly on a shelf in the culture room, in a total of
six treatments with 18 repetitions of each. After three
days, the seedlings were transferred to glass flasks
with MS medium, where they were kept for another
27
days in the conditions described above, with a 12 h
photoperiod. The treatments were arranged completely
randomly on a shelf in the culture room, keeping the
six treatments, but with 10 repetitions of each
treatment.
3.5
Evaluation of development of cabbage seedlings
The seedlings were evaluated for percentage of
germination and for length of the seedlings three days
after they had been inoculated in the medium for
germination. At the first evaluation stage, the
germinated seedlings were measured with the help of
a V8 stereoscopic microscope, using the program
AxioVision, both from Zeiss. At the second stage, 27
days after transferring the seedlings to the glass flask,
10
seedlings were individually removed from the
medium, taking care not to damage their roots, and
were washed in water to remove the culture medium
and traces of agar; they were then dried with
absorbent paper. After this procedure, each of the
seedlings was weighed, the number of leaves was
counted and the leaf area and length of roots and aerial
part were measured. After drying them in a stove at
50
℃
for 16 hours, each seedling was weighed again
to determine the weight of dry material. The design
was completely random and the above variables were
compared by analysis of variance followed by the
Student Newman-Keuls test to compare averages;
when the data did not fulfill the necessary premises
non-parametric analysis of variance (Kruskal-Wallis)
was instead used, and the differences between
averages were compared by Dunn’s test (P<0.05) with
the help of the Sigma Stat program (Kuo et al., 1992).
3.6
Scanning Electron Microscopy
Samples of roots, stems and leaves from the plants
cultivated
in vitro
were fixed with glutaraldehyde
2.5%,
formaldehyde 2.5% and sodium cacodylate
buffer 0.1 M pH 7.2 for 24 h at 4
℃
.
After this stage,
the samples were submitted to three washing sessions
of 15 min in cacodylate buffer 0.1 M, followed by
